Daniel B. Rifkin, Ph.D.

An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-beta Activation in the Bone Microenvironment
Thiolloy, Sophie; Edwards, James R; Fingleton, Barbara; Rifkin, Daniel B; Matrisian, Lynn M; Lynch, Conor C
2012 ;7(1):e29862-e29862, PLoS ONE
BACKGROUND: Breast to bone metastases frequently induce a 'vicious cycle' in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment. METHODOLOGY/PRINCIPAL FINDINGS: To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (muCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFbeta, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays). CONCLUSION/SIGNIFICANCE: Collectively, these studies identify a novel 'mini-vicious cycle' between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFbeta would be beneficial for the treatment of bone metastases
— id: 149981, year: 2012, vol: 7, page: e29862, stat: Journal Article,

LTBPs, more than just an escort service
Todorovic, Vesna; Rifkin, Daniel B
2012 Feb;113(2):410-418, Journal of cellular biochemistry
Latent transforming growth factor beta (TGF-beta) binding proteins (LTBPs) are large extracellular glycoproteins structurally similar to fibrillins. They perform intricate and important roles in the extracellular matrix (ECM) and perturbations of their function manifest as a wide range of diseases. LTBPs are major regulators of TGF-beta bioavailability and action. In addition, LTBPs interact with other ECM proteins-from cytokines to large multi-factorial aggregates like microfibrils and elastic fibers, affecting their genesis, structure, and performance. In the present article, we review recent advancements in the field and relate the complex roles of LTBP in development and homeostasis. J. Cell. Biochem. 113: 410-418, 2012. (c) 2011 Wiley Periodicals, Inc
— id: 149808, year: 2012, vol: 113, page: 410, stat: Journal Article,

Binding of Anti-SSA Antibodies to Apoptotic Fetal Cardiocytes Stimulates Urokinase Plasminogen Activator (uPA)/uPA Receptor-Dependent Activation of TGF-beta and Potentiates Fibrosis
Briassouli, Paraskevi; Rifkin, Daniel; Clancy, Robert M; Buyon, Jill P
2011 Nov 15;187(10):5392-5401, Journal of immunology
In congenital heart block (CHB), binding of maternal anti-SSA/Ro Abs to fetal apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases urokinase plasminogen activator (uPA)/uPA receptor (uPAR)-dependent plasmin activation. Because the uPA/uPAR system plays a role in TGF-beta activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF-beta, thereby promoting a profibrosing phenotype. Supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes bound by IgG from a mother whose child had CHB (apoptotic-CHB-IgG [apo-CHB-IgG]) exhibited significantly increased levels of active TGF-beta compared with supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor. Treatment of the culture medium with anti-TGF-beta Ab or TGF-beta inhibitor (SB431542) abrogated the luciferase response, thereby confirming TGF-beta dependency. Increased uPA levels and activity were present in supernatants generated from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes compared with healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor, respectively. Treatment of apo-CHB-IgG cardiocytes with anti-uPAR or anti-uPA Abs or plasmin inhibitor aprotinin prior to coculturing with healthy cardiocytes attenuated TGF-beta activation. Supernatants derived from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes promoted Smad2 phosphorylation and fibroblast transdifferentiation, as evidenced by increased smooth muscle actin and collagen expression, which decreased when fibroblasts were treated with supernatants from cocultures pretreated with uPAR Abs. These data suggested that binding of anti-Ro Abs to apoptotic cardiocytes triggers TGF-beta activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promotes myofibroblast transdifferentiation and scar
— id: 140530, year: 2011, vol: 187, page: 5392, stat: Journal Article,

Control of lung development by latent TGF-beta binding proteins
Dabovic, Branka; Chen, Yan; Choi, Jiwon; Davis, Elaine C; Sakai, Lynn Y; Todorovic, Vesna; Vassallo, Melinda; Zilberberg, Lior; Singh, Amanjot; Rifkin, Daniel B
2011 Jun;226(6):1499-1509, Journal of cellular physiology
The latent TGF-beta binding proteins (LTBP-1 -3, and -4) assist in the secretion and localization of latent TGF-beta molecules. Ltbp3(-/-) and Ltbp4S(-/-) mice have distinct phenotypes and only in the lungs does deficiency of either Ltbp-3 or Ltbp-4 cause developmental abnormalities. To determine if these two LTBPs have additional common functions, we generated mice deficient for both Ltbp-3 and Ltbp-4S. The only novel defect in Ltbp3(-/-);Ltbp4S(-/-) mice was an early lethality compared to mice with single mutations. In addition lung abnormalities were exacerbated and the terminal air sac septation defect was more severe in Ltbp3(-/-);Ltbp4S(-/-) mice than in Ltbp4S(-/-) mice. Decreased cellularity of Ltbp3(-/-);Ltbp4S(-/-) lungs was correlated with higher rate of apoptosis in newborn lungs of Ltbp3(-/-);Ltbp4S(-/-) animals compared to WT, Ltbp3(-/-), and Ltbp4S(-/-) mice. No differences in the maturation of the major lung cell types were discerned between the single and double mutant mice. However, the distribution of type 2 cells and myofibroblasts was abnormal, and myofibroblast segregation in some areas might be an indication of early fibrosis. We also observed differences in ECM composition between Ltbp3(-/-);Ltbp4S(-/-) and Ltbp4S(-/-) lungs after birth, reflected in decreased incorporation of fibrillin-1 and -2 in Ltbp3(-/-);Ltbp4S(-/-) matrix. The function of the lungs of Ltbp3(-/-);Ltbp4S(-/-) mice after the first week of life was potentially further compromised by macrophage infiltration, as proteases secreted from macrophages might exacerbate developmental emphysema. Together these data indicate that LTBP-3 and -4 perform partially overlapping functions only in the lungs
— id: 138140, year: 2011, vol: 226, page: 1499, stat: Journal Article,

Development of a novel multiplexed-TGFbeta assay
Hardee M.E.; Marciscano A.E.; Rifkin D.B.; Barcellos-Hoff M.
2011 ;81(2 SUPPL 1):S755-S755, International journal of radiation oncology biology physics
Purpose/Objective(s): Transforming growth factor-beta (TGFbeta) levels are increased by radiation therapy. Pre-and post-radiotherapy systemic levels of TGFb measured by ELISA, mink lung epithelial cell luciferase assay (MLEC), and cell-based biological assays have been correlated with treatment outcomes and radiation-induced toxicity. However, aspects of these assays and TGFbeta biology can make TGFb measurements difficult to interpret. TGFbeta is secreted along with the latency associated peptide (LAP) that is activated extracellularly when needed; available assays measure active TGFbeta using artificial activation by acid or heat. Specimen contamination by platelets is another source of variability since latent TGFbeta is present in platelets in large quantities. Our goal is to develop an assay that eliminates artificial activation, includes an internal control for platelet contamination, and can reproducibly measure both active and latent TGFbeta. Materials/Methods: To measure active TGFbeta1, a commercially available TGFbeta1 ELISA using the Meso Scale Discovery (MSD) platform was used. MSD uses electrochemiluminescent detection that provides sensitivity over a concentration range of 5 logs and allows multiplexing of up to 10 assays. An ELISA for LAP-b1, a measure of latent TGFbeta1 was developed. Different preparations of plasma and serum were tested and contamination from platelet degranulation was measured using an ELISA of platelet factor-4 (PF4), which is released from a-granules of activated platelets. Results: LAP-s1 and PF4 assays using the MSD platform were successfully developed using commercially available antibodies. LAP-b1 ELISA was a highly reproducible measurement of total TGFbeta, as verified using MLEC assay of exogenously activated samples. Measurement of total TGFb in serum (19,085-156,058 pg/mL) was high and variable due to contamination by platelet activation as measured by PF4. By spiking plasma with increasing volumes of serum (to replicate platelet contamination), we determined a PF4 cutoff value of 1.5 mg/mL, below which total TGFbeta levels were constant. Collection of plasma in EDTA containing tubes (vs heparin tubes) and double centrifugation was most effective at reducing platelet contamination and lead to the most reproducible TGFbeta measurements that averaged 344 pg/ml.We determined that plasma contains undetectable levels of active TGFbeta but that as little as 62 pg/mL exogenous TGFbeta could be detected in plasma. Conclusions:We developed an improved TGFbeta assay for biological fluids that simultaneously measures active and latent TGFbeta. By avoiding the uncertainty introduced by exogenous activation in currently available assays and including an internal control for platelet contamination, this assay will be more robust for future correlative studies of systemic TGFbeta levels and radiation treatment outcomes and toxicity
— id: 150889, year: 2011, vol: 81, page: S755, stat: Journal Article,

Active and total transforming growth factor-{beta}1 are differentially regulated by dopamine and estradiol in the pituitary
Recouvreux, M Victoria; Guida, M Clara; Rifkin, Daniel B; Becu-Villalobos, Damasia; Diaz-Torga, Graciela
2011 Jul;152(7):2722-2730, Endocrinology
Dopamine, acting through the dopamine type 2 receptor (Drd2), is the main inhibitor of pituitary prolactin (PRL) secretion and lactotroph proliferation. TGF-beta1 is involved, at least in part, in mediating these actions. It was described that TGF-beta1 synthesis in rat pituitary lactotrophs is up-regulated by dopamine and down-regulated by estradiol. TGF-beta1 is secreted as a large latent complex. The local regulation of cytokine activation in the pituitary has not yet been explored. In this work, we studied pituitary active and total TGF-beta1 content, as well as TGF-beta1 mRNA, and the in vivo role of dopamine and estradiol on pituitary TGF-beta1 levels. Adult female mice (wild type), and female mice with a null mutation in the Drd2 (Drd2(-/-)), were used. The loss of dopaminergic tone induced a decrease in TGF-beta1 mRNA expression, in active and total cytokine content, and in TGF-beta type II receptor expression. Dopamine regulation of pituitary TGF-beta1 activation process was inferred by the inhibition of active cytokine by in vivo sulpiride treatment. Interestingly, in the absence of dopaminergic tone, estradiol induced a strong increase in active TGF-beta1. PRL secretion correlated with active, but not total cytokine. TGF-beta1 inhibitory action on lactotroph proliferation and PRL secretion was decreased in Drd2(-/-) pituitary cells, in correlation with decreased TGF-beta type II receptor. The study of the TGF-beta1 activation process and its regulation is essential to understand the cytokine activity. As an intermediary of dopamine inhibition of lactotroph function, TGF-beta1 and local activators may be important targets in the treatment of dopamine agonist-resistant prolactinomas
— id: 136472, year: 2011, vol: 152, page: 2722, stat: Journal Article,

Long form of latent TGF-beta binding protein 1 (Ltbp1L) regulates cardiac valve development
Todorovic, Vesna; Finnegan, Erin; Freyer, Laina; Zilberberg, Lior; Ota, Mitsuhiko; Rifkin, Daniel B
2011 Jan;240(1):176-187, Developmental dynamics
Transforming Growth Factor beta (TGF-beta) is crucial for valve development and homeostasis. The long form of Latent TGF-beta binding protein 1 (LTBP1L) covalently binds all TGF-beta isoforms and regulates their bioavailability. Ltbp1L expression analysis during valvulogenesis revealed two patterns of Ltbp1L production: an early one (E9.5-11.5) associated with endothelial-to-mesenchymal transformation (EMT); and a late one (E12.5 to birth) contemporaneous with valve remodeling. Similarly, histological analysis of Ltbp1L(-/-) developing valves identified two different pathologies: generation of hypoplastic endocardial cushions in early valvulogenesis, followed by development of hyperplastic valves in late valvulogenesis. Ltbp1L promotes valve EMT, as Ltbp1L absence yields hypoplastic endocardial cushions in vivo and attenuated EMT in vitro. Ltbp1L(-/-) valve hyperplasia in late valvuogenesis represents a consequence of prolonged EMT. We demonstrate that Ltbp1L is a major regulator of Tgf-beta activity during valvulogenesis since its absence results in a perturbed Tgf-beta pathway that causes all Ltbp1L(-/-) valvular defects. Developmental Dynamics, 2011. (c) 2010 Wiley-Liss, Inc
— id: 116226, year: 2011, vol: 240, page: 176, stat: Journal Article,

Binding of apoptotic fetal cardiocytes by anti-Ro/La antibodies stimulates uPA/uPAR-dependent activation of TGFbeta and potentiates fibrosis
Briassouli P.; Rifkin D.; Buyon J.P.; Clancy R.M.
2010 ;62:489-489, Arthritis & rheumatism
Purpose: Organ injury induced by antibodies characteristic of Sjogren Syndrome and Systemic Lupus Erythematosus, while varied in the adult and fetus, may share in common a link between apoptosis and ultimate fibrosis. In congenital heart block (CHB), binding of maternal anti-Ro/La antibodies to apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases uPA/uPAR-dependent plasmin activation. Immunological staining of CHB hearts reveals AV node TGFb staining and TGFb activation promotes fibroblast trans-differentiation, a scarring phenotype. Since the uPA/uPAR system plays a role in TGFb activation, this study evaluated whether anti-Ro/La binding to apoptotic cardiocytes via plasmin activation stimulates TGFb and promotes a profibrosing phenotype. Methods and Results: Initial analysis showed increased TGFb in supernatants from co-cultures of healthy cards and apoptotic cards incubated with IgG fractions from mothers whose sera contain anti-Ro/La antibodies and who had a child with CHB (apo-CHB-IgG) compared to co-cultures of healthy cards and apoptotic cards incubated with control IgG (apo-nl-IgG). Using a luciferase bioassay of active TGFb, supernatants from co-cultures of healthy cards and apo-CHB-IgG cards exhibited increased levels of active TGFb compared to those cocultured with apo-nl-IgG cards (511pg/ml CHB-IgG RLU vs 217 nl-IgG RLU; p=0.007; n=7). Abrogation of RLU via anti-TGFb antibody or TGFb inhibitor (SB431542) confirmed TGFb activation was solely due to TGFb. Significantly increased uPA levels (903 pg/ml vs 508 pg/ml; p =0.01; n=5) and uPA activity (0.8 units vs 0.04 units; p=0.005; n=3) were demonstrated in supernatants generated from coculture of healthy cards and apo CHB-IgG cards compared to healthy cards and apo nl-IgG, respectively. To determine whether uPA activity was responsible for TGFb activation, coculture experiments were conducted in which the apo-CHB-IgG cards were treated with anti-uPAR or anti-uPA antibodies or the plasmin inhibitor aprotinin prior to coculturing with healthy cards. In all instances treatments attenuated TGFb activation and uPA activity. To evaluate the profibrotic role of the observed TGFb activation, supernatants from either apo-CHB-IgG or apo-nl-IgG cocultures with healthy cards were applied to serum deprived cardiac fibroblasts. Only supernatants derived from cocultures of healthy cards and apo-CHB-IgG cardiocytes promoted transdifferentiation as evidenced by increased SMAc staining, an effect that was decreased when fibroblasts were treated with supernatants where cocultures were pretreated with uPAR antibodies. Supporting the hypothesis that increased uPA activity is causally related to the pathogenesis of CHB, cord blood samples from 12 of 18 children with CHB exhibited increased uPA activity and uPAR cleavage compared to 4 of 14 non CHB children exposed to maternal anti-Ro antibodies. Conclusions: These data suggest that binding of anti-Ro/La antibodies to apoptotic cardiocytes by virtue of increased uPAR-dependent uPA activity trigger TGFb activation thus initiating and amplifying a cascade of events that promote myofibroblast transdifferentiation and scar
— id: 130926, year: 2010, vol: 62, page: 489, stat: Journal Article,

Microfibril Structure Masks Fibrillin-2 in Postnatal Tissues
Charbonneau, NL; Jordan, CD; Keene, DR; Lee-Arteaga, S; Dietz, HC; Rifkin, DB; Ramirez, F; Sakai, LY
2010 JUN 25 ;285(26):20242-20251, Journal of biological chemistry
Fibrillin microfibrils are polymeric structures present in connective tissues. The importance of fibrillin microfibrils to connective tissue function has been demonstrated by the multiple genetic disorders caused by mutations in fibrillins and in microfibril-associated molecules. However, knowledge of microfibril structure is limited, largely due to their insolubility. Most previous studies have focused on how fibrillin-1 is organized within microfibril polymers. In this study, an immunochemical approach was used to circumvent the insolubility of microfibrils to determine the role of fibrillin-2 in postnatal microfibril structure. Results obtained from studies of wild type and fibrillin-1 null tissues, using monoclonal and polyclonal antibodies with defined epitopes, demonstrated that N-terminal fibrillin-2 epitopes are masked in postnatal microfibrils and can be revealed by enzymatic digestion or by genetic ablation of Fbn1. From these studies, we conclude that fetal fibrillin polymers form an inner core within postnatal microfibrils and that microfibril structure evolves as growth and development proceed into the postnatal period. Furthermore, documentation of a novel cryptic site present in EGF4 in fibrillin-1 underscores the molecular complexity and tissue-specific differences in microfibril structure
— id: 110676, year: 2010, vol: 285, page: 20242, stat: Journal Article,

Differential effects of alendronate and losartan therapy on osteopenia and aortic aneurysm in mice with severe Marfan syndrome
Nistala, Harikiran; Lee-Arteaga, Sui; Carta, Luca; Cook, Jason R; Smaldone, Silvia; Siciliano, Gabriella; Rifkin, Aaron N; Dietz, Harry C; Rifkin, Daniel B; Ramirez, Francesco
2010 Dec 15;19(24):4790-4798, Human molecular genetics
Reduced bone mineral density (osteopenia) is a poorly characterized manifestation of pediatric and adult patients afflicted with Marfan syndrome (MFS), a multisystem disorder caused by structural or quantitative defects in fibrillin-1 that perturb tissue integrity and TGFbeta bioavailability. Here we report that mice with progressively severe MFS (Fbn1(mgR/mgR) mice) develop osteopenia associated with normal osteoblast differentiation and bone formation. In vivo and ex vivo experiments, respectively, revealed that adult Fbn1(mgR/mgR) mice respond more strongly to locally induced osteolysis and that Fbn1(mgR/mgR) osteoblasts stimulate pre-osteoclast differentiation more than wild-type cells. Greater osteoclastogenic potential of mutant osteoblasts was largely attributed to Rankl up-regulation secondary to improper TGFbeta activation and signaling. Losartan treatment, which lowers TGFbeta signaling and restores aortic wall integrity in mice with mild MFS, did not mitigate bone loss in Fbn1(mgR/mgR) mice even though it ameliorated vascular disease. Conversely, alendronate treatment, which restricts osteoclast activity, improved bone quality but not aneurysm progression in Fbn1(mgR/mgR) mice. Taken together, our findings shed new light on the pathogenesis of osteopenia in MFS, in addition to arguing for a multifaceted treatment strategy in this congenital disorder of the connective tissue
— id: 140040, year: 2010, vol: 19, page: 4790, stat: Journal Article,

Bone matrix to growth factors: location, location, location
Rifkin, Daniel B; Todorovic, Vesna
2010 Sep 20;190(6):949-951, Journal of cell biology
The demonstration that fibrillin-1 mutations perturb transforming growth factor (TGF)-beta bioavailability/signaling in Marfan syndrome (MFS) changed the view of the extracellular matrix as a passive structural support to a dynamic modulator of cell behavior. In this issue, Nistala et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201003089) advance this concept by demonstrating how fibrillin-1 and -2 regulate TGF-beta and bone morphogenetic protein (BMP) action during osteoblast maturation
— id: 112559, year: 2010, vol: 190, page: 949, stat: Journal Article,

E-selectin ligand-1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-beta
Yang, Tao; Mendoza-Londono, Roberto; Lu, Huifang; Tao, Jianning; Li, Kaiyi; Keller, Bettina; Jiang, Ming Ming; Shah, Rina; Chen, Yuqing; Bertin, Terry K; Engin, Feyza; Dabovic, Branka; Rifkin, Daniel B; Hicks, John; Jamrich, Milan; Beaudet, Arthur L; Lee, Brendan
2010 Jul 1;120(7):2474-2485, Journal of clinical investigation
The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-beta signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-beta signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-beta is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand-1 (ESL-1), acts as a negative regulator of TGF-beta production by binding TGF-beta precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-beta by a furin-like protease, leading to reduced secretion of mature TGF-beta by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-beta/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-beta/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-beta during skeletal development and homeostasis
— id: 146002, year: 2010, vol: 120, page: 2474, stat: Journal Article,

F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-beta activation
Attur, Mukundan G; Palmer, Glyn D; Al-Mussawir, Hayf E; Dave, Mandar; Teixeira, Cristina C; Rifkin, Daniel B; Appleton, C Thomas G; Beier, Frank; Abramson, Steven B
2009 Jan;23(1):79-89, FASEB journal
In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (approximately 7-fold) expression of F-spondin, a neuronal extracellular matrix glycoprotein, in human OA cartilage (P<0.005). OA-specific up-regulation of F-spondin was also demonstrated in rat knee cartilage following surgical menisectomy. F-spondin treatment of OA cartilage explants caused a 2-fold increase in levels of the active form of TGF-beta1 (P<0.01) and a 10-fold induction of PGE2 (P<0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF-beta and the thrombospondin domain of the F-spondin molecule. F-spondin addition to cartilage explant cultures also caused a 4-fold increase in collagen degradation (P<0.05) and a modest reduction in proteoglycan synthesis (approximately 20%; P<0.05), which were both TGF-beta and PGE2 dependent. F-spondin treatment also led to increased secretion and activation of MMP-13 (P<0.05). Together these studies identify F-spondin as a novel protein in OA cartilage, where it may act in situ at lesional areas to activate latent TGF-beta and induce cartilage degradation via pathways that involve production of PGE2
— id: 92153, year: 2009, vol: 23, page: 79, stat: Journal Article,

p38 MAPK is an early determinant of promiscuous Smad2/3 signaling in the aortas of fibrillin-1 (Fbn1)-null mice
Carta, Luca; Smaldone, Silvia; Zilberberg, Lior; Loch, David; Dietz, Harry C; Rifkin, Daniel B; Ramirez, Francesco
2009 Feb 27;284(9):5630-5636, Journal of biological chemistry
Excessive transforming growth factor-beta (TGF-beta) signaling characterizes the progression of aortic aneurysm in mouse models of Marfan syndrome, a systemic disorder of the connective tissue that is caused by mutations in the gene encoding the extracellular matrix protein fibrillin-1. Fibrillin-1 mutations are believed to promote abnormal Smad2/3 signaling by impairing the sequestration of latent TGF-beta complexes into the extracellular matrix. Here we report that promiscuous Smad2/3 signaling is the cell-autonomous phenotype of primary cultures of vascular smooth muscle cells (VSMC) explanted from the thoracic aortas of Fbn1 mutant mice with either neonatal onset or progressively severe aortic aneurysm. This cellular phenotype was characterized in VSMC isolated from Fbn1-null (mgN/mgN) mice, which recapitulate the most severe form of Marfan syndrome. We found that loss of fibrillin-1 deposition promotes the production of intracellular reactive oxygen species and abnormal accumulation of phosphorylated TGF-beta-activated kinase 1 and p38 MAPK, in addition to increasing the levels of endogenous phospho-Smad2. We showed that improper Smad2/3 signaling in Fbn1-null VSMC is in part stimulated by phospho-p38 MAPK, which is in turn activated in response to signals other than those mediated by the kinase activity of the ALK5 receptor. Consistent with these cell culture data, in vivo analyses documented that phospho-p38 MAPK accumulates earlier than phospho-Smad2 in the aortic wall of mgN/mgN mice and that systemic inhibition of phospho-p38 MAPK activity lowers the levels of phospho-Smad2 in this tissue. Collectively, these findings indicate that improper activation of p38 MAPK is a precursor of constitutive Smad2/3 signaling in the aortic wall of a mouse model of neonatal lethal Marfan syndrome
— id: 135228, year: 2009, vol: 284, page: 5630, stat: Journal Article,

Dual functions for LTBP in lung development: LTBP-4 independently modulates elastogenesis and TGF-beta activity
Dabovic, Branka; Chen, Yan; Choi, Jiwon; Vassallo, Melinda; Dietz, Harry C; Ramirez, Francesco; von Melchner, Harald; Davis, Elaine C; Rifkin, Daniel B
2009 Apr;219(1):14-22, Journal of cellular physiology
The latent TGF-beta binding proteins (LTBP) -1, -3, and -4 are extracellular proteins that assist in the secretion and localization of latent TGF-beta. The null mutation of LTBP-4S in mice causes defects in the differentiation of terminal air-sacs, fragmented elastin, and colon carcinomas. We investigated lung development from embryonic day 14.5 (E14.5) to day 7 after birth (P7) in order to determine when the defects in elastin organization initiate and to further examine the relation of TGF-beta signaling levels and air-sac septation in Ltbp4S-/- lungs. We found that defects in elastogenesis are visible as early as E14.5 and are maintained in the alveolar walls, in blood vessel media, and subjacent airway epithelium. The air-sac septation defect was associated with excessive TGF-beta signaling and was reversed by lowering TGF-beta2 levels. Thus, the phenotype is not directly reflective of a change in TGF-beta1, the only TGF-beta isoform known to complex with LTBP-4. Reversal of the air-sac septation defect was not associated with normalization of the elastogenesis indicating two separate functions of LTBP-4 as a regulator of elastic fiber assembly and TGF-beta levels in lungs
— id: 92147, year: 2009, vol: 219, page: 14, stat: Journal Article,

Latent Transforming Growth Factor beta-binding Proteins and Fibulins Compete for Fibrillin-1 and Exhibit Exquisite Specificities in Binding Sites
Ono, RN; Sengle, G; Charbonneau, NL; Carlberg, V; Bachinger, HP; Sasaki, T; Lee-Arteaga, S; Zilberberg, L; Rifkin, DB; Ramirez, F; Chu, ML; Sakai, LY
2009 JUN 19 ;284(25):16872-16881, Journal of biological chemistry
Latent transforming growth factor (TGF) beta-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGF beta. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit 'exquisite specificities,' a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive
— id: 100448, year: 2009, vol: 284, page: 16872, stat: Journal Article,

Extracellular microfibrils: contextual platforms for TGFbeta and BMP signaling
Ramirez, Francesco; Rifkin, Daniel B
2009 Oct;21(5):616-622, Current opinion in cell biology
The extracellular matrix plays a key role in organ formation and tissue homeostasis. Recent studies have revealed that fibrillin assemblies (microfibrils) confer both tissue integrity and regulate signaling events that instruct cell performance and that perturbation of either function manifests in disease. These analyses have also indicated that fibrillin assemblies impart contextual specificity to TGFbeta and BMP signaling. Moreover, correlative evidence suggests functional coupling between cell-directed assembly of microfibrils and targeting of TGFbeta and BMP complexes to fibrillins. Hence, the emerging view is that fibrillin-rich microfibrils are molecular integrators of structural and instructive signals with TGFbetas and BMPs as nodal points that convert extracellular inputs into discrete and context-dependent cellular responses
— id: 133719, year: 2009, vol: 21, page: 616, stat: Journal Article,

Mutations in LTBP4 cause a syndrome of impaired pulmonary, gastrointestinal, genitourinary, musculoskeletal, and dermal development
Urban, Zsolt; Hucthagowder, Vishwanathan; Schurmann, Nura; Todorovic, Vesna; Zilberberg, Lior; Choi, Jiwon; Sens, Carla; Brown, Chester W; Clark, Robin D; Holland, Kristen E; Marble, Michael; Sakai, Lynn Y; Dabovic, Branka; Rifkin, Daniel B; Davis, Elaine C
2009 Nov;85(5):593-605, American journal of human genetics
We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4
— id: 120525, year: 2009, vol: 85, page: 593, stat: Journal Article,

Latent transforming growth factor-beta-binding protein-4 regulates transforming growth factor-beta1 bioavailability for activation by fibrogenic lung fibroblasts in response to bleomycin
Zhou, Yong; Koli, Katri; Hagood, James S; Miao, Mi; Mavalli, Mahendra; Rifkin, Daniel B; Murphy-Ullrich, Joanne E
2009 Jan;174(1):21-33, American journal of pathology
Recent evidence suggests that subsets of lung fibroblasts differentially contribute to fibrogenic progression. We have previously shown that a subset of rat lung fibroblasts with fibrogenic characteristics [Thy-1 (-) fibroblasts] responds to stimuli (bleomycin, interleukin-4, etc) with increased latent transforming growth factor (TGF)-beta activation, whereas non-fibrogenic Thy-1-expressing [Thy-1 (+)] fibroblasts do not. Activation of latent TGF-beta1 by interstitial lung fibroblasts is critical for fibrogenic responses. To better understand the susceptibility of fibrogenic fibroblasts to the stimulation of TGF-beta activation, we examined the role of latent TGF-beta-binding proteins (LTBPs), key regulators of TGF-beta bioavailability and activation, in TGF-beta1 activation by these fibroblasts. Treatment of fibroblasts with bleomycin up-regulated LTBP-4 mRNA, protein, and soluble LTBP-4-bound large latent TGF-beta1 complexes in Thy-1 (-) fibroblasts to significantly higher levels than in Thy-1 (+) fibroblasts. Bleomycin-induced TGF-beta1 activation required LTBP-4, since lung fibroblasts deficient in LTBP-4 did not activate TGF-beta1. Expression of LTBP-4 restored TGF-beta1 activation in response to bleomycin, but expression either of LTBP-4 lacking the TGF-beta-binding site or only the TGF-beta-binding domain did not. Bleomycin treatment of mice increased LTBP-4 expression in the lung. Thy-1 knockout mice had increased levels of both LTBP-4 expression and TGF-beta activation, as well as enhanced Smad3 phosphorylation compared with wild-type mice. Together, these data identify a critical role for LTBP-4 in the regulation of latent TGF-beta1 activation in bleomycin-induced lung fibrosis
— id: 135218, year: 2009, vol: 174, page: 21, stat: Journal Article,

In vitro and in vivo evidence for shear-induced activation of latent transforming growth factor-beta 1
Ahamed, J; Burg, N; Yoshinaga, K; Janczak, CA; Rifkin, DB; Coller, BS
2008 NOV 1 ;112(9):3650-3660, Blood
Transforming growth factor-beta 1 (TGF-beta 1) has potent physiologic and pathologic effects on a variety of cell types at subnanomolar concentrations. Platelets contain 40 times as much TGF-beta 1 as other cells and secrete it as an inactive ( latent) form in complex with latency-associated peptide ( LAP), which is disulfide bonded via Cys33 to latent TGF-beta binding protein 1 (LTBP-1). Little is known about how latent TGF-beta 1 becomes activated in vivo. Here we show that TGF-beta 1 released from platelets or fibroblasts undergoes dramatic activation when subjected to stirring or shear forces, providing a potential mechanism for physiologic control. Thioldisulfide exchange appears to contribute to the process based on the effects of thiol-reactive reagents and differences in thiol labeling of TGF-beta 1 before and after stirring or shear. Activation required the presence of LTBP, as TGF-beta 1 contained in complex with only LAP could not be activated by stirring when studied as either a recombinant purified protein complex or in the platelet releasates or sera of mice engineered to contain an LAP C33S mutation. Release and activation of latent TGF-beta 1 in vivo was demonstrated in a mouse model 5 minutes after thrombus formation. These data potentially provide a novel mechanism for in vivo activation of TGF-beta 1. (Blood. 2008; 112: 3650-3660)
— id: 90068, year: 2008, vol: 112, page: 3650, stat: Journal Article,

F-spondin, a neuroregulatory protein, is upregulated in human and surgically-induced osteoarthritis: Evidence for regulation of cartilage metabolism via latent tgf-b1 activation
Attur, M; Palmer, G; Al-Mussawir, HE; Rifkin, DB; Teixeira, CC; Appleton, CTG; Beier, F; Abramson, SB
2008 SEP ;58(9):S895-S895, Arthritis & rheumatism
— id: 88578, year: 2008, vol: 58, page: S895, stat: Journal Article,

Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine
Ivanov, Ivaylo I; Frutos, Rosa de Llanos; Manel, Nicolas; Yoshinaga, Keiji; Rifkin, Daniel B; Sartor, R Balfour; Finlay, B Brett; Littman, Dan R
2008 Oct 16;4(4):337-349, Cell Host & Microbe
The requirements for in vivo steady state differentiation of IL-17-producing T-helper (Th17) cells, which are potent inflammation effectors, remain obscure. We report that Th17 cell differentiation in the lamina propria (LP) of the small intestine requires specific commensal microbiota and is inhibited by treating mice with selective antibiotics. Mice from different sources had marked differences in their Th17 cell numbers and animals lacking Th17 cells acquired them after introduction of bacteria from Th17 cell-sufficient mice. Differentiation of Th17 cells correlated with the presence of cytophaga-flavobacter-bacteroidetes (CFB) bacteria in the intestine and was independent of toll-like receptor, IL-21 or IL-23 signaling, but required appropriate TGF-beta activation. Absence of Th17 cell-inducing bacteria was accompanied by increase in Foxp3+ regulatory T cells (Treg) in the LP. Our results suggest that composition of intestinal microbiota regulates the Th17:Treg balance in the LP and may thus influence intestinal immunity, tolerance, and susceptibility to inflammatory bowel diseases
— id: 93379, year: 2008, vol: 4, page: 337, stat: Journal Article,

Report of the National Heart, Lung, and Blood Institute and National Marfan Foundation working group on research in Marfan syndrome and related disorders
Pearson, GD; Devereux, R; Loeys, B; Maslen, C; Milewicz, D; Pyeritz, R; Ramirez, F; Rifkin, D; Sakai, L; Svensson, L; Wessels, A; Van Eyk, J; Dietz, HC
2008 AUG 12 ;118(7):785-791, Circulation
— id: 86820, year: 2008, vol: 118, page: 785, stat: Journal Article,

Perturbation of transforming growth factor (TGF)-beta1 association with latent TGF-beta binding protein yields inflammation and tumors
Yoshinaga, Keiji; Obata, Hiroto; Jurukovski, Vladimir; Mazzieri, Roberta; Chen, Yan; Zilberberg, Lior; Huso, David; Melamed, Jonathan; Prijatelj, Petra; Todorovic, Vesna; Dabovic, Branka; Rifkin, Daniel B
2008 Dec 2;105(48):18758-18763, Proceedings of the National Academy of Sciences of the United States of America
Transforming growth factor-beta (TGF-beta) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-beta is often released as part of an inactive tripartite complex consisting of TGF-beta, the TGF-beta propeptide, and a molecule of latent TGF-beta binding protein (LTBP). The interaction of TGF-beta and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-beta from this state is crucial for signaling. To examine the contribution of LTBP to TGF-beta function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1(C33S/C33S) mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-beta1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1(-/-) animals. Tgfb1(C33S/C33S) mice exhibited decreased levels of active TGF-beta1, decreased TGF-beta signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-beta complex is important for proper TGF-beta1 function and that Tgfb1(C33S/C33S) mice are hypomorphs for active TGF-beta1. Moreover, although mechanisms exist to activate latent TGF-beta1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP
— id: 92146, year: 2008, vol: 105, page: 18758, stat: Journal Article,

Extracellular microfibrils in development and disease
Ramirez, F; Sakai, LY; Rifkin, DB; Dietz, HC
2007 SEP ;64(18):2437-2446, Cellular & molecular life sciences: CMLS
Fibrillins are the structural components of extracellular microfibrils that impart physical properties to tissues, alone or together with elastin as elastic fibers. Genetic studies in mice have revealed that fibrillin-rich microfibrils are also involved in regulating developmental programs and homeostatic processes through the modulation of TGF-beta/BMP signaling events. A new paradigm has thus emerged whereby the spatiotemporal organization of microfibrils dictates both the cellular activities and physical properties of connective tissues. These observations have paved the way to novel therapeutic approaches aimed at counteracting the life-threatening complications in human conditions caused by dysfunctions of fibrillin-rich microfibrils
— id: 74193, year: 2007, vol: 64, page: 2437, stat: Journal Article,

Long form of latent TGF-{beta} binding protein 1 (Ltbp1L) is essential for cardiac outflow tract septation and remodeling
Todorovic, Vesna; Frendewey, David; Gutstein, David E; Chen, Yan; Freyer, Laina; Finnegan, Erin; Liu, Fangyu; Murphy, Andrew; Valenzuela, David; Yancopoulos, George; Rifkin, Daniel B
2007 Oct;134(20):3723-3732, Development
Latent TGF-beta binding protein 1 (LTBP1) is a member of the LTBP/fibrillin family of extracellular proteins. Due to the usage of different promoters, LTBP1 exists in two major forms, long (L) and short (S), each expressed in a temporally and spatially unique fashion. Both LTBP1 molecules covalently interact with latent TGF-beta and regulate its function, presumably via interaction with the extracellular matrix (ECM). To explore the in vivo role of Ltbp1 in mouse development, at the time when only the L isoform is expressed, we mutated the Ltbp1L locus by gene targeting. Ltbp1L-null animals die shortly after birth from defects in heart development, consisting of the improper septation of the cardiac outflow tract (OFT) and remodeling of the associated vessels. These cardiac anomalies present as persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA), which are associated with the faulty function of cardiac neural crest cells (CNCCs). The lack of Ltbp1L in the ECM of the septating OFT and associated vessels results in altered gene expression and function of CNCCs and decreased Tgf-beta activity in the OFT. This phenotype reveals a crucial role for Ltbp1L and matrix as extracellular regulators of Tgf-beta activity in heart organogenesis
— id: 74213, year: 2007, vol: 134, page: 3723, stat: Journal Article,

