Metabolic regulation of collagen I in fibroblasts isolated from normal peritoneum and adhesions by dichloroacetic acid.
OBJECTIVE: We have previously demonstrated that collagen I, a major component of postoperative adhesions, is differentially regulated in fibroblasts isolated from normal human peritoneum and adhesions. Collagen I messenger RNA (mRNA) levels are significantly lower in fibroblasts from normal peritoneum compared with levels from adhesion fibroblasts. This variation is further accentuated by hypoxia. Because adhesions provide a means of supplying oxygen and nutrients to postsurgical ischemic tissue, we sought to examine the role of aerobic metabolism in the differential expression of collagen I. To examine this issue, we used a compound, dichloroacetic acid (DCA), that stimulates pyruvate dehydrogenase, causing pyruvate to be metabolized in the Kreb's cycle rather than converted into lactate, thereby switching anaerobic to aerobic metabolism. Specifically, we have exposed human fibroblast cultures from normal peritoneum and adhesions to DCA (0, 50, and 100 microg/mL) for 24 hours under normal and hypoxic (2% oxygen) conditions. STUDY DESIGN: Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) of collagen I and beta-actin was performed by using mRNA extracted from all treatment points. Analysis of PCR-amplified products was performed by fractionation over a 2% agarose gel, followed by ethidium bromide staining of DNA bands. A scanning densimeter was used to determine the ratio of intensity of each band relative to beta-actin. Densimetric analysis of gel bands was performed by using the National Institutes of Health image analysis program. RESULTS: Although DCA stimulated peritoneal fibroblast collagen I mRNA expression under normoxic conditions, its expression was reduced during hypoxia. In adhesion fibroblasts, DCA treatment consistently lowered collagen I mRNA expression; this effect was manifested to a greater extent under hypoxic conditions. CONCLUSION: In summary, these findings confirm that fibroblasts from adhesions are characterized by excessive collagen I production, which is further accentuated by hypoxia. These observations are extended to show the stimulation of oxidative metabolism by DCA increases collagen I production; in contrast DCA inhibits collagen I production by normoxic adhesion fibroblasts as well as under hypoxic conditions in both types of fibroblasts. Thus, regulation of metabolic activity of peritoneal cells may provide a target for future interventions for reduction of postoperative adhesions.
Diamond, MP; El-Hammady, E; Wang, R; Saed, G
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