Thorsten Kirsch, Ph.D.

Stem cells in orthopaedics and fracture healing
Alwattar, Basil J; Schwarzkopf, Ran; Kirsch, Thorsten
2011 ;69(1):6-10, Bulletin of the NYU Hospital for Joint Diseases
Stem cell application is a burgeoning field of medicine that is likely to influence the future of orthopaedic surgery. Stem cells are associated with great promise and great controversy. For the orthopaedic surgeon, stem cells may change the way that orthopaedic surgery is practiced and the overall approach of the treatment of musculoskeletal disease. Stem cells may change the field of orthopaedics from a field dominated by surgical replacements and reconstructions to a field of regeneration and prevention. This review will introduce the basic concepts of stem cells pertinent to the orthopaedic surgeon and proceed with a more in depth discussion of current developments in the study of stem cells in fracture healing
— id: 133180, year: 2011, vol: 69, page: 6, stat: Journal Article,

The role of pyrophosphate/phosphate homeostasis in terminal differentiation and apoptosis of growth plate chondrocytes
Kim, Hyon Jong; Delaney, John D; Kirsch, Thorsten
2010 Sep;47(3):657-665, Bone
Extracellular inorganic phosphate (P(i)) concentrations are the highest in the growth plate just before the onset of mineralization. The study reported here demonstrates that P(i) not only is required for hydroxyapatite mineral formation but also modulates terminal differentiation and apoptosis of growth plate chondrocytes. Extracellular P(i) stimulated terminal differentiation marker gene expression, including the progressive ankylosis gene (ank), alkaline phosphatase (APase), matrix metalloproteinase-13 (MMP-13), osteocalcin, and runx2, mineralization, and apoptosis of growth plate chondrocytes. The stimulatory effect of extracellular P(i) on terminal differentiation and apoptosis events of growth plate chondrocytes was dependent on the concentration, the expression levels of type III Na(+)/P(i) cotransporters, and ultimately P(i) uptake. A high extracellular P(i) concentration was required for the stimulation of apoptosis, whereas lower P(i) concentrations were required for the most effective stimulation of terminal differentiation events, including terminal differentiation marker gene expression and mineralization. Suppression of Pit-1 was sufficient to inhibit the stimulatory effects of extracellular P(i) on terminal differentiation events. On the other hand, increasing the local extracellular P(i) concentration by overexpressing ANK, a protein transporting intracellular PP(i) to the extracellular milieu where it is hydrolyzed to P(i) in the presence of APase, resulted in marked increases of hypertrophic and early terminal differentiation marker mRNA levels, including APase, runx2 and type X collagen, and slight increase of MMP-13 mRNA levels, but decreased osteocalcin mRNA level, a late terminal differentiation markers. In the presence of levamisole, a specific APase inhibitor to prevent hydrolysis of extracellular PP(i) to P(i), ANK overexpression of growth plate chondrocytes resulted in decreased mRNA levels of hypertrophic and terminal differentiation markers but increased MMP-13 mRNA levels. In conclusion, with extracellular PP(i) inhibiting and extracellular P(i) stimulating hypertrophic and terminal differentiation events, a precise regulation of PP(i)/P(i) homeostasis is required for the spatial and temporal control of terminal differentiation events of growth plate chondrocytes
— id: 112036, year: 2010, vol: 47, page: 657, stat: Journal Article,

Progressive ankylosis protein (ANK) in osteoblasts and osteoclasts controls bone formation and bone remodeling
Kim, Hyon Jong; Minashima, Takeshi; McCarthy, Edward F; Winkles, Jeffrey A; Kirsch, Thorsten
2010 Aug;25(8):1771-1783, Journal of bone & mineral research
The progressive ankylosis gene (ank) encodes a transmembrane protein that transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu. ank/ank mice, which express a truncated nonfunctional ANK, showed a markedly reduced bone mass, bone-formation rate, and number of tartrate-resistant acid phosphatase-positive (TRAP(+)) multinucleated osteoclasts. ANK function deficiency suppressed osteoblastic differentiation of ank/ank bone marrow stromal cells, as indicated by the decrease in the expression of bone marker genes, including osterix, reduced alkaline phosphatase activity, and mineralization. Runx2 gene expression levels were not altered. Conversely, overexpression of ANK in the preosteoblastic cell line MC3T3-E1 resulted in increased expression of bone marker genes, including osterix. Whereas runx2 expression was not altered in ANK-overexpressing MC3T3-E1 cells, runx2 transcriptional activity was increased. Extracellular PP(i) or P(i) stimulated osteoblastogenic differentiation of MC3T3-E1 cells or partially rescued delayed osteoblastogenic differentiation of ank/ank bone marrow stromal cells. A loss of PP(i) transport function ANK mutation also stimulated osteoblastogenic differentiation of MC3T3-E1 cells. Furthermore, ANK function deficiency suppressed the formation of multinucleated osteoclasts from ank/ank bone marrow cells cultured in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-kappaB ligand. In conclusion, ANK is a positive regulator of osteoblastic and osteoclastic differentiation events toward a mature osteoblastic and osteoclastic phenotype. (c) 2010 American Society for Bone and Mineral Research
— id: 111580, year: 2010, vol: 25, page: 1771, stat: Journal Article,

Quantitative assessment of the bone morphogenetic protein expression from alternate bone graft harvesting sites
Takemoto, Richelle C; Fajardo, Marc; Kirsch, Thorsten; Egol, Kenneth A
2010 Sep;24(9):564-566, Journal of orthopaedic trauma
OBJECTIVE:: Bone morphogenetic proteins (BMPs) play important roles in the stimulation of osteogenesis and osteoinduction during bone fracture healing and their expression levels may be important for bone graft efficacy. The objective of this study was to determine if there are variations in the expression of BMPs and their receptors in various bone graft harvesting sites. We analyzed autogenous marrow aspirates obtained from three different graft sites for the mRNA levels of BMPs and their receptors. METHODS:: Using real-time polymerase chain reaction, we analyzed the mRNA levels of BMPs and their receptors in autogenous bone marrow aspirates obtained from three different bone graft sites of 10 different human subjects. Collection of autogenous bone marrow from the iliac crest, the proximal humerus, and the proximal tibia was performed using standard sterile techniques in the operating room as part of surgery to treat an established fracture nonunion. RESULTS:: The mRNA levels of BMP-2 and BMP-5 were the highest in the bone marrow aspirates from the three different sites, whereas the mRNA levels of the other osteoinductive BMPs (BMP-4, -5, -6, -7, -8, and -9) were lower. The mRNA levels of BMP-3, an inhibitor of osteogenesis, were the lowest in the bone marrow aspirates of all three different sites. There were no statistical significant differences in the mRNA levels of any of the BMPs or their receptors investigated in this study in the bone marrow of the three different sites. CONCLUSION:: Because no statistical significant differences in the mRNA levels of the BMPs and their receptors were detected in the bone marrow aspirates from the three different sites, our findings suggest that potential differences of various graft sites in the augmentation of bone healing does not result from different expression levels of BMPs
— id: 111983, year: 2010, vol: 24, page: 564, stat: Journal Article,

Progressive Ankylosis Gene (ank) Regulates Osteoblast Differentiation
Kirsch, Thorsten; Kim, Hyon Jong; Winkles, Jeffrey A
2009 ;189(1-4):158-162, Cells tissues organs
The progressive ankylosis gene (ank) is a transmembrane protein that transports intracellular pyrophosphate to the extracellular milieu. Human mutations of ank lead to craniometaphyseal dysplasia, a disease which is characterized by the overgrowth of craniofacial bones and osteopenia in long bones, suggesting that ANK plays a regulatory role in osteoblast differentiation. To determine the role of ANK in osteoblast differentiation, we suppressed ANK expression in the osteoblastic MC3T3-E1 cell line using siRNA and determined the expression of osteoblastic marker genes and the transcription factors osterix and runx2. In addition, we determined the osteoblastic differentiation of bone marrow stromal cells isolated from the bone marrow of ank/ank mice, which express a truncated, nonfunctional ANK protein, or wild-type littermates. Suppression of ANK expression in MC3T3-E1 cells led to a decrease in bone marker gene expression, including alkaline phosphatase, bone sialoprotein, osteocalcin and type I collagen. In addition, osterix gene expression was decreased in ANK expression-suppressed MC3T3 cells, whereas runx2 expression was increased. Bone marrow stromal cells isolated from ank/ank mice cultured in the presence of ascorbate-2-phosphate for up to 35 days showed markedly reduced mineralization compared to the mineralization of bone marrow stromal cells isolated from wild-type littermates. In conclusion, these findings suggest that ANK is a positive regulator of differentiation events towards a mature osteoblastic phenotype
— id: 83034, year: 2009, vol: 189, page: 158, stat: Journal Article,

Collagen / annexin V interactions regulate chondrocyte mineralization
Kim, Hyon Jong; Kirsch, Thorsten
2008 Apr 18;283(16):10310-10317, Journal of biological chemistry
Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Since mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca2+ influx resulting in an increased intracellular Ca2+ concentration, [Ca2+]i, and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca2+]i, alkaline phosphatase activity and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-L-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N-terminus deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca2+ channel activities) decreased [Ca2+]i, alkaline phosphatase activity and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Since annexin V is upregulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization
— id: 76623, year: 2008, vol: 283, page: 10310, stat: Journal Article,

Determinants of pathologic mineralization
Kirsch, Thorsten
2008 ;18(1):1-9, Critical reviews in eukaryotic gene expression
Physiologic mineralization is necessary for the formation of skeletal tissues and for their appropriate functions during adulthood. Mineralization has to be controlled and restricted to specific regions. If the mineralization process occurs in regions that normally do not mineralize, there can be severe consequences (pathologic or ectopic mineralization). Recent findings have indicated that physiologic and pathologic mineralization events are initiated by matrix vesicles, membrane-enclosed particles released from the plasma membranes of mineralization-competent cells. The understanding of how these vesicles are released from the plasma membrane and initiate the mineralization process may provide novel therapeutic strategies to prevent pathologic mineralization. In addition, other regulators (activators and inhibitors) of physiologic mineralization have been identified and characterized, and there is evidence that the same factors also contribute to the regulation of pathologic mineralization. Finally, programmed cell death (apoptosis) may be a contributor to physiologic mineralization and if occurring after tissue injury may induce pathologic mineralization and mineralization-related differentiation events in the injured and surrounding areas. This review describes how the understanding of mechanisms and factors regulating physiologic mineralization can be used to develop new therapeutic strategies to prevent pathologic or ectopic mineralization events
— id: 76624, year: 2008, vol: 18, page: 1, stat: Journal Article,

Collagen/annexin v interactions regulate growth plate chondroyte mineralization
Kim, H; Kirsch, T
2007 ;22(7):S33-S33, Journal of bone & mineral research
— id: 83088, year: 2007, vol: 22, page: S33, stat: Journal Article,

Loss of annexin VI affects endochondral bone formation
Kim, H; Moss, SE; Kirsch, T
2007 ;22(7):S263-S263, Journal of bone & mineral research
— id: 83090, year: 2007, vol: 22, page: S263, stat: Journal Article,

Physiological and pathological mineralization: A complex multifactorial process
Kirsch T.
2007 ;18(5):425-427, Current opinion in orthopaedics
— id: 83069, year: 2007, vol: 18, page: 425, stat: Journal Article,

Editorial introductions
Kirsch T.; Tosi L.L.
2007 ;18(5):vii-viii, Current opinion in orthopaedics
— id: 83070, year: 2007, vol: 18, page: vii, stat: Journal Article,

Progressive ankylosis gene (ank) regulates osteoblast differentiation
Kirsch, T; Kim, H; Winkles, JA
2007 ;22(7):S93-S93, Journal of bone & mineral research
— id: 83089, year: 2007, vol: 22, page: S93, stat: Journal Article,

