ID: 19: Increased heparanase expression in keratinocytes promotes dermal fibrosis in scleroderma

Abstract

Interactions between keratinocyte and dermal fibroblast via paracrine loop play an important role in wound repair and keloid formation. In this study, we investigated heparanase expression in activated keratinocyte, and tested its effect on the survival of dermal fibroblasts. Plasma heparanase levels were measured in scleroderma patients, and heparanase expression was evaluated in the skin of bleomycin-induced fibrosis mice and HaCaT keratinocyte (HaCaT). Dermal fibroblasts were cocultured with HaCaT separated by transwell insert under serum starvation, and apoptosis was determined using APOPercentage assay. Plasma heparanase levels were significantly higher in 26 scleroderma patients than in 10 healthy subjects, and positively correlated with plasma TGF- β levels. In bleomycin-induced fibrosis mice, increased heparanase expression was observed in keratinocyte layer, but not in dermal layer. Treatment of HaCaT with hypoxia resulted in significant increase in heparanase expression, and this increase was accompanied by concomitant increase of matrix metalloproteinase-9, both of which are known to degrade epidermal basement membrane components. Coculture of dermal fibroblasts and HaCaT in the presence of hypoxia significantly protected the apoptosis of dermal fibroblasts induced by serum starvation, but it was abolished by anti-heparanase antibody or transfection of HaCaT with heparanase siRNA. Dermal fibroblasts cocultured with HaCaT exposed to hypoxia exhibited increased Akt phosphorylation, and pretreatment of dermal fibroblasts with LY294002, an inhibitor of phosphatidylinositol 3-kinase, significantly abolished anti-apoptotic effect of heparanase on dermal fibroblasts. These data indicate that hypoxia, caused possibly by microvascular alteration, increases heparanase production in keratinocytes, which may promote fibrosis in scleroderma by inhibiting the apoptosis of dermal fibroblasts.