Abstract
Introduction
Osteoarthritis (OA) involves pathological change in all joint tissues, including cartilage degradation and synovitis. Synovial inflammation is significantly associated with pain severity and incidence in knee OA. It is becoming evident that synovitis also plays an active role in the initiation and progression of cartilage erosion in OA, through direct secretion of catabolic enzymes as well as factors that stimulate chondrocyte catabolic activity. Therapeutic agents that target both synovitis and cartilage pathology are likely to be maximally beneficial in treating pain and slowing cartilage breakdown in OA. We have previously shown that an amide-derivative of HA (HYMOVIS™) was superior to native HA of the same MW in improving gait, and reducing synovial hyperplasia in a sheep OA model. In the present study the mechanisms whereby the chemically modified HA may be beneficial were examined using chondrocytes and synovial fibroblasts from knees of OA patients.
Patients & Methods
Chondrocytes (HAC, n=6) and synovial fibroblasts (HSF, n=6) were isolated from OA patients at the time of knee replacement. HYMOVIS™ (0, 0.5, 1.0 or 1.5mg/mL) was added to simultaneously or 1 hour before interleukin-1β (IL1, 2ng/mL). Cultures were terminated 30 minutes later for Bioplex® quantitation of p-JNK, p-NFκB and p-p38; or 24 hours later for RNA isolation and analysis of gene expression by real time RT-PCR, and measurement of MMP13 activity in the media. Only statistically significant results are reported.
Results
In HAC in the absence of IL1, HYMOVIS™ decreased MMP13, ADAMTS5, PTGS2 and IL6 and increased COL2A1 mRNA (2–10fold). In HSF in absence of IL1, HYMOVIS™ decreased TIMP1, TIMP3, CD44, IL6 and increased PTGS2 (2–3fold). In HAC and HSF, IL1 increased expression of MMP1, MMP13, PTGS2, IL6 (>100fold), ADAMTS4 (∼10 fold), all phosphoproteins (3–10fold), and APMA-activated MMP13 activity in media. IL1 increased expression of ADAMTS5 (∼10fold) only in HSF. As expected, IL1 reduced expression of the key matrix proteins in HAC (2–3 fold decrease in COL2A1 and ACAN) and HSF (2 fold decrease in COL1A1). When added simultaneously with IL1, HYMOVIS™ decreased expression of MMP13, ADAMTS5, PTGS2, IL6 expression, and normalised matrix protein expression in both HAC and HAS. Pre-incubation with HYMOVIS™ for 1 hour inhibited IL1-stimulated p-JNK, p-NFκB and p-p38 in both cell types (excluding p-JNK in HSF). In HAC, HYMOVIS™ pre-incubation was superior to simultaneous addition in reducing expression of MMP1, MMP13, ADAMTS4, PTGS2, and IL6 expression. There was a less dramatic effect of HYMOVIS™ pre-incubation on gene expression in HSF compared with HAC. The inhibitory effects of HYMOVIS™ on IL1 stimulated gene expression in HAC and HSF was partially ameliorated by pre-incubation with a CD-44 blocking antibody.
Discussion/Conclusions
The present studies have demonstrated several potential key mechanisms whereby the intra-articular injection of a hexadecylamide-derivative of HA (HYMOVIS™) may have both symptom and disease-modifying effects in OA. The previously described increased joint retention of the hexadecylamide-derivative, might act in a similar manner to the pre-incubation studies in our cell culture studies, to reduce the initiation of degradative events with recurrent/cyclic inflammatory episodes that typify OA.