Deletion of protein kinase C-epsilon signaling pathway induces glomerulosclerosis and tubulointerstitial fibrosis in vivo
Meier, Matthias; Menne, Jan; Park, Joon-Keun; Holtz, Marcel; Gueler, Faikah; Kirsch, Thorsten; Schiffer, Mario; Mengel, Michael; Lindschau, Carsten; Leitges, Michael; Haller, Hermann
2007 Apr;18(4):1190-1198, Journal of the American Society of Nephrology
Protein kinase C (PKC), a family of 12 distinct serine-threonine kinases, is an important intracellular signaling pathway involved in various cellular functions, such as proliferation, hypertrophy, apoptosis, and adhesion. PKC-epsilon, a novel PKC isoform that is activated in the diabetic kidney, has been demonstrated to have a central role in the underlying signaling infrastructure of myocardial ischemia and hypertrophy. The renal phenotype of PKC-epsilon(-/-) mice was studied with regard to renal hypertrophy and fibrosis. PKC-epsilon(-/-) deficient knockout mice were generated and then killed after 6, 16, and 26 wk of life. Kidney/body weight ratio did not show any significant group difference compared with appropriate wild-type controls. Urinary albumin/creatinine ratio remained normal in wild-type mice, whereas PKC-epsilon(-/-) mice after 6 and 16 wk showed elevated albuminuria. Masson-Goldner staining revealed that tubulointerstitial fibrosis and mesangial expansion were significantly increased in PKC-epsilon(-/-) mice. However, this profibrotic phenotype was not observed in other organs, such as liver and lung. Immunohistochemistry of the kidneys from PKC-epsilon(-/-) mice showed increased renal fibronectin and collagen IV expression that was further aggravated in the streptozotocin-induced diabetic stress model. Furthermore, TGF-beta(1), phospho-Smad2, and phospho-p38 mitogen-activate protein kinase expression was increased in PKC-epsilon(-/-) mice, suggesting a regulatory role of PKC-epsilon in TGF-beta(1) and its signaling pathway in the kidney. These results indicate that deletion of PKC-epsilon mediates renal fibrosis and that TGF-beta1 and its signaling pathway might be involved. Furthermore, these data suggest that activation of PKC-epsilon in the diabetic state may rather represent a protective response to injury than be a mediator of renal injury
— id: 76625, year: 2007, vol: 18, page: 1190, stat: Journal Article,

Cell-cell and cell-matrix interactions during development and pathogenesis
Kirsch T.
2006 ;17(5):387-389, Current opinion in orthopaedics
— id: 83071, year: 2006, vol: 17, page: 387, stat: Journal Article,

Determinants of pathological mineralization
Kirsch, Thorsten
2006 Mar;18(2):174-180, Current opinion in rheumatology
PURPOSE OF REVIEW: Physiological mineralization is necessary for the formation of skeletal tissues and for their appropriate functions during adulthood. Pathological or ectopic mineralization of soft tissues, including articular cartilage and cardiovascular tissues, leads to morbidity and mortality. Recent findings suggest that the mechanisms and factors regulating physiological mineralization may be identical or similar to those regulating ectopic mineralization. Therefore, the purpose of this review is to describe the current knowledge of mechanisms and determinants that regulate physiological mineralization and how these determinants can be used to understand ectopic mineralization better. RECENT FINDINGS: Recent findings have indicated that physiological and pathological mineralization are initiated by matrix vesicles, membrane-enclosed particles released from the plasma membrane of mineralization-competent cells. An understanding of how these vesicles initiate the physiological mineralization process may provide novel therapeutic strategies to prevent ectopic mineralization. In addition, other regulators (activators and inhibitors) of physiological mineralization have been identified and characterized, and evidence indicates that the same factors also contribute to the regulation of ectopic mineralization. Finally, programmed cell death (apoptosis) may be a contributor to physiological mineralization, and if occurring after tissue injury may induce ectopic mineralization and mineralization-related differentiation events in the injured area and surrounding areas. SUMMARY: This review describes how the understanding of mechanisms and factors regulating physiological mineralization can be used to develop new therapeutic strategies to prevent pathological or ectopic mineralization events
— id: 76627, year: 2006, vol: 18, page: 174, stat: Journal Article,

Cellular and molecular mechanisms of tissue protection by lipophilic calcium channel blockers
Menne, J; Park, JK; Agrawal, R; Lindschau, C; Kielstein, JT; Kirsch, T; Marx, A; Muller, D; Bahlmann, FH; Meier, M; Bode-Boger, SM; Haller, H; Fliser, D
2006 ;20(7):994-+, FASEB journal
Long-acting third-generation dihydropyridine calcium channel blockers (CCBs) improve endothelial dysfunction and prevent cardiovascular events in humans, but their cellular and molecular mechanisms of tissue protection are not elucidated in detail. We assessed organ (renal) protection by the highly lipophilic CCB lercanidipine in a double-transgenic rat (dTGR) model with overexpression of human renin and angiotensinogen genes. We randomly treated dTGR with lercanidipine (2.5 mg/ kg/ day; n = 20) or vehicle (n = 20) for 3 wk. Furthermore, we explored the influence of lercanidipine on protein kinase C (PKC) signaling in vivo and in vitro using endothelial and vascular smooth muscle cell cultures. Cumulative mortality was 60% in untreated dTGR, whereas none of the lercanidipine-treated animals died (P < 0.001). We found significantly less albuminuria and improved renal function in lercanidipine-treated dTGR (both P < 0.05). Lercanidipine treatment also significantly (P < 0.05) reduced blood levels of the endogenous NOS inhibitor asymmetric dimethylarginine. On histological examination, we observed significantly less tissue inflammation and fibrosis in lercanidipine-treated animals (both P < 0.05). Lercanidipine significantly inhibited angiotensin (ANG) I-mediated PKC-alpha and -delta activation in vivo and in vitro, partly due to reduced intracellular calcium flux. As a result, lercanidipine improved endothelial cell permeability in vitro. Lercanidipine prevents tissue injury and improves survival in a model of progressive organ damage. These effects may result, at least in part, from inhibition of tissue inflammation as well as improved NO bioavailability. Modulation of PKC activity may be an important underlying intracellular mechanism. $$:
— id: 83085, year: 2006, vol: 20, page: 994, stat: Journal Article,

Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes
Wang, Wei; Kirsch, Thorsten
2006 Oct 13;281(41):30848-30856, Journal of biological chemistry
Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C alpha (PKCalpha), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKCalpha play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKCalpha was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKCalpha led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKCalpha stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis
— id: 76626, year: 2006, vol: 281, page: 30848, stat: Journal Article,

Annexins - their role in cartilage mineralization
Kirsch, Thorsten
2005 Jan 1;10:576-581, Frontiers in biosciences
Annexins II, V and VI are highly expressed by hypertrophic and terminally differentiated growth plate chondrocytes and by osteoblasts. Because of the localization of annexins in areas of cartilage and bone mineralization, we hypothesized that these annexins play a regulatory role in the mineralization process. In this article we review the function of annexins II, V and VI in physiological mineralization of skeletal tissues and in pathological mineralization of articular cartilage
— id: 76632, year: 2005, vol: 10, page: 576, stat: Journal Article,

Syndecan-3: a cell-surface heparan sulfate proteoglycan important for chondrocyte proliferation and function during limb skeletogenesis
Pacifici, Maurizio; Shimo, Tsuyoshi; Gentili, Chiara; Kirsch, Thorsten; Freeman, Theresa A; Enomoto-Iwamoto, Motomi; Iwamoto, Masahiro; Koyama, Eiki
2005 ;23(3):191-199, Journal of bone & mineral metabolism
Syndecans are single-pass integral membrane components that serve as co-receptors for growth factors and cytokines and can elicit signal transduction via their cytoplasmic tails. We review here previous studies from our groups on syndecan-3 biology and function in the growth plates of developing long bones in chick and mouse embryos. Gain- and loss-of-function data indicate that syndecan-3 has important roles in restricting mitotic activity to the proliferative zone of growth plate and may do so in close cooperation and interaction with the signaling molecule Indian hedgehog (IHH). Biochemical and protein-modeling data suggest a dimeric/oligomeric syndecan-3 configuration on the chondrocyte's cell surface. Analyses of embryos misexpressing syndecan-3 or lacking IHH provide further clues on syndecan-3/IHH interdependence and interrelationships. The data and the conclusions reached provide insights into mechanisms fine-tuning chondrocyte proliferation, maturation, and function in the developing and growing skeleton and into how abnormalities in these fundamental mechanisms may subtend human congenital pathologies, including osteochondromas in hereditary multiple exostoses syndrome
— id: 76628, year: 2005, vol: 23, page: 191, stat: Journal Article,

The conserved core domains of annexins A1, A2, A5, and B12 can be divided into two groups with different Ca2+-dependent membrane-binding properties
Patel, Darshana R; Isas, J Mario; Ladokhin, Alexey S; Jao, Christine C; Kim, Yujin E; Kirsch, Thorsten; Langen, Ralf; Haigler, Harry T
2005 Mar 1;44(8):2833-2844, Biochemistry
The hallmark of the annexin super family of proteins is Ca(2+)-dependent binding to phospholipid bilayers, a property that resides in the conserved core domain of these proteins. Despite the structural similarity between the core domains, studies reported herein showed that annexins A1, A2, A5, and B12 could be divided into two groups with distinctively different Ca(2+)-dependent membrane-binding properties. The division correlates with the ability of the annexins to form Ca(2+)-dependent membrane-bound trimers. Site-directed spin-labeling and Forster resonance energy transfer experimental approaches confirmed the well-known ability of annexins A5 and B12 to form trimers, but neither method detected self-association of annexin A1 or A2 on bilayers. Studies of chimeras in which the N-terminal and core domains of annexins A2 and A5 were swapped showed that trimer formation was mediated by the core domain. The trimer-forming annexin A5 and B12 group had the following Ca(2+)-dependent membrane-binding properties: (1) high Ca(2+) stoichiometry for membrane binding ( approximately 12 mol of Ca(2+)/mol of protein); (2) binding to membranes was very exothermic (> -60 kcal/ mol of protein); and (3) binding to bilayers that were in the liquid-crystal phase but not to bilayers in the gel phase. In contrast, the nontrimer-forming annexin A1 and A2 group had the following Ca(2+)-dependent membrane-binding properties: (1) lower Ca(2+) stoichiometry for membrane binding (<or=4 mol of Ca(2+)/mol of protein); (2) binding to membranes was relatively less exothermic (< -33 kcal/ mol of protein); and (3) binding to bilayers that were in either the liquid-crystal phase or gel phase. The biological implications of this subdivision are discussed
— id: 76630, year: 2005, vol: 44, page: 2833, stat: Journal Article,

Role of the progressive ankylosis gene (ank) in cartilage mineralization
Wang, Wei; Xu, Jinping; Du, Bin; Kirsch, Thorsten
2005 Jan;25(1):312-323, Molecular & cellular biology
Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PP(i) concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PP(i) concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PP(i) levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (P(i)) and gene expression of the type III Na(+)/P(i) cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PP(i) transport to the extracellular milieu, initial hydrolysis of PP(i) to P(i), P(i)-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PP(i), and subsequent mineralization
— id: 76631, year: 2005, vol: 25, page: 312, stat: Journal Article,

