Biomedical subjects

T Kirsch

Publications and source records attributed to T Kirsch.

At least 19 recordsLinked to original sources

PubMed2006

Nephrin loss in experimental diabetic nephropathy is prevented by deletion of protein kinase C alpha signaling in-vivo.

Albuminuria in diabetic nephropathy is due to endothelial dysfunction, a loss of negative charges in the basement membrane, and changes a of the slit-membrane diaphragm composition. We have recently shown that protein kinase C alpha (PKCalpha)-deficient mice are protected against the development of albuminuria under diabetic conditions. We here tested the hypothesis that PKCalpha mediates the hyperglycemia-induced downregulation of the slit-diaphragm protein nephrin. After 8 weeks of streptozotocin (STZ)-induced hyperglycemia the expression of glomerular nephrin was significantly reduced. In contrast, other slit-diaphragm proteins such as podocin and CD2AP were unaltered in diabetic state. In PKCalpha-/- mice, hyperglycemia-induced downregulation of nephrin was prevented. Podocin and CD2AP remained unchanged. In addition, the nephrin messenger RNA expression was also reduced in hyperglycemic wild-type mice but remained unaltered in PKCalpha-/- mice. We postulate that the underlying mechanism of the hyperglycemia-induced regulation of various proteins of the glomerular filtration barrier is a PKCalpha-dependent regulation of the Wilms' Tumor Suppressor (WT1) which previously has been shown to act as a direct transcription factor on the nephrin promoter. Our data suggest that PKCalpha activation may be an important intracellular signaling pathway in the regulation of nephrin expression and glomerular albumin permeability in the diabetic state.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol.

BACKGROUND: Circulating endothelial cells (CECs) have been identified as markers of vascular damage in a variety of disorders, such as myocardial infarction, vasculitis, and transplantation. CD146-driven immunomagnetic isolation has gained widespread use, but the technique is hampered by the lack of a definition of CECs and the absence of a consensus for their enumeration. AIM: To evaluate several variables influencing immunomagnetic isolation of CECs, formulate a definition for CECs and propose a consensus protocol for their enumeration. METHODS: We devised a protocol based on CD146-driven immunomagnetic isolation and a subsequent confirmatory step with Ulex-Europaeus-Lectin-1 staining. In a multi-center effort, we evaluated the preanalytical and analytical phases of this protocol. We evaluated the effects of storage, anticoagulation and density centrifugation, and compiled previous experience with this technique. RESULTS: Our protocol permitted unequivocal identification of CECs with acceptable reproducibility. There was an effect of storage time in that median cell numbers declined to only 87.5% of their baseline values during 24 h of storage at 4 degrees C. Recovery was lower with citrate than with ethylene-diamine tetra-acetic acid after 4 h of storage; density centrifugation was also associated with lower recovery. We provide a comprehensive list of technical recommendations and potential pitfalls. Finally, based on our experience with this protocol and a recent consensus workshop, we formulated a working definition for CECs. CONCLUSION: Our work represents an important step toward consensus regarding the CECs. Our recommendations represent the experience of three major centers and should now be scrutinized by others in the field.

CD146 Antigen↗

PubMed2005

Circulating endothelial cells in relapse and limited granulomatous disease due to ANCA associated vasculitis.

