Thrombin and plasmin induced proteoglycan release in human cartilage is par-dependent

Abstract

s / Osteoarthritis and Cartilage 20 (2012) S54–S296 S122 media. Neither fatty acids nor TNFa were cytotoxic. The increase in COX2 gene expression of cultured chondrocytes in response to TNFa was coun teracted by DHA (p<0.05) and oleic acid (p<0.05). MMP1 expression was also increased in response to TNFa and this effect was counteracted by oleic acid (p<0.05) and palmitic acid (p<0.05). GAG release by cartilage explants was decreased when cultured in oleic acid (p<0.05). Gene expression of MMP3, MMP13 and ADAMTS4 in cell cultures and NO production by cartilage explants did not significantly change in response to any fatty acid. Exposure of chondrocytes and explants to linoleic acid (n 6) in absence or presence of TNFa did not influence any of the parameters measured. Conclusions: DHA (n 3), oleic acid (n 9), and palmitic acid (saturated) are able to counteract some of the effects induced by TNFa in cartilage explants or chondrocyte cultures. This was already known for DHA, but reported for the first time for oleic acid and palmitic acid. Since fatty acids influence inflammation and degradation of cartilage, they can be regarded potential therapeutic targets in OA prevention and treatment. Figure1. The effect of fatty acids on COX2 gene expression in chondrocytes in absence or presence of 10 ng/ml TNFa (n1⁄46, from 2 donors). Acknowledgment: This study was performed within the framework of the Dutch Top Institute Pharma project #T1-213. 230 THROMBIN AND PLASMIN INDUCED PROTEOGLYCAN RELEASE IN HUMAN CARTILAGE IS PAR DEPENDENT L. Nieuwenhuizen, R.E. Schutgens, G. Roosendaal, S.C. Mastbergen, D.H. Biesma, F.P. Lafeber. Univ. Med. Ctr. Utrecht, Utrecht, Netherlands Purpose: Osteoarthritis (OA) and Rheumatoid Arthrisis (RA) are charac terized by degradation of the cartilage. Proteases of the coagulation cascade and the fibrinolytic system, such as thrombin and plasmin, are elevated in both plasma and synovial fluid of OA and RA patients and are able to induce cartilage degradation. Cross talking between coagulation and inflammation is mediated by protease activated receptors (PARs), which are expressed at increased level in OA and RA cartilage. These receptors are activated through cleavage by serine proteases, such as thrombin and plasmin. RNA interference is a process inwhich genes can be silenced sequence specific. This can be invoked by transfection of tissue/ cells with small interfering RNA (siRNA). Our aimwas to study whether the thrombin and plasmin induced cartilage damage in human cartilage was PAR dependent. Methods: Full thickness OA human articular cartilage tissue was obtained during total knee surgery. Slices of cartilage were cut asepti cally from the articular surface. Within 1 hour of dissection the slices were cut into square pieces, weighed aseptically (range, 5.0 to 15.0 mg) and each sample was individually put into culture. Cartilage was cultured for 4 days in the presence of different concentrations thrombin (10, 30, or 100nM), or plasmin (10, 30, or 100nM). In addition, cartilage was transfected with PAR1 4 small interfering RNA (600nM) or control siRNA (600nM), and cultured with thrombin (100nM) or plasmin (100nM). Cartilage matrix turnover, in terms of proteoglycan release, was determined at day 4. To investigate the silencing effect of the siRNA transfection, cartilage RNA was extracted and PAR1 4 mRNA expression was analyzed with RT PCR. Results: Thrombin and plasmin increased proteoglycan release in human cartilage in a dose dependent and statistically significant manner (500% for thrombin at 100nM; 217% for plasmin at 100nM). Thrombin and plasmin induced proteoglycan release was statistically significant reduced with PAR1 4 siRNA (60% for thrombin at 100nM; 54% for plasmin at 100nM). Control siRNA failed to reduce thrombin and plasmin induced proteoglycan release. Transfectionwith PAR1 4 siRNA resulted in complete suppression of PAR1 4 mRNA expression, whereas no effect of control siRNA on PAR1 4 mRNA expression was noted. Conclusions: