THU0016 Heparanase Is Active in Human Osteoarthritic Cartilage and Drives Catabolic Responses in Primary Human Chondrocytes

Background Osteoarthritis (OA) affects hundreds of millions of people worldwide, resulting in significant morbidity. Articular cartilage homeostasis is governed by resident chondrocytes that can either contribute to cartilage anabolism by producing tissue constituents or drive catabolic events by secreting aggrecanases and collagenases. The chondrocytes9 pericellular matrix (PCM) acts as a mechanosensor by sequestering growth factors that are bound to heparan sulfate (HS) proteoglycans, such as perlecan. Heparanase is the sole mammalian enzyme with HS degrading activities. Heparanase enzymatic activity releases HS-bound growth factors, cytokines, chemokines and other ligands stored within the extracellular matrix. Through both enzymatic and non-enzymatic activities, heparanase influences the expression of a number of important genes and has been implicated in numerous processes involved in cancer progression, inflammation and remodeling of the ECM. Little is known about whether heparanase plays a role in cartilage homeostasis. Objectives To ascertain whether heparanase may have a role in modulating the anabolic or catabolic responses of human articular chondrocytes. Methods Heparanase enzymatic activity was measured in collagenase digests of osteoarthritic articular cartilage using solid-phase-bound radiolabeled heparan sulfate as substrate. Primary human chondrocytes obtained from OA patients undergoing joint replacement surgery, were incubated with or without recombinant human pro-heparanase. In some experiments, the heparanase inhibitor, PG545, was also added. Heparanase was also added together with fibroblast growth factor 2 (FGF-2) or interleukin 1 (IL-1). The association of pro-heparanase with the chondrocytes and its conversion to active heparanase was evaluated by Western blot. Induction of the catabolic genes MMP13 , ADAMTS4 and ADAMTS5 and the anabolic genes ACAN (encoding the aggrecan core protein) and COL2A1 (encoding the alpha chain of type II collagen), was analyzed by real time reverse-transcriptase PCR. MMP13 enzymatic activity in the culture medium was measured with a specific fluorescent assay. ERK phosphorylation was evaluated by Western blot. Results The collagenase-digested cartilage (but not the collagenase blend alone), liberated small fragments compatible with HS-degradation products, indicated active heparanase is present in osteoarthritis cartilage. Cultured chondrocytes rapidly associated with the inactive 62-kDa pro-heparanase, which was then cleaved to the active 50-kDa enzyme. Addition of heparanase to cultured human chondrocytes activated the catabolic genes MMP13 and ADAMTS4 by 4 hours and reduced the expression of anabolic genes ACAN and COL2A1 by 20 hours. Heparanase also induced human chondrocytes to secrete active MMP13 to the culture medium by 20 hours and induced intracellular ERK phosphorylation by 30 minutes. A synergistic effect was observed between heparanase and FGF-2, but not between heparanase and IL-1. PG545, a heparan sulfate mimetic, reversed the effects of heparanase. Conclusions Heparanase is active in osteoarthritic cartilage and induces catabolic responses in cultured primary human chondrocytes. This response seems to be due, at least in part, to the release of heparan sulfate-bound growth factors such as FGF-2. Disclosure of Interest None declared