Abstract Body: Impaired endothelial wound healing in blood vessels is a main contributor to vascular stent failure. Identifying the mechanisms of delayed endothelial repair is essential for improving vessel patency and patient survival rates. As such, it is vital to identify regulators of endothelial healing. Retrospective RNAseq data analysis from in vivo non-injured vs injured endothelial cells (EC, GEO: GSE115618) showed a significant increase in the expression of the gap junction protein connexin 43 (Cx43) in healing EC. Gap junctions have been implicated as regulators of wound healing in other tissues, yet Cx43 is not typically expressed in conduit artery EC. We hypothesize that injury-induced upregulation of Cx43 gap junction signaling is essential for EC healing. To test this, we developed a temporary mouse carotid artery ligation model which results in EC denudation injury. Confocal imaging of VE-cadherin was used to confirm EC loss following a one-minute ligation, and wound closure was almost complete within 24 hours in mice. Western blot of flushed carotids demonstrated an upregulation of EC Cx43, and en-face staining indicated Cx43 expression is primarily localized to the immediate wound edge EC (within 100 μm). We generated endothelial-specific (CDH5crePac) Cx43 knockout mice (CDH5-Cx43KO) to test the requirement of Cx43. We confirmed a loss of Cx43 in EC of mice fed a tamoxifen diet for four weeks following ligation. Wound healing was significantly reduced in CDH5-Cx43KO mice. Single cell RNA sequencing was performed on whole carotids following ligation injury in Cx43fl/fl vs. CDH5-Cx43KO mice to determine Cx43-dependent effects on EC phenotypic change during wound healing. As Cx43 channel functions are regulated by phosphorylation, we used phosphorylation mutation mice (alanine knock-in) that target key Cx43 C-terminal domains. We found that preventing Cx43 phosphorylation by mitogen-activated protein kinase (Cx43-MAPK), which is associated with gap junction turnover and cell proliferation, resulted in similar delays in healing to EC-Cx43 knockout. These data suggest that EC injury-induced Cx43 upregulation is required for normal EC wound healing after vascular injury and that gap junctional signaling via Cx43-MAPK interactions may play a mechanistic role. Further studies are ongoing to understand the specific role of Cx43 gap junction signaling in EC wound healing.