Paolo G Mignatti, M.D.

Protein targets of inflammatory serine proteases and cardiovascular disease
Sharony, Ram; Yu, Pey-Jen; Park, Joy; Galloway, Aubrey C; Mignatti, Paolo; Pintucci, Giuseppe
2010 ;7:45-45, Journal of inflammation (London, England)
ABSTRACT: Serine proteases are a key component of the inflammatory response as they are discharged from activated leukocytes and mast cells or generated through the coagulation cascade. Their enzymatic activity plays a major role in the body's defense mechanisms but it has also an impact on vascular homeostasis and tissue remodeling. Here we focus on the biological role of serine proteases in the context of cardiovascular disease and their mechanism(s) of action in determining specific vascular and tissue phenotypes. Protease-activated receptors (PARs) mediate serine protease effects; however, these proteases also exert a number of biological activities independent of PARs as they target specific protein substrates implicated in vascular remodeling and the development of cardiovascular disease thus controlling their activities. In this review both PAR-dependent and -independent mechanisms of action of serine proteases are discussed for their relevance to vascular homeostasis and structural/functional alterations of the cardiovascular system. The elucidation of these mechanisms will lead to a better understanding of the molecular forces that control vascular and tissue homeostasis and to effective preventative and therapeutic approaches
— id: 112200, year: 2010, vol: 7, page: 45, stat: Journal Article,

Transforming growth factor-beta 1 (TGF-beta1) induces angiogenesis through vascular endothelial growth factor (VEGF)-mediated apoptosis
Ferrari, Giovanni; Cook, Brandoch D; Terushkin, Vitaly; Pintucci, Giuseppe; Mignatti, Paolo
2009 May;219(2):449-458, Journal of cellular physiology
VEGF and TGF-beta1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF-beta1 induces apoptosis. We have previously shown that VEGF/VEGF receptor-2 (VEGFR2) signaling mediates TGF-beta1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF-mediated apoptosis is required for TGF-beta1 induction of angiogenesis. In vitro the apoptotic effect of TGF-beta1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF-beta1. Inhibition of VEGF/VEGFR2 signaling abrogates formation of cord-like structures by TGF-beta1 with an effect comparable to that of z-VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF-beta1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF-beta1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF-beta1 induction of both apoptosis and angiogenesis, an effect similar to that of z-VAD. Thus, TGF-beta1 induction of angiogenesis requires a rapid and transient apoptotic effect mediated by VEGF/VEGFR2. This novel, unexpected role of VEGF and VEGFR2 indicates VEGF-mediated apoptosis as a potential target to control angiogenesis
— id: 97035, year: 2009, vol: 219, page: 449, stat: Journal Article,

Topical Mitogen-Activated Protein Kinases Inhibition Reduces Intimal Hyperplasia in Arterialized Vein Grafts
Gulkarov, Iosif; Bohmann, Katja; Cinnante, Karma M; Pirelli, Luigi; Yu, Pey-Jen; Grau, Juan B; Pintucci, Giuseppe; Galloway, Aubrey C; Mignatti, Paolo
2009 Jun 1;154(1):150-156, Journal of surgical research
OBJECTIVE: Vein graft arterialization results in activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases-1 and -2 (ERK1/2), which have been implicated in cell proliferation, migration, and apoptosis. The goal of our study was to characterize the effect of MAPK inhibition on intimal hyperplasia (IH) in arterialized vein grafts in hypercholesterolemic rabbits. METHODS: Reversed bilateral jugular vein to common carotid artery interposition grafts were constructed in 16 New Zealand White rabbits. The veins were incubated for 30 min prior to grafting with either the synthetic ERK1/2 activation inhibitor UO126 or the control vehicle. Vein graft and control jugular vein were harvested 3 h, 1 d, and 28 d after arterialization for histological and biochemical analyses. RESULTS: Treatment with UO126 was associated with 31% reduction in mean intimal area (1.68 +/- 0.78 mm(2)versus 2.44 +/- 1.65 mm(2); mean +/- SD; P = 0.036) relative to controls. The intima-to-media ratio of UO126-treated vein grafts decreased by 29% (0.53 +/- 0.04 versus 0.74 +/- 0.06; mean +/- SD; P < 0.01) compared to controls, vehicle-treated vein grafts. There was also significant increase in apoptosis in UO126-treated vein graft medial cell layer at 1 d. CONCLUSION: Topical administration of UO126 before vein grafting significantly decreases IH in arterialized vein grafts in hypercholesterolemic rabbits. These results may have significant implications for the development of strategies aimed at blocking or reducing IH in bypass grafts. Therefore, further evaluation of this simple strategy to improve vein graft patency following coronary artery or peripheral vascular bypass surgery is warranted
— id: 96446, year: 2009, vol: 154, page: 150, stat: Journal Article,

Inhibition of smooth muscle cell migration and neointima formation in vein grafts by overexpression of matrix metalloproteinase-3
Kallenbach, Klaus; Salcher, Rolf; Heim, Albert; Karck, Matthias; Mignatti, Paolo; Haverich, Axel
2009 Mar;49(3):750-758, Journal of vascular surgery
OBJECTIVE: Saphenous vein grafts suffer from neointima formation following bypass surgery. Matrix metalloproteinases (MMPs) play important roles in this process. We examined MMP-3 for its therapeutic potential to prevent smooth muscle cell migration and neointima formation in venous bypass grafts using adenovirus-mediated gene transfer. METHODS: Human aortic smooth muscle cells (HASMC) were transduced with adenoviral vectors encoding ss-galactosidase (AVEssgal) or human MMP-3 (hMMP-3), and characterized for migration in the amniotic membrane stroma as an in vitro model of the vascular wall. Cholesterol-fed New Zealand white rabbits underwent jugular vein bypass grafting into carotid arteries. Before insertion, grafts were incubated ex vivo with either AVEssgal or hMMP-3. Transgene expression was characterized by immunohistochemistry and in situ zymography. Grafts (n = 6) were explanted after 28 days and intimal hyperplasia was quantified. RESULTS: Migration of HASMC was significantly reduced when transduced with hMMP-3 compared to controls (P < .001). Immunocytochemistry of hMMP-3 transduced venous grafts localized this protein to the intima. In situ-zymography showed increased MMP activity in the intima of hMMP-3 transfected grafts. Stenosis degree (P = .001), intima/media-ratio (P = .023) and lesion thickness (P = .003) were significantly reduced in grafts transduced with Ad.MMP-3 in comparison to controls. There was no difference inside control groups. CONCLUSION: MMP-3 overexpression inhibits formation of intimal hyperplasia in arterialized vein grafts. Adenovirus mediated gene transfer of MMP-3 may be of clinical use to prevent vein graft stenosis following bypass surgery
— id: 135230, year: 2009, vol: 49, page: 750, stat: Journal Article,

Correlation between plasma osteopontin levels and aortic valve calcification: potential insights into the pathogenesis of aortic valve calcification and stenosis
Yu, Pey-Jen; Skolnick, Adam; Ferrari, Giovanni; Heretis, Katherine; Mignatti, Paolo; Pintucci, Giuseppe; Rosenzweig, Barry; Diaz-Cartelle, Juan; Kronzon, Itzhak; Perk, Gila; Pass, Harvey I; Galloway, Aubrey C; Grossi, Eugene A; Grau, Juan B
2009 Jul;138(1):196-199, Journal of thoracic & cardiovascular surgery
OBJECTIVE: The inflammatory process of aortic stenosis involves the differentiation of aortic valve myofibroblasts into osteoblasts. Osteopontin, a proinflammatory glycoprotein, both stimulates differentiation of myofibroblasts and regulates the deposition of calcium by osteoblasts. Osteopontin levels are increased in patients with such conditions as end-stage renal disease, ectopic calcification, and autoimmune disease. We hypothesized that increased plasma osteopontin levels might be associated with the presence of aortic valve calcification and stenosis. METHODS: Venous blood from volunteers older than 65 years undergoing routine echocardiographic analysis or aortic valve surgery for aortic stenosis was collected. Plasma osteopontin levels were measured by means of enzyme-linked immunosorbent assay. The presence of aortic stenosis was defined as an aortic valve area of less than 2.0 cm(2). Aortic valve calcification was assessed by using a validated echocardiographic grading system (1, none; 2, mild; 3, moderate; 4, severe). Comparisons were performed with nonpaired t tests. RESULTS: Aortic stenosis was present in 23 patients (mean age, 78 years) and was absent in 7 patients (mean age, 72 years). Aortic valve calcification scores were 3.5 +/- 0.6 and 1.3 +/- 0.5 in patients with and without aortic stenosis, respectively (P < .001). Patients with no or mild aortic valve calcification had lower osteopontin levels compared with patients with moderate or severe aortic valve calcification (406.1 +/- 165.8 vs 629.5 +/- 227.5 ng/mL, P = .01). Similarly, patients with aortic stenosis had higher osteopontin levels compared with patients without aortic stenosis (652.2 +/- 218.7 vs 379.7 +/- 159.9 ng/mL, P < .01). CONCLUSION: Increased levels of plasma osteopontin are associated with the presence of aortic valve calcification and stenosis. These findings suggest that osteopontin might play a functional role in the pathogenesis of calcific aortic stenosis
— id: 100629, year: 2009, vol: 138, page: 196, stat: Journal Article,

