Accelerated Endothelial to Mesenchymal Transition Increased Fibrosis via Deleting Notch Signaling in Wound Vasculature

Skin wound healing in adults is characterized by a peak of angiogenesis followed by regression of the excessive vasculature in parallel with collagen deposition and fibrosis in the wound. We hypothesized that regressing vessels in healing wounds were in fact entering an endothelial to mesenchymal transition contributing to scarring. Using vascular-specific fate tracking (Cdh5-creERt2/ROSA-YFP mice), full-thickness excisional wounds were analyzed to reveal a time-dependent transition from endothelial phenotype characterized by vascular endothelial-cadherin, CD31, and CD34 toward a mesenchymal phenotype characterized by alpha-smooth muscle actin and fibroblast-specific protein 1 expression. We next conditionally ablated RBPJ in the vasculature (Rbpjfl/fl/Cdh5-creERt2ROSA-YFP) to evaluate the role of canonical Notch signaling in this process. Endothelial to mesenchymal transition was clearly accelerated after the loss of Notch signaling within the vasculature. The acceleration of endothelial to mesenchymal transition resulted in delayed wound healing, increased fibrosis, and extensive scar tissue formation, with the rapid loss of key endothelial genes and proteins and upregulation of mesenchymal protein expression (alpha-smooth muscle actin and fibroblast-specific protein 1) in vessels. Our findings here uncover a cellular contributor to skin wound scarring through the process of endothelial to mesenchymal transition in skin wounds and demonstrate the importance of Notch signaling in regulating this critical process during healing.