转录因子Id1对内皮祖细胞增殖、迁移的影响及在损伤血管修复中作用的研究
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摘要
1.背景与目的:
血管内皮损伤是动脉粥样硬化、高血压、糖尿病、介入治疗后再狭窄等多种血管性疾病共同的病理生理基础,尽早促进受损血管再内皮化、恢复内皮功能为抑制血管损伤不良修复、预防或防治血管再狭窄及血栓形成的有效策略。因此,揭示内皮再生机理,促进内皮细胞有益再生是损伤性血管疾病治疗中亟待解决的问题。既往认为血管损伤主要依靠损伤部位邻近的内皮细胞再生修复,但病损情况下邻近内皮的增殖能力有限。近年研究显示除了血管损伤局部附近内皮细胞再生外,不同来源的血管前体细胞同样是参与损伤内皮修复的重要力量。业已证实,内皮前体细胞即所称的内皮祖细胞(Endothelial precusor cells, EPCs),能“归巢”于血管损伤处定向分化为内皮细胞并通过旁分泌机制促进内皮修复和血管新生。然而,目前对于调控EPCs增殖、迁移等生理功能的机制或某些关键因子在其中的作用尚不完全清楚。
分化抑制因子(Inhibitor of DNA binding/differentiation, Id)属于螺旋-环-螺旋(Helix-loop-helix, HLH)转录因子家族,Id包括Id1-Id4四个亚型,各成员均包括高度保守的HLH结构区,但缺乏碱性DNA结合区。Id与其他碱性HLH蛋白(如E2A,E12,E47,c-Myc)形成异二聚体,从而抑制了这些碱性HLH蛋白与DNA及其他组织特异性碱性LHL转录因子结合,影响特异性蛋白的表达,抑制细胞分化。目前发现,Id参与了包括表皮、肌肉、神经等多种类细胞增殖分化及及肿瘤形成,且Id1与骨髓EPCs动员等密切相关,但Id是否也对EPCs功能有调控作用并参与血管损伤修复尚不清楚。研究结果发现:①Id与血管系统发生密切相关,胚胎发育中Id1-Id4表达呈复杂的时空模式,但Id1、Id3广泛表达且重叠贯穿整个胚胎脑血管系统,Id1/Id3双基因敲除小鼠由于明显的血管畸形、分叉障碍导致胚胎E13.5期死亡;②Id参与肿瘤血管新生:Id在内皮细胞过表达导致血管新生,缺失导致促血管生成基因如FGFR-1的下调,而野生型骨髓内皮前体细胞高表达Id1、Id3,移植后掺合到肿瘤血管床参与血管新生,且Id1、Id3阳性表达与血管密度显著正相关;③体外细胞实验中,Id能促进人脐静脉内皮细胞(HUVEC)的增殖、激活及管样形成,促血管生长因子VEGF、TGF-β则诱导HUVEC及骨髓源EPCs表达Id1和Id3,提示Id1、Id3可能是VEGF等促血管生长作用的下游关键靶点。研究结果提示:Id1可能表达于EPCs,并有调控其增殖、血管新生的功效,推测Id1为决定EPCs增殖的一类开关基因,受上游特殊信号刺激Id1的表达左右着增殖重要基因的转录,在EPCs介导的血管损伤修复中发挥重要作用。
在本课题中,我们将分别从细胞及在体动物水平探讨Id1在EPCs增殖、迁移以及血管损伤修复中的作用。为研究EPCs生理功能的调控机制及EPCs在血管损伤修复中的作用提供实验依据,为深入探讨促进血管损伤后内皮有益再生提供新的思路。
2.方法:
2.1重组腺病毒Ad-Id1的构建
由大鼠组织中提取RNA,经RT-PCR扩增得到目的基因Id1片段,经pMD19T-Simple和AdEasy细菌内同源重组系统构建Id1的病毒过表达载体,即Ad-Id1,通过测序、PCR和酶切鉴定重组病毒Ad-Id1的构建。
2.2 Id1对EPCs增殖、迁移功能的影响
用密度梯度离心法及选择性培养的方法,体外分离、培养小鼠脾源性EPCs,通过细胞形态学、表面分子标志以及Dil-acLDL/FITC-UEA-I双阳性等方法进行鉴定;转染Ad- Id1以及si-RNA-Id1,观察过表达或沉默Id1基因对EPCs增殖、迁移的作用。
2.3观察Id1在血管损伤后局部血管壁的表达及参与血管损伤修复过程的作用
用体重为20g-30g的雄性昆明小鼠复制颈动脉损伤模型,通过荧光定量RT-PCR以及Western blot分别观察Id1在血管损伤修复过程中mRNA和蛋白表达水平的变化;将过表达Id1的EPCs注入颈动脉损伤小鼠尾静脉,14d后观察损伤血管局部再内皮化及新生内膜增殖程度。
3.结果;
3.1.重组腺病毒Ad-Id1的构建
