Hypoxia-generated superoxide induces the development of the adhesion phenotype

Adhesion fibroblasts exhibit higher TGF-beta1 and type I collagen expression as compared to normal peritoneal fibroblasts. Furthermore, exposure of normal peritoneal fibroblasts to hypoxia results in an irreversible increase in TGF-beta1 and type I collagen. We postulated that the mechanism by which hypoxia induced the adhesion phenotype is through the production of superoxide either directly or through the formation of peroxynitrite. To test this hypothesis, normal peritoneal and adhesion fibroblasts were treated with superoxide dismutase (SOD), a superoxide scavenger, and xanthine/xanthine oxidase, a superoxide-generating system, under normoxic and hypoxic conditions. Also, cells were treated with peroxynitrite. TGF-beta1 and type I collagen expression was determined before and after all treatments using real-time RT/PCR. Hypoxia treatment resulted in a time-dependent increase in TGF-beta1 and type I collagen mRNA levels in both normal peritoneal and adhesion fibroblasts. Similarly, treatment with xanthine oxidase, to endogenously generate superoxide, resulted in higher mRNA levels of TGF-beta1 and type I collagen in both normal peritoneal and adhesion fibroblasts. In contrast, treatment with SOD, to scavenge endogenous superoxide, resulted in a decrease in TGF-beta1 and type I collagen expression in adhesion fibroblasts to levels seen in normal peritoneal fibroblasts; no effect on the expression of these molecules was seen in normal peritoneal fibroblasts. Exposure to hypoxia in the presence of SOD had no effect on mRNA levels of TGF-beta1 and type I collagen in either normal peritoneal or adhesion fibroblasts. Peroxynitrite treatment alone significantly induced both adhesion phenotype markers. In conclusion, hypoxia, through the production of superoxide, causes normal peritoneal fibroblasts to acquire the adhesion phenotype. Scavenging superoxide, even in the presence of hypoxia, prevented the development of the adhesion phenotype. These findings further support the central role of free radicals in the development of adhesions.