Myofibroblast contraction activates latent TGF-beta 1 from the extracellular matrix
Wipff, PJ; Rifkin, DB; Meister, JJ; Hinz, B
2007 DEC 17 ;179(6):1311-1323, Journal of cell biology
The conjunctive presence of mechanical stress and active transforming growth factor beta 1 (TGF-beta 1) is essential to convert fibroblasts into contractile myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that myofibroblast contraction functions as a mechanism to directly activate TGF-beta 1 from self-generated stores in the ECM. Contraction of myofi broblasts and myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-beta 1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-beta 1. In myofibroblast-populated wounds, activation of the downstream targets of TGF-beta 1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-beta 1 and its receptor. We propose activation of TGF-beta 1 via integrin-mediated myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of myofibroblasts to a stiffened ECM
— id: 75633, year: 2007, vol: 179, page: 1311, stat: Journal Article,

E-Selectin Ligand 1 negatively regulates TGF beta in the Golgi during skeletogenesis
Yang, T; Mendoza-Londono, R; Lu, H; Li, K; Keller, B; Jiang, M; Chen, Y; Bertin, T; Dabovic, B; Rifkin, DB; Hick, J; Beaudet, AL; Lee, B
2007 SEP ;22(1):S105-S105, Journal of bone & mineral research
— id: 75797, year: 2007, vol: 22, page: S105, stat: Journal Article,

A rapid and sensitive bioassay to measure bone morphogenetic protein activity
Zilberberg, Lior; ten Dijke, Peter; Sakai, Lynn Y; Rifkin, Daniel B
2007 ;8:41-41, BMC Cell Biology
BACKGROUND: Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily and were originally identified as proteins that induce ectopic bone formation. BMPs were shown subsequently to be involved in several biological processes during development and in adult tissues through the regulation of the growth, differentiation and apoptosis of various cell types. An alkaline phosphatase (ALP)-based assay is the most widely used assay to evaluate BMP activity. However, the ALP assay is not rapid and not sensitive enough to measure BMP activity at physiological concentrations. In this paper, we describe a highly sensitive, rapid, and specific cell-based assay for the quantification of BMP activity. RESULTS: Two cells lines, C2C12 and HepG2 were stably transfected with a reporter plasmid consisting of BMP-responsive elements from the Id1 promoter fused to a luciferase reporter gene. Exposure of cells containing this construct to BMPs induces the expression of luciferase, which can be quantified with a luminometer. The bioassay is specific for BMPs and can detect BMP-4 activity at a concentration as low as 3 pM. Related family members, such as TGF-beta1, TGF-beta2 and TGF-beta3, do not induce the reporter gene. CONCLUSION: The assay is rapid (less than 24 hours) and can be used, as described in this paper, to measure BMP activity in complex solutions and in cell culture in a simple and efficient way
— id: 75377, year: 2007, vol: 8, page: 41, stat: Journal Article,

Phenotype of mice that cannot form TGF beta 1 large latent complex
Dabovic, B; Jurukovski, V; Obata, H; Chen, Y; Zilberberg, L; Mazzieri, R; Yoshinaga, K; Rifkin, D
2006 NOV ;25(2):S87-S87, Matrix biology
— id: 70621, year: 2006, vol: 25, page: S87, stat: Journal Article,

Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome
Habashi, JP; Judge, DP; Holm, TM; Cohn, RD; Loeys, BL; Cooper, TK; Myers, L; Klein, EC; Liu, GS; Calvi, C; Podowski, M; Neptune, ER; Halushka, MK; Bedja, D; Gabrielson, K; Rifkin, DB; Carta, L; Ramirez, F; Huso, DL; Dietz, HC
2006 APR 7 ;312(5770):117-121, Science
Aortic aneurysm and dissection are manifestations of Marfan syndrome (MFS), a disorder caused by mutations in the gene that encodes fibrillin-1. Selected manifestations of MFS reflect excessive signaling by the transforming growth factor-beta (TGF-beta) family of cytokines. We show that aortic aneurysm in a mouse model of MFS is associated with increased TGF-beta signaling and can be prevented by TGF-beta antagonists such as TGF-beta-neutralizing antibody or the angiotensin II type 1 receptor (AT1) blocker, losartan. AT1 antagonism also partially reversed noncardiovascular manifestations of MFS, including impaired alveolar septation. These data suggest that losartan, a drug already in clinical use for hypertension, merits investigation as a therapeutic strategy for patients with MFS and has the potential to prevent the major life-threatening manifestation of this disorder
— id: 63603, year: 2006, vol: 312, page: 117, stat: Journal Article,

Isoform-specific activation of latent transforming growth factor beta (LTGF-beta) by reactive oxygen species
Jobling, Michael F; Mott, Joni D; Finnegan, Monica T; Jurukovski, Vladimir; Erickson, Anna C; Walian, Peter J; Taylor, Scott E; Ledbetter, Steven; Lawrence, Catherine M; Rifkin, Daniel B; Barcellos-Hoff, Mary Helen
2006 Dec;166(6):839-848, Radiation research
The three mammalian transforming growth factor beta (TGF-beta) isoforms are each secreted in a latent complex in which TGF-beta homodimers are non-covalently associated with homodimers of their respective pro-peptide called the latency-associated peptide (LAP). Release of TGF-beta from its LAP, called activation, is required for binding of TGF-beta to cellular receptors, making extracellular activation a critical regulatory point for TGF-beta bioavailability. Our previous work demonstrated that latent TGF-beta1 (LTGF-beta1) is efficiently activated by ionizing radiation in vivo and by reactive oxygen species (ROS) generated by Fenton chemistry in vitro. In the current study, we determined the specific ROS and protein target that render LTGF-beta1 redox sensitive. First, we compared LTGF-beta1, LTGF-beta2 and LTGF-beta3 to determine the generality of this mechanism of activation and found that redox-mediated activation is restricted to the LTGF-beta1 isoform. Next, we used scavengers to determine that ROS activation was a function of OH(.) availability, confirming oxidation as the primary mechanism. To identify which partner of the LTGF-beta1 complex was functionally modified, each was exposed to ROS and tested for the ability to form a latent complex. Exposure of TGF-beta1 did not alter its ability to associate with LAP, but exposing LAP-beta1 to ROS prohibited this phenomenon, while treatment of ROS-exposed LAP-beta1 with a mild reducing agent restored its ability to neutralize TGF-beta1 activity. Taken together, these results suggest that ROS-induced oxidation in LAP-beta1 triggers a conformational change that releases TGF-beta1. Using site-specific mutation, we identified a methionine residue at amino acid position 253 unique to LAP-beta1 as critical to ROS-mediated activation. We propose that LTGF-beta1 contains a redox switch centered at methionine 253, which allows LTGF-beta1 to act uniquely as an extracellular sensor of oxidative stress in tissues
— id: 83229, year: 2006, vol: 166, page: 839, stat: Journal Article,

Cardiac outflow tract defects in mice lacking LTBP-1L
Todorovic, V; Freyer, L; Gutstein, D; Frendewey, D; Rifkin, D
2006 NOV ;25(2):S26-S26, Matrix biology
— id: 70620, year: 2006, vol: 25, page: S26, stat: Journal Article,

Amino acid requirements for formation of the TGF-beta-latent TGF-beta binding protein complexes
Chen, Yan; Ali, Tariq; Todorovic, Vesna; O'leary, Joanne M; Kristina Downing, A; Rifkin, Daniel B
2005 Jan 7;345(1):175-186, Journal of molecular biology
Transforming growth factor beta (TGF-beta) is secreted primarily as a latent complex consisting of the TGF-beta homodimer, the TGF-beta propeptides (called the latency-associated protein or LAP) and the latent TGF-beta binding protein (LTBP). Mature TGF-beta remains associated with LAP by non-covalent interactions that block TGF-beta from binding to its receptor. Complex formation between LAP and LTBP is mediated by an intramolecular disulfide exchange between the third 8-cysteine (8-Cys3) domain of LTBP with a pair of cysteine residues in LAP. Only the third 8-Cys domains of LTBP-1, -3, and -4 bind LAP. From comparison of the 8-Cys3(LTBP-1) structure with that of the non-TGF-beta-binding 8-Cys6(fibrillin-1), we observed that a two-residue insertion in 8-Cys3(LTBP-1) increased the potential for disulfide exchange of the 2-6 disulfide bond. We further proposed that five negatively charged amino acid residues surrounding this bond mediate initial protein-protein association. To validate this hypothesis, we monitored binding by fluorescence resonance energy transfer (FRET) analysis and co-expression assays with TGF-beta1 LAP (LAP-1) and wild-type and mutant 8-Cys3 domains. FRET experiments demonstrated ionic interactions between LAP-1 and 8-Cys3. Mutation of the five amino acid residues revealed that efficient complex formation is most dependent on two of these residues. Although 8-Cys3(LTBP-1) binds proTGF-betas effectively, the domain from LTBP-4 does so poorly. We speculated that this difference was due to the substitution of three acidic residues by alanine, serine, and arginine in the LTBP-4 sequence. Additional experiments with 8-Cys3(LTBP-4) indicated that enhanced binding of LAP to 8-Cys3(LTBP-4) is achieved if the residues A, S, and R are changed to those in 8-Cys3(LTBP1) (D, D, and E) and the QQ dipeptide insertion of LTBP-4 is changed to the FP in 8-Cys3(LTBP-1). These studies identify surface residues that contribute to the interactions of 8-Cys3 and LAP-1 and may yield information germane to the interaction of 8-Cys domains and additional TGF-beta superfamily propeptides, an emerging paradigm for growth factor regulation
— id: 48106, year: 2005, vol: 345, page: 175, stat: Journal Article,

Lung alveolar septation defects in Ltbp-3-null mice
Colarossi, Cristina; Chen, Yan; Obata, Hiroto; Jurukovski, Vladimir; Fontana, Laura; Dabovic, Branka; Rifkin, Daniel B
2005 Aug;167(2):419-428, American journal of pathology
Latent transforming growth factor (TGF)-beta binding proteins (LTBPs) modulate the secretion and activation of latent TGF-beta. To explore LTBP function in vivo, we created an Ltbp-3(-/-) mouse that has developmental emphysema with decreased septation in terminal alveoli. Differences in distal airspace enlargement were obvious at day 6 after birth. Secondary septation was inhibited, so by days 21 to 28 the mean linear intercept was approximately twofold greater in mutant versus control lungs. There were no differences in lung collagen and elastin, visualized by immunohistochemistry, or in myofibroblast numbers, determined by alpha-smooth muscle actin-positive cells, between mutant or wild-type lungs as the animals aged, other than differences associated with altered lung structure in mutant animals. However, from day 10 there was twice the number of alveolar type II cells in mutant alveoli compared to controls. At days 6 and 10, a transient enhancement in cell proliferation in the mutant lungs was observed by both 5-bromo-2'-deoxy-uridine and proliferating cell nuclear antigen labeling, accompanied by enhanced numbers of terminal dUTP nick-end labeling-positive cells at days 4, 6, and 10. Finally, there was a transient decrease in TGF-beta signaling at days 4 to 6 in Ltbp-3(-/-) lungs. These results indicate that in the absence of Ltbp-3, a temporary decrease in TGF-beta signaling in the lungs at days 4 to 6 alters cell proliferation, correlating with inhibition of septation and developmental emphysema
— id: 58066, year: 2005, vol: 167, page: 419, stat: Journal Article,

Osteopetrosis-like phenotype in latent TGF-beta binding protein 3 deficient mice
Dabovic, B; Levasseur, R; Zambuto, L; Chen, Y; Karsenty, G; Rifkin, D B
2005 Jul;37(1):25-31, Bone
LTBPs are extracellular matrix proteins resembling fibrillins. LTBP-1, 3, and 4 covalently bind latent TGF-beta and modulate tissue levels of this potent cytokine through regulation of its secretion, localization, and/or activation. To address LTBP function in vivo, we generated Ltbp-3 null mice. Ltbp-3-/- animals developed craniofacial abnormalities due to early ossification of the skull base synchondroses and displayed reduced body size. In addition, histological examination of Ltbp-3-/- skeletons revealed an increase in bone mass. The osteoblast numbers and mineral apposition rates were decreased in Ltbp-3-/- mice, whereas the osteoclast numbers were similar in null and wild type mice. Histological examination revealed persistence of cartilage remnants in Ltbp-3-/- trabecular bone. Taken together, these results indicate that the Ltbp-3-/- high bone mass phenotype was due to a defect in bone resorption. We hypothesize that lack of Ltbp-3 results in decreased levels of TGF-beta in bone and cartilage, which leads to compromised osteoclast function and decreased bone turnover
— id: 146003, year: 2005, vol: 37, page: 25, stat: Journal Article,

Fibronectin is required for integrin alphavbeta6-mediated activation of latent TGF-beta complexes containing LTBP-1
Fontana, Laura; Chen, Yan; Prijatelj, Petra; Sakai, Takao; Fassler, Reinhard; Sakai, Lynn Y; Rifkin, Daniel B
2005 Nov;19(13):1798-1808, FASEB journal
Transforming growth factor-betas (TGF-beta) are secreted as latent complexes consisting of the TGF-beta dimer, the TGF-beta propeptide dimer, and the latent TGF-beta binding protein (LTBP). Although the bonds between TGF-beta and its propeptide are cleaved intracellulary, the propeptide associates with TGF-beta by electrostatic interactions, thereby conferring latency to the complex. We reported that a specific sequence of LTBP-1 is required for latent TGF-beta activation by the integrin alphavbeta6. Here we describe a 24 amino acid sequence from the hinge domain required for activation. The LTBP-1 polypeptide rL1N, which includes the hinge, associates with fibronectin in binding assays. We present evidence that fibronectin null cells minimally activate latent TGF-beta and poorly incorporate the active hinge sequence into their matrix. In addition, cells missing the fibronectin receptor alpha5beta1 exhibit defective activation of latent TGF-beta by alphavbeta6 and decreased matrix incorporation. The results indicate specificity for integrin-mediated latent TGF-beta activation that include unique sequences in LTBP-1 and an appropriate matrix molecule
— id: 62742, year: 2005, vol: 19, page: 1798, stat: Journal Article,

Methods for measuring TGF-b using antibodies, cells, and mice
Jurukovski, Vladimir; Dabovic, Branka; Todorovic, Vesna; Chen, Yan; Rifkin, Daniel B
2005 ;117:161-175, Methods in molecular medicine
The transforming growth factor (TGF)-betas are essential in pre- and postnatal development, differentiation and morphogenesis of higher organisms. Quantitation of the levels of TGF-beta synthesis, secretion, and activation are crucial for grasping the mechanisms that control these events and ultimately control TGF-beta action. Rather than presenting a single method, we describe several methods for measuring active TGF-beta in different experimental situations. This is possible as a result of advances in transgenic mice technology that allow in vivo TGF-beta measurements in addition to the more established in vitro approaches
— id: 67921, year: 2005, vol: 117, page: 161, stat: Journal Article,

A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2
Loeys, BL; Chen, JJ; Neptune, ER; Judge, DP; Podowski, M; Holm, T; Meyers, J; Leitch, CC; Katsanis, N; Sharifi, N; Xu, FL; Myers, LA; Spevak, PJ; Cameron, DE; De Backer, J; Hellemans, J; Chen, Y; Davis, EC; Webb, CL; Kress, W; Coucke, P; Rifkin, DB; De Paepe, AM; Dietz, HC
2005 MAR ;37(3):275-281, Nature genetics
We report heterozygous mutations in the genes encoding either type I or type II transforming growth factor beta receptor in ten families with a newly described human phenotype that includes widespread perturbations in cardiovascular, craniofacial, neurocognitive and skeletal development. Despite evidence that receptors derived from selected mutated alleles cannot support TGFbeta signal propagation, cells derived from individuals heterozygous with respect to these mutations did not show altered kinetics of the acute phase response to administered ligand. Furthermore, tissues derived from affected individuals showed increased expression of both collagen and connective tissue growth factor, as well as nuclear enrichment of phosphorylated Smad2, indicative of increased TGFbeta signaling. These data definitively implicate perturbation of TGFbeta signaling in many common human phenotypes, including craniosynostosis, cleft palate, arterial aneurysms, congenital heart disease and mental retardation, and suggest that comprehensive mechanistic insight will require consideration of both primary and compensatory events
— id: 48922, year: 2005, vol: 37, page: 275, stat: Journal Article,

Expression of truncated latent TGF-beta-binding protein modulates TGF-beta signaling
Mazzieri, Roberta; Jurukovski, Vladimir; Obata, Hiroto; Sung, Joanne; Platt, Alec; Annes, Eric; Karaman-Jurukovska, Nevena; Gleizes, Pierre-Emmanuel; Rifkin, Daniel B
2005 May 15;118(Pt 10):2177-2187, Journal of cell science
Transforming growth factor-beta is released from most cells as an inactive complex consisting of transforming growth factor-beta, the transforming growth factor-beta propeptide and the latent transforming growth factor-beta-binding protein. We studied the role of latent transforming growth factor-beta-binding protein in modulating transforming growth factor-beta availability by generating transgenic mice that express a truncated form of latent transforming growth factor-beta-binding protein-1 that binds to transforming growth factor-beta but is missing the known N- and C-terminal matrix-binding sequences. As transforming growth factor-beta is an inhibitor of keratinocyte proliferation and is involved in the control of hair cycling, we over-expressed the mutated form of latent transforming growth factor-beta-binding protein under the control of the keratin 14-promoter. Transgenic animals displayed a hair phenotype due to a reduction in keratinocyte proliferation, an abbreviated growth phase and an early initiation of the involution (catagen) phase of the hair cycle. This phenotype appears to result from excess active transforming growth factor-beta, as enhanced numbers of pSmad2/3-positive nuclei are observed in transgenic animal skin. These data suggest that the truncated form of latent transforming growth factor-beta-binding protein-1 competes with wild-type latent transforming growth factor-beta-binding protein for binding to latent transforming growth factor-beta, resulting in latent transforming growth factor-beta complexes that fail to be targeted correctly in the extracellular matrix. The mis-localization of the transforming growth factor-beta results in inappropriate activation and premature initiation of catagen, thereby illustrating the significance of latent transforming growth factor-beta-binding protein interaction with transforming growth factor-beta in the targeting and activation of latent transforming growth factor-beta in addition to previously reported effects on small latent complex secretion
— id: 56144, year: 2005, vol: 118, page: 2177, stat: Journal Article,

Latent transforming growth factor-beta (TGF-beta) binding proteins: orchestrators of TGF-beta availability
Rifkin, Daniel B
2005 Mar 4;280(9):7409-7412, Journal of biological chemistry
— id: 50631, year: 2005, vol: 280, page: 7409, stat: Journal Article,

Latent TGF-beta binding proteins
Todorovic, V; Jurukovski, V; Chen, Y; Fontana, L; Dabovic, B; Rifkin, D B
2005 Jan;37(1):38-41, International journal of biochemistry & cell biology
Latent TGF-beta binding proteins are multidomain proteins with a common, highly repetitive structural organization and partially overlapping expression patterns. Latent TGF-beta binding protein-1, -3 and -4 bind latent TGF-beta. TGF-betas are normally secreted as latent complexes, consisting of the mature TGF-beta dimer non-covalently bound to its processed propeptide dimer plus a latent TGF-beta binding protein. The latent TGF-beta binding protein is covalently bound to the propeptide. These binding proteins may perform at least two functions: structural, as components of the matrix, and regulatory, as modulators of TGF-beta availability
— id: 48101, year: 2005, vol: 37, page: 38, stat: Journal Article,

Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1
Annes, Justin P; Chen, Yan; Munger, John S; Rifkin, Daniel B
2004 Jun 7;165(5):723-734, Journal of cell biology
Transforming growth factor-betas (TGF-beta) are secreted as inactive complexes containing the TGF-beta, the TGF-beta propeptide, also called the latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-beta regulation. We have investigated the role of LTBP in modulating TGF-beta generation by the integrin alphaVbeta6. We show that even though alphavbeta6 recognizes an RGD on LAP, LTBP-1 is required for alphaVbeta6-mediated latent TGF-beta activation. The domains of LTBP-1 necessary for activation include the TGF-beta propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in alphaVbeta6-mediated latent TGF-beta activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-beta activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix
— id: 44942, year: 2004, vol: 165, page: 723, stat: Journal Article,

A genetic screen to identify latent transforming growth factor beta activators
Annes, Justin; Vassallo, Melinda; Munger, John S; Rifkin, Daniel B
2004 Apr 1;327(1):45-54, Analytical biochemistry
The mechanisms by which latent transforming growth factor beta (TGFbeta) is converted to the active cytokine are largely unknown. Here we present a genetic screen that combines retroviral mutagenesis and cDNA expression cloning to reveal proteins involved in the extracellular regulation of latent TGFbeta activation. The screen employs a cell line engineered to express green fluorescent protein (GFP) in response to TGFbeta. The cells produce their own latent TGFbeta. Therefore, after transduction with a retroviral cDNA library that contains an insert for an activator of latent TGFbeta, cells expressing the activator are GFP-bright. These cells are enriched by fluorescence-activated cell sorting and grown as individual clones. The isolated clones are cocultured with a second TGFbeta reporter cell line that produces luciferase in response to TGFbeta. Cells that have acquired the ability to activate latent TGFbeta induce luciferase expression in the absence but not in the presence of neutralizing antibodies to TGFbeta. The activator expressed by the positive clones can be identified by retrieval of the retrovirus cDNA insert
— id: 42347, year: 2004, vol: 327, page: 45, stat: Journal Article,

Annexin II-mediated plasmin generation activates TGF-beta3 during epithelial-mesenchymal transformation in the developing avian heart
Krishnan, Suba; Deora, Arunkumar B; Annes, Justin P; Osoria, Jerelyn; Rifkin, Daniel B; Hajjar, Katherine A
2004 Jan 1;265(1):140-154, Developmental biology (Orlando)
Epithelial-mesenchymal transformation (EMT), the process by which epithelial cells are converted into motile, invasive mesenchymal cells, is critical to valvulogenesis. Transforming growth factor-beta3 (TGF-beta3), an established mediator of avian atrioventricular (AV) canal EMT, is secreted as a latent complex. In vitro, plasmin-mediated proteolysis has been shown to release active TGF-betas from the latent complex. Annexin II, a co-receptor for tissue plasminogen activator (tPA) and plasminogen, promotes cell-surface generation of the serine protease plasmin. Here, we show that annexin II-mediated plasmin activity regulates release of active TGF-beta3 during chick AV canal EMT. Primary embryonic endocardial-derived cells express annexin II which promotes plasminogen activation in vitro. Incubation of heart explant cultures with either alpha(2)antiplasmin (alpha(2)AP), a major physiological plasmin inhibitor, or anti-annexin II IgG, blocked EMT by approximately 80%, and 50%, respectively. Anti-annexin II IgG-mediated inhibition of EMT was overcome by the addition of recombinant TGF-beta3. Upon treatment with anti-annexin II IgG or alpha(2)AP, conditioned medium from heart explant cultures showed absence of the active fragment of TGF-beta3 by Western blot analysis and a approximately 50% decrease in TGF-beta specific bioactivity. Our results suggest that annexin II-mediated plasmin activity regulates the release of active TGF-beta during cardiac valve development in the avian heart
— id: 42348, year: 2004, vol: 265, page: 140, stat: Journal Article,

Fibrillin microfibrils: multipurpose extracellular networks in organismal physiology
Ramirez, F; Sakai, LY; Dietz, HC; Rifkin, DB
2004 OCT 4 ;19(2):151-154, Physiological genomics
Organismal physiology depends significantly on the proper assembly of extracellular matrix (ECM) macroaggregates that impart structural integrity to the connective tissue. Recent genetic studies in mice have unraveled unsuspected new functions of architectural matrix components in regulating signaling events that modulate patterning, morphogenesis, and growth of several organ systems. As a result, a new paradigm has emerged whereby tissue-specific organization of the ECM dictates not only the physical properties of the connective tissue, but also the ability of the matrix to direct a broad spectrum of cellular activities through the regulation of growth factor signaling. These observations pave the way to novel therapeutic approaches aimed at counteracting the deleterious consequences of perturbations of connective tissue homeostasis
— id: 46487, year: 2004, vol: 19, page: 151, stat: Journal Article,

Making sense of latent TGFbeta activation
Annes, Justin P; Munger, John S; Rifkin, Daniel B
2003 Jan 15;116(Pt 2):217-224, Journal of cell science
TGFbeta is secreted as part of a latent complex that is targeted to the extracellular matrix. A variety of molecules, 'TGFbeta activators,' release TGFbeta from its latent state. The unusual temporal discontinuity of TGFbeta synthesis and action and the panoply of TGFbeta effects contribute to the interest in TGF-beta. However, the logical connections between TGFbeta synthesis, storage and action are obscure. We consider the latent TGFbeta complex as an extracellular sensor in which the TGFbeta propeptide functions as the detector, latent-TGFbeta-binding protein (LTBP) functions as the localizer, and TGF-beta functions as the effector. Such a view provides a logical continuity for various aspects of TGFbeta biology and allows us to appreciate TGFbeta biology from a new perspective
— id: 35175, year: 2003, vol: 116, page: 217, stat: Journal Article,

Growth retardation as well as spleen and thymus involution in latent TGF-beta binding protein (Ltbp)-3 null mice
Chen, Yan; Dabovic, Branka; Colarossi, Cristina; Santori, Fabio R; Lilic, Mirjana; Vukmanovic, Stanislav; Rifkin, Daniel B
2003 Aug;196(2):319-325, Journal of cellular physiology
The latent TGF-beta binding protein (LTBP)-3 is an extracellular matrix (ECM) protein that binds the small latent complex (SLC) of TGF-beta. Disruption of the Ltbp-3 gene by homologous recombination in mice yields mutant animals that display multiple skeletal abnormalities. In addition, these mice have retarded growth. On an inbred 129 SvEv background, half of the Ltbp-3 mutant mice die between 3 and 4 weeks after birth. These mice show severe involution of the thymus and spleen and a sharp reduction in the numbers of CD4/CD8 double positive T-cells in the thymus. The thymus and spleen defect is caused by elevated corticosterone levels in the serum and can be reversed by injection of aminoglutethimide (AMG), an inhibitor of steroid synthesis. This result indicates that the thymus and spleen defect is a secondary defect due to high corticosterone levels probably induced by stress of unknown etiology
— id: 39195, year: 2003, vol: 196, page: 319, stat: Journal Article,

Latent transforming growth factor beta-binding protein 1 interacts with fibrillin and is a microfibril-associated protein
Isogai, Zenzo; Ono, Robert N; Ushiro, Shin; Keene, Douglas R; Chen, Yan; Mazzieri, Roberta; Charbonneau, Noe L; Reinhardt, Dieter P; Rifkin, Daniel B; Sakai, Lynn Y
2003 Jan 24;278(4):2750-2757, Journal of biological chemistry
Latent transforming growth factor beta-binding protein 1 (LTBP-1) targets latent complexes of transforming growth factor beta to the extracellular matrix, where the latent cytokine is subsequently activated by several different mechanisms. Fibrillins are extracellular matrix macromolecules whose primary function is architectural: fibrillins assemble into ultrastructurally distinct microfibrils that are ubiquitous in the connective tissue space. LTBPs and fibrillins are highly homologous molecules, and colocalization in the matrix of cultured cells has been reported. To address whether LTBP-1 functions architecturally like fibrillins, microfibrils were extracted from tissues and analyzed immunochemically. In addition, binding studies were conducted to determine whether LTBP-1 interacts with fibrillins. LTBP-1 was not detected in extracted beaded-string microfibrils, suggesting that LTBP-1 is not an integral structural component of microfibrils. However, binding studies demonstrated interactions between LTBP-1 and fibrillins. The binding site was within three domains of the LTBP-1 C terminus, and in fibrillin-1 the site was defined within four domains near the N terminus. Immunolocalization data were consistent with the hypothesis that LTBP-1 is a fibrillin-associated protein present in certain tissues but not in others. In tissues where LTBP-1 is not expressed, LTBP-4 may substitute for LTBP-1, because the C-terminal end of LTBP-4 binds equally well to fibrillin. A model depicting the relationship between LTBP-1 and fibrillin microfibrils is proposed
— id: 42351, year: 2003, vol: 278, page: 2750, stat: Journal Article,

Solution structure of the third TB domain from LTBP1 provides insight into assembly of the large latent complex that sequesters latent TGF-beta
Lack, Jeremy; O'Leary, Joanne M; Knott, Vroni; Yuan, Xuemei; Rifkin, Daniel B; Handford, Penny A; Downing, A Kristina
2003 Nov 21;334(2):281-291, Journal of molecular biology
Almost all TGF-beta is secreted as part of a large latent complex. This complex is formed from three molecules, a latent transforming growth factor-beta binding protein (LTBP), which plays roles in targeting and activation, a latency associated peptide (LAP), which regulates latency, and the TGF-beta cytokine. LAP is the TGF-beta pro-peptide that is cleaved intracellularly prior to secretion, and TGF-beta binds non-covalently to LAP. Formation of the large latent complex is important for the efficient secretion of TGF-beta. Previous studies have revealed that the LTBP-LAP interaction is mediated by intracellular exchange of a single disulphide bond within the third, and only the third, TB domain (TB3) with LAP. We have previously reported the structure of a homologous TB domain from fibrillin-1. However, TB3 contains a two amino acid insertion, not found in fibrillin-1 TB domains, which is not amenable to molecular modelling. In order to clarify the basis of TB domain function, we have determined the solution NMR structure of TB3(LTBP1). Comparison with the fibrillin-1 TB domain reveals that the two-residue insertion is associated with a significant increase in solvent accessibility of one of the disulphide bonds (linking the second and sixth cysteine residues). Site-directed mutagenesis and NMR studies indicate that this is the only disulphide bond that can be removed without perturbing the TB domain fold. Furthermore, a ring of negatively charged residues has been identified that surrounds this disulphide bond. Homology modelling suggests that the surface properties of TB3 domains from different LTBP isoforms correlate with binding activities. This research provides testable hypotheses regarding the molecular basis of complex formation between LTBPs and LAPs
— id: 42349, year: 2003, vol: 334, page: 281, stat: Journal Article,

Molecular cloning of the mouse Ltbp-1 gene reveals tissue specific expression of alternatively spliced forms
Noguera, Irene; Obata, Hiroto; Gualandris, Anna; Cowin, Pamela; Rifkin, Daniel B
2003 Apr 10;308(5642):31-41, Gene
Latent transforming growth factor binding proteins (Ltbp-1, -2, -3 and -4) and fibrillins (Fbn-1 and -2) are structurally related cysteine-rich extracellular matrix proteins that localize to the 10 nm microfibrils. Ltbp-1 is thought to promote the secretion and proper folding of the small latent transforming growth factor beta (TGF-beta) complex (TGF-beta plus its propeptide) and is implicated in sequestering it in the extracellular matrix. Here we report the isolation of the mouse Ltbp-1 complementary DNA (cDNA) and gene. The longer form of the Ltbp-1 cDNA encodes a predicted 1713 amino acid protein containing 18 epidermal growth factor-like repeats, four 8-cysteine domains and several motifs that suggest interactions with alpha(IV)beta(1) and alpha(9)beta(1) integrins. Northern blotting analyses indicate that long and short Ltbp-1 transcripts are widely expressed in adult mouse tissues and most abundantly expressed in heart. Ltbp-1 is a single copy gene that maps to chromosome 17, band E (1-3) and encompasses more than 212 kb. The Ltbp-1 gene contains 34 exons and shows a similar organization to the LTBP-2 gene, suggesting that these genes originated from a common ancestral gene
— id: 38125, year: 2003, vol: 308, page: 31, stat: Journal Article,

Cell signaling events: a view from the matrix
Ramirez, Francesco; Rifkin, Daniel B
2003 Apr;22(2):101-107, Matrix biology
Cellular activities are primarily initiated, modulated and sustained by multifunctional molecules (cytokines and growth factors) that are secreted into the extracellular space and that signal through membrane-bound, high-affinity receptors. In contrast to the fairly well understood mechanisms that mediate the specificity of signal transduction within the confined and compartmentalized environment of the cell, significantly less is known about the mechanisms that regulate the availability of signaling molecules in the extracellular milieu. Recent findings have implicated the participation of extracellular protein macroaggregates in signaling events controlling patterning and morphogenesis. The results suggest a functional coupling between the tissue-specific organization of collagenous and elastic macroaggregates and their ability to perform instructive as well as structural functions. These observations open the way to a novel understanding in these poorly understood and critically important areas of cell and developmental biology
— id: 42350, year: 2003, vol: 22, page: 101, stat: Journal Article,

The latent-TGFbeta-binding-protein-1 (LTBP-1) is expressed in the organizer and regulates nodal and activin signaling
Altmann, Curtis R; Chang, Chenbei; Munoz-Sanjuan, Ignacio; Bell, Esther; Heke, Michael; Rifkin, Daniel B; Brivanlou, Ali H
2002 Aug 1;248(1):118-127, Developmental biology (Orlando)
The latent TGF-beta binding proteins (LTBP) are believed to control the availability of TGF-beta in the extracellular milieu. To gain insight into the potential roles of LTBP in early development, we isolated the Xenopus LTBP-1 (xLTBP-1) cDNA. The cDNA encodes a protein similar to the mammalian LTBP-1 in both size and domain structure. In addition, we found a novel longer splice isoform of xLTBP. The RNAs for both forms of xLTBP displayed temporal regulation and the shorter transcript is expressed maternally. Both transcripts also display spatial regulation and are found in the dorsal mesoderm of the organizer. In animal cap experiments, LTBP-1 potentiates the activity of activin and nodal. The activity of LTBP-1 did not appear to require covalent association with activin as the addition of medium containing activin and LTBP-1 to animal caps enhanced the activin effect. These results indicate that LTBP-1 may be part of the regulatory system that establishes the threshold of morphogen activity for activins and nodals in the dorsal side of the embryo during gastrulation
— id: 42353, year: 2002, vol: 248, page: 118, stat: Journal Article,

The integrin alphaVbeta6 binds and activates latent TGFbeta3
Annes, Justin P; Rifkin, Daniel B; Munger, John S
2002 Jan 30;511(1-3):65-68, FEBS letters
Transforming growth factors-beta (TGFbeta1, 2 and 3) are secreted in a complex with their propeptides (latency-associated peptide 1 (LAP1), 2 and 3). TGFbeta signaling requires the dissociation of LAP and TGFbeta, a process termed latent TGFbeta activation. This process is a critical but incompletely understood step in the regulation of TGFbeta function. In particular, the extent to which activation mechanisms differ among the three TGFbeta isoforms is relatively unexplored. We show here that alphaVbeta6 binds and activates latent TGFbeta3
— id: 28180, year: 2002, vol: 511, page: 65, stat: Journal Article,

Latent TGF-beta binding protein-3 (LTBP-3) requires binding to TGF-beta for secretion
Chen, Yan; Dabovic, Branka; Annes, Justin P; Rifkin, Daniel B
2002 Apr 24;517(1-3):277-280, FEBS letters
Latent transforming growth factor-beta (TGF-beta) binding protein (LTBP)-1, which is easily secreted, has been shown to enhance the secretion of TGF-beta. Here we show that another member of the LTBP family, LTBP-3, is not secreted by several cell types, but secretion occurs after coexpression with TGF-beta. The secretion of LTBP-3 requires complexing of LTBP-3 with Cys33 of the TGF-beta propeptide
— id: 39630, year: 2002, vol: 517, page: 277, stat: Journal Article,

Bone defects in latent TGF-beta binding protein (Ltbp)-3 null mice; a role for Ltbp in TGF-beta presentation
Dabovic, B; Chen, Y; Colarossi, C; Zambuto, L; Obata, H; Rifkin, D B
2002 Oct;175(1):129-141, Journal of endocrinology
The latent transforming growth factor (TGF)-beta binding proteins (LTBP)-1, -3 and -4 bind the latent form of the multipotent cytokine TGF-beta. To examine the function of the LTBPs, we made a null mutation of Ltbp-3 by gene targeting. The homozygous mutant animals developed cranio-facial malformations by 12 days. By three months, there was a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. The mutant animals developed osteosclerosis of the long bones and vertebrae as well as osteoarthritis between 6 and 9 months of age. These latter phenotypic changes were similar to those described for mice that have impaired TGF-beta signaling. Thus, we suggest that Ltbp-3 plays an important role in regulating TGF-beta bioavailability as the phenotype of the Ltbp-3 null mouse appears to result from decreased TGF-beta signaling. Histological examination of the skulls from null animals revealed no effects on calvarial suture closure. However, the synchondroses in the skull base were obliterated within 2 weeks of birth. This is in contrast to the wild-type synchondroses, which remain unossified throughout the life of the animal and enable growth of the skull base through endochondral ossification. Histological changes in mutant basooccipital-basosphenoid synchondrosis were observed 1.5 days after birth. Compared with wild-type or heterozygous littermates, the basooccipital-basosphenoid synchondrosis of Ltbp-3 null mice contained increased numbers of hypertrophic chondrocytes. The expression of bone sialoprotein-1 (a marker for osteoblasts) was observed in cells surrounding the synchondrosis at postnatal day 1.5 indicating ectopic ossification. The expression of Indian hedgehog (Ihh) (a marker for chondrocytes committed to hypertrophic differentiation) was found through the basooccipital-basosphenoid synchondrosis, whereas the expression of parathyroid hormone related protein (PTHrP), which inhibits chondrocyte differentiation, appeared to be diminished in Ltbp-3 null mice. This suggests that Ltbp-3 may control chondrocyte differentiation by regulating TGF-beta availability. TGF-beta may regulate PTHrP expression either downstream of Ihh or independently of Ihh signaling
— id: 39576, year: 2002, vol: 175, page: 129, stat: Journal Article,