OBJECTIVES: To evaluate numbers of circulating endothelial cells (CECs) in ANCA associated vasculitis and compare vasculitic relapse with limited granulomatous disease. METHODS: Sixteen patients with vasculitic relapse of ANCA associated vasculitis and 12 patients with limited granulomatous disease due to Wegener's granulomatosis (WG) were studied. Six patients with newly diagnosed vasculitic disease and six patients with vasculitis with infectious complications were also studied. Twenty two patients in remission were studied, as were 20 healthy controls. Counting of CECs was performed with anti-CD146 driven immunomagnetic isolation and staining with Ulex Europaeus lectin 1(UEA-1). RESULTS: Patients with vasculitic relapse had markedly increased numbers of circulating endothelial cells (12-800 cells/ml, median 88 cells/ml) as did patients with newly diagnosed systemic vasculitis (20-216 cells/ml, median 56 cells/ml). Patients with limited granulomatous disease due to WG had only slightly increased cell numbers (4-44 cells/ml, median 20 cells/ml), which were similar to those of patients in remission (4-36 cells/ml, median 16 cells/ml). Numbers of CECs in patients with granulomatous disease were significantly lower than in those patients with relapse or new onset vasculitis (p<0.001). Cell numbers in patients with relapse and new onset vasculitis declined with immunosuppressive treatment. Patients with infection had 4-36 cells/ml (median 10 cells/ml). A cut off value of 20 cells/ml for a positive result yielded 64% specificity and 95% sensitivity for active systemic vasculitis; the positive predictive value was 63% and the negative predictive value 95%. CONCLUSION: Markedly increased numbers of CECs discriminate active vasculitis from limited granulomatous disease and remission. These findings add further proof to the concept of CECs as a marker of ANCA associated small vessel vasculitis.

Acute Disease↗

PubMed2005

Biocompatibility parameters of different dialysis membranes assessed during systemic inflammation.

BACKGROUND: We explored whether biocompatible dialyzer membranes modulate the inflammatory response during blood contact in patients with systemic inflammation. METHODS: 15 patients with end-stage renal disease and systemic inflammation (mean serum C-reactive protein 86 +/- 4 mg/l) were randomly treated with Cuprophan (CU), polyamide (PA) and vitamin-E coated (VEC) membrane-based dialyzers. RESULTS: Changes in blood pressure, capillary blood oxygen saturation and differential blood counts during the hemodialysis session were not significantly different between the three dialyzers. Baseline blood levels of activated circulating complement (C3a) were more than 100 times above normal, and unlike expected they decreased during hemodialysis treatments (CU: from 7,389 +/- 783 to 5,423 +/- 761 ng/ml; PA: from 7,379 +/- 980 to 5,690 +/- 714 ng/ml; VEC: from 7.377 +/- 714 to 5,360 +/- 1,005 ng/ml; all n.s.). No significant differences between treatments were found with respect to changes in blood concentrations of TNF-alpha, interleukin-6 and interleukin-1 receptor antagonist as well as ICAM-1 (CU: from 451 +/- 41 to 477 +/- 41 ng/ml; PA: from 437 +/- 42 to 449 +/- 40 ng/ml; VEC: from 461 +/- 43 to 460 +/- 47 ng/ml). Furthermore, generation of reactive oxygen species by mononuclear blood cells was comparable during hemodialysis with the CU, PA and VEC dialyzer. CONCLUSION: The choice of dialyzer membrane material does not affect most aspects of biocompatibility when patients have significant systemic inflammation. This confounding variable should be taken into account in studies exploring the effects of biocompatible dialyzer membranes.

Female↗

PubMed2005

Compartment-specific quantitative gene expression analysis after laser microdissection from archival renal allograft biopsies.

BACKGROUND: Various immunological and non-immunological pathomechanisms are responsible for the cellular damage in renal allografts. Since the kidney is an anatomically complex organ with functional and morphological heterogeneous compartments (interstitium, tubuli, vessels, glomeruli), the local response to injury maybe variable, therefore, the identification of local pathomechanisms is important. AIM: To elucidate any discrepancies in quantitative mRNA expression profiles between a total specimen analysis and a cell-specific evaluation after laser microdissection. METHODS: Real-time RT-PCR was performed for complement component C3 and heme oxygenase-1 (HO-1) genes compared to the housekeeping gene beta-actin using whole section RNA extracted from formalin-fixed and paraffin-embedded archival material of 16 explanted, rejected renal allografts. Ten non-transplant nephrectomies served as controls. For five cases from each group, five different compartments of the organs (interstitium, proximal tubuli, distal tubuli, vessels, glomeruli) were microdissected and quantitative analysis for C3 and HO-1 was performed identically. RESULTS: Whole section mRNA expression analysis: the data showed a constant expression of the housekeeping gene beta-actin, a 7-fold increased expression of C3 and a 3-fold decreased expression of HO-1 in the allograft group as compared to the control group. mRNA expression results from microdissected compartments: in the control group, C3 and HO-1 expression could only be detected in the proximal tubuli of all cases whereas all five compartments analyzed from the rejecting kidneys showed expression of the two genes. In the allografts, expression levels of the investigated genes varied considerably not only among the different compartments but between individual cases as well. CONCLUSION: Laser microdissection combined with real-time RT-PCR is a feasible approach for retrospective quantitative gene expression analysis in formalin-fixed and paraffin-embedded renal allograft specimens. As shown for C3 and HO-1, cell-specific expression patterns ofpathogenetically relevant genes vary considerably between individual cases. A close correlation of morphology and cell-specific gene expression analysis will contribute to the elucidation of the complex pathogenesis of chronic renal allograft nephropathy.

