Background Cyclic phosphatidic acid (cPA) is one of bioactive lipid, has been implicated as a mediator of various biological effects including inhibitory effects of proliferation, invasion and metastasis of cancer cells. cPA is naturally occurring mediator even exists in human serum. 2-carba-cPA (2ccPA) is structurally modified formula of cPA and has shown improved bioactivity. We have previously confirmed that 2ccPA stimulated HAS-2 production on human osteoarthritic chondrocytes and synovial fibroblasts (SFs) in vitro. Furthermore, intra-articular administration of 2ccPA has shown its suppressing effect of pain, swelling, and articular cartilage destruction in rabbit experimental osteoarthritis (OA). We have shown that 2ccPA might had direct inhibitory effect of cartilage degrading enzymes on rheumatoid synovial fibroblasts (SFs) in vitro. Inflammatory arthritis such as rheumatoid arthritis (RA) and early stage of OA involves synovial inflammation and subsequent production of cartilage degrading enzymes also from chondrocytes. 2ccPA may be possible another therapeutic option for degenerative arthritis.
Objectives The aim of this study was to evaluate the direct effects of 2ccPA on cartilage matrix degrading enzymes using SFs and articular chondrocytes under influence of inflammatory cytokines.
Methods In vitro studies were performed using SFs and chondrocytes obtained from arthritis patients (RA and OA) at joint replacement surgery. First, 2ccPA 0–25 μM was added to cell cultures and effects of 2ccPA on ADAMTS-4, -5, MMP-3, 9, -13 expression was assessed by real time PCR using specific primers to corresponding genes. Beta-actin was used as endogenous expression control for PCR. As we confirmed that 2ccPA had dose-dependent inhibitory effects on expression of above enzymes, the second experiment was performed. SFs or chondrocytes were pre-cultured with IL-1β (1 ng/ml) and/or TNF-α (10 ng/ml) for 24 hours, then added 10 μM 2ccPA to study attenuated effect of 2ccPA on synthesis of above cartilage matrix degrading enzymes. Newly synthesized MMP-3, -13 from SFs or chondrocytes in cultured media after 24 hours of 2ccPA addition were measured by sandwich ELISA.
Results 2ccPA itself repressed cartilage degrading enzymes, ADAMTS-4, ADAMTS-5, MMP-3, MMP-9 and MMP-13 expression in both SFs and chondrocytes was all repressed by low dose of 2ccPA. Even after cells had been stimulated by cytokines, 10 μM 2ccPA repressed expression of cartilage degenerating enzymes in SFs or chondrocytes. Expression of MMP-13 were repressed more in chondrocytes by 2ccPA. ELISA results also confirmed the inhibitory effect of 2ccPA on MMP-13 production in RA SFs (n=5) by 52% or in RA chondrocytes (n=3) by 43%. In OA, MMP-13 production was reduced by 31% in OA SFs (n=6) and 34% in OA chondrocytes (n=4). However, not significant reduction of MMP-3 in both SFs or chondrocytes. MMP-9 expression by ELISA was under the detectable limit.
Conclusions The in vitro results confirmed that 2ccPA had suppressing effect of cartilage degrading enzymes expression on SFs and chondrocytes, supports the hypothesis that 2ccPA might have played direct role to suppress inflammation and also protect articular cartilage in arthritic condition.
Disclosure of Interest I. Masuda Grant/research support from: SANSHO, K. Okada: None declared, H. Yamanaka: None declared, S. Momohara: None declared
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