Bone abnormalities in latent TGF-[beta] binding protein (Ltbp)-3-null mice indicate a role for Ltbp-3 in modulating TGF-[beta] bioavailability
Dabovic, Branka; Chen, Yan; Colarossi, Cristina; Obata, Hiroto; Zambuto, Laura; Perle, Mary Ann; Rifkin, Daniel B
2002 Jan 21;156(2):227-232, Journal of cell biology
The TGF-betas are multifunctional proteins whose activities are believed to be controlled by interaction with the latent TGF-beta binding proteins (LTBPs). In spite of substantial effort, the precise in vivo significance of this interaction remains unknown. To examine the role of the Ltbp-3, we made an Ltbp-3-null mutation in the mouse by gene targeting. Homozygous mutant animals develop cranio-facial malformations by day 10. At 2 mo, there is a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. Histological examination of the skulls from null animals revealed ossification of the synchondroses within 2 wk of birth, in contrast to the wild-type synchondroses, which never ossify. Between 6 and 9 mo of age, mutant animals also develop osteosclerosis and osteoarthritis. The pathological changes of the Ltbp-3-null mice are consistent with perturbed TGF-beta signaling in the skull and long bones. These observations give support to the notion that LTBP-3 is important for the control of TGF-beta action. Moreover, the results provide the first in vivo indication for a role of LTBP in modulating TGF-beta bioavailability
— id: 27277, year: 2002, vol: 156, page: 227, stat: Journal Article,

Overexpression of the 18 kDa and 22/24 kDa FGF-2 isoforms results in differential drug resistance and amplification potential
Dini, Germana; Funghini, Silvia; Witort, Ewa; Magnelli, Lucia; Fanti, Elena; Rifkin, Daniel B; Del Rosso, Mario
2002 Oct;193(1):64-72, Journal of cellular physiology
We investigated the role of low molecular weight (LMW) and high molecular weight (HMW) isoforms of basic fibroblast growth factor 2 (FGF-2) in the expression of transformation-related phenotypic alterations, drug sensitivity modulation, and gene amplification potential. For this purpose, we used NIH 3T3 and A31 cells transfected with different cDNA FGF-2 constructs allowing expression of the different proteins. Both cell lines showed marked phenotypic alterations when expressing the LMW FGF-2 or the four HMW FGF-2 isoforms: they acquired a transformed morphology, grew at higher saturation densities in 10% serum, and exhibited anchorage-independent growth and increased invasive potential. However, HMW FGF-2-expressing cells also grew in 1% serum and their invasive potential was lower than in cells expressing all FGF-2 forms or LMW FGF-2 alone. We have grown the different cell lines under a selective pressure of N-(phosphonacetyl)-l-aspartate (PALA), a drug which specifically inhibits the aspartate transcarbamylase activity of the multifunctional carbamyl-P-synthetase/aspartate transcarbamylase/dihydro-orotase genes (CAD) enzyme (and thus inhibits de novo pyrimidine biosynthesis) and selects for cells with amplified copies of the CAD gene. Our results demonstrate that aberrant expression of the LMW FGF-2 and/or HMW FGF-2 isoforms differently modulates drug resistance and gene amplification properties in the NIH 3T3 and A31 cell lines by differential amplification of the CAD gene. Coexpression of all isoforms appears to be necessary to obtain cumulative effects and nuclear-targeted HMW FGF-2 has a pivotal role in such a cooperation
— id: 42352, year: 2002, vol: 193, page: 64, stat: Journal Article,

Tamoxifen and Estrogen Effects on TGF-beta Formation: Role of Thrombospondin-1, alphavbeta3, and Integrin-Associated Protein
Harpel JG; Schultz-Cherry S; Murphy-Ullrich JE; Rifkin DB
2001 Jun 1;284(1):11-14, Biochemical & biophysical research communications
We have found that the enhanced activation of latent TGF-beta by human breast carcinoma cell lines either treated with tamoxifen or deprived of estrogen is dependent upon thrombospondin-1 (TSP-1) since activation was blocked by anti-TSP-1 antibodies or by a TSP antagonist peptide. However, TGF-beta formation upon tamoxifen exposure to estrogen withdrawal is associated with decreased levels of soluble TSP-1. A concomitant increase in the expression of the TSP-1 receptors alphavbeta3 and integrin-associated protein (IAP) occurs under these conditions, and antibodies to TSP-1 or to these receptors inhibit increased TGF-beta formation. Therefore, increased cell surface associated TSP-1 enhances latent TGF-beta activation.
— id: 20644, year: 2001, vol: 284, page: 11, stat: Journal Article,

Fibroblast growth factors
Moscatelli D; Rifkin DB
Tumor angiogenesis and microcirculation New York : Dekker, 2001,
— id: 2767, year: 2001, vol: , page: 227, stat: Chapter,

TGF-beta activation; normal and pathological consequence
Rifkin, DB
2001 Dec;41(6):1568-1568, American zoologist
— id: 27511, year: 2001, vol: 41, page: 1568, stat: Journal Article,

Evidence that the integrin alpha(v)beta(6) activates TGF-beta 3
Annes, JP; Rifkin, DB; Munger, JS
2000 DEC ;11(2):263A-264A, Molecular biology of the cell
— id: 55207, year: 2000, vol: 11, page: 263A, stat: Journal Article,

The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium
Gualandris A; Annes JP; Arese M; Noguera I; Jurukovski V; Rifkin DB
2000 Dec;11(12):4295-4308, Molecular biology of the cell
The latent transforming growth factor-beta-binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-beta-masking protein, LTBP-1 is involved both in the sequestration of latent TGF-beta in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-beta or the latency associated peptide, which neutralize TGF-beta. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-beta 1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-beta-dependent mechanism
— id: 39508, year: 2000, vol: 11, page: 4295, stat: Journal Article,

Biochemical analysis of the arginine methylation of high molecular weight fibroblast growth factor-2
Klein S; Carroll JA; Chen Y; Henry MF; Henry PA; Ortonowski IE; Pintucci G; Beavis RC; Burgess WH; Rifkin DB
2000 Feb 4;275(5):3150-3157, Journal of biological chemistry
The post-translational methylation of the N-terminally extended or high molecular weight (HMW) forms of fibroblast growth factor-2 (FGF-2) has been shown to affect the nuclear accumulation of the growth factor. In this study, we determined the extent and position of methyl groups in HMW FGF-2. Using mass spectrometry and amino acid sequence analysis, we have shown that the 22- and 22.5-kDa forms of HMW FGF-2 contain five dimethylated arginines located at positions -22, -24, -26, -36, and -38 using the methionine residue normally used to initiate the 18-kDa form as position 0. The 24-kDa form of HMW FGF-2 contains seven to eight dimethylated arginines located at positions -48, -50, and -52, in addition to positions -22, -24, -26, -36, and -38. In vitro methylation reactions demonstrate that the N-terminal extension of HMW FGF-2 acts as a specific substrate for yeast Hmt1p and human HRMT1L2 arginine methyltransferases. These findings indicate that HMW FGF-2, with the presence of five or more dimethylated Gly-Arg-Gly repeats, contains an RGG box-like domain, which may be important for protein-protein and/or protein-RNA interactions
— id: 8561, year: 2000, vol: 275, page: 3150, stat: Journal Article,

Measurement of active TGF-beta generated by cultured cells
Mazzieri R; Munger JS; Rifkin DB
2000 ;142:13-27, Methods in molecular biology
— id: 11706, year: 2000, vol: 142, page: 13, stat: Journal Article,

Blocking LTBP-mediated matrix incorporation in vivo results in increased TGF-beta activation in mouse epidermis and hair follicles
Mazzieri, R; Obata, H; Sung, JJ; Gleizes, PE; Platt, AB; Dabovic, B; Rifkin, DB
2000 DEC ;11(2):28A-28A, Molecular biology of the cell
— id: 55206, year: 2000, vol: 11, page: 28A, stat: Journal Article,

Nonenzymatic interactions between proteinases and the cell surface: novel roles in normal and malignant cell physiology
Mignatti P; Rifkin DB
2000 ;78(1):103-157, Advances in cancer research
— id: 27868, year: 2000, vol: 78, page: 103, stat: Journal Article,

LTBP-1 is an organizer gene which acts as an activin agonist in mesoderm induction and axis formation
Quarto, N; Rifkin, DB; Hemmati-Brivanlou, A
2000 DEC ;11(2):275A-275A, Molecular biology of the cell
— id: 55208, year: 2000, vol: 11, page: 275A, stat: Journal Article,

Hybrid and complex glycans are linked to the conserved N-glycosylation site of the third eight-cysteine domain of LTBP-1 in insect cells
Rudd PM; Downing AK; Cadene M; Harvey DJ; Wormald MR; Weir I; Dwek RA; Rifkin DB; Gleizes PE
2000 Feb 22;39(7):1596-1603, Biochemistry
Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia niTN-5B1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells. Sf9 cells mainly processed the fucosylated paucomannosidic structure (GlcNAc)(2)(Mannose)(3)Fucose, although hybrid and complex N-glycosylations were also detected. In High-Five cells, the peptide was found to be modified with a wide variety of hybrid and complex sugars in addition to paucomanosidic oligosaccharides. Most glycans had one or two fucose residues bound through alpha1,3 and alpha1,6 linkages to the innermost GlcNAc. On the basis of these results and on the structure of an eight-cysteine domain from fibrillin-1, we present a model of glycosylated CR3 and discuss the role of glycosylation in eight-cysteine domain protein-protein interactions
— id: 57557, year: 2000, vol: 39, page: 1596, stat: Journal Article,

Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals
Arese M; Chen Y; Florkiewicz RZ; Gualandris A; Shen B; Rifkin DB
1999 May;10(5):1429-1444, Molecular biology of the cell
Human basic fibroblast growth factor (FGF-2) occurs in four isoforms: a low molecular weight (LMW FGF-2, 18 kDa) and three high molecular weight (HMW FGF-2, 22, 22.5, and 24 kDa) forms. LMW FGF-2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF-2s are nuclear and exert activities through an intracrine, perhaps nuclear, pathway. Selective overexpression of HMW FGF-2 forms in fibroblasts promotes growth in low serum, whereas overexpression of LMW FGF-2 does not. The HMW FGF-2 forms have two functional domains: an amino-terminal extension and a common 18-kDa amino acid sequence. To investigate the role of these regions in the intracrine signaling of HMW FGF-2, we produced stable transfectants of NIH 3T3 fibroblasts overexpressing either individual HMW FGF-2 forms or artificially nuclear-targeted LMW FGF-2. All of these forms of FGF-2 localize to the nucleus/nucleolus and induce growth in low serum. The nuclear forms of FGF-2 trigger a mitogenic stimulus under serum starvation conditions and do not specifically protect the cells from apoptosis. These data indicate the existence of a specific role for nuclear FGF-2 and suggest that LMW FGF-2 represents the biological messenger in both the autocrine/paracrine and intracrine FGF-2 pathways
— id: 6112, year: 1999, vol: 10, page: 1429, stat: Journal Article,

Melanoma growth in wild-type, urokinase-type plasminogen activator knockout and tissue-type plasminogen activator knockout mice
Chuang N; Shamamian P; Roses DF; Rifkin DB; Shapiro RL
1999 ;60:367-368, Surgical forum
— id: 25206, year: 1999, vol: 60, page: 367, stat: Journal Article,

[Activation of latent TGF-beta. A required mechanism for vascular integrity]
Gleizes PE; Rifkin DB
1999 Apr;47(4):322-329, Pathologie biologie
Recent molecular genetics studies in humans and mice showed that transforming growth factor-beta (TGF-beta) is involved in vasculogenesis and maintenance of blood vessel integrity. These results confirm earlier in vitro studies demonstrating generation of active TGF-beta when endothelial cells are cocultured with smooth muscle cells or pericytes. TGF-beta is secreted as a latent, inactive complex and becomes active only when released. Latent TGF-beta binds covalently to proteins (LTBP) that target it to the extracellular matrix. Thus, the latency of TGF-beta is essential to the regulation of the bioavailability and activity of this cytokine. The development of methods for measuring activation of latent TGF-beta in cell cultures and identification of the proteins contained in the latent TGF-beta complex have shed new light on the mechanism of activation of latent TGF-beta possibly involved in vasculogenesis, angiogenesis, and other processes
— id: 42355, year: 1999, vol: 47, page: 322, stat: Journal Article,

Modulation of cell growth and transformation by doxycycline-regulated FGF-2 expression in NIH-3T3 cells
Gualandris A; Arese M; Shen B; Rifkin DB
1999 Nov;181(2):273-284, Journal of cellular physiology
The single-copy fibroblast growth factor 2 (FGF-2) gene encodes four coexpressed isoforms of different molecular masses. The 18-kDa FGF-2 is primarily localized in the cytoplasm, whereas the higher molecular mass isoforms (HMW FGF-2) localize to the nucleus and nucleolus. The overexpression of either 18-kDa FGF-2 or HMW FGF-2 promotes cell transformation in a dose-dependent manner. In NIH 3T3 cells, the selective overexpression of HMW FGF-2 but not of 18-kDa FGF-2 confers upon the cells the unique phenotype of growth in low serum-containing medium. Thus, the distinct intracellular localization and the level of expression of FGF-2 are pivotal requirements for the differential effects of FGF-2 isoforms on the cellular phenotype. On this basis, we established a doxycycline-regulatable FGF-2 expression system that permitted us to regulate the expression of each isoform in a time- and dose-dependent manner. We analyzed the growth properties of cells in the presence and absence of doxycycline in both normal and low serum-containing medium and in soft agar. The doxycycline-activated expression of 18-kDa FGF-2 did not allow growth in low serum medium. The growth of cells expressing HMW FGF-2 was increased by doxycycline under all three conditions, and a relationship between the level of HMW FGF-2 expression and cell growth was observed for all three conditions. This doxycycline-regulatable FGF-2 expression system provides a mechanism to analyze changes in FGF-2 targeted pathways and genes and to characterize pathways specifically activated by either the 18-kDa FGF-2 or the HMW FGF-2 isoforms.
— id: 6212, year: 1999, vol: 181, page: 273, stat: Journal Article,

Diltiazem reduces retinal neovascularization in a mouse model of oxygen induced retinopathy
Higgins RD; Yu K; Sanders RJ; Nandgaonkar BN; Rotschild T; Rifkin DB
1999 Jan;18(1):20-27, Current eye research
PURPOSE: To assess the effect of diltiazem, a calcium channel blocking agent, on oxygen induced retinopathy (OIR) in a mouse model using neovascular nuclei quantitation and a quantitative scoring system based on examining fluorescein perfused retinal whole mount preparations. METHODS: The mouse model of oxygen induced retinopathy consisting of a 5 day exposure to 75% oxygen from postnatal day 7 to 12 was used to produce retinal neovascularization. Fluorescein conjugated dextran angiography of retinal vasculature was performed and retinal whole mounts were prepared to score features of retinopathy. The parameters that were scored in a masked fashion included blood vessel growth, blood vessel tuft formation, extra retinal neovascularization, degree of central vasoconstriction, retinal hemorrhage, and tortuosity of vessels. Diltiazem (0.05-0.5 mg/kg/day subcutaneously for five days) was administered to mice pups during exposure to oxygen to determine if calcium channel blockade altered retinopathy. In addition, quantification of retinal neovascular nuclei was performed in a masked fashion with periodic acid Schiff (PAS) staining of frozen eye sections. RESULTS: Animals that were exposed to hyperoxia for five days had a median (25th, 75th quartile) retinopathy score of 9 (8,11) versus control animals that had a retinopathy score of 1 (0,1) with p<0.001. Subscores for blood vessel growth, blood vessel tufts, extra-retinal neovascularization, central vasoconstriction, hemorrhage, and blood vessel tortuosity were all significantly different between control and treated animals. In addition, quantification of neovascular nuclei showed a significant increase in the number of nuclei extending beyond the inner limiting membrane into the vitreous in hyperoxic treated animals. Diltiazem at doses of 0.2 and 0.5 mg/kg/day improved the retinopathy as measured by the total retinopathy score [5 (4,6) and 4 (3.75,5.25), respectively]. The average number of extraretinal neovascular nuclei per retinal section (mean +/-standard deviation) was significantly decreased by diltiazem at doses of 0.2 and 0.5 mg/kg/day (31.4+/-18.8 and 20.9+/-6.9, respectively) when compared to hyperoxic treated animals (56.1+/-21.5). CONCLUSIONS: Diltiazem reduces oxygen induced retinopathy in the mouse as measured by a scoring system based on a retinal whole mount method of retinal neovascularization and by quantification of extra retinal neovascular nuclei
— id: 7345, year: 1999, vol: 18, page: 20, stat: Journal Article,

The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis
Munger JS; Huang X; Kawakatsu H; Griffiths MJ; Dalton SL; Wu J; Pittet JF; Kaminski N; Garat C; Matthay MA; Rifkin DB; Sheppard D
1999 Feb 5;96(3):319-328, Cell
Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin
— id: 7411, year: 1999, vol: 96, page: 319, stat: Journal Article,

Proteolytic control of growth factor availability
Rifkin DB; Mazzieri R; Munger JS; Noguera I; Sung J
1999 Jan;107(1):80-85, Apmis
Most growth factors are released from cells in a form that does not permit immediate interaction with their high affinity receptors. An important mechanism for presentation of these released latent growth factors is activation by the plasminogen activator-plasmin system. The involvement of this system in the biology of Transforming Growth Factor-beta (TGF-beta) is reviewed
— id: 6074, year: 1999, vol: 107, page: 80, stat: Journal Article,

Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression
Wilhelm OG; Wilhelm S; Escott GM; Lutz V; Magdolen V; Schmitt M; Rifkin DB; Wilson EL; Graeff H; Brunner G
1999 Aug;180(2):225-235, Journal of cellular physiology
The glycosylphosphatidylinositol (GPI)-anchored, multifunctional receptor for the serine proteinase, urokinase plasminogen activator (uPAR, CD87), regulates plasminogen activation and cell migration, adhesion, and proliferation. uPAR occurs in functionally distinct, membrane-anchored and soluble isoforms (s-uPAR) in vitro and in vivo. Recent evidence indicates that s-uPAR present in the circulation of cancer patients correlates with tumor malignancy and represents a valuable prognostic marker in certain types of cancer. We have therefore analyzed the mechanism of uPAR shedding in vitro. We present evidence that uPAR is actively released from ovarian cancer cells since the rate of receptor shedding did not correlate with uPAR expression. While s-uPAR was derived from the cell surface, it lacked the hydrophobic portion of the GPI moiety indicating anchor cleavage. We show that uPAR release is catalyzed by cellular GPI-specific phospholipase D (GPI-PLD), an enzyme cleaving the GPI anchor of the receptor. Thus, recombinant GPI-PLD expression increased receptor release up to fourfold. Conversely, a 40% reduction in GPI-PLD activity by GPI-PLD antisense mRNA expression inhibited uPAR release by more than 60%. We found that GPI-PLD also regulated uPAR expression, possibly by releasing a GPI-anchored growth factor. Our data suggest that cellular GPI-PLD might be involved in the generation of circulating prognostic markers in cancer and possibly regulate the function of GPI-anchored proteins by generating functionally distinct, soluble counterparts
— id: 35194, year: 1999, vol: 180, page: 225, stat: Journal Article,

Inhibition of angiogenesis and matrix metalloproteinase-2 activity by dexrazoxane (zinecard) may mediate its antitumor effects
Chuang JN; Ren CJ; Mendoza S; Shamamian P; Roses DF; Rifkin DB; Chachoua A; Shapiro RL
1998 ;49:424-426, Surgical forum
— id: 25210, year: 1998, vol: 49, page: 424, stat: Journal Article,

Hyperoxia stimulates endothelin-1 secretion from endothelial cells; modulation by captopril and nifedipine
Higgins RD; Hendricks-Munoz KD; Caines VV; Gerrets RP; Rifkin DB
1998 May;17(5):487-493, Current eye research
PURPOSE: Retinopathy of prematurity (ROP) is a vasoproliferative condition that can result in severe visual impairment and blindness in preterm babies. Two conditions seen very early in radioimmunoassay (ROP) are vasoconstriction and vaso-obliteration. A potent vasoconstrictor secreted by endothelial cells is endothelin-1 (ET-1). Premature birth results in a relative systemic hyperoxia, compared to the in utero oxygen milieu. We tested the hypothesis that hyperoxia increases ET-1 expression as a possible mechanism for vasoconstriction in the retinal vasculature. METHODS: Bovine retinal endothelial cells and adrenal capillary endothelial cells were isolated and maintained in culture. Cells were exposed to control or hyperoxic culture conditions for 24 h, with and without addition of captopril and nifedipine. Media was collected and assayed for ET-1 by ROP. In addition, cell counts and secreted LDH assays were performed. RESULTS: Conditioned media from cultured bovine retinal and adrenal endothelial cells exposed to hyperoxic culture conditions for 24 h were found to have higher levels of ET-1 than conditioned media from normoxic control cells. Captopril (10(-6) M and 10(-4) M) and nifedipine (10(-6) M and 10(-4) M) inhibited ET-1 release from hyperoxia-exposed endothelial cells. Under normoxic conditions, ET-1 release was inhibited by 10(-4) M captopril or 10(-4) M nifedipine. CONCLUSIONS: These results demonstrate that (1) hyperoxia stimulates in vitro ET-1 secretion in bovine retinal and adrenal capillary endothelial cells, and (2) captopril and nifedipine downregulate ET-1 secretion under normoxic and hyperoxic culture conditions, in a dose-dependent fashion. We speculate that ET-1 may be involved in retinal vessel vasoconstriction seen early in the development of ROP. Further, ACE inhibitors and calcium-channel blocking agents, such as captopril and nifedipine, may provide an avenue for blocking vasoconstriction in ROP
— id: 42356, year: 1998, vol: 17, page: 487, stat: Journal Article,

Transforming growth factor-beta 1 latency associated peptide binds to and signals through the integrin alpha v beta 6
Kawakatsu, H; Munger, JS; Rifkin, DB; Sheppard, D
1998 NOV ;9(11):297A-297A, Molecular biology of the cell
— id: 53646, year: 1998, vol: 9, page: 297A, stat: Journal Article,

Interactions between growth factors and integrins: latent forms of transforming growth factor-beta are ligands for the integrin alphavbeta1
Munger JS; Harpel JG; Giancotti FG; Rifkin DB
1998 Sep;9(9):2627-2638, Molecular biology of the cell
The multipotential cytokine transforming growth factor-beta (TGF-beta) is secreted in a latent form. Latency results from the noncovalent association of TGF-beta with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-beta-binding protein (LTBP) produces the most common form of latent TGF-beta, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-beta. LTBP and the LAP propeptides of TGF-beta (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-beta function in the ECM, we determined whether latent TGF-beta1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits alphav and beta1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. alphavbeta1 eluted with EDTA. After purification in the presence of Mn2+, a small amount of alphavbeta5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was alphavbeta1 dependent. These results establish alphavbeta1 as a LAP-beta1 receptor. Interactions between latent TGF-beta and alphavbeta1 may localize latent TGF-beta to the surface of specific cells and may allow the TGF-beta1 gene product to initiate signals by both TGF-beta receptor and integrin pathways
— id: 12078, year: 1998, vol: 9, page: 2627, stat: Journal Article,

Cells expressing the integrin alpha v beta g can activate latent TGF-beta
Munger, JS; Kawakatsu, H; Dalton, S; Rifkin, DB; Sheppard, D
1998 NOV ;9(11):417A-417A, Molecular biology of the cell
— id: 53648, year: 1998, vol: 9, page: 417A, stat: Journal Article,

Structure and activation of the large latent transforming growth factor-Beta complex
Nunes I; Munger J; Harpel JG; Nagano Y; Shapiro R; Gleizes PE; Rifkin DB
1998 Oct;69(10):643-648, Journal of the American Optometric Association
BACKGROUND: Many cytokines regulate processes involved in the pathogenesis of proliferative vitreoretinopathy. Transforming growth factor-beta (TGF-beta) is an example of a pluripotent growth factor that regulates cell proliferation, extracellular matrix (ECM) deposition, cell migration, and differentiation--all biological activities involved in the formation and progression of proliferative vitreoretinopathies. METHODS: A review of experimental results that demonstrate how vascular cells generate biologically active TGF-beta is presented. Most cell types--including endothelial cells and pericytes, which form the retinal microvasculature--express TGF-beta as a large latent TGF-beta complex. Mature TGF-beta, the biologically active form, must be generated from the large latent complex before it can signal by binding to its high affinity cell surface receptors. RESULTS: A critical step in regulating TGF-beta effects may be the activation of the large latent TGF-beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that co-culturing bovine smooth muscle cells or pericytes and endothelial cells generates active TGF-beta. CONCLUSION: The mechanism of latent TGF-beta activation self-regulates through effectors of plasmin generation. Studying TGF-beta generation by co-cultures of pericytes and endothelial cells can provide us with insights into how disruption of latent TGF-beta activation may lead to unregulated endothelial proliferation, ECM deposition, and cellular infiltration, as observed clinically in neovascular- and fibrotic-related pathologies
— id: 6041, year: 1998, vol: 69, page: 643, stat: Journal Article,

Irsogladine maleate inhibits angiogenesis in wild-type and plasminogen activator-deficient mice
Ren CJ; Ueda F; Roses DF; Harris MN; Mignatti P; Rifkin DB; Shapiro RL
1998 Jul 1;77(2):126-131, Journal of surgical research
BACKGROUND: The activation of the zymogen plasminogen to the serine protease plasmin by urokinase-type (uPA) and tissue-type (tPA) plasminogen activators (PA) is an important event in a variety of physiologic and pathophysiologic processes in mammals. Enhanced PA activity occurs during angiogenesis and has been correlated in vitro and in vivo with increased tumor aggressiveness and is an indicator of poor prognosis in a variety of tumors in humans. Preliminary studies suggest that the antiulcer drug irsogladine maleate (IM) diminishes PA activity in vitro and may inhibit angiogenesis in vivo. To define the precise mechanism of angiogenesis inhibition by IM in vivo, we tested the ability of IM to blunt angiogenesis in a mouse cornea neovascularization model performed in wild-type and PA-knockout mice. METHODS: Three days prior to pellet implantation, groups of C57Bl/6 wild-type, uPA-deficient (upA-/-), and tPA-deficient (tPA-/-) mice received IM (300 mg/kg), IM (500 mg/kg), or vehicle (0.5% carboxymethylcellulose) via oral gavage. After 3 days of treatment, hydron polymer-coated pellets of sucrose aluminum sulfate containing 100 ng of basic fibroblast growth factor (bFGF) were inserted into surgically created pockets in the cornea of each mouse. On postoperative day 6, the neovascularization of each cornea was evaluated by a blinded observer using slit lamp microscopy and photographed. Angiogenesis was quantified by calculating vascular area (mm2) +/- SEM using a modified formula for a half ellipse that incorporates calibrated vessel measurements [Vessel length (mm) x Clock hours x pi x 0.2]. RESULTS: IM treatment (300 and 500 mg/kg/day) resulted in a dose-dependent reduction of angiogenesis in wild-type mice by 21 and 45.3% (P < 0.02, P < 0.001), in tPA-deficient mice by 42.6 and 46% (P < 0.001, P < 0.001), and in uPA-deficient mice by 27.2 and 46% (P < 0.05, p < 0.001), respectively. No quantitative differences in neovascularization were observed in either treatment group between transgenic mouse strains. No toxicity was noted in any group. CONCLUSION: IM inhibits bFGF-induced angiogenesis in wild-type, tPA-knockout, and uPA-knockout mice. The observation that IM significantly diminishes angiogenesis in both PA-deficient mice and wild-type mice suggests that the mechanism of action of IM may be independent of plasminogen activation.
— id: 12076, year: 1998, vol: 77, page: 126, stat: Journal Article,

Activation of latent TGF-beta: Participation of proteases
Rifkin, DB; Munger, J; Mazzieri, R; Sung, J; Harpel, JG
1998 NOV ;9(11):6A-6A, Molecular biology of the cell
— id: 53639, year: 1998, vol: 9, page: 6A, stat: Journal Article,

Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis
Seghezzi G; Patel S; Ren CJ; Gualandris A; Pintucci G; Robbins ES; Shapiro RL; Galloway AC; Rifkin DB; Mignatti P
1998 Jun 29;141(7):1659-1673, Journal of cell biology
FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2-induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-Mr FGF-2 (22-24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF
— id: 7787, year: 1998, vol: 141, page: 1659, stat: Journal Article,

Intracellular association of FGF-2 with the ribosomal protein L6/TAXREB107
Shen B; Arese M; Gualandris A; Rifkin DB
1998 Nov 18;252(2):524-528, Biochemical & biophysical research communications
By using the yeast two-hybrid system, we identified the ribosomal protein L6/TAXREB107 as an intracellular partner for FGF-2. L6/TAXREB107 also mediates the DNA binding of the HTLV-1 transactivator Tax. In vitro binding experiments indicated that both the high-molecular-weight forms (HMW) and the 18-kDa form of FGF-2 bind to L6/TAXREB107. Deletion analysis suggested that L6/TAXREB107 has two binding sites for HMW FGF-2 and one binding site for 18-kDa FGF-2, implying that the unique N-terminal extension of the HMW FGF-2 is one of the binding domains for L6/TAXREB107. Transfection assays showed that high expression of either HMW or 18 kDa FGF-2 stimulates Tax-mediated transactivation in NIH 3T3 cells. This result suggests a possible role of FGF-2 in Tax-mediated HTLV-1 transformation as well as FGF-2 binding to ribosomes and/or their precursors.
— id: 7791, year: 1998, vol: 252, page: 524, stat: Journal Article,

Biological roles of fibroblast growth factor-2
Bikfalvi A; Klein S; Pintucci G; Rifkin DB
1997 Feb;18(1):26-45, Endocrine reviews
— id: 9010, year: 1997, vol: 18, page: 26, stat: Journal Article,

TGF-beta latency: biological significance and mechanisms of activation
Gleizes PE; Munger JS; Nunes I; Harpel JG; Mazzieri R; Noguera I; Rifkin DB
1997 ;15(3):190-197, Stem cells
Transforming growth factor (TGF-) beta is secreted as a latent complex in which the mature growth factor remains associated with its propeptide. In order to elicit a biological response, the cytokine must be released from the latent complex, a process termed latent TGF-beta activation or TGF-beta formation. Although latent TGF-beta activation is a critical step in the regulation of its activity, little is known about the molecular mechanisms that lead to the production of active TGF-beta. In this article, we present an overview of the data available on this topic, and we propose a tentative model for the mechanism of TGF-beta formation based upon the observations with different cell systems and on recent findings on the structure of the latent TGF-beta complex
— id: 7154, year: 1997, vol: 15, page: 190, stat: Journal Article,

Fibroblast growth factors as angiogenesis factors: new insights into their mechanism of action
Klein S; Roghani M; Rifkin DB
1997 ;79:159-192, Experientia. Supplementum
— id: 12428, year: 1997, vol: 79, page: 159, stat: Journal Article,

Latent transforming growth factor-beta: structural features and mechanisms of activation
Munger JS; Harpel JG; Gleizes PE; Mazzieri R; Nunes I; Rifkin DB
1997 May;51(5):1376-1382, Kidney international
Transforming growth factor-beta are cytokines with a wide range of biological effects. They play a pathologic role in inflammatory and fibrosing diseases such as nephrosclerosis. TGF-beta s are secreted in a latent form due to noncovalent association with latency associated peptide (LAP), which is a homodimer formed from the propeptide region of TGF-beta. LAP is disulfide linked to another protein, latent TGF-beta binding protein (LTBP). LTBP has features in common with extracellular matrix proteins, and targets latent TGF-beta to the matrix. Activation of latent TGF-beta can be accomplished in vitro by denaturing treatments, plasmin digestion, ionizing radiation and interaction with thrombospondin. The mechanisms by which latent TGF-beta is activated physiologically are not well understood. Results to date suggest an important role for proteases, particularly plasmin, although other mechanisms probably exist. A general model of activation is proposed in which latent TGF-beta is released from the extracellular matrix by proteases, localized to cell surfaces, and activated by cell-associated plasmin
— id: 35177, year: 1997, vol: 51, page: 1376, stat: Journal Article,

Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta
Nunes I; Gleizes PE; Metz CN; Rifkin DB
1997 Mar 10;136(5):1151-1163, Journal of cell biology
Transforming growth factor-beta (TGF-beta) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-beta, the TGF-beta propeptide, and the latent TGF-beta binding protein (LTBP). To interact with its cell surface receptors, TGF-beta must be released from the latent complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-beta. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S-transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in cross-linking and formation of TGF-beta by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-beta generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (deltaN293) or 441 (deltaN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that deltaN293 LTBP-1S was matrix associated via a transglutaminase-dependent reaction, whereas deltaN441 LTBP-1S was not. This suggests that residues 294-441 are critical to the transglutaminase reactivity of LTBP-1S
— id: 12354, year: 1997, vol: 136, page: 1151, stat: Journal Article,

Plasminogen/plasminogen activator and growth factor activation
Rifkin DB; Gleizes PE; Harpel J; Nunes I; Munger J; Mazzieri R; Noguera I
1997 ;212:105-115, CIBA Foundation symposium
The plasminogen/plasminogen activator system is widely used in extracellular proteolysis. In this review the involvement of this system in tumour invasion, cell migration, growth factor presentation and inhibition of angiogenesis are discussed
— id: 12147, year: 1997, vol: 212, page: 105, stat: Journal Article,

Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: An autocrine mechanism of angiogenesis
Seghezzi, G; Patel, S; Ren, CJ; Pintucci, G; Gualandris, A; Robbins, E; Shapiro, RL; Galloway, AC; Rifkin, DB; Mignatti, P
1997 NOV ;8(5):1335-1335, Molecular biology of the cell
— id: 53166, year: 1997, vol: 8, page: 1335, stat: Journal Article,

Urokinase-type plasminogen activator-deficient mice are predisposed to staphylococcal botryomycosis, pleuritis, and effacement of lymphoid follicles
Shapiro RL; Duquette JG; Nunes I; Roses DF; Harris MN; Wilson EL; Rifkin DB
1997 Jan;150(1):359-369, American journal of pathology
Urokinase-type plasminogen activator (uPA) is thought to be an important mediator in the proteolytic degradation of extracellular matrix components observed in a wide variety of normal physiological and pathological conditions. However, the phenotype of a recently developed strain of urokinase-deficient (uPA-/-) mice appears to be normal when maintained under ideal nonstressful conditions. We report an outbreak of botryomycosis, an unusual staphylococcal infection, in a colony of uPA-deficient mice. A detailed histological examination of these uPA-deficient animals also revealed a variety of previously unreported phenotypic abnormalities such as pleuritis and the effacement of lymphoid follicles in the regional lymph nodes and spleen. Additional phenotypic abnormalities such as dystrophic calcifications and rectal prolapse were also observed in the uPA-deficient population. These abnormalities were also noted in ostensibly healthy uPA-deficient animals. Botryomycosis did not affect a colony of wild-type (uPA+/+) animals maintained concurrently under identical conditions in the same room. The peculiar predisposition of the uPA-deficient animals to this rare bacterial infection and the development of phenotypic abnormalities associated with the targeted disruption the uPA gene suggests that uPA contributes significantly to the cutaneous microenvironment and is additional evidence of the extensive involvement of the plasminogen activators in mammalian physiology
— id: 12426, year: 1997, vol: 150, page: 359, stat: Journal Article,

Inhibition of glycosylphosphatidylinositol (GPI) phospholipase D by suramin-like compounds
Brunner G; Zalkow L; Burgess E; Rifkin DB; Wilson EL; Gruszecka-Kowalik E; Powis G
1996 Sep-Oct;16(5A):2513-2516, Anticancer research
A number of proteins are found attached to the plasma membrane of mammalian cells by a glycosylphosphatidylinositol (GPI) anchor that can be cleaved by GPI specific phospholipase D (GPI-PLD). There are no known specific inhibitors of GPI-PLD. We examined some inhibitors of phosphatidylinositol specific phospholipase C (PI-PLC) for their ability to inhibit human serum and human bone marrow cell GPI-PLD. Azo analogues of suramin were found to be potent inhibitors of GPI-PLD. One compound had an IC50 of 3.7 microM that was 10-fold lower than the IC50 required to inhibit PI-PLC. The azo suramin analogues inhibited cancer cell growth at concentrations similar to those required to inhibit GPI-PLD, and below concentrations required to inhibit growth factor binding. It is possible that inhibition of cell growth might be related to the ability of the compounds to inhibit GPI-PLD
— id: 12546, year: 1996, vol: 16, page: 2513, stat: Journal Article,

