Foxc2 Overexpression In Endothelial Progenitor Cells Enhances Re-endothelialization Following Arterial Injury By Increased CXCR4-dependent Homing

Endothelial disruption or dysfunction is vital to the initiation and progression of atherosclerosis and postangioplasty restenosis. Thus, maintenance of endothelial integrity and promotion of early re-endothelialization are of paramount importance for reducing cardiovascular diseases and the post-intervention complications. Accumulating evidences suggest that endothelial progenitor cells (EPCs) mobilized or transfused are capable of facilitating endothelial repair through direct differentiation into endothelial cells and/or via the paracrine mechanisms. The vasoregenerative effects of EPCs depend on their homing to the vascular injury sites. Indeed, the number of recruited EPCs appears to be related to their circulating numbers but also the functional properties of EPCs homing. Studies show that CXCR4 is a key molecule in regulating EPCs homing and recruitment. Emerging evidences exist for the impaired CXCR4-dependent homing and the low engraftment of EPCs due to some risk factors for coronary artery disease. These underline the need for new strategies capable of increasing the CXCR4-mediated homing potential of EPCs.The Foxc2 protein, a member of the Forkhead/Fox transcription factor family, is essential for the cardiovascular system. Foxc2 plays an important role in the combinatorial regulation of endothelial gene expression and vasculogenesis during embryonic development. Foxc2 is recognized as a novel regulator of angiogenesis via induction of integrinβ3 and angiopoietin-2 expression. Foxc2 might be involved in the angiogenesis under pathologic conditions. Just as recent studies reveal that Foxc2 expression is associated with the human heart failure and increased in periinfarcted zones of the rat left ventricle. Notably, Foxc2 directly induces the expression of CXCR4 in endothelial cells. Recently, it is accepted that the phenotypic and functional behavior of endothelial colony forming cells (ECFCs, also called "late" EPCs) is very similar to mature endothelial cells. Until now there are no data showing the effects of Foxc2 on the functional properties of EPCs. Based on the effects of Foxc2 on the properties of endothelial cells, we sought to determine whether Foxc2 may affect CXCR4 expression and the homing capacity of EPCs as well as the EPCs-mediated endothelial repair.Part One Isolation and Characterization of Mouse Bone Marrow-derived Endothelial Progenitor CellsObjective:To isolate and culture mouse bone marrow-derived EPCs, and to identify the morphological, immunophenotypic and functional characteristics.Methods:Isolated mononuclear cells were cultured in endothelial cell basal medium-2 (EBM-2) supplemented with endothelial growth medium (EGM). After 24 h, nonadherent cells were removed. Adherent cells were cultured for further 15-21 days. Morphological characteristics were observed by inverted microscope. Immunofluorescence, western blot and flow cytometry were used to detect the endothelial cell markers and the hematopoietic markers. The functional characteristics were assessed by uptake of Dil-acLDL and Matrigel tube formation assay.Results:Isolated bone marrow derived-mononuclear cells were cultured for 15-21 days, which exhibited a cobblestone-like morphology. Immunofluorescence and western blot analysis demonstrated that the adherent cells expressed the endothelial cell markers, including CD31, VE-cad, FLK-1 and vWF. Flow cytometric analysis revealed that only very few cells expressed the hematopoietic marker CD34 or leukocytic marker CD45. Moreover, the majority of adherent cells were found positive for uptake of DiI-acLDL. In addition, these cells were capable of assembling into tube-like structures when plated in matrigel. On the basis of the morphological, immunophenotypic and functional characteristics, the cells in our study were confirmed EPCs characterized as ECFCs or late EPCs.Conclusions:Isolated bone marrow derived-mononuclear cells are cultured in EBM-2 supplemented with EGM, which give EPCs.Part Two Effects of Foxc2 Overexpression on Migration and Adhesion Potential of Endothelial Progenitor CellsObjective:To investigate the effects and mechanisms of Foxc2 overexpression on the in vitro