The angiogenic capacity of local tissue critically regulates the response to ischemic injury. Elevated reactive oxygen species production, commonly associated with ischemic injury, has been shown to promote phosphorylation of the vascular endothelial growth factor receptor 2 (VEGFR2), a critical regulator of angiogenesis. Previous data from our lab demonstrated that diminished levels of the antioxidant glutathione positively augment ischemic angiogenesis. Here, we sought to determine the relationship between glutathione levels and oxidative stress in VEGFR2 signaling. We reveal that decreasing the ratio of GSH to GSSG with diamide leads to enhanced protein S-glutathionylation, increased reactive oxygen species (ROS) production, and enhanced VEGFR2 activation. However, increasing ROS alone was insufficient in activating VEGFR2, while ROS enhanced VEGF-stimulated VEGFR2 activation at supraphysiological levels. We also found that inhibiting glutathione reductase activity is sufficient to increase VEGFR2 activation and sensitizes cells to ROS-dependent VEGFR2 activation. Taken together, these data suggest that regulation of the cellular GSH:GSSG ratio critically regulates VEGFR2 activation. This work represents an important first step in separating thiol mediated signaling events from ROS dependent signaling.

Arterial and venous endothelial cells exhibit differential responses to oxidant stress, and decreases in GSH:GSSG are more exacerbated in venous endothelial cells. Specific pathogenesis in these vascular conduits, with respect to oxidant stress handling, warrants further study, especially considering surgical interventions such as Coronary artery bypass grafting that use both interchangeably.

Molecular oxygen (O2) is an essential component for survival and development. Variation in O2 levels leads to changes in molecular signaling and ultimately affects the physiological functions of many organisms. Nitric oxide (NO) and hydrogen sulfide (H2S) are two gaseous cellular signaling molecules that play key roles in several physiological functions involved in maintaining vascular homeostasis including vasodilation, anti-inflammation, and vascular growth. Apart from the aforementioned functions, NO and H2S are believed to mediate hypoxic responses and serve as O2 chemosensors in biological systems. In this literature review, we briefly discuss NO and H2S and their roles during hypoxia.

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.

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