FGF suppresses apoptosis and induces differentiation of fiber cells in the mouse lens
Chow, RL; Roux, GD; Roghani, M; Palmer, MA; Rifkin, DB; Moscatelli, DA; Lang, RA
1996 FEB 15 ;37(3):4509-4509, Investigative ophthalmology & visual science. IOVS
— id: 53037, year: 1996, vol: 37, page: 4509, stat: Journal Article,

The regulation of TGF-beta activity by hematopoietic cells
Coetzee, S; Burger, P; Rifkin, D; Wilson, EL
1996 NOV 15 ;88(10):2150-2150, Blood
— id: 52712, year: 1996, vol: 88, page: 2150, stat: Journal Article,

Identification and characterization of an eight-cysteine repeat of the latent transforming growth factor-beta binding protein-1 that mediates bonding to the latent transforming growth factor-beta1
Gleizes PE; Beavis RC; Mazzieri R; Shen B; Rifkin DB
1996 Nov 22;271(47):29891-29896, Journal of biological chemistry
Most cultured cell types secrete small latent transforming growth factor-beta (TGF-beta) as a disulfide-bonded complex with a member of the latent TGF-beta binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-beta1. Coexpression in Sf9 cells of small latent TGF-beta1 with deletion mutants of LTBP-1 showed that the third eight-cysteine repeat of LTBP-1 is necessary and sufficient for covalent interaction with small latent TGF-beta1. Analysis by mass spectrometry of this eight-cysteine repeat, produced as a recombinant peptide in Sf9 cells, confirmed that it was N-glycosylated, as expected from the primary sequence. No other post-translational modifications of this domain were detected. Alkylation of the recombinant peptide with vinyl pyridine failed to reveal any free cysteines, indicating that, in the absence of small latent TGF-beta, the eight cysteines of this domain are engaged in intramolecular bonds. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-beta1 through a mechanism that does not require any specific post-translational modification of this domain. They also suggest that this domain adopts different conformations depending on whether it is free or bound to small latent TGF-beta
— id: 12468, year: 1996, vol: 271, page: 29891, stat: Journal Article,

Integrin regulation by endogenous expression of 18-kDa fibroblast growth factor-2
Klein S; Bikfalvi A; Birkenmeier TM; Giancotti FG; Rifkin DB
1996 Sep 13;271(37):22583-22590, Journal of biological chemistry
The three high molecular weight (HMW) forms of fibroblast growth factor-2 (FGF-2) have a distinct intracellular localization and differentially affect cell mobility and growth compared with the fourth 18-kDa form. To characterize further the effects of the 18-kDa and HMW forms of FGF-2, we have examined their ability to modulate integrin expression. Transfected NIH 3T3 cells expressing only 18-kDa FGF-2 exhibited increased cell surface levels of alpha5beta1, whereas cells expressing only HMW FGF-2 exhibited cell surface alpha5beta1 levels similar to parental cells. When cells synthesizing 18-kDa FGF-2 were transfected with a cDNA encoding a dominant negative FGF receptor, alpha5beta1 cell surface levels decreased. Immunoprecipitation of biosynthetically labeled cells indicated that expression of 18-kDa FGF-2 increased the biosynthesis and rate of maturation of alpha5. Northern blot analysis showed that 18-kDa FGF-2 increases the level of the alpha5 subunit mRNA but does not affect beta1 subunit transcript levels. Experiments utilizing luciferase reporter gene activity revealed increased alpha5 promoter activity in cells expressing 18-kDa FGF-2 indicating that the enhanced alpha5 transcript level is due to modulation of the transcription rate. Therefore, interaction of 18-kDa FGF-2 with FGF receptors results in changes in alpha5beta1 biosynthesis and processing. In contrast, endogenous expression of HMW FGF-2 does not mediate this effect
— id: 8012, year: 1996, vol: 271, page: 22583, stat: Journal Article,

Characterization of fibroblast growth factor-2 binding to ribosomes
Klein S; Morimoto T; Rifkin DB
1996 ;13(3-4):219-228, Growth factors
We have examined the binding of FGF-2 to ribosomes and have found that in NIH 3T3 cells that synthesize high amounts of all FGF-2 forms, both 18 kDa and HMW forms of FGF-2 bind to ribosomes. Ribosomes purified from these cells were treated with RNase or puromycin to identify the binding site of FGF-2 on the ribosome. Neither RNase nor puromycin treatment affected the in vivo binding of FGF-2 to ribosomes suggesting that FGF-2 binds ribosomal protein or rRNA, but not mRNA. The stoichiometry of binding in these cells was approximately 1 FGF-2 molecule bound per 1 ribosome. Binding was unaffected by high salt treatment indicating that FGF-2 tightly associates with polysomes. An in vitro binding experiment performed with purified ribosomes and recombinant FGF-2 suggested that the binding site is saturable. HBNF, a protein with similar charge and size to FGF-2, bound 15-fold less than FGF-2 to purified ribosomes. These results indicate that the binding of FGF-2 to ribosomes is specific
— id: 12671, year: 1996, vol: 13, page: 219, stat: Journal Article,

Characterization of different forms of cell-associated matrix metalloproteinase-9 (MMP-9)
Mazzieri, R; Zanetta, L; Monea, S; Galloway, AC; Rifkin, DB; Mignatti, P
1996 DEC ;7(3):350-350, Molecular biology of the cell
— id: 53347, year: 1996, vol: 7, page: 350, stat: Journal Article,

Plasminogen activators and angiogenesis
Mignatti P; Rifkin DB
1996 ;213 ( Pt 1)(3-4):33-50, Current topics in microbiology & immunology
— id: 42357, year: 1996, vol: 213 ( Pt 1), page: 33, stat: Journal Article,

Plasminogen activators and matrix metalloproteinases in angiogenesis
Mignatti P; Rifkin DB
1996 ;49(1-3):117-137, Enzyme & protein
In the initial stages of capillary formation (angiogenesis) microvascular endothelial cells of preexisting blood vessels locally degrade the underlying basal lamina and invade into the stroma of the tissue to be vascularized. A consistent body of experimental evidence has shown that this process requires a wide array of dedradative enzymes. Components of the plasminogen activator (PA)-plasmin system and of the matrix metalloproteinase (MMP) family play important roles. PAs trigger a proteinase cascade that results is the generation of high local concentrations of plasmin and active MMPs. This increase in proteolytic activity has three major consequences: it permits endothelial cell degradation and invasion of the vessel basal lamina, generates extracellular matrix (ECM) degradation products that are chemotactic for endothelial cells, and activates and mobilizes growth factors localized in the ECM. In addition, urokinase-type PA modulates some endothelial cell functions, including proliferation and migration, with a mechanism independent of proteolytic activity. PA and MMP activities are modulated in endothelial cells by complex mechanisms, including transcriptional regulation by a variety of growth factors and cytokines with angiogenic activity, extracellular control of the proteolytic activities by tissue inhibitors, and interaction with binding sites on the cell membrane and ECM
— id: 57369, year: 1996, vol: 49, page: 117, stat: Journal Article,

Latency-associated peptide (LAP), which binds and confers latency upon transforming growth factor-beta (TGF-beta), is a substrate for protein kinase C and thrombin-stimulated platelet kinase(s)
Munger, JS; Harpel, JG; Rom, WN; Rifkin, DB
1996 MAR ;44(3):A231-A231, Journal of investigative medicine
— id: 52943, year: 1996, vol: 44, page: A231, stat: Journal Article,

Effects of endogenously activated transforming growth factor-beta on growth and differentiation of retinoic acid-treated HL-60 cells
Nunes I; Kojima S; Rifkin DB
1996 Feb 1;56(3):495-499, Cancer research
Retinoic acid (RA)-treated HL-60 cells were used as a model to study differentiation of granulocytic leukemias. RA induces these cells to mature into granulocytes and to decrease growth. Mediators of these RA effects have not been identified definitively, but transforming growth factor-beta (TGF-beta) has been implicated in regulating proliferation and differentiation of myelogenous leukemic cells. The role of TGF-beta in RA-dependent differentiation and cessation of growth was examined by adding neutralizing anti-TGF-beta IgG to RA-treated HL-60 cells, followed by assessing cell growth and markers of granulocytic differentiation over 5 days. After addition of neutralizing anti-TGF-beta IgG, growth of RA-treated HL-60 cells was maintained at control levels, but granulocytic differentiation continued. These experiments demonstrated that the antiproliferative activity of RA was TGF-beta dependent but that differentiation was not. Because most cell types secrete TGF-beta in a biologically inactive complex, a TGF-beta-dependent effect requires cells to activate the latent form of TGF-beta. Active and total TGF-beta levels were quantitated in media harvested from control and RA-treated cells using a luciferase-based bioassay for TGF-beta activity. Similar levels of total TGF-beta were observed between control and RA-treated cells. RA-treated cells produced active TGF-beta (18-24 pg/ml) after 1, 2, and 3 days of treatment, whereas negligible levels were produced by control cultures. Activation of endogenous latent TGF-beta by RA-treated cells occurred through a plasmin-independent mechanism
— id: 56835, year: 1996, vol: 56, page: 495, stat: Journal Article,

Structure and activation of the large latent transforming growth factor-beta complex
Nunes I; Munger JS; Harpel JG; Nagano Y; Shapiro RL; Gleizes PE; Rifkin DB
1996 Mar;20 Suppl 3:S4-S8, International journal of obesity & related metabolic disorders
Most cell types express transforming growth factor-beta (TGF-beta) as a large latent TGF-beta complex that must be converted to an active form before TGF-beta can interact with cell surface TGF-beta receptors. This conversion involves the release of mature TGF-beta from the complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide, latency associated peptide. A critical step in regulating TGF-beta effects may be the activation of the large latent TGF-beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that coculturing bovine endothelial and smooth muscle cells generates active TGF-beta. Coculture activation of the large latent TGF-beta complex occurs through a plasmin-dependent mechanism that requires concentration of reactants on the cell surface and/or extracellular matrix. The mechanism of latent TGF-beta activation self-regulates through effectors of plasmin generation
— id: 12639, year: 1996, vol: 20 Suppl 3, page: S4, stat: Journal Article,

Angiogenesis and the fibrinolytic system
Pintucci G; Bikfalvi A; Klein S; Rifkin DB
1996 ;22(6):517-524, Seminars in thrombosis & hemostasis
Angiogenesis is defined as the formation of capillaries from preexisting blood vessels. This involves a balanced spatiotemporal modulation of endothelial cell adhesion, migration, and proliferation. An array of proteolytic enzymes expressed from endothelial cells including those of the plasminogen activator/plasmin system are required for angiogenesis to occur. In this review we focus on the growth factors that are involved in the angiogenic process and that modulate the expression and/or the activity of the plasminogen activator/plasmin system. The elucidation of the interactions between angiogenic growth factors, endothelial cell proteolytic enzymes, and the extracellular environment could ultimately lead to the therapeutic approaches to block angiogenesis and the pathophysiological conditions associated with it
— id: 9012, year: 1996, vol: 22, page: 517, stat: Journal Article,

Methylation of high molecular weight fibroblast growth factor-2 determines post-translational increases in molecular weight and affects its intracellular distribution
Pintucci G; Quarto N; Rifkin DB
1996 Aug;7(8):1249-1258, Molecular biology of the cell
The high molecular weight (HMW) forms (24, 22.5, and 22 kDa) of basic fibroblast growth factor-2 (FGF-2) contain an N-terminal extension responsible for their predominantly nuclear localization. These forms of FGF-2 are post-translationally modified, resulting in a 1- to 2-kDa increase in apparent molecular mass. Here we show that this post-translational modification is inhibited by methionine starvation and by the methyltransferase inhibitors 5'-deoxy-5'-methylthioadenosine (MTA) and 3-deaza-adenosine. Inhibition of the methylation-dependent modification results in a significant decrease in HMW FGF-2 nuclear accumulation, suggesting that methylation is relevant to the intracellular distribution of these forms of FGF-2. Treatment with MTA does not affect either the synthesis or the intracellular fate of another nuclear protein, the SV40 large T antigen, demonstrating that this drug does not have a generalized effect on nuclear protein accumulation. These results link HMW FGF-2 post-translational modification to its intracellular distribution
— id: 9011, year: 1996, vol: 7, page: 1249, stat: Journal Article,

Autocrine regulation of vascular endothelial growth factor (VEGF) expression by fibroblast growth factor-2 (FGF-2)
Seghezzi, G; Patel, S; Pintucci, G; Galloway, A; Rifkin, D; Mignatti, P
1996 DEC ;7(3):2045-2045, Molecular biology of the cell
— id: 53358, year: 1996, vol: 7, page: 2045, stat: Journal Article,

Induction of primary cutaneous melanocytic neoplasms in urokinase-type plasminogen activator (uPA)-deficient and wild-type mice: cellular blue nevi invade but do not progress to malignant melanoma in uPA-deficient animals
Shapiro RL; Duquette JG; Roses DF; Nunes I; Harris MN; Kamino H; Wilson EL; Rifkin DB
1996 Aug 1;56(15):3597-3604, Cancer research
Evidence suggests that the plasminogen activators (PAs), in particular urokinase-type PA (uPA), play a pivotal role in tumor invasion and metastasis. We studied the contribution of the PAs to the malignant phenotype through the chemical induction of melanocytic neoplasms in uPA-deficient mice. Primary tumors were induced and promoted concurrently in 35 uPA-/- deficient and 35 uPA+/+ wild-type mice using a single application of 7,12-dimethylbenz(a)anthracene followed by repetitive applications of croton oil. Animals were sacrificed at 60-day intervals for 1 year. At necropsy, the four largest pigmented lesions in each animal were excised, characterized histologically, and evaluated microscopically for evidence of invasion. The regional lymph nodes, lungs, and solid abdominal visceral organs were sectioned and examined microscopically for evidence of metastatic disease. Cellular blue nevi were induced in 100% of uPA-/- and uPA+/+ promoted animals. Although a reduction in the radial and vertical progression of these lesions was noted in the uPA-deficient mice compared with the wild-type group, more than 95% of cellular blue nevi induced in both groups of animals invaded the underlying tissues. These lesions did not metastasize to the regional lymph nodes. Malignant melanoma arose in 5 of 35 (14.3%) of promoted wild-type mice. These tumors were locally aggressive, produced tissue-type PA, but were not metastatic to the regional nodes, lungs, or abdominal viscera. These results indicate that the invasive capability of melanocytic lesions may depend more on tissue-type PA than uPA activity. No melanomas were induced in the uPA-/- mice. The resistance of the uPA -/- strain to melanoma induction suggests that uPA contributes to malignant progression. We propose that the absence of uPA negatively affects tumorigenesis by decreasing the liberation and availability of growth factors such as basic fibroblast growth factor
— id: 12575, year: 1996, vol: 56, page: 3597, stat: Journal Article,

Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms
Bikfalvi A; Klein S; Pintucci G; Quarto N; Mignatti P; Rifkin DB
1995 Apr;129(1):233-243, Journal of cell biology
To study possible functional differences of the 18-kD and high molecular weight forms of basic fibroblast growth factor (bFGF), we have examined the effect of endogenous production of different bFGF forms on the phenotype of NIH 3T3 cells. Cells transfected with cDNAs coding for either 18-kD bFGF (18-kD bFGF) or all four molecular forms (18, 22, 22.5, 24 kD; wild type [WT] bFGF) exhibit increased migration and decreased FGF receptor number compared to parental cells. However, migration and FGF receptor number of cells transfected with a cDNA coding only for high molecular weight bFGF (22, 22.5, and 24 kD; HMW bFGF) were similar to that of parental cells transfected with vector alone. Cells expressing HMW, 18 kD, or WT bFGF grew to high saturation densities in 10% serum. However, only cells expressing HMW or WT bFGF grew in low serum. Cell surface or metabolic labeling of the different cell types followed by immunoprecipitation with anti-bFGF antibody showed primarily cell surface-associated 18-kD bFGF. In addition, when cells expressing exclusively HMW bFGF were transfected with a cDNA coding for 18-kD bFGF, migration was increased, bFGF receptors were down-regulated, and 18-kD bFGF was found on the cell surface. Cells expressing 18-kD bFGF transfected with a cDNA encoding FGF receptor-2 lacking the COOH-terminal domain (dominant negative bFGF receptor) exhibited a flat morphology and decreases in migration and saturation density. Cells expressing HMW bFGF transfected with the dominant negative bFGF receptor continued to grow to a high saturation density, proliferated in low serum, and exhibited no morphological changes. These results indicate that increased cell migration and FGF receptor down-regulation are mediated by the extracellular interaction of 18-kD bFGF with its cell surface receptor. Growth in low serum may be stimulated by the intracellular action of HMW bFGF through mechanisms independent of the presence of a cell surface receptor. Thus, the different molecular forms of bFGF may act through distinct but convergent pathways
— id: 6578, year: 1995, vol: 129, page: 233, stat: Journal Article,

FGF suppresses apoptosis and induces differentiation of fibre cells in the mouse lens
Chow RL; Roux GD; Roghani M; Palmer MA; Rifkin DB; Moscatelli DA; Lang RA
1995 Dec;121(12):4383-4393, Development
To determine whether fibroblast growth factor (FGF) has a role in lens development, we have generated transgenic mice expressing a dominant-negative form of the murine FGF receptor-1 (FGFRDN) in the lens. Using the fibre cell-specific alpha A-crystallin promoter to express the FGFRDN, we have asked whether FGF is required for fibre cell differentiation. The transgenic mice display diminished differentiation of fibre cells as indicated by their reduced elongation. In addition, transgenic lenses have an unusual refractile anomaly that morphological and biochemical data show results from the apoptosis of fibre cells in the central region of the lens. These results show that lens fibre cells are dependent on FGF for their survival and differentiation, and demonstrate that growth factor deprivation in vivo can lead to apoptosis
— id: 6894, year: 1995, vol: 121, page: 4383, stat: Journal Article,

Transforming growth factor-beta 1 regulates axon/Schwann cell interactions
Einheber S; Hannocks MJ; Metz CN; Rifkin DB; Salzer JL
1995 Apr;129(2):443-458, Journal of cell biology
We have investigated the potential regulatory role of TGF-beta in the interactions of neurons and Schwann cells using an in vitro myelinating system. Purified populations of neurons and Schwann cells, grown alone or in coculture, secrete readily detectable levels of the three mammalian isoforms of TGF-beta; in each case, virtually all of the TGF-beta activity detected is latent. Expression of TGF-beta 1, a major isoform produced by Schwann cells, is specifically and significantly downregulated as a result of axon/Schwann cell interactions. Treatment of Schwann cells or Schwann cell/neuron cocultures with TGF-beta 1, in turn, has dramatic effects on proliferation and differentiation. In the case of purified Schwann cells, treatment with TGF-beta 1 increases their proliferation, and it promotes a pre- or nonmyelinating Schwann cell phenotype characterized by increased NCAM expression, decreased NGF receptor expression, inhibition of the forskolin-mediated induction of the myelin protein P0, and induction of the Schwann cell transcription factor suppressed cAMP-inducible POU protein. Addition of TGF-beta 1 to the cocultures inhibits many of the effects of the axon on Schwann cells, antagonizing the proliferation induced by contact with neurons, and, strikingly, blocking myelination. Ultrastructural analysis of the treated cultures confirmed the complete inhibition of myelination and revealed only rudimentary ensheathment of axons. Associated defects of the Schwann cell basal lamina and reduced expression of laminin were also detected. These effects of TGF-beta 1 on Schwann cell differentiation are likely to be direct effects on the Schwann cells themselves which express high levels of TGF-beta 1 receptors when cocultured with neurons. The regulated expression of TGF-beta 1 and its effects on Schwann cells suggest that it may be an important autocrine and paracrine mediator of neuron/Schwann cell interactions. During development, TGF-beta 1 could serve as an inhibitor of Schwann cell proliferation and myelination, whereas after peripheral nerve injury, it may promote the transition of Schwann cells to a proliferating, nonmyelinating phenotype, and thereby enhance the regenerative response
— id: 7896, year: 1995, vol: 129, page: 443, stat: Journal Article,

POTENT 2'-AMINO-2'-DEOXYPYRIMIDINE RNA INHIBITORS OF BASIC FIBROBLAST GROWTH-FACTOR
JELLINEK, D; GREEN, LS; BELL, C; LYNOTT, CK; GILL, N; VARGEESE, C; KIRSCHENHEUTER, G; MCGEE, DPC; ABESINGHE, P; PIEKEN, WA; SHAPIRO, R; RIFKIN, DB; MOSCATELLI, D; JANJIC, N
1995 SEP 12 ;34(36):11363-11372, Biochemistry
Screening of random oligonucleotide libraries with SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L. (1990) Science 249, 595-510] has emerged as a powerful method for identifying high-affinity nucleic acid ligands for a wide range of molecular targets, Nuclease sensitivity of unmodified RNA and DNA, however, imposes considerable restrictions on their use as therapeutics or diagnostics. Modified RNA in which pyrimidine 2'-hydroxy groups have been substituted with 2'-amino groups (2'-aminopyrimidine RNA) is known to be substantially more resistant to serum nucleases, We report here on the use of SELEX to identify high-affinity 2'-aminopyrimidine RNA ligands to a potent angiogenic factor, basic fibroblast growth factor(bFGF). High-affinity ligands with the same consensus primary structure have been isolated from two independent libraries of approximately 6 x 10(14) molecules containing 30 or 50 randomized positions. Compared to unmodified RNA with the same sequence, 2'-aminopyrimidine ligands are at least 1000-fold more stable in 90% human serum. The sequence information required for high-affinity binding to bFGF is contained within 24-26 nucleotides. The minimal ligand m21A (5'-GGUGUGUGGAAGACAGCGGGUGGUUC-3'; G = guanosine, A = adenosine, C = 2'-amino-2'-deoxycytidine, U = 2'-amino-2'-deoxyuridine, and C = 2'-amino-2'-deoxycytidine or deoxycytidine) binds to bFGF with an apparent dissociation constant (K-d) of (3.5 +/- 0.3) x 10(-10) M at 37 degrees C in phosphate-buffered saline (pH 7.4). Dissociation of m21A from bFGF is adequately described with a first-order rate constant of (1.96 +/- 0.08) x 10(-3) s(-1) (t(1/2) = 5.9 min). The calculated value for the association rate constant (k(on) = k(off)/K-d) was 5.6 x 10(6) M(-1) s(-1). Highly specific binding of m21A to bFGF was observed: binding to denatured bFGF, five proteins from the FGF family (acidic FGF, FGF-4, FGF-5, FGF-6, and FGF-7), and four other heparin binding proteins is substantially weaker under the same conditions with K-d(bFGF)/K-d(protein) values ranging from (4.1 +/- 1.4) x 10(-2) to > 10(-6). Heparin but not chondroitin sulfate competed for binding of m21A to bFGF. In cell culture, m21A inhibited [I-125]bFGF binding to both low-affinity sites (ED(50) approximate to 1 nM) and high-affinity sites (ED(50) approximate to 3 nM) on CHO cells expressing transfected FGF receptor-1. Basic FGF-dependent migration of bovine aortic endothelial cells as well as bFGF-induced proliferation of human umbilical vein endothelial cells was also inhibited by m21A in a concentration-dependent manner with ED(50) values of 50-100 nM. The 2'-aminopyrimidine RNA ligand m21A therefore represents a useful lead compound in our efforts to develop potent oligonucleotide-based angiogenesis antagonists
— id: 86739, year: 1995, vol: 34, page: 11363, stat: Journal Article,

Lipopolysaccharide inhibits activation of latent transforming growth factor-beta in bovine endothelial cells
Kojima S; Vernooy R; Moscatelli D; Amanuma H; Rifkin DB
1995 Apr;163(1):210-219, Journal of cellular physiology
The activation of latent transforming growth factor-beta (TGF-beta) by vascular endothelial cells (ECs) is regulated by cellular plasminogen activator (PA)/plasmin, transglutaminase (TGase), and latent TGF-beta levels. Because lipopolysaccharide (LPS) has been reported to reduce EC surface plasmin levels by increasing the production of the inhibitor of PA, PA inhibitor-1 (PAI-1), we have tested whether LPS might suppress latent TGF-beta activation in ECs using two different systems, namely, bovine aortic ECs (BAECs) cocultured with smooth muscle cells (SMCs) and BAECs treated with retinol. BAECs were either cocultured with SMCs after treatment with 15 ng/ml LPS or were treated with 2 microM retinol and/or 10 ng/ml LPS, and the expression of PA, surface plasmin, TGase, and the amounts of active and latent TGF-beta secreted into the culture medium were measured. The downregulation of surface PA/plasmin levels with LPS was accompanied by a profound decline of both TGase and latent TGF-beta expression as well as the suppression of surface activation of latent TGF-beta. The effect was dependent on the concentration of LPS and on treatment time. The formation of TGF-beta did not occur in cells maintained in LPS-contaminated culture medium
— id: 6662, year: 1995, vol: 163, page: 210, stat: Journal Article,

Characterization of latent TGF-beta activation by murine peritoneal macrophages
Nunes I; Shapiro RL; Rifkin DB
1995 Aug 1;155(3):1450-1459, Journal of immunology
Transforming growth factor-beta (TGF-beta) is secreted by most cells as a biologically inactive complex, called the large latent TGF-beta complex. The complex is comprised of latent TGF-beta binding protein (LTBP) and latent TGF-beta, which is mature TGF-beta associated noncovalently with its amino-terminal propeptides. LTBP is disulfide-linked to the amino-terminal propeptide of latent TGF-beta. Active TGF-beta is generated by release of TGF-beta from the complex. Generation of active TGF-beta by macrophages has been reported, but the activation mechanism has not been described. Latent TGF-beta activation by macrophages was characterized using serum-free cultures of resident and thioglycollate-elicited murine peritoneal macrophages that were either unstimulated or LPS-stimulated in vitro. Serum-free conditioned medium was assayed for TGF-beta using a quantitative luciferase-based bioassay. LPS-stimulated thioglycollate-elicited macrophages activated endogenous latent TGF-beta, whereas non-LPS-stimulated thioglycollate-elicited and resident macrophages generated undetectable levels of TGF-beta. Latent TGF-beta activation required plasmin and urokinase (uPA), uPA binding to the uPA receptor, interaction with the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor, tissue type II transglutaminase, and LTBP. A time-course analysis of latent TGF-beta activation revealed that maximal TGF-beta was generated after 24 h (25 +/- 5 pg/ml). TGF-beta formed within the initial 24 h modulated the plasminogen activator system by down-regulating uPA, suggesting that TGF-beta temporally modulated its own formation by regulating cell-associated uPA
— id: 6852, year: 1995, vol: 155, page: 1450, stat: Journal Article,

Basic fibroblast growth factor (bFGF) transfected stromal cells promote the growth of Dami leukemic cells
Ogilvie, V; Coetzee, S; Rifkin, D; Quesenberry, P; Wilson, EL
1995 NOV 15 ;86(10):1229-1229, Blood
— id: 53118, year: 1995, vol: 86, page: 1229, stat: Journal Article,

Effects of a single administration of fibroblast growth factor on vascular wall reaction to injury
Parish MA; Grossi EA; Baumann FG; Asai T; Rifkin DB; Colvin SB; Galloway AC
1995 Apr;59(4):948-954, Annals of thoracic surgery
Expansion of the vascular wall through formation of neointimal fibromuscular lesions is the basic mechanism underlying stenosis of vascular grafts, restenosis of arteries treated by balloon angioplasty, and other major cardiovascular problems. This study examined the effect of a single, systemic, low dose of basic fibroblast growth factor (bFGF) on formation of neointimal fibromuscular lesions in response to injury. New Zealand white rabbits (n = 76) were subjected to balloon injury of the abdominal aorta. Forty-five rabbits were given a single intravenous dose of bFGF (0.5 microgram/kg) immediately after injury, and 31 rabbits were given only the vehicle solution as controls. After 2 (n = 15), 5 (n = 21), 14 (n = 29), or 28 (n = 11) days the rabbits were sacrificed. Those rabbits receiving the single administration of bFGF exhibited significantly greater intimal thickening (intima/media ratio) than the control group at 5 days (mean +/- standard error of the mean, 0.091 +/- 0.009 versus 0.058 +/- 0.006; p < 0.002), but not at 14 or 28 days. These results were achieved without any significant differences in mitotic indices, as determined by a mitostatic method, between the two groups at any postinjury interval examined. The findings suggest that a single systemic dose of exogenous bFGF has a relatively long term effect on enhancing the neointimal response to vascular injury. Therefore, local control of endogenous bFGF may be useful in limiting formation of vascular neointimal fibromuscular lesions, thus improving the long-term results of vascular grafts, balloon angioplasty, and other cardiovascular procedures
— id: 56694, year: 1995, vol: 59, page: 948, stat: Journal Article,

An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct
Abe M; Harpel JG; Metz CN; Nunes I; Loskutoff DJ; Rifkin DB
1994 Feb 1;216(2):276-284, Analytical biochemistry
Transforming growth factor-beta (TGF-beta) is a potent regulator of cellular differentiation, proliferation, migration, and protein expression. These properties have been exploited to create a variety of bioassays for detecting the mature growth factor. In this paper, we describe a highly sensitive and specific, nonradioactive quantitative bioassay for TGF-beta based on its ability to induce plasminogen activator inhibitor-1 (PAI-1) expression. Mink lung epithelial cells (MLEC) were stably transfected with an expression construct containing a truncated PAI-1 promoter fused to the firefly luciferase reporter gene. Addition of TGF-beta (0.2 to > 30 pM) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. Although responsive to TGF-beta, this promoter fragment was only minimally influenced by other known inducers of PAI-1 expression. When compared to the widely used MLEC assay, this assay demonstrated greater sensitivity and specificity, allowing quantification of TGF-beta in complex biological solutions
— id: 6305, year: 1994, vol: 216, page: 276, stat: Journal Article,

An endogenous glycosylphosphatidylinositol-specific phospholipase D releases basic fibroblast growth factor-heparan sulfate proteoglycan complexes from human bone marrow cultures
Brunner G; Metz CN; Nguyen H; Gabrilove J; Patel SR; Davitz MA; Rifkin DB; Wilson EL
1994 Apr 15;83(8):2115-2125, Blood
Basic fibroblast growth factor (bFGF) is a hematopoietic cytokine that stimulates stromal and stem cell growth. It binds to a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan on human bone marrow (BM) stromal cells. The bFGF-proteoglycan complex is biologically active and is released by addition of exogenous phosphatidylinositol-specific phospholipase C. In this study, we show the presence of an endogenous GPI-specific phospholipase D (GPI-PLD) that releases the bFGF-binding heparan sulfate proteoglycan and the variant surface glycoprotein (a model GPI-anchored protein) from BM cultures. An involvement of proteases in this process is unlikely, because released proteoglycan contained the GPI anchor component, ethanol-amine, and protease inhibitors did not diminish the release. The mechanism of release is likely to involve a GPI-PLD and not a GPI-specific phospholipase C, because the release of variant surface glycoprotein did not reveal an epitope called the cross-reacting determinant that is exposed by phospholipase C-catalyzed GPI anchor cleavage. In addition, phosphatidic acid (which is specifically a product of GPI-PLD-catalyzed anchor cleavage) was generated during the spontaneous release of the GPI-anchored variant surface glycoprotein. We also detected GPI-PLD-specific enzyme activity and mRNA in BM cells. Therefore, we conclude that an endogenous GPI-PLD releases bFGF-heparan sulfate proteoglycan complexes from human BM cultures. This mechanism of GPI anchor cleavage could be relevant for mobilizing biologically active bFGF in BM. An endogenous GPI-PLD could also release other GPI-anchored proteins important for hematopoiesis and other physiologic processes
— id: 56502, year: 1994, vol: 83, page: 2115, stat: Journal Article,

GROWTH-FACTOR REGULATION OF THE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-SPECIFIC PHOSPHOLIPASE-D IN BONE-MARROW STROMAL CELLS
BRUNNER, G; GULATI, M; GABRILOVE, JL; RIFKIN, DB; WILSON, EL
1994 NOV 15 ;84(10):A564-A564, Blood
— id: 52291, year: 1994, vol: 84, page: A564, stat: Journal Article,

BASIC FIBROBLAST GROWTH-FACTOR (BFGF) TRANSFECTED STROMAL CELLS SUPPORT SURVIVAL OF MO7E CELLS
COETZEE, S; OGILVIE, V; BRUNNER, G; QUARTO, N; RIFKIN, D; QUESENBERRY, P; WILSON, EL
1994 NOV 15 ;84(10):A279-A279, Blood
— id: 52284, year: 1994, vol: 84, page: A279, stat: Journal Article,

MODULATION OF THE PRODUCTION AND THE ACTIVATION OF LATENT TGF-BETA BY RETINOID AND LIPOPOLYSACCHARIDE IN BOVINE ENDOTHELIAL-CELLS
KOJIMA, S; RIFKIN, DB
1994 JAN 4 ;269(6):317-317, Journal of cellular biochemistry
— id: 52587, year: 1994, vol: 269, page: 317, stat: Journal Article,

Release of GPI-anchored membrane proteins by a cell-associated GPI-specific phospholipase D
Metz CN; Brunner G; Choi-Miura NH; Nguyen H; Gabrilove J; Caras IW; Altszuler N; Rifkin DB; Wilson EL; Davitz MA
1994 Apr 1;13(7):1741-1751, EMBO journal
Although many glycosylphosphatidylinositol (GPI)-anchored proteins have been observed as soluble forms, the mechanisms by which they are released from the cell surface have not been demonstrated. We show here that a cell-associated GPI-specific phospholipase D (GPI-PLD) releases the GPI-anchored, complement regulatory protein decay-accelerating factor (DAF) from HeLa cells, as well as the basic fibroblast growth factor-binding heparan sulfate proteoglycan from bone marrow stromal cells. DAF found in the HeLa cell culture supernatants contained both [3H]ethanolamine and [3H]inositol, but not [3H]palmitic acid, whereas the soluble heparan sulfate proteoglycan present in bone marrow stromal cell culture supernatants contained [3H]ethanolamine. 125I-labeled GPI-DAF incorporated into the plasma membranes of these two cell types was released in a soluble form lacking the fatty acid GPI-anchor component. GPI-PLD activity was detected in lysates of both HeLa and bone marrow stromal cells. Treatment of HeLa cells with 1,10-phenanthroline, an inhibitor of GPI-PLD, reduced the release of [3H]ethanolamine-DAF by 70%. The hydrolysis of these GPI-anchored molecules is likely to be mediated by an endogenous GPI-PLD because [3H]ethanolamine DAF is constitutively released from HeLa cells maintained in serum-free medium. Furthermore, using PCR, a GPI-PLD mRNA has been identified in cDNA libraries prepared from both cell types. These studies are the first demonstration of the physiologically relevant release of GPI-anchored proteins from cells by a GPI-PLD
— id: 7885, year: 1994, vol: 13, page: 1741, stat: Journal Article,

Mammary artery versus saphenous vein grafts: assessment of basic fibroblast growth factor receptors
Nguyen HC; Grossi EA; LeBoutillier M 3rd; Steinberg BM; Rifkin DB; Baumann FG; Colvin SB; Galloway AC
1994 Aug;58(2):308-310, Annals of thoracic surgery
Neointimal hyperplasia limits the long-term patency of saphenous vein grafts (SVGs), but is notably absent from most internal mammary artery (IMA) grafts. Basic fibroblast growth factor (bFGF) is a local endothelial and vascular smooth muscle mitogen known to be involved in the pathogenesis of neointimal hyperplasia. This study used an animal model to compare the number of available high-affinity (HAR) and low-affinity (LAR) bFGF receptors in SVGs and IMA grafts and to determine whether distention injury causes an increase in receptor availability. The IMA and SVG specimens were harvested from 12 dogs and distended at 25 or 200 mm Hg for 15 minutes, and then the bFGF receptor uptake was measured in them using iodine 125-labeled bFGF. In the IMA conduits distended at low pressure, there were 2.54 +/- 0.10 (mean +/- standard error of the mean) HARs per mm2 of intimal surface area available and 5.19 +/- 0.40 LARs per mm2. High-pressure distention significantly (p < 0.001) increased the number of available HARs to 5.06 +/- 0.27 per mm2 and of LARs to 7.27 +/- 0.042 per mm2. At low pressure, the SVGs had significantly (p < 0.001) more HARs (9.14 +/- 0.84 per mm2) and LARs (18.2 +/- 0.57 per mm2) available than did the IMA conduits, and high pressure significantly (p < 0.001) increased the number of HARs available in SVGs to 24.1 +/- 2.43 per mm2 and the number of LARs to 44.7 +/- 2.34 per mm2.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 12923, year: 1994, vol: 58, page: 308, stat: Journal Article,