migration and adhesion potential of EPCsMethods:EPCs were transfected by using lipidosome transfection reagent with Foxc2 expression vector (Foxc2-EPCs) or with empty control vector (Ctrl-EPCs) and examined 48 hours later. Foxc2 expression of EPCs was detected by western blot and quantitative RT-PCR (qRT-PCR). CXCR4 expression of EPCs was detected by immunofluorescence, western blot, flow cytometry and qRT-PCR. The migration of EPCs toward SDF-la was evaluated in a transwell migration assay, and the adhesion to fibronectin was determined using a static adhesion assay.Results:qRT-PCR and Western blot showed that the transfection with Foxc2 expression vector efficiently induced the up-regulation of Foxc2 mRNA and protein in EPCs. Flow cytometry showed that Foxc2 overexpression significantly increased surface expression of CXCR4 on EPCs. Fluorescence microscopy and western blot analysis further confirmed that Foxc2 overexpression up-regulated CXCR4 expression of EPCs. qRT-PCR analysis showed CXCR4 mRNA expression in Foxc2-EPCs was about 2-fold of Ctrl-EPCs (P<0.05). Foxc2-EPCs demonstrated an increased response to SDF-1α-mediated chemotaxis and adhesion. However, the increased effects were reduced by pretreatment of Foxc2-EPCs with the AMD3100 or LY294002.Conclusions:Foxc2 overexpression may markedly increase CXCR4 expression in EPCs and enhance the in vitro migration and adhesion capacities of EPCs. Moreover, the enhanced in vitro function by Foxc2 overexpression is associated with the up-regulation of CXCR4 and the activation of PI3K/Akt signal pathway.Part Three Effects of Foxc2 Overexpression on Homing and Re-endothelialization Potential of Endothelial Progenitor CellsObjective:To investigate the effects of Foxc2 overexpression on the in vivo homing and the re-endothelialization potential of EPCs.Methods:Mouse carotid injury was established using a 0.014-inch-PTCA flexible guide wire. The mice subjected to carotid injury were randomly assigned into experimental groups. EPCs and GFP/EPCs were isolated and cultured respectively from wild type mice and GFP/mice. EPCs and GFP/EPCs were transfected with Foxc2 expression vector (Foxc2-EPCs or Foxc2-GFP/EPCs) or with empty control vector (Ctrl-EPCs or Ctrl-GFP/EPCs). The mice subjected to carotid injury received PBS, Ctrl-EPCs or Foxc2-EPCs by tail vein injection. To examine the homing capacity, at 3 days after GFP/EPCs delivery, the recruited GFP+ cells to the injury sites were detected by fluorescent microscopy. For assessment of re-endothelialization, animals were perfused with Evans blue dye at 7 days after EPCs delivery. At 14 days, the percent of GFP positive endothelial cells in the cross-section of injured arteries was estimated to observe the incorporation of the delivered EPCs into the regenerative endothelial layer. Neointimal formation was assessed by the morphometric analysis for neointima and media area ratio (N/M) at 28 days after EPCs transfusion. Results:At 3 days after GFP/EPCs delivery, GFP+ cells were strictly restricted to the injury sites. 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). At 7 days after EPCs delivery, the degree of re-endothelialization was significantly higher in mice transfused with Ctrl-EPCs compared with PBS. Nevertheless, Foxc2-EPCs delivery further increased the degree of re-endothelialization relative to Ctrl-EPCs (90.3±1.6% vs.57.2±1.3%, P<0.05). More GFP cells were incorporated into the CD31 positive endothelial layer at 14 days after Foxc2-GFP/EPCs compared with Ctrl-EPCs delivery (46.7±7.1% vs.31.5±5.3%, P<0.05). At 28 days, Ctrl-EPCs delivery led to a 65% reduction in N/M compared with PBS control (P<0.05). However, the inhibitory effect of Foxc2-EPCs delivery was greater than Ctrl-EPCs (0.38±0.03 vs.0.67±0.05, P<0.05). Finally, preincubation with AMD3100 or LY294002 significantly attenuated the enhanced homing and therapeutic potential of Foxc2-EPCs for promoting re-endothelialization and inhibiting neointimal formation.Conclusions:Foxc2 overexpression may increase EPCs homing and recruitment to the sites of vascular injury, and thereby enhance the therapeutic benefit of EPCs for facilitating re-endothelialization and inhibiting neointimal hyperplasia. Moreover, the enhanced in vivo function by Foxc2 overexpression is associated with the CXCR4/PI3K/Akt signal pathway.