These results demonstrate for the first time that thrombin and plasmin induced proteoglycan release in human cartilage is PAR dependent and offer promise for the use of siRNA as a new strategy for therapeutic intervention in OA and RA. 231 BONE SIALOPROTEIN : A KEY MEDIATOR OF THE ANGIOGENIC ACTIVITY OF HYPERTROPHIC OSTEOARTHRITIC CHONDROCYTES L. Pesesse , C. Sanchez , C. Baudouin , P. Msika , Y. Henrotin . Univ. of Liege, Liege, Belgium; 2 Laboratoires ExpanSci., IRD Direction, Epernon, France Purpose: Hypertrophic differentiation of chondrocytes in osteoarthritis (OA) is a pathological process leading to vascularization and mineraliza tion of the cartilage. The pathogenesis of OA is thought to reiterate changes that occur during endochondral ossification in which angiogenesis is required to initiate chondrocyte hypertrophic differentiation. We previ ously demonstrated that Bone Sialoprotein (BSP) production is associated with chondrocyte hypertrophy and the severity of osteoarthritic lesions. In this work, we investigated the impact of hypertrophic differentiation on the chondrocytes capacity to promote vascularization. We also speculated that BSP is a key mediator of cartilage vascularization. Methods: In alginate beads, OA chondrocytes cultured in the presence of serum undergo hypertrophic differentiation in long term culture. Using this model, we tested the effects of hypertrophic chondrocytes condi tioned medium after 24 hours serum deprivation on the invasion and migration of endothelial cells using two different models : the real time follow up of the cells performed with the xCELLigence system (Roche) and high end microscopic analysis of living endothelial cells in a wound healing assay. We also studied BSP gene expression (by RT PCR) and production (by western blot) during chondrocyte hypertrophic differentiation. The effects of IL 1b (170 pg/ml) and TNFa (25 ng/ml) were tested on the synthesis of BSP by hypertrophic chondrocytes. These cytokines were added to the culture medium before (day 7) or after (day 21) hypertrophic phenotype was reached. Finally, the effect of increased concentration of recombinant BSP (25 ng/ml to 400 ng/ml) on the production of a proangiogenic factor, interleukin 8 (IL8) and an anti angiogenic factor, thrombospondin 1 (TSP1) was studied by immunoassays. Results: Hypertrophic OA chondrocytes conditionned medium showed a higher stimulating effect on endothelial cells migration and invasion than serum containing medium (positive control). We demonstrated that BSP gene expression and protein productionwere associated with markers of hypertrophy collagen type 10 (COL X), alkaline phosphatase (AP), nucleoside triphosphate pyrophosphohydrolase (NTPPPH) in OA but this association was not observed in normal chondrocyte cultures. IL 1b or TNFa in the culture medium decreased gene expression of BSP when added at day 7 or at day 21 (p<0.05). In the same manner, both cytokines Abstracts / Osteoarthritis and Cartilage 20 (2012) S54–S296 S123s / Osteoarthritis and Cartilage 20 (2012) S54–S296 S123 inhibited BSP protein production. BSP increased IL 8 but decreased TSP1 protein expression by OA chondrocytes in a dose dependent manner (IL8 : r1⁄40.58, p1⁄40.0047 ; TSP1 : r1⁄4 0.95, p<0,0001). Conclusions: Hypertrophic chondrocytes stimulated migration and inva sion of endothelial cells indicating that hypertrophic chondrocytes expressed a pro angiogenic phenotype. BSP expression is associated with hypertrophic differentiation of OA chondrocytes suggesting that it could be a keymediator of the hypertrophic chondrocytes induced angiogenesis. This hypothesis is supported by the inhibiting effect of BSP on TSP 1, a anti angiogenic factor. To control or reverse chondrocyte hypertrophic differ entiation is a promising way for the treatment of OA. In this context, BSP is a potential target for drugs. 232 AKT PHOSPHORYLATION IN HUMAN CHONDROCYTES IS DOWN REGULATED VIA P53R2 IN RESPONSE TO MECHANICAL STRESS K. Kawakita, T. Nishiyama, T. Fujishiro, S. Hayashi, S. Hashimoto, K. Iwasa, S. Sakata, M. Kurosaka. Kobe Univ. graduate school of medicine,