Suppression of neointimal lesions after vascular injury: a role for polyclonal anti-basic fibroblast growth factor antibody
Nguyen HC; Steinberg BM; LeBoutillier M 3rd; Baumann FG; Rifkin DB; Grossi EA; Galloway AC
1994 Aug;116(2):456-461, Surgery
BACKGROUND. Basic fibroblast growth factor (bFGF) is a potent local promoter of vascular smooth muscle cell migration and proliferation and may play a major role in the pathogenesis of intimal fibromuscular lesions. Preliminary studies have shown that exogenous bFGF localizes to injured rabbit aorta and suggest that this interaction might be inhibited by anti-bFGF immunoglobulin (Ig) G. This study was designed to evaluate the possible role of polyclonal anti-bFGF IgG in reducing intimal fibromuscular lesion formation in the injured rabbit aorta. METHODS. The abdominal aortic endothelium was subjected to balloon injury in 13 rabbits. Six rabbits received intravenous rabbit anti-bFGF IgG, and seven received irrelevant rabbit IgG (16 micrograms/kg) 30 minutes before injury and daily for 5 days after injury. At 14 days after injury the aorta was fixed and sectioned, and the intimal and medial areas were measured by computerized digital morphometry with the intimal/medial ratio as an index of neointimal lesion formation. RESULTS. In the control group the intimal/medial ratio was 0.538 +/- 0.046 (mean +/- SEM), which was significantly greater than the anti-bFGF-treated group value of 0.148 +/- 0.021 (p < 0.001). CONCLUSIONS. These results show that daily doses of intravenous polyclonal anti-bFGF IgG for 5 days after balloon aortic injury significantly inhibit intimal fibromuscular lesion formation at 14 days. The results suggest that the process of intimal fibromuscular lesion formation may be susceptible to modification by antagonists to bFGF
— id: 12925, year: 1994, vol: 116, page: 456, stat: Journal Article,

Requirement for receptor-bound urokinase in plasmin-dependent cellular conversion of latent TGF-beta to TGF-beta
Odekon LE; Blasi F; Rifkin DB
1994 Mar;158(3):398-407, Journal of cellular physiology
The role of receptor-bound urokinase-type plasminogen activator (uPA) in cellular activation of latent transforming growth factor-beta (LTGF-beta) was investigated in a model system of mouse LB6 cells transfected with either a human uPA receptor cDNA (LhuPAR+), a human prouPA cDNA (LhuPA), or a control neomycin-resistance cDNA (Lneo). When LhuPAR+ cells were co-cultured with LhuPA cells, the plasmin-dependent fibrinolytic activity generated was more than that observed in either homotypic cultures with fivefold greater number of LhuPA cells or co-cultures containing LhuPA and Lneo cells instead of the LhuPAR+ cells. The preferential activation of TGF-beta by co-cultures with the greatest plasmin-generation potential, LhuPAR+ and LhuPA cells, was confirmed by three independent bioassays. In the first assay, a 48% decrease in PA activity, a measure of active TGF-beta production, was observed with BAE cells treated with conditioned medium (CM) from co-cultures of LhuPA and LhuPAR+ cells. Inclusion of neutralizing antibodies to TGF-beta abrogated the inhibitory effect of CM on PA activity demonstrating that the inhibitory molecule was TGF-beta. Addition of the amino terminal fragment of uPA (ATF) or omission of plasminogen from co-cultures blocked both the fibrinolytic activity and the generation of TGF-beta activity in the CM. In the second assay, CM from co-cultures of LhuPA and LhuPAR+ cells inhibited the migration of BAE cells in a wound assay. Controls with anti-TGF-beta IgG indicated that the inhibition was due to TGF-beta. In the third assay, proliferation of mink lung epithelial cells was inhibited by CM generated by co-cultures of LhuPA and LhuPAR+ cells as compared to CM from the same cells cultured in the absence of plasminogen or to CM from a co-culture of LhuPA with LhuPAR- cells. Excess mannose-6-phosphate (M6P) blocked the generation of TGF-beta as assayed by both the BAE migration and PA assays, presumably because it interfered with cell-surface localization of LTGF-beta. Additionally, small numbers of LhuPA and LhuPAR+ cells co-cultured with BAE cells inhibited the BAE cell PA activity via the paracrine action of TGF-beta. These results support the conclusion that plasmin-dependent activation LTGF-beta by LB6 cells is promoted by the surface localization of uPA by its receptor
— id: 6473, year: 1994, vol: 158, page: 398, stat: Journal Article,

Tumor cells secrete an angiogenic factor that stimulates basic fibroblast growth factor and urokinase expression in vascular endothelial cells
Peverali FA; Mandriota SJ; Ciana P; Marelli R; Quax P; Rifkin DB; Della Valle G; Mignatti P
1994 Oct;161(1):1-14, Journal of cellular physiology
Culture medium conditioned by human SK-Hep1 hepatoma cells or mouse S180 sarcoma cells rapidly up-regulates endothelial cell expression of basic fibroblast growth factor (bFGF) and induces formation of capillary-like structures by vascular endothelial cells grown on three-dimensional fibrin gels (in vitro angiogenesis). Incubation of endothelial cells with the tumor cell-conditioned media also results in increased expression of urokinase plasminogen activator (uPA), a key component of the proteolytic system required for cell invasion and capillary formation. Although the tumor cell-conditioned media contain no bFGF, addition of anti-recombinant bFGF IgG abolishes the up-regulation of uPA and blocks in vitro angiogenesis. This indicates that both the increase in uPA production and formation of capillary-like structures are mediated by endogenous bFGF expressed by the endothelial cells. Both the bFGF/uPA-inducing activity and the angiogenic activity of SK-Hep1 cell-conditioned medium copurify with a relatively acid-resistant peptide that has moderate affinity for heparin and M(r) < 18 kDa > 3.5 kDa. Known cytokines with similar biochemical features do not possess the same biological activity. These findings indicate that angiogenesis can be mediated by endothelial cell bFGF through an autocrine mechanism and that the bFGF-inducing peptide may represent a novel tumor-derived angiogenic factor that modulates in endothelial cells the concerted expression of cytokines and proteolytic enzymes required for capillary formation
— id: 42358, year: 1994, vol: 161, page: 1, stat: Journal Article,

Studies on FGF-2: nuclear localization and function of high molecular weight forms and receptor binding in the absence of heparin
Rifkin DB; Moscatelli D; Roghani M; Nagano Y; Quarto N; Klein S; Bikfalvi A
1994 Sep;39(1):102-104, Molecular reproduction & development
Multiple forms of FGF-2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF-2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF-2 as the AUG codon is 3' to the CUG codons. Although the 18 kDa form FGF-2 is localized primarily in the cytosol, a significant fraction of the HMW FGF-2 has a nuclear location. The nuclear localization of HMW FGF-2 is determined by amino acid residues in the amino-terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino-terminal extension of HMW FGF-2. The nuclear localization of HMW FGF-2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF-2, or all forms of FGF-2, we have found that HMW FGF-2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF-2. These cells are transformed by what appears to be the intracellular action of HMW FGF-2. The interaction of FGF-2 with heparin has also been examined.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 6730, year: 1994, vol: 39, page: 102, stat: Journal Article,

MECHANISMS AND CONSEQUENCE OF TGF-BETA FORMATION BY VASCULAR CELLS
RIFKIN, DB
1994 JAN 4 ;269(6):307-307, Journal of cellular biochemistry
— id: 52586, year: 1994, vol: 269, page: 307, stat: Journal Article,

Heparin increases the affinity of basic fibroblast growth factor for its receptor but is not required for binding
Roghani M; Mansukhani A; Dell'Era P; Bellosta P; Basilico C; Rifkin DB; Moscatelli D
1994 Feb 11;269(6):3976-3984, Journal of biological chemistry
The role of heparin or heparan sulfates in the interaction of basic fibroblast growth factor (bFGF) with its high affinity receptor were investigated using purified extracellular ligand-binding region of FGF receptor-1 (FGFR-1) and intact receptors expressed in a myeloid cell line (32D) that does not express detectable levels of heparan sulfate proteoglycans or in Chinese hamster ovary (CHO) cell mutants defective in heparan sulfate synthesis. The purified extracellular domain of FGFR-1 formed complexes with 125I-bFGF both in the presence or absence of heparin. Intact FGFR-1 expressed in 32D cells also bound the same amount of 125I-bFGF in the presence or absence of heparin when saturating concentrations of bFGF were used. Varying the concentration of 125I-bFGF showed that heparin increased the amount of 125I-bFGF bound at low bFGF concentrations and increased the affinity of bFGF for its receptor by about 3-fold. To eliminate the possibility of alteration of bFGF properties through the chemical modification reactions, bFGF was labeled biosynthetically. The binding of biosynthetically labeled bFGF to FGFR-1 also did not require heparin. When FGFR-1 or FGFR-2 were expressed in mutant CHO cells deficient in heparan sulfate synthesis, the cells also bound 125I-bFGF in the absence of heparin, and the addition of heparin increased the affinity of bFGF for its receptors 2-3-fold. Thus, heparin or heparan sulfate is not required for the binding of bFGF to its receptors but increases the binding affinity to a moderate degree. Finally, the requirement for heparin in signal transduction through the receptor was investigated. Expression of c-fos mRNA was induced by bFGF in 32D cells expressing FGFR-1 to the same extent in the presence or absence of heparin
— id: 6499, year: 1994, vol: 269, page: 3976, stat: Journal Article,

Mechanism of action of angiostatic steroids: suppression of plasminogen activator activity via stimulation of plasminogen activator inhibitor synthesis
Blei F; Wilson EL; Mignatti P; Rifkin DB
1993 Jun;155(3):568-578, Journal of cellular physiology
Recently, a novel class of angiostatic steroids which block angiogenesis in several systems has been described. Since the elaboration of proteases is believed to be an important component of angiogenesis, we tested whether these steroids blocked the fibrinolytic response of endothelial cells to the angiogenic protein, basic fibroblast growth factor [bFGF]). Cultured bovine aortic endothelial (BAE) cells were incubated with bFGF and/or medroxyprogesterone acetate (MPA), an angiostatic steroid which has been shown to inhibit vascularization, collagenolysis, and tumor growth. When bFGF (3 ng/ml) was added to confluent monolayers of BAE cells, plasminogen activator (PA) activity in the medium was increased threefold. In contrast, MPA at 10(-6) M, 10(-7) M, 10(-8) M, and 10(-9) M decreased PA levels in the medium by 83%, 83%, 75%, and 39%, respectively. The stimulation of PA levels in BAE cells by bFGF (3 ng/ml) was abrogated by the presence of 10(-6) M MPA. This decrease in PA activity was found to be mediated by a significant increase in plasminogen activator inhibitor type-1 (PAI-1) production. MPA, therefore, negated one of the important enzymatic activities associated with the angiogenic process. In contrast to the decreased levels of secreted PA in cultures exposed simultaneously to MPA and bFGF, cell-associated PA levels remained high, consistent with earlier observations indicating that PAI-1 does not inhibit cell-associated PA. Thus, angiostatic steroids may exert their inhibitory effects on angiogenesis by increasing the synthesis of PAI-1. This, in turn, inhibits PA activity and, therefore, plasmin generation, which is essential for the invasive aspect of angiogenesis
— id: 8234, year: 1993, vol: 155, page: 568, stat: Journal Article,

Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells
Brunner G; Nguyen H; Gabrilove J; Rifkin DB; Wilson EL
1993 Feb 1;81(3):631-638, Blood
We have shown previously that basic fibroblast growth factor (bFGF) is a mitogen for human bone marrow (BM) stromal cells and that bFGF stimulates myelopoiesis in primary BM cultures. In this article, we demonstrate the presence of bFGF in two cell lineages in human BM and peripheral blood as well as the deposition of bFGF into the extracellular matrix of BM stromal cell cultures. In immunofluorescence experiments on BM and peripheral blood smears, megakaryocytes and platelets stained strongly for bFGF, whereas weaker staining was observed in immature and mature cells of the granulocyte series. The presence of bFGF in platelets was confirmed by enzyme-linked immunosorbent assay as well as by immunoprecipitation followed by immunoblotting. bFGF was synthesized by BM stromal cell cultures and was found either cell associated or localized in the nucleus and the nucleoli, and its location was dependent on the fixation procedure used. Addition of exogenous bFGF to stromal cells showed the presence of extracellular binding molecules for this cytokine. bFGF could be released from these sites by soluble heparin or phosphatidylinositol-specific phospholipase C. This study supports the role of bFGF as a stromal cell mitogen and stimulator of myelopoiesis. The data indicate that the stromal cells produce bFGF and that their extracellular matrix can serve as a reservoir for this growth factor. In addition, the results suggest a possible involvement of bFGF in platelet function as well as in megakaryocytopoiesis
— id: 13270, year: 1993, vol: 81, page: 631, stat: Journal Article,

MOBILIZATION OF BFGF-PROTEOGLYCAN COMPLEXES IN HUMAN BONE-MARROW CULTURES BY A GPI-SPECIFIC PHOSPHOLIPASE-D
BRUNNER, G; NGUYEN, H; RIFKIN, DB; GABRILOVE, J; WILSON, EL
1993 NOV 15 ;82(10):A370-A370, Blood
— id: 52146, year: 1993, vol: 82, page: A370, stat: Journal Article,

Role of the latent TGF-beta binding protein in the activation of latent TGF-beta by co-cultures of endothelial and smooth muscle cells
Flaumenhaft R; Abe M; Sato Y; Miyazono K; Harpel J; Heldin CH; Rifkin DB
1993 Feb;120(4):995-1002, Journal of cell biology
Transforming growth factor beta (TGF-beta) is released from cells in a latent form consisting of the mature growth factor associated with an aminoterminal propeptide and latent TGF-beta binding protein (LTBP). The endogenous activation of latent TGF-beta has been described in co-cultures of endothelial and smooth muscle cells. However, the mechanism of this activation remains unknown. Antibodies to native platelet LTBP and to a peptide fragment of LTBP inhibit in a dose-dependent manner the activation of latent TGF-beta normally observed when endothelial cells are cocultured with smooth muscle cells. Inhibition of latent TGF-beta activation was also observed when cells were co-cultured in the presence of an excess of free LTBP. These data represent the first demonstration of a function for the LTBP in the extracellular regulation of TGF-beta activity and indicate that LTBP participates in the activation of latent TGF-beta, perhaps by concentrating the latent growth factor on the cell surface where activation occurs
— id: 13266, year: 1993, vol: 120, page: 995, stat: Journal Article,

Activation of latent transforming growth factor beta
Flaumenhaft R; Kojima S; Abe M; Rifkin DB
1993 ;24:51-76, Advances in pharmacology (New York)
— id: 13286, year: 1993, vol: 24, page: 51, stat: Journal Article,

High-affinity RNA ligands to basic fibroblast growth factor inhibit receptor binding
Jellinek D; Lynott CK; Rifkin DB; Janjic N
1993 Dec 1;90(23):11227-11231, Proceedings of the National Academy of Sciences of the United States of America
We have isolated RNA ligands with low-nanomolar affinity and high specificity to basic fibroblast growth factor from a pool of 10(14) molecules containing 30 randomized positions by the systematic evolution of ligands by exponential enrichment (SELEX) procedure. High-affinity ligands could be classified into two families based on sequence and secondary structure similarities. Representative RNA ligands from the two families compete with one another as well as with heparin for binding to the protein. Furthermore, we show that these ligands inhibit the first step in the signaling pathway of basic fibroblast growth factor: binding of the growth factor to its cell-surface receptors. These findings emphasize the general usefulness of SELEX as a tool for discovering potent, specific oligonucleotide antagonists of target proteins
— id: 42359, year: 1993, vol: 90, page: 11227, stat: Journal Article,

Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells
Klein S; Giancotti FG; Presta M; Albelda SM; Buck CA; Rifkin DB
1993 Oct;4(10):973-982, Molecular biology of the cell
During angiogenesis capillary endothelial cells undergo a coordinated set of modifications in their interactions with extracellular matrix components. In this study we have investigated the effect of the prototypical angiogenic factor basic fibroblast growth factor (bFGF) on the expression and function of several integrins in microvascular endothelial cells. Immunoprecipitation experiments with antibodies to individual subunits indicated that microvascular cells express at their surface several integrins. These include the alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 laminin/collagen receptors; the alpha 6 beta 1 laminin receptor; the alpha 5 beta 1 and alpha v beta 1 fibronectin receptors; the alpha 6 beta 4 basement membrane receptor; and the alpha v beta 3 and alpha v beta 5 vitronectin receptors. Treatment with bFGF caused a significant increase in the surface expression of the alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha 6 beta 4, and alpha v beta 5 integrins. In contrast, the level of expression of the alpha 1 beta 1 and alpha v beta 3 integrins was decreased in bFGF-treated cells. Immunoprecipitation of metabolically labeled cells indicated that bFGF increases the biosynthesis of the alpha 3, alpha 5, alpha 6, beta 4, and beta 5 subunits and decreases the production of the alpha v and beta 3 subunits. These results suggest that bFGF modulates integrin expression by altering the biosynthesis of individual alpha or beta subunits. In accordance with the upregulation of several integrins observed in bFGF-treated cells, these cells adhered better to fibronectin, laminin, vitronectin, and type I collagen than did untreated cells. The largest differences in beta 1 integrin expression occurred approximately 72 h after exposure to bFGF, at a time when the expression of the endothelial cell-to-cell adhesion molecule endoCAM was also significantly upregulated. In contrast, a shorter exposure to bFGF (24-48 h) was required for the maximal induction of plasminogen activator production in the same cells. Taken together, these results show that bFGF causes significant changes in the level of expression and function of several integrins in microvascular endothelial cells
— id: 56551, year: 1993, vol: 4, page: 973, stat: Journal Article,

Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells
Kojima S; Nara K; Rifkin DB
1993 Apr;121(2):439-448, Journal of cell biology
A hitherto unknown function for transglutaminase (TGase; R-glutaminyl-peptide: amine gamma-glutamyltransferase, EC 2.3.2.13) was found in the conversion of latent transforming growth factor-beta (LTGF-beta) to active TGF-beta by bovine aortic endothelial cells (BAECs). The cell-associated, plasmin-mediated activation of LTGF-beta to TGF-beta induced either by treatment of BAECs with retinoids or by cocultures of BAECs and bovine smooth muscle cells (BSMCs) was blocked by seven different inhibitors of TGase as well as a neutralizing antibody to bovine endothelial cell type II TGase. Control experiments indicated that TGase inhibitors and/or a neutralizing antibody to TGase did not interfere with the direct action of TGF-beta, the release of LTGF-beta from cells, or the activation of LTGF-beta by plasmin or by transient acidification. After treatment with retinoids, BAECs expressed increased levels of TGase coordinate with the generation of TGF-beta, whereas BSMCs and bovine embryonic skin fibroblasts, which did not activate LTGF-beta after treatment with retinoids, did not. Furthermore, both TGase inhibitors and a neutralizing antibody to TGase potentiated the effect of retinol in enhancing plasminogen activator (PA) levels in cultures of BAECs by suppressing the TGF-beta-mediated enhancement of PA inhibitor-1 (PAI-1) expression. These results indicate that type II TGase is a component required for cell surface, plasmin-mediated LTGF-beta activation process and that increased expression of TGase accompanies retinoid-induced activation of LTGF-beta
— id: 8221, year: 1993, vol: 121, page: 439, stat: Journal Article,

Mechanism of retinoid-induced activation of latent transforming growth factor-beta in bovine endothelial cells
Kojima S; Rifkin DB
1993 May;155(2):323-332, Journal of cellular physiology
Cell-associated plasmin is a putative physiological activator of latent transforming growth factor-beta (LTGF-beta). Since retinoids enhance the production of plasminogen activator (PA) and thereby increase cell-associated plasmin activity, we tested the possibility that retinoids might induce the activation of LTGF-beta using bovine endothelial cells (ECs) as a model system. ECs treated with physiological concentrations of retinol or retinoic acid formed active TGF-beta in the culture media in a dose- and time-dependent fashion. Cells were treated with 2 microM retinol for 24 h, and the amount of TGF-beta produced during a subsequent 12-h incubation period was measured. Out of a total of 14 pM LTGF-beta secreted, 0.7 pM was converted to active TGF-beta. Northern blot analyses showed that mRNA levels for TGF-beta 2 but not for TGF-beta 1 increased in cells treated with retinol. Inclusion of either inhibitors of PA or of plasmin or antibody against PA in the culture medium as well as depletion of plasminogen from the serum blocked the formation of TGF-beta, suggesting that PA, plasminogen, and the resulting plasmin are essential for activation of LTGF-beta in retinoid-stimulated cells. Antibody against the LTGF-beta binding protein blocked activation implying that localization of LTGF-beta through its binding protein may be important. However, inhibition of binding of LTGF-beta to the cell surface mannose 6-phosphate receptor did not prevent activation. These data indicate that retinoids up-regulate the production of LTGF-beta in ECs and induce activation of LTGF-beta, perhaps, by increasing PA and plasmin levels. Thus, TGF-beta might be a local mediator of some of the biological activities of retinoids both in vivo and in vitro
— id: 13175, year: 1993, vol: 155, page: 323, stat: Journal Article,

Bovine urokinase-type plasminogen activator and its receptor: cloning and induction by retinoic acid
Kratzschmar J; Haendler B; Kojima S; Rifkin DB; Schleuning WD
1993 Mar 30;125(2):177-183, Gene
Full-length cDNAs encoding bovine urokinase-type plasminogen activator (u-PA) and urokinase receptor (u-PAR) were cloned from an aortic endothelial cell cDNA library using PCR-amplified cDNA fragments as probes. Bovine u-PA amino acid identity ranges from 79.5 to 70.9% when compared to its pig, human, baboon and mouse analogues, while bovine u-PAR is 61.8 and 59.6% identical to its human and mouse counterparts, respectively. All Cys residues previously found in mature u-PA and u-PAR from these different species are also conserved in the bovine molecules. Bovine u-PA and its cell-surface receptor display one and six potential sites of N-linked glycosylation, respectively. Northern blot hybridization demonstrated a moderate induction of u-PA and u-PAR mRNA in bovine aortic endothelial cells after treatment with 10 nM and 1 microM retinoic acid for 8 hours
— id: 42360, year: 1993, vol: 125, page: 177, stat: Journal Article,

Biology and biochemistry of proteinases in tumor invasion
Mignatti P; Rifkin DB
1993 Jan;73(1):161-195, Physiological reviews
— id: 56423, year: 1993, vol: 73, page: 161, stat: Journal Article,

INTERLEUKIN-1 (IL-1) INHIBITS THE MIGRATORY RESPONSE OF HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS (HUVEC) TO BASIC FIBROBLAST GROWTH-FACTOR (BFGF)
ODEKON, LE; ROGHANI, M; MOSCATELLI, DA; LEVIN, RI; RIFKIN, DB
1993 FEB 19 ;7(3):A193-A193, FASEB journal
— id: 54346, year: 1993, vol: 7, page: A193, stat: Journal Article,

TGF-beta: structure, function, and formation
Rifkin DB; Kojima S; Abe M; Harpel JG
1993 Jul 1;70(1):177-179, Thrombosis & haemostasis
— id: 56578, year: 1993, vol: 70, page: 177, stat: Journal Article,

MECHANISM OF ACTIVATION OF LATENT TGF-BETA
RIFKIN, DB; KOJIMA, S; ABE, M; HARPEL, J; NUNES, I
1993 MAR 29 ;43(4):105-105, Journal of cellular biochemistry
— id: 54219, year: 1993, vol: 43, page: 105, stat: Journal Article,

PHOSPHOLIPASE-C RELEASE OF BIOLOGICALLY-ACTIVE BASIC FIBROBLAST GROWTH FACTOR-HEPARAN SULFATE PROTEOGLYCAN COMPLEXES FROM HUMAN BONE-MARROW CULTURES
BRUNNER, G; GABRILOVE, J; RIFKIN, DB; WILSON, EL
1992 JAN ;20(1):107-107, Experimental hematology
— id: 52124, year: 1992, vol: 20, page: 107, stat: Journal Article,

Basic fibroblast growth factor-induced activation of latent transforming growth factor beta in endothelial cells: regulation of plasminogen activator activity
Flaumenhaft R; Abe M; Mignatti P; Rifkin DB
1992 Aug;118(4):901-909, Journal of cell biology
Exposure of bovine aortic or capillary endothelial cells to basic FGF (bFGF) for 1 h resulted in an approximately sixfold increase in plasminogen activator (PA) activity by 18 h that returned nearly to basal levels by 36 h. We hypothesized that the decrease in PA activity following bFGF stimulation was mediated by transforming growth factor beta (TGF-beta) formed from its inactive precursor. Conditioned medium collected from endothelial cells 36 h after a 1-h exposure to bFGF, but not control medium, inhibited basal levels of PA activity when transferred to confluent monolayers of bovine aortic endothelial cells. Antibody to TGF-beta neutralized the inhibitory activity of this conditioned medium, indicating that the medium contained active TGF-beta. Northern blot analysis and quantitation of acid activatable latent TGF-beta in conditioned medium demonstrated that bFGF exposure did not increase the amount of transcription or secretion of latent TGF-beta by the endothelial cells. Both aprotinin, an inhibitor of plasmin, and anti-urokinase type PA IgG blocked the generation of active TGF-beta in cultures exposed to bFGF. These results demonstrated that plasmin generated by uPA activity is required for the activation of latent TGF-beta in endothelial cell cultures treated with bFGF. Activation of TGF-beta by endothelial cells exposed to bFGF appears to limit both the degree and duration of PA stimulation. Thus, in bFGF-stimulated endothelial cell cultures, PA levels are controlled by a negative feedback loop: PA, whose expression is stimulated by bFGF, contributes to the formation of TGF-beta, which in turn opposes the effects of bFGF by limiting PA synthesis and activity. These studies suggest a role for TGF-beta in reversing the invasive stage of angiogenesis and contributing to the formation of quiescent capillaries
— id: 13507, year: 1992, vol: 118, page: 901, stat: Journal Article,

Cell density dependent effects of TGF-beta demonstrated by a plasminogen activator-based assay for TGF-beta
Flaumenhaft R; Rifkin DB
1992 Jul;152(1):48-55, Journal of cellular physiology
Transforming growth factor-beta 1 (TGF-beta 1) induces a decrease in plasminogen activator (PA) expression in confluent cultures of bovine aortic endothelial (BAE) cells. We describe an assay using the suppression of PA expression in confluent BAE cells by TGF-beta 1 which detects concentrations of the growth factor ranging from 5 to 200 pg/ml and has an ED50 of 15-20 pg/ml. The assay can be performed in 96-well plates and requires a minimum of 35 ul of solution per sample, thereby limiting the amount of reagents required and allowing many samples to be tested in a single assay. Here we demonstrate that the effect of TGF-beta 1 on PA expression in BAE cells depends on the length of time the cells are exposed to the growth factor and the density at which the cells are plated. In cells plated at a high density (3.5 x 10(5) cells/cm2), both 4 h and 24 h exposures to TGF-beta 1 suppress PA expression. However, with cells plated sparsely (3.5 x 10(4) cells/cm2), a 4 h exposure to TGF-beta 1 increases PA expression 2-fold, whereas a 24 h exposure results in an 85% inhibition of basal PA expression. The paradoxical stimulation of PA expression in cells at a sparse density upon 4 h exposure to TGF-beta 1 occurs in a dose-dependent manner with an ED50 of 15-20 pg/ml. This bifunctional response of PA production in cells exposed to TGF-beta 1 may have implications with regard to the role of TGF-beta 1 in angiogenesis
— id: 13527, year: 1992, vol: 152, page: 48, stat: Journal Article,

The extracellular regulation of growth factor action
Flaumenhaft R; Rifkin DB
1992 Oct;3(10):1057-1065, Molecular biology of the cell
— id: 13406, year: 1992, vol: 3, page: 1057, stat: Journal Article,

THE REGULATION OF PLASMINOGEN-ACTIVATOR ACTIVITY IN HUMAN BONE-MARROW STROMAL CELLS
HANNOCKS, MJ; RIFKIN, DB; OLIVER, L; GABRILOVE, J; WILSON, EL
1992 JAN ;20(1):108-108, Experimental hematology
— id: 52125, year: 1992, vol: 20, page: 108, stat: Journal Article,

Control of transforming growth factor-beta activity: latency vs. activation
Harpel JG; Metz CN; Kojima S; Rifkin DB
1992 ;4(4):321-335, Progress in growth factor research
Transforming growth factor-beta is a pluripotent regulator of cell growth and differentiation. The growth factor is expressed as a latent complex that must be converted to an active form before interacting with its ubiquitous high affinity receptors. This conversion involves the release of the mature growth factor through disruption of the non-covalent interactions with its pro-peptide or latency associated peptide. The mechanisms for this release in vivo have not been fully characterized but appear to be cell specific and might involve processes such as acidification or proteolysis. Although several factors including transcriptional regulation, receptor modulation and scavenging of the active growth factor have been implicated, the critical step controlling the biological effects of transforming growth factor-beta may be the activation of the latent molecule
— id: 56539, year: 1992, vol: 4, page: 321, stat: Journal Article,

Immunohistochemical localization of basic fibroblast growth factor in wound healing sites of mouse skin
Kurita Y; Tsuboi R; Ueki R; Rifkin DB; Ogawa H
1992 ;284(4):193-197, Archives of dermatological research
The immunohistochemical localization of basic fibroblast growth factor (bFGF) was examined during wound healing in mouse skin. Frozen sections taken from the rounded skin defects were reacted with polyclonal anti-human recombinant bFGF IgG followed by incubation with FITC-conjugated IgG. The basal layer keratinocytes and hair bulbs at the wound edge were strongly stained with this antibody. In the reepithelized area, several layers of keratinocytes from the basal layer were positively stained regardless of the time after wounding. These findings suggest that germinative keratinocytes which express bFGF function as leading cells in the covering of the wound defect. However, dermal granulation tissue, including capillary endothelial cells, fibroblasts and macrophages unexpectedly did not demonstrate any immunoreactivity throughout the process of wound healing. Simultaneous histochemical investigation using cultivated mouse keratinocytes and bovine aortic endothelial cells showed primarily cytoplasmic fluorescence. The discrepancy in the staining patterns of endothelial cells in vivo and in vitro suggests that immunoreactive bFGF is either not expressed in vivo, or is processed or masked
— id: 42363, year: 1992, vol: 284, page: 193, stat: Journal Article,

Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex
Mignatti P; Morimoto T; Rifkin DB
1992 Apr;151(1):81-93, Journal of cellular physiology
Basic fibroblast growth factor (bFGF) modulates functions of a variety of cell types. Whereas bFGF is known to act extracellularly, the protein lacks a transient signal peptide. No defined mechanism for bFGF secretion has been characterized besides release from dead or injured cells. To study this problem we devised an experimental system to examine bFGF-mediated migration of isolated single cells. Under these conditions individual cells are not affected by bFGF derived from other cells. By this method we have previously shown that bFGF released by NIH 3T3 cells transfected with bFGF cDNA modulates migration in an autocrine manner. We have now examined the effects on cell motility of drugs or treatments known to affect various pathways of protein secretion. Drugs that block secretion via the endoplasmic reticulum (ER)-Golgi complex or via multidrug resistance proteins did not inhibit cell motility. Migration was enhanced by the calcium ionophore A23187, which stimulates exocytosis, and was inhibited by methylamine, serum-free, and low temperature (18 degrees C) conditions, which block endo- and exocytosis. The reversal of these effects by the concomitant addition of affinity-purified anti-bFGF IgG or recombinant bFGF showed that the alterations in cell migration were mediated by changes in bFGF externalization. Thus bFGF can be released via a mechanism of exocytosis independent of the ER-Golgi pathway
— id: 42362, year: 1992, vol: 151, page: 81, stat: Journal Article,

Urokinase-type plasminogen activator mediates basic fibroblast growth factor-induced bovine endothelial cell migration independent of its proteolytic activity
Odekon LE; Sato Y; Rifkin DB
1992 Feb;150(2):258-263, Journal of cellular physiology
The dependence of urokinase-type plasminogen activator (uPA) induction on endogenous basic fibroblast growth factor (bFGF) activity during endothelial cell migration was investigated utilizing a combination of wounded endothelial cell monolayers and substrate overlay techniques. Purified polyclonal rabbit immunoglobulin G (IgG) against bFGF blocked the appearance of uPA-dependent lytic activity normally observed at the edge of a wounded bovine aortic endothelial (BAE) cell monolayer. Additionally, the migration of cells into the denuded area was inhibited 30-50% by antibodies either to bFGF or to bovine uPA. Incubation of wounded monolayers with either purified bovine uPA or agents able to induce PA activity, such as phorbol myristate acetate (PMA), vanadate, or bFGF, resulted in enhanced migration of cells (28-50%). Anti-bovine uPA IgG blocked a significant fraction (25%) of BAE cell migration induced by exposure to exogenous bFGF. The role of uPA in migration of wounded BAE cells was not dependent on plasmin generation. Furthermore, the amino terminal fragment (ATF) of human recombinant (hr) uPA, which is enzymatically inactive, stimulated endothelial cell movement in the presence of anti-bFGF IgG. These results suggest that BAE cell migration from the edge of a wounded monolayer is dependent upon local increases of uPA mediated by endogenous bFGF. Moreover, the data support the conclusion that migration is stimulated via a signalling mechanism dependent upon occupancy of the uPA receptor but independent of uPA-mediated proteolysis
— id: 13697, year: 1992, vol: 150, page: 258, stat: Journal Article,

Plasminogen activator expression and matrix degradation
Rifkin DB
1992 ;1:20-22, Matrix. Supplement
The plasminogen activator (PA)-plasmin system is discussed in respect to the major interacting proteins. The various mechanisms controlling this system are described including physical properties of the reactants, molecules affecting PA synthesis, and receptors. Data is presented showing that for cell invasion to take place not only must the PA-plasmin system be operative but also specific metalloproteases must be activated. Indirect evidence indicates that this activation occurs through the action of the PA-plasmin system
— id: 13771, year: 1992, vol: 1, page: 20, stat: Journal Article,

A wound healing model using healing-impaired diabetic mice
Tsuboi R; Shi CM; Rifkin DB; Ogawa H
1992 Nov;19(11):673-675, Journal of dermatology
A quantitative histological approach was employed to evaluate the effects of basic fibroblast growth factor (bFGF) in healing-impaired diabetic mice. The dorsal areas of female mutant diabetic mice, C57BL KsJ db/db (Jackson Lab.), were given two 6 mm-size full thickness wounds with a punch biopsy instrument. After application of bFGF, the wounds were left open. 8 days after wounding, the mice were sacrificed, and histological sections were evaluated using several histological parameters, such as the degree of wound closure, granulation tissue thickness, matrix density, and capillary numbers. Application of 5 micrograms of bFGF for 5 days induced significant responses by all of these dermal parameters when compared to those of non-treated db/db mice (p < 0.001). A minimum of 0.5 micrograms bFGF per day was required for a significant effect. Time-course experiments indicated that the granulation response in bFGF-treated mice peaked between 8 and 12 days and decreased after 12 days, while matrix density continued to increase until the 18th day
— id: 42361, year: 1992, vol: 19, page: 673, stat: Journal Article,

ANGIOSTATIC STEROIDS INHIBIT ENDOTHELIAL-CELL PLASMINOGEN-ACTIVATOR ACTIVITY
BLEI, F; WILSON, EL; RIFKIN, DB
1991 APR ;29(4):A137-A137, Pediatric research
— id: 51662, year: 1991, vol: 29, page: A137, stat: Journal Article,

Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan
Brunner G; Gabrilove J; Rifkin DB; Wilson EL
1991 Sep;114(6):1275-1283, Journal of cell biology
Basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow stromal cells and stimulates haematopoiesis in vitro. We report here that primary human bone marrow cultures contain bFGF and express heparin-like bFGF binding sites on the cell surface and in the extracellular matrix (ECM). bFGF bound predominantly to a 200-kD cell surface heparan sulfate proteoglycan (HSPG), which was also found in conditioned medium. bFGF was released from bone marrow cultures by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) and, less efficiently, by plasmin. Solubilized bFGF was found as a complex with the 200-kD HSPG. The complex was biologically active as shown by its ability to stimulate plasminogen activator production in bovine aortic endothelial cells. bFGF-HSPG complexes of bovine endothelial cells, however, were not released by PI-PLC. While only trace amounts of the bFGF-binding 200-kD HSPG were released spontaneously from bone marrow cultures, incubation with PI-PLC solubilized almost all of the 200-kD HSPG. The HSPG could be metabolically labeled with ethanolamine or palmitate, which was partially removed by treatment with PI-PLC. These findings indicate linkage of the HSPG to the cell surface via a phosphatidylinositol anchor. Plasmin released the 200-kD HSPG less efficiently than PI-PLC. We conclude that HSPGs of human bone marrow serve as a reservoir for bFGF, from which it can be released in a biologically active form via a dual mechanism; one involving a putative endogenous phospholipase, the other involving the proteolytic cascade of plasminogen activation
— id: 13933, year: 1991, vol: 114, page: 1275, stat: Journal Article,

