The role of endothelial connexins in vascular wound repair

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|| Project Summary . Impaired endothelial wound healing in blood vessels like the coronary artery and saphenous vein is a primary contributor to vascular stent failure and deadly coronary thrombosis. Identifying the mechanism of delayed endothelial repair is essential for improving vessel patency and patient survival rates. As such, this proposal focuses on identifying regulators of normal endothelial healing and understanding how these factors are dysregulated when healing is impaired. Previous studies have shown connexin 43 (Cx43) gap junctions may regulate wound healing. However, the specific role of connexin 43 in endothelial cell (EC) healing is unknown. Preliminary data in this proposal indicates that ligation-induced vascular injury promotes increases in the expression of EC Cx43 surrounding the damaged area in the aorta and carotid arteries in mice. Cx43-mediated gap junction intracellular communication is controlled by phosphorylation at the Cx43 C-terminus. My preliminary data demonstrate that specific Cx43 phosphorylation at its serine (s) 368, associated with gap junction closure, is present during the final stages of EC wound healing in vivo. Cx43-s368 also reduced the rate of wound closure in cultured human EC. These findings inform the hypothesis that Cx43 expression and channel functions are critical for EC wound healing in large arteries. The aims of this proposal are to define the role of connexin 43 in endothelial wound healing (Aim 1), investigate if a loss of Cx43 limits healing in vivo (Aim 1), and test if channel functions, regulated by posttranslational modifications, improve/delay vascular repair (Aim 2). This investigation will be completed using a novel mouse carotid EC injury survival surgery in mice I developed, which will allow for the assessment of Cx43 expression in carotid EC during the healing process. Both genetic and pharmacological strategies will be used to alter Cx43 expression and phosphorylation in mouse injury models and in cultured human EC, and the impact of these modifications on the rate and quality of EC healing will be quantified. Additionally, RNAseq approaches will be used to identify Cx43 gap junction-dependent signaling in the regenerating endothelium. These aims will be accomplished under the mentorship of researchers with extensive experience in vascular biology, Dr. Scott Johnstone, Dr. Robert Gourdie, and Dr. Brant Isakson, at Virginia Tech’s Fralin Biomedical Research Institute (FBRI). The FBRI is home to research equipment including but not limited to animal facilities, confocal microscopes, flow cytometers, and cell culture equipment. This state- of-the-art research environment will allow for a detailed and mechanistic investigation. Ultimately, this study will characterize a novel role for Cx43 in vascular EC. This project is designed to identify new potential therapeutic targets in vascular disease that will promote the mission of the NIH to enhance patient health and to promote longer life free of illness.