由大鼠组织提取RNA,进行RT-PCR获得含酶切位点的Id1基因全CDS区,将目的基因连接到pMD19T-Simple载体中进行扩增,经酶切、连接、转化等步骤后得到pAdTrack-Id1,与骨架质粒pAdEasy-1在BJ5183细菌内进行同源重组,通过筛选、293T细胞包装后获得重组病毒Ad-Id1,经鉴定、扩增获得高滴度的Ad-Id1病毒颗粒以用于下一步的实验。Ad-Id1的病毒滴度约为1.2×1010 -2.8×1011 pfu/ml。
3.2 Id1对体外培养脾源性EPCs增殖、迁移功能的影响
3.2.1脾源性EPCs鉴定
分离培养的脾源性EPCs经过诱导分化后向内皮细胞表型转变,流式细胞检测Scal-1、VEGFR-2在培养细胞中的阳性率分别为83.5%、57.6%,Dil-acLDL/FITC- UEA-I双阳性细胞约占90%,说明所培养脾源性细胞是EPCs。
3.2.2 Id1在脾源性EPCs的表达情况
Id1在脾源性EPCs呈低水平表达,而当受到血清或VEGF刺激后,Id1的基因及蛋白水平均明显增高;静止状态下Id1定位表达于EPCs细胞浆内。
3.2.3基因转染脾源性EPCs
转染后细胞状态良好,贴壁生长,无变圆、缩小或脱落等病理迹象,经过荧光倒置显微镜、RT-PCR以及Western blot观察发现Ad- Id1的转染效率在第24小时约60%,48-72小时达高峰,约80%。si-RNA-Id1转染后4天,Id1在EPCs的表达被显著抑制,转染率约50%。
3.2.4 Ad- Id1对EPCs增殖的作用
采用MTT法分析发现,Ad- Id1对EPCs增殖显著影响。无论与未转染对照组还是Ad-GFP对照组比较,Ad- Id1对EPCs的增殖有统计学差异(均* P<0.05),提示Ad- Id1对EPCs有促进增殖的作用。
3.2.5 Ad- Id1对EPCs迁移的作用
外源性Id1过表达显著促进EPCs迁移。在Ad- Id1作用诱导下,平均每个视野中迁移EPCs的数目从7.1±1.8增加到26.1±2.8,增加了近3倍(* P<0.01)。加入Id1封闭性抗体Id1-Ab后,Ad- Id1促迁移作用明显减弱(# P<0.05),提示EPCs迁移能力的增强是过表达Id1导致。
3.2.6利用小片段RNA(si-RNA)干扰沉默Ad- Id1对EPCs增殖、迁移的影响
结果表明si-RNA- Id1介导的Id1基因的沉默明显抑制了EPCs的增殖、迁移功能,与未干预组及阴性对照组比较均有显著差异(* p<0.05)。
3.3 Id1在血管损伤修复中的表达及作用
3.3.1小鼠颈动脉损伤模型的建立
经损伤血管组织切片H&E染色证实,本研究成功复制了小鼠颈动脉损伤模型,镜下观察到血管损伤后7天局部内膜有增生,14天时新生内膜增生明显,到28天新生的内膜几乎堵塞整个血管腔。内膜/中膜比值(IA/MA)14d组为1.30±0.15,28d组为4.10±0.20较损伤7d组的0.28±0.02显著增加(分别为P<0.01,P=0.000)。
3.3.2 Id1在血管损伤局部血管壁的表达
免疫组化实验显示Id1在损伤血管的新生内膜、中膜以及外膜组织均有表达。Id1mRNA在正常血管组织低表达,血管损伤后表达迅速上升,114d即达到高峰,之后逐渐下降维持到血管损伤后第28天仍较对照组高。Western blot检测到Id1蛋白表达在血管损伤后第7d开始上调,14d时达到高峰,然后逐渐回落,在损伤后第28天仍有表达。
3.3.3移植过表达Id1的EPCs对损伤血管再内皮化的影响
损伤14d时Ad-Id1-EPCs转染组再内皮化率为68.36±4.51%,而Ad-GFP-EPCs转染组及未转染组再内皮化率分别为43.1±6.59%、40.5±7.82%,两者之间无统计学差异,但与Ad-Id1-EPCs转染组比较均存在明显差异(P<0.05),说明转染Id1过表达的EPCs可促进第14d时损伤血管再内皮化。
3.3.4移植过表达Id1的EPCs对损伤血管局部新生内膜增殖的影响
血管损伤后第14d,Ad-Id1-EPCs转染组小鼠损伤颈动脉内、中膜比值为1.08±0.15,结果与Ad-GFP-EPCs组(1.16±0.14)及未转染组(1.15±0.17)比较三组之间比较无明显差异(P>0.05),而三组中膜面积均无明显差异(P>0.05),提示转移植表达Id1的EPCs未减轻血管损伤后新生内膜的增殖程度。
4.结论:
4.1 Id1在静止状态的脾源性EPCs呈低表达,定位于细胞浆内;
