Wound fluid inhibits wound fibroblast nitric oxide synthesis

Abstract

Fibroblast-derived nitric oxide (NO) is an autocrine stimulator of collagen synthesis by wound fibroblasts. Little is known about the in vivo regulation of wound fibroblast NO synthesis. We investigated the net effect of wound environment on wound fibroblast NO production and characterized a soluble factor mediating this effect. Wound fibroblasts and acellular wound fluid (pool of 100 Lewis rats) were isolated from subcutaneously implanted polyvinyl alcohol sponges harvested 10 days post-wounding. Fibroblasts were incubated in the presence of 10% (v/v) wound fluid. Nitrite, an index of NO synthesis, was measured in supernatants by Griess reagent. Wound fibroblasts spontaneously synthesize large amounts of NO. Spontaneous NO synthesis was further increased by LPS + IFN-gamma (P < 0.001). Wound fluid significantly inhibited both spontaneous and LPS plus IFN-gamma-stimulated NO synthesis (by 88 and 55%, respectively; P < 0.01). Wound fluid from 5- to 35-day-old wounds equally suppressed NO synthesis. Separation by Sephadex G-100 gel filtration identified the active factor in wound fluid to have a molecular weight of about 100 kDa. Characterization of this factor showed it to be a heat-resistant (56 degrees C, 30 min), trypsin-sensitive, and neuraminidase-resistant protein (ammonium sulfate precipitation). The isoelectric point appeared to be 7.0, as determined by ion exchange chromatography. Addition of high arginine did not restore the effect of wound fluid on fibroblast NO synthesis, suggesting that substrate is not a limiting factor. Our data demonstrate that following postoperative day 5 the wound environment contains a high molecular weight protein that inhibits NO synthesis by wound fibroblasts.