Annexin V and terminal differentiation of growth plate chondrocytes
Wang, Wei; Xu, Jinping; Kirsch, Thorsten
2005 Apr 15;305(1):156-165, Experimental cell research
Terminal differentiation and mineralization are the final events in endochondral bone formation and allow the replacement of cartilage by bone. Retinoic acid (RA) stimulates these events, including upregulation of expression and activity of alkaline phosphatase (APase), expression of annexins II, V, and VI proteins, which bind to membranes and form Ca(2+) channels, expression of osteocalcin and runx2, another mineralization-related protein and terminal differentiation-related transcription factor, and ultimately mineralization. Chelating cytosolic Ca(2+) with BAPTA-AM, interfering with annexin Ca(2+) channel activities using K-201, a specific annexin Ca(2+) channel blocker, or suppression of annexin V expression using siRNA inhibited these events. Overexpression of annexin V in embryonic chicken growth plate chondrocytes resulted in an increase of cytoplasmic Ca(2+) concentration, [Ca(2+)](i) similar to [Ca(2+)](i) increase in RA-treated cultures. Overexpression of annexin V also resulted in upregulation of annexin II, annexin VI, osteocalcin, and runx2 gene expression, expression and activity of APase, and ultimately stimulation of mineralization. K-201 inhibited upregulation of osteocalcin and runx2 gene expression, APase expression and activity, and mineralization in annexin V-overexpressing growth plate chondrocytes. These findings indicate that annexins II, V, and VI alter Ca(2+) homeostasis in growth plate chondrocytes thereby regulating terminal differentiation and mineralization events. Overexpression of annexin V is sufficient to stimulate these terminal differentiation events in growth plate chondrocytes, whereas suppression of annexin V expression inhibits these events
— id: 76629, year: 2005, vol: 305, page: 156, stat: Journal Article,

Effect of COX-2-specific inhibition on fracture-healing in the rat femur
Brown, Karen M; Saunders, Marnie M; Kirsch, Thorsten; Donahue, Henry J; Reid, J Spence
2004 Jan;86-A(1):116-123, Journal of bone & joint surgery (American volume)
BACKGROUND: Nonsteroidal anti-inflammatory medications have been shown to delay fracture-healing. COX-2-specific inhibitors such as celecoxib have recently been approved for human use. Our goal was to determine, mechanically, histologically, morphologically, and radiographically, whether COX-2-specific inhibition affects bone-healing. METHODS: A nondisplaced unilateral fracture was created in the right femur of fifty-seven adult male rats. Rats were given no drug, indomethacin (1 mg/kg/day), or celecoxib (3 mg/kg/day) daily, starting on postoperative day 1. Fractures were analyzed at four, eight, and twelve weeks after creation of the fracture. Callus and bridging bone formation was assessed radiographically. The amounts of fibrous tissue, cartilage, woven bone, and mature bone formation were determined histologically. Morphological changes were assessed to determine fibrous healing, callus formation, and bone-remodeling. Callus strength and stiffness were assessed biomechanically with three-point bending tests. RESULTS: At four weeks, only the indomethacin group showed biomechanical and radiographic evidence of delayed healing. Although femora from rats treated with celecoxib appeared to have more fibrous tissue than those from untreated rats at four and eight weeks, radiographic signs of callus formation, mechanical strength, and stiffness did not differ significantly between the groups. By twelve weeks, there were no significant differences among the three groups. CONCLUSIONS: Postoperative administration of celecoxib, a COX-2-specific inhibitor, did not delay healing as seen at twelve weeks following fracture in adult rat femora. At four and eight weeks, fibrous healing predominated in the celecoxib group as compared with the findings in the untreated group; however, mechanical strength and radiographic signs of healing were not significantly inhibited. Clinical Relevance: Many orthopaedists rely on narcotic analgesia for postfracture and postoperative pain, despite deleterious side effects and morbidity. Traditional nonsteroidal anti-inflammatory medications have been shown to delay fracture union. This effect may be smaller with COX-2-specific inhibitors
— id: 76633, year: 2004, vol: 86-A, page: 116, stat: Journal Article,

The role of the progressive ankylosis gene (ank) in cartilage mineralization
Wang, W; Xu, J; Du, B; Kirsch, T
Chemistry and biology of mineralized tissues Toronto : University of Toronto Press, 2004,
— id: 4804, year: 2004, vol: , page: 43, stat: Chapter,

Osteoarthritis: A cellular differentiation defect?
Kirsch T.
2003 ;14(5):356-361, Current opinion in orthopaedics
Purpose of review: Osteoarthritis is the most common form of arthritis, affecting a large population of mostly elderly people. No cure for osteoarthritis currently exists. Ultimate treatment is joint replacement. Understanding the mechanisms causing onset and progression is critical. This review describes recent findings that provide new insights into changes of cellular phenotype in osteoarthritis as a possible reason for tissue failure. Recent findings: Recent findings suggest that articular chondrocytes, when stimulated, can undergo hypertrophic and terminal differentiation events similar to those occurring during endochondral bone formation. Interestingly, collagenase-3 (matrix metalloproteinase-13), a main matrix-degrading enzyme in osteoarthritis, is expressed only in terminally differentiated chondrocytes during normal development. Summary: Although terminal differentiation events are required for endochondral bone formation, they lead to cartilage destruction when occurring in articular chondrocytes. Maintaining the articular chondrocyte phenotype and preventing these cells from undergoing hypertrophic and terminal differentiation might provide novel therapeutic targets to prevent onset or progression of osteoarthritis. copyright 2003 Lippincott Williams & Wilkins. <5>
— id: 83072, year: 2003, vol: 14, page: 356, stat: Journal Article,

Annexins and tissue mineralization -- matrix vesicles, ion channel activity of annexins and anexin V-collagen interactions
Kirsch, T
Annexins : biological importance and annexin-related pathologies Georgetown TX : Landes Bioscience/Eurekah.com, 2003,
— id: 4803, year: 2003, vol: , page: 172, stat: Chapter,

Functional differences between growth plate apoptotic bodies and matrix vesicles
Kirsch, Thorsten; Wang, Wei; Pfander, David
2003 Oct;18(10):1872-1881, Journal of bone & mineral research
Mineralization often occurs in areas of apoptotic changes. Our findings indicate that physiological mineralization is mediated by matrix vesicles. These matrix vesicles use mechanisms to induce mineralization that are different from the mechanisms used by apoptotic bodies released from apoptotic cells. Therefore, different therapeutic approaches must be chosen to inhibit pathological mineralization depending on the mechanism of mineralization (matrix vesicles versus apoptotic bodies). INTRODUCTION: Physiological mineralization in growth plate cartilage is restricted to regions of terminally differentiated and apoptotic chondrocytes. Pathological mineralization of tissues also often occurs in areas of apoptosis. We addressed the question of whether apoptotic changes control mineralization events or whether both events are regulated independently. METHODS: To induce mineralization, we treated growth plate chondrocytes with retinoic acid (RA); apoptosis in these cells was induced by treatment with staurosporine, anti-Fas, or TNFalpha. The degrees of mineralization and apoptosis were determined, and the structure and function of matrix vesicles and apoptotic bodies were compared. RESULTS: Release of matrix vesicles and mineralization in vivo in the growth plate occurs earlier than do apoptotic changes. To determine the functional relationship between apoptotic bodies and matrix vesicles, growth plate chondrocytes were treated with RA to induce matrix vesicle release and with staurosporine to induce release of apoptotic bodies. After 3 days, approximately 90% of staurosporine-treated chondrocytes were apoptotic, whereas only 2-4% of RA-treated cells showed apoptotic changes. RA- and staurosporine-treated chondrocyte cultures were mineralized after 3 days. Matrix vesicles isolated from RA-treated cultures and apoptotic bodies isolated from staurosporine-treated cultures were associated with calcium and phosphate. However, matrix vesicles were bigger than apoptotic bodies. Furthermore, matrix vesicles but not apoptotic bodies contained alkaline phosphatase and Ca2+ channel-forming annexins II, V, and VI. Consequently, matrix vesicles but not apoptotic bodies were able to take up Ca2+ and form the first mineral phase inside their lumen. Mineralization of RA-treated cultures was inhibited by antibodies specific for annexin V but not mineralization of staurosporine-treated cultures. CONCLUSION: Physiological mineralization of growth plate chondrocytes is initiated by specialized matrix vesicles and requires alkaline phosphatase and annexins. In contrast, mineral formation mediated by apoptotic bodies occurs by a default mechanism and does not require alkaline phosphatase and annexins
— id: 76634, year: 2003, vol: 18, page: 1872, stat: Journal Article,

Annexin-mediated Ca2+ influx regulates growth plate chondrocyte maturation and apoptosis
Wang, Wei; Xu, Jinping; Kirsch, Thorsten
2003 Feb 7;278(6):3762-3769, Journal of biological chemistry
Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca(2+) influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca(2+), whereas K-201, a specific annexin Ca(2+) channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca(2+) influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca(2+) influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes
— id: 76635, year: 2003, vol: 278, page: 3762, stat: Journal Article,

Molecular regulation of cartilage and bone mineralization
Kirsch T.
2002 ;13(5):382-387, Current opinion in orthopaedics
Biomineralization is a cell-regulated process. Matrix vesicles, which are released from mineralization-competent cells, initiate the mineralization process. These particles contain channel-forming annexins II, V and VI, and an Na⁺/Pi symport system, which enable Ca²⁺ and inorganic phosphate (Pi) influx into the vesicles. Rapid Ca²⁺ and Pi influxes are required for the formation of the first mineral phase inside the vesicles. Furthermore, matrix vesicles contain plasma cell membrane glycoprotein 1 (PC-1), an enzyme which generates pyrophosphate (PPi). Extracellular PPi is also regulated by ank, a transmembrane protein that transports intracellular PPi to the extracellular milieu. PPi is an inhibitor of mineralization; however, matrix vesicle-associated alkaline phosphatase (TNAP) may degrade PPi and thus may not only provide Pi but more importantly remove an inhibitor of mineralization. The release of mineralization-competent matrix vesicles is regulated by annexin-mediated alteration of Ca²⁺ homeostasis, suggesting that targeting annexin functions might provide a novel therapeutic strategy to prevent pathological mineralization. Furthermore, the possible inhibitory function of other factors, such as matrix gla protein (MGP), in mineralization and cell differentiation is discussed. copyright 2002 Lippincott Williams & Wilkins, Inc
— id: 83073, year: 2002, vol: 13, page: 382, stat: Journal Article,

Syndecan-3 is a selective regulator of chondrocyte proliferation
Kirsch, Thorsten; Koyama, Eiki; Liu, Mufei; Golub, Ellis E; Pacifici, Maurizio
2002 Nov 1;277(44):42171-42177, Journal of biological chemistry
Chondrocyte proliferation is important for skeletal development and growth, but the mechanisms regulating it are not completely clear. Previously, we showed that syndecan-3, a cell surface heparan sulfate proteoglycan, is expressed by proliferating chondrocytes in vivo and that proliferation of cultured chondrocytes in vitro is sensitive to heparitinase treatment. To further establish the link between syndecan-3 and chondrocyte proliferation, additional studies were carried out in vivo and in vitro. We found that the topographical location of proliferating chondrocytes in developing chick long bones changes with increasing embryonic age and that syndecan-3 gene expression changes in a comparable manner. For in vitro analysis, mitotically quiescent chondrocytes were exposed to increasing amounts of fibroblast growth factor-2 (FGF-2). Proliferation was stimulated by as much as 8-10-fold within 24 h; strikingly, this stimulation was significantly prevented when the cells were treated with both fibroblast growth factor-2 (FGF-2) and antibodies against syndecan-3 core protein. This neutralizing effect was dose-dependent and elicited a maximum of 50-60% inhibition. To establish specificity of neutralizing effect, cultured chondrocytes were exposed to FGF-2, insulin-like growth factor-1, or parathyroid hormone, all known mitogens for chondrocytes. The syndecan-3 antibodies interfered only with FGF-2 mitogenic action, but not that of insulin-like growth factor-1 or parathyroid hormone. Protein cross-linking experiments indicated that syndecan-3 is present in monomeric, dimeric, and oligomeric forms on the chondrocyte surface. In addition, molecular modeling indicated that contiguous syndecan-3 molecules might form stable complexes by parallel pairing of beta-sheet segments within the ectodomain of the core protein. In conclusion, the results suggest that syndecan-3 is a direct and selective regulator of the mitotic behavior of chondrocytes and its role may involve formation of dimeric/oligomeric structures on their cell surface
— id: 76636, year: 2002, vol: 277, page: 42171, stat: Journal Article,