4.2 Id1影响EPCs增殖、迁移功能:过表达Id1可促进EPCs的增殖、迁移,干扰Id1则抑制EPCs的增殖、迁移;
4.3 Id1在损伤血管局部呈动态表达,14d为其表达高峰期;
4.4移植过表达Id1的EPCs到颈动脉损伤动物模型,可促进14d后损伤血管再内皮化,但未明显抑制局部新生内膜的增殖。
1. Background and Objective:
Enhancement of reendothelialization is a critical therapeutic option to repair injured blood vessels. Regeneration of injured endothelium has been attributed to the migration and proliferation of neighboring endothelial cells (ECs). Mature ECs are terminally differentiated cells with a low proliferative potential, and their capacity to substitute damaged endothelium is limited. Increasing evidence suggests that circulating endothelial progenitor cells (EPCs), which can home to sites of tissue injury and differentiate into mature ECs and participate in re-endothelialization after vascular injury, may be an endogenous repair mechanism to maintain the integrity of the endothelial monolayer by replacing denuded parts of the artery. EPCs also secrete various cytoprotective or pro-angiogenic factors in a paracrine manner to promote the survival and proliferation of ECs. The physiological and therapeutic significance of EPCs in reendothelialization processes is currently the subject of intensive investigation.
Mobilization of EPCs by cytokines, growth factors or drugs; infusion ex-vivo expanded EPCs; and EPC-based gene therapy have been suggested to contribute to re-endothelialization and to inhibit intimal hyperplasia after vascular injury. A critical limitation for therapeutic application of post-natal EPCs is their low number in the circulation (which is even lower in patients with cardiovascular risk factors). The approaches mentioned above have the potential to overcome the problem. The mechanisms underlying reendothelialization by EPCs must be intensively investigated and these mechanisms understood before novel strategies for EPC therapy are translated into clinical applications.
