KLF4调控EPCs分化及修复损伤血管的作用研究
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摘要
1.背景与目的:
内皮功能紊乱是血管性疾病的重要的病理生理基础,再血管化内皮和再内皮化是介入治疗和药物支架后阻止再狭窄和血栓形成的重要环节。大量的证据表明,骨髓源内皮前体细胞(endothelial precusor cells, EPCs)在血管内皮修复和内皮功能替代上起了重要的作用。更进一步研究表明,EPCs的增殖与分化是影响内皮修复的重要环节, EPCs在体内能够分化成有功能的内皮细胞,参与缺血组织的血管新生并整合到损伤血管壁的新生内膜中参与损伤血管的再内皮化。但是,EPCs的归巢、增殖、分化,其受多种因素影响,其内在调控因素尚不完全清楚。
转录因子KLF4通过对其下游基因的调控,在细胞增殖、分化、血管生成和肿瘤发生发展等多种生理、病理过程中起着重要作用。近几年,研究表明KLF4调控多种细胞的增殖分化,可能是调节血管功能和疾病的重要“分子开关”。KLF4最早发现于内脏和上皮,具有促进细胞终端分化、成长、循环的功能。其为血流及炎症因子激活,KLF4高表达能够上调eNOS、TM,具有抗炎、抗血栓作用。
在本研究我们主要通过构建携带KLF4基因的腺病毒载体,过表达KLF4或小RNA干扰转染实验,阐明KLF4在EPCs分化及修复损伤血管中的作用及其机制,期望为深入认识KLF4的生物学功能及EPCs的调控机制提供新的实验依据,为进一步促进血管损伤后内皮再生、血管良性修复提供新的策略和靶点。
2.方法:
2.1 KLF4在EPCs分化中的表达
通过密度梯度离心及选择性培养的方法在体外分离、培养大鼠骨髓源EPCs,经过细胞形态学、表面分子标志以及Dil-acLDL/FITC-UEA-I双阳性鉴定后,通过RT-PCR以及Western blot分别观察KLF4在EPCs分化过程中mRNA和蛋白表达水平的表达。
2.2重组腺病毒Ad-KLF4的构建
从大鼠组织提取RNA,经RT-PCR扩增得到目的基因KLF4片段,首先构建重组穿梭质粒pTOPO-KLF4。将pTOPO-KLF4与腺病毒载pAd在重组酶的作用下,pTOPO-KLF4的attL1-attL2序列与腺病毒载体的attR1-attR2序列发生同源重组。重组子命名为pAd-KLF4。pAd-KLF4和空载体pAd经PacI酶切线性化,暴露出载体的反向终末重复序列,将重组子转染293细胞进行包装,扩增后进行滴度的测定。
2.3 KLF4在EPCs分化中的作用
在EPCs通过Ad-KLF4过表达或siRNA沉默KLF4观察EPCs分化的表型改变,通过免疫荧光、流式细胞学、RT-PCR以及Western blot分别观察成熟血管内皮表型CD31、VWF在EPCs分化过程中免疫荧光、mRNA和蛋白水平的表达。
2.4 KLF4在EPCs分化中的分子机制
在EPCs通过Ad-KLF4过表达或siRNA沉默KLF4对下游靶基因eNOS的在转录、蛋白表达水平的影响,进一步观察了eNOS抑制剂L-NAME,对Ad-KLF4诱导的EPCs分化的影响,以明确eNOS是否参与Ad-KLF4介导调控的EPCs分化。
2.5 Ad-KLF4转染的EPCs与平滑肌细胞共培养对平滑肌的增殖及迁移的影响
培养大鼠血管平滑肌细胞和内皮细胞,将Ad-KLF4转染的EPCs接种于下室,分别将平滑肌细胞接种于上室,建立细胞共培养体系,通过3~H-TdR参入法检测平滑肌细胞的增殖作用,用迁移计数方法检测平滑肌细胞。
2.6转染KLF4基因的EPCs移植在血管内膜修复中的作用
建立大鼠颈动脉球囊损伤模型,将Ad-KLF4转染的EPCs通过尾静脉移植至大鼠体内。2周后在局部血管,通过荧光定量RT-PCR以及Western blot分别观察KLF4在血管损伤修复过程中mRNA和蛋白表达,行HE染色、伊文氏蓝染色计算内膜/中膜面积比及损伤血管再内皮化情况。
3.结果:
3.1 KLF4在EPCs分化中的表达
3.1.1 EPCs鉴定