Actins↗

PubMed2001

Altered gene expression in cerebral capillaries of stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats (SHRSP) are a well-characterized, genetic model for stroke. We showed earlier that the structure and function of the tight junctions in SHRSP blood-brain barrier endothelial cells is disturbed prior to stroke. To investigate the molecular events leading to endothelial dysfunction in SHRSP cerebral capillaries, we carried out suppression subtractive hybridization (SSH) in combination with a cDNA filter screening step. We identified two cDNA fragments that were upregulated in SHRSP, compared to stroke-resistant spontaneously hypertensive rats (SHR), and found open reading frames of 133 and 138 amino acids, respectively. These peptides did not match any known proteins in public databases. A third upregulated SHRSP cDNA fragment was identified as the rat sulfonylurea receptor 2B (SUR2B). We also isolated and cloned the cDNA of the rat homologue for the mouse G-protein signaling 5 (RGS5) regulator. This regulator was downregulated in SHRSP. We used in situ hybridization to show that rat RGS5 is expressed in the brain capillary endothelium and in the choroid plexus. Our findings may lead to the identification of new stroke-related genes.

ATP-Binding Cassette Transporters↗

PubMed2001

Authentic matrix vesicles contain active metalloproteases (MMP). a role for matrix vesicle-associated MMP-13 in activation of transforming growth factor-beta.

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.

Cell Membrane↗

PubMed2001

Type IIA procollagen: expression in developing chicken limb cartilage and human osteoarthritic articular cartilage.

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.

Aged↗

PubMed2001

Expression of early and late differentiation markers (proliferating cell nuclear antigen, syndecan-3, annexin VI, and alkaline phosphatase) by human osteoarthritic chondrocytes.

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.

Alkaline Phosphatase↗

PubMed2001

Vascular endothelial growth factor in articular cartilage of healthy and osteoarthritic human knee joints.

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.

Cartilage, Articular↗

PubMed2001

The crystal structure of the BMP-2:BMPR-IA complex and the generation of BMP-2 antagonists.

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 BMP2: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-II 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.

Amino Acid Sequence↗

PubMed2000

Structural alterations of tight junctions are associated with loss of polarity in stroke-prone spontaneously hypertensive rat blood-brain barrier endothelial cells.

The mechanisms leading to stroke in stroke-prone spontaneously hypertensive rats (SHRSP) are not well understood. We tested the hypothesis that the endothelial tight junctions of the blood-brain barrier are altered in SHRSP prior to stroke. We investigated tight junctions in 13-week-old SHRSP, spontaneously hypertensive stroke-resistant rats (SHR) and age-matched Wistar-Kyoto rats (WKY) by electron microscopy and immunocytochemistry. Ultrathin sections showed no difference in junction structure of cerebral capillaries from SHRSP, SHR and WKY, respectively. However, using freeze-fracturing, we observed that the blood-brain barrier specific distribution of tight junction particles between P- and E-face in WKY (58.7+/-3.6%, P-face; 41.2+/-5.59%, E-face) and SHR (53.2+/-19. 3%, P-face; 55.6+/-13.25%, E-face) was changed to an 89.4+/-9.9% predominant E-face association in cerebral capillaries from SHRSP. However, the expression of the tight junction molecules ZO-1, occludin, claudin-1 and claudin-5 was not changed in capillaries of SHRSP. Permeability of brain capillaries from SHRSP was not different compared to SHR and WKY using lanthanum nitrate as a tracer. In contrast, analysis of endothelial cell polarity by distribution of the glucose-1 transporter (Glut-1) revealed that its abluminal:luminal ratio was reduced from 4:1 in SHR and WKY to 1:1 in endothelial cells of cerebral capillaries of SHRSP. In summary, we demonstrate that early changes exist in cerebral capillaries from a genetic model of hypertension-associated stroke. We suggest that a disturbed fence function of the tight junctions in SHRSP blood-brain barrier endothelial cells may lead to subtle changes in polarity. These changes may contribute to the pathogenesis of stroke.