During reendothelialization, migration and proliferation of EPCs are the key steps regulated by various mechanisms and signals. The inhibitor of DNA binding 1 (Id1) proteins, an important subfamily member of helix-loop-helix (HLH) transcriptional factors, has been implicated in regulating the growth, proliferation, migration and differentiation of cells. Several studies have focused on the Id1 transcription factor because Id1 knockout mice (Id1+/– Id3–/–) exhibit impaired tumor growth associated with impaired recruitment of EPCs. Restraint of the expression of the cyclin-dependent kinase inhibitor p21 by Id1 is one key element of its activity in facilitating EPC generation in the bone marrow. Recent studies also demonstrated tumor-induced expression of Id1 in EPCs, and conditional Id1 suppression resulted in impaired mobilization of EPCs. These studies shed light on the relationship between Id1 and the functional regulation of EPCs, including recruitment, population and mobilization of EPCs.
Based on the available data concerning Id1 on EPC behavior, Id1 may exert potential effects on the migration and proliferation of EPCs, contributing to the neovascularization and vascular repair process after injury. In this study, we overexpressed Id1 and si-RNA to evaluate the possible role of Id1 on EPCs proliferation, migration, and participation in vascular regeneration. Our findings provide a novel insight into understanding of biological function of Id1 and the molecular mechanisms behind EPCs-mediated vascular regeneration.
2. Methods:
2.1 Construction of recombinant adenoviral vectors
Adenoviral vectors repectively expressing Id1 were generated using the AdEasy system. Briefly, full-length rat Id1 cDNA were generated by RT-PCR using total RNA from Sprague–Dawley (SD) rat heart and spleen. The cDNA was first TA-cloned into pMD19-T simple vector and then subcloned into pAdTrack-CMV, resulting in pAdTrack-Id1. The shuttle vectors were used to generate recombinant adenoviruses according to the manufacturer’s protocol. All PCR-amplified fragments and cloning junctions were verified by DNA sequencing and enzymatic digestion. An adenovirus encoding green fluorescent protein (GFP; Ad-GFP) was used as control.
2.2 Effecs of Id1 on spleen derived EPCs proliferation and migration in vitro
spleen derived 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 mouse Scal-1 and VEGFR-2. To investigate the effect of Id1 on spleen derived EPCs proliferation and migaration in vitro, we transduced Ad- Id1 and pGenesil- Id1 into EPCs that were cultured in serum- and VEGF- free medium.
2.3 Expression and function of Id1 during vascular repair following mice carotid artery injured
To evaluate the role of Id1 in vascular repair in vivo, firstly, we examined the expression of Id1 in injured mice carotid artery, using PCR and Westen blot analysis. Secondly, we transduced Ad- Id1 and pGenesil- Id1 into EPCs. Thirdly, these transduced EPCs were injected by intravenous tail vein after induction of arterial injury. The injured segments were isolated 14 days after EPCs transplantation. Overexpression of Id1 was confirmed by immunohistochemistry. No observable adverse side effect (mortality or any other clinical signs of distress/morbidity) was found in experimental animals. Evans Blue dye was administered to evaluate reendothelialization at 14 day after injury, and the neointimal formation was assessed at 14 day following vascular injury.
3. Results:
3.1 Recombinant adenoviral vectors expressing Id1
Full length cDNA encoding Id1 was amplified by RT-PCR using total RNA from Sprague–Dawley (SD) rat heart or spleen. The cDNA was first TA-cloned into pMD19-T simple vector and then subcloned into adenoviral shuttle vector pAdTrack-CMV. Recombinant adenovirus Ad-Id1 were generated and purified according to the manufacturer’s protocol. The adenovirus virus titer was about 1.2×1010-2.8×1011 plaque-forming units per millilitre (pfu /ml), as determined by plaque assay.
3.2 Effecs of Id1 on spleen derived EPCs proliferation and migration in vitro
3.2.1 spleen derived EPCs isolation and characterization
After 4-7 days of culture, adherent EPCs were characterized by immunofluorescence and flow cytometry analysis (FACS). The majority of cells (>90%) stained positive for DiI-AcLDL and lectin, and expressed endothelial/stem cell markers, including Scal-1 (83.5%) and VEGFR-2 (57.6 %) confirming the cell type of EPCs.