TGF-beta1 induces rearrangement of FLK-1-VE-cadherin-beta-catenin complex at the adherens junction through VEGF-mediated signaling
Cook, Brandoch D; Ferrari, Giovanni; Pintucci, Giuseppe; Mignatti, Paolo
2008 Dec 15;105(6):1367-1373, Journal of cellular biochemistry
VEGF and TGF-beta1 induce angiogenesis but have opposing effects on vascular endothelial cells: VEGF promotes survival; TGF-beta1 induces apoptosis. We have previously shown that TGF-beta1 induces endothelial cell apoptosis via up-regulation of VEGF expression and activation of signaling through VEGF receptor-2 (flk-1). In context with TGF-beta1, VEGF signaling is transiently converted from a survival into an apoptotic one. VEGF promotes cell survival in part via activation of PI3K/Akt by a mechanism dependent on the formation of a multi-protein complex that includes flk-1 and the adherens junction proteins VE-cadherin and beta-catenin. Here we report that TGF-beta1 induces rearrangement of the adherens junction complex by separating flk-1 from VE-cadherin and increasing beta-catenin association with both flk-1 and VE-cadherin. This rearrangement is caused neither by changes in adherens junction mRNA or protein expression nor by post-translational modification, and requires VEGF signaling through flk-1. These results show that the adherens junction is an important regulatory component of TGF-beta1-VEGF interaction in endothelial cells
— id: 92170, year: 2008, vol: 105, page: 1367, stat: Journal Article,

Tissue inhibitor of metalloproteinases-2 binding to membrane-type 1 matrix metalloproteinase induces MAPK activation and cell growth by a non-proteolytic mechanism
D'Alessio, Silvia; Ferrari, Giovanni; Cinnante, Karma; Scheerer, William; Galloway, Aubrey C; Roses, Daniel F; Rozanov, Dmitri V; Remacle, Albert G; Oh, Eok-Soo; Shiryaev, Sergey A; Strongin, Alex Y; Pintucci, Giuseppe; Mignatti, Paolo
2008 Jan 4;283(1):87-99, Journal of biological chemistry
Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation
— id: 79292, year: 2008, vol: 283, page: 87, stat: Journal Article,

Thrombin cleaves the high molecular weight forms of basic fibroblast growth factor (FGF-2): a novel mechanism for the control of FGF-2 and thrombin activity
Yu, P-J; Ferrari, G; Pirelli, L; Galloway, A C; Mignatti, P; Pintucci, G
2008 Apr 17;27(18):2594-2601, Oncogene
The fgf-2 gene encodes low molecular weight (LMW, 18 kDa) and high molecular weight (HMW, 22-24 kDa) forms that originate from alternative translation of a single mRNA and exhibit diverse biological functions. HMW fibroblast growth factor-2 (FGF-2) inhibits cell migration and induces cell transformation or growth arrest in a cell type- and dose-dependent fashion. Conversely, LMW FGF-2 upregulates both cell proliferation and migration in most cell types. Although transcriptional and translational regulation of HMW and LMW FGF-2 has been extensively investigated, little is known about post-translational control of their relative expression. Here we report that thrombin, a key coagulation factor and inflammatory mediator, cleaves HMW FGF-2 into an LMW FGF-2-like form that stimulates endothelial cell migration and proliferation. The effect of thrombin on these cell functions requires HMW FGF-2 cleavage. This post-translational control mechanism adds a novel level of complexity to the regulation of FGF-2, and links the activities of thrombin and FGF-2 in patho-physiological processes in which both molecules are expressed
— id: 79088, year: 2008, vol: 27, page: 2594, stat: Journal Article,

TGF-beta 1 induced angiogenesis follows a time dependent sequence: apoptosis precedes cellular proliferation
Terushkin, Vitaly; Mignatti, Paolo
2007 ;1:33-33, Probe: the publication of research on biomedical endeavors
— id: 75324, year: 2007, vol: 1, page: 33, stat: Journal Article,

Basic fibroblast growth factor (FGF-2): the high molecular weight forms come of age
Yu, Pey-Jen; Ferrari, Giovanni; Galloway, Aubrey C; Mignatti, Paolo; Pintucci, Giuseppe
2007 Apr 1;100(5):1100-1108, Journal of cellular biochemistry
After over thirty years from its discovery, research on basic fibroblast growth factor (FGF-2) keeps revealing new aspects of the complexity of its gene expression as it evolved in the eukaryotic organisms. The discovery of multiple forms of FGF-2 generated by alternative translation from AUG and non-canonical CUG codons on the same mRNA transcript has led to the characterization of a low molecular weight (LMW) FGF-2 form and various high molecular weight (HMW) forms (four in humans). In this review, we discuss the biochemical features and biological activities of the different FGF-2 forms. In particular, we focus on the properties that are unique to the HMW forms and its biological functions
— id: 72034, year: 2007, vol: 100, page: 1100, stat: Journal Article,

Vascular injury and modulation of MAPKs: A targeted approach to therapy of restenosis
Yu, Pey-Jen; Ferrari, Giovanni; Pirelli, Luigi; Gulkarov, Iosif; Galloway, Aubrey C; Mignatti, Paolo; Pintucci, Giuseppe
2007 Jul;19(7):1359-1371, Cellular signalling
Cardiovascular interventions that restore blood circulation to ischemic areas are accompanied by significant tissue damage, which triggers a vascular remodeling response that may result in restenosis of blood conduits. Early endothelial dysfunction and/or impairment is the early event of a cascade that leads, through an inflammatory response and dedifferentiation of medial smooth muscle cells with abundant deposition of extracellular matrix, to intimal hyperplasia. Here we present the molecular and cellular mechanisms of intimal hyperplasia secondary to vascular injury and discuss the potential role of therapeutic modulation of the intracellular signaling pathways that differentially effect vascular endothelial and smooth muscle cells. The role of mitogen-activated protein kinases (MAPKs) and the outcome of their modulation in these processes are highlighted here as they provide a promising therapeutic target for prevention of restenosis
— id: 72033, year: 2007, vol: 19, page: 1359, stat: Journal Article,

VEGF, a prosurvival factor, acts in concert with TGF-beta1 to induce endothelial cell apoptosis
Ferrari, Giovanni; Pintucci, Giuseppe; Seghezzi, Graziano; Hyman, Kevin; Galloway, Aubrey C; Mignatti, Paolo
2006 Nov 14;103(46):17260-17265, Proceedings of the National Academy of Sciences of the United States of America
VEGF and TGF-beta1 are potent angiogenesis inducers with opposing effects on endothelial cells. TGF-beta1 induces apoptosis; VEGF protects endothelial cells from apoptosis. We found that TGF-beta1 promotes endothelial cell expression of FGF-2, which up-regulates VEGF synthesis. Inhibition of VEGF signaling through VEGF receptor 2 (flk-1) abrogates TGF-beta1-induced apoptosis and p38(MAPK) activation. Inhibition of p38(MAPK) blocks TGF-beta1-induced apoptosis, showing that VEGF/flk-1-mediated activation of p38(MAPK) is required for TGF-beta1 induction of apoptosis. In the absence of TGF-beta1, VEGF activates p38(MAPK) and promotes endothelial cell survival. However, in context with TGF-beta1, VEGF/flk-1-mediated activation of p38(MAPK) results in apoptosis. Thus, cross-talk between TGF-beta1 and VEGF signaling converts VEGF/flk-1-activated p38(MAPK) into a proapoptotic signal. This finding illustrates an unexpected role of VEGF and indicates that VEGF can be pharmacologically converted into an apoptotic factor, a novel approach to antiangiogenesis therapy
— id: 69698, year: 2006, vol: 103, page: 17260, stat: Journal Article,

Mechanisms of c-reactive protein up-regulation in arterialized vein grafts
Gulkarov, Iosif; Pintucci, Giuseppe; Bohmann, Katja; Saunders, Paul C; Sullivan, Raymond F; Ferrari, Giovanni; Mignatti, Paolo; Galloway, Aubrey C
2006 Feb;139(2):254-262, Surgery
BACKGROUND: C-reactive protein (CRP), an acute phase reactant, is an independent predictor of coronary artery syndromes and a mediator of the vascular response to injury. CRP has been found in arterialized vein grafts and has been linked to atherogenesis; however, its involvement in vein graft early failure or intimal hyperplasia has not been assessed. This study was designed to investigate the mechanism(s) of CRP up-regulation in arterialized vein grafts. METHODS: Carotid artery bypass with arterialized jugular vein grafts (AVG) was performed in 18 dogs. AVG were harvested at 3, 8, and 24 hours and 4, 14, and 28 days, using the femoral vein obtained at the time of AVG harvest as a control. Serum CRP levels were characterized by enzyme-linked immunosorbent assay; AVG expression of CRP was studied by immunofluorescence, Western blotting, in situ hybridization, Northern blotting, and quantitative RT-PCR. RESULTS: CRP levels peaked at 24 hours in serum and AVG but remained at baseline in control veins. By double immunofluorescence, CRP was associated with the media and adventitia of AVG. However, Northern blotting analysis showed no CRP mRNA expression in AVG. Reverse transcriptase polymerase chain reaction analysis confirmed the lack of up-regulation of CRP in AVG. CONCLUSION: CRP levels are increased in AVG, peaking 24 hours after arterialization. However, no significant production of CRP was detected in AVG. Therefore, increased CRP levels within AVG appear to originate mostly from CRP diffusion from the systemic circulation. These results have significant implications for the development of strategies aimed at blocking CRP up-regulation in bypass grafts
— id: 72035, year: 2006, vol: 139, page: 254, stat: Journal Article,

