Deficiency of Endothelial Cxcr4 Reduces Reendothelialization and Enhances Neointimal Hyperplasia After Vascular Injury in Atherosclerosis-Prone Mice

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Abstract

Objective—

The Cxcl12/Cxcr4 chemokine ligand/receptor axis mediates the mobilization of smooth muscle cell progenitors, driving injury-induced neointimal hyperplasia. This study aimed to investigate the role of endothelial Cxcr4 in neointima formation.

Approach and Results—

β-Galactosidase staining using bone marrow x kinase (Bmx)-CreERT2 reporter mice and double immunofluorescence revealed an efficient and endothelial-specific deletion of Cxcr4 in Bmx-CreERT2+ compared with Bmx-CreERT2−Cxcr4-floxed apolipoprotein E–deficient (Apoe−/−) mice (referred to as Cxcr4EC-KOApoE−/− and Cxcr4EC-WTApoE−/−, respectively). Endothelial Cxcr4 deficiency significantly increased wire injury–induced neointima formation in carotid arteries from Cxcr4EC-KOApoE−/− mice. The lesions displayed a higher number of macrophages, whereas the smooth muscle cell and collagen content were reduced. This was associated with a significant reduction in reendothelialization and endothelial cell proliferation in injured Cxcr4EC-KOApoE−/− carotids compared with Cxcr4EC-WTApoE−/− controls. Furthermore, stimulation of human aortic endothelial cells with chemokine (C-X-C motif) ligand 12 (CXCL12) significantly enhanced their wound-healing capacity in an in vitro scratch assay, an effect that could be reversed with the CXCR4 antagonist AMD3100. Also, flow cytometric analysis showed a reduced mobilization of Sca1+Flk1+Cd31+ and of Lin−Sca1+ progenitors in Cxcr4EC-KOApoE−/− mice after vascular injury, although Cxcr4 surface expression was unaltered. No differences could be detected in plasma concentrations of Cxcl12, vascular endothelial growth factor, sphingosine 1-phosphate, or Flt3 (fms-related tyrosine kinase 3) ligand, all cytokines with an established role in progenitor cell mobilization. Nonetheless, double immunofluorescence revealed a significant reduction in local endothelial Cxcl12 staining in injured carotids from Cxcr4EC-KOApoE−/− mice.

Conclusions—

Endothelial Cxcr4 is crucial for efficient reendothelialization after vascular injury through endothelial wound healing and proliferation, and through the mobilization of Sca1+Flk1+Cd31+ cells, often referred to as circulating endothelial progenitor cells.

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