3.2.2 Expression and location of Id1 in EPCs
Id1 was present at fairly low levels in quiescent EPCs, but was rapidly upregulated upon stimulation with serum and VEGF (a strong growth factor of EPCs) and was detected via mRNA in RT-PCR or by protein in western blotting. To analyze the subcellular localization of Id1, EPCs were fixed and subjected to DAB staining with anti-Id1 multiclonal antibody by immunocytochemical staining: Id1 was localized predominantly in the cytoplasm.
3.2.3 EPCs transfection
Adenovirus-mediated Id1 expression was confirmed by fluorescence, RT-PCR, and Western blot analysis. The transfection efficiency was 60% at 24h post-transfection and 80% at 48-72h post-transfection. Four days after introduction of pGenesil1-Id1, an approximate 60% of Id1 expression loss was shown, as measured by Western blot and RT-PCR.
3.2.4 Effect of Ad- Id1 on EPCs proliferation
The proliferation of EPCs was not ehanced significantly by Ad- Id1. Despite a decrease observed in cells transfected with Ad- Id1 as compared with Ad-GFP (p<0.05), it has significant meaning in compare with untransfected control (p<0.05).
3.2.5 Effect of Ad- Id1 on EPCs migration
Over expression of exogenous Id1 extensively improved the migration of EPCs, and in fact, the average migrated cell number of the EPCs increased by approximately 3-fold, from 7.1±1.8 to 6.1±2.8 (* P<0.01) compared to that of the control cells.
3.2.6 Role of siRNA- Id1
After introduction of si-RNA- Id1 EPCs exhibited a decrease in cell proliferation and migration when compared with negative control siRNA transfected cells or untransfected cells (* p< 0.05).
3.3 Expression and function of Id1 during vascular repair following mice carotid artery injured
3.3.1 Mice carotid injury model
Histological analysis and H&E staining demonstrated that neointimal formation was initiated at 7days and obviously developed at 28 day after vascular injury in control mouse. In addition, after transfection with Ad- Id1 or Ad-GFP, green fluorescence was detected with vascular tissues under a fluorescence microscope at 48 h post-vascular injury, indicating the efficiency of adenovirus transfection in mice carotid arteries.
3.3.2 Expression of Id1 during vascular repair process
Id1 mRNA expression was detected at low levels in normal uninjured control arteries, whereas following vascular injury Id1 mRNA level was rapidly enhanced with a peak at 14 d which gradually declined thereafter in 14 days and remained elevated for up to at least 28days. Id1 protein expression was assessed by Western blotting and was consistently found to be up-regulated. Further, immunohistochemistry showed that Id1 was detected in the intima and media of local vessels.
3.3.3 Effect of Ad- Id1-EPCs transplantation on vascular reendothelialization
Evans Blue dye was administered to evaluate reendothelialization at 14 days after injury. Nonendothelialized lesions were marked blue about 100% at injured vessels, whereas the reendothelialized area appeared white at uninjured vessels. The reendothelialized area in the Ad- Id1-EPCs infected arteries was significantly larger than that in Ad-GFP-EPCs, and non-infected groups (reendothelialized area /totle vessel injured area ratio was 68.36±4.51, 43.1±6.59 and 40.5±7.82, respectively, p<0.05), suggesting that transplantation of Ad-Id1-EPCs effectively promoted reendothelialization after vascular injury.
3.3.4 Effect of Ad- Id1-EPCs transplantation on neointimal formation
No marked decrease in the I/M ratio was shown in Ad- Id1-EPCs group compared with Ad-GFP-EPCs or non-infected group at day 14 (1.08±0.1, 1.16±0.14 and 1.15±0.17, respectively, p>0.05).
4. Conclusions:
4.1 Id1 was present at fairly low levels in quiescent EPCs and was localized mainly in the cytoplasm. Id1 was rapidly upregulated upon stimulation with serum and VEGF;
4.2 Overexpression of Id1 stimulated spleen derived EPCs proliferation and migration, and that was reversed by si-RNA-mediated silencing of Id1 expression;
4.3 Id1 was dynamically expressed in vascular lesions, and attained the peak expression in14 day after injure.
4.4 Ad-Id1-EPCs transplantation could home into the vascular injury site and accelerate reendothelialization of denuded vessel, howere, could not inhibit the neointima formation at the same period after vascular injury.
