Nitric Oxide Promotion of Collagen Expression by Normal/Diabetic Fibroblasts

Objectives: Chronic wounds are responsible for nearly 70% of all non-traumatic amputations. The non-healing diabetic foot wound displays pathologically reduced collagen expression and nitric oxide concentration. The purpose of this study was to examine Type I and III collagen expression by normal and diabetic skin fibroblasts after treatment with short- and long-acting nitric oxide (NO) donors. Culture of the cells was conducted under both normoxic and hypoxic conditions to simulate a normal and diabetic wound environment. Our hypothesis is that nitric oxide up-regulates collagen expression by hypoxic diabetic wound fibroblasts. Methods: Human skin fibroblasts in vitro and mouse skin wounds in vivo were treated with nitric oxide donor compounds. Changes in type I and III collagen expression and rate of wound closure were measured. Short-acting (NOR-3; ½-life = 40 min) and long-acting (SNOG; ½-life = 80 hr) nitric oxide donors were used and collagen gene expression by fibroblasts cultured under normoxic (20% O2) or hypoxic (2% O2) conditions for 1, 3 or 7 days was examined by real-time PCR. We examined the effects of these compounds on wound healing by assessing rate of wound closure between different experimental treatment groups and between normal and diabetic mice. Results: Type I and III collagen expression were significantly lower under hypoxic conditions and by diabetic fibroblasts. SNOG increased Type III collagen expression by diabetic fibroblasts on days 3 and 7 under both normoxic and hypoxic conditions; and Type I collagen expression on day 7. Additionally, SNOG accelerated wound healing in diabetic mice to a rate comparable with that of normal mice. NOR-3 had no significant effect on collagen expression or wound healing. Conclusion: The ability to control collagen expression by regulating nitric oxide availability could provide a novel and effective means of promoting wound repair in diabetes. Supported by Juvenile Diabetes Research Foundation.