Animals↗

PubMed2000

The roles of annexins and types II and X collagen in matrix vesicle-mediated mineralization of growth plate cartilage.

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.

Animals↗

PubMed2000

BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II.

Bone morphogenetic protein-2 (BMP-2) induces bone formation and regeneration in adult vertebrates and regulates important developmental processes in all animals. BMP-2 is a homodimeric cysteine knot protein that, as a member of the transforming growth factor-beta (TGF-beta) superfamily, signals by oligomerizing type I and type II receptor serine-kinases in the cell membrane. The binding epitopes of BMP-2 for BMPR-IA (type I) and BMPR-II or ActR-II (type II) were characterized using BMP-2 mutant proteins for analysis of interactions with receptor ectodomains. A large epitope 1 for high-affinity BMPR-IA binding was detected spanning the interface of the BMP-2 dimer. A smaller epitope 2 for the low-affinity binding of BMPR-II was found to be assembled by determinants of a single monomer. Symmetry-related pairs of the two juxtaposed epitopes occur near the BMP-2 poles. Mutations in both epitopes yielded variants with reduced biological activity in C2C12 cells; however, only epitope 2 variants behaved as antagonists partially or completely inhibiting BMP-2 activity. These findings provide a framework for the molecular description of receptor recognition and activation in the BMP/TGF-beta superfamily.

Amino Acid Sequence↗

PubMed2000

Isolation of recombinant BMP receptor IA ectodomain and its 2:1 complex with BMP-2.

Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily which induces bone formation and regeneration, and important steps during early embryonic development. BMP-2 signals via oligomerization of type I and type II serine/threonine kinase receptors. We report here expression of the extracellular domain of the human type IA receptor for BMP-2 (BMPR-IA) in Escherichia coli. This soluble form of BMPR-IA (sBMPR-IA) was purified employing a BMP-2 affinity column. Gel filtration experiments and analysis of gel filtration fractions by polyacrylamide electrophoresis and densitometry reveal that BMP-2 forms a defined 1:2 complex with sBMPR-IA that can be purified and hopefully used for crystallization studies.

Binding Sites↗

PubMed2000

Matrix vesicles mediate mineralization of human thyroid cartilage.

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.

Adult↗

PubMed2000

Crystal structure of the BMP-2-BRIA ectodomain complex.

Bone morphogenetic proteins (BMPs) belong to the large transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. BMP-2 can induce ectopic bone and cartilage formation in adult vertebrates and is involved in central steps in early embryonal development in animals. Signaling by these cytokines requires binding of two types of transmembrane serine/threonine receptor kinase chains classified as type I and type II. Here we report the crystal structure of human dimeric BMP-2 in complex with two high affinity BMP receptor IA extracellular domains (BRIAec). The receptor chains bind to the 'wrist' epitopes of the BMP-2 dimer and contact both BMP-2 monomers. No contacts exist between the receptor domains. The model reveals the structural basis for discrimination between type I and type II receptors and the variability of receptor-ligand interactions that is seen in BMP-TGF-beta systems.

Amino Acid Sequence↗

PubMed2000

Activation of annexin II and V expression, terminal differentiation, mineralization and apoptosis in human osteoarthritic 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.

Adult↗