血管内皮损伤是动脉粥样硬化、高血压、糖尿病、介入治疗后再狭窄等多种血管性疾病共同的病理生理基础,尽早促进受损血管再内皮化、恢复内皮功能为抑制血管损伤不良修复、预防或防治血管再狭窄及血栓形成的有效策略。因此,揭示内皮再生机理,促进内皮细胞有益再生是损伤性血管疾病治疗中亟待解决的问题。既往认为血管损伤主要依靠损伤部位邻近的内皮细胞再生修复,但病损情况下邻近内皮的增殖能力有限。近年研究显示除了血管损伤局部附近内皮细胞再生外,不同来源的血管前体细胞同样是参与损伤内皮修复的重要力量。业已证实,内皮前体细胞即所称的内皮祖细胞(Endothelial precusor cells, EPCs),能“归巢”于血管损伤处定向分化为内皮细胞并通过旁分泌机制促进内皮修复和血管新生。然而,目前对于调控EPCs增殖、迁移等生理功能的机制或某些关键因子在其中的作用尚不完全清楚。
分化抑制因子(Inhibitor of DNA binding/differentiation, Id)属于螺旋-环-螺旋(Helix-loop-helix, HLH)转录因子家族,Id包括Id1-Id4四个亚型,各成员均包括高度保守的HLH结构区,但缺乏碱性DNA结合区。Id与其他碱性HLH蛋白(如E2A,E12,E47,c-Myc)形成异二聚体,从而抑制了这些碱性HLH蛋白与DNA及其他组织特异性碱性LHL转录因子结合,影响特异性蛋白的表达,抑制细胞分化。目前发现,Id参与了包括表皮、肌肉、神经等多种类细胞增殖分化及及肿瘤形成,且Id1与骨髓EPCs动员等密切相关,但Id是否也对EPCs功能有调控作用并参与血管损伤修复尚不清楚。研究结果发现:①Id与血管系统发生密切相关,胚胎发育中Id1-Id4表达呈复杂的时空模式,但Id1、Id3广泛表达且重叠贯穿整个胚胎脑血管系统,Id1/Id3双基因敲除小鼠由于明显的血管畸形、分叉障碍导致胚胎E13.5期死亡;②Id参与肿瘤血管新生:Id在内皮细胞过表达导致血管新生,缺失导致促血管生成基因如FGFR-1的下调,而野生型骨髓内皮前体细胞高表达Id1、Id3,移植后掺合到肿瘤血管床参与血管新生,且Id1、Id3阳性表达与血管密度显著正相关;③体外细胞实验中,Id能促进人脐静脉内皮细胞(HUVEC)的增殖、激活及管样形成,促血管生长因子VEGF、TGF-β则诱导HUVEC及骨髓源EPCs表达Id1和Id3,提示Id1、Id3可能是VEGF等促血管生长作用的下游关键靶点。研究结果提示:Id1可能表达于EPCs,并有调控其增殖、血管新生的功效,推测Id1为决定EPCs增殖的一类开关基因,受上游特殊信号刺激Id1的表达左右着增殖重要基因的转录,在EPCs介导的血管损伤修复中发挥重要作用。
在本课题中,我们将分别从细胞及在体动物水平探讨Id1在EPCs增殖、迁移以及血管损伤修复中的作用。为研究EPCs生理功能的调控机制及EPCs在血管损伤修复中的作用提供实验依据,为深入探讨促进血管损伤后内皮有益再生提供新的思路。
2.方法:
2.1重组腺病毒Ad-Id1的构建
由大鼠组织中提取RNA,经RT-PCR扩增得到目的基因Id1片段,经pMD19T-Simple和AdEasy细菌内同源重组系统构建Id1的病毒过表达载体,即Ad-Id1,通过测序、PCR和酶切鉴定重组病毒Ad-Id1的构建。
2.2 Id1对EPCs增殖、迁移功能的影响