Regulated production of mineralization-competent matrix vesicles by terminally differentiated chondrocytes
Wang, W; Kirsch, T
The growth plate Amsterdam : IOS Press, 2002,
— id: 4802, year: 2002, vol: , page: 151, stat: Chapter,

Retinoic acid stimulates annexin-mediated growth plate chondrocyte mineralization
Wang, Wei; Kirsch, Thorsten
2002 Jun 10;157(6):1061-1069, Journal of cell biology
Biomineralization is a highly regulated process that plays a major role during the development of skeletal tissues. Despite its obvious importance, little is known about its regulation. Previously, it has been demonstrated that retinoic acid (RA) stimulates terminal differentiation and mineralization of growth plate chondrocytes (Iwamoto, M., I.M. Shapiro, K. Yagumi, A.L. Boskey, P.S. Leboy, S.L. Adams, and M. Pacifici. 1993. Exp. Cell Res. 207:413-420). In this study, we provide evidence that RA treatment of growth plate chondrocytes caused a series of events eventually leading to mineralization of these cultures: increase in cytosolic calcium concentration, followed by up-regulation of annexin II, V, and VI gene expression, and release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles. Cotreatment of growth plate chondrocytes with RA and BAPTA-AM, a cell permeable Ca2+ chelator, inhibited the up-regulation of annexin gene expression and mineralization of these cultures. Interestingly, only matrix vesicles isolated from RA-treated cells that contained annexins, were able to take up Ca2+ and mineralize, whereas vesicles isolated from untreated or RA/BAPTA-treated cells, that contained no or only little annexins were not able to take up Ca2+ and mineralize. Cotreatment of chondrocytes with RA and EDTA revealed that increases in the cytosolic calcium concentration were due to influx of extracellular calcium. Interestingly, the novel 1,4-benzothiazepine derivative K-201, a specific annexin Ca2+ channel blocker, or antibodies specific for annexin II, V, or VI inhibited the increases in cytosolic calcium concentration in RA-treated chondrocytes. These findings indicate that annexins II, V, and VI form Ca2+ channels in the plasma membrane of terminally differentiated growth plate chondrocytes and mediate Ca2+ influx into these cells. The resulting increased cytosolic calcium concentration leads to a further up-regulation of annexin II, V, and VI gene expression, the release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles, and the initiation of mineralization by these vesicles
— id: 76637, year: 2002, vol: 157, page: 1061, stat: Journal Article,

Authentic matrix vesicles contain active metalloproteases (MMP). a role for matrix vesicle-associated MMP-13 in activation of transforming growth factor-beta
D'Angelo, M; Billings, P C; Pacifici, M; Leboy, P S; Kirsch, T
2001 Apr 6;276(14):11347-11353, Journal of biological chemistry
Matrix vesicles (MV) play a key role in the initiation of cartilage mineralization. Although many components in these microstructures have been identified, the specific function of each component is still poorly understood. In this study, we show that metalloproteases (MMP), MMP-2, -9, and -13 are associated with MV isolated from growth plate cartilage. In addition, we provide evidence that MV contain transforming growth factor-beta (TGF-beta) and that MV-associated MMP-13 is capable of activating latent TGF-beta. To determine whether MMPs are associated directly with MV, vesicles isolated from growth plate cartilage were sequentially treated with hyaluronidase, NaCl, and bacterial collagenase to remove matrix proteins and other components attached to their outer surface. Finally, the vesicles were incubated with detergent to rupture the MV membrane and expose components that are inside the vesicles. Each treated MV fraction was subjected to substrate zymography, immunoblotting, and substrate activity assay. Whereas active MMP-13 was lost after combined treatment with hyaluronidase and NaCl, MMP-2 and -9 activities were still retained in the pellet fraction even after detergent treatment, suggesting that the gelatinases, MMP-2 and -9, are integral components of MV. In addition, MV contain TGF-beta in the small latent complex, and MMP-13 associated with the MV surface was responsible for activation of TGF-beta. Since the amount of TGF-beta activated by hypertrophic chondrocytes increased with mineral appearance in serum-free chondrocyte cultures, a role for active MV-associated MMPs is suggested in activation of TGF-beta seen during late chondrocyte hypertrophy and mineralization of growth plate cartilage
— id: 83041, year: 2001, vol: 276, page: 11347, stat: Journal Article,

Type IIA procollagen: expression in developing chicken limb cartilage and human osteoarthritic articular cartilage
Nah, H D; Swoboda, B; Birk, D E; Kirsch, T
2001 Apr;220(4):307-322, Developmental dynamics
Type IIA procollagen is an alternatively spliced product of the type II collagen gene and uniquely contains the cysteine (cys)-rich globular domain in its amino (N)-propeptide. To understand the function of type IIA procollagen in cartilage development under normal and pathologic conditions, the detailed expression pattern of type IIA procollagen was determined in progressive stages of development in embryonic chicken limb cartilages (days 5-19) and in human adult articular cartilage. Utilizing the antibodies specific for the cys-rich domain of the type IIA procollagen N-propeptide, we localized type IIA procollagen in the pericellular and interterritorial matrix of condensing pre-chondrogenic mesenchyme (day 5) and early cartilage (days 7-9). The intensity of immunostaining was gradually lost with cartilage development, and staining became restricted to the inner layer of perichondrium and the articular cap (day 12). Later in development, type IIA procollagen was re-expressed at the onset of cartilage hypertrophy (day 19). Different from type X collagen, which is expressed throughout hypertrophic cartilage, type IIA procollagen expression was transient and restricted to the zone of early hypertrophy. Immunoelectron microscopic and immunoblot analyses showed that a significant amount of the type IIA procollagen N-propeptide, but not the carboxyl (C)-propeptide, was retained in matrix collagen fibrils of embryonic limb cartilage. This suggests that the type IIA procollagen N-propeptide plays previously unrecognized roles in fibrillogenesis and chondrogenesis. We did not detect type IIA procollagen in healthy human adult articular cartilage. Expression of type IIA procollagen, together with that of type X collagen, was activated by articular chondrocytes in the upper zone of moderately and severely affected human osteoarthritic cartilage, suggesting that articular chondrocytes, which normally maintain a stable phenotype, undergo hypertrophic changes in osteoarthritic cartilage. Based on our data, we propose that type IIA procollagen plays a significant role in chondrocyte differentiation and hypertrophy during normal cartilage development as well as in the pathogenesis of osteoarthritis
— id: 83042, year: 2001, vol: 220, page: 307, stat: Journal Article,

The crystal structure of the BMP-2 : BMPR-IA complex and the generation of BMP-2 antagonists
Nickel, J; Dreyer, MK; Kirsch, T; Sebald, W
2001 ;83A(10):S7-S14, Journal of bone & joint surgery (American volume)
Background: Bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) belong to the large transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. Signaling of the BMPs requires the binding of the BMP to the BMP cell surface receptors BMPR-IA, BMPR-IB, and BMPR-II. Similar to other cytokines, members of the TGF-beta superfamily exhibit stringent specificity in their ligand-receptor interactions, which may be a reason for the qualitative and quantitative differences in cellular responses. To understand how BMPs and GDFs activate their receptors, it is important to determine structure and binding mechanisms of ligand-receptor complexes. We have used BMP-2 as a key representative of the BMPs to identify the epitopes for type I and type II receptor binding by mutational interaction analyses and have solved the crystal structure of a BMP-2:BMPR-IA receptor ectodomain complex. Methods: To identify amino acid side chains involved in receptor binding, a collection of in vitro mutagenized human BMP-2 variants was prepared and subjected to interaction analyses with use of the receptor ectodomains of BMPR-IA, BMPR-II, and ActR-Il immobilized on a biosensor system. The biological activity of the BMP-2 variants was measured by BMP-2 dependent expression of alkaline phosphatase (ALP) in C2C12 cells. For crystallization, a complex of BMP-2 and the ectodomain of BMPR-IA was formed in solution, purified, and crystallized as described(12). Results: The ligand-receptor interaction analysis of the BMP-2 variants identified distinct epitopes for type I and type II receptor binding. Because the structure of TGF-beta -like proteins has been compared with that of an open hand, the binding epitope for the type I receptor was-on the basis of its location-termed 'wrist' epitope. The crystal structure of the BMP-2:BMPR-IA ectodomain complex revealed a key feature of the ligand-receptor interaction: a large hydrophobic residue (Phe85) within a hydrophobic patch of BMPR-IA fit into a hydrophobic pocket composed of residues of both BMP-2 monomers. A second epitope identified by alanine mutagenesis scanning was termed the 'knuckle' epitope on the basis of its location on the outer side of the 'finger' segments of BMP-2. Mutations in either the wrist epitope or the knuckle epitope produced variants with altered biological activities. Variants with antagonistic properties were exclusively generated by mutations in the knuckle epitope of BMP-2. Conclusions and Clinical Relevance: The identification and characterization of the two receptor binding epitopes in BMP-2 provide new insight into the primary steps of BMP-receptor activation. Because of the structural similarities between members of the TGF-beta superfamily, it can be assumed that the data presented in this work are transferable to other TGF-beta receptor systems. Because of the association with various diseases, the generation of antagonists of other TGF-beta superfamily members might generate potent tools for basic research and therapeutic approaches. $$:
— id: 83091, year: 2001, vol: 83A, page: S7, stat: Journal Article,

Vascular endothelial growth factor in articular cartilage of healthy and osteoarthritic human knee joints
Pfander, D; Kortje, D; Zimmermann, R; Weseloh, G; Kirsch, T; Gesslein, M; Cramer, T; Swoboda, B
2001 Nov;60(11):1070-1073, Annals of rheumatic diseases
OBJECTIVE: To determine the levels of vascular endothelial growth factor (VEGF) mRNA and protein expression in normal and osteoarthritic (OA) human articular cartilage, and whether VEGF expression alters during the progression of OA. METHODS: Sections from normal and OA human knee cartilage were immunotained with a polyclonal antibody recognising VEGF. In addition, total RNA was isolated from normal and osteoarthritic human knee cartilage and analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) for VEGF mRNA expression. RESULTS: VEGF was found to be present in normal and OA human knee cartilage in all cartilage layers. A significant increase of VEGF immunopositive chondrocytes to up to approximately 82% was detected in severe OA cartilage compared with normal articular cartilage (approximately 56% of immunopositive chondrocytes). RT-PCR analysis showed the expression of VEGF also on the mRNA level. CONCLUSIONS: VEGF is expressed by articular chondrocytes in normal and OA human knee cartilage. The percentage of VEGF immunopositive chondrocytes significantly increases in late stages of the disease. The VEGF transcript levels encoding all four isoforms shows a big variability in samples from different donors, suggesting a distinct regulation of the expression of the four VEGF isoforms in normal and OA cartilage
— id: 83043, year: 2001, vol: 60, page: 1070, stat: Journal Article,

Expression of early and late differentiation markers (proliferating cell nuclear antigen, syndecan-3, annexin VI, and alkaline phosphatase) by human osteoarthritic chondrocytes
Pfander, D; Swoboda, B; Kirsch, T
2001 Nov;159(5):1777-1783, American journal of pathology
Although osteoarthritis is characterized by a progressive loss of the extracellular cartilage matrix, very little is known about the fate of articular chondrocytes during the progression of the disease. In this study we examined the expression of syndecan-3, a marker of early chondrocyte differentiation, and annexin VI, a marker of late chondrocyte differentiation, in mammalian embryonic growth plate cartilage and normal and osteoarthritic human articular cartilage. Whereas syndecan-3 was expressed in the proliferative and hypertrophic zones of growth platecartilage, immunostaining for annexin VI waspredominately found in the hypertrophic and mineralizing zones of fetal bovine growth plate cartilage. Approximately 20% of chondrocytes were immunopositive for syndecan-3 in normal human articular cartilage, the number of syndecan-3-expressing chondrocytes significantly increased during the progression of osteoarthritis with more than 80% syndecan-3-positive cells in the upper zone of severely affected osteoarthritic cartilage. Similarly, the number of annexin VI-expressing cells significantly increased in the upper cartilage zones during the progression of osteoarthritis. Furthermore, immunostaining for proliferating cell nuclear antigen, a marker for cell proliferation, was detected in chondrocytes in the upper zone of osteoarthritic cartilage. Double-labeling experiments with antibodies against syndecan-3 and annexin VI revealed chondrocytes that expressed only syndecan-3, and cells that expressed both syndecan-3 and annexin VI. These results suggest that the expression of early (proliferating cell nuclear antigen, syndecan-3) and late differentiation markers (annexin VI, alkaline phosphatase) is activated in chondrocytes of osteoarthritic cartilage
— id: 83044, year: 2001, vol: 159, page: 1777, stat: Journal Article,