分离培养的EPCs经过诱导分化后向内皮细胞表型转变,流式细胞检测及VEGFR-2和CD133在培养细胞中的阳性率分别为82.35%±2.04%,62.13±3.05%, Dil-acLDL/FITC- UEA-I双阳性细胞约占80%,说明所培养细胞是EPCs。
3.1.2 EPCs向ECs分化的特征
形态学变化:细胞在培养第3~4天可见部分贴壁细胞的多个细胞集落,呈圆形或不规则形,随之梭形细胞数量逐渐增加;第6~10天贴壁的梭形细胞从细胞团边缘出芽生长,呈放射状分布;14d后可观察到梭形细胞首尾相连形成条索状结构,梭形细胞基本接近融合,具有成索状或网状血管样生长趋势;21天细胞增多,融合后呈铺路石状。利用细胞流式分析技术(FACS)对EPCs的分子表面标记检测,发现在MNCs培养过程随时间的变化,在EPCs分化过程中,干细胞表型CD133逐渐减低,而成熟ECs表型逐渐增高。
3.1.3 KLF4在EPCs分化中的表达
在培养7天的MNCs细胞,免疫组化染色,阳性细胞成棕黄色,胞核为苏木精淡染,KLF4表达在EPCs的细胞核。在EPCs分化过程中,KLF4 mRNA在第4天较低,第7天表达逐渐增强,14天表达明显增强,分别为(0.41±0.025,0.1±0.008,0.720±0.017,1.223±0.013)各组间比较(P<0.01)。KLF4蛋白表达在第4天较低,第7天表达逐渐增强,14天表达明显增强(分别为0.41±0.025,0.1±0.008,0.72±0.017,1.22±0.013)各组间比较(P<0.01),14d时表达最强。
3.2.重组腺病毒Ad-KLF4的构建
从大鼠组织提取RNA,经RT-PCR扩增得到目的基因KLF4片段,首先构建重组穿梭质粒pTOPO-KLF4。将pTOPO-KLF4与腺病毒载pAd在重组酶的作用下,pTOPO-KLF4的attL1-attL2序列与腺病毒载体的attR1-attR2序列发生同源重组。重组子命名为pAd-KLF4。pAd-KLF4和空载体pAd经PacI酶切线性化,暴露出载体的反向终末重复序列,将重组子转染293细胞进行包装,扩增后进行滴度的测定,病毒滴度约为1.0×10~9 pfu/ml。
3.3 KLF4在EPCs-ECs分化中的作用
3.3.1 Ad-KLF4转染EPCs
Ad-KLF4转染后,检测EPCs中KLF4mRNA及蛋白表达发现Ad-KLF4明显高于转染Ad-GFP和未转染组以及对照组,Ad-GFP转染和未转染组之间无显著差异,表明Ad-KLF4的有效转染EPCs呈现高表达。
3.3.2 Ad-KLF4转染EPCs对EPCs-ECs分化影响
Ad-KLF4转染EPCs与Ad-GFP组相比,形态特征呈现出细胞贴壁伸展的更多,呈长梭形或多角形分化形态,向内皮形态变化。流式细胞学检测、免疫荧光、定量PCR及蛋白表达检测提示,Ad-KLF4转染与Ad-GFP转染组比较,Dil-ac-LDL/FITC-UEA-1摄取阳性细胞计数增加,CD133、CD34mRNA显著降低,而成熟内皮表型vWF、CD31表型明显增高,表明转染Ad-KLF4促进了EPCs向成熟ECs表型的分化。
3.3.3 KLF4-siRNA转染EPCs对EPCs-ECs分化影响
经过KLF4siRNA内源性的干扰实验,发现在KLF4蛋白水平与阴性对照组比较,几乎不表达,表明进行了内源性的有效干扰,(0.98±0.03 vs 0.02±0.01 ,P<0.01, n=3)
KLF4siRNA转染72小时后与阴性对照组相比,在EPCs分化过程中,成熟内皮表型CD31的蛋白表达与阴性对照组比较明显降低((1.43±0.04 vs 1.01±0.02 ,P<0.05,n=3),VWF的蛋白表达与阴性对照组比较也明显降低((1.21±0.06 vs 0.87±0.02 ,P<0.05,n=3)。
3.4 KLF4在EPCs分化中的机制
3.4.1 KLF4对eNOS表达的影响
在转染Ad-KLF4后eNOS mRNA和蛋白表达比较Ad-GFP组明显升高,与未转染组比较也显著升高。在进行小RNA干扰阻断内源性的KLF4研究中发现,KLF4 siRNA转染eNOS mRNA及蛋白表达与阴性对照组比较,明显降低,与未转染组比较也表现降低。