Anti-proliferative and anti-inflammatory effects of topical MAPK inhibition in arterialized vein grafts
Pintucci, Giuseppe; Saunders, Paul C; Gulkarov, Iosif; Sharony, Ram; Kadian-Dodov, Daniella L; Bohmann, Katja; Baumann, F Gregory; Galloway, Aubrey C; Mignatti, Paolo
2006 Feb;20(2):398-400, FASEB journal
Vein graft failure following bypass surgery is a frequent and important clinical problem. The vascular injury caused by arterialization is responsible for vein graft intimal hyperplasia, a lesion generated by medial smooth muscle cell proliferation and migration into the intima, increased extracellular matrix deposition, and formation of a thick neointima. Development of the neointima into a typical atherosclerotic lesion and consequent stenosis ultimately result in vein graft failure. Endothelial damage, inflammation, and intracellular signaling through mitogen-activated protein kinases (MAPKs) have been implicated in the early stages of this process. We therefore investigated the effects of topical inhibition of ERK-1/2 MAPK activation on vascular cell proliferation and apoptosis, and on the inflammatory response in a canine model of vein graft arterialization. For this purpose, vein grafts were incubated with the MEK-1/2 inhibitor, UO126, ex vivo for 30 min before grafting. This treatment effectively abolished arterialization-induced ERK-1/2 activation, decreased medial cell proliferation, and increased apoptosis. UO126 treatment also inhibited the vein graft infiltration by myeloperoxidase-positive inflammatory cells that follows vein graft arterialization. Thus, topical ex vivo administration of MAPK inhibitors can provide a pharmacological tool to prevent or reduce the vascular cell responses that lead to vein graft intimal hyperplasia and graft failure
— id: 62809, year: 2006, vol: 20, page: 398, stat: Journal Article,

Inhibition of mitogen-activated protein kinases (MAPKs) as a strategy to prevent intimal hyperplasia following cardiovascular interventions
Pirelli L; Yu P-J; Gulkarov I; Galloway AC; Mignatti P; Pintucci G
2006 ;3(3):173-183, Vascular Disease Prevention
Upon injury, blood vessels undergo a significant remodeling characterized by intimal damage and dedifferentiation of medial smooth muscle cells. Normally quiescent medial cells lose their contractile phenotype and begin to proliferate, migrate, and secrete abundant extracellular matrix. The resulting neointima formation, also referred to as intimal hyperplasia, precedes atherosclerosis of the vascular conduits. Restenosis greatly limits the success of percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass grafting (CABG), two common procedures widely used to restore circulation in occluded vascular districts. Growth factors, cytokines, inflammatory mediators, and oxidative and shear stress are among the culprits that initiate this process. More recent studies have been directed towards the intracellular sensors of these stimuli in the hope of discovering the common mechanisms that control the response to injury. A group of enzymes called mitogen-activated protein kinases (MAPKs) play a central role in relaying extracellular stimuli to the cellular core, the nucleus. The discovery that MAPK intracellular signaling pathways control processes as diverse as cell proliferation, migration, and survival via fine modulation of gene expression has prompted a number of studies on MAPK involvement in the response to vascular injury. Here we review the studies that characterized MAPK activation upon arterial or vein graft injury and its involvement in vascular remodeling. The experimental findings indicate that the MAPK signaling pathways are suitable targets for novel therapies to prevent restenosis of blood conduits and extend their life span. copyright 2006 Bentham Science Publishers Ltd
— id: 67520, year: 2006, vol: 3, page: 173, stat: Journal Article,

Matrix metalloproteinase expression in vein grafts: role of inflammatory mediators and extracellular signal-regulated kinases-1 and -2
Sharony, Ram; Pintucci, Giuseppe; Saunders, Paul C; Grossi, Eugene A; Baumann, F Gregory; Galloway, Aubrey C; Mignatti, Paolo
2006 Apr;290(4):H1651-H1659, American journal of physiology. Heart & circulatory physiology
Matrix metalloproteinases (MMPs) play key roles in vascular remodeling. We characterized the role of inflammatory mediators and extracellular signal-regulated kinases (ERKs) in the control of arterialized vein graft expression of MMP-9, MMP-2, and membrane-type 1-MMP (MT1-MMP) and of the tissue inhibitor of metalloproteinases-2 (TIMP-2). For this purpose we used a canine model of jugular vein to carotid artery interposition graft and analyzed the vein grafts at various postoperative times (30 min to 28 days) using the contralateral vein as a control. To study the role of ERK-1/2, veins were incubated with the mitogen-activated protein kinase kinase (MEK-1/2) inhibitor UO126 for 30 min before being grafted. Vein graft extracts were analyzed for MMPs, TIMP-2, tumor necrosis factor-alpha (TNF-alpha), polymorphonuclear neutrophil (PMN) infiltration, myeloperoxidase (MPO), and thrombin activity, and for ERK-1/2 activation. Vein graft arterialization resulted in rapid and sustained (8 h to 28 days) upregulation of vein graft-associated MMP-9, MMP-2, MT1-MMP, thrombin activity, and TNF-alpha levels with concomitant TIMP-2 downregulation. MMP-2 activation preceded MT1-MMP upregulation. PMN infiltration and vein graft-associated MPO activity increased within hours after arterialization, indicating a prompt, local inflammatory response. In cultured smooth muscle cells, both thrombin and TNF-alpha upregulated MT1-MMP expression; however, only thrombin activated MMP-2. Inhibition of ERK-1/2 activation blocked arterialization-induced upregulation of MMP-2, MMP-9, and MT1-MMP. Thus, thrombin, inflammatory mediators, and activation of the ERK-1/2 pathway control MMP and TIMP-2 expression in arterialized vein grafts
— id: 72036, year: 2006, vol: 290, page: H1651, stat: Journal Article,

Proteases and extracellular environment
Del Rosso, M; Fibbi, G; Schmitt, M; Mignatti, P
2005 FEB ;93(2):190-191, Thrombosis & haemostasis
— id: 48670, year: 2005, vol: 93, page: 190, stat: Journal Article,

PDGF-BB induces vascular smooth muscle cell expression of high molecular weight FGF-2, which accumulates in the nucleus
Pintucci, Giuseppe; Yu, Pey-Jen; Saponara, Fiorella; Kadian-Dodov, Daniella L; Galloway, Aubrey C; Mignatti, Paolo
2005 Aug 15;95(6):1292-1300, Journal of cellular biochemistry
Basic fibroblast growth factor (FGF-2) and platelet-derived growth factor (PDGF) are implicated in vascular remodeling secondary to injury. Both growth factors control vascular endothelial and smooth muscle cell proliferation, migration, and survival through overlapping intracellular signaling pathways. In vascular smooth muscle cells PDGF-BB induces FGF-2 expression. However, the effect of PDGF on the different forms of FGF-2 has not been elucidated. Here, we report that treatment of vascular aortic smooth muscle cells with PDGF-BB rapidly induces expression of 20.5 and 21 kDa, high molecular weight (HMW) FGF-2 that accumulates in the nucleus and nucleolus. Conversely, PDGF treatment has little or no effect on 18 kDa, low-molecular weight FGF-2 expression. PDGF-BB-induced upregulation of HMW FGF-2 expression is controlled by sustained activation of extracellular signal-regulated kinase (ERK)-1/2 and is abolished by actinomycin D. These data describe a novel interaction between PDGF-BB and FGF-2, and indicate that the nuclear forms of FGF-2 may mediate the effect of PDGF activity on vascular smooth muscle cells. (c) 2005 Wiley-Liss, Inc
— id: 56377, year: 2005, vol: 95, page: 1292, stat: Journal Article,