Regulatory roles of cytosolic calcium and annexins in the initiation of mineralization in skeletal tissues
Kirsch, T
2000 ;15(2):S339-S339, Journal of bone & mineral research
— id: 83092, year: 2000, vol: 15, page: S339, stat: Journal Article,

Matrix vesicles mediate mineralization of human thyroid cartilage
Kirsch, T; Claassen, H
2000 Apr;66(4):292-297, Calcified tissue international
Mineralization and ossification of human thyroid cartilage first starts after the end of adolescence when the previously cartilaginous human skeleton has become ossified and the epiphyseal discs are in the process of closing. However, the mechanisms involved in mineralization and ossification of human thyroid cartilage are not well understood. Ultrastructural analysis of human thyroid cartilage revealed that mineralization started close to cartilage canals in a matrix containing gigantic collagen fibers (asbestoid fibers). Matrix vesicles were detected in mineralized areas and were often associated with needle-like crystals. For the first time we were able to isolate matrix vesicles from human thyroid cartilage by mild enzymatic digestions and ultracentrifugation. These particles were oval and varied in size; some were heavily calcified. They were enriched in alkaline phosphatase, calcium, and inorganic phosphate, suggesting that the particles contain Ca2+-Pi complexes. Immunoblot analysis of these vesicles revealed the presence of annexins II, V, and VI, membrane-associated, channel-forming proteins, which allow influx of Ca2+ into the vesicles and intralumenal crystal growth. In addition, the vesicles were associated with types II and X collagen, suggesting that this association not only anchors the vesicles to the extracellular matrix, but, as shown previously, also stimulates Ca2+ influx into these particles. In conclusion, matrix vesicles isolated from human thyroid cartilage contain all the components, enabling them to initiate and mediate the mineralization process in human thyroid cartilage
— id: 83037, year: 2000, vol: 66, page: 292, stat: Journal Article,

The roles of annexins and types II and X collagen in matrix vesicle-mediated mineralization of growth plate cartilage
Kirsch, T; Harrison, G; Golub, E E; Nah, H D
2000 Nov 10;275(45):35577-35583, Journal of biological chemistry
Annexins II, V, and VI are major components of matrix vesicles (MV), i.e. particles that have the critical role of initiating the mineralization process in skeletal tissues. Furthermore, types II and X collagen are associated with MV, and these interactions mediated by annexin V stimulate Ca(2+) uptake and mineralization of MV. However, the exact roles of annexin II, V, and VI and the interaction between annexin V and types II and X collagen in MV function and initiation of mineralization are not well understood. In this study, we demonstrate that annexin II, V, or VI mediate Ca(2+) influx into phosphatidylserine (PS)-enriched liposomes, liposomes containing lipids extracted from authentic MV, and intact authentic MV. The annexin Ca(2+) channel blocker, K-201, not only inhibited Ca(2+) influx into fura-2-loaded PS-enriched liposomes mediated by annexin II, V, or VI, but also inhibited Ca(2+) uptake by authentic MV. Types II and X collagen only bound to liposomes in the presence of annexin V but not in the presence of annexin II or VI. Binding of these collagens to annexin V stimulated its Ca(2+) channel activities, leading to an increased Ca(2+) influx into the liposomes. These findings indicate that the formation of annexin II, V, and VI Ca(2+) channels in MV together with stimulation of annexin V channel activity by collagen (types II and X) binding can explain how MV are able to rapidly take up Ca(2+) and initiate the formation of the first crystal phase
— id: 83040, year: 2000, vol: 275, page: 35577, stat: Journal Article,

Regulatory roles of zinc in matrix vesicle-mediated mineralization of growth plate cartilage
Kirsch, T; Harrison, G; Worch, K P; Golub, E E
2000 Feb;15(2):261-270, Journal of bone & mineral research
Zinc (Zn2+) has long been known to play important roles in mineralization and ossification of skeletal tissues, but the mechanisms of Zn2+ action are not well understood. In this study we investigated the effects of Zn2+ on mineralization in a cell culture system in which terminal differentiation and mineralization of hypertrophic growth plate chondrocytes was induced by retinoic acid (RA) treatment. Addition of Zn2+ to RA-treated cultures decreased mineralization in a dose-dependent manner without affecting alkaline phosphatase (APase) activity. Characterization of matrix vesicles (MVs), particles that initiate the mineralization process, revealed that vesicles isolated from RA-treated and RA/Zn2+-treated cultures showed similar APase activity, but vesicles from RA/Zn2+-treated cultures contained significantly less Ca2+ and Pi. MVs isolated from RA-treated cultures were able to take up Ca2+ and mineralize in vitro, whereas vesicles isolated from RA/Zn2+-treated cultures were not able to do so. Detergent treatment, which ruptures the MV membrane and exposes preformed intravesicular Ca2+-Pi-phospholipid complexes, did not restore the Ca2+ uptake abilities of MVs isolated from RA/Zn2+-treated cultures, suggesting that vesicles from RA/Zn2+-treated cultures did not contain functional Ca2+-Pi-phospholipid complexes. Zn2+ treatment did not affect the content of annexins II, V, and VI in MVs or the Ca2+-dependent, EDTA-reversible binding of these molecules to the membrane surface. However, Zn2+ treatment did affect the EDTA-nonreversible binding of these molecules to the MV membrane, suggesting that Zn2+ interferes with the assembly of annexins in the MV membrane. In addition, Zn2+ inhibited annexin II-, V-, and VI-mediated Ca2+ influx into liposomes. In conclusion, Zn2+ inhibits the mineralizing competence of intravesicular Ca2+-Pi-phospholipid complexes and function of annexin channels, thereby controlling Ca2+ influx into MVs, the formation of the first crystal phase inside the vesicles and initiation of mineralization
— id: 83036, year: 2000, vol: 15, page: 261, stat: Journal Article,

Activation of annexin II and V expression, terminal differentiation, mineralization and apoptosis in human osteoarthritic cartilage
Kirsch, T; Swoboda, B; Nah, H
2000 Jul;8(4):294-302, Osteoarthritis & cartilage
OBJECTIVE: To test the hypothesis that terminal differentiation of chondrocytes in human osteoarthritic cartilage might lead to the failure of repair mechanisms and might cause progressive loss of structure and function of articular cartilage. DESIGN: Markers for terminally differentiated chondrocytes, such as alkaline phosphatase, annexin II, annexin V and type X collagen, were detected by immunohistochemical analysis of human normal and osteoarthritic knee cartilage from medial and lateral femoral condyles. Apoptosis in these specimens was detected using the TUNEL labeling. Mineralization and matrix vesicles were detected by alizarin red S staining and electron microscopic analysis. RESULTS: Alkaline phosphatase, annexin II, annexin V and type X collagen were expressed by chondrocytes in the upper zone of early stage and late stage human osteoarthritic cartilage. However, these proteins, which are typically expressed in hypertrophic and calcifying growth plate cartilage, were not detectable in the upper, middle and deep zones of healthy human articular cartilage. TUNEL labeling of normal and osteoarthritic human cartilage sections provided evidence that chondrocytes in the upper zone of late stage osteoarthritic cartilage undergo apoptotic changes. In addition, mineral deposits were detected in the upper zone of late stage osteoarthritic cartilage. Needle-like mineral crystals were often associated with matrix vesicles in these areas, as seen in calcifying growth plate cartilage. CONCLUSION: Human osteoarthritic chondrocytes adjacent to the joint space undergo terminal differentiation, release alkaline phosphatase-, annexin II- and annexin V-containing matrix vesicles, which initiate mineral formation, and eventually die by apoptosis. Thus, these cells resume phenotypic changes similar to terminal differentiation of chondrocytes in growth plate cartilage culminating in the destruction of articular cartilage in osteoarthritis
— id: 83039, year: 2000, vol: 8, page: 294, stat: Journal Article,

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development
Nah, H D; Pacifici, M; Gerstenfeld, L C; Adams, S L; Kirsch, T
2000 Mar;15(3):522-533, Journal of bone & mineral research
Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments
— id: 83038, year: 2000, vol: 15, page: 522, stat: Journal Article,

The function of types II and X collagen in matrix vesicle-mediated mineralization or skeletal tissues
Kirsch, T; Golub, EE; Nah, HD
1999 ;10(2):146A-146A, Molecular biology of the cell
— id: 83083, year: 1999, vol: 10, page: 146A, stat: Journal Article,

Activation of annexin II, annexin V and type IIA procollagen expression, mineralization terminal differentiation and apoptosis in human osteoarthritic cartilage
Kirsch, T; Swoboda, B; Nah, HD
1999 ;42(9):S255-S255, Arthritis & rheumatism
— id: 83097, year: 1999, vol: 42, page: S255, stat: Journal Article,

Retinoid signaling is required for chondrocyte maturation and endochondral bone formation during limb skeletogenesis
Koyama, E; Golden, E B; Kirsch, T; Adams, S L; Chandraratna, R A; Michaille, J J; Pacifici, M
1999 Apr 15;208(2):375-391, Developmental biology (Orlando)
Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues
— id: 83035, year: 1999, vol: 208, page: 375, stat: Journal Article,

Involvement of tenascin-c and syndecan-3 in the development of chick limb diarthrodial joints
Pacifici, M; Koyama, E; Kirsch, T; Leatherman, JL; Golden, EB
Biology of the synovial joint Amsterdam : Harwood Academic Publishers, 1999,
— id: 4805, year: 1999, vol: , page: 23, stat: Chapter,

Regulatory effects of zinc on matrix vesicle-mediated mineralization
Grande, EA; Kirsch, T
1998 ;77(A):248-248, Journal of dental research
— id: 83074, year: 1998, vol: 77, page: 248, stat: Journal Article,

The role of collagen-annexin V interactions in mineralization of skeletal tissues
Kirsch, T; Nah, HD; Pacifici, M
1998 ;17(2):162-162, Matrix biology
— id: 83080, year: 1998, vol: 17, page: 162, stat: Journal Article,

Retinoids and their nuclear receptors promote the completion of chondrocyte maturation during limb skeletogenesis
Koyama, E; Golden, EB; Kirsch, T; Adams, SL; Pacifici, M
1998 ;17(2):155-155, Matrix biology
— id: 83081, year: 1998, vol: 17, page: 155, stat: Journal Article,

Reexpression of type IIA procollagen in adult osteoarthritic
Nah, HD; Swoboda, B; Koyama, E; Suh, JY; Croll-Halpern, W; Kirsch, T
1998 ;17(2):159-159, Matrix biology
— id: 83082, year: 1998, vol: 17, page: 159, stat: Journal Article,

Type IIA procollagen is an early marker for osteoarthritic cartilage
Nah, HD; Swoboda, B; Koyama, E; Suh, JY; Croll-Halpern, W; Kirsch, T
1998 ;77(A):724-724, Journal of dental research
— id: 83075, year: 1998, vol: 77, page: 724, stat: Journal Article,

