Abstract 286: Role of Glutathione in the Regulation of VEGFR2 in Human Aortic Endothelial Cells

Abstract

Tissue ischemia due to blood vessel occlusion favors new blood vessel formation or angiogenesis. Although hypoxia is one of the important regulators of angiogenesis, angiogenesis is a rare event in many cardiovascular diseases (CVD). This decreased angiogenesis could be attributed to many factors including altered glutathione (GSH) redox state. Previously, our laboratory has shown an intricate relationship between decreases in GSH in ischemic tissues and angiogenesis. Therefore, we hypothesized that altered GSH redox state regulates angiogenesis by affecting the vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR2) pathway. We used human aortic endothelial cells (HAEC) and treated with diamide (DA), an oxidant that oxidizes GSH to GSSG, thereby altering GSH redox state. DA treatment decreased GSH and increased GSSG concentrations resulting in decreased GSH/GSSG. Using western blotting, we found that decreased GSH/GSSG activates VEGFR2. To identify the mechanism of VEGFR2 activation after DA treatment, we measured VEGF release into the media using ELISA. There was no difference in extracellular VEGF between the groups suggesting that DA activates VEGFR2 ligand-independently. We then investigated the role of protein tyrosine phosphatases (PTP) using a pan-PTP inhibitor, sodium orthovanadate, which did not alter VEGFR2 activation after DA treatment suggesting no role for PTPs in this pathway. We also studied the role of intracellular H 2 O 2 in DA-induced activation using PEG-catalase to quench H 2 O 2 and then treated with DA. PEG-catalase pretreatment did not affect VEGFR2 activation showing H 2 O 2 is not involved in DA-induced VEGFR2 activation. In order to validate the DA-induced receptor activation, we inhibited glutathione reductase using 2-AAPA to increase intracellular GSSG levels. 2-AAPA treatment alone activated VEGFR2, confirming receptor activation after altering GSH/GSSG. Lastly, altering GSH/GSSG decreased the proliferation of HAECs. Our results elucidate a novel role of glutathione especially GSSG in ligand-independent VEGFR2 activation and subsequent inhibition of cell proliferation. Based on our data, GSSG could be a possible mechanism for decreased angiogenesis in CVD.