Direct evidence for methylation of arginine residues in high molecular weight forms of basic fibroblast growth factor
Burgess WH; Bizik J; Mehlman T; Quarto N; Rifkin DB
1991 Feb;2(2):87-93, Cell regulation
Basic fibroblast growth factor (bFGF) is a heparin-binding angiogenic polypeptide mitogen. Protein sequence analysis of bFGF isolated from tissue sources initially established that it is composed of 146 amino acids (apparent Mr 18,000). More recently larger apparent molecular weight forms have been identified and partially characterized. In addition, these high molecular weight forms (apparent Mr 22,000 and 25,000) have been shown to localize preferentially to nuclear fractions of transfected cells. In this report we demonstrate that the higher molecular weight, amino terminally extended forms of bFGF contain methylated arginine residues. The demonstration is based on 1) amino acid sequence analysis of a protein known to contain methylated arginine (myelin basic protein) and a comparison with amino acid sequence analysis of trypsin-derived fragments of the high molecular weight bFGF purified from guinea pig brain and 2) the ability to label in vivo the high molecular weight forms of bFGF with S-adenosyl-L-(methyl-3H)-methionine, the substrate of arginine-protein methylase I. These results are suggestive of a role of arginine methylation in directing nuclear localization of certain forms of bFGF
— id: 42368, year: 1991, vol: 2, page: 87, stat: Journal Article,

Cellular activation of latent transforming growth factor beta requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor
Dennis PA; Rifkin DB
1991 Jan 15;88(2):580-584, Proceedings of the National Academy of Sciences of the United States of America
The activation of latent transforming growth factor beta (LTGF-beta) normally seen in cocultures of bovine aortic endothelial and bovine smooth muscle cells can be inhibited by coculturing the cells with either mannose 6-phosphate (Man-6-P) or antibodies directed against the cation-independent Man-6-P/insulin-like growth factor type II receptor (anti-Man-6-PR). This result was established by measuring the ability of coculture conditioned medium (formed with or without Man-6-P or anti-Man-6-PR) to suppress bovine aortic endothelial cell migration and protease production, activities previously shown to be related to transforming growth factor beta activity. The inhibition by Man-6-P is dose dependent, with maximal inhibition seen at 100 microM and is specific because mannose 1-phosphate and glucose 6-phosphate do not interfere with activation of LTGF-beta. The inhibitory effect of anti-Man-6-PR is also specific and dose dependent; maximal inhibition of activation occurs at 400 micrograms/ml. Control experiments indicate that Man-6-P and anti-Man-6-PR do not interfere with the basal level of migration of bovine aortic endothelial cells, the migration observed when exogenous transforming growth factor beta is added, the activation of transforming growth factor beta by plasmin or transient acidification, and the release of LTGF-beta. Thus, binding to the cation-independent Man-6-P/insulin-like growth factor type II receptor appears to be a requirement for activation of LTGF-beta
— id: 14155, year: 1991, vol: 88, page: 580, stat: Journal Article,

Extracellular matrix regulation of growth factor and protease activity
Flaumenhaft R; Rifkin DB
1991 Oct;3(5):817-823, Current opinion in cell biology
Extracellular matrices bind many growth factors, proteases, and protease inhibitors. These interactions not only localize these molecules to the pericellular environment, but also modulate their biological activities. Recent evidence suggests that some growth factors may be active in vivo primarily in complexes with extracellular matrix molecules and that this interaction may be essential to their activity
— id: 13877, year: 1991, vol: 3, page: 817, stat: Journal Article,

Lipoprotein (a) inhibits the generation of transforming growth factor beta: an endogenous inhibitor of smooth muscle cell migration
Kojima S; Harpel PC; Rifkin DB
1991 Jun;113(6):1439-1445, Journal of cell biology
Conditioned medium (CM) derived from co-cultures of bovine aortic endothelial cells (BAECs) and bovine smooth muscle cells (BSMCs) contains transforming growth factor-beta (TGF-beta) formed via a plasmin-dependent activation of latent TGF-beta (LTGF beta), which occurs in heterotypic but not in homotypic cultures (Sato, Y., and D. B. Rifkin. 1989. J. Cell Biol. 107: 1199-1205). The TGF-beta formed is able to block the migration of BSMCs or BAECs. We have found that the simultaneous addition to heterotypic culture medium of plasminogen and the atherogenic lipoprotein, lipoprotein (a) (Lp(a)), which contains plasminogen-like kringles, inhibits the activation of LTGF-beta in a dose-dependent manner. The inclusion of LDL in the culture medium did not show such an effect. Control experiments indicated that Lp(a) does not interfere with the basal level of cell migration, the activity of exogenous added TGF-beta, the release of LTGF-beta from cells, the activation of LTGF-beta either by plasmin or by transient acidification, or the activity of plasminogen activator. The addition of Lp(a) to the culture medium decreased the amount of plasmin found in BAECs/BSMCs cultures. Similar results were obtained using CM derived from cocultures of human umbilical vein endothelial cells and human foreskin fibroblasts. These results suggest that Lp(a) can inhibit the activation of LTGF-beta by competing with the binding of plasminogen to cell or matrix surfaces. Therefore, high plasma levels of Lp(a) might enhance smooth muscle cell migration by decreasing the levels of the migration inhibitor TGF-beta thus contributing to generation of the atheromatous lesions
— id: 14009, year: 1991, vol: 113, page: 1439, stat: Journal Article,

Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor
Mignatti P; Mazzieri R; Rifkin DB
1991 Jun;113(5):1193-1201, Journal of cell biology
Basic fibroblast growth factor, a potent angiogenesis inducer, stimulates urokinase (uPA) production by vascular endothelial cells. In both basic fibroblast growth factor-stimulated and -nonstimulated bovine capillary endothelial and human umbilical vein endothelial cells single-chain uPA binding is mediated by a membrane protein with a Mr of 42,000. Exposure of bovine capillary or endothelial human umbilical vein endothelial cells to pmolar concentrations of basic fibroblast growth factor results in a dose-dependent, protein synthesis-dependent increase in the number of membrane receptors for uPA (19,500-187,000) and in a parallel decrease in their affinity (KD = 0.144-0.790 nM). With both cells, single-chain uPA binding is competed by synthetic peptides whose sequence corresponds to the receptor-binding sequence in the NH2-terminal domain of uPA. Exposure of bovine capillary endothelial cells to transforming growth factor beta 1, which inhibits uPA production and upregulates type 1 plasminogen activator inhibitor, the major endothelial cell plasminogen activator inhibitor, has no effect on uPA receptor levels. These results show that basic fibroblast growth factor, besides stimulating uPA production by vascular endothelial cells, also increases the production of receptors, which modulates their capacity to focalize this enzyme on the cell surface. This effect may be important in the degradative processes that occur during angiogenesis
— id: 42364, year: 1991, vol: 113, page: 1193, stat: Journal Article,

Basic fibroblast growth factor released by single, isolated cells stimulates their migration in an autocrine manner
Mignatti P; Morimoto T; Rifkin DB
1991 Dec 15;88(24):11007-11011, Proceedings of the National Academy of Sciences of the United States of America
Basic fibroblast growth factor (bFGF), a protein with angiogenic, mitogenic, and chemotactic properties, lacks a signal sequence and is not secreted via the classical secretory pathway. However, the growth factor is known to act extracellularly. Since no defined mechanism for bFGF release has been described, it has been suggested that this growth factor is released from dead or damaged cells. To test this hypothesis we characterized the effect of exogenously added bFGF and neutralizing antibody on the migration of single, isolated NIH 3T3 cells transfected with bFGF cDNA. Under these conditions the observed cell cannot be affected by bFGF derived from other cells. Cells were seeded onto colloidal gold-coated coverslips at a density of one cell per coverslip. A cell migrating on this substrate produces a track free of refringent gold particles that is measured by an image analyzer. The results showed that cell motility directly correlated with the amount of bFGF released from the migrating cells. Affinity-purified anti-bFGF antibody, but not irrelevant IgG, reduced the level of migration of the bFGF transfectants to that of the control cells transfected with the vector alone, showing that bFGF stimulates migration of the cell that releases it. Thus, bFGF is secreted by viable cells and mediates cell functions via a 'true' autocrine mechanism
— id: 13815, year: 1991, vol: 88, page: 11007, stat: Journal Article,

Release of basic fibroblast growth factor, an angiogenic factor devoid of secretory signal sequence: a trivial phenomenon or a novel secretion mechanism?
Mignatti P; Rifkin DB
1991 Nov;47(3):201-207, Journal of cellular biochemistry
Basic fibroblast growth factor (bFGF), a potent angiogenesis inducer, lacks a signal sequence. Therefore, it has been proposed that bFGF is primarily released from dead or damaged cells. Other proteins devoid of secretion signals, interleukin 1 beta (IL-1 beta) and the muscle lectin L-14, have been shown to be released via exocytosis, a novel secretion pathway independent of the 'classic' endoplasmic reticulum-Golgi route. In the light of these findings and of our own recent results, we discuss evidence that bFGF can be released from single, uninjured cells and mediate functions in an autocrine manner. As is the case for IL-1 beta and L-14, externalization of bFGF may occur via exocytosis, a pathway utilized during development and differentiation
— id: 13859, year: 1991, vol: 47, page: 201, stat: Journal Article,

Immunoreactive basic fibroblast growth factor-like proteins in chromaffin granules
Presta M; Rifkin DB
1991 Mar;56(3):1087-1088, Journal of neurochemistry
— id: 42366, year: 1991, vol: 56, page: 1087, stat: Journal Article,

The NH2-terminal extension of high molecular weight bFGF is a nuclear targeting signal
Quarto N; Finger FP; Rifkin DB
1991 May;147(2):311-318, Journal of cellular physiology
Basic fibroblast growth factor (bFGF) is a member of the heparin-binding growth factor (HBGF) family that includes at least seven species. These proteins are potent regulators of a number of cellular processes, including cell division and angiogenesis. Multiple forms of bFGF exist differing only in the length of their NH2-terminal extensions. These species of bFGF also have unique subcellular distributions. The smallest form (18 kD) occurs predominantly in the cytosol, while the higher molecular weight forms (22, 22.5, 24 kD) are associated with the nucleus and ribosomes. Here we report that the nuclear localization of the higher molecular weight forms of bFGF derives specifically from the amino acid sequences within the NH2-terminal extension. This has been demonstrated by constructing a chimeric protein containing the NH2-terminal extension of the highest molecular weight form of bFGF fused to beta-galactosidase (beta-gal). After transfection in a transient expression system, the chimeric protein accumulated in the nuclei of transfected cells, while the wild-type beta-gal was found predominantly in the cytoplasm
— id: 42365, year: 1991, vol: 147, page: 311, stat: Journal Article,

Selective expression of high molecular weight basic fibroblast growth factor confers a unique phenotype to NIH 3T3 cells
Quarto N; Talarico D; Florkiewicz R; Rifkin DB
1991 Sep;2(9):699-708, Cell regulation
The phenotypes of NIH 3T3 cells transfected with basic fibroblast growth factor (bFGF) cDNAs that express only the high molecular weight (HMW) forms of bFGF, the 18-kDa form, or all forms were examined. Cells producing the 18 kDa or all forms of bFGF were transformed at high levels of growth factor expression but were nontransformed at low levels. Cell producing low levels of HMW forms of bFGF were growth impaired when compared with the parental cells. These cells tended to form multinucleated giant cells, did not grow in soft agar, were nontumorigenic, had a normal bFGF receptor number, and had a nontransformed morphology. Cells expressing high levels of HMW bFGFs had a transformed morphology and were tumorigenic. These data suggest a specific functional role for HMWbFGF
— id: 13931, year: 1991, vol: 2, page: 699, stat: Journal Article,

Mechanisms controlling the extracellular activity of basic fibroblast growth factor and transforming growth factor
Rifkin DB; Moscatelli D; Flaumenhaft R; Sato Y; Saksela O; Tsuboi R
1991 ;614:250-258, Annals of the New York Academy of Sciences
— id: 14164, year: 1991, vol: 614, page: 250, stat: Journal Article,

New observations on the intracellular localization and release of bFGF
Rifkin DB; Quarto N; Mignatti P; Bizik J; Moscatelli D
1991 ;638:204-206, Annals of the New York Academy of Sciences
— id: 14216, year: 1991, vol: 638, page: 204, stat: Journal Article,

The stimulatory effect of PDGF on vascular smooth muscle cell migration is mediated by the induction of endogenous basic FGF
Sato Y; Hamanaka R; Ono J; Kuwano M; Rifkin DB; Takaki R
1991 Feb 14;174(3):1260-1266, Biochemical & biophysical research communications
The migration of arterial smooth muscle cells from the media to the intima is a crucial event for the development of the atherosclerotic lesion, and platelet derived growth factor (PDGF) is thought to play an important role in this process. Here we report that the spontaneous migration of bovine smooth muscle (BSM) cells is dependent on endogenously produced basic fibroblast growth factor (bFGF). PDGF stimulates the migration of BSM cells and its effect is abolished by affinity purified anti-bFGF antibody. PDGF induces bFGF mRNA in BSM cells. These results indicate that the effect of PDGF on the migration of BSM cells may be mediated by the induction of endogenous bFGF
— id: 42367, year: 1991, vol: 174, page: 1260, stat: Journal Article,

Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures
Wilson EL; Rifkin DB; Kelly F; Hannocks MJ; Gabrilove JL
1991 Mar 1;77(5):954-960, Blood
We previously showed that basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow (BM) stromal cells and significantly delays their senescence. In the present study, we demonstrated that low concentrations of bFGF (0.2 to 2 ng/mL) enhance myelopoiesis in long-term human BM culture. Addition of bFGF to long-term BM cultures resulted in an increase in (a) the number of nonadherent cells (sixfold), particularly those of the neutrophil granulocyte series; (b) the number of nonadherent granulocyte colony-stimulating factor (G-CSF)- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive progenitor cells; (c) the number of adherent foci of hematopoietic cells (10-fold); and (d) the number of progenitor cells in the adherent stromal cell layer. These effects were not noted with higher concentrations of bFGF (20 ng/mL). Thus, low concentrations of bFGF effectively augment myelopoiesis in human long-term BM cultures, and bFGF may therefore be a regulator of the hematopoietic system in vitro and in vivo
— id: 57423, year: 1991, vol: 77, page: 954, stat: Journal Article,

Expression of tissue factor procoagulant activity: regulation by cytosolic calcium
Bach R; Rifkin DB
1990 Sep;87(18):6995-6999, Proceedings of the National Academy of Sciences of the United States of America
Intact bovine fibroblasts, pericytes, and kidney cells manifested significantly less tissue factor procoagulant activity than their disrupted counterparts. Addition of calcium ionophore A23187 rapidly and reversibly enhanced the cell-surface expression of tissue factor in intact cells up to the level achieved by disruption. Inhibitors of calmodulin blocked the ionophore-dependent enhancement of procoagulant activity. Similar kinetic parameters were obtained for factor X hydrolysis by tissue factor-factor VIIa on unperturbed pericytes and phosphatidylcholine vesicles. Increase in Vmax and decrease in apparent Km for this reaction were seen after either disruption or ionophore stimulation of the pericytes. Addition of phosphatidylserine to the reconstituted phospholipid vesicles also increased the Vmax and decreased the apparent Km for factor X hydrolysis. These data agree with the hypothesis that the expression of tissue factor procoagulant activity on cell surfaces is modulated by calcium-mediated changes in the asymmetric distribution of phosphatidylserine in plasma membrane
— id: 42369, year: 1990, vol: 87, page: 6995, stat: Journal Article,

Studies on the role of basic fibroblast growth factor in vivo: inability of neutralizing antibodies to block tumor growth
Dennis PA; Rifkin DB
1990 Jul;144(1):84-98, Journal of cellular physiology
Affinity-purified polyclonal rabbit antibodies prepared against recombinant basic fibroblast growth factor (bFGF) neutralized the ability of bFGF to stimulate plasminogen activator (PA) production and endothelial cell migration in vitro. After iodination and intraperitoneal injection of the antibodies in mice, approximately 76% of the maximum circulating level of 125I-anti-bFGF antibodies (AF) was found as intact IgG after 24 hr. Furthermore, the circulating 125I-AF retained the ability to bind bFGF. Studies were performed to determine whether the growth of three different murine tumors (CT26, EHS, or B16/BL6) could be inhibited with affinity-purified neutralizing antibodies against bFGF. Tumors were injected subcutaneously in syngeneic mice, and neutralizing antibodies against bFGF were injected daily into the peritoneum. All studies, which varied in tumor burden, antibody dose, and study length, indicated that neutralizing antibodies against bFGF had no effect on tumor size, tumor growth, or tumor histology
— id: 42373, year: 1990, vol: 144, page: 84, stat: Journal Article,

Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor
Flaumenhaft R; Moscatelli D; Rifkin DB
1990 Oct;111(4):1651-1659, Journal of cell biology
The radius of diffusion of basic FGF (bFGF) in the presence and in the absence of the glycosaminoglycans heparin and heparan sulfate was measured. Iodinated 125I-bFGF diffuses further in agarose, fibrin, and on a monolayer of bovine aortic endothelial (BAE) cells in the presence of heparin than in its absence. Heparan sulfates affected the diffusion of 125I-bFGF in a manner similar to, though less pronounced than, heparin. When applied at the center of a monolayer of BAE cells, bFGF plus heparin stimulated morphological changes at a 10-fold greater radius than bFGF alone. These results suggest that bFGF-heparin and/or heparan sulfate complexes may be more effective than bFGF alone in stimulating cells located away from the bFGF source because the bFGF-glycosaminoglycan complex partitions into the soluble phase rather than binding to insoluble glycosaminoglycans in the extracellular matrix. Thus, the complex of bFGF and glycosaminoglycan may represent one of the active forms of bFGF in vivo
— id: 25414, year: 1990, vol: 111, page: 1651, stat: Journal Article,

THE EFFECT OF RECOMBINANT BASIC FIBROBLAST GROWTH-FACTOR ON THE WOUND-HEALING AND ITS LOCALIZATION IN HEALING IMPAIRED DB DB MICE
Kurita, Y; Tsuboi, R; Ogawa, H; Rifkin, DB
1990 Apr;94(4):545-545, Journal of investigative dermatology
— id: 31999, year: 1990, vol: 94, page: 545, stat: Journal Article,

THE EFFECT OF RECOMBINANT BASIC FIBROBLAST GROWTH-FACTOR ON THE WOUND-HEALING AND ITS LOCALIZATION IN HEALING IMPAIRED DB/DB MICE
Kurita, Y; Tsuboi, R; Ogawa, H; Rifkin, DB
1990 Apr;38(2):A611-A611, Clinical research
— id: 31979, year: 1990, vol: 38, page: A611, stat: Journal Article,

UREMIC LEVELS OF OXALIC-ACID SUPPRESS REPLICATION OF HUMAN ENDOTHELIAL-CELLS INDUCED BY FIBROBLAST GROWTH-FACTORS
Lewin, RI; Moscatelli, DA; Recht, PA; Lee, SY; Rifkin, DB; Ekundayo, CI
1990 Apr;38(2):A233-A233, Clinical research
— id: 32064, year: 1990, vol: 38, page: A233, stat: Journal Article,

Long-term culture of human bone marrow stromal cells in the presence of basic fibroblast growth factor
Oliver LJ; Rifkin DB; Gabrilove J; Hannocks MJ; Wilson EL
1990 ;3(3):231-236, Growth factors
Basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow stromal cells. Normally, large numbers of human bone marrow stromal cells are difficult to obtain. However, nanogram/ml concentrations of bFGF stimulate the growth of passaged bone marrow stromal cells both in media formulated for optimal growth of stromal cells and in a simple mixture of RPMI-1640 and 10% fetal calf serum facilitating the successive expansion of stromal cells through multiple passages. bFGF also greatly accelerates the formation of a primary stromal cell layer following inoculation of newly harvested bone marrow cells into dishes. In the presence of bFGF, the stromal cells attain high densities, lose their contact inhibition and grow in multilayered sheets. Heparin greatly potentiates the stimulatory effect of low concentrations of bFGF. The effects of bFGF are fully reversible: cells cultured in the presence of this factor for multiple passages revert to normal growth rates following trypsinization and subculture. A short (4 h) exposure of the cells to bFGF elicits profound growth stimulation. This supports the hypothesis that this factor binds to glycosaminoglycans in the cell matrix which act as a storage reservoir for this cytokine
— id: 35203, year: 1990, vol: 3, page: 231, stat: Journal Article,

Nuclear and cytoplasmic localization of different basic fibroblast growth factor species
Renko M; Quarto N; Morimoto T; Rifkin DB
1990 Jul;144(1):108-114, Journal of cellular physiology
The subcellular distribution of basic fibroblastic growth factor (bFGF) was analyzed by subcellular fractionation and immunofluorescence to gain insight into potential mechanisms for its release from cells. Subcellular fractionation of either SK-Hep-1 cells or NIH 3T3 cells transfected with a bFGF cDNA revealed that the 18 kd form of bFGF was found primarily in the cytosolic fraction, whereas the 22 and 24 kd forms of bFGF were found preferentially in ribosomal and nuclear fractions. Analysis of bFGF distribution by immunofluorescence using an antibody that recognized all forms of bFGF indicated both cytoplasmic and nuclear localization but failed to reveal any growth factor in structures representing secretory vesicles. Therefore, bFGF has a distribution inconsistent with that of a secretory protein
— id: 42374, year: 1990, vol: 144, page: 108, stat: Journal Article,

Growth factor control of extracellular proteolysis
Rifkin DB; Moscatelli D; Bizik J; Quarto N; Blei F; Dennis P; Flaumenhaft R; Mignatti P
1990 Dec 2;32(3):313-318, Cell differentiation & development
The involvement of proteases and growth factors in angiogenesis is complex. The angiogenic factor basic fibroblast growth factor (bFGF) induces increased synthesis of both plasminogen activator and collagenase in endothelial cells. In addition, bFGF increases the number of plasminogen activator receptors on the cell surface. Increased production of plasmin may be responsible for the release of soluble complexes of heparan sulfate-bFGF which may be the active form of bFGF. The activity of a negative regulator of angiogenesis, transforming growth factor beta (TGF-beta), is also regulated by proteases since the released latent form of TGF-beta is activated by a surface proteolytic assembly plasminogen activator and plasmin. Since TGF-beta induces an inhibitor of plasminogen activator, the activation reaction is self-regulatory
— id: 14245, year: 1990, vol: 32, page: 313, stat: Journal Article,

Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity
Saksela O; Rifkin DB
1990 Mar;110(3):767-775, Journal of cell biology
Cultured bovine capillary endothelial (BCE) cells synthesize heparan sulfate proteoglycans (HSPG), which are both secreted into the culture medium and deposited in the cell layer. The nonsoluble HSPGs can be isolated as two predominant species: a larger 800-kD HSPG, which is recovered from preparations of extracellular matrix, and a 250-kD HSPG, which is solubilized by nonionic detergent extraction of the cells. Both HSPG species bind bFGF. 125I-bFGF bound to BCE cell cultures is readily released by either heparinase or plasmin. When released by plasmin, the growth factor is recovered from the incubation medium as a complex with the partly degraded high molecular mass HSPG. Endogenous bFGF activity is released by a proteolytic treatment of cultured BCE cells. The bFGF-binding HSPGs are also released when cultures are incubated with the inactive proenzyme plasminogen. Under such experimental conditions, the release of the extracellular proteoglycans can be enhanced by treating the cells either with bFGF, which increases the plasminogen activating activity expressed by the cells, or decreased by treating the cells with transforming growth factor beta, which decreases the plasminogen activating activity of the cells. Specific immune antibodies raised against bovine urokinase also block the release of HSPG from BCE cell cultures. We propose that this plasminogen activator-mediated proteolysis provides a mechanism for the release of biologically active bFGF-HSPG complexes from the extracellular matrix and that bFGF release can be regulated by the balance between factors affecting the pericellular proteolytic activity
— id: 42375, year: 1990, vol: 110, page: 767, stat: Journal Article,

Characterization of the activation of latent TGF-beta by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self-regulating system
Sato Y; Tsuboi R; Lyons R; Moses H; Rifkin DB
1990 Aug;111(2):757-763, Journal of cell biology
The conversion of latent transforming growth factor beta (LTGF-beta) to the active species, transforming growth factor beta (TGF-beta), has been characterized in heterotypic cultures of bovine aortic endothelial (BAE) cells and bovine smooth muscle cells (SMCs). The formation of TGF-beta in co-cultures of BAE cells and SMCs was documented by a specific radioreceptor competition assay, while medium from homotypic cultures of BAE cells or SMCs contained no active TGF-beta as determined by this assay. The concentration of TGF-beta in the conditioned medium of heterotypic co-cultures was estimated to be 400-1,200 pg/ml using the inhibition of BAE cell migration as an assay. Northern blotting of poly A+ RNA extracted from both homotypic and heterotypic cultures of BAE cells and SMCs revealed that BAE cells produced both TGF-beta 1 and TGF-beta 2, while SMCs produced primarily TGF-beta 1. No change in the expression of these two forms of TGF-beta was apparent after 24 h in heterotypic cultures. Time course studies on the appearance of TGF-beta indicated that most of the active TGF-beta was generated within the first 12 h after the establishment of co-cultures. The generation of TGF-beta in co-cultures stimulated the production of the protease inhibitor plasminogen activator inhibitor-1 (PAI-1). The inclusion of neutralizing antibodies to TGF-beta in the co-culture medium blocked the observed increase in PAI-1 levels. The increased expression of PAI-1 subsequent to TGF-beta formation blocked the activation of the protease required for conversion of LTGF-beta to TGF-beta as the inclusion of neutralizing antibodies to PAI-1 in the co-culture medium resulted in prolonged production of TGF-beta. This effect was lost upon removal of the PAI-1 antibodies. Thus, the activation of LTGF-beta appears to be a self-regulating system
— id: 42370, year: 1990, vol: 111, page: 757, stat: Journal Article,

Bimodal relationship between invasion of the amniotic membrane and plasminogen activator activity
Tsuboi R; Rifkin DB
1990 Jul 15;46(1):56-60, International journal of cancer
Three human tumor cell lines, Bowes' melanoma, HT1080 and Osmond cells, were characterized for their ability to invade the amniotic membrane and their production of plasminogen activator. Bowes' melanoma cells, which release large amounts of tissue plasminogen activator (tPA), were poorly invasive on the amniotic membrane. The addition of plasmin inhibitors, anti-tPA antibody or tissue inhibitor of metalloproteinase (TIMP) to the amnion assay enhanced invasiveness. The depletion of plasminogen from the growth medium also enhanced the degree of invasiveness. Similarly, HT1080 cells, which produce high levels of urokinase-type plasminogen activator (uPA), were poorly invasive under standard conditions but invasion was enhanced by plasmin inhibitors or anti-uPA antibodies. Conversely, Osmond cells, which produce low levels of uPA, were very invasive on the amniotic membrane. Invasion by these cells was blocked by the addition of plasmin inhibitors or anti-uPA antibodies to the amnion assay. These results suggest that invasion requires only a minimum level of PA activity and that, as PA production exceeds this optimal level, the degree of invasion decreases. We propose that high levels of plasmin, generated by the tPA or uPA secreted by the cells, may cause uncontrolled matrix degradation and interrupt the interaction of cells and matrix in the early stages of invasion. The inhibition of excessive plasmin activity may stabilize and increase cell matrix contacts and result in an enhancement of invasion
— id: 42371, year: 1990, vol: 46, page: 56, stat: Journal Article,

Recombinant basic fibroblast growth factor stimulates wound healing in healing-impaired db/db mice
Tsuboi R; Rifkin DB
1990 Jul 1;172(1):245-251, Journal of experimental medicine
The stimulatory effect of recombinant basic fibroblast growth factor (bFGF) on wound healing was assessed using healing-impaired (db/db) mice. Full-thickness wounds were made in female diabetic C57BL/KsJ db/db mice, and their normal (db/+) littermates with a punch biopsy instrument. Recombinant bFGF was applied locally to the open wound once a day. The mice were later killed and histological sections of the wounds were prepared. The degree of wound healing was evaluated using several histological parameters such as degree of reepithelialization, granulation tissue thickness, matrix density, number of infiltrated cells, and number of capillaries. Wounds from normal mice displayed good reepithelialization rates and granulation tissue formation, while wounds from db/db mice had poor responses, especially in the dermal parameters. Although the application of bFGF to wounds in the normal (db/+) mice had little effect, application of bFGF to wounds in db/db mice induced significant responses in all of the dermal parameters compared with nontreated db/db mice (p less than 0.001). In the presence of bFGF, these parameters approximated those observed in nontreated littermates. A minimum of 0.5 microgram bFGF in either single or multiple applications was required for a significant effect. bFGF that was either boiled or pretreated with neutralizing antibody had little stimulatory effect. Time-course experiments indicated that the granulation response in bFGF-treated mice peaked between 8 and 12 d, and decreased after 12 d, while matrix density continued to increase until the 18th day (p less than 0.05). The breaking strength of healed linear wounds in db/db mice was also decreased when compared with heterozygous littermates. This parameter was also improved by the administration of bFGF to the wounds (p less than 0.05)
— id: 42372, year: 1990, vol: 172, page: 245, stat: Journal Article,

Correlation of cell migration, cell invasion, receptor number, proteinase production, and basic fibroblast growth factor levels in endothelial cells
Tsuboi R; Sato Y; Rifkin DB
1990 Feb;110(2):511-517, Journal of cell biology
The levels of endogenous basic fibroblast growth factor (bFGF) in seven clones of cultured bovine capillary endothelial (BCE) cells were assayed, and their relation to cell morphology, bFGF receptor number, cell migration, amniotic membrane invasivity, and proteinase levels were studied. Immunoblotting experiments with anti-bFGF IgG demonstrated that cells from these clones contained different amounts of bFGF. The cells containing high levels of bFGF had a spindle or elongated appearance at confluence and a low number of high affinity receptors for bFGF. The cells containing low levels of bFGF had a cobblestone-like appearance and a higher number of high affinity receptors. When exposed to 10 ng/ml bFGF, cells containing a low level of bFGF took on an elongated appearance with a crisscross pattern similar to that seen with the high producer bFGF cells. The endogenous bFGF levels of the BCE cell clones correlated with the extent of cell migration after wounding of a monolayer and the degree of invasion of the human amniotic membrane. Cells from the clone with the highest endogenous bFGF level migrated well, invaded the amnion membrane without the addition of exogenous bFGF, and were relatively unaffected by the addition of bFGF. Cells from the clone containing the lowest level of bFGF did not migrate or invade under normal conditions. However, the addition of bFGF to the culture medium strongly enhanced both of these processes. The inclusion of anti-bFGF IgG in the media suppressed cell migration and invasion. The plasminogen activator (PA) activities of cell lysates of the clones, assayed by the 125I-fibrin plate technique, indicated that the PA levels did not correlate with the bFGF levels. Metalloproteinase activities in the conditioned medium, assayed by gelatin zymography, correlated with the endogenous bFGF levels, suggesting that the degree of expression of metalloproteinases might be critical for cell migration and invasion. These data suggest that endogenous bFGF may have an important role for migration and invasion of BCE cells during neovascularization via the induction and/or activation of specific metalloproteinases
— id: 42376, year: 1990, vol: 110, page: 511, stat: Journal Article,

INVERSE RELATIONSHIP BETWEEN INVASION OF THE AMNIOTIC MEMBRANE AND PLASMINOGEN-ACTIVATOR ACTIVITY
Tsuboi, R; Ogawa, H; Rifkin, DB
1990 Apr;38(2):A614-A614, Clinical research
— id: 31981, year: 1990, vol: 38, page: A614, stat: Journal Article,

INVERSE RELATIONSHIP BETWEEN INVASION OF THE AMNIOTIC MEMBRANE AND PLASMINOGEN-ACTIVATOR ACTIVITY
Tsuboi, R; Ogawa, H; Rifkin, DB
1990 Apr;94(4):586-586, Journal of investigative dermatology
— id: 32003, year: 1990, vol: 94, page: 586, stat: Journal Article,

Immunohistochemical localization of basic fibroblast growth factor in astrocytomas
Zagzag D; Miller DC; Sato Y; Rifkin DB; Burstein DE
1990 Nov 15;50(22):7393-7398, Cancer research
Because of the prominent neovascularization observed in the growth of brain tumors, we studied the occurrence of basic fibroblast growth factor (bFGF), a potent angiogenic factor in astrocytomas, the most aggressive of which often have marked vascular hyperplasia. Using immunohistochemical methods, we examined 21 examples of such tumors, 7 glioblastomas multiforme, 7 anaplastic astrocytomas, and 7 low grade astrocytomas. Using polyclonal and affinity-purified rabbit antisera to human bFGF, we detected immunoreactive bFGF in all cases of glioblastoma multiforme. bFGF was present in both endothelial cells and neoplastic astrocytes. In 4 of 7 anaplastic astrocytomas, the tumor astrocytes had bFGF immunoreactivity and, in 5 of 7 cases, endothelial cells were also immunopositive. In glioblastomas multiforme and anaplastic astrocytomas, capillaries adjacent to tumor showed bFGF immunoreactivity, whereas capillaries distant from the tumors were not immunostained. In low grade astrocytomas, astrocytic cells were weakly immunoreactive in 2 of 7 cases, and in only 1 of the 7 cases capillaries were immunostained. In each grade, reactive astroglial cells showed variable bFGF immunoreactivity. The immunostaining was not seen with the flow-through fraction obtained after affinity purification of the bFGF antiserum with pure recombinant bFGF. These results suggest a possible role for bFGF in tumor growth and in angiogenesis in astrocytomas
— id: 8224, year: 1990, vol: 50, page: 7393, stat: Journal Article,

Monospecific antibodies implicate basic fibroblast growth factor in normal wound repair
Broadley KN; Aquino AM; Woodward SC; Buckley-Sturrock A; Sato Y; Rifkin DB; Davidson JM
1989 Nov;61(5):571-575, Laboratory investigation
Exogenous polypeptide growth factors influence the rate of wound healing and other biological processes, but there is no direct evidence that these peptides have an intrinsic role. To test whether basic fibroblast growth factor is involved in wound repair, rats were implanted with subcutaneous polyvinyl alcohol sponges containing slow-release pellets releasing either a polyclonal neutralizing antiserum directed against basic fibroblast growth factor, preimmune IgG, or nothing. Histological and biochemical evaluation of the granulation tissue that infiltrated the sponges showed anti-basic fibroblast growth factor to cause significant reductions in DNA, protein, and collagen content when compared with either preimmune IgG or placebo at the early stages of wound repair
— id: 42377, year: 1989, vol: 61, page: 571, stat: Journal Article,

Alpha 2-macroglobulin is a binding protein for basic fibroblast growth factor
Dennis PA; Saksela O; Harpel P; Rifkin DB
1989 May 5;264(13):7210-7216, Journal of biological chemistry
After incubation with human serum or plasma, 125I-basic fibroblast growth factor (bFGF) (molecular mass 18.5 kDa) exhibits molecular mass forms greater than 200 kDa as determined by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. These high molecular mass forms of bFGF are immunoprecipitable with antiserum raised against alpha 2-macroglobulin (alpha 2M). Purified alpha 2M and 125I-bFGF form a covalent complex in a specific, saturable manner. Excess unlabeled bFGF competes with 125I-bFGF for complex formation. Complex formation is complete after 4 h and is inhibited by pretreating alpha 2M with dithiothreitol, iodoacetamide, iodoacetic acid, and N-ethylmaleimide. The complex is resistant to acidic conditions and denaturants such as urea. Heparin, which binds bFGF, has no effect on complex formation. Methylamine, which blocks protease binding to alpha 2M, increases the amount of 125I-bFGF that can be bound 2-fold. Plasmin and trypsin treatment of alpha 2M has no effect on 125I-bFGF binding. The ability of growth factors to compete for binding is specific, as aFGF and TGF-beta compete for binding to alpha 2M, whereas platelet-derived growth factor does not. 125I-bFGF.alpha 2M complexes do not bind to low affinity bFGF binding sites and bind poorly to high affinity bFGF binding sites on BHK-21 cells. In addition, 125I-bFGF bound to alpha 2M has decreased ability to stimulate plasminogen activator production in bovine capillary epithelial cells
— id: 10622, year: 1989, vol: 264, page: 7210, stat: Journal Article,

Role of extracellular matrix in the action of basic fibroblast growth factor: matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis
Flaumenhaft R; Moscatelli D; Saksela O; Rifkin DB
1989 Jul;140(1):75-81, Journal of cellular physiology
When bovine capillary endothelial (BCE) cells were treated with 10 ng/ml of basic fibroblast growth factor (bFGF) for 10 or 30 minutes at 37 degrees C, washed extensively with phosphate-buffered saline (PBS) and incubated in bFGF-free medium, plasminogen activator (PA) production was stimulated to the same extent as in cells exposed continuously to bFGF. Three methods of removing bFGF from heparin-like binding sites in the extracellular matrix, but not from bFGF receptors, abolished this long-term effect of a brief exposure to bFGF. First, BCE cells exposed to bFGF for 30 minutes were washed with 2M NaCl and incubated in bFGF-free medium. Second, BCE cells were incubated with bFGF for 10 minutes in the presence of heparin, and cells were washed with PBS and incubated in bFGF-free medium. Third, BCE cell cultures were treated with heparinase and exposed to bFGF. Each of these treatments abolished the long-term (24-48 hours) stimulation of PA production normally observed after brief exposure to bFGF. In each of these experiments, incubation of cells in bFGF-containing medium after the treatments resulted in normal stimulation of PA production, demonstrating that the treatments did not harm the cells. Stimulation of DNA synthesis was observed when cells were exposed to bFGF for 2 hours at 4 degrees C, incubated in bFGF-free medium for 24 hours at 37 degrees C, and assayed for 3H-thymidine incorporation. However, no stimulation was observed if the 2 hours incubation at 4 degrees C was carried out in the presence of heparin. Thus, long-term stimulation of PA activity and DNA synthesis after a brief exposure to bFGF seems to be a consequence of bFGF binding to the extracellular matrix. The extracellular matrix may act as a physiologic buffer, binding bFGF when concentrations are high and releasing it later for interaction with its receptor. This interaction with matrix may be required for the in vivo action of bFGF
— id: 10556, year: 1989, vol: 140, page: 75, stat: Journal Article,