3.4.2 L-NAME对Ad-KLF4促进的EPCs分化表型的影响
L-NAME加入Ad-KLF4转染72小时后检测蛋白表达分析,发现L-NAME干预组在EPCs分化为成熟内皮表型CD31、VWF的蛋白表达与Ad-KLF4降低,与未转染组相比也降低。培养液NO含量检测,发现Ad-KLF4组较和L-NAME干预组NO含量高,也明显高于对照组,L-NAME与Ad-GFP组比较无明显差异。体外血管生成,提示Ad-KLF4组较和L-NAME干预组数量多。
3.5 Ad-KLF4转染EPCs与平滑肌共培养对平滑肌增殖和迁移的作用
3.5.1 Ad-KLF4转染EPCs与平滑肌共培养对平滑肌增殖的作用
Ad-KLF4转染的EPCs与Ad-GFP及为未转染组比较明显抑制血管平滑肌细胞3~H-TdR掺入,在Ad-KLF4及为未转染组之间血管平滑肌细胞3~H-TdR掺入无显著差异。应用siRNA阻断内源性的KLF4与阴性对照组比较平滑肌细胞增殖明显,3~H-TdR掺入与未转染组比较3~H-TdR掺入。在阴性对照组和未转染组之间比较无差异。
3.5.2 Ad-KLF4转染EPCs与平滑肌共培养对平滑肌迁移的作用
在进行平滑肌细胞迁移检测,结果表明,共培养6 h,Ad-KLF4转染的EPCs组明显抑制平滑肌细胞的迁移数,Ad-GFP与对照组比较比较差异无显著性。KLF4 siRNA转染EPCs组与阴性对照组比较平滑肌细胞迁移数明显增加。在阴性对照组和未转染组之间比较无差异。
3.5.3 Ad-KLF4转染EPCs与平滑肌共培养对平滑肌P53蛋白的作用
应用Ad-KLF4转染的EPCs与对照组比较平滑肌细胞的P53蛋白增加。Ad-GFP与对照组比较比较差异无显著性。KLF4 siRNA转染EPCs组与阴性对照组比较P53蛋白明显降低。在阴性对照组和未转染组之间比较无差异。
3.6 Ad-GFP-EPCs移植在血管损伤修复中的作用
3.6.1 Ad-GFP-EPCs移植对再内皮化的作用
通过Evans blue染色发现, Ad-KLF4转染的EPCs移植组再内皮化率明显高于单纯,而Ad-GFP-EPCs移植组也高于球囊损伤组,各组之间比较,P<0.01,存在显著差异。
3.6.2 Ad-GFP-EPCs移植对新生内膜的作用
Ad-EPCs移植组在损伤2周后血管新生内膜、血管腔狭窄程度比较损伤未移植组和Ad-GFP组明显减轻,Ad-EPCs移植组新生内膜/中膜的比值较其他两组低。
3.6.3 Ad-GFP-EPCs移植组局部损伤处KLF4的表达
进行损伤血管的免疫组化检测。结果表明,正常血管KLF4表达极低,在Ad-KLF4转染的EPCs移植组,KLF4在新生内膜、中膜以及外膜组织均有表达,表达较强,而Ad-GFP-EPCs移植组表达降低,但较正常组明显增高,球囊损伤组KLF4表达比Ad-GFP-EPCs移植组减低,但均高于正常组。WesternBlot法进行损伤血管的KLF4蛋白检测。结果表明,正常血管KLF4蛋白表达极低,在Ad-KLF4转染的EPCs移植组,KLF4蛋白表达高于正常组、Ad-GFP-EPCs及球囊损伤组,其中KLF4表达球囊损伤组较Ad-GFP-EPCs移植组低,但均高于正常组,各组之间P<0.01。
4.结论:
4.1在EPCs分化过程中KLF4在分化晚期表达,逐渐增高。
4.2过表达KLF4促进EPCs向成熟内皮分化。
4.3过表达KLF4促进EPCs的eNOS表达,培养液的NO增多,eNOS抑制剂,L-NAME减低了KLF4在EPCs分化为成熟内皮表型CD31、VWF的蛋白表达,抑制了血管新生。
4.4 KLF4基因修饰EPCs与平滑肌共培养明显抑制平滑肌的增殖和迁移,同时使平滑肌的P53蛋白表达增高。
4.5 KLF4基因修饰EPCs移植抑制了损伤血管新生内膜,促进了再内皮化,在局部损伤处表达增高。
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 vectors
