Role Of KLF4 On EPCs Differentiation And The Repair Vascular After Injury

1. Background and Objective:Dysfunction of the vascular endothelium is a vital factor in the pathogenesis of vascular disease. Neovascularization and re-endothelialization are important repair mechanisms that might prevent the occurrence of in-stent restenosis and late thrombosis with drug-eluting stents after angioplasty. A growing body of evidence demonstrates that endothelial progenitor cells (EPCs) might play an important part in endothelial repair and the replacement of dysfunctional endothelium. Furthermore,the proliferation and differentiation of EPCs can be enhanced or inhibited by the regulation of genes that encode transcription factors and enzymes. However, the underlying mechanisms remain completely unknown.KLF4,zinc finger transcription factors, was involved in the regulation of numerous biological processes including proliferation, differentiation, development, and apoptosis. Recent experiments have indicated that KLF4 functions in response to upstream signals to promote the specification and differentiation of epidermal cells and intestinal epithelium. Many studies have confirmed that KLF4 is expressed in endothelial cells and is induced by proinflammatory stimuli and shear stress. Therefore, KLF4 plays an important role in the biology of endothelial cells . However, a potential role for KLF4 in EPCs, and the mechanisms by which it might act, have yet to be elucidated.In this study, we performed overexpressed KLF4 and siRNA transfection experiments to evaluate whether KLF4 regulates the differentiation of EPCs ,and participation in vascular regeneration,and to investigate the molecular mechanisms involved.Our findings provide a novel role in biological function of KLF4 and the molecular mechanisms behind EPCs-mediated re-endothelialization.2. Methods:2.1 KLF4 expression during differentiation of EPCs into endothelial cells (ECs).EPCs were isolated by density gradient centrifugation and cultured in low glucose DMEM supplemented with 10% FCS and 10ng/mL VEGF. To confirm the EPCs phenotype, cells were incubated with DiI-acLDL for 4 hours, fixed with 4% paraformaldehyde and then incubated with FITC-labeled lectin (UEA-1) for 1 hour. Dual-stained cells positive for both DiI-acLDL and UEA-1 were identified as EPCs. Additionally, flow cytometry (FACS) analysis was performed using antibodies against rat CD133, and VEGFR-2. We tested whether KLF4 was expressed during the differentiation of EPCs by performing RT-PCR to detect mRNA expression and western blotting to detect protein expression.2.2 Construction of recombinant adenoviral vectorsAdenoviral vectors repectively expressing KLF4 were generated using the AdEasy system. Briefly, full-length rat KLF4 cDNA were generated by RT-PCR using total RNA from Sprague–Dawley (SD) rat heart. Recombinant shuttle plasmid(pTOPO-KLF4)was constructed,and then homologous recombination was performed between attL1-attL2 sequence in TOPO-KLF4 and attR1-attR2 sequence in pAd.The recombinant adenovirus named as pAd-KLF4.The pAd-KLF4 and pAd linearized byPacI was transfected into 293 cells,and packaged,respectively.2.3 Effecs of KLF4 on EPCs differentiation into ECsTo investigate the effect of KLF4 on EPCs differentiation into ECs, we transduced Ad-KLF4 and KLF4siRNA into EPCs that were cultured in serum- and VEGF- free medium.We tested the changes of CD31and VWF during the differentiation of EPCs by performing RT-PCR to detect mRNA expression and western blotting to detect protein expression.2.4 Molecular mechanisms underlying KLF4 effects on EPCsTo identify the molecular mechanism that might be involved in the effect of KLF4 on EPCs, we next examined the effect of KLF4 on eNOS expression of mRNA expression and protein expression by performing RT-PCR and western blotting . Moreover, L-NAME, which is an inhibitor of NOS, to further confirm the impact of eNOS in KLF4-induced differentiation of EPCs.2.5. effect of KLF4 gene modified EPCs on ECs and VSMCs migration and proliferation 3~H-TdRIn the cell coculture system, KLF4 gene modified EPCs were seeded in the lower chamber and VSMCs in the upper chamber. 3~H-TdR incorporation were used to determine the effects of EPCs on the proliferation of VSMCs . The number of VSMCs or ECs migration was counted. Moreover ,we examined the protein expression of P53 on VSMCs.2.6 effect of Ad-KLF4 transduced EPCs transplantation on vascular endothelium repairThe carotid arteries intima injury model were made by balloon damage in SD rats.Ad-KLF4 transduced EPCs were injected by intravenous tail vein after induction of arterial injury. The injured segments were isolated 2 weeks after EPCs transplantation. RT-PCR and Western blot were used to detect the level of KLF4 mRNA and protein in balloon-injured rat carotid artery. Evans Blue dye was administered to evaluate reendothelialization at 14 days after injury,The morphology of arterial intima and media was studied by optical microscopy and image analysing system.3. Results:3.1 Characterization of EPCs and KLF4 expression during differentiation of EPCs into endothelial cells (ECs).3.1.1 EPCs isolation and characterizationAfter 7 days of culture, adherent EPCs were characterized by immunofluorescence and flow cytometry analysis (FACS). The majority of cells (>80%) stained positive for DiI-AcLDL and lectin, and expressed endothelial/stem cell markers, including VEGFR-2 (82.35%±2.04%), and CD133 (62.13±3.05%), confirming the cell type of EPCs.3.1.2 Characterization of during differentiation of EPCs into ECsTotal BM-MNCs were isolated. After 4 days in culture, when induced to differentiate, some cells formed foci or cord-like structures and after 7 days spindle-like cells had begun to sprout from the foci. After 14 days, some cells had formed tubular-like structures, whereas after 21 days, many cells had assumed a“cobblestone-like”morphology. We found that the cells that formed foci during the early stages of differentiation expressed markers for endothelial progenitors, including CD133, but did not express CD31and eNOS. After 14 days, the cells that had begun to sprout from the periphery of foci expressed CD31 but not CD133 by immunostaining and FACS analysis.3.1.3 KLF4 expression during differentiation of EPCs into endothelial cells (ECs).The levels of KLF4 mRNA and protein were low during the early stages of differentiation but had increased by day 7 and had increased significantly by day 14 of culture. In order to analyze the subcellular localization of KLF4, we performed immunostaining, which revealed that KLF4 was localized predominantly in the nucleus of EPCs and confirmed that KLF4 was expressed in these cells..3.2 Recombinant adenoviral vectors expressing KLF4Full length cDNA encoding either KLF4 was amplified by RT-PCR using total RNA from Sprague–Dawley (SD) rat heart . The cDNA was first TA-cloned into pTOPO-KLF4 vector and then subcloned into adenoviral shuttle vector pAdTrack-CMV. Recombinant adenovirus Ad-KLF4 were generated and purified according to the manufacturer's protocol. The adenovirus virus titer was about 1×10~9 plaque-forming units per millilitre (pfu /ml), as determined by plaque assay.3.3 effect of KLF4 enhances the expression of EC markers in EPCsThe expression of KLF4 was increased markedly in EPCs infected with Ad-KLF4. The expression of KLF4 protein in the Ad-KLF4 group was significantly higher than that in the Ad-GFP or control group. Quantitative real-time PCR analysis revealed that the expression of CD133 and CD34 was decreased, but the expression of vWF and CD31 was increased. Western blotting revealed that overexpression of KLF4 upregulated the expression of CD31and VWF, which are markers of mature ECs by immunofluorescence staining. In addition, overexpression of KLF4 increased the percentage of Dil-ac-LDL/FITC-UEA-1 double-positive cells dramatically, as compared with the Ad-GFP or control group. The level of KLF4 protein was reduced effectively in the EPCs after transfection with the KLF4 siRNA oligonucleotide as compared with the NS siRNA Importantly, protein expression of the mature EC markers CD31 and VWF was also inhibited dramatically in the cells transfected with the KLF4-specific siRNA.3.4 KLF4 mediates the differentiation of EPCs into ECs by promoting eNOS expressionWestern blotting and RT-PCR revealed that overexpression of KLF4 upregulated the expression of eNOS protein and gene. Moreover, the expression of eNOS protein was inhibited by the KLF4 siRNA but not by the NS siRNA.Overexpression of KLF4 enhanced NO levels in the medium as compared with the control and Ad-GFP. Moreover, L-NAME, which is an inhibitor of NOS, inhibited the induction of protein expression by KLF4 of CD31 and VWF in the EPCs and nearly attenuated the protein expression of these factors. Moreover,the tube formation was significantly attenuated on treatment with L-NAME.3.5 effect of KLF4 gene modified EPCs on VSMCs migration and proliferationIn the cell coculture system,KLF4 gene modified EPCs can inhibit VSMCs proliferation and and migration in Ad-klf4-EPCs group than control group. Compared with the control group, the protein expression of P53 in VSMCs was increased.3.6 effect of KLF4 gene modified EPCs transplantation on vascular repair after injuryThe injured segments were isolated 2 weeks after EPCs transplantation. Findings with RT-PCR and Western blot in injured artery showed that KLF4 was overexpressed in Ad-KLF4-EPCs transplantation group. Immunohistochemistry detection showed that KLF4 expression was significantly enhanced in Ad-KLF4-EPCs transplantation group. Further, immunohistochemistry showed that KLF4 was detected in the intima, media, and adventitia of local vessels.A marked decrease in the neointimal area and I/M ratio was shown in Ad-KLF4-EPCs treated rats compared with that of control group at day 14. Nonendothelialized lesions were marked blue about 95% at injured vessels, whereas the reendothelialized area appeared white at uninjured vessels.The reendothelialized area in the Ad-KLF4-EPCs treated arteries was significantly larger than that in Ad-GFP infected arteries.4. Conclusions:4.1 Overexpression of KLF4 stimulated EPCs differentiation into Ecs.4.2 Overexpression of KLF4 upregulated the expression of eNOS protein and gene. The expression of eNOS protein was inhibited by the KLF4 siRNA .Moreover, L-NAME, which is an inhibitor of NOS, inhibited the induction of protein expression by KLF4 of CD31 and VWF in the EPCs and nearly attenuated the protein expression of these factors. The tube formation was significantly attenuated on treatment with L-NAME.4.3 In the cell coculture system,KLF4 gene modified EPCs can inhibit VSMCs proliferation and and migration,and increase the protein exproeeion of P53 in VSMCs .4.4 KLF4 expression was significantly enhanced and larger the reendothelialized area in the Ad-KLF4-EPCs treated arteries after injury .