Nitric Oxide Donors Selectively Reduce the Expression of Matrix Metalloproteinases-8 and -9 by Human Diabetic Skin Fibroblasts

Abstract

Chronic, nonhealing skin wounds are a common ailment in uncontrolled diabetes and are associated with significant morbidity. The nonhealing diabetic foot wound displays pathologically elevated matrix metalloproteinase (MMP) activity. In contrast, the concentration of nitric oxide (NO) is significantly reduced in these chronic ulcers. Addition of NO to diabetic wounds improves wound healing, but the mechanism for this effect is poorly understood. Diabetic and nondiabetic human skin fibroblasts were cultured to confluence and then treated with 0, 1, 10, and 100 nm concentrations of three NO donors (NOR-3, SNAP, and SNOG) with varying half-lives for 1, 3, and 7 days. At harvest, the cultures were analyzed for their production of NO and the effect of NO donor treatment on cell proliferation (cell number) and MMP expression (MMP-1, -2, -8, -9, and -13). The NO donor with the shortest half-life (NOR-3) produced a rise in NO on day 1 in both normal and diabetic fibroblasts at the highest concentration used; there was a corresponding decrease in both MMP-8 and MMP-9 expression in the diabetic fibroblasts and a decrease in only MMP-9 expression in the normal fibroblasts. After longer times in culture or at lower concentrations, NOR-3 was without effect on NO production or MMP expression. Further, NOR-3 had no effect on cell proliferation. In contrast to NOR-3, NO donors with longer half-lives (SNAP and SNOG) significantly (P < 0.05) increased NO production by both normal and diabetic fibroblasts during the entire course of the experiment and even after a media change lacking additional NO donor at day 3. SNAP and SNOG dose-dependently reduced MMP-8 and -9 mRNA expression in both normal and diabetic fibroblasts through day 7. The expression of MMP-1, -2, and -13 was not significantly affected by any of the NO donor treatments. These experiments show distinct deficits in NO production and elevations in MMP-8 and -9 expression in diabetic human skin fibroblasts compared to normal. In addition, treating these cultures with NO donor compounds with half-lives greater than 5 h selectively raised NO production by the cells, increased cell proliferation, and decreased MMP-8 and -9 expression in a dose-dependent manner. There was no effect of the NO donor compounds on MMP-1, -2, or -13 expression. One possible mechanism to account for NO's beneficial effect on wound healing may involve stimulation of cell proliferation and down-regulation of MMP expression.