In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases
Mignatti P; Tsuboi R; Robbins E; Rifkin DB
1989 Feb;108(2):671-682, Journal of cell biology
The role of basic fibroblast growth factor-(bFGF) induced proteinases in basement membrane (BM) invasion by bovine capillary endothelial (BCE) cells was studied using a quantitative in vitro assay previously described (Mignatti et al., 1986). 125I-iododeoxyuridine-labeled BCE cells were grown for 72 h on the human amnion BM, and cell invasion was determined by measuring the radioactivity associated with the tissue after removal of the noninvasive cell layer. BCE cells were noninvasive under normal conditions. Addition of human bFGF to either the BM or to the stromal aspect of the amnion induced BCE cell invasion with a dose-dependent response. This effect was maximal in the presence of 70 ng/ml bFGF, and was inhibited by anti-FGF antibody. Transforming growth factor beta, as well as plasmin inhibitors and anti-tissue type plasminogen activator antibody inhibited BCE cell invasion. The tissue inhibitor of metalloproteinases, 1-10 phenanthroline, anti-type IV and anti-interstitial collagenase antibodies had the same effect. On the contrary, anti-stromelysin antibody and Eglin, an inhibitor of elastase, were ineffective. The results obtained show that both the plasminogen activator-plasmin system and specific collagenases are involved in the invasive process occurring during angiogenesis
— id: 10740, year: 1989, vol: 108, page: 671, stat: Journal Article,

Transformation by basic fibroblast growth factor requires high levels of expression: comparison with transformation by hst/K-fgf
Quarto N; Talarico D; Sommer A; Florkiewicz R; Basilico C; Rifkin DB
1989 ;5(2):101-110, Oncogene research
Basic fibroblast growth factor is a potent mitogen for a variety of cell types and has been suggested to have transforming activity. To test this hypothesis, we have introduced a human bFGF cDNA into NIH 3T3 cells either by DNA transfection or by retrovirus infection. We have compared the properties of cell lines obtained with cells prepared similarly but expressing the hst/K-fgf growth factor. While bFGF does not contain an amino terminal signal sequence and is not secreted from cells in which it is synthesized, hst/K-fgf does contain a signal sequence and is secreted from cells. Our results show that the transformed phenotype correlates directly with the level of bFGF expression, since all transformed clones expressed high levels of bFGF, while nontransformed clones expressed comparatively low levels of bFGF. In contrast, even low levels of hst/K-fgf expression resulted in a transformed phenotype. These results suggest that bFGF is an inefficient transforming protein and that this may relate to its lack of secretion
— id: 10813, year: 1989, vol: 5, page: 101, stat: Journal Article,

Recent developments in the cell biology of basic fibroblast growth factor
Rifkin DB; Moscatelli D
1989 Jul;109(1):1-6, Journal of cell biology
— id: 10568, year: 1989, vol: 109, page: 1, stat: Journal Article,

The role of proteases in matrix breakdown during cellular invasion
Rifkin DB; Tsuboi R; Mignatti P
1989 Oct;140(4):1112-1113, American review of respiratory disease
— id: 10476, year: 1989, vol: 140, page: 1112, stat: Journal Article,

GROWTH-FACTOR CONTROL OF ENDOTHELIAL-CELL FIBRINOLYSIS
Rifkin, DB
1989 Aug 19;62(1):458-458, Thrombosis & haemostasis
— id: 31666, year: 1989, vol: 62, page: 458, stat: Journal Article,

Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture
Sato Y; Rifkin DB
1989 Jul;109(1):309-315, Journal of cell biology
When a confluent monolayer of bovine aortic endothelial (BAE) cells is wounded with a razor blade, endothelial cells (ECs) spontaneously move into the denuded area. If bovine pericytes or smooth muscle cells (SMCs) are plated into the denuded area at low density, they block the movement of the ECs. This effect is dependent upon the number of cells plated into the wound area and contact between ECs and the plated cells. Antibodies to transforming growth factor-beta 1 (TGF-beta 1) abrogate the inhibition of BAE cell movement by pericytes or SMCs. TGF-beta 1, if added to wounded BAE cell monolayers, also inhibits cell movement. When cultured separately, BAE cells, pericytes, and SMCs each produce an inactive TGF-beta 1-like molecule which is activated in BAE cell-pericyte or BAE cell-SMC co-cultures. The activation appears to be mediated by plasmin as the inhibitory effect on cell movement in co-cultures of BAE cells and pericytes is blocked by the inclusion of inhibitors of plasmin in the culture medium
— id: 10572, year: 1989, vol: 109, page: 309, stat: Journal Article,

An amino-terminally extended and post-translationally modified form of a 25kD basic fibroblast growth factor
Sommer A; Moscatelli D; Rifkin DB
1989 May 15;160(3):1267-1274, Biochemical & biophysical research communications
Basic fibroblast growth factor (bFGF) is a heparin-binding angiogenic polypeptide mitogen. bFGF proteins characteristically have a molecular weight of 18,000 which is consistent with the predicted primary translation product of 155 amino acids from the cDNA. More recently, higher molecular weight forms of bFGF have been identified but their structural relationship to the commonly known 18kD bFGFs has not been established. We now show that a 25kD bFGF purified from guinea pig brain tissue is an N-terminally extended and post-translationally modified form of the growth factor. Although the exact nature of the post-translational modifications has not been determined, circumstantial evidence suggests that they may be methylated arginines
— id: 25416, year: 1989, vol: 160, page: 1267, stat: Journal Article,

Interaction of heparin with human basic fibroblast growth factor: protection of the angiogenic protein from proteolytic degradation by a glycosaminoglycan
Sommer A; Rifkin DB
1989 Jan;138(1):215-220, Journal of cellular physiology
Fibroblast growth factors (FGF) are a family of heparin-binding angiogenic polypeptide mitogens. In the presence of heparin, recombinant human basic fibroblast growth factor (bFGF) is fully protected from tryptic digestion and partially protected from chymotryptic digestion. Complete protection of bFGF by heparin is achieved at ratios of growth factor:heparin of approximately 10 or less (w/w). The protection requires bioactive bFGF because inactivated bFGF is rapidly degraded by trypsin or chymotrypsin in the presence of heparin. The bFGF-heparin interaction forms hydrophobic complexes which become insoluble in aqueous buffers at bFGF:heparin ratios of 8 to 10 (w/w). The heparin was found to bind up to a tenfold excess of bFGF on a weight basis. bFGF in the presence of heparin is as active as bFGF alone in inducing 3H-thymidine incorporation into Swiss 3T3 fibroblast DNA
— id: 42378, year: 1989, vol: 138, page: 215, stat: Journal Article,

COORDINATE CONTROL OF CELL-MIGRATION AND INVASION IN CAPILLARY ENDOTHELIAL-CELLS BY ENDOGENOUS BASIC FIBROBLAST GROWTH-FACTOR
Tsuboi, R; Sato, Y; Rifkin, DB
1989 Apr;37(2):A641-A641, Clinical research
— id: 31721, year: 1989, vol: 37, page: A641, stat: Journal Article,

COORDINATE CONTROL OF CELL-MIGRATION AND INVASION IN CAPILLARY ENDOTHELIAL-CELLS BY ENDOGENOUS BASIC FIBROBLAST GROWTH-FACTOR
Tsuboi, R; Sato, Y; Rifkin, DB
1989 Mar;92(3):534-534, Journal of investigative dermatology
— id: 31740, year: 1989, vol: 92, page: 534, stat: Journal Article,

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential
Delli-Bovi P; Curatola AM; Newman KM; Sato Y; Moscatelli D; Hewick RM; Rifkin DB; Basilico C
1988 Jul;8(7):2933-2941, Molecular & cellular biology
We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences
— id: 11053, year: 1988, vol: 8, page: 2933, stat: Journal Article,

In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor
Hatten ME; Lynch M; Rydel RE; Sanchez J; Joseph-Silverstein J; Moscatelli D; Rifkin DB
1988 Feb;125(2):280-289, Developmental biology (Orlando)
When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation
— id: 11190, year: 1988, vol: 125, page: 280, stat: Journal Article,

The development of a quantitative RIA for basic fibroblast growth factor using polyclonal antibodies against the 157 amino acid form of human bFGF. The identification of bFGF in adherent elicited murine peritoneal macrophages
Joseph-Silverstein J; Moscatelli D; Rifkin DB
1988 Jun 13;110(2):183-192, Journal of immunological methods
Polyclonal antibodies which have the capacity to neutralize the biological activity of basic fibriblast growth factor (bFGF) in vitro, have been raised in rabbits against the 157 amino acid form of bFGF purified from human placenta. In a dot blot assay the anti-bFGF antibodies do not recognize the acidic form of FGF (aFGF) with which the basic form shares significant amino acid sequence homology. As determined by immunoblotting, bFGF antibodies recognized only bFGF in a mixture of placentally derived heparin-binding proteins, demonstrating the specificity of these antibodies. Using the anti-human bFGF antibodies, we have developed a solid-phase competitive radioimmunoassay sensitive to 7.8 ng/ml (0.4 pmol/ml) for bFGF. aFGF does not compete with bFGF for binding to the antibodies in the radioimmunoassay even at 2.04 micrograms/ml. The specificity of the assay was further demonstrated by a lack of competition of cytochrome C, myoglobin, epidermal growth factor or bovine serum albumin with bFGF for binding to the antibodies. We have identified bFGF in extracts of adherent thioglycollate-stimulated mouse peritoneal macrophages by immunological criteria including the ability of the extract to compete with 125I-bFGF for binding to affinity-purified anti-human bFGF antibodies in the RIA and the ability of these antibodies in inhibit the bFGF-like biological activity of the macrophage extract
— id: 11065, year: 1988, vol: 110, page: 183, stat: Journal Article,

Multiple forms of an angiogenesis factor: basic fibroblast growth factor
Moscatelli D; Joseph-Silverstein J; Presta M; Rifkin DB
1988 Jan;70(1):83-87, Biochimie
An angiogenesis factor has been isolated from human placenta and human hepatoma cells on the basis of its ability to stimulate protease production in cultured capillary endothelial cells. The purified angiogenesis factor also stimulated DNA synthesis and motility in capillary endothelial cells and induced angiogenesis in vivo. Amino acid sequence data revealed that the angiogenesis factor was human basic fibroblast growth factor (bFGF). Other angiogenesis factors isolated on the basis of their ability to stimulate endothelial cell proliferation have also been identified as bFGFs. The bFGFs that have been sequenced show variability in their N-termini. These different forms of bFGF may be naturally occurring processed forms or may be generated by proteases released during the isolation procedure. Recently a bFGF with a large N-terminal extension has been identified. This Mr 25,000 bFGF has the same biological activity and the same affinity for the bFGF receptor as the typical Mr 18,000 bFGFs. The Mr 25,000 bFGF can be converted into an Mr 18,000 form by treatment with low concentrations of trypsin, suggesting that it may be a precursor to the Mr 18,000 bFGF
— id: 11285, year: 1988, vol: 70, page: 83, stat: Journal Article,

Membrane and matrix localization of proteinases: a common theme in tumor cell invasion and angiogenesis
Moscatelli D; Rifkin DB
1988 Aug 3;948(1):67-85, Biochimica & biophysica acta
— id: 10990, year: 1988, vol: 948, page: 67, stat: Journal Article,

New aspects of blood vessel growth: tumor and tissue-derived angiogenesis factors
Presta M; Rifkin DB
1988 ;18(1):6-17, Haemostasis
Angiogenesis factors have been recently purified from both tumors and nonneoplastic tissues. The biological properties shown by these factors in vivo and on cultured endothelial cells will be discussed
— id: 11290, year: 1988, vol: 18, page: 6, stat: Journal Article,

Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation
Saksela O; Moscatelli D; Sommer A; Rifkin DB
1988 Aug;107(2):743-751, Journal of cell biology
Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion
— id: 11021, year: 1988, vol: 107, page: 743, stat: Journal Article,

Cell-associated plasminogen activation: regulation and physiological functions
Saksela O; Rifkin DB
1988 ;4:93-126, Annual review of cell biology
— id: 11259, year: 1988, vol: 4, page: 93, stat: Journal Article,

Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis
Sato Y; Rifkin DB
1988 Sep;107(3):1199-1205, Journal of cell biology
We have found that the spontaneous migration of bovine aortic endothelial cells from the edge of a denuded area in a confluent monolayer is dependent upon the release of endogenous basic fibroblast growth factor (bFGF). Cell movement is blocked by purified polyclonal rabbit IgG to bFGF as well as affinity purified anti-bFGF IgG and anti-bFGF F(ab')2 fragments. The inhibitory effect of the immunoglobulins is dependent upon antibody concentration, is reversible, is overcome by the addition of recombinant bFGF, and is removed by affinity chromatography of the antiserum through a column of bFGF-Sepharose. Cell movement is also reversibly inhibited by the addition of protamine sulfate and suramin; two agents reported to block bFGF binding to its receptor. The addition of recombinant bFGF to wounded monolayers accelerates the movement of cells into the denuded area. Transforming growth factor beta which has been shown to antagonize several other effects of bFGF also inhibits cell movement. The anti-bFGF IgG prevents the movement of bovine capillary endothelial cells, BHK-21, NIH 3T3, and human skin fibroblasts into a denuded area. Antibodies to bFGF, as well as suramin and protamine sulfate also suppress the basal levels of plasminogen activator and DNA synthesis in bovine aortic endothelial cells
— id: 10962, year: 1988, vol: 107, page: 1199, stat: Journal Article,

Endothelial cell growth factors and the vessel wall
Joseph-Silverstein J; Rifkin DB
1987 Oct;13(4):504-513, Seminars in thrombosis & hemostasis
The role of endothelial cell growth factors in the maintenance of the blood vessel wall is, as we have described here, much more complex than merely stimulating the mitogenesis of endothelial cells. The FGFs are capable of eliciting an array of responses in endothelial cells, some, or all, of which are important for neovascularization and the control of clot dissolution. These endothelial cell responses include protease elaboration, chemotaxis, and mitogenesis. That these growth factors seem neither to be constitutively released into the medium of cultured cells that synthesize bFGF, nor released into the bloodstream in vivo suggests that the temporal and local control of neovascularization may involve the regulation of growth factor release from cells such as endothelial cells, fibroblasts, and macrophages. Although there is no known example of this for bFGF, it is well known that both thrombin and Factor Xa stimulate the release of a mitogenic protein from endothelial cells and that low oxygen tension stimulates the release of macrophage-derived angiogenesis factor. In addition, both TGF beta and heparin alone appear to play a role in wound healing and vessel wall maintenance. The work of Roberts et al suggests that TGF beta is not only angiogenic, but also stimulates the growth of fibrotic tissue as well. Studies on mast cells demonstrated that released heparin is chemotactic for endothelial cells and can potentiate tumor angiogenesis. An attractive hypothesis is that these molecules not only act as FGF potentiators or inhibitors but that they also may exert their angiogenic effects by inducing FGF release from cells. Perhaps angiogenin, an angiogenic molecule with no mitogenic activity, works in this way. However, no evidence as yet exists concerning this point. A second level of control of neovascularization may involve the interaction of FGF with other molecules released into the same microenvironment. For example, thrombin and TGF beta released from platelets, as well as heparin released from mast cells, have all been demonstrated to affect bFGF activity in vitro and may act as modifiers of FGF activity in vivo. Since bFGF can modulate fibrinolytic activity, one could imagine that its release into a wound region of the vasculature could have detrimental effects on clot formation and subsequent wound healing. Thus, the transient inhibition of bFGF activity by TGF beta would allow clot formation before the induction of neovascularization by bFGF, TGF beta thereby playing a role in the regulation of the sequence in which events occur.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 11355, year: 1987, vol: 13, page: 504, stat: Journal Article,

Angiogenesis : mechanisms and pathobiology
Klagsbrun, Michael; Rifkin, Daniel B
Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory, 1987,
— id: 61, year: 1987, vol: , page: , stat: ,

Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor
Moscatelli D; Joseph-Silverstein J; Manejias R; Rifkin DB
1987 Aug;84(16):5778-5782, Proceedings of the National Academy of Sciences of the United States of America
A Mr 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig brain along with the typical Mr 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The Mr 25,000 form, like the Mr 18,000 form, was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The Mr 25,000 guinea pig protein stimulated plasminogen activator production by cultured bovine capillary endothelial cells in a dose-dependent manner at concentrations of 0.1-10 ng/ml, the same range that was effective for guinea pig and human Mr 18,000 bFGFs. The binding of human 125I-labeled bFGF to baby hamster kidney cells is inhibited equally by the Mr 25,000 guinea pig protein and the Mr 18,000 guinea pig and human bFGFs. Polyclonal antibodies raised against human bFGF recognize both the Mr 25,000 and 18,000 guinea pig proteins in an immunoblot analysis. In a radioimmunoassay, both the Mr 25,000 and Mr 18,000 guinea pig proteins compete equally well with iodinated human bFGF for binding to the anti-human bFGF antibodies. When treated with low concentrations of trypsin, the Mr 25,000 guinea pig bFGF was converted to a Mr 18,000 protein. These results show that the two molecules are closely related and suggest that the Mr 25,000 protein shares substantial homology with the Mr 18,000 bFGF
— id: 25417, year: 1987, vol: 84, page: 5778, stat: Journal Article,

The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells
Saksela O; Moscatelli D; Rifkin DB
1987 Aug;105(2):957-963, Journal of cell biology
Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis in vivo, stimulates the production of both urokinase- and tissue-type plasminogen activators (PAs) in cultured bovine capillary endothelial cells. The observed increase in proteolytic activity induced by bFGF was effectively diminished by picogram amounts of transforming growth factor beta (TGF beta), but could not be abolished by increasing the amount of TGF beta. However, the inhibition by TGF beta was greatly enhanced if the cells were pretreated with TGF beta before addition of bFGF. After prolonged incubation of cultures treated simultaneously with bFGF and TGF beta, the inhibitory effect of TGF beta diminished and the stimulatory effect of the added bFGF dominated as assayed by PA levels. TGF beta did not alter the receptor binding of labeled bFGF, nor did a 6-h pretreatment with TGF beta reduce the amount of bFGF bound. The major difference between the effects of bFGF and TGF beta was that while bFGF effectively enhanced PA activity expressed by the cells, TGF beta decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production. Both bFGF and TGF beta increased the secretion of the endothelial-type plasminogen activator inhibitor
— id: 25418, year: 1987, vol: 105, page: 957, stat: Journal Article,

THE OPPOSING EFFECTS OF BASIC FIBROBLAST GROWTH-FACTOR AND TRANSFORMING GROWTH-FACTOR-BETA ON THE REGULATION OF PLASMINOGEN-ACTIVATOR ACTIVITY IN CAPILLARY ENDOTHELIAL-CELLS
Saksela, O; Moscatelli, D; Rifkin, DB
1987 Jul 6;58(1):505-505, Thrombosis & haemostasis
— id: 31143, year: 1987, vol: 58, page: 505, stat: Journal Article,

A form of human basic fibroblast growth factor with an extended amino terminus
Sommer A; Brewer MT; Thompson RC; Moscatelli D; Presta M; Rifkin DB
1987 Apr 29;144(2):543-550, Biochemical & biophysical research communications
The amino acid sequence of a human placental bFGF was determined by a combination of protein and cDNA sequencing. The placental bFGF consists of 157 amino acid residues with a calculated molecular weight of 17,464 and is highly homologous to bovine pituitary bFGF. The human protein contains an amino terminal extension when compared to the sequence established for bovine bFGF (Esch et al., 1985) and to the sequence of the predicted translation product based on human bFGF cDNA clones (Abraham et al., 1986)
— id: 25419, year: 1987, vol: 144, page: 543, stat: Journal Article,

Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade
Mignatti P; Robbins E; Rifkin DB
1986 Nov 21;47(4):487-498, Cell
To understand the role of proteinases in tumor invasion, the effects of inhibitors of metallo-, serine-, and cysteine-proteinases on this process were studied using 125I-iododeoxyuridine-labeled B16/BL6 cells grown on human amnion basement membrane. Cellular invasion was quantitated by measuring the radioactivity associated with the amniotic membrane after the B16/BL6 cells on the basement membrane were removed by lysis followed by scraping. The results obtained with proteinase inhibitors showed that inhibitors of collagenase and plasmin prevented invasion of the amnion. Tissue invasion was also blocked by antiurokinase antibodies. On the contrary, cysteine-proteinase inhibitors and anti-tissue plasminogen activator antiserum were ineffective. Mersalyl, a compound known to activate collagenase, stimulated invasion under conditions where plasmin formation or activity were inhibited. Evidence for the role of a plasminogen activator-plasmin-collagenase activation cascade in B16 invasion is provided
— id: 42379, year: 1986, vol: 47, page: 487, stat: Journal Article,

A PROTEASE CASCADE IN MELANOMA INVASION
MIGNATTI, P; RIFKIN, DB
1986 APR ;34(2):A768-A768, Clinical research
— id: 41419, year: 1986, vol: 34, page: A768, stat: Journal Article,

A PROTEASE CASCADE IN MELANOMA INVASION
MIGNATTI, P; RIFKIN, DB
1986 APR ;86(4):494-494, Journal of investigative dermatology
— id: 41473, year: 1986, vol: 86, page: 494, stat: Journal Article,

INHIBITORS OF SERINE PROTEINASES AND METALLOPROTEINASES BLOCK INVITRO INVASION BY B-16 MELANOMA-CELLS
Mignatti, P; Rifkin, DB
1986 Mar;27(2):57-57, Proceedings (American Association for Cancer Research)
— id: 31055, year: 1986, vol: 27, page: 57, stat: Journal Article,

Identification of a pituitary factor responsible for enhancement of plasminogen activator activity in breast tumor cells
Mira-y-Lopez R; Joseph-Silverstein J; Rifkin DB; Ossowski L
1986 Oct;83(20):7780-7784, Proceedings of the National Academy of Sciences of the United States of America
Sheep pituitary glands contain a protein that stimulates plasminogen activator (PA) activity 3- to 20-fold in serum-free cultures of T47D, MTW9/PL, and SC115 breast tumor cells. This protein was found to be similar to basic fibroblast growth factor (bFGF) in size, cationic nature, and affinity for heparin. Purified human placental bFGF, a homologue of human and bovine pituitary bFGF, was effective in stimulating mammary tumor cell PA at a concentration of 1 ng/ml. Antibodies to placental bFGF blocked the PA stimulatory activity of sheep pituitary extracts. Because of these properties, the active protein in sheep pituitary glands was identified as bFGF. This represents another function of bFGF, indicating that its spectrum of target cells is wider than previously thought. Because of previously established correlations between PA production in vitro and tumor growth in vivo, we suggest that bFGF may contribute to the growth of breast carcinomas in vivo. Other types of carcinomas may also be affected by bFGF
— id: 42380, year: 1986, vol: 83, page: 7780, stat: Journal Article,

Both normal and tumor cells produce basic fibroblast growth factor
Moscatelli D; Presta M; Joseph-Silverstein J; Rifkin DB
1986 Nov;129(2):273-276, Journal of cellular physiology
We have previously purified from human placenta a basic fibroblast growth factor (FGF)-like molecule which stimulates the production of plasminogen activator (PA) and collagenase, induces DNA synthesis, produces an increase in motility in cultured bovine capillary endothelial (BCE) cells, and induces angiogenesis in vivo. The ability of basic FGF to stimulate PA production in BCE cells was used as an assay for the presence of basic FGF-like molecules in extracts of both normal and tumor-derived cultured cells. The identity of the PA-stimulatory activity with basic FGF was confirmed by its high affinity for heparin and by its cross-reactivity with antibodies to human placental basic FGF. Basic FGF-like molecules were identified in eight of ten cell lines tested, and the amount of FGF-like activity present in these cells bore no relation to their origin from normal or tumor tissue. The test cells, BCE cells, had one of the highest levels of FGF-like activity, suggesting that it may have an autocrine role in these cells
— id: 25422, year: 1986, vol: 129, page: 273, stat: Journal Article,

Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis, and migration
Moscatelli D; Presta M; Rifkin DB
1986 Apr;83(7):2091-2095, Proceedings of the National Academy of Sciences of the United States of America
A protein that stimulates the production of plasminogen activator and latent collagenase in cultured bovine capillary endothelial cells has been purified 10(6)-fold from term human placenta by using a combination of heparin affinity chromatography, ion-exchange chromatography, and gel chromatography. The purified molecule has a molecular weight of 18,700 as determined by NaDodSO4/PAGE under both reducing and nonreducing conditions. The purified molecule stimulates the production of plasminogen activator and latent collagenase in a dose-dependent manner between 0.1 and 10 ng of protein/ml. The purified protein also stimulates DNA synthesis and chemotaxis in capillary endothelial cells in the same concentration range. Thus, this molecule has all of the properties predicted for an angiogenic factor
— id: 25424, year: 1986, vol: 83, page: 2091, stat: Journal Article,

Purification and biological activities of an angiogenesis factor from human placenta
Moscatelli DA; Presta M; Mignatti P; Mullins DE; Crowe RM; Rifkin DB
1986 Jul-Aug;6(4):861-863, Anticancer research
Several crude angiogenesis preparations, as well as a purified angiogenesis factor from human placenta, were tested for their ability to stimulate the production of plasminogen activator (PA) and collagenase activities, motility, chemotaxis, DNA synthesis and clonal growth in cultured endothelial cells. Treatment of capillary endothelial cells resulted in a stimulation of all the above activities, whereas endothelial cells derived from large vessels did not respond. These cellular activities are postulated to be responsible for capillary growth in vivo
— id: 25423, year: 1986, vol: 6, page: 861, stat: Journal Article,

BASIC FIBROBLAST GROWTH-FACTOR (BFGF)-LIKE MOLECULES ARE PRODUCED BY A VARIETY OF CULTURED-CELLS
MOSCATELLI, D; PRESTA, M; JOSEPHSILVERSTEIN, J; RIFKIN, DB
1986 MAY ;45(6):1718-1718, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 41448, year: 1986, vol: 45, page: 1718, stat: Journal Article,

Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration
Presta M; Moscatelli D; Joseph-Silverstein J; Rifkin DB
1986 Nov;6(11):4060-4066, Molecular & cellular biology
A 17,500-dalton protein which stimulates plasminogen activator production in cultured bovine capillary endothelial cells has been purified from a SK-Hep-1 human hepatoma cell lysate by using heparin affinity chromatography and fast protein-liquid ion exchange chromatography. The purified molecule stimulated plasminogen activator production in a dose-dependent manner between 0.01 and 1 ng/ml. It also stimulated collagenase synthesis, DNA synthesis, and motility in capillary endothelial cells in the same concentration range. This molecule was identified as a basic fibroblast growth factor-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with a polyclonal antibody raised against the human placental basic fibroblast growth factor
— id: 25421, year: 1986, vol: 6, page: 4060, stat: Journal Article,

MOLECULES CONTROLLING CELLULAR INVASION
Rifkin, DB
1986 Aug;58(8):539-539, Seikagaku
— id: 31014, year: 1986, vol: 58, page: 539, stat: Journal Article,

PROTEINS REGULATING CELLULAR INVASION
Rifkin, DB
1986 Nov;14(3):235-235, Journal of cellular biochemistry
— id: 31090, year: 1986, vol: 14, page: 235, stat: Journal Article,

IDENTIFICATION OF BFGF IN CONDITIONED MEDIA AND EXTRACTS OF CULTURED-CELLS - DEVELOPMENT OF A SOLID-PHASE RIA FOR BFGF USING ANTI-HUMAN BFGF IGG
SILVERSTEIN, JJ; MOSCATELLI, D; RIFKIN, DB
1986 NOV ;103(5):A297-A297, Journal of cell biology
— id: 41321, year: 1986, vol: 103, page: A297, stat: Journal Article,

MECHANISMS FOR REGULATING TISSUE FACTOR INITIATED COAGULATION BY CULTURED BOVINE CELLS
Bach, R; Rifkin, DB
1985 ;54(1):225-225, Thrombosis & haemostasis
— id: 30718, year: 1985, vol: 54, page: 225, stat: Journal Article,

Production of latent collagenase by human umbilical vein endothelial cells in response to angiogenic preparations
Moscatelli DA; Rifkin DB; Jaffe EA
1985 Feb;156(2):379-390, Experimental cell research
Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells
— id: 25426, year: 1985, vol: 156, page: 379, stat: Journal Article,

PURIFICATION AND BIOLOGICAL-ACTIVITIES OF AN ANGIOGENIC FACTOR FROM HUMAN-PLACENTA
Moscatelli, DA; Presta, M; Mignetti, P; Ossowski, L; Mullins, DE; Crowe, RM; Rifkin, DB
1985 ;5(6):618-618, Anticancer research
— id: 30821, year: 1985, vol: 5, page: 618, stat: Journal Article,

Purification and characterization of a low molecular mass cysteine proteinase inhibitor from human amniotic fluid
Rohrlich ST; Levy H; Rifkin DB
1985 ;180(2):239-241, Progress in clinical & biological research
— id: 42382, year: 1985, vol: 180, page: 239, stat: Journal Article,

Purification and characterization of a low molecular mass cysteine proteinase inhibitor from human amniotic fluid
Rohrlich ST; Levy H; Rifkin DB
1985 Feb;366(2):147-155, Biological chemistry Hoppe-Seyler
We have purified the human low molecular mass cysteine proteinase inhibitor in good yield from amniotic fluid, using ultrafiltration through 100-kDa and 1-kDa cut-off filters, chromatography on Ultrogel AcA 54, and affinity chromatography on alkylated papain-agarose. Approximately 1-4 mg/l of this inhibitor are present in amniotic fluid. The purified inhibitor had an apparent molecular mass of 10.5-12 kDa, as judged by its electrophoretic behavior. Amino acid analysis showed it to be rich in acidic and aliphatic residues and in cysteine. No carbohydrate side-chains could be demonstrated. The purified inhibitor inhibited papain, ficin, cathepsins B, C, and H, the cathepsin B-like enzyme from B16 melanoma cells, and a bovine chromaffin granule enkephalin-converting activity. No inhibition of Ca2-dependent neutral cysteine proteinase, serine- or metallo-proteinases was seen. Analysis of the purified inhibitor by isoelectric focusing revealed 7 major bands with pI values of 7.95, 7.0, 6.7, 6.55, 6.25, 5.5, and 5.2, all of which inhibited papain
— id: 42381, year: 1985, vol: 366, page: 147, stat: Journal Article,

GENERATION OF PLASMIN-INHIBITOR-PLASMIN COMPLEXES IN PLASMA BY TISSUE PLASMINOGEN-ACTIVATOR AND UROKINASE
HARPEL, PC; WEIL, D; LEVIN, RI; RIFKIN, DB
1984 ;14(1):67-67, Haemostasis
— id: 40843, year: 1984, vol: 14, page: 67, stat: Journal Article,

Aspirin inhibits vascular plasminogen activator activity in vivo. Studies utilizing a new assay to quantify plasminogen activator activity
Levin RI; Harpel PC; Weil D; Chang TS; Rifkin DB
1984 Aug;74(2):571-580, Journal of clinical investigation
Vascular or tissue-type plasminogen activator (TPA) is a key enzyme in physiologic fibrinolysis. To study the role of prostaglandins in modulating the synthesis and release of TPA in vivo, we prospectively studied the effect of aspirin (650 mg/d X 2) on TPA activity in 13 human subjects before and after 10 min of forearm venous occlusion. TPA activity was quantified by a newly developed enzyme-linked immunosorbent assay that both measures and differentiates between TPA and urokinase (UK)-like plasminogen activator activity. This assay is based on the observation that the concentration of alpha 2-plasmin inhibitor-plasmin complexes in Reptilase-clotted plasma increases linearly in proportion to the amount of activator added. Resting TPA activity was higher in women than in men (0.56 +/- 0.59 vs. 0.15 +/- 0.11 U/ml, P = 0.049). Venous occlusion induced an eightfold rise in TPA activity in women (to 4.5 U/ml, P = 0.006) and a 15-fold rise in men (to 2.28 U/ml, P = 0.004), whereas UK activity was not detected. Aspirin inhibited the rise in TPA activity after venous occlusion by 69% in men (P = 0.004) and 70% in women (P = 0.014). In contrast, aspirin had no effect on pre- or post-occlusion hematocrits or Factor VIII-related antigen levels. There was no correlation between plasma salicylate level and percentage inhibition of TPA. Neither exogenous aspirin (0-1 microgram/ml) nor salicylate (0-70 micrograms/ml) inhibited the generation of alpha 2-plasmin inhibitor-plasmin complexes by exogenous TPA or interfered with the assay system. We conclude that aspirin may have an antifibrinolytic effect in man that has not been previously described
— id: 57531, year: 1984, vol: 74, page: 571, stat: Journal Article,

Synthesis of collagenase and plasminogen activator by endothelial cells
Moscatelli D; Gross JL; Jaffe EA; Rifkin DB
Biology of endothelial cells Boston : Kluwer, 1984,
— id: 2766, year: 1984, vol: , page: 429, stat: Chapter,

Stimulation of motility in cultured bovine capillary endothelial cells by angiogenic preparations
Mullins DE; Rifkin DB
1984 May;119(2):247-254, Journal of cellular physiology
Several angiogenic preparations that have been shown to stimulate plasminogen activator (PA) and collagenase production by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate BCE cell motility in the phagokinetic track assay. Bovine retinal extract, medium conditioned by 3T3-F442A differentiated mouse adipocytes, SK HEP-1 human hepatoma cell lysate, mouse sarcoma 180 cell lysate, and medium conditioned by mouse sarcoma 180 cells stimulated motility 68.7%, 48.5%, 140.9%, 56.5%, and 102.1%, respectively, relative to untreated cells. The motility-stimulating activity of these preparations was dose dependent and linear over the 16-h assay period. Several hormones and growth factors were tested for BCE cell motility-stimulating activity, including insulin, vasopressin, fibroblast growth factor, and a partially purified preparation of sarcoma growth factor, and were found to be ineffective. 12-0-tetradecanoyl-phorbol-acetate (TPA), a potent stimulator of both PA and collagenase activities in BCE cells, also did not stimulate motility, indicating that protease production is not sufficient to stimulate BCE cell motility in this assay. Neither SK HEP-1 hepatoma cell lysate nor TPA was effective in stimulating motility in bovine aortic endothelial (BAE) cells. The inability of SK HEP-1 hepatoma cell lysate to stimulate movement in BAE cells is consistent with the observation that angiogenesis occurs by sprouting of capillaries, not large vessels
— id: 42383, year: 1984, vol: 119, page: 247, stat: Journal Article,

PROTEASE PRODUCTION BY CULTURED ENDOTHELIAL-CELLS
RIFKIN, D; MOSCATELLI, D
1984 ;43(3):587-587, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 41010, year: 1984, vol: 43, page: 587, stat: Journal Article,

PLASMINOGEN-ACTIVATOR PRODUCTION BY ENDOTHELIAL-CELLS EXPOSED TO ANGIOGENIC STIMULI
RIFKIN, DB; MOSCATELLI, D
1984 ;14(1):128-128, Haemostasis
— id: 40994, year: 1984, vol: 14, page: 128, stat: Journal Article,

A polypeptide secreted by transformed cells that modulates human plasminogen activator production
Davies RL; Rifkin DB; Tepper R; Miller A; Kucherlapati R
1983 Jul 8;221(4606):171-173, Science
A diffusible factor produced and secreted by malignant murine cells was capable of inducing plasminogen activator production by normal diploid human fibroblasts. The factor's ability to induce plasminogen activator was insensitive to treatment with nucleases, but its activity was destroyed by digestion with proteases. It is proposed that such a factor would play a role in malignancy if it would recruit normal cells that were adjacent to transformed cells to produce plasminogen activator which could result in tumor-promoted proteolysis
— id: 42384, year: 1983, vol: 221, page: 171, stat: Journal Article,

PURIFICATION AND CHARACTERIZATION OF A CARTILAGE DERIVED COLLAGENASE INHIBITOR
GABRIELIDES, C; RIFKIN, DB
1983 ;97(5):A460-A460, Journal of cell biology
— id: 40611, year: 1983, vol: 97, page: A460, stat: Journal Article,

