Activated Protein C synergises with IL-1 to drive collagenolysis from equine articular cartilage

Introduction: Osteoarthritis (OA) is a chronic, debilitating, degenerative joint disease in which degradation of the ECM macromolecules aggrecan and type II collagen are a major feature. Invitro models of degradation using Oncostatin M (OSM) in combination with Interleukin-1β (IL-1) stimulate collagen breakdown in some species [1, 2]. However, in our preliminary findings, this was not observed in equine cartilage. Activated Protein C (APC) is a plasma serine protease with roles in the clotting cascade and inflammation suppression. Increased levels of APC were found in synovial fluid from joints of human patients diagnosed with OA, [3] and it was proposed that MMP-2 is co-coordinately regulated and tightly bound to APC in synovial fluid. Previous in-vitro work has identified a role for APC in collagen degradation in ovine articular cartilage [4]. We investigated whether APC synergises with pro-inflammatory cytokines in stimulating equine cartilage degeneration and investigated the ability of commonly used corticosteroids to prevent collagen and proteoglycan release in this invitro model. Methods: Articular cartilage explant cultures (n=3) were treated with IL-1 (10ng/ml), Tumor Necrosis Factor-α (TNFα) (10ng/ml), OSM (50ng/ml) or OSM (50ng/ml) + IL-1 (1ng/ml) and ± APC (1-20μg/ml). A sub-group (n=3) were also treated with dexamethasone (10–10), triamcinalone acetate (10–10) or methylprednisolone acetate (10– 10) – corticosteroids commonly used therapeutically for equine OA. Glycosaminoglycan (GAG) loss, hydroxyproline loss, DNA content and gene expression were measured after 4 days. Selected explants treated ± IL-1 ± APC were treated with an MMP inhibitor, or inactive racemate (both at 25μM). Statistically significant differences between control and cytokine-treated cartilage were identified using mixed effects linear regression models. Results: IL-1 (10ng/ml) caused significant GAG release from explants over a 4 day culture period, whilst APC (20μg/ml) alone had no significant effect. In combination with IL-1, APC had no additional effect on GAG release. There was a trend for APC to increase collagen breakdown when used alone (p=0.064). However, at doses of 520μg/ml, APC, in combination with IL-1, caused significant collagen loss from explants (p=0.006) within the 4 day culture period, and which continued to increase throughout longer culture periods (data not shown). At lower doses of APC (1μg/ml), this effect was not observed (Fig.1 A, B). At a concentration of 10μg/ml, APC also synergized with TNFα, caused significant hydroxyproline loss, within 4 days (p<0.01) No synergy occurred with OSM or OSM+IL-1 (data not shown).