title: Foxc2 overexpression enhances benefit of endothelial progenitor cells for inhibiting neointimal formation by promoting CXCR4-dependent homing.
abstract: OBJECTIVE
  Endothelial progenitor cells (EPCs) are capable of enhancing re-endothelialization and attenuating neointimal formation. However, inefficient homing limits the therapeutic efficacy of EPCs transplantation. CXCR4 plays a critical role in regulating EPCs homing. Here, we studied the effect of Foxc2 overexpression on CXCR4 expression and the homing capacity of EPCs as well as the EPCs-mediated therapeutic benefit after artery injury.
  
  
  METHODS
  Bone marrow-derived EPCs were transfected with Foxc2 expression vector (Foxc2-EPCs) or empty control vector (Ctrl-EPCs) and examined 48 hours later. CXCR4 expression of EPCs was detected by flow cytometry and quantitative reverse transcriptase-polymerase chain reaction. The migration of EPCs toward SDF-1α was evaluated in a transwell migration assay, and the adhesion to fibronectin was determined using a static adhesion assay. For in vivo studies, EPCs were injected intravenously into the mice subjected to carotid injury. At 3 days after green fluorescent protein (GFP)/EPCs delivery, the recruited cells to the injury sites were detected by fluorescent microscopy. Re-endothelialization and neointimal formation were, respectively, assessed by Evans blue dye at 7 days and by the morphometric analysis for neointima and media area ratio (N/M) at 28 days after EPCs transfusion.
  
  
  RESULTS
  Foxc2 overexpression significantly increased the surface expression of CXCR4 on EPCs (about 1.9-fold of Ctrl-EPCs, P < .05). Foxc2-EPCs showed an increased migration toward SDF-1α (P < .05); Foxc2 overexpression increased also the adhesion capacity of EPCs (P < .05). In vivo, the number of recruited GFP cells was significantly higher in the mice transfused with Foxc2-GFP/EPCs compared with Ctrl-GFP/EPCs (about 2-fold of Ctrl-GFP/EPCs). The degree of re-endothelialization was higher in mice transfused with Foxc2-EPCs compared with Ctrl-EPCs (90.3% ± 1.6% vs 57.2% ± 1.3%; P < .05). Foxc2-EPCs delivery resulted in a greater inhibition of neointimal hyperplasia than Ctrl-EPCs administration (N/M: 0.38 ± 0.03 vs 0.67 ± 0.05, P < .05). Preincubation with CXCR4-Ab, AMD3100, or LY294002 significantly attenuated the enhanced in vitro and in vivo effects of Foxc2-EPCs.
  
  
  CONCLUSIONS
  Our findings indicate that Foxc2 overexpression increases CXCR4 expression of EPCs and efficiently enhances the homing potential of EPCs, thereby improving EPCs-mediated therapeutic benefit after endothelial injury. Foxc2 may be a novel molecular target for improving the therapeutic efficacy of EPCs transplantation.
authors.affiliation: Institute of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Dujuan
authors.initials: D
authors.lastname: Li
authors.forename: Dan
authors.initials: D
authors.lastname: Yan
authors.forename: Weishuo
authors.initials: W
authors.lastname: Liu
authors.forename: Mincai
authors.initials: M
authors.lastname: Li
authors.forename: Jun
authors.initials: J
authors.lastname: Yu
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Zhiling
authors.initials: Z
authors.lastname: Qu
authors.forename: Qiurong
authors.initials: Q
authors.lastname: Ruan
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D054377:Chemokine CXCL12 / Q000378:metabolism; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism* / Q000502:physiology; D051858:Forkhead Transcription Factors / Q000096:biosynthesis*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D058426:Neointima / Q000517:prevention & control*; D019718:Receptors, CXCR4 / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism* / Q000502:physiology; D015854:Up-Regulation
chemical_list: C514936:CXCR4 protein, mouse; D054377:Chemokine CXCL12; D051858:Forkhead Transcription Factors; D019718:Receptors, CXCR4; C082078:mesenchyme fork head 1 protein