Increased content of type-VI collagen epitopes in human osteoarthritic cartilage: quantitation by inhibition ELISA
Swoboda, B; Pullig, O; Kirsch, T; Kladny, B; Steinhauser, B; Weseloh, G
1998 Jan;16(1):96-99, Journal of orthopaedic research
Type-VI collagen is an integral part of the extracellular cartilage matrix. However, the exact amounts of type-VI collagen in normal and osteoarthritic human cartilage still are not known. In this study, we describe an inhibition enzyme-linked immunosorbent assay that was developed to quantitate type-VI collagen epitopes found in guanidinium chloride extracts from normal and osteoarthritic human cartilage. In 31 cartilage samples from various localizations of healthy adult human knees, type-VI collagen epitopes accounted for approximately 0.40% of the total collagen content. Interestingly, type-VI collagen epitopes increased about 4-fold in osteoarthritic cartilage. A statistically significant increase of type-VI collagen epitopes was found during early stages of the disease, with only a superficial roughening of the cartilage surface and a loss of proteoglycans. Thus, these findings indicate that type-VI collagen is a minor component of normal human articular cartilage and that the amount of type-VI collagen epitopes increases significantly during early stages of osteoarthritis
— id: 83068, year: 1998, vol: 16, page: 96, stat: Journal Article,

Role of annexin II and V in matrix vesicle-mediated biomineralization
Worch, KP; Pacifici, M; Polson, AM; Kirsch, T
1998 ;77(A):269-269, Journal of dental research
— id: 83076, year: 1998, vol: 77, page: 269, stat: Journal Article,

Role of annexin II and V in matrix vesicle-mediated biomineralization
Worch, KP; Pacifici, M; Polson, AM; Kirsch, T
1998 ;77(A):927-927, Journal of dental research
— id: 83077, year: 1998, vol: 77, page: 927, stat: Journal Article,

Type IIA procollagen expression during cartilage development
Halpern, W; Kirsch, T; Nah, HD
1997 ;76(A):1632-1632, Journal of dental research
— id: 83078, year: 1997, vol: 76, page: 1632, stat: Journal Article,

Retinoic acid regulates matrix vesicle-mediated mineralization of growth plate cartilage
Kirsch, T; Worch, KP; Pacifici, M
1997 ;12(3):F271-F271, Journal of bone & mineral research
— id: 83093, year: 1997, vol: 12, page: F271, stat: Journal Article,

Annexin V-mediated calcium flux across membranes is dependent on the lipid composition: implications for cartilage mineralization
Kirsch, T; Nah, H D; Demuth, D R; Harrison, G; Golub, E E; Adams, S L; Pacifici, M
1997 Mar 18;36(11):3359-3367, Biochemistry
Annexin V is a major component of matrix vesicles and has a role in mediating the influx of Ca2+ into these vesicles, thus promoting the initiation of hypertrophic cartilage matrix mineralization. However, the mechanisms and factors regulating annexin V-mediated Ca2+ influx into these vesicles are not well understood. Since the lipid composition of matrix vesicles differs from that of the plasma membrane of chondrocytes and is rich in phosphatidylserine, we asked whether the lipid composition may regulate annexin V function. We prepared liposomes containing different concentrations of phosphatidylserine and determined how the lipid composition affected (a) the interactions between annexin V and liposomes and (b) annexin V-mediated Ca2+ influx into the liposomes. We found that annexin V was able to bind to every liposome tested. However, we observed the most prominent increases in tryptophan 187 emission intensity, a measure of the degree of interactions between annexin V and lipid bilayers, only with liposomes containing a high concentration of phosphatidylserine. In addition, a significant fraction of annexin V associated with phosphatidylserine-rich liposomes was not extractable by EDTA treatment but required a detergent, indicating that annexin V inserts into bilayers of these liposomes. Chemical cross-linking analysis revealed that matrix vesicles and phosphatidylserine-rich liposomes induced the formation of the annexin V hexamer. Interestingly, a significant Ca2+ influx in the presence of annexin V occurred only in liposomes containing a high phosphatidylserine content. Moreover, annexin V-mediated Ca2+ influx into these liposomes was inhibited (i) by anti-annexin V antibodies and (ii) by treatment with zinc and cadmium, indicating the essential role of the protein in Ca2+ influx. The results of this study indicate that phosphatidylserine-rich bilayers induce the formation of a hexameric annexin V, possibly leading to a Ca2+-dependent insertion of annexin V into the bilayer and establishment of annexin V-mediated Ca2+ influx into matrix vesicles or liposomes. The phosphatidylserine-rich membrane of matrix vesicles in vivo may thus offer an ideal specialized environment in which the biological function of annexin V is optimized, leading to rapid Ca2+ influx, intralumenal crystal growth, and cartilage matrix mineralization
— id: 83067, year: 1997, vol: 36, page: 3359, stat: Journal Article,

Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes
Kirsch, T; Nah, H D; Shapiro, I M; Pacifici, M
1997 Jun 2;137(5):1149-1160, Journal of cell biology
Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca2+ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro-osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix
— id: 83066, year: 1997, vol: 137, page: 1149, stat: Journal Article,

Retinoic acid regulates matrix vesicle mediated mineralization of skeletal tissues
Worch, KP; Polson, AM; Kirsch, T
1997 ;76(A):2616-2616, Journal of dental research
— id: 83079, year: 1997, vol: 76, page: 2616, stat: Journal Article,

Localization of collagens and alkaline phosphatase activity during mineralization and ossification of human first rib cartilage
Claassen, H; Kampen, W U; Kirsch, T
1996 Mar;105(3):213-219, Histochemistry & cell biology
The localization of type X collagen and alkaline phosphatase activity was examined in order to gain a better understanding of tissue remodelling during development of human first rib cartilage. First rib cartilages from children and adolescents showed no staining for type X collagen and alkaline phosphatase activity. After onset of mineralization in the late second decade, a peripheral ossification process preceded by mineralized fibrocartilage could be distinguished from a more central one preceded by mineralized hyaline cartilage. No immunostaining for type X collagen was found in either type of cartilage. However, strong staining for alkaline phosphatase activity was detected around chondrocyte-like cells within fibrocartilage adjacent to the peripheral mineralization front, while a weaker staining pattern was observed around chondrocytes of hyaline cartilage near the central mineralization front. In addition, the territorial matrix of some chondrocytes within the hyaline cartilage revealed staining for type I collagen, suggesting that these cells undergo a dedifferentiation process, which leads to a switch from type II to type I collagen synthesis. The study provides evidence that mineralization of the hyaline cartilage areas in human first rib cartilage occurs in the absence of type X collagen synthesis but in the presence of alkaline phosphatase. Thus, mineralization of first rib cartilage seems to follow a different pattern from endochondral ossification in epiphyseal discs
— id: 83064, year: 1996, vol: 105, page: 213, stat: Journal Article,

Cartilage canals in human thyroid cartilage characterized by immunolocalization of collagen types I, II, pro-III, IV and X
Claassen, H; Kirsch, T; Simons, G
1996 Aug;194(2):147-153, Anatomy & embryology
In this study the collagenous composition of cartilage canals in human thyroid cartilage, which are perichondral invaginations of blood vessels and connective tissue, and the surrounding cartilage matrix were investigated by immunolabelling with specific antibodies against type I, II, pro-III, IV and X collagen. During childhood and early adolescence no cartilage canals were detected in thyroid cartilage, and immunolabelling for type IV collagen was restricted to basal lamina components of blood vessels in the perichondrium. First immunolabelling for type IV collagen, belonging to blood vessels in cartilage canals, in both sexes was detected about the end of the second decade; it was localized in the dorsal part of the thyroid cartilage plate. At this time thyroid cartilage has already reached its final form and size. As revealed by von Kossa staining, vascularization preceded mineralization and ossification. In contrast to the male thyroid cartilage plate, no immunostaining for type IV collagen and no ossification was detected in the ventral half of female thyroid cartilage even in advanced age. The extracellular matrix of cells in cartilage canals showed positive immunostaining for collagen types I and pro-III as well as for collagen type II, indicating that the cells in the canal possess fibroblastic and chondrogenic properties. The extracellular matrix of hypertrophic chondrocytes adjacent to cartilage canals showed strong immunoreactivity for type X collagen. First mineralization was detected close to cartilage canals, suggesting that mineralization in human thyroid cartilage starts in the extracellular matrix adjacent to cartilage canals
— id: 83063, year: 1996, vol: 194, page: 147, stat: Journal Article,

Changes in annexin V and type I collagen expression during cartilage mineralization
Kirsch, T; Pacifici, M; Nah, HD
1996 ;11(3):M465-M465, Journal of bone & mineral research
— id: 83094, year: 1996, vol: 11, page: M465, stat: Journal Article,

Transient expression of a subset of cartilage-characteristic genes during intramembranous bone formation
Nah, HD; Pacifici, M; Kirsch, T
1996 ;11(3):P223-P223, Journal of bone & mineral research
— id: 83095, year: 1996, vol: 11, page: P223, stat: Journal Article,

Syndecan-3 and the control of chondrocyte proliferation during endochondral ossification
Shimazu, A; Nah, H D; Kirsch, T; Koyama, E; Leatherman, J L; Golden, E B; Kosher, R A; Pacifici, M
1996 Nov 25;229(1):126-136, Experimental cell research
During endochondral ossification, chondrocytes progress through several stages of maturation before they are replaced by bone cells. Chondrocyte proliferation, the first step in this complex multistage process, is strictly controlled both spatially and temporally but its underlying mechanisms of regulation remain unclear. In this study we asked whether chondrocytes produce syndecan-3, a cell surface receptor for growth factors such as fibroblast growth factor 2 (FGF-2), and whether syndecan-3 may play a role in proliferation during chondrocyte maturation. We found that proliferating immature cartilage from chick embryo tibia and sternum contained significant amounts of syndecan-3 mRNA, whereas mature hypertrophic cartilage contained markedly lower transcript levels. Immunohistochemical analyses on sections of Day 18 chick embryo tibia revealed that syndecan-3 was spatially restricted and indeed detectable only in immature proliferating chondrocytes in the top zone of growth plate. These syndecan-3-rich proliferating chondrocytes lay beneath developing articular chondrocytes rich in their typical matrix protein tenascin-C, resulting in a striking boundary between these two populations of chondrocytes. Immature proliferating chondrocyte populations reared in growth-promoting culture conditions displayed strong continuous syndecan-3 gene expression; upon induction of maturation by vitamin C treatment, syndecan-3 gene expression was markedly down-regulated. Treatment with FGF-2 for 24 h stimulated both syndecan-3 gene expression and chondrocyte proliferation; this growth stimulation was counteracted by cotreatment with heparinase I or III. The results of the study indicate that syndecan-3 participates in the maturation of chondrocytes during endochondral ossification and represents a regulator of the proliferative phase of this multistage process
— id: 83065, year: 1996, vol: 229, page: 126, stat: Journal Article,

Localization of type I and II collagen during development of human first rib cartilage
Claassen, H; Kampen, W U; Kirsch, T
1995 Oct;192(4):329-334, Anatomy & embryology
The localization of fibrillar type I and II collagen was investigated by immunofluorescence staining with specific antibodies in order to obtain a better understanding of tissue remodelling during the development of first rib cartilage. In childhood and early adolescence type I collagen was found to be restricted to the perichondrium of first rib cartilage, while type II collagen was localized in the matrix of hyaline cartilage. However, in advanced age type I collagen was also found in the territorial matrix of intermediate and central chondrocytes of first rib cartilage. The matrix of subperichondrial chondrocytes was negative for type I collagen. This suggests that some chondrocytes in first rib cartilage undergo a modulation to type I collagen-producing cells. The first bone formation was observed in rib cartilages of 20- to 25-year-old adults. Interestingly, the ossification began peripherally, adjacent to the innermost layer of the perichondrium where areas of fibrocartilage had developed. The newly formed bone matrix showed strong immunostaining for type I collagen. Fibrocartilage bordering peripherally on bone matrix revealed only a faint staining for type I collagen, but strong immunoreactivity to type II collagen. The interterritorial matrix of the central chondrocytes failed to react with the type II collagen antibody, in both men and women, from the end of the second decade. These observations indicate that major matrix changes occur at the same time in male and female first rib cartilages. Thus, our findings indicate that ossification in human first rib cartilage does not follow the same pattern as that observed in endochondral ossification of epiphyseal discs or sternal cartilage
— id: 83062, year: 1995, vol: 192, page: 329, stat: Journal Article,

