Antioxidants inhibit fatty acid and glucose-mediated induction of neutral endopeptidase gene expression in human microvascular endothelial cells1

Abstract

Neutral endopeptidase (NEP) is a membrane-bound metallopeptidase that degrades tachykinins and may regulate their role in wound repair. NEP enzyme activity is increased in diabetic wounds and skin compared with normal controls. We have shown that unsaturated fatty acids and glucose upregulate NEP activity in human microvascular endothelial cells (HMECs) and that vitamins E and C reduce this effect. To determine whether these changes involve NEP gene expression regulation, we analyzed NEP mRNA levels in HMECs cultured with elevated glucose (40 mM) and fatty acids oleate (40 microM) and linoleate (40 microM) for 48 hours or 1 month. Cells were exposed for an additional 48 hours to antioxidants vitamins E or C or N-acetylcysteine. Total RNA was extracted and analyzed for NEP mRNA using real-time reverse transcriptase polymerase chain reaction. NEP gene expression was standardized to beta-actin mRNA and results were analyzed using ANOVA. Elevated glucose, oleate, and linoleate upregulated NEP mRNA in short and longterm HMEC cultures, but did not alter rate of NEP mRNA degradation. Vitamins E and C and N-acetylcysteine blocked glucose- and fatty acid-induced NEP mRNA (p < or = 0.05). The potential role of oxidative stress in NEP activation was confirmed by demonstrating that elevated glucose and fatty acids increase H(2)O(2) levels in HMECs. Regulation of NEP enzyme activity by glucose and fatty acids appears to include gene expression transcription as well as modulation of enzyme activity. Our results also suggest that oxidative stress may be involved in upregulation of NEP by fatty acids and glucose.