Adenoviral 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 ECs
To 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 EPCs
To 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-TdR
In 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 repair
The 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 characterization
After 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 ECs
Total 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 KLF4
Full 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 EPCs
The 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 expression
Western 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 proliferation
In 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 injury
The 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 .
内皮功能紊乱是血管性疾病的重要的病理生理基础,再血管化内皮和再内皮化是介入治疗和药物支架后阻止再狭窄和血栓形成的重要环节。大量的证据表明,骨髓源内皮前体细胞(endothelial precusor cells, EPCs)在血管内皮修复和内皮功能替代上起了重要的作用。更进一步研究表明,EPCs的增殖与分化是影响内皮修复的重要环节, EPCs在体内能够分化成有功能的内皮细胞,参与缺血组织的血管新生并整合到损伤血管壁的新生内膜中参与损伤血管的再内皮化。但是,EPCs的归巢、增殖、分化,其受多种因素影响,其内在调控因素尚不完全清楚。
转录因子KLF4通过对其下游基因的调控,在细胞增殖、分化、血管生成和肿瘤发生发展等多种生理、病理过程中起着重要作用。近几年,研究表明KLF4调控多种细胞的增殖分化,可能是调节血管功能和疾病的重要“分子开关”。KLF4最早发现于内脏和上皮,具有促进细胞终端分化、成长、循环的功能。其为血流及炎症因子激活,KLF4高表达能够上调eNOS、TM,具有抗炎、抗血栓作用。
在本研究我们主要通过构建携带KLF4基因的腺病毒载体,过表达KLF4或小RNA干扰转染实验,阐明KLF4在EPCs分化及修复损伤血管中的作用及其机制,期望为深入认识KLF4的生物学功能及EPCs的调控机制提供新的实验依据,为进一步促进血管损伤后内皮再生、血管良性修复提供新的策略和靶点。
2.方法:
2.1 KLF4在EPCs分化中的表达
通过密度梯度离心及选择性培养的方法在体外分离、培养大鼠骨髓源EPCs,经过细胞形态学、表面分子标志以及Dil-acLDL/FITC-UEA-I双阳性鉴定后,通过RT-PCR以及Western blot分别观察KLF4在EPCs分化过程中mRNA和蛋白表达水平的表达。
2.2重组腺病毒Ad-KLF4的构建
从大鼠组织提取RNA,经RT-PCR扩增得到目的基因KLF4片段,首先构建重组穿梭质粒pTOPO-KLF4。将pTOPO-KLF4与腺病毒载pAd在重组酶的作用下,pTOPO-KLF4的attL1-attL2序列与腺病毒载体的attR1-attR2序列发生同源重组。重组子命名为pAd-KLF4。pAd-KLF4和空载体pAd经PacI酶切线性化,暴露出载体的反向终末重复序列,将重组子转染293细胞进行包装,扩增后进行滴度的测定。
