Regulation of metabolic activity of peritoneal fibroblasts by dichloroacetate provides a potential target for interventions to reduce postoperative adhesions

Abstract

OBJECTIVE: Adhesion fibroblasts manifested lower apoptosis as compared to normal peritoneal fibroblasts. Since adhesions provide oxygen and nutrients to postsurgical ischemic tissue, we sought to examine the role of aerobic metabolism in regulating apoptosis. Utilizing a compound, dichloroacetic acid (DCA), which stimulates pyruvate dehydrogenase, causing pyruvate to be converted in the Kreb's cycle rather than into lactate, thereby converting anaerobic to aerobic metabolism.DESIGN: Primary cultures of fibroblasts from normal peritoneal and adhesion tissues from the same patients (n=5).MATERIALS AND METHODS: Adhesion and normal peritoneal fibroblasts were treated with DCA (0, 100 mg/ml) for 2 hours, under normal (20% O2) and hypoxic (2%, O2) conditions. Apoptosis of fibroblasts was assessed by: 1) the TUNEL assay that measures nuclear DNA fragmentation by incorporating fluorescein-12-dUTP at the 3′-OH ends, and 2) the caspase 3 assay kit (Sigma) that measures caspase 3 activity.RESULTS: DCA treatment, under normoxic conditions, resulted in significant increases in caspase 3 activity from 1.62 to 7.34 and from 1.11 to 8.03 micromoles of pNA/min/mL in normal peritoneal and adhesion fibroblasts, respectively (p<0.05). In addition, DCA treatment, under hypoxic conditions, resulted in significant increases in caspase 3 activity from 1.62 to 5.67 and from 1.11 to 6.17 micromoles of pNA/min/mL in normal peritoneal and adhesion fibroblasts, respectively (p<0.05). These results were supported by the observed increase in nuclear DNA fragmentation that occurred in the DCA treated fibroblasts under normal and hypoxic conditions.CONCLUSIONS: These findings confirm that fibroblasts from adhesions are characterized by lower apoptosis, which is further accentuated by hypoxia. Stimulation of oxidative metabolism by DCA is associated with induction of apoptosis. These support the suggestion that regulation of metabolic activity of peritoneal cells may provide a target for interventions designed to reduce postoperative adhesions. OBJECTIVE: Adhesion fibroblasts manifested lower apoptosis as compared to normal peritoneal fibroblasts. Since adhesions provide oxygen and nutrients to postsurgical ischemic tissue, we sought to examine the role of aerobic metabolism in regulating apoptosis. Utilizing a compound, dichloroacetic acid (DCA), which stimulates pyruvate dehydrogenase, causing pyruvate to be converted in the Kreb's cycle rather than into lactate, thereby converting anaerobic to aerobic metabolism. DESIGN: Primary cultures of fibroblasts from normal peritoneal and adhesion tissues from the same patients (n=5). MATERIALS AND METHODS: Adhesion and normal peritoneal fibroblasts were treated with DCA (0, 100 mg/ml) for 2 hours, under normal (20% O2) and hypoxic (2%, O2) conditions. Apoptosis of fibroblasts was assessed by: 1) the TUNEL assay that measures nuclear DNA fragmentation by incorporating fluorescein-12-dUTP at the 3′-OH ends, and 2) the caspase 3 assay kit (Sigma) that measures caspase 3 activity. RESULTS: DCA treatment, under normoxic conditions, resulted in significant increases in caspase 3 activity from 1.62 to 7.34 and from 1.11 to 8.03 micromoles of pNA/min/mL in normal peritoneal and adhesion fibroblasts, respectively (p<0.05). In addition, DCA treatment, under hypoxic conditions, resulted in significant increases in caspase 3 activity from 1.62 to 5.67 and from 1.11 to 6.17 micromoles of pNA/min/mL in normal peritoneal and adhesion fibroblasts, respectively (p<0.05). These results were supported by the observed increase in nuclear DNA fragmentation that occurred in the DCA treated fibroblasts under normal and hypoxic conditions. CONCLUSIONS: These findings confirm that fibroblasts from adhesions are characterized by lower apoptosis, which is further accentuated by hypoxia. Stimulation of oxidative metabolism by DCA is associated with induction of apoptosis. These support the suggestion that regulation of metabolic activity of peritoneal cells may provide a target for interventions designed to reduce postoperative adhesions.