Investigating the Effect of Inhibiting 'Reactive Oxygen Species' on Intracellular Signaling Pathways in Endothelial Cells

Abstract

ObjectiveElevated Reactive Oxygen Species (ROS) in Endothelial Cells (EC's) have been implicated in the pathogenesis of cardiovascular diseases, and much research has gone into elucidating the mechanisms through which oxidative stress leads to these diseases. Research in the last decade, however, has uncovered the central role lower levels of ROS play in physiological intracellular signaling pathways, such as those stimulated by Vascular Endothelial Growth Factor (VEGF) and Tumour Necrosis Factor‐α (TNF‐α) in EC's. Our objective in this study was to determine the effect of a lack of ROS in EC's on the intracellular signaling pathways stimulated by VEGF and TNF‐α.MethodsThe activity of these pathways can be measured by the expression of key downstream proteins that are common to these two pathways such as Akt, ERK‐1/2, IKKα/B, AMPK, eNOS, mTOR and FOXO1. To investigate these pathways in vitro, Bovine Endothelial Aortic Cells (BAEC's) were treated with ROS inhibitors (NAC, DPI) and then exposed to VEGF or TNF‐α to stimulate their respective pathways. Protein was then extracted and transferred to a membrane by Western Blotting then visualized by immunoflourescence.ResultsWestern blots demonstrated that reduction of ROS levels by NAC inhibited VEGF‐induced Akt phosphorylation but not phosphorylation of ERK1/2 in BAEC. The findings of TNF‐induced signaling activation were not conclusive.ConclusionWhereas VEGF‐mediated activation of PI3K‐Akt activation requires ROS, ERK1/2 activation is independent of ROS in ECs.This research was funded by National Institutes of Health (NIH) and American Heart Association (AHA) grants.