2.3 KLF4在EPCs分化中的作用
在EPCs通过Ad-KLF4过表达或siRNA沉默KLF4观察EPCs分化的表型改变,通过免疫荧光、流式细胞学、RT-PCR以及Western blot分别观察成熟血管内皮表型CD31、VWF在EPCs分化过程中免疫荧光、mRNA和蛋白水平的表达。
2.4 KLF4在EPCs分化中的分子机制
在EPCs通过Ad-KLF4过表达或siRNA沉默KLF4对下游靶基因eNOS的在转录、蛋白表达水平的影响,进一步观察了eNOS抑制剂L-NAME,对Ad-KLF4诱导的EPCs分化的影响,以明确eNOS是否参与Ad-KLF4介导调控的EPCs分化。
2.5 Ad-KLF4转染的EPCs与平滑肌细胞共培养对平滑肌的增殖及迁移的影响
培养大鼠血管平滑肌细胞和内皮细胞,将Ad-KLF4转染的EPCs接种于下室,分别将平滑肌细胞接种于上室,建立细胞共培养体系,通过3~H-TdR参入法检测平滑肌细胞的增殖作用,用迁移计数方法检测平滑肌细胞。
2.6转染KLF4基因的EPCs移植在血管内膜修复中的作用
建立大鼠颈动脉球囊损伤模型,将Ad-KLF4转染的EPCs通过尾静脉移植至大鼠体内。2周后在局部血管,通过荧光定量RT-PCR以及Western blot分别观察KLF4在血管损伤修复过程中mRNA和蛋白表达,行HE染色、伊文氏蓝染色计算内膜/中膜面积比及损伤血管再内皮化情况。
3.结果:
3.1 KLF4在EPCs分化中的表达
3.1.1 EPCs鉴定
分离培养的EPCs经过诱导分化后向内皮细胞表型转变,流式细胞检测及VEGFR-2和CD133在培养细胞中的阳性率分别为82.35%±2.04%,62.13±3.05%, Dil-acLDL/FITC- UEA-I双阳性细胞约占80%,说明所培养细胞是EPCs。
3.1.2 EPCs向ECs分化的特征
形态学变化:细胞在培养第3~4天可见部分贴壁细胞的多个细胞集落,呈圆形或不规则形,随之梭形细胞数量逐渐增加;第6~10天贴壁的梭形细胞从细胞团边缘出芽生长,呈放射状分布;14d后可观察到梭形细胞首尾相连形成条索状结构,梭形细胞基本接近融合,具有成索状或网状血管样生长趋势;21天细胞增多,融合后呈铺路石状。利用细胞流式分析技术(FACS)对EPCs的分子表面标记检测,发现在MNCs培养过程随时间的变化,在EPCs分化过程中,干细胞表型CD133逐渐减低,而成熟ECs表型逐渐增高。
3.1.3 KLF4在EPCs分化中的表达
在培养7天的MNCs细胞,免疫组化染色,阳性细胞成棕黄色,胞核为苏木精淡染,KLF4表达在EPCs的细胞核。在EPCs分化过程中,KLF4 mRNA在第4天较低,第7天表达逐渐增强,14天表达明显增强,分别为(0.41±0.025,0.1±0.008,0.720±0.017,1.223±0.013)各组间比较(P<0.01)。KLF4蛋白表达在第4天较低,第7天表达逐渐增强,14天表达明显增强(分别为0.41±0.025,0.1±0.008,0.72±0.017,1.22±0.013)各组间比较(P<0.01),14d时表达最强。
3.2.重组腺病毒Ad-KLF4的构建
从大鼠组织提取RNA,经RT-PCR扩增得到目的基因KLF4片段,首先构建重组穿梭质粒pTOPO-KLF4。将pTOPO-KLF4与腺病毒载pAd在重组酶的作用下,pTOPO-KLF4的attL1-attL2序列与腺病毒载体的attR1-attR2序列发生同源重组。重组子命名为pAd-KLF4。pAd-KLF4和空载体pAd经PacI酶切线性化,暴露出载体的反向终末重复序列,将重组子转染293细胞进行包装,扩增后进行滴度的测定,病毒滴度约为1.0×10~9 pfu/ml。
3.3 KLF4在EPCs-ECs分化中的作用
3.3.1 Ad-KLF4转染EPCs
Ad-KLF4转染后,检测EPCs中KLF4mRNA及蛋白表达发现Ad-KLF4明显高于转染Ad-GFP和未转染组以及对照组,Ad-GFP转染和未转染组之间无显著差异,表明Ad-KLF4的有效转染EPCs呈现高表达。
