Objective: Connective tissue growth factor (CTGF) has been proposed to play a role in fibrotic process of systemic sclerosis. Since hypoxia was known to be associated with fibrosis in several profibrogenic conditions, we investigated whether CTGF expression in dermal fibroblast is regulated by hypoxia caused by microvascular loss. Methods: Dermal fribroblasts from patient with systemic sclerosis and normal controls were cultured in the presence of cobalt chloride (CoCl₂), a chemical inducer of HIF-1α or hypoxic culture conditions. Expression of HIF-1α, VEGF and CTGF was evaluated by semiquantitative reverse transcription-polymerase chain reaction and Western blotting. Results: Scleroderma fibroblasts expressed increased levels of HIF-1α, VEGF and CTGF compared to normal dermal fibroblasts. Dermal fibroblasts exposed to various concentration of CoCl₂ (1∼100μM) enhanced the expression of CTGF mRNA in dose-dependent fashion. Actinomycin D significantly blocked the hypoxia-mediated up-regulation of CTGF mRNA expression, whereas cycloheximide did not block the up-regulation. Up-regulation of CTGF by hypoxia was not mediated by endogenous production of transforming growth factor (TGF)-β. In time-kinetics study, dermal fibroblasts from scleroderma patients exhibited earlier peak expression of CTGF mRNA than those from normal dermal fibroblasts. In addition, simultaneous treatment of suboptimal concentration of CoCl₂ and TGF-β exhibited the up-regulation of CTGF mRNA in additive fashion. Interferon-γ did not modulate the expression of CTGF mRNA induced by CoCl₂, while the up-regulation of CTGF by TGF-β was downregulated by Interferon-γ in a dose-dependent fashion. Conclusion: These data indicate that hypoxia up-regulates the expression of CTGF in dermal fibroblasts and provide the evidence that hypoxia caused by microvascular alterations contributes the progression of fibrosis in systemic sclerosis by up-regulation of CTGF.

Keywords: Systemic sclerosis, Hypoxia, Connective tissue growth factor, Dermal fibroblast, Transforming gronth factor-β