Background Osteoarthritis (OA) and Rheumatoid Arthrisis (RA) are characterized 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 in which genes can be silenced sequence-specific. This can be invoked by transfection of tissue/cells with small interfering RNA (siRNA).
Objectives Our aim was 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 aseptically 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, 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. Transfection with 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.
Disclosure of Interest L. Nieuwenhuizen Grant/Research support from: unrestricted grant from Novo Nordisk, R. Schutgens: None Declared, G. Roosendaal: None Declared, S. Mastbergen: None Declared, D. Biesma: None Declared, F. Lafeber: None Declared
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