Vein graft arterialization causes differential activation of mitogen-activated protein kinases
Saunders, Paul C; Pintucci, Giuseppe; Bizekis, Costas S; Sharony, Ram; Hyman, Kevin M; Saponara, Fiorella; Baumann, F Gregory; Grossi, Eugene A; Colvin, Stephen B; Mignatti, Paolo; Galloway, Aubrey C
2004 May;127(5):1276-1284, Journal of thoracic & cardiovascular surgery
OBJECTIVE: Vascular injury results in activation of the mitogen-activated protein kinases-extracellular-signal regulated kinases, c-jun N-terminal kinase, and p38(MAPK)-which have been implicated in cell proliferation, migration, and apoptosis. The goal of this study was to characterize mitogen-activated protein kinase activation in arterialized vein grafts. METHODS: Carotid artery bypass using reversed external jugular vein was performed in 29 dogs. Vein grafts were harvested after 30 minutes and 3, 8, and 24 hours, and 4, 7, 14, and 28 days. Contralateral external jugular vein and external jugular vein interposition vein-to-vein grafts were used as controls. Vein graft extracts were analyzed for extracellular-signal regulated kinases, c-jun N-terminal kinase, and p38(MAPK) activation. Proliferating cell nuclear antigen expression was investigated as a parameter of cell proliferation. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling staining and intimal hyperplasia by morphometric examination of tissue sections. RESULTS: Significant intimal hyperplasia was observed at 28 days. Over the time points studied, vein graft arterialization resulted in bimodal activation of both extracellular-signal regulated kinase and p38(MAPK) (30 minutes through 3 hours; 4 days) but did not induce activation of c-jun N-terminal kinase. Proliferating cell nuclear antigen expression increased from days 1 through 28, and apoptosis increased between 8 and 24 hours. CONCLUSION: Vein graft arterialization induces bimodal activation of extracellular-signal regulated kinase and p38(MAPK); however, in contrast with what is described in arterial injury, it does not induce c-jun N-terminal kinase activation. These results provide the first comprehensive characterization of the mitogen-activated protein kinase signaling pathways activated in vein graft arterialization and identify mitogen-activated protein kinases as potential mediators of vein graft remodeling and subsequent intimal hyperplasia
— id: 45314, year: 2004, vol: 127, page: 1276, stat: Journal Article,

Activation of mitogen-activated protein kinases during preparation of vein grafts and modulation by a synthetic inhibitor
Bizekis, Costas; Pintucci, Giuseppe; Derivaux, Christopher C; Saponara, Fiorella; Kim, Jin-Hee; Hyman, Kevin M; Sharony, Ram; Grossi, Eugene A; Baumann, F Gregory; Mignatti, Paolo; Galloway, Aubrey C
2003 Sep;126(3):659-665, Journal of thoracic & cardiovascular surgery
OBJECTIVE: Long-term durability of saphenous vein grafts used for coronary artery bypass grafting is limited by neointimal formation. Arterial vascular injury is known to activate intracellular mitogen-activated protein kinases, including extracellular signal-regulated kinases and c-jun N-terminal kinases, that affect cell differentiation, proliferation, migration, and apoptosis. This study tests the hypothesis that these mitogen-activated protein kinases are activated in saphenous veins during preparation for coronary artery bypass grafting. METHODS: Saphenous veins were harvested from 10 patients undergoing coronary artery bypass grafting. A specimen from each vein was placed in ice-cold lysis buffer immediately after harvesting (t = 0). The remaining tissue was incubated at room temperature in normal saline, 0.1% dimethylsulfoxide (vehicle), or 50 mmol/L PD98059 (mitogen-activated protein kinase kinase-1/2 inhibitor) until the vein was grafted (mean 50 minutes). To study kinetics of intracellular signaling pathways, canine saphenous veins were harvested, and mitogen-activated protein kinases and PI-3 kinase pathways were studied after different incubation time intervals. Extracted proteins were analyzed by Western blotting or in vitro kinase assay. RESULTS: The human saphenous veins showed elevated levels of active extracellular signal-regulated kinase after harvesting (t = 0) and prior to implant (t = 1). Incubation with PD98059 resulted in decreased activation of extracellular signal-regulated kinase. Kinetics of canine saphenous veins showed extracellular signal-regulated kinase and c-jun N-terminal kinase activation, in a time-dependent manner, along with activation of the growth factor-regulated PI3 kinase pathway. CONCLUSIONS: This study characterizes activation of extracellular signal-regulated kinases and c-jun N-terminal kinases during vein graft preparation and demonstrates the ability to inhibit extracellular signal-regulated kinase activation by simple incubation with a specific inhibitor. Further studies are needed to evaluate the significance of these findings with respect to graft durability
— id: 39061, year: 2003, vol: 126, page: 659, stat: Journal Article,

A quantitative in vitro model of smooth muscle cell migration through the arterial wall using the human amniotic membrane
Kallenbach, Klaus; Fernandez, Harold A; Seghezzi, Graziano; Baumann, F Gregory; Patel, Sundeep; Grossi, Eugene A; Galloway, Aubrey C; Mignatti, Paolo
2003 Jun 1;23(6):1008-1013, Arteriosclerosis, thrombosis, & vascular biology
OBJECTIVE: The development of intimal hyperplasia involves smooth muscle cell (SMC) migration into the intima and proliferation. Matrix metalloproteinases and their tissue inhibitors play important roles in this process. In this study, we describe a novel in vitro model for studying SMC migration through the vessel wall. METHODS AND RESULTS: Human aortic SMCs (hASMCs) labeled with 125I-iododeoxyuridine or unlabeled were grown on the stromal aspect of the human amniotic membrane. Mechanical damage to endothelial cells grown on the basement membrane and addition of growth factors or platelets were characterized for their effect on SMC migration into the stroma both by histological methods and by measuring the radioactivity associated with the membrane after removal of noninvasive SMCs. To assess the reliability of the model, the cells were infected with a recombinant adenovirus encoding the tissue inhibitor of metalloproteinase-1 (TIMP-1). Addition of a platelet-derived growth factor gradient stimulated hASMC infiltration into the stroma. This effect was abolished with TIMP-1-transduced hASMC, confirming that TIMP-1 overexpression blocks SMC invasion of the stroma. CONCLUSIONS: This in vitro model of SMC migration in the vessel wall provides an inexpensive, quantitative, and reliable tool to study the molecular and cellular mechanisms of intimal hyperplasia
— id: 39254, year: 2003, vol: 23, page: 1008, stat: Journal Article,

Induction of stromelysin-1 (MMP-3) by fibroblast growth factor-2 (FGF-2) in FGF-2-/- microvascular endothelial cells requires prolonged activation of extracellular signal-regulated kinases-1 and -2 (ERK-1/2)
Pintucci, Giuseppe; Yu, Pey-Jen; Sharony, Ram; Baumann, F Gregory; Saponara, Fiorella; Frasca, Antonio; Galloway, Aubrey C; Moscatelli, David; Mignatti, Paolo
2003 Dec 1;90(5):1015-1025, Journal of cellular biochemistry
Basic fibroblast growth factor (FGF-2) and matrix metalloproteinases (MMPs) play key roles in vascular remodeling. Because FGF-2 controls a number of proteolytic activities in various cell types, we tested its effect on vascular endothelial cell expression of MMP-3 (stromelysin-1), a broad-spectrum proteinase implicated in coronary atherosclerosis. Endothelial cells (EC) from FGF-2-/- mice are highly responsive to exogenous FGF-2 and were therefore used for this study. The results showed that treatment of microvascular EC with human recombinant FGF-2 results in strong induction of MMP-3 mRNA and protein expression. Upregulation of MMP-3 mRNA by FGF-2 requires de novo protein synthesis and activation of the ERK-1/2 pathway. FGF-2 concentrations (5-10 ng/ml) that induce rapid and prolonged (24 h) activation of ERK-1/2 upregulate MMP-3 expression. In contrast, lower concentrations (1-2 ng/ml) that induce robust but transient (<8 h) ERK-1/2 activation are ineffective. Inhibition of ERK-1/2 activation at different times (-0.5 h to +8 h) of EC treatment with effective FGF-2 concentrations blocks MMP-3 upregulation. Thus, FGF-2 induces EC expression of MMP-3 with a threshold dose effect that requires sustained activation of the ERK-1/2 pathway. Because FGF-2 controls other EC functions with a linear dose effect, these features indicate a unique role of MMP-3 in vascular remodeling
— id: 44759, year: 2003, vol: 90, page: 1015, stat: Journal Article,

Shedding of membrane vesicles mediates fibroblast growth factor-2 release from cells
Taverna, Simona; Ghersi, Giulio; Ginestra, Angela; Rigogliuso, Salvatrice; Pecorella, Sonia; Alaimo, Giovanna; Saladino, Francesca; Dolo, Vincenza; Dell'Era, Patrizia; Pavan, Antonio; Pizzolanti, Giuseppe; Mignatti, Paolo; Presta, Marco; Vittorelli, Maria Letizia
2003 Dec 19;278(51):51911-51919, Journal of biological chemistry
Fibroblast growth factor-2 (FGF-2), a polypeptide with regulatory activity on cell growth and differentiation, lacks a conventional secretory signal sequence, and its mechanism of release from cells remains unclear. We characterized the role of extracellular vesicle shedding in FGF-2 release. Viable cells released membrane vesicles in the presence of serum. However, in serum-free medium vesicle shedding was dramatically down-regulated, and the cells did not release FGF-2 activity into their conditioned medium. Addition of serum to serum-starved cells rapidly induced intracellular FGF-2 clustering under the plasma membrane and into granules that colocalized with patches of the cell membrane with typical features of shed vesicle membranes. Shed vesicles carried three FGF-2 isoforms (18, 22, 24 kDa). Addition of vesicles to endothelial cells stimulated chemotaxis and urokinase plasminogen activator production, which were blocked by anti-FGF-2 antibodies. Treatment of intact vesicles with 2.0 m NaCl or heparinase, which release FGF-2 from membrane-bound proteoglycans, did not abolish their stimulatory effect on endothelial cells, indicating that FGF-2 is carried inside vesicles. The comparison of the stimulatory effects of shed vesicles and vesicle-free conditioned medium showed that vesicles represent a major reservoir of FGF-2. Thus, FGF-2 can be released from cells through vesicle shedding
— id: 134708, year: 2003, vol: 278, page: 51911, stat: Journal Article,

