Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition in Atherosclerosis
Background: Protein-tyrosine-phosphatase CD45 is expressed in all nucleated cells of the hematopoietic system but has also been detected in mitral valve endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndoMT). Interestingly, our recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony-forming cells (ECFCs) also induced expression of multiple EndoMT marker genes. We hypothesize that CD45 may contribute to atherosclerosis, a prevalent cardiovascular disease dependent on EndoMT.  However, the detailed molecular mechanisms underlying how CD45 contributes to EndoMT and the impact of therapeutic manipulation of CD45 expression in atherosclerosis are entirely unknown. Method: We generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE-deficient (ApoE-/-/C57BL/6) background and fed them a Western diet to produce atherosclerosis. We enriched mouse aortic ECs with anti-CD31 beads to perform single-cell RNA sequencing. Cellular, biochemical,  and molecular approaches were used to investigate the effect of endothelial CD45-specific deletion on EndoMT and lesion development in ApoE-/- model of atherosclerosis. Results: Endothelial ApoE-/- CD45-deficient mice showed reduced lesion development, plaque macrophages, and expression of cell adhesion molecules when compared to ApoE-/- controls. Single-cell RNA sequencing revealed that loss of endothelial CD45 reduces EndoMT marker expression and TGF-ß signaling in atherosclerotic mice, which is associated with the reduction of lesions in the ApoE-/- mouse model. Mechanistically, the loss of CD45 results in increased FGFR2 in mouse aortic ECs and KLF2 expression in the aortic root. Consistently, endothelial CD45-deficiency inhibits EndoMT and TGF-ß signaling. Conclusions: These findings demonstrate that genetic depletion of endothelial CD45 protects against EndoMT-driven atherosclerosis by promoting FGFR2 and KLF2 expression while inhibiting TGFß signaling and EndoMT. Thus, targeting endothelial CD45 may be a novel therapeutic strategy to reduce  EndoMT and treat atherosclerosis. Overall design: We generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE-deficient (ApoE-/-/C57BL/6) background and fed them a Western diet to produce atherosclerosis. We enriched mouse aortic ECs with anti-CD31 beads to perform single-cell RNA sequencing (10X Genomics). Cellular, biochemical,  and molecular approaches were used to investigate the effect of endothelial CD45-specific deletion on EndoMT and lesion development in ApoE-/- model of atherosclerosis.