Xylosyltransferase-I Regulates Glycosaminoglycan Synthesis during the Pathogenic Process of Human Osteoarthritis

Loss of glycosaminoglycan (GAG) chains of proteoglycans (PGs) is an early event of osteoarthritis (OA) resulting in cartilage degradation that has been previously demonstrated in both huma and experimental OA models. However, the mechanism of GAG loss and the role of xylosyltransferase-I (XT-I) that initiates GAG biosynthesis onto PG molecules in the pathogenic process of human OA are unknown. In this study, we have characterized XT-I expression and activity together with GAG synthesis in human OA cartilage obtained from different regions of the same joint, defined as “normal”, “late-stage” or adjacent to “late-stage”. The results showed that GAG synthesis and content increased in cartilage from areas flanking OA lesions compared to cartilage from macroscopically “normal” unaffected regions, while decreased in “late-stage” OA cartilage lesions. This increase in anabolic state was associated with a marked upregulation of XT-I expression and activity in cartilage “next to lesion” while a decrease in the “late-stage” OA cartilage. Importantly, XT-I inhibition by shRNA or forced-expression with a pCMV-XT-I construct correlated with the modulation of GAG anabolism in human cartilage explants. The observation that XT-I gene expression was down-regulated by IL-1β and up-regulated by TGF-β1 indicates that these cytokines may play a role in regulating GAG content in human OA. Noteworthy, expression of IL-1β receptor (IL-1R1) was down-regulated whereas that of TGF-β1 was up-regulated in early OA cartilage. Theses observations may account for upregulation of XT-I and sustained GAG synthesis prior to the development of cartilage lesions during the pathogenic process of OA

Anticorps antiphospholipides (Applications des méthodes de purification et d'un modèle murin, à l'étude de leurs spécificités antigéniques et de leurs activités catalytiques)

Les anticorps antiphospholipides constituent une famille hétérogène d'immunoglobulines. Ils reconnaissent des protéines cofacteurs comme la b2glycoprotéine I.. Une réaction croisée a été mise en évidence entre les anticorps anti-b2GPI et anti-PF4-héparine chez des patients SAPL jamais exposé à l'héparine. Cette réaction croisée a été reproduite pour la première fois dans un modèle murin de SAPL crée en exploitant le réseau idiotypique. Ce modèle animal a montré aussi l'implication des anticorps murins induits anti-b2GPI dans la thrombose spontanée et de fabriquer des anticorps monoclonaux anti-b2GPI murins. Deux méthodes chromatographiques de pseudoaffinité ont été envisagées pour purifier ces anticorps et étudier leurs activités catalytiques. En exploitant la présence des molécules d'adhésion cellulaire solubles (E-sélectines, ICAM-1, VCAM-1) dans le sérum de 114 patients SAPL. Les taux des E-sélectines se sont révélés les meilleurs marqueurs de risque thrombotique chez ces patients.The antiphospholipid antibodies constitute a heterogeneous family of immunoglobulins. They recognize protein cofactors like the b2-glycoprotein I. A cross reactivity was shown between the anti-b2GPI antibodies and anti-PF4-heparin among antiphospholipid syndrome (APS) patients never exposed to heparin. This cross reactivity was reproduced for the first time using a murine model of APS created by exploiting the idiotypic network. This animal model also showed the implications of the murine antibodies anti-b2GPI in spontaneous thrombosis (in mice) and also produced monoclonal anti-b2GPI antibodies. Two pseudoaffinity chromatographic methods were modulled to purify these antibodies and their catalytic activities were characterised. By exploiting the presence of soluble cellular molecule adhesion (E-selectines, ICAM-1, VCAM-1) using the sera of 114 APS patients it was concluded, that the E-selectines rates appeared the best markers of thrombotic risk among these patients.COMPIEGNE-BU (601592101) / SudocSudocFranceF

Cyclic stretch-induced thrombin generation by rat vascular smooth muscle cells is mediated by the integrin alpha(v)beta(3) pathway