Trophic effects of platelets on cultured endothelial cells are mediated by platelet-associated fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF)
Green, D; Rafii, S; Froum, S; Pinnell, J; Mignatti, P; Pintucci, G
2002 NOV 16 ;100(11):681A-681A, Blood
— id: 37109, year: 2002, vol: 100, page: 681A, stat: Journal Article,

Transforming growth factor-beta1 induces apoptosis in vascular endothelial cells by activation of mitogen-activated protein kinase
Hyman, Kevin M; Seghezzi, Graziano; Pintucci, Giuseppe; Stellari, Giulia; Kim, Jee Hyun; Grossi, Eugene A; Galloway, Aubrey C; Mignatti, Paolo
2002 Aug;132(2):173-179, Surgery
BACKGROUND: Vascular endothelial cell apoptosis is central in atherosclerosis and intimal hyperplasia. Transforming growth factor (TGF)-beta1 induces endothelial cell apoptosis through unidentified mechanism(s). Although TGF-beta1 signals through the Smad proteins, in some nonendothelial cell types it also activates the mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK [p38(MAPK)]). p38(MAPK) relays apoptotic signals in several cell types. We hypothesized that TGF-beta1 activates endothelial cell MAPKs and induces apoptosis through p38(MAPK) activation. METHODS: Human umbilical vein or bovine capillary endothelial cells were incubated with TGF-beta1 for 0.5 to 12 hours. MAPK activation was characterized by Western blotting with antibodies to phosphorylated extracellular signal-regulated kinase 1/2, p38(MAPK), or c-Jun N-terminal kinases 1/2. To study apoptosis, extracts of cells incubated with TGF-beta1 for 6 hours with or without MAPK inhibitors were characterized by Western blotting analysis of poly (ADP-Ribose) polymerase degradation. RESULTS: TGF-beta1 induced p38(MAPK), extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase 1/2 activation and increased apoptosis. Inhibition of p38(MAPK) significantly reduced TGF-beta1-induced apoptosis. In contrast, inhibition of other signaling pathways was ineffective. CONCLUSIONS: TGF-beta1 induces endothelial cell apoptosis through p38(MAPK) activation. Because TGF-beta1 is upregulated in vascular remodeling, p38(MAPK) is a potential target to prevent endothelial cell apoptosis during this process
— id: 33331, year: 2002, vol: 132, page: 173, stat: Journal Article,

Increased membrane type 1 matrix metalloproteinase expression from adenoma to colon cancer: a possible mechanism of neoplastic progression
Malhotra, Sandeep; Newman, Elliot; Eisenberg, David; Scholes, John; Wieczorek, Rosemary; Mignatti, Paolo; Shamamian, Peter
2002 Apr;45(4):537-543, Diseases of the colon & rectum
PURPOSE: Membrane type 1 matrix metalloproteinase is a membrane-associated matrix metalloproteinase central to the degradation of basement membrane components via the activation of matrix metalloproteinase-2. Although membrane type 1 matrix metalloproteinase is overexpressed in invasive colon cancer, its expression in colonic polyps and carcinoma in situ has not been defined. In addition, the association of membrane type 1 matrix metalloproteinase expression by a primary tumor and recurrence of colon cancers has not been examined. METHODS: Immunoperoxidase staining was performed on randomly selected specimens containing adenoma (n = 17), carcinoma in situ (n = 9), or metastatic colon carcinoma (n = 8) with mouse monoclonal antibody to human membrane type 1 matrix metalloproteinase. Similar staining was also performed on randomly selected node-negative colon cancers that recurred within five years of resection (n = 17), matched for age, gender, stage, grade, and vascular, lymphatic, and perineural invasion, and node-negative colon cancers that did not recur within five years of resection (n = 17). Staining for membrane type 1 matrix metalloproteinase was graded. Mean scores for the groups were compared by Wilcoxon test. RESULTS: We found a progressive and significant increase in the mean score of membrane type 1 matrix metalloproteinase from normal mucosa to adenoma (P < 0.001), carcinoma in situ (P < 0.006), and invasive cancer (P < 0.009). However, there was no difference in membrane type 1 matrix metalloproteinase expression between the recurrent and nonrecurrent groups of node-negative colon cancer (P = not significant). CONCLUSIONS: These data suggest that membrane type 1 matrix metalloproteinase expression increases with progression from normal mucosa to invasive adenocarcinoma; however, it cannot be used as a prognostic indicator on which adjuvant therapy is based in node-negative colon cancer because of its failure to predict recurrence in this patient group
— id: 27560, year: 2002, vol: 45, page: 537, stat: Journal Article,

Plasmin activates pro-matrix metalloproteinase-2 with a membrane-type 1 matrix metalloproteinase-dependent mechanism
Monea, S; Lehti, K; Keski-Oja, J; Mignatti, P
2002 AUG ;192(2):160-170, Journal of cellular physiology
Membrane-type 1 matrix metalloproteinase (MT1-MMP) has been implicated as a physiological activator of progelatinase A (MMP-2). We previously reported that plasmin treatment of cells results in proMMP-2 activation and increased type IV collagen degradation. Here, we analysed the role of MT1-MMP in plasmin activation of MMP-2 using HT-1080 cells transfected with MT1-MMP sense or antisense cDNA. Control, vector-transfected cells that expressed endogenous MT1-MMP, and antisense cDNA transfectants with very low levels of MT1-MMP did not activate proMMP-2. Conversely, cells transfected with sense MT1-MMP cDNA expressed high MT1-MMP levels and processed proMMP-2 to 68/66-kDa intermediate activation products. Control cells and MT1-MMP transfectants had much higher levels of cell-associated MMP-2 than antisense cDNA transfectants. Addition of plasmin(ogen) to control or MT1-MMP-transfected cells generated active, 62-kDa MMP-2, but was ineffective with antisense cDNA transfectants. The effect of plasmin(ogen) was prevented by inhibitors of plasmin, but not by metalloproteinase inhibitors, implicating plasmin as a mechanism for proMMP-2 activation independent of the activity of MT1-MMP or other MMPs. Plasmin-mediated activation of proMMP-2 did not result from processing of proMT1-MMP and did not correlate with alpha(v)beta(3) integrin or TIMP-2 levels. Thus, plasmin can activate proMMP-2 only in the presence of MT1-MMP; however, this process does not require the catalytic activity of MT1-MMP. (C) 2002 Wiley-Liss, Inc
— id: 55301, year: 2002, vol: 192, page: 160, stat: Journal Article,

Trophic effects of platelets on cultured endothelial cells are mediated by platelet-associated fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF)
Pintucci, Giuseppe; Froum, Scott; Pinnell, Jared; Mignatti, Paolo; Rafii, Shahin; Green, David
2002 Nov;88(5):834-842, Thrombosis & haemostasis
In addition to their role in primary hemostasis, platelets serve to support and maintain the vascular endothelium. Platelets contain numerous growth factors including the potent angiogenic inducers VEGF and FGF-2. To characterize the function of these two platelet-associated growth factors, the effects of the addition of purified platelets to cultured endothelial cells were examined. The survival and proliferation of endothelial cells were markedly stimulated (2-3-fold and 5-15-fold respectively) by the addition of gel-filtered platelets. Acetylsalicylic acid-treated or lyophilized fixed-platelets were ineffective in supporting endothelial cell proliferation. In Transwell assays, the stimulatory effect of platelets on endothelial cells was preserved, consistent with an effect mediated by secreted factors. The combined inhibition of VEGF and FGF-2 by neutralizing antibodies, in contrast to inhibition of either alone, abrogated both platelet-induced endothelial cell survival and proliferation. FGF-2 isoforms were detected in platelet lysates, as well as in the releases of agonist-stimulated platelets. Megakaryocytes generated by ex vivo expansion of hematopoietic progenitor cells with kit ligand and thrombopoietin were analyzed for expression of FGF-2. Punctate cytoplasmic staining but no nuclear staining was observed by immunocytochemistry consistent with possible localization of the growth factor to cytoplasmic granules. The addition of platelets to cultured endothelial cells activated extracellular signal-regulated kinase (ERK) in a dose and time-dependent manner. This effect was abrogated by both anti-FGF-2 and anti-VEGF antibody. Since FGF-2 and VEGF are potent angiogenic factors and known endothelial cell survival factors, their release by platelets provides a plausible mechanism for the platelet support of vascular endothelium
— id: 97036, year: 2002, vol: 88, page: 834, stat: Journal Article,