3.3.2 Ad-KLF4转染EPCs对EPCs-ECs分化影响
Ad-KLF4转染EPCs与Ad-GFP组相比,形态特征呈现出细胞贴壁伸展的更多,呈长梭形或多角形分化形态,向内皮形态变化。流式细胞学检测、免疫荧光、定量PCR及蛋白表达检测提示,Ad-KLF4转染与Ad-GFP转染组比较,Dil-ac-LDL/FITC-UEA-1摄取阳性细胞计数增加,CD133、CD34mRNA显著降低,而成熟内皮表型vWF、CD31表型明显增高,表明转染Ad-KLF4促进了EPCs向成熟ECs表型的分化。
3.3.3 KLF4-siRNA转染EPCs对EPCs-ECs分化影响
经过KLF4siRNA内源性的干扰实验,发现在KLF4蛋白水平与阴性对照组比较,几乎不表达,表明进行了内源性的有效干扰,(0.98±0.03 vs 0.02±0.01 ,P<0.01, n=3)
KLF4siRNA转染72小时后与阴性对照组相比,在EPCs分化过程中,成熟内皮表型CD31的蛋白表达与阴性对照组比较明显降低((1.43±0.04 vs 1.01±0.02 ,P<0.05,n=3),VWF的蛋白表达与阴性对照组比较也明显降低((1.21±0.06 vs 0.87±0.02 ,P<0.05,n=3)。
3.4 KLF4在EPCs分化中的机制
3.4.1 KLF4对eNOS表达的影响
在转染Ad-KLF4后eNOS mRNA和蛋白表达比较Ad-GFP组明显升高,与未转染组比较也显著升高。在进行小RNA干扰阻断内源性的KLF4研究中发现,KLF4 siRNA转染eNOS mRNA及蛋白表达与阴性对照组比较,明显降低,与未转染组比较也表现降低。
3.4.2 L-NAME对Ad-KLF4促进的EPCs分化表型的影响
L-NAME加入Ad-KLF4转染72小时后检测蛋白表达分析,发现L-NAME干预组在EPCs分化为成熟内皮表型CD31、VWF的蛋白表达与Ad-KLF4降低,与未转染组相比也降低。培养液NO含量检测,发现Ad-KLF4组较和L-NAME干预组NO含量高,也明显高于对照组,L-NAME与Ad-GFP组比较无明显差异。体外血管生成,提示Ad-KLF4组较和L-NAME干预组数量多。
3.5 Ad-KLF4转染EPCs与平滑肌共培养对平滑肌增殖和迁移的作用
3.5.1 Ad-KLF4转染EPCs与平滑肌共培养对平滑肌增殖的作用
Ad-KLF4转染的EPCs与Ad-GFP及为未转染组比较明显抑制血管平滑肌细胞3~H-TdR掺入,在Ad-KLF4及为未转染组之间血管平滑肌细胞3~H-TdR掺入无显著差异。应用siRNA阻断内源性的KLF4与阴性对照组比较平滑肌细胞增殖明显,3~H-TdR掺入与未转染组比较3~H-TdR掺入。在阴性对照组和未转染组之间比较无差异。
3.5.2 Ad-KLF4转染EPCs与平滑肌共培养对平滑肌迁移的作用
在进行平滑肌细胞迁移检测,结果表明,共培养6 h,Ad-KLF4转染的EPCs组明显抑制平滑肌细胞的迁移数,Ad-GFP与对照组比较比较差异无显著性。KLF4 siRNA转染EPCs组与阴性对照组比较平滑肌细胞迁移数明显增加。在阴性对照组和未转染组之间比较无差异。
3.5.3 Ad-KLF4转染EPCs与平滑肌共培养对平滑肌P53蛋白的作用
应用Ad-KLF4转染的EPCs与对照组比较平滑肌细胞的P53蛋白增加。Ad-GFP与对照组比较比较差异无显著性。KLF4 siRNA转染EPCs组与阴性对照组比较P53蛋白明显降低。在阴性对照组和未转染组之间比较无差异。
3.6 Ad-GFP-EPCs移植在血管损伤修复中的作用
3.6.1 Ad-GFP-EPCs移植对再内皮化的作用
通过Evans blue染色发现, Ad-KLF4转染的EPCs移植组再内皮化率明显高于单纯,而Ad-GFP-EPCs移植组也高于球囊损伤组,各组之间比较,P<0.01,存在显著差异。
3.6.2 Ad-GFP-EPCs移植对新生内膜的作用