Areas of asbestoid (amianthoid) fibers in human thyroid cartilage characterized by immunolocalization of collagen types I, II, IX, XI and X
Claassen, H; Kirsch, T
1995 May;280(2):349-354, Cell & tissue research
The distribution of type I, II, IX, XI and X collagens in and close to areas of asbestoid (amianthoid) fibers in thyroid cartilages of various ages was investigated in this study. Asbestoid fibers were first detected in thyroid cartilage from a 3-year-old male child. Areas of asbestoid fibers functionally appear to serve as guide rails for vascularization of thyroid cartilage. Alcian blue staining in the presence of 0.3 M MgCl2 revealed a loss of glycosaminoglycans in areas of asbestoid fibers. In addition, the fibers reacted positively with antibodies against collagen types II, IX and XI, but showed no staining with antibodies to collagen types I and X. Territorial matrix of adjacent chondrocytes showed the same staining pattern. In addition to staining for type II, IX and XI collagens, asbestoid fibers showed strong immunostaining for type I collagen after puberty but not for type X collagen. However, groups of chondrocytes within areas of asbestoid fibers reacted strongly with antibodies to type X collagen, suggesting that this collagen plays an important role in matrix of highly differentiated chondrocytes. The finding that these type X collagen-positive chondrocytes also revealed immunostaining for type I collagen confirms previous studies showing that hypertrophic chondrocytes can further differentiate into cells that are characterized by the synthesis of type X and I collagens
— id: 83055, year: 1995, vol: 280, page: 349, stat: Journal Article,

Mineralization and osteogenesis in the human first rib cartilage
Kampen, W U; Claassen, H; Kirsch, T
1995 Mar;177(2):171-177, Annals of anatomy = Anatomischer Anzeiger
Mineralization and osteogenesis in the human first rib cartilage were studied radiologically and by means of normal and polarized light microscopy. Onset of mineralization occurs at the end of puberty and is located directly beneath the perichondrium. Bone is formed in a typical spur-like pattern, arising medially from the upper edge of the manubrium sterni and laterally from the caudal rim of the bony part of the rib. From the middle of the second decade, large cartilage canals with several blood vessels and loose perivascular connective tissue are seen in central areas of the first costal cartilage. These parts are the last to be mineralized and ossified in old age. The type of osteogenesis cannot be classified according to common patterns. In spite of the subperichondral localization it cannot be intramembranous, because the new bone is separated from the perichondrium by a layer of mineralized cartilage. Osteogenesis cannot be called endochondral compared with the epiphyseal plate for the following reasons: there are no hypertrophied chondrocytes; an immunoreactivity for collagen type X is missing; areas where bone is formed directly on hyaline cartilage could be proved. Vascularization and onset of osteogenesis are separated in time and localization. Mineralization and osteogenesis in human first rib cartilage are physiological age-related changes, which cannot be regarded as degenerative processes. Ossification is not directly correlated with the invasion of blood vessels and cannot be classified as one of the classical concepts of intramembranous or endochondral osteogenesis
— id: 83054, year: 1995, vol: 177, page: 171, stat: Journal Article,

LIPID-COMPOSITION AND COLLAGEN-BINDING MODULATE CALCIUM-CHANNEL PROPERTIES OF ANNEXIN-V
KIRSCH, T; GOLUB, EE; PACIFICI, M
1995 ;10(3):S238-S238, Journal of bone & mineral research
— id: 83096, year: 1995, vol: 10, page: S238, stat: Journal Article,

SPECIFICITY OF A POTENTIAL VACUOLAR TARGETING RECEPTOR FOR VACUOLAR TARGETING INFORMATION
KIRSCH, T; SAALBACH, G; RAIKHEL, NV; BEEVERS, L
1995 ;6(2):605-605, Molecular biology of the cell
— id: 83084, year: 1995, vol: 6, page: 605, stat: Journal Article,

Immunolocalization of type X collagen before and after mineralization of human thyroid cartilage
Claassen, H; Kirsch, T
1994 Jan;101(1):27-32, Histochemistry
In this study the distribution of type X collagen in thyroid cartilages of various ages is described. Fetal and juvenile thyroid cartilage was negative for type X collagen, but showed a strong staining reaction for type II collagen. Type X collagen and calcium deposition were first detected in thyroid cartilage of 18-to 21-year-old adults. Type X collagen was restricted to large chondrocytes near or in mineralized cartilage, confirming the notion that type X collagen precedes mineralization. From these observations it was concluded that chondrocytes in thyroid cartilage undergo differentiation steps that are similar, but much slower, compared to cells in growth plate and sternal cartilage. Some type X collagen-positive areas also showed staining for type I collagen, suggesting that there is a further differentiation of chondrocytes to cells which are characterized by the simultaneous synthesis of type X and I collagen. However, a dedifferentiation process during aging of thyroid cartilage where cells switch from synthesis of type II to type I collagen cannot be excluded
— id: 83056, year: 1994, vol: 101, page: 27, stat: Journal Article,

Temporal and spatial localization of type I and II collagens in human thyroid cartilage
Claassen, H; Kirsch, T
1994 Mar;189(3):237-242, Anatomy & embryology
Thyroid cartilages of various ages were investigated by immunofluorescence staining for localization of the fibrillar collagen types I and II in order to understand the tissue remodeling occurring during the mineralization and ossification of thyroid cartilage. In fetal and juvenile thyroid cartilages, type I collagen was restricted to the inner and outer perichondrium, while type II collagen was localized in the matrix of hyaline cartilage. However, in advanced ages, type I collagen was also localized in the pericellular and in the interterritorial matrix of intermediate and central chondrocytes of thyroid cartilage. The matrix of peripheral chondrocytes was negative for type I collagen. This suggest that some chondrocytes in thyroid cartilage undergo a differentiation to type I collagen-producing chondrocytes. At the beginning of ossification, bone-related type I collagen was chiefly detected in the central cartilage layer, but was never deposited first from the perichondrium in the direction to the subperichondrial cartilage. This observation confirmed previous findings showing that osteogenesis mainly follows an endochondral ossification pattern. Interterritorial matrix failed to react with the type II collagen antibody in men from the beginning of the third decade, and later still in women, even after treatment with hyaluronidase. These observations indicate that major matrix changes occur faster in male than in female thyroid cartilage
— id: 83053, year: 1994, vol: 189, page: 237, stat: Journal Article,

Roles of the nucleational core complex and collagens (types II and X) in calcification of growth plate cartilage matrix vesicles
Kirsch, T; Ishikawa, Y; Mwale, F; Wuthier, R E
1994 Aug 5;269(31):20103-20109, Journal of biological chemistry
Matrix vesicles (MV) were shown to initiate mineralization in cartilage and other vertebrate tissues. However, the factors that drive this process remain to be fully elucidated. Recent studies have shown that a preformed nucleational core consisting mainly of a Ca(2+)-phosphatidylserine-Pi complex, is necessary for the accumulation of Ca2+ by MV. In addition, the collagens attached to the MV surface were shown to play an important role in stimulating Ca2+ uptake. In this study, we extend this knowledge by showing that both, the nucleational core and the collagens (types II and X), are co-requirements for rapid influx of Ca2+ into intact MV. MV to which collagen fragments were attached were released from hypertrophic chicken cartilage by trypsin and collagenase digestion (trypsin/collagenase-released MV (TCRMV), while 'collagen-free' MV were released by hyaluronidase and collagenase digestion (hyaluronidase/collagenase-released MV (HCRMV). In contrast to TCRMV which showed active uptake of Ca2+, HCRMV showed only little uptake. However, binding of native type II collagen to HCRMV stimulated uptake of Ca2+. Sucrose gradients separated TCRMV and HCRMV into three different density fractions: a low density top fraction (SI), an intermediate density middle fraction (SII), and a high density pellet fraction (SIII). The SIII fractions of TCRMV and HCRMV contained significantly higher levels of mineral ions than did the SI and SII fractions. Only the SIII fraction of TCRMV which contained a stable nucleational core and surface-attached collagens, showed active Ca2+ uptake; all other sucrose fractions of TCRMV and HCRMV showed little or no uptake. Detergent treatment to purposely rupture the membrane greatly enhanced Ca2+ uptake by the SIII fraction of HCRMV, presumably by exposing the internal nucleational core. Addition of either native type II or type X collagen to the intact SIII fraction of HCRMV stimulated Ca2+ uptake to a level similar to that of the SIII fraction of TCRMV; however, incubation of the SI and SII fractions of either TCRMV or HCRMV with type II or X collagen did not activate Ca2+ uptake. These findings indicate that both a functional nucleational core and surface-attached collagens need to be present to support active mineralization of MV
— id: 83057, year: 1994, vol: 269, page: 20103, stat: Journal Article,

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage
Aigner, T; Reichenberger, E; Bertling, W; Kirsch, T; Stoss, H; von der Mark, K
1993 ;63(4):205-211, Virchows archiv B. Cell pathology including molecular pathology
Type X collagen is a short chain, non-fibril-forming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic alpha 1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, alpha 1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression
— id: 83060, year: 1993, vol: 63, page: 205, stat: Journal Article,

EFFECT OF COLLAGENS ON THE CALCIUM-UPTAKE OF CRMV (COLLAGENASE RELEASED MATRIX VESICLES)
KIRSCH, T; WUTHIER, RE
1993 ;7(7):A1307-A1307, FASEB journal
— id: 83086, year: 1993, vol: 7, page: A1307, stat: Journal Article,

A POSSIBLE REGULATORY FUNCTION OF AMYLIN ON THE MINERALIZATION OF GROWTH PLATE CHONDROCYTES
MWALE, F; KIRSCH, T; ISHIKAWA, Y; WUTHIER, R
1993 ;7(7):A1238-A1238, FASEB journal
— id: 83087, year: 1993, vol: 7, page: A1238, stat: Journal Article,

Immunohistochemical detection of interstitial collagens in bone and cartilage tissue remnants in an infant Peruvian mummy
Nerlich, A G; Parsche, F; Kirsch, T; Wiest, I; von der Mark, K
1993 Jul;91(3):279-285, American journal of physical anthropology
We investigated the immunohistochemical presence of various collagen types in bone and cartilage tissue from an infant Peruvian mummy dating between 500 and 1000 A.D. which had been excavated at the necropolis of Las Trancas in the Nazca region in Peru. Following careful rehydration and decalcification of the tissue, the mummy tissue showed morphologically good preservation of the matrix, which could be shown to be composed of various collagen types in a typical pattern. Bone consisted of a collagen I matrix with a small rim of collagen III and V at the endosteal lining and a pericellular collagen V staining around osteocytic holes. In the hypertrophic cartilage of the epiphyseal growth plate, a typical pattern of collagen types II and X could be found. These observations provide evidence that in well-preserved mummy tissue the antigenic determinants of major matrix components are still adequately preserved for an immunohistochemical analysis. This technique may thus be a very helpful tool for the analysis of pathologic processes of historic bone tissue. It may also allow in certain circumstances a distinction between pseudopathologic tissue destruction and pathologic tissue alteration
— id: 83058, year: 1993, vol: 91, page: 279, stat: Journal Article,