Lack of ERK activation and cell migration in FGF-2-deficient endothelial cells
Pintucci, Giuseppe; Moscatelli, David; Saponara, Fiorella; Biernacki, Peter R; Baumann, F Gregory; Bizekis, Costas; Galloway, Aubrey C; Basilico, Claudio; Mignatti, Paolo
2002 Apr;16(6):598-600, FASEB journal
The formation of blood capillaries from preexisting vessels (angiogenesis) and vascular remodeling secondary to atherosclerosis or vessel injury are characterized by endothelial cell migration and proliferation. Numerous growth factors control these cell functions. Basic fibroblast growth factor (FGF-2), a potent angiogenesis inducer, stimulates endothelial cell proliferation, migration, and proteinase production in vitro and in vivo. However, mice genetically deficient in FGF-2 have no apparent vascular defects. We have observed that endothelial cell migration in response to mechanical damage in vitro is accompanied by activation of the extracellular signal-regulated kinase (ERK) pathway, which can be blocked by neutralizing anti-FGF-2 antibodies. Endothelial cells from mice that are genetically deficient in FGF-2 neither migrate nor activate ERK in response to mechanical wounding. Addition of exogenous FGF-2 restores a normal cell response, which shows that impaired migration results from the genetic deficiency of this growth factor. Injury-induced ERK activation in endothelial cells occurs only at the edge of the wound. In addition, FGF-2-induced ERK activation mediates endothelial cell migration in response to wounding without a significant effect on proliferation. These data show that FGF-2 is a key regulator of endothelial cell migration during wound repair
— id: 34522, year: 2002, vol: 16, page: 598, stat: Journal Article,

Trophic effect of platelets on cultured endothelial cells is mediated by FGF-2 and VEGF
Pintucci, G; Froum, S; Rafii, S; Mignatti, P; Green, D
2001 NOV 16 ;98(11):56B-56B, Blood
— id: 55348, year: 2001, vol: 98, page: 56B, stat: Journal Article,

Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis
Shamamian P; Schwartz JD; Pocock BJ; Monea S; Whiting D; Marcus SG; Mignatti P
2001 Nov;189(2):197-206, Journal of cellular physiology
Gelatinase A (MMP-2), a matrix metalloproteinase (MMP) involved in tumor invasion and angiogenesis, is secreted as an inactive zymogen (proMMP-2) and activated by proteolytic cleavage. Here we report that polymorphonuclear neutrophil (PMN)-derived elastase, cathepsin G, and proteinase-3 activate proMMP-2 through a mechanism that requires membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Immunoprecipitation of human PMN-conditioned medium with a mixture of antibodies to elastase, cathepsin G, and proteinase-3 abolished proMMP-2 activation, whereas individual antibodies were ineffective. Incubation of HT1080 cells with either purified PMN elastase or cathepsin G or proteinase-3 resulted in dose-and time-dependent proMMP-2 activation. Addition of PMN-conditioned medium to MT1-MMP expressing cells resulted in increased proMMP-2 activation and in vitro invasion of extracellular matrix (ECM), but had no effect with cells that express no MT1-MMP. MMP-2 activation by PMN-conditioned medium or purified elastase was blocked by the elastase inhibitor alpha(1)-antitrypsin but not by Batimastat, an MMP inhibitor, showing that elastase activation of MMP-2 is not mediated by MMP activities. The PMN-conditioned medium-induced increase in cell invasion was blocked by Batimastat as well as by alpha(1)-antitrypsin, showing that PMN serine proteinases trigger a proteinase cascade that entails proMMP-2 activation: this gelatinase is the downstream effector of the proinvasive activity of PMN proteinases. These findings indicate a novel role for PMN-mediated inflammation in a variety of tissue remodeling processes including tumor invasion and angiogenesis
— id: 26640, year: 2001, vol: 189, page: 197, stat: Journal Article,

Depletion of polymorphonuclear neutrophis inhibits angiogenesis in vivo
Chuang N; Shapiro RL; Mignatti P; Roses DF; Shamamian P
2000 ;61:246-248, Surgical forum
— id: 25207, year: 2000, vol: 61, page: 246, stat: Journal Article,

Monoclonal antibody to vascular endothelial-cadherin is a potent inhibitor of angiogenesis, tumor growth, and metastasis
Liao, F; Li, YW; O'Connor, W; Zanetta, L; Bassi, R; Santiago, A; Overholser, J; Hooper, A; Mignatti, P; Dejana, E; Hicklin, DJ; Bohlen, P
2000 DEC 15 ;60(24):6805-6810, Cancer research
Vascular endothelial-cadherin (VE-cad) is an endothelial cell-specific adhesion molecule that is crucial for proper assembly of vascular tubes. Here we show that a monoclonal antibody (BV13) directed to the extracellular region of VE-cad inhibits formation of adherens junctions and capillary-like structures by endothelial cells and blocks angiogenesis In the mouse cornea and in Matrigel plugs in vivo. Systemic administration of BV13 markedly decreases the growth of s.c. Lewis lung or human A431 epidermoid tumors and strongly suppresses the growth of Lewis lung metastases. These data demonstrate that VE-cad is essential for postnatal angiogenesis and thus validate VE-cad as a novel target for antiangiogenesis agents
— id: 55256, year: 2000, vol: 60, page: 6805, 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,

Transforming growth factor beta 1 (TGF-b1) induces a growth factor cascade that results in extracellular-regulated kinase (ERK) and p38 mitogen-activated protein kinase activation in endothelial cells
Seghezzi, G; Pintucci, G; Yun, J; Steinberg, BM; Ferdinand, B; Grossi, EA; Baumann, FG; Colvin, SB; Galloway, AC; Mignatti, P
2000 FEB ;35(2):296A-296A, Journal of the American College of Cardiology
— id: 33427, year: 2000, vol: 35, page: 296A, stat: Journal Article,

Neutrophil-derived serine proteinases enhance membrane type-1 matrix metalloproteinase-dependent tumor cell invasion
Shamamian P; Pocock BJ; Schwartz JD; Monea S; Chuang N; Whiting D; Marcus SG; Galloway AC; Mignatti P
2000 Feb;127(2):142-147, Surgery
BACKGROUND: Matrix metalloproteinase-2 degrades a variety of basement membrane components and is essential for tumor invasion. We have previously reported that membrane type-1 matrix metalloproteinase (MT1-MMP) cooperates with neutrophil-derived serine proteinases (NDPs; elastase, cathepsin G, protease-3) to activate matrix metalloproteinase-2. We therefore hypothesized that NDPs enhance tumor-cell invasion. METHODS: Clones of human HT1080 fibrosarcoma cells transfected with MT1-MMP sense (HT-SE) or antisense CDNA (HT-AS) were used. These cells express either high (HT-SE) or extremely low levels (HT-AS) of MT1-MMP relative to nontransfected HT1080 cells (HT-WT). The cells were incubated in the presence or absence of purified NDP, with or without alpha 1-antitrypsin or the MMP inhibitor batimastat. Cell invasion was measured with the use of Boyden chambers with polycarbonate membranes coated with a reconstituted extracellular matrix. RESULTS: Under control conditions HT-WT and HT-SE cells were 4-fold more invasive than HT-AS cells. The addition of NDP increased HT-WT and HT-SE cell invasion 60% to 100% but had no effect on HT-AS cells. alpha 1-antitrypsin or batimastat did not decrease the baseline invasiveness of HT-WT and HT-SE cells; however, they abrogated the stimulatory effect of NDP. CONCLUSIONS: HT1080 cell invasion depends on MT1-MMP expression. MT1-MMP overexpression does not increase invasiveness by itself. NDPs increase invasion by MT1-MMP expressing cells by activating matrix metalloproteinase-2
— id: 9013, year: 2000, vol: 127, page: 142, stat: Journal Article,

Expression of Von Willebrand factor, an endothelial cell marker, is up-regulated by angiogenesis factors: a potential method for objective assessment of tumor angiogenesis
Zanetta L; Marcus SG; Vasile J; Dobryansky M; Cohen H; Eng K; Shamamian P; Mignatti P
2000 Jan 15;85(2):281-288, International journal of cancer
von Willebrand factor (vWF), a glycoprotein produced uniquely by endothelial cells and megakaryocytes, is routinely used to identify vessels in tissue sections. Vessel density in tumor specimens, as determined by immuno-histochemical staining for vWF or other endothelial cell markers, is a negative prognostic factor for many solid tumors. vWF is heterogeneously distributed throughout the vasculature, transcriptional control in response to the tissue microenvironment being responsible for local variations in endothelial cell levels of vWF. Here, we report that fibroblast growth factor-2 and vascular endothelial growth factor, potent angiogenesis inducers expressed in a variety of tumors, up-regulate expression of vWF mRNA and protein in cultured endothelial cells with a synergistic effect. Our data support the measurement of vWF mRNA in tumors to detect activated endothelium or angiogenesis. For this purpose, we developed a semi-quantitative RT-PCR for vWF mRNA. Preliminary results obtained with specimens from colon carcinoma and the corresponding normal colonic mucosa showed higher vWF mRNA levels in most tumors than in their normal counterparts. The differences in vWF mRNA levels were much larger than the differences in vessel counts between a tumor and the corresponding normal mucosa, indicating that high vWF mRNA levels in tumors may indeed be an early sign of activation of the endothelium. The rapidity, objectivity, sensitivity and specificity of this technique make it suitable for routine clinical application to identify aggressive, highly angiogenic tumors.
— id: 9014, year: 2000, vol: 85, page: 281, stat: Journal Article,

