Deficiency of endothelial CXCR4 reduces reendothelialization and enhances neointimal hyperplasia after vascular injury in atherosclerosis-prone mice.
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.


RESULTS
β-Galactosidase staining using bone marrow x kinase (Bmx)-CreER(T2) reporter mice and double immunofluorescence revealed an efficient and endothelial-specific deletion of Cxcr4 in Bmx-CreER(T2+) compared with Bmx-CreER(T2-) Cxcr4-floxed apolipoprotein E-deficient (Apoe(-/-)) mice (referred to as Cxcr4(EC-KO)ApoE(-/-) and Cxcr4(EC-WT) ApoE(-/-), respectively). Endothelial Cxcr4 deficiency significantly increased wire injury-induced neointima formation in carotid arteries from Cxcr4(EC-KO)ApoE(-/-) 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 Cxcr4(EC-KO)ApoE(-/-) carotids compared with Cxcr4(EC-WT)ApoE(-/-) 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 Cxcr4(EC-KO) ApoE(-/-) 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 Cxcr4(EC-KO)ApoE(-/-) 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.
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.). kreislaufinstitut@med.uni-muenchen.de hnoels@ukaachen.de.
Heidi
H
Noels
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Baixue
B
Zhou
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Pathricia V
PV
Tilstam
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Wendy
W
Theelen
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Xiaofeng
X
Li
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Lukas
L
Pawig
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Corinna
C
Schmitz
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Shamima
S
Akhtar
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Sakine
S
Simsekyilmaz
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Erdenechimeg
E
Shagdarsuren
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Andreas
A
Schober
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Ralf H
RH
Adams
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Jürgen
J
Bernhagen
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Elisa A
EA
Liehn
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.).
Yvonne
Y
Döring
From the Institute for Molecular Cardiovascular Research (H.N., B.Z., P.V.T., W.T., X.L., L.P., S.A., S.S., E.S., E.A.L.) and Institute of Biochemistry and Molecular Cell Biology (C.S., J.B.), University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany; Institute for Cardiovascular Prevention (A.S., Y.D., C.W.) and August-Lenz-Stiftung, Institute for Cardiovascular Research (J.B.), Ludwig-Maximilians-University Munich, Munich, Germany; Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany (R.H.A.); Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands (C.W.); and German Centre for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Forschung), partner site Munich Heart Alliance, Munich, Germany (C.W.). kreislaufinstitut@med.uni-muenchen.de hnoels@ukaachen.de.
Christian
C
Weber
D000818:Animals; D000950:Antigens, Ly / Q000502:physiology; D001057:Apolipoproteins E / Q000502:physiology; D050197:Atherosclerosis / Q000473:pathology* / Q000503:physiopathology; D020212:Carotid Artery Injuries / Q000473:pathology*; D002465:Cell Movement; D054377:Chemokine CXCL12 / Q000502:physiology; D042783:Endothelial Cells / Q000502:physiology*; D005260:Female; D006965:Hyperplasia; D008565:Membrane Proteins / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D058426:Neointima / Q000473:pathology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D019718:Receptors, CXCR4 / Q000502:physiology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000502:physiology
D000950:Antigens, Ly; D001057:Apolipoproteins E; C514936:CXCR4 protein, mouse; D054377:Chemokine CXCL12; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D019718:Receptors, CXCR4; C492366:Bmx protein, mouse; D011505:Protein-Tyrosine Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2
chemokine Cxcl12; progenitor cells; receptors, Cxcr4; restenosis; vascular biology