Outer membrane protein YadA of enteropathogenic yersiniae mediates specific binding to cellular but not plasma fibronectin
Schulze-Koops, H; Burkhardt, H; Heesemann, J; Kirsch, T; Swoboda, B; Bull, C; Goodman, S; Emmrich, F
1993 Jun;61(6):2513-2519, Infection & immunity
The binding of bacteria or bacterial products to host proteins of tissue extracellular matrix may be a mechanism of tissue adherence. We investigated interactions of the plasmid-encoded outer membrane protein YadA, which confers pathogenic functions on enteropathogenic yersiniae, with fibronectin. Attachment of YadA-positive and YadA-negative recombinant Yersinia enterocolitica strains to cartilage-derived human cellular fibronectin and human plasma fibronectin in the solid phase revealed that YadA mediates binding of yersiniae to cellular fibronectin in a saturable, concentration-dependent manner. The interaction could be inhibited by an anti-YadA-specific anti-serum. An anti-beta 1-integrin antibody and the synthetic peptide G-R-G-D-S-P, representing the binding site for alpha 5 beta 1-integrin on fibronectin, did not block attachment of YadA-positive yersiniae to cellular fibronectin, indicating a binding site for YadA on cellular fibronectin independent of the R-G-D-S-containing site. By contrast, YadA failed to mediate binding to plasma fibronectin immobilized on nitrocellulose or plastic surfaces. These observations provide evidence for the hypothesis that the binding region for YadA in cellular fibronectin is not present in plasma fibronectin. This study is the first report on differential binding of bacteria to splicing variants of fibronectin. Further experiments might answer the question whether binding of YadA to cellular fibronectin contributes to the pathogenesis of yersiniae, both to the initial adhesion of the bacteria to the matrices of the host and to the arthritogenic potential of enteropathogenic yersiniae
— id: 83059, year: 1993, vol: 61, page: 2513, stat: Journal Article,

ENTEROPATHOGENIC YERSINIAE SPECIFICALLY ADHERE TO CELLULAR BUT NOT PLASMA FIBRONECTIN VIA THE PLASMID-ENCODED OUTER-MEMBRANE PROTEIN YADA
SCHULZEKOOPS, H; BURKHARDT, H; HEESEMANN, J; KIRSCH, T; GOODMAN, S; EMMRICH, F
1993 ;36(9):S226-S226, Arthritis & rheumatism
— id: 83098, year: 1993, vol: 36, page: S226, stat: Journal Article,

Characterization of the nucleational core complex responsible for mineral induction by growth plate cartilage matrix vesicles
Wu, L N; Yoshimori, T; Genge, B R; Sauer, G R; Kirsch, T; Ishikawa, Y; Wuthier, R E
1993 Nov 25;268(33):25084-25094, Journal of biological chemistry
The factors that drive mineralization of matrix vesicles (MV) have proven difficult to elucidate; in the present studies, various detergent, chemical, and enzyme treatments were used to reveal the nature of the nucleational core. Incubation with detergents that permeabilized the membrane enhanced calcification of treated MV incubated in synthetic cartilage lymph. While detergents removed most of the membrane lipid, they left significant amounts of the MV annexins and nearly all of the Ca2+, Pi, and Zn2+. Extraction with 1 M NaCl removed much of the Ca2+ and Pi present in MV, markedly reducing Ca2+ accumulation; these effects could be prevented by low levels of Ca2+ and Pi in the NaCl extractant. Treatment with chymotrypsin appeared to damage proteins required for MV mineralization; further treatment with detergents to bypass the membrane reactivated MV mineralization. Treatment of MV with pH 6 citrate removed Ca2+ and Pi, destroying their ability to mineralize; subsequent treatment with detergents did not reactivate these MV. Incubation of the detergent-resistant core with o-phenanthroline complexed Zn2+ and stimulated mineralization; addition of Zn2+ to synthetic cartilage lymph blocked the ability of the core to mineralize. These studies show that once the nucleational core complex is formed, the membrane-enclosed domain is no longer essential for MV calcification. Our findings indicate that the MV core contains two main components as follows: a smaller membrane-associated complex of Ca2+, Pi, phosphatidylserine, and the annexins that nucleates crystalline mineral formation, and a larger pool of Ca2+ and Pi bound to lumenal proteins. These proteins appear to bind large amounts of mineral ions, stabilize the nucleational complex, and aid its transformation to the first crystalline phase. Once nucleated, the crystalline phase appears to feed on protein-bound mineral ions until external ions enter through the MV ion channels. Zn2+ appears to regulate gating of the ion channels and conversion of the nucleational complex to the crystalline state
— id: 83061, year: 1993, vol: 268, page: 25084, stat: Journal Article,

Selective binding of anchorin CII (annexin V) to type II and X collagen and to chondrocalcin (C-propeptide of type II collagen). Implications for anchoring function between matrix vesicles and matrix proteins
Kirsch, T; Pfaffle, M
1992 Sep 28;310(2):143-147, FEBS letters
Anchorin CII is a collagen binding protein of the annexin family associated with plasma membranes of chondrocytes, osteoblasts, and many other cells. As a major constituent of cartilage-derived matrix vesicles it has been shown to bind to native type II and X collagen. In accordance with this observation, here we show the localization of anchorin CII in the extracellular matrix of calcifying cartilage in the fetal human growth plate, and that it was restricted to the chondrocyte surface in proliferating and resting cartilage. Furthermore, we present evidence, using a slot blot assay, that anchorin CII not only binds to native type II and X collagen, but also to chondrocalcin, the carboxy-terminal extension of type II procollagen, in a calcium-independent manner. Pepsin digestion of type II collagen results in loss of anchorin CII binding, confirming our previous notion that the telopeptide region of type II collagen carries anchorin CII binding sites
— id: 83046, year: 1992, vol: 310, page: 143, stat: Journal Article,

Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and X collagen and matrix mineralization
Kirsch, T; Swoboda, B; von der Mark, K
1992 Dec;52(1):89-100, Differentiation
In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type X collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hypertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentiation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or X collagen antibodies revealed that type X collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and X collagen, indicating further differentiation of chondrocytes to a new type of 'post-hypertrophic' cell. This cell type, descending from a type X collagen producing chondrocyte, is different from the previously described 'dedifferentiated' or 'modulated' types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type X collagen synthesis. First calcium deposits were detected by alizarine red S staining in type X collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type X collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis
— id: 83045, year: 1992, vol: 52, page: 89, stat: Journal Article,

Remodelling of collagen types I, II and X and calcification of human fetal cartilage
Kirsch, T; von der Mark, K
1992 Aug;18(2):107-117, Bone & mineral
Evidence from recent studies on type X collagen in hypertrophic chick cartilage suggests that it may be involved in cartilage calcification. Here we compare the distribution of type X collagen with that of calcium mineral deposition in fetal human growth plate cartilages of long bones and ribs. Using a specific antibody we demonstrate the presence of type X collagen in a narrow, sharply defined zone of hypertrophic chondrocytes. Type X collagen was also localized in the calcifying cartilage remaining within spongy bone trabecules. Calcium deposits were, however, detected by alizarine red S only in the lower hypertrophic zone and in bone, confirming the notion that type X collagen is deposited in the hypertrophic cartilage before mineral deposition. By immunofluorescence double staining we demonstrate codistribution of type II and X collagen in the hypertrophic zone, while type I collagen was absent from hypertrophic cartilage matrix; it was detected only in the perichondrium, in vascular cavities, and in osteoid and bone. From these observations we conclude that the sequence of events leading to cartilage mineralization begins with chondrocyte hypertrophy, followed by type X collagen synthesis and finally by deposition of calcium mineral
— id: 83048, year: 1992, vol: 18, page: 107, stat: Journal Article,

Localization of collagen X in human fetal and juvenile articular cartilage and bone
Nerlich, A G; Kirsch, T; Wiest, I; Betz, P; von der Mark, K
1992 Dec;98(5):275-281, Histochemistry
The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilage-bone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often 'spotty' distribution of collagen X with increasing cartilage 'degeneration' associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and 'spotty' collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected
— id: 83047, year: 1992, vol: 98, page: 275, stat: Journal Article,

The fate of chondrocytes in osteoarthritic cartilage: regeneration, dedifferentiation or hypertrophy?
von der Mark, K; Kirsch, T; Aigner, T; Reichenberger, E; Nerlich, A; Weseloh, G; Stoess, H
Articular cartilage and osteoarthritis New York : Raven Press, 1992,
— id: 4801, year: 1992, vol: , page: 221, stat: Chapter,

Type X collagen synthesis in human osteoarthritic cartilage. Indication of chondrocyte hypertrophy
von der Mark, K; Kirsch, T; Nerlich, A; Kuss, A; Weseloh, G; Gluckert, K; Stoss, H
1992 Jul;35(7):806-811, Arthritis & rheumatism
OBJECTIVE: To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OA) cartilage, using type X collagen as a specific marker. METHODS. The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with 3H-proline, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and II collagen was analyzed immunohistochemically. RESULTS. Immunostaining revealed an irregular distribution of type X collagen, which was localized around chondrocyte clusters in fibrillated OA cartilage, but was absent from the noncalcified region of normal articular cartilage. Freshly isolated OA chondrocytes synthesized predominantly type X collagen, while control chondrocytes synthesized mostly type II collagen. CONCLUSION. Our findings indicate focal premature chondrocyte differentiation to hypertrophic cells in OA cartilage
— id: 83049, year: 1992, vol: 35, page: 806, stat: Journal Article,

Ca2+ binding properties of type X collagen
Kirsch, T; von der Mark, K
1991 Dec 2;294(1-2):149-152, FEBS letters
Type X collagen is a developmentally regulated collagen that is only synthesized by chondrocytes of the hypertrophic and calcifying zone in fetal cartilage. There is evidence in the literature that type X collagen may be involved in cartilage calcification. Here we show that type X collagen synthesis precedes calcium deposition in nodules of fetal human chondrocytes forming in cell culture and present evidence that type X collagen binds calcium in a specific and dose dependent manner. In an assay using bovine type X collagen coupled to beads and 45Ca2+ we determined a total of about 15 binding sites per alpha 1(X) chain with a dissociation of 32 microM
— id: 83050, year: 1991, vol: 294, page: 149, stat: Journal Article,

Isolation of human type X collagen and immunolocalization in fetal human cartilage
Kirsch, T; von der Mark, K
1991 Mar 28;196(3):575-580, European journal of biochemistry
Type X collagen was extracted with 1 M NaCl and 10 mM dithiothreitol at neutral pH from fetal human growth plate cartilage and purified to homogeneity by gel filtration and anion-exchange chromatography. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 66,000 under reducing conditions, and as a high-Mr oligomer under non-reducing conditions. Purified collagenase digests most of the molecule; pepsin digestion at 4 degrees C decreases the Mr of the monomer to 53,000. A rabbit antiserum was raised against purified human type X collagen; the IgG fraction was specific for this collagen by criteria of ELISA and immunoblotting after absorption with collagen types I, II, VI, IX and XI. Immunohistological studies localized type X collagen exclusively in the zone of hypertrophic and calcifying cartilage
— id: 83051, year: 1991, vol: 196, page: 575, stat: Journal Article,

Isolation of bovine type X collagen and immunolocalization in growth-plate cartilage
Kirsch, T; von der Mark, K
1990 Jan 15;265(2):453-459, Biochemical journal
Type X collagen was extracted with 1 M-NaCl and 10 mM-dithiothreitol at neutral pH from fetal-bovine growth cartilage and purified to homogeneity by using f.p.l.c. gel filtration on a Superose 12 column, followed by ion-exchange chromatography on a Mono Q column. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 58,000 under reducing conditions and as a high-Mr oligomer in its unreduced form. The amino acid composition is similar to the published composition of chick type X collagen. Pepsin digestion at 4 degrees C decreases the Mr of the monomer to 43,000; purified bacterial collagenase digests most of the molecule, leaving a non-collagenous domain of apparent Mr 15,000, which probably represents the C-terminal globular domain. The IgG fraction from a rabbit antiserum raised against purified bovine type X collagen was specific for this collagen by the criteria of e.l.i.s.a. and immunoblotting after immunoabsorption with collagen types I, II, IX and XI. Immunofluorescence localization of type X collagen in sections of fetal-bovine and human cartilage was possible after acetone fixation of sections and hyaluronidase treatment. Type X collagen was restricted to the zone of hypertrophic and calcified cartilage inside the bone spicules of the growth plate
— id: 83052, year: 1990, vol: 265, page: 453, stat: Journal Article,