Inhibition of endothelial cell migration by gene transfer of tissue inhibitor of metalloproteinases-1
Fernandez HA; Kallenbach K; Seghezzi G; Grossi E; Colvin S; Schneider R; Mignatti P; Galloway A
1999 Apr;82(2):156-162, Journal of surgical research
BACKGROUND. Angiogenesis requires degradation of the vessel's basal lamina and endothelial cell migration into the tissue stroma. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play important roles in this process. MMP activity is tightly regulated during vessel growth. This work was designed to characterize the effect of TIMP-1 upregulation on endothelial cell invasion of the extracellular matrix. METHODS. We constructed replication-deficient recombinant adenoviruses that encode either TIMP-1 (Ad.TIMP-1) or Escherichia coli lac Z (Ad.beta gal) cDNA. Bovine aortic endothelial (BAE) cells were infected with 100 infectious particles/cell. Gene expression was assessed by Northern and Western blotting. TIMP-1 activity in cell-conditioned media was measured by a resorufin-labeled casein protease assay. BAE cell migration was measured by Boyden chamber assays with 0.2% gelatin-coated, 8. 0-mcm polycarbonate membranes. RESULTS. TIMP-1 was overexpressed by Ad.TIMP-1-infected BAE cells relative to control, Ad. beta gal-infected or uninfected cells. TIMP-1 activity in Ad.TIMP-1 cell-conditioned medium was 2.8-fold higher than in control cells. By Boyden chamber assays with gelatin-coated membranes, Ad. TIMP-1-infected BAE cells showed 89.97 +/-1.64% (mean +/- SEM) reduction in migration relative to Ad.beta gal-infected cells (P < 0. 02) and 90.53 +/- 1.12% relative to uninfected cells (P < 0.02). Without gelatin coating, migration was equivalent in all groups. CONCLUSION. The replication-deficient recombinant adenovirus we constructed affords rapid and efficient upregulation of functional TIMP-1 in endothelial cells. Infection results in a dramatic decrease in cell migration and invasion of extracellular matrix. Thus, such a recombinant vector may provide a useful tool for the gene therapy of vascular remodeling and inhibition of angiogenesis.
— id: 12038, year: 1999, vol: 82, page: 156, stat: Journal Article,

Roles of MT1-MMP in the regulation of cell surface proteolysis
Monea S; Roberts B; Marcus SG; Shamamian P; Mignatti P
1999 Jun 30;878:703-706, Annals of the New York Academy of Sciences
— id: 9016, year: 1999, vol: 878, page: 703, stat: Journal Article,

Mechanical endothelial damage results in basic fibroblast growth factor-mediated activation of extracellular signal-regulated kinases
Pintucci G; Steinberg BM; Seghezzi G; Yun J; Apazidis A; Baumann FG; Grossi EA; Colvin SB; Mignatti P; Galloway AC
1999 Aug;126(2):422-427, Surgery
BACKGROUND: Endothelial damage, such as that associated with balloon angioplasty or preparation of veins for bypass grafts, results in intimal hyperplasia. Growth factors and cytokines that modulate endothelial cell functions through various intracellular signaling pathways mediate rapid endothelial repair, which may prevent or reduce restenosis. Here we investigated the effect of mechanical injury of endothelial cells on the mitogen-activated kinase signaling pathways, extracellular-signal-regulated kinases (ERKs), C-Jun N-terminal kinase (JNK/SAPK), and p38. METHODS: Confluent human umbilical vein endothelial cells or bovine aortic endothelial cells were wounded with a razor blade; mitogen-activated kinase activation was monitored by immunoblotting with antibodies to active ERK, JNK/SAPK, or p38. RESULTS: Wounding of human umbilical vein endothelial cell or bovine aortic endothelial cell monolayers resulted in rapid (5-minute) activation of ERK-1 and -2, which was abolished by monoclonal antibody to basic fibroblast growth factor (FGF-2). This antibody or an inhibitor of ERK activation, PD98059, also blocked endothelial cell migration after the wounding. Thus FGF-2-induced ERK activation mediates the endothelial response to wounding. CONCLUSIONS: ERK-1 and -2 are activated by FGF-2 released from endothelial cells in response to injury. Therapeutic strategies aimed at reducing FGF-2-induced intimal hyperplasia should preserve ERK activation in endothelial cells while abolishing it in smooth muscle cells
— id: 8488, year: 1999, vol: 126, page: 422, stat: Journal Article,

Modulation of matrix metalloproteinase activity in human saphenous vein grafts using adenovirus-mediated gene transfer
Fernandez HA; Kallenbach K; Seghezzi G; Mehrara B; Apazidis A; Baumann FG; Grossi EA; Colvin S; Mignatti P; Galloway AC
1998 Aug;124(2):129-136, Surgery
BACKGROUND: Neointima formation after human saphenous vein grafting (hSVG) involves several matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). This study assessed the feasibility of modulating MMP activity in hSVGs by adenovirus-mediated gene transfer. METHODS: First, 1 x 10(9) plaque-forming units (pfu) of replication-deficient recombinant adenoviruses encoding either beta-galactosidase (ad beta gal), MMP-3 (AdMMP-3), or TIMP-1 (AdTIMP-1) were added into the lumen of hSVGs for 1 hour. After incubation at 37 degrees C for 24 hours, specimens were analyzed by immunohistochemistry, in situ zymography, and X-gal staining. RESULTS: By X-gal staining ad beta gal-infected hSVGs stained positively in the intima and occasionally in the media. Immunohistochemistry of AdMMP-3- and AdTIMP-1-infected hSVGs localized these proteins to the intima. In situ zymography showed increased MMP activity in the intima of AdMMP-3-infected hSVGs relative to AdTIMP-1- or Ad beta gal-infected vessels. CONCLUSIONS: MMP-3 and TIMP activity can be regulated in hSVGs by replication-deficient recombinant adenoviruses. We have previously demonstrated that MMP-3 or TIMP-1 transduction, or both, inhibit SMC migration in an in vitro reconstituted vessel wall. Modulation of MMP activity may thus afford high patency rates in genetically engineered hSVGs. However, adenovirus-mediated gene delivery is limited to the vessel's intima; strategies to infect medial smooth muscle cells need to be developed
— id: 7562, year: 1998, vol: 124, page: 129, stat: Journal Article,

Roles of MT1-MMP in the regulation of cell surface proteolysis
Monea, S; Roberts, B; Marcus, S; Shamamian, P; Mignatti, P
1998 NOV ;9(11):175A-175A, Molecular biology of the cell
— id: 53644, year: 1998, vol: 9, page: 175A, stat: Journal Article,

Matrix metalloproteinase (MMP) 2 and 9 activity in experimental acute pancreatitis
Patel, S; Schwartz, J; Chaung, N; Marcus, SG; Pachter, HL; Deutsch, E; Galloway, AC; Eng, K; Mignatti, P; Shamamian, P
1998 APR 15 ;114(4):A1416-A1416, Gastroenterology
— id: 53478, year: 1998, vol: 114, page: A1416, 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,

Soluble factor(s) released from neutrophils activates endothelial cell matrix metalloproteinase-2
Schwartz JD; Monea S; Marcus SG; Patel S; Eng K; Galloway AC; Mignatti P; Shamamian P
1998 Apr;76(1):79-85, Journal of surgical research
OBJECTIVE: Polymorphonuclear leukocyte (PMN) infiltration and microvascular injury are hallmarks of the tissue remodeling associated with multiple organ failure. These processes require the concerted action of various proteolytic enzymes, including serine and matrix metalloproteinases (MMPs). Matrix metalloproteinase-2 (MMP-2) plays an important role in the turnover of various ECM components, including type IV collagen, fibronectin, and gelatins. Like all MMPs, MMP-2 is secreted as an inactive zymogen (proMMP-2) and activated extracellularly by limited proteolytic cleavage. The physiologic mechanism(s) of proMMP-2 activation remains unclear. This study was designed to characterize the effect of PMNs on the activation of proMMP-2 produced by endothelial cells. METHODS: PMNs and human umbilical vein endothelial cells (HUVECs) were grown either separately or together for 2-16 h. To evaluate the role of cell-cell contact, cocultures were also established in which the two cell types were separated by a semipermeable polycarbonate membrane. Alternatively, PMN-conditioned medium was added to HUVEC cultures with or without various proteinase inhibitors (aprotinin, 1,10-phenanthroline, Batimastat, E-64, eglin c peptide, or pepstatin A). After incubation, the culture supernatants were analyzed by gelatin zymography to characterize the gelatinases. RESULTS: HUVECs produce MMP-2 in its inactive (72 kDa) form. PMNs produce high levels of MMP-9 (gelatinase B, 92 kDa) but no MMP-2. Coculture of PMNs with or addition of PMN-conditioned medium to HUVECs results in the production of active (62 kDa) MMP-2. ProMMP-2 activation by PMN-conditioned medium is not blocked by inhibitors of plasmin, cysteine-, acid-, or metalloproteinases. CONCLUSION: PMNs release a soluble factor that activates endothelial cell MMP-2 through a novel mechanism independent of cell-cell contact and not attributable to the activities of plasmin, cysteine-, acid-, or metalloproteinases. These findings may provide insight into the tissue remodeling that accompanies PMN-mediated microvascular injury
— id: 9018, year: 1998, vol: 76, page: 79, stat: Journal Article,

