NITRIC-OXIDE, AN AUTOCRINE REGULATOR OF WOUND FIBROBLAST SYNTHETIC FUNCTION

Nitric oxide (NO) is synthesized in wounds, but its exact role and cel lular source are not known. Wound fibroblasts (WF) are phenotypically characterized by increased collagen synthesis and contractility. We hy pothesized that WF may be also phenotypically altered during wound hea ling to synthesize NO. WF were isolated from polyvinyl alcohol sponges implanted in male Lewis rats and harvested 10 days later. Proliferati on in response to 10% fetal bovine serum was assessed by [H-3]thymidin e incorporation in a microculture system. A fibroblast-populated colla gen lattice was used for assaying contractility. Collagen synthesis wa s determined by measuring the collagenase-sensitive fraction of protei n-incorporated [H-3]proline. Fibroblasts were incubated in the presenc e or the absence of 0.5 mM S-methyl-isothio-uronium or 0.5 mM N-monome thyl-L-arginine, both competitive inhibitors of NO synthase. WF sponta neously synthesize and release NO (4.60 +/- 0.29 nmol nitrite/mu g DNA /48 h). Normal dermal fibroblasts do not synthesize NO. WF NO synthesi s was limited to the first and second passages postharvest and was inh ibitable by S-methyl-isothio-uronium (96%) and N-monomethyl-L-arginine (84%). In vivo iNOS expression by WF was confirmed by in situ hybridi zation and immunohistochemistry. Inhibition of endogenous NO synthesis had no effect on fibroblast proliferation. However, fibroblast-mediat ed collagen contraction was enhanced (p < 0.01), and collagen synthesi s was significantly decreased (p < 0.05) by inhibiting NO synthase. Th e data show that WF are phenotypically altered during the healing proc ess to synthesize NO, which, in turn, regulates their collagen synthet ic and contractile activities.