Abstract 344: A20 Increases Endothelial Nitric Oxide Synthase Expression and Activity to Sustain Endothelial Cell Function under Inflammatory Insults
Nitric oxide generated by endothelial nitric oxide synthase (eNOS) acts at different levels to sustain proper endothelial cell (EC) function. Pro-inflammatory molecules associated with various pathological conditions negatively affect eNOS expression and activity, altering vascular homeostasis. Decreased eNOS levels and activity are the pathognomonic features of EC dysfunction and a prelude to atherosclerotic remodeling of the vasculature. We have demonstrated that A20 maintains EC homeostasis by inhibiting NF-kappa B activation and protecting from apoptosis. The aim of this study is to explore whether A20 impacts eNOS expression/activity.
A20 was overexpressed in human coronary artery EC (HCAEC) by recombinant adenovirus-mediated gene transfer. Effects of A20 on eNOS expression and phosphorylation were analyzed by quantitative PCR, human eNOS promoter-luciferase reporter assay, chromatin immunoprecipitation (ChIP) and Western blot. Our data demonstrate that over-expression of A20 in HCAEC significantly increased eNOS mRNA and protein levels by promoting eNOS transcription, as demonstrated by ChIP, and eNOS promoter analysis using Luciferase reporter assays. Importantly, A20-induced upregulation of eNOS expression was sustained in HCAEC treated with tumor necrosis factor that inhibits eNOS transcription and function. Moreover, A20 enhanced phosphorylation of eNOS (Ser-1177), a surrogate marker of its activity, by promoting the activation of its upstream kinase AKT.
Our data demonstrate that A20 upregulates and activates eNOS, even in EC exposed to inflammation. A20’s ability to promote and sustain eNOS activity could be critical to its cytoprotective effect in EC. Further, it supports our pursuit of A20-based therapies to maintain vascular homeostasis, preventing vascular diseases such as atherosclerosis, transplant arteriosclerosis and diabetic vasculopathy.
- © 2013 by American Heart Association, Inc.