Abstract Body (Do not enter title and authors here): Introduction: Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells (ECs). However, a recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells induced expression of EndMT marker genes. Also, the CD45 phosphatase inhibitor blocked EndMT activities in TGFβ1-treated ovine mitral valve endothelial cells. EndMT contributes to atherosclerotic pathobiology and is associated with more complex plaques. We identified CD45-positive ECs undergoing EndMT in atherosclerotic ApoE-deficient (ApoE^-/-) mice. Here, we aim to investigate whether endothelial CD45-specific deletion can prevent EndMT in a mouse model of atherosclerosis.
Methods and Results: We generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE^-/- background (C57BL/6) and fed these mice a Western diet for 16 weeks. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA-seq. Cells populated 15 major clusters after integrating the single-cell transcriptomic profiles. In the EC-iCD45KO group, the population of endothelial, resident macrophage, and mesenchymal-endothelial transition cells increased while vascular smooth muscle, EndMT, and fibroblast cells decreased. In EndMT cells, EC markers (Cdh5, Cldn5, Pecam1) increased, but SMC markers (Acta2) and EndMT markers (Col1a2 and Col3a1）decreased. In addition, we characterized EndMT cells into two sub-clusters: 1 and 2. EC-specific CD45 deletions reduced the cell numbers in the EndMT2 sub-cluster in atherosclerotic mice but had less impact on the EndMT1 sub-cluster. FGFR pathways were potentiated while TGFβ1 and Tgfbr2 decreased in EndMT1 sub-cluster， and the genes controlling endothelial cell development were upregulated but the genes controlling actin cytoskeleton organization were downregulated in both EndMT1 and 2 sub-clusters.
Conclusion: Our single-cell analysis indicates that the loss of endothelial CD45 protects against EndMT-driven atherosclerosis, inhibiting TGFβ and EndoMT marker expression while stimulating FGFR pathways. Consistently, EC-iCD45KO/ApoE^-/- mice showed reduced plaque macrophages, expression of cell adhesion molecules, foam cell formation, and lesion development when compared to ApoE^-/- controls. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndMT and atherosclerosis.