Down-regulation of epidermal growth factor receptor correlates with plasminogen activator activity in human A431 epidermoid carcinoma cells
Gross JL; Krupp MN; Rifkin DB; Lane MD
1983 Apr;80(8):2276-2280, Proceedings of the National Academy of Sciences of the United States of America
Human A431 epidermoid carcinoma cells in culture exhibit epidermal growth factor (EGF)-induced 'down-regulation' of cell-surface and total cellular (Triton X-100 extractable) EGF receptors caused entirely by an enhanced rate (4-fold) of receptor inactivation [Krupp, M. N., Connolly, D. T. & Lane, M. D. (1982) J. Biol. Chem. 257, 11489-11496]. The following observations show that this enhanced rate of EGF receptor inactivation is closely correlated with an increased cellular activity of plasminogen activator (PA), a serine protease. First, EGF-induced down-regulation of cell-surface and total cellular EGF receptors and the concomitant increase in cellular PA activity occur with identical kinetics, the t 1/2 for both processes being 3-3.5 hr. Second, the EGF dose-response curves for down-regulation of total cellular EGF receptor and increased PA activity are similar. The EGF concentrations for half-maximal responses of both processes are 10-15 nM and 20 nM, respectively. Third, the removal of EGF from previously down-regulated cells results in the recovery of total cellular EGF binding activity with a concurrent loss of cellular PA activity. Fourth, blocking PA synthesis or activity with cycloheximide or dexamethasone prevents down-regulation of the EGF receptor. Fifth, the addition of leupeptin, an inhibitor of PA and plasmin action, blocks EGF-induced receptor down-regulation as well as the increase of PA activity. That EGF receptor down-regulation is independent of plasminogen per se in the culture medium suggests that PA-mediated events may initiate the rapid inactivation of the EGF receptor that occurs during down-regulation
— id: 42387, year: 1983, vol: 80, page: 2276, stat: Journal Article,

Increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro
Gross JL; Moscatelli D; Rifkin DB
1983 May;80(9):2623-2627, Proceedings of the National Academy of Sciences of the United States of America
Bovine capillary endothelial (BCE) cells produce increased amounts of the proteases plasminogen activator (PA) and latent collagenase when cultured in the presence of the following preparations which are known to contain angiogenic activities: bovine retinal extract, mouse adipocyte conditioned medium, and human hepatoma cell lysate. These preparations stimulated both BCE cell PA and collagenase activities in a dose-dependent manner. Both activities were increased to about the same level by these preparations as by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. Mitogens that are not angiogenic, such as insulin, epidermal and fibroblast growth factors, and endothelial cell growth supplement, had no effect on BCE cell PA and collagenase activities. Two of the angiogenic preparations (retinal extract and mouse adipocyte-conditioned medium) had no effect on PA activity in endothelial cells derived from bovine aortae (BAE cells). The angiogenic preparations had little (human hepatoma cell lysate, mouse adipocyte-conditioned medium) or no (bovine retinal extract) effect on BAE cell collagenase activities. In the bovine system, the induction of high levels of both PA and collagenase activities by angiogenic preparations is limited to capillary endothelial cells
— id: 27429, year: 1983, vol: 80, page: 2623, stat: Journal Article,

Plasminogen activator in differentiating mouse keratinocytes
Isseroff RR; Fusenig NE; Rifkin DB
1983 Apr;80(4):217-222, Journal of investigative dermatology
The activity of the serine protease plasminogen activator (PA) was measured in cell lysates from primary mouse keratinocyte cultures as well as from a number of established mouse keratinocyte lines. Enzyme activity was generally higher in the transformed lines than in the primary cultures; however, among the lines tested, those that expressed the highest degree of morphologic differentiation had the highest levels of cell-associated PA. In both the normal (primary) and transformed (established) keratinocyte cultures, PA activity increased when cultures reached confluence and morphologic evidence of differentiation was noted. The highest specific activity of the enzyme was found in cells shed from differentiating cultures, which consisted predominantly of detergent-resistant cornified envelopes. As the cultures differentiated and these cells were shed from the culture surface, the total cell-associated PA activity of the culture decreased accordingly. In both the normal and transformed keratinocyte cultures, peak PA activity occurred at a time when DNA synthesis was declining. These findings indicate that as keratinocytes differentiate, their intracellular levels of PA increase. The modulation of this endogenous keratinocyte enzyme may play an important, although as yet undefined, role in the normal maturation and terminal differentiation of these cells
— id: 42385, year: 1983, vol: 80, page: 217, stat: Journal Article,

Plasminogen is present in the basal layer of the epidermis
Isseroff RR; Rifkin DB
1983 Apr;80(4):297-299, Journal of investigative dermatology
This study was undertaken to determine whether plasminogen was present within the epidermis. Cryostat sections of normal human skin were incubated with either whole rabbit antihuman plasminogen serum, the IgG fraction thereof, or the immune-specific IgG fraction thereof, purified by affinity chromatography on plasminogen-Sepharose. Standard indirect immunofluorescent techniques using rhodamine-conjugated goat antirabbit IgG were subsequently employed. Fluorescence was localized to the basal layer of the epidermis. Nonimmune serum and immune serum depleted of its specific anti-plasminogen IgG by prior affinity chromatography yielded negative results, demonstrating the specificity of staining for plasminogen. Our findings indicate that plasminogen is present within the epidermis, localized to the peripheral intracytoplasmic area of basal keratinocytes. This finding supports the hypothesis that a proteolytic event mediated by plasminogen activator with the subsequent generation of plasmin may be involved in normal keratinocyte physiology
— id: 42386, year: 1983, vol: 80, page: 297, stat: Journal Article,

PRODUCTION OF A UROKINASE INHIBITOR BY HUMAN CHORIOCARCINOMA CELLS
MULLINS, DE; RIFKIN, DB
1983 ;97(5):A117-A117, Journal of cell biology
— id: 40495, year: 1983, vol: 97, page: A117, stat: Journal Article,

Proteases, angiogenesis, and invasion
Rifkin DB; Moscatelli D; Gross J; Jaffe E
1983 ;36(9):187-200, Symposium on fundamental cancer research
— id: 27430, year: 1983, vol: 36, page: 187, stat: Journal Article,

Plasminogen activator and collagenase production by cultured capillary endothelial cells
Gross JL; Moscatelli D; Jaffe EA; Rifkin DB
1982 Dec;95(3):974-981, Journal of cell biology
— id: 27434, year: 1982, vol: 95, page: 974, stat: Journal Article,

Binding of developing mouse cerebellar cells to fibronectin: a possible mechanism for the formation of the external granular layer
Hatten ME; Furie MB; Rifkin DB
1982 Sep;2(9):1195-1206, Journal of neuroscience
The role of the matrix glycoprotein fibronectin in the formation of the external granular layer of the developing mouse cerebellum was investigated by in vitro studies of the binding of cerebellar cells to a fibronectin-coated culture substratum and by in vivo immunocytochemical localization of antiplasma fibronectin antiserum in cerebellar tissue. The adhesion of cells dissociated from embryonic and early postnatal mouse cerebellum is developmental stage-specific when the cells are plated on tissue culture substrata derivatized with human plasma fibronectin. Cells dissociated from mouse cerebellum at embryonic day 13 form cellular aggregates on insoluble plasma fibronectin. In contrast, cells dissociated from embryonic day 16 through postnatal day 7 cerebellum form a monolayer. Time-lapse video recordings reveal extensive cell movement of late embryonic and early postnatal cerebellar cells on insoluble plasma fibronectin. Late embryonic and early postnatal cerebellar cells bind to fibronectin but do not degrade the fibronectin substratum. Immunocytochemical studies of the binding of antiplasma fibronectin antisera to cryostat sections of intact embryonic and early postnatal cerebellar tissue reveal a brightly stained region of endogenous fibronectin along the route of granule cell migration from the lateral caudal part of the neuroepithelium lining the fourth ventricle up onto the external surface of the cerebellar anlage. When the formation of the external granular layer is completed, the intense region of fibronectin is no longer visible
— id: 42388, year: 1982, vol: 2, page: 1195, stat: Journal Article,

Biochemistry of granule cell migration in developing mouse cerebellum
Hatten ME; Rifkin DB; Furie MB; Mason CA; Liem RK
1982 ;85 Pt B(9):509-519, Progress in clinical & biological research
— id: 42389, year: 1982, vol: 85 Pt B, page: 509, stat: Journal Article,

PLASMINOGEN IS PRESENT IN THE BASAL LAYER OF THE EPIDERMIS
Isseroff, RR; Rifkin, DB
1982 ;30(2):A605-A605, Clinical research
— id: 30563, year: 1982, vol: 30, page: A605, stat: Journal Article,

PLASMINOGEN IS PRESENT IN THE BASAL LAYER OF THE EPIDERMIS
Isseroff, RR; Rifkin, DB
1982 ;78(4):359-359, Journal of investigative dermatology
— id: 30565, year: 1982, vol: 78, page: 359, stat: Journal Article,

EGF-RECEPTOR DOWN-REGULATION IS CORRELATED WITH PLASMINOGEN-ACTIVATOR SYNTHESIS
Krupp, MN; Gross, JL; Rifkin, DB; Lane, MD
1982 ;95(2):A416-A416, Journal of cell biology
— id: 30363, year: 1982, vol: 95, page: A416, stat: Journal Article,

Phorbol esters stimulate protease production by human endothelial cells
Moscatelli D; Jaffe EA; Rifkin DB
1982 ;7(3):401-404, Carcinogenesis: a comprehensive survey
— id: 27435, year: 1982, vol: 7, page: 401, stat: Journal Article,

COLLAGENASE PRODUCTION BY HUMAN UMBILICAL VEIN ENDOTHELIAL- CELLS IN RESPONSE TO ANGIOGENIC PREPARATIONS
Moscatelli, D; Rifkin, DB; Jaffe, EA
1982 ;95(2):A206-A206, Journal of cell biology
— id: 30351, year: 1982, vol: 95, page: A206, stat: Journal Article,

STIMULATION OF MOTILITY IN BOVINE CAPILLARY ENDOTHELIAL-CELLS BY ANGIOGENIC PREPARATIONS
Mullins, DE; Moscatelli, DA; Rifkin, DB
1982 ;95(2):A187-A187, Journal of cell biology
— id: 30513, year: 1982, vol: 95, page: A187, stat: Journal Article,

COLLAGENASE AND PLASMINOGEN-ACTIVATOR PRODUCTION BY CULTURED CAPILLARY ENDOTHELIAL-CELLS
Gross, JL; Moscatelli, D; Jaffe, EA; Rifkin, DB
1981 ;91(2):A34-A34, Journal of cell biology
— id: 30402, year: 1981, vol: 91, page: A34, stat: Journal Article,

PLASMINOGEN-ACTIVATOR IN DIFFERENTIATING KERATINOCYTES
Isseroff, RR; Rifkin, DB; Fusenig, NE
1981 ;29(2):A600-A600, Clinical research
— id: 30271, year: 1981, vol: 29, page: A600, stat: Journal Article,

PLASMINOGEN-ACTIVATOR IN DIFFERENTIATING KERATINOCYTES
Isseroff, RR; Rifkin, DB; Fusenig, NE
1981 ;76(4):332-333, Journal of investigative dermatology
— id: 30258, year: 1981, vol: 76, page: 332, stat: Journal Article,

ANGIOGENIC FACTORS STIMULATE PLASMINOGEN-ACTIVATOR AND COLLAGENASE PRODUCTION BY CAPILLARY ENDOTHELIAL-CELLS
Moscatelli, D; Gross, JL; Rifkin, DB
1981 ;91(2):A201-A201, Journal of cell biology
— id: 30405, year: 1981, vol: 91, page: A201, stat: Journal Article,

The involvement of proteases and protease inhibitors in neovascularization
Rifkin DB; Gross JL; Moscatelli D; Gabrielides C
1981 ;40(10-11):1259-1263, Acta biologica & medica Germanica
Bovine capillary endothelial cells have been found to respond to several stimuli by producing increased amounts of plasminogen activator and latent collagenase. These stimulators include the tumor promoter tetradecanoyl phorbol-13-acetate as well as crude preparations from a human hepatoma, bovine retinae, and mouse adipocytes, all of which are known to contain angiogenic factors. Endothelial cells and skin fibroblasts do not respond to these stimuli in the same way, indicating a specificity of the response. In addition, inhibitors of plasmin and vertebrate collagenase have been isolated from cartilage, a tissue resistant to neovascularization. We have proposed that these specific protease inhibitors confer on cartilage its antiangiogenic properties
— id: 27436, year: 1981, vol: 40, page: 1259, stat: Journal Article,

Isolation of the major serine protease inhibitor from the 5-day serum-free conditioned medium of human embryonic lung cells and demonstration that it is fetuin
Rohrlich ST; Rifkin DB
1981 Oct;109(1):1-15, Journal of cellular physiology
Human embryonic lung (HuEL) cells in culture produce large amounts of the enzyme, plasminogen activator, and thus generate substantial amounts of active plasmin. Despite the presence of plasmin, however, HuEL cells grow in ordered, flattened, adherent sheets. It seemed of interest to characterize protease inhibitors that might be present in HuEL cultures and which might account for this apparent contradiction. This paper reports the isolation and purification of the major serine protease inhibitor in 5-day serum-free conditioned medium (CM) from HuEL cells, and the purification of an identical molecule from fetal bovine serum (FBS). Both the CM-derived inhibitor and the FBS-derived inhibitor are identical with fetuin, the major glycoprotein of FBS. The CM-derived inhibitor is apparently derived from the FBS used to supplement the growth medium of HuEL cells between serum-free CM collection periods. It is not labeled metabolically with 3H-leucine. Its electrophoretic behavior is indistinguishable from that of standard fetuin in SDS-PAGE, non-SDS basic pH,PAGE, and isoelectric focusing. The CM-derived inhibitor and standard fetuin inhibit trypsin and plasmin with similar efficiencies, but neither inhibits chymotrypsin, pancreatic elastase, or plasminogen activator. They are immunologically indistinguishable. The suggestion is made that fetuin, and possibly other protease inhibitors present in HuEL cell cultures, may be concentrated locally by HuEL cells and gradually released back into the medium in the absence of serum. These molecules may serve to protect HuEL cells against proteases they generate
— id: 42390, year: 1981, vol: 109, page: 1, stat: Journal Article,

SV40 mutants with an altered small-t protein are tumorigenic in newborn hamsters
Topp WC; Rifkin DB; Sleigh MJ
1981 Jun;111(2):341-350, Virology
— id: 42391, year: 1981, vol: 111, page: 341, stat: Journal Article,

Characterization of a temperature-sensitive membrane alteration in chick embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus
Eger, R; Rifkin, D
1980 Aug 4;600(2):313-319, Biochimica & biophysica acta
The intramembrane particles of freeze-fractured chick embryo fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (TS68) are distributed differently at the permissive and non-permissive temperatures if, and only if, the cells are treated with glycerol before fixation. Few aggregates of intramembrane particles are present in glycerol-treated cells grown at the permissive temperature for transformation (36 degrees C), while numerous large aggregates of particles are present at the non-permissive temperature (41 degrees C). Changes in the distribution of particles after cells are shifted from 36 to 41 degrees C are observed after 20 min, while a temperature shift from 41 to 26 degrees C causes changes in glycerol-induced redistributions after 1 h. The changes observed in temperature shifts from 36 to 41 degrees C and from 41 to 36 degrees D do not require protein synthesis or RNA synthesis
— id: 135297, year: 1980, vol: 600, page: 313, stat: Journal Article,

Requirement for the large T and small T proteins of SV40 in the maintenance of the transformed state
Frisque RJ; Rifkin DB; Topp WC
1980 ;44 Pt 1,(7):325-331, Cold Spring Harbor symposia on quantitative biology
— id: 42399, year: 1980, vol: 44 Pt 1,, page: 325, stat: Journal Article,

Transformation of primary hamster brain cells with JC virus and its DNA
Frisque RJ; Rifkin DB; Walker DL
1980 Jul;35(1):265-269, Journal of virology
— id: 42395, year: 1980, vol: 35, page: 265, stat: Journal Article,

Location of a gelatin-binding region of human plasma fibronectin
Furie MB; Frey AB; Rifkin DB
1980 May 25;255(10):4391-4394, Journal of biological chemistry
— id: 42396, year: 1980, vol: 255, page: 4391, stat: Journal Article,

Proteolytically derived fragments of human plasma fibronectin and their localization within the intact molecule
Furie MB; Rifkin DB
1980 Apr 10;255(7):3134-3140, Journal of biological chemistry
— id: 42397, year: 1980, vol: 255, page: 3134, stat: Journal Article,

Early mutants of polyoma virus (dl8 and dl23) with altered transformation properties: is polyoma virus middle T antigen a transforming gene product?
Griffin BE; Ito Y; Novak U; Spurr N; Dilworth S; Smolar N; Pollack R; Smith K; Rifkin DB
1980 ;44 Pt 1,(7):271-283, Cold Spring Harbor symposia on quantitative biology
— id: 42400, year: 1980, vol: 44 Pt 1,, page: 271, stat: Journal Article,

Transformation of rat embryo fibroblasts by cloned polyoma virus DNA fragments containing only part of the early region
Hassell JA; Topp WC; Rifkin DB; Moreau PE
1980 Jul;77(7):3978-3982, Proceedings of the National Academy of Sciences of the United States of America
Recombinant plasmids containing either the entire polyoma viral genome or one or the other of the two HindIII fragments of polyoma virus DNA were constructed and cloned in Escherichia coli X1776, and their DNAs were individually tested for the capacity to transform an established line of rat cells. The recombinant plasmids containing the entire polyoma genome and those containing the HindIII-1 fragment of polyoma DNA (45-1.4 map units) efficiently transform rat cells, whereas the plasmids containing the HindIII-2 fragment (1.4-45.0 map units) do not. The properties of many independent transformed cell lines established by infection with the cloned HindIII-1 fragment were determined. In contrast to the parent cell line, rat cells transformed with the cloned HindIII-1 fragment grow to high saturation densities, form colonies with high efficiency in dilute agar suspension, produce high levels of plasminogen activator, and display a disorganized arrangement of actin cables. By all criteria examined, these cells transformed by fragments are indistinguishable from cells transformed by whole polyoma viral DNA. Cellular DNA prepared from many HindIII-1 fragment-transformed cell lines was analyzed for the presence and arrangement of polyoma viral sequences by Southern blot-hybridization. In all cases examined, only those viral sequences contained within the HindIII-1 fragment of polyoma DNA were detected. These data establish a strong correlation between polyoma DNA sequences mapping within a restricted portion of the early region and the induction and maintenance of the transformed phenotype
— id: 42394, year: 1980, vol: 77, page: 3978, stat: Journal Article,

PHENOTYPE OF POLYOMA-INDUCED HAMSTER TUMOR-CELL LINES
Israel, MA; Martin, MA; Miyamura, T; Takemoto, KK; Rifkin, D; Pollack, R
1980 ;35(1):252-255, Journal of virology
— id: 27982, year: 1980, vol: 35, page: 252, stat: Journal Article,

PLASMINOGEN-ACTIVATOR IN DIFFERENTIATING KERATINOCYTES
Isseroff, RR; Rifkin, DB; Fusenig, NE
1980 ;87(2):A28-A28, Journal of cell biology
— id: 28086, year: 1980, vol: 87, page: A28, stat: Journal Article,

Tetradecanoyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells
Moscatelli D; Jaffe E; Rifkin DB
1980 Jun;20(2):343-351, Cell
— id: 27437, year: 1980, vol: 20, page: 343, stat: Journal Article,

Regulation of plasminogen activator synthesis in chick embryo fibroblasts infected with avian retroviruses
Rifkin DB
1980 ;44 Pt 1,(7):665-668, Cold Spring Harbor symposia on quantitative biology
— id: 42398, year: 1980, vol: 44 Pt 1,, page: 665, stat: Journal Article,

Studies on the control of plasminogen activator production by cultured human embryonic lung cells: requirements for inhibition by corticosteroids
Rifkin DB; Crowe RM
1980 Dec;105(3):417-422, Journal of cellular physiology
The ability of actinomycin D to interfere with the dexamethasone-mediated inhibition of plasminogen activator (PA) production by human-embryonic lung (HuEL) cells has been examined. The enzyme produced by HuEL cells in the presence of both dexamethasone and actinomycin D appears to be the product of de novo protein synthesis, as determined by the dependence of PA production on active protein synthesis and the net increase in total PA during the course of an experiment. Inhibition of RNA synthesis must be continuous to maintain PA production in the presence of dexamethasone, since reinitiation of RNA synthesis causes an immediate loss of PA activity in the cells. Cordycepin and alpha-amanitin also prevent dexamethasone-mediated inhibition of PA in HuEL cells, indicating that the RNA whose synthesis must be prevented is of the mRNA class. These experiments imply that PA production in HuEL cells may be under translational as well as transcriptional control
— id: 42392, year: 1980, vol: 105, page: 417, stat: Journal Article,

The small-t protein of SV40 is required for loss of actin cable networks and plasminogen activator synthesis in transformed rat cells
Topp WC; Rifkin DB
1980 Oct 30;106(2):282-291, Virology
— id: 42393, year: 1980, vol: 106, page: 282, stat: Journal Article,

REQUIREMENT FOR THE LARGE T-PROTEINS AND SMALL T-PROTEINS OF SV40 IN THE MAINTENANCE OF THE TRANSFORMED STATE
Frisque, RJ; Rifkin, DB; Topp, WC
1979 ;44(2):325-331, Cold Spring Harbor symposia on quantitative biology
— id: 28055, year: 1979, vol: 44, page: 325, stat: Journal Article,

PROTEOLYTICALLY-DERIVED FRAGMENTS OF HUMAN-PLASMA FIBRONECTIN AND THEIR LOCALIZATION WITHIN THE INTACT MOLECULE
Furie, MB; Rifkin, DB
1979 ;32(4):193-193, Journal of supramolecular structure
— id: 30028, year: 1979, vol: 32, page: 193, stat: Journal Article,

EARLY MUTANTS OF POLYOMA-VIRUS (DL8 AND DL23) WITH ALTERED TRANSFORMATION PROPERTIES - IS POLYOMA-VIRUS MIDDLE T-ANTIGEN A TRANSFORMING GENE-PRODUCT
Griffin, BE; Ito, Y; Novak, U; Spurr, N; Dilworth, S; Smolar, N; Pollack, R; Smith, K; Rifkin, DB
1979 ;44(2):271-283, Cold Spring Harbor symposia on quantitative biology
— id: 28054, year: 1979, vol: 44, page: 271, stat: Journal Article,

The effect of avian retroviruses on limb bud chondrogenesis in vitro
Gross JL; Rifkin DB
1979 Nov;18(3):707-718, Cell
Mesenchymal cells isolated from stage 24 embryonic chicken limb buds were infected with the temperature-sensitive transformation mutants of Rous sarcoma virus tsNY68, tsNY10 and tsLA25 at the nonpermissive temperature for transformation (41 degrees C). Virus infection greatly inhibited subsequent limb bud chondrogenesis under nontransforming conditions, as indicated by a reduction in the rate of 35SO4 incorporation into cell-associated proteoglycans. The inhibition of chondrogenesis was directly related to the percentage of cells infected with tsNY68 at 41 degrees C. The observed inhibition of chondrogenesis was independent of src gene expression since this effect was also caused by many viruses which lack the src gene, including the leukosis viruses RAV-1, RAV-2 and MAV-2(0); the src deletion mutant RSVtd107; and the reticuloendotheliosis viruses REV-T and SNV. Infection of mesenchymal cells with tsNY68 under nontransforming conditions did not cause changes in parameters such as the rate of thymidine incorporation, total cell DNA and total cell protein. Infection with tsNY68 at 41 degrees C resulted in altered kinetics of 35SO4 incorporation into cell-associated proteoglycans and a corresponding reduction in 35SO4-labeled proteoglycans extracted from the cell layer. There were no apparent quantitative effects on the rate of accumulation of proteoglycans in the culture medium. The proteoglycans extracted from the cells and the collected medium of tsNY68-infected cultures were smaller than those of uninfected cultures, as shown by agarose gel chromatography
— id: 42401, year: 1979, vol: 18, page: 707, stat: Journal Article,

Tumor promoters induce changes in the chick embryo fibroblast cytoskeleton
Rifkin DB; Crowe RM; Pollack R
1979 Oct;18(2):361-368, Cell
We have examined the effect of the tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), on the actin-containing elements of the cytoskeleton of chick embryo fibroblasts (CEF). TPA at concentrations as low as 7.3 times 10-10M indices a reversible change in the cytoskeleton as visualized by indirect immunofluorescence using anti-actin antibodies. Cells incubated with TPA lose the ordered actin-containing structures found in normal cells and resemble Rous sarcoma virus-transformed cells in that the immunofluorescent actin pattern is diffuse. The TPA effects are both dose-and time-dependent. Analogs of TPA which are inactive as tumor promoters do not induce cytoskeletal changes at the concentrations tested, while a second tumor promoter, PDD, is also able to cause alterations in actin-containing structures. The action of TPA requires de novo synthesis of both RNA and protein. The direct cytoskeletal changes are neither plasmin-dependent nor subject to inhibition by incubating the cells with high levels of protease inhibitors during the exposure to TPA. However, plasminogen does increase the sensitivity of cells to TPA
— id: 42402, year: 1979, vol: 18, page: 361, stat: Journal Article,

REGULATION OF PLASMINOGEN-ACTIVATOR SYNTHESIS IN CHICK-EMBRYO FIBROBLASTS INFECTED WITH AVIAN RETROVIRUSES
Rifkin, DB
1979 ;44(2):665-668, Cold Spring Harbor symposia on quantitative biology
— id: 28056, year: 1979, vol: 44, page: 665, stat: Journal Article,

TUMORIGENICITY OF REVERTANTS FROM AN SV40-TRANSFORMED LINE
Steinberg, BM; Rifkin, D; Shin, S; Boone, C; Pollack, R
1979 ;11(4):539-546, Journal of supramolecular structure
— id: 28076, year: 1979, vol: 11, page: 539, stat: Journal Article,

Fibronectin from chicken embryo fibroblasts contains covalently bound phosphate
Teng MH; Rifkin DB
1979 Mar;80(3):784-791, Journal of cell biology
Fibronectin isolated from cultures of chicken embryo fibroblasts (CEF) contains phosphorus linked to serine and threonine by monoester bonds. Normal and Rous sarcoma virus (RSV)-transformed cells were incubated with [32P]orthophosphate, and fibronectin was isolated from the cell surfaces and conditioned media. 32P was stably associated with fibronectin during immunoprecipitation, SDS-polyacrylamide gel electrophoresis, phospholipid solvent extraction, and hot acid but not alkaline treatment. After a limited acid hydrolysis of fibronectin, both phosphoserine and phosphothreonine were found. The specific radioactivity of the 32P-labeled fibronectin from the conditioned medium of normal CEF was higher than that from the cultures of transformed CEF
— id: 42403, year: 1979, vol: 80, page: 784, stat: Journal Article,

ORDERING OF PROTEOLYTIC FRAGMENTS OF HUMAN COLD-INSOLUBLE GLOBULIN (CIG)
Furie, M; Rifkin, DB
1978 ;79(2):A54-A54, Journal of cell biology
— id: 29870, year: 1978, vol: 79, page: A54, stat: Journal Article,

Clonal sublines of rat neurotumor RT4 and cell differentiation. II. A conversion coupling of tumorigenicity and a glial property
Imada M; Sueoka N; Rifkin DB
1978 Sep;66(1):109-116, Developmental biology (Orlando)
— id: 42405, year: 1978, vol: 66, page: 109, stat: Journal Article,

Plasminogen activator synthesis by cultured human embryonic lung cells: characterization of the suppressive effect of corticosteroids
Rifkin DB
1978 Dec;97(3 Pt 2 Suppl 1):421-428, Journal of cellular physiology
The synthesis of plasminogen activator (PA) by cultured human embryonic lung (HuEL) cells has been examined. The production of PA by these cells was found to be reversibly inhibited by physiological levels of glucocorticoids. The suppression of PA synthesis in HuEL cells was not accompanied by an inhibition of cell growth. Moreover, the glucocorticoid induced deinduction of plasminogen activator synthesis occurred in both growing and non-growing cells. The inhibition of PA production by corticosteroids appeared to have a requirement for DNA-dependent RNA synthesis since the inhibition of DNA-dependent RNA synthesis at the time of exposure of cells to corticosteroids prevented the deinduction of PA
— id: 42404, year: 1978, vol: 97, page: 421, stat: Journal Article,

The preparation and use of pyridoxal [32P]phosphate as a labeling reagent for proteins on the outer surface of membranes
Eger R; Rifkin DB
1977 Oct 3;470(1):70-83, Biochimica & biophysica acta
Pyridoxal [32P] phosphate was prepared using [gamma-32P] ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [32P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [32P] phosphate. The Schiff base formed between pyridoxal [32P] phosphate and protein amino groups was reduced with NaBH4. The distribution of pyridoxal [32P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin
— id: 42407, year: 1977, vol: 470, page: 70, stat: Journal Article,

A sensitive assay for elastase employing radioactive elastin coupled to sepharose
Rifkin DB; Crowe RM
1977 May 1;79(1-2):268-275, Analytical biochemistry
— id: 42409, year: 1977, vol: 79, page: 268, stat: Journal Article,

Isolation of a protease inhibitor from tissues resistant to tumor invasion
Rifkin DB; Crowe RM
1977 Dec;358(12):1525-1531, Hoppe-Seylers zeitschrift fur physiologische Chemie
We have purified to homogeneity the major trypsin inhibitors from both bovine cartilage and aorta, two tissues reported to be highly resistant to invasion. The two inhibitors appear to be identical and they resemble the Kunitz inhibitor with respect to molecular weight, amino acid composition, range of susceptible proteases, and antigenicity. Each of these inhibitors accounts for 100% of the antitrypsin activity found in extracts of bovine cartilage and aorta
— id: 42406, year: 1977, vol: 358, page: 1525, stat: Journal Article,

Production of plasminogen activator by established cell lines of mouse origin
Rifkin DB; Pollack R
1977 Apr;73(1):47-55, Journal of cell biology
The correlation between malignant transformation and increased plasminogen activator synthesis has been studied in a variety of established cell lines. In contrast to the behavior of secondary mouse embryo cultures, which always show increased fibrinolytic activity when transformed, no such correlation was found within the BALB/c 3T3 line and its transformed derivatives. Cell lines were established from tumors initiated in BALB/c mice by several transformed cell lines. These lines were generally found to contain no more plasminogen activator than the cells used for inoculation. A correlation was found between transformation and plasminogen activator synthesis within Swiss 3T3 cell lines. However, the correlation was not maintained by serum revertants of transformed Swiss 3T3 cells
— id: 42410, year: 1977, vol: 73, page: 47, stat: Journal Article,

Patterns of plasminogen activator production in cultured normal embryonic cells
Rohrlich ST; Rifkin DB
1977 Oct;75(1):31-42, Journal of cell biology
Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor
— id: 42408, year: 1977, vol: 75, page: 31, stat: Journal Article,

The organization of the proteins of vesicular stomatitis virions: labeling with pyridoxal phosphate
Eger R; Compans RW; Rifkin DB
1975 Aug;66(2):610-615, Virology
— id: 42412, year: 1975, vol: 66, page: 610, stat: Journal Article,

Plasma membrane alteration associated with malignant transformation in culture
Gilula NB; Eger RR; Rifkin DB
1975 Sep;72(9):3594-3598, Proceedings of the National Academy of Sciences of the United States of America
The intramembrane organization of the plasma membranes of nonmalignant cells in culture has been compared by freeze-fracturing with that of virally-transformed malignant cells. No dramatic differences are present in the distribution of intramembrane particles in the plasma membranes of these cells when the cells are examined without fixation or with mild fixation (glutaraldehyde treatment) prior to freezing. However, a redistribution of intramembrane particles into aggregates occurs in the membranes of nontransformed cells after treatment with glycerol. The aggregation of particles is extensive in normal chick embryo fibroblasts, and less extensive in mouse 3T3 cells. The glycerol-induced particle redistribution is not inhibited at 4 degrees, but it is inhibited by pretreatment with 2.5% glutaraldehyde. A significant number of the cells remain viable after the glycerol treatment, and the process is reversible. Particle aggregation does not appear to be related to either growth rate or cell density. Transformed Rous sarcoma virus/chick embryo fibroblasts and simian virus 40/3T3 cells have few particle aggregates after glycerol treatment. The plasma membranes of chick embryo fibroblasts transformed with a mutant of Rous sarcoma virus (TS-68) that is temperature sensitive for transformation, have few particle aggregates when grown at the permissive temperature (37 degrees). Extremely prominent particle aggregates are present in the plasma membranes of cells grown at the nonpermissive temperature (41 degrees). These observations indicate that there is an alteration in the plasma membrane associated with viral transformation which is related to a glycerol-sensitive mechanism that controls the distribution of intramembrane particles
— id: 42411, year: 1975, vol: 72, page: 3594, stat: Journal Article,

Proteases and biological control
Reich, Edward; Rifkin, Daniel B.; Shaw, Elliott
[Cold Spring Harbor, N.Y.] : Cold Spring Harbor Laboratory, 1975,
— id: 215, year: 1975, vol: , page: , stat: ,

Properties of plasminogen activators formed by neoplastic human cell cultures
Rifkin DB; Loeb JN; Moore G; Reich E
1974 May 1;139(5):1317-1328, Journal of experimental medicine
— id: 42413, year: 1974, vol: 139, page: 1317, stat: Journal Article,

Virus-induced modification of cellular membranes related to viral structure
Rifkin DB; Quigley JP
1974 ;28(0):325-351, Annual review of microbiology
— id: 42414, year: 1974, vol: 28, page: 325, stat: Journal Article,

Studies of Rous sarcoma virus. Effects of nucleoside analogues on virus synthesis
Brdar B; Rifkin DB; Reich E
1973 Apr 10;248(7):2397-2408, Journal of biological chemistry
— id: 42416, year: 1973, vol: 248, page: 2397, stat: Journal Article,

An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. II. Mammalian fibroblast cultures transformed by DNA and RNA tumor viruses
Ossowski L; Unkeless JC; Tobia A; Quigley JP; Rifkin DB; Reich E
1973 Jan 1;137(1):112-126, Journal of experimental medicine
— id: 42418, year: 1973, vol: 137, page: 112, stat: Journal Article,

The project-grant application of the National Institutues of Health. A beginning scientist's first project-grant application
Rifkin DB
1973 May;32(5):1543-1543, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 42415, year: 1973, vol: 32, page: 1543, stat: Journal Article,

An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. I. Chick embryo fibroblast cultures transformed by avian RNA tumor viruses
Unkeless JC; Tobia A; Ossowski L; Quigley JP; Rifkin DB; Reich E
1973 Jan 1;137(1):85-111, Journal of experimental medicine
— id: 42417, year: 1973, vol: 137, page: 85, stat: Journal Article,

Specificity of Rous sarcoma virus synthesis
Brdar B; Rifkin DB; Reich E
1972 Aug 15;24(3):347-350, FEBS letters
— id: 42354, year: 1972, vol: 24, page: 347, stat: Journal Article,

Isolation and characterization of ribonucleoprotein substructures from Rous sarcoma virus
Quigley JP; Rifkin DB; Compans RW
1972 Oct;50(1):65-75, Virology
— id: 42421, year: 1972, vol: 50, page: 65, stat: Journal Article,

Determination of phospholipid composition of RNA tumor viruses by 32 P labeling of infected cell cultures
Quigley JP; Rifkin DB; Einhorn MH
1972 Jun;47(2):614-619, Analytical biochemistry
— id: 42422, year: 1972, vol: 47, page: 614, stat: Journal Article,

Lipid studies of Rous sarcoma virus and host cell membranes
Quigley JP; Rifkin DB; Reich E
1972 Nov;50(2):550-557, Virology
— id: 42419, year: 1972, vol: 50, page: 550, stat: Journal Article,

A specific labeling procedure for proteins on the outer surface of membranes
Rifkin DB; Compans RW; Reich E
1972 Oct 25;247(20):6432-6437, Journal of biological chemistry
— id: 42420, year: 1972, vol: 247, page: 6432, stat: Journal Article,

Phospholipid composition of Rous sarcoma virus, host cell membranes and other enveloped RNA viruses
Quigley, J P; Rifkin, D B; Reich, E
1971 Oct;46(1):106-116, Virology
— id: 42423, year: 1971, vol: 46, page: 106, stat: Journal Article,

Selective lysis of cells transformed by Rous Sarcoma virus
Rifkin, D B; Reich, E
1971 Jul;45(1):172-181, Virology
— id: 42424, year: 1971, vol: 45, page: 172, stat: Journal Article,

A possible ambiguity in the coding of mouse hemoglobin
Rifkin DB; Hirsh DI; Rifkin MR; Konigsberg WK
1966 ;31(3):715-718, Cold Spring Harbor symposia on quantitative biology
— id: 42427, year: 1966, vol: 31, page: 715, stat: Journal Article,

Isolation and amino acid composition of the tryptic peptides from the beta chain of C57BL-6 mouse hemoglobin
Rifkin DB; Rifkin M; Konigsberg W
1966 Sep 26;116(1):284-292, Archives of biochemistry & biophysics. ABB
— id: 42425, year: 1966, vol: 116, page: 284, stat: Journal Article,

The presence of two major hemoglobin components in an inbred strain of mice
Rifkin DB; Rifkin MR; Konigsberg W
1966 Mar;55(3):586-592, Proceedings of the National Academy of Sciences of the United States of America
— id: 42426, year: 1966, vol: 55, page: 586, stat: Journal Article,