International audienceVascular smooth muscle cell (VSMC) phenotypic modulation plays a pivotal role in atherothrombotic diseases. Thrombin generation at the surface of VSMCs and activation of integrin mechanotransduction pathways represent potential mechanisms. Here, we examine whether mechanical stretch increases thrombin generation on cultured rat aortic VSMCs. The integrin (v3) antagonist peptide (cRGDPV) dose-dependently decreased thrombin generation without stretch. Static stretch (5, 1 Hz) failed to modify the thrombin-forming capacity of VSMCs, whereas 10 cyclic stretch during 60 and 360 min enhanced integrin (v3) expression and thrombin generation at the surface of VSMCs by 30 without inducing apoptosis. Cyclic stretch also stimulated Src phosphorylation, cleavage of talin, and binding of prothrombin to VSMCs. Upregulation of (v3) expression, Src phosphorylation, and enhanced thrombin generation by cyclic stretch were abolished by cRGDPV and silencing RNA (siRNA) against (v) as well as by selective inhibition of integrin (v3) inside-out signalling by a talin-siRNA. Complete abolition of stretch-induced VSMC-supported thrombin generation by the RGT peptide, which disrupts the interaction of Src with the (3) cytoplasmic tail, demonstrates the link between outside-in pathways involving (3)-Src interaction and thrombin activity dependent on inside-out signalling. These data show that the contribution of cyclic stretch to VSMC-supported thrombin generation is driven by the integrin (v3) signalling pathway and suggest a role for pulsatility-induced intramural thrombin in VSMC-dependent vascular remodelling

Expression of pro-inflammatory and anti-inflammatory cytokines in human OA cartilage.

<p>Total RNA was isolated from human cartilage samples obtained from early OA and late OA regions and the expression of target genes were analyzed by qPCR. Target genes in early OA were compared to late OA and expressed as fold induction. Values are mean ± SD of 3 experiments per parameter per joint per patient.</p

Representative sample of femoral condyle from human OA knee and toluidine blue stained sections.

<p>(A) Femoral condyle from human OA knee joint indicating three sites biopsied for further biochemical and molecular analysis; (a) cartilage sample taken from macroscopically unaffected area and distant from the lesion was termed “normal” (b) cartilage sample isolated from areas immediately close to lesion was termed “Next to lesion”; (c) cartilage specimen excised from lesion was termed “late-stage OA”; Cartilage samples were isolated using a 6 mm-biopsy punch. (B) Representative photomicrographs of cartilage sections showing areas from where samples were recovered for toluidine blue staining; (a) cartilage section from a normal area showing a relatively smooth articular surface; (b) section from areas close to lesion showing dense staining for PGs in mid zone of the cartilage where chondrocyte proliferation and activation occurs; (c) cartilage section from lesions showing degraded articular surface, loss of PG staining and chondrocyte cloning (original magnification ×40).</p

Effect of XT-I overexpression on GAG anabolism in chondrocytes and cartilage.

<p>(A) Immunoblot of XT-I protein in pCMV- or pCMV-XT-I-transfected chondrocytes 48 h after transfection. XT-I recombinant protein was indicated by the arrow. (B) GAG synthesis in primary human chondrocytes or (C) in cartilage explants from late OA region of human femoral condyle transfected with pCMV- (control) or pCMV-XT-I vector. XT-I- or mock-transfected cells or cartilage were labeled with ³⁵S-sulfate, and the amount of ³⁵S-labeled GAGs was measured 48 h posttransfection. Data are mean ± SD of 3 experiments per parameter, per joint, per patient. **,*Significantly (<i>P</i><0.01, <i>P</i><0.05, respectively) higher than controls.</p

Analysis of GAG synthesis and expression of XT-I and aggrecan genes.

<p>(A) GAG synthesis analysis in cartilage from “normal”, early OA and late-stage OA areas for each patient studied by ³⁵S-sulfate incorporation. Insert shows the mean of GAG synthesis rate values corresponding to different areas of cartilage sample from all patients. Values are mean ± SEM of 3 experiments per parameter, per joint, per patient. (B) and (C) Analyses of the expression of XT-I and aggrecan genes in cartilage from normal, early OA and late-stage OA areas for each patient. The expression level of the genes was analyzed by qPCR. Measurements were normalized to the control “normal”. Inserts in (B) and (C) show the mean of XT-I and aggrecan gene expression level values corresponding to different areas of cartilage sample from all patients. Values are mean ± SD of 3 experiments per parameter, per joint, per patient. **significantly (<i>P</i><0.01) higher than normal and late groups; §significantly (<i>P</i><0.05) higher than normal and late groups; *significantly (<i>P</i><0.05) lower than normal.</p

Effect of IL-1β (A) and TGF-β1 (B) on XT-I gene expression in normal articular cartilage.

<p>Cartilage samples isolated from normal regions of human femoral condyle was exposed to IL-1β or TGF-β1 and then XT-I gene expression was analyzed by qPCR. Measurements were normalized to control (non treated). Each value represents the mean ± SD of 3 experiments per parameter per joint per patient. *Significantly (<i>P</i><0.05) lower than control group; #significantly (<i>P</i><0.01) higher than control group.</p