Ad-EPCs移植组在损伤2周后血管新生内膜、血管腔狭窄程度比较损伤未移植组和Ad-GFP组明显减轻,Ad-EPCs移植组新生内膜/中膜的比值较其他两组低。
3.6.3 Ad-GFP-EPCs移植组局部损伤处KLF4的表达
进行损伤血管的免疫组化检测。结果表明,正常血管KLF4表达极低,在Ad-KLF4转染的EPCs移植组,KLF4在新生内膜、中膜以及外膜组织均有表达,表达较强,而Ad-GFP-EPCs移植组表达降低,但较正常组明显增高,球囊损伤组KLF4表达比Ad-GFP-EPCs移植组减低,但均高于正常组。WesternBlot法进行损伤血管的KLF4蛋白检测。结果表明,正常血管KLF4蛋白表达极低,在Ad-KLF4转染的EPCs移植组,KLF4蛋白表达高于正常组、Ad-GFP-EPCs及球囊损伤组,其中KLF4表达球囊损伤组较Ad-GFP-EPCs移植组低,但均高于正常组,各组之间P<0.01。
4.结论:
4.1在EPCs分化过程中KLF4在分化晚期表达,逐渐增高。
4.2过表达KLF4促进EPCs向成熟内皮分化。
4.3过表达KLF4促进EPCs的eNOS表达,培养液的NO增多,eNOS抑制剂,L-NAME减低了KLF4在EPCs分化为成熟内皮表型CD31、VWF的蛋白表达,抑制了血管新生。
4.4 KLF4基因修饰EPCs与平滑肌共培养明显抑制平滑肌的增殖和迁移,同时使平滑肌的P53蛋白表达增高。
4.5 KLF4基因修饰EPCs移植抑制了损伤血管新生内膜,促进了再内皮化,在局部损伤处表达增高。
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 vectors
Adenoviral 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 ECs
To 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 EPCs
To 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-TdR
In 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 repair
The 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 characterization
After 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 ECs
Total 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 KLF4
Full 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 EPCs
The 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 expression
Western 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 proliferation
In 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 injury
The 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 .
引文
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5. Atkins GB, Jain MK. Role of Krüppel-like transcription factors in endothelial biology.Circ Res. 2007 , 100(12):1686-1695.
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