The incidence of tissue ischemia resulting from progressive vascular occlusion is on the rise, and leads to several cardiovascular pathologies characterized by arterial blockage such as peripheral artery disease. Revascularization of tissue is time sensitive and essential to restore adequate blood flow. Decreases in antioxidant capacity such as decreases in the reduced form of glutathione (GSH) concentrations and corresponding increases in oxidant stress are hallmarks of disease progression and endothelial cell dysfunction. Decreases in glutathione are thought to correspond with a linear increase in disease severity that is a poorly understood relationship. The current proposal seeks to: (a) determine the influence of changing GSH:GSSG levels on protein glutathionylation driving vascular endothelial growth factor receptor 2 (VEGFR2) signaling in arteriogenesis, (b) determine the role of glutathionylation in oxidative and shear stress induced endothelial cell NF-?B signaling, (c) study in vivo arteriogenesis in murine models that have mutations in the GSH synthesis pathway, and are undergoing ligations to mimic acute and chronic peripheral artery disease, and (d) restore defective arteriogenesis progression by stimulating a more reductive cellular environment to improve endothelial cell function. We will test the central hypothesis that a critical balance between the reductive and oxidative cellular environments drives optimal VEGFR2 signaling to mediate arteriogenic remodeling in response to increased shear and oxidant stress. The proposed aims will utilize in vitro cultures of endothelial cells isolated from our glutathione synthesis mutant murine animals to generate data focusing on glutathionylation of proteins driving VEGFR2 specific signaling. The proposed aims also include our in vivo mouse models of arterial blockage as clinically relevant models of vascular remodeling.
Specific Aim 1 will focus on determining the role of glutathionylation in VEGFR2 activation during endothelial cell arteriogenic signaling.
Specific Aim 2 will assess the role of low level oxidant stress and its control over glutathionylation driving arteriogenic signaling. We will utilize in vitro cultures of endothelial cells isolated from our glutathione synthesis mutant murine animals to study signaling in aims 1 and 2.
Specific Aim 3 will assess the role of the oxidative/reductive balance in arteriogenesis remodeling in vivo. Here we will use our in vivo mouse models of arterial blockage. Successful completion of this project will provide new insights into the mechanism by which glutathione regulates arteriogenesis in a physiologic range of GSH:GSSG following arterial ligation. Such information could be the basis for new intervention therapies developed to precisely control arteriogenesis following artery blockage. Enhancing the vascular remodeling potential of tissue through manipulation of glutathione and protein glutathionylation may represent a critical first step in attenuating tissue damage due to vascular occlusion.
The gradual occlusion of blood vessels such as that reported in peripheral artery disease is increasing in frequency, due to a variety of risk factors including several cardiovascular pathologies such as diabetes, obesity, etc. This project addresses the novel relationship between the antioxidant glutathione, its oxidized form (GSSG) and the protein modifications (glutathionylation) that influence the progression of arteriogenesis following the occlusion of conduit arteries. The proposed research will uncover the underlying mechanisms by which levels of glutathione and corresponding protein modification can be manipulated to enhance the progression of arteriogenesis following ischemic insult; providing potential avenues to the development of novel vascular therapeutics.