Activation of tumor cell matrix metalloproteinase-2 by neutrophil proteinases requires expression of membrane-type 1 matrix metalloproteinase
Schwartz JD; Shamamian P; Monea S; Whiting D; Marcus SG; Galloway AC; Mignatti P
1998 Aug;124(2):232-238, Surgery
BACKGROUND: Matrix metalloproteinase-2 (MMP-2), an enzyme involved in tumor invasion, is secreted as an inactive proenzyme and requires interaction with membrane-type 1 MMP (MT1-MMP) for activation. We have previously demonstrated that polymorphonuclear neutrophils (PMNs) release a soluble factor(s) that activates pro-MMP-2. Therefore, we tested the hypothesis that PMN-derived proteinases act in concert with MT1-MMP to activate pro-MMP-2. METHODS: Human HT-1080 cells transfected with MT1-MMP cDNA (HT-SE) or the corresponding antisense cDNA (HT-AS) or an empty vector (HT-V), which expressed differing levels of MT1-MMP, were incubated with serum-free, human PMN-conditioned medium with or without proteinase inhibitors. The culture supernatants were analyzed by gelatin zymography. RESULTS: Ht-1080 cells expressing basal (HT-V) or low levels (HT-AS) of MT1-MMP secreted MMP-2 in proenzyme from (72 kd). Ht-1080 cells with high levels of MT1-MMP (HT-SE) secreted pro MMP-2 and a 68 kd intermediate activation product. Addition of PMN-conditioned medium to either HT-SE or HT-V clones resulted in dose-dependent generation of active, 62 kd MMP-2. In contrast, when PMN-conditioned medium was added to HT-AS clones, no MMP-2 activation occurred. CONCLUSIONS: PMN-derived serine proteinases act in concert with MT1-MMP to activate proMMP-2. This finding indicates a potential role for inflammatory cells in promoting extracellular matrix breakdown during tumor invasion
— id: 9017, year: 1998, vol: 124, page: 232, 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,

Urokinase plasminogen activator and gelatinases are associated with membrane vesicles shed by human HT1080 fibrosarcoma cells
Ginestra A; Monea S; Seghezzi G; Dolo V; Nagase H; Mignatti P; Vittorelli ML
1997 Jul 4;272(27):17216-17222, Journal of biological chemistry
Membrane vesicles are shed by tumor cells both in vivo and in vitro. Although their functions are not well understood, it has been proposed that they may play multiple roles in tumor progression. We characterized membrane vesicles from human HT1080 fibrosarcoma cell cultures for the presence of proteinases involved in tumor invasion. By gelatin zymography and Western blotting, these vesicles showed major bands corresponding to the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2) and to the MMP-9. tissue inhibitor of metalloproteinase 1 complex. Both gelatinases appeared to be associated with the vesicle membrane. HT1080 cell vesicles also showed a strong, plasminogen-dependent fibrinolytic activity in 125I fibrin assays; this activity was associated with urokinase plasminogen activator, as shown by casein zymography and Western blotting. Urokinase was bound to its high affinity receptor on the vesicle membrane. Addition of plasminogen resulted in activation of the progelatinases associated with the vesicles, indicating a role of the urokinase-plasmin system in MMP-2 and MMP-9 activation. We propose that vesicles shed by tumor cells may provide a large membrane surface for the activation of membrane-associated proteinases involved in extracellular matrix degradation and tissue invasion
— id: 8328, year: 1997, vol: 272, page: 17216, stat: Journal Article,

Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants
Mazzieri R; Masiero L; Zanetta L; Monea S; Onisto M; Garbisa S; Mignatti P
1997 May 1;16(9):2319-2332, EMBO journal
The urokinase-type plasminogen activator (uPA) and the matrix-degrading metalloproteinases MMP-2 and MMP-9 (type IV collagenases/gelatinases) have been implicated in a variety of invasive processes, including tumor invasion, metastasis and angiogenesis. MMP-2 and MMP-9 are secreted in the form of inactive zymogens that are activated extracellularly, a fundamental process for the control of their activity. The physiological mechanism(s) of gelatinase activation are still poorly understood; their comprehension may provide tools to control cell invasion. The data reported in this paper show multiple roles of the uPA-plasmin system in the control of gelatinase activity: (i) both gelatinases are associated with the cell surface; binding of uPA and plasmin(ogen) to the cell surface results in gelatinase activation without the action of other metallo- or acid proteinases; (ii) inhibition of uPA or plasminogen binding to the cell surface blocks gelatinase activation; (iii) in soluble phase plasmin degrades both gelatinases; and (iv) gelatinase activation and degradation occur in a dose- and time-dependent manner in the presence of physiological plasminogen and uPA concentrations. Thus, the uPA-plasmin system may represent a physiological mechanism for the control of gelatinase activity
— id: 8327, year: 1997, vol: 16, page: 2319, stat: Journal Article,

Requirement for plasmin and membrane type I matrix metalloproteinase in the cell surface activation of gelatinase A (MMP-2)
Monea, S; Lehti, K; Schwartz, J; Shamamian, P; Marcus, S; Galloway, AC; KeskiOja, J; Mignatti, P
1997 NOV ;8(5):434-434, Molecular biology of the cell
— id: 53159, year: 1997, vol: 8, page: 434, stat: Journal Article,

Activation of endothelial or tumor cell progelatinase A (MMP-2) by human polymorphonuclear neutrophils (PMN)
Schwartz, JD; Monea, S; Shamamian, P; Marcus, SG; Whiting, D; Galloway, AC; Mignatti, P
1997 NOV ;8(5):435-435, Molecular biology of the cell
— id: 53160, year: 1997, vol: 8, page: 435, 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,

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,

Vesicles shed by tumor cells possess membrane-bound urokinase plasminogen activator and type IV collagenase
Monea, S; Ginestra, A; Seghezzi, G; Vittorelli, ML; Mignatti, P
1996 DEC ;7(3):352-352, Molecular biology of the cell
— id: 53348, year: 1996, vol: 7, page: 352, stat: Journal Article,

Tumor cell-conditioned medium stimulates expression of the urokinase receptor in vascular endothelial cells
Seghezzi, G; Marelli, R; Mandriota, S J; Nolli, M L; Mazzieri, R; Mignatti, P
1996 Nov;169(2):300-308, Journal of cellular physiology
We have previously reported that culture medium conditioned by human SK-Hep1 hepatoma cells or mouse S180 sarcoma cells induces in vitro angiogenesis and stimulates production of urokinase plasminogen activator (uPA) in vascular endothelial cells. These activities are mediated by a 3.5-10 kDa, heparin-binding peptide that upregulates endothelial cell expression of basic fibroblast growth factor (bFGF; Peverali et al., 1994, J. Cell. Physiol. 161:1-14.) We now report that SK-Hep 1 or S180 cell-conditioned medium rapidly induces a 4- to 5-fold increase in cell-bound uPA activity and in the high-affinity binding of 125I-prouPA to vascular endothelial cells. Ligand blotting and purification experiments show an equivalent increase in the synthesis of a cell surface protein corresponding to the endothelial cell uPA receptor (uPAR) on the basis of M, (45-50 kDa) and sensitivity to phosphatidylinositol-specific phospholipase C (PI-PLC). The tumor cell-conditioned media also upregulate uPAR mRNA levels in endothelial cells. Thus, the increase in uPA binding capacity of endothelial cells is mediated by an increased expression of uPAR. The uPAR-inducing activity of SK-Hep 1 or S180 cell-conditioned medium is not neutralized by antibodies to bFGF, and is associated with a peptide that has a M, higher than 10 kDa and no affinity for heparin. Therefore, it appears to be distinct from the bFGF/uPA-inducing factor secreted by the same cells, and from other heparin-binding cytokines that upregulate uPAR expression in endothelial cells
— id: 133234, year: 1996, vol: 169, page: 300, 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,

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,

Vascular endothelial growth factor increases urokinase receptor expression in vascular endothelial cells
Mandriota SJ; Seghezzi G; Vassalli JD; Ferrara N; Wasi S; Mazzieri R; Mignatti P; Pepper MS
1995 Apr 28;270(17):9709-9716, Journal of biological chemistry
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and endothelial cell-specific mitogen that stimulates urokinase-type plasminogen activator (uPA) activity in vascular endothelial cells. Here, we report that VEGF increases the high affinity binding of uPA to the same cells and that this binding is prevented by a peptide corresponding to the uPA receptor (uPAR) binding growth factor-like domain of uPA. Ligand cross-linking, ligand blotting, and uPA-Sepharose affinity chromatography revealed an increase in a cell surface uPA binding protein that corresponds to the uPAR on the basis of its affinity for uPA, M(r) of 50,000-55,000, and phosphatidylinositol-specific phospholipase C sensitivity. By Scatchard analysis, VEGF increased the number of uPAR molecules by 2.8-3.5-fold and concomitantly decreased their affinity for uPA. By northern blotting uPAR mRNA was increased in a dose- and time-dependent manner in response to VEGF. Taken together, these findings demonstrate that VEGF-induced angiogenesis is accompanied by increased uPAR expression and uPA activity on the endothelial cell surface. These observations are consistent with the notion that the uPA-uPAR interaction facilitates cellular invasion
— id: 8441, year: 1995, vol: 270, page: 9709, 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,

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,

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,

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,

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,

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,

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,

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,

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,

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,

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,

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,