用密度梯度离心法及选择性培养的方法,体外分离、培养小鼠脾源性EPCs,通过细胞形态学、表面分子标志以及Dil-acLDL/FITC-UEA-I双阳性等方法进行鉴定;转染Ad- Id1以及si-RNA-Id1,观察过表达或沉默Id1基因对EPCs增殖、迁移的作用。
2.3观察Id1在血管损伤后局部血管壁的表达及参与血管损伤修复过程的作用
用体重为20g-30g的雄性昆明小鼠复制颈动脉损伤模型,通过荧光定量RT-PCR以及Western blot分别观察Id1在血管损伤修复过程中mRNA和蛋白表达水平的变化;将过表达Id1的EPCs注入颈动脉损伤小鼠尾静脉,14d后观察损伤血管局部再内皮化及新生内膜增殖程度。
3.结果;
3.1.重组腺病毒Ad-Id1的构建
由大鼠组织提取RNA,进行RT-PCR获得含酶切位点的Id1基因全CDS区,将目的基因连接到pMD19T-Simple载体中进行扩增,经酶切、连接、转化等步骤后得到pAdTrack-Id1,与骨架质粒pAdEasy-1在BJ5183细菌内进行同源重组,通过筛选、293T细胞包装后获得重组病毒Ad-Id1,经鉴定、扩增获得高滴度的Ad-Id1病毒颗粒以用于下一步的实验。Ad-Id1的病毒滴度约为1.2×1010 -2.8×1011 pfu/ml。
3.2 Id1对体外培养脾源性EPCs增殖、迁移功能的影响
3.2.1脾源性EPCs鉴定
分离培养的脾源性EPCs经过诱导分化后向内皮细胞表型转变,流式细胞检测Scal-1、VEGFR-2在培养细胞中的阳性率分别为83.5%、57.6%,Dil-acLDL/FITC- UEA-I双阳性细胞约占90%,说明所培养脾源性细胞是EPCs。
3.2.2 Id1在脾源性EPCs的表达情况
Id1在脾源性EPCs呈低水平表达,而当受到血清或VEGF刺激后,Id1的基因及蛋白水平均明显增高;静止状态下Id1定位表达于EPCs细胞浆内。
3.2.3基因转染脾源性EPCs
转染后细胞状态良好,贴壁生长,无变圆、缩小或脱落等病理迹象,经过荧光倒置显微镜、RT-PCR以及Western blot观察发现Ad- Id1的转染效率在第24小时约60%,48-72小时达高峰,约80%。si-RNA-Id1转染后4天,Id1在EPCs的表达被显著抑制,转染率约50%。
3.2.4 Ad- Id1对EPCs增殖的作用
采用MTT法分析发现,Ad- Id1对EPCs增殖显著影响。无论与未转染对照组还是Ad-GFP对照组比较,Ad- Id1对EPCs的增殖有统计学差异(均* P<0.05),提示Ad- Id1对EPCs有促进增殖的作用。
3.2.5 Ad- Id1对EPCs迁移的作用
外源性Id1过表达显著促进EPCs迁移。在Ad- Id1作用诱导下,平均每个视野中迁移EPCs的数目从7.1±1.8增加到26.1±2.8,增加了近3倍(* P<0.01)。加入Id1封闭性抗体Id1-Ab后,Ad- Id1促迁移作用明显减弱(# P<0.05),提示EPCs迁移能力的增强是过表达Id1导致。
3.2.6利用小片段RNA(si-RNA)干扰沉默Ad- Id1对EPCs增殖、迁移的影响
结果表明si-RNA- Id1介导的Id1基因的沉默明显抑制了EPCs的增殖、迁移功能,与未干预组及阴性对照组比较均有显著差异(* p<0.05)。
3.3 Id1在血管损伤修复中的表达及作用
3.3.1小鼠颈动脉损伤模型的建立
经损伤血管组织切片H&E染色证实,本研究成功复制了小鼠颈动脉损伤模型,镜下观察到血管损伤后7天局部内膜有增生,14天时新生内膜增生明显,到28天新生的内膜几乎堵塞整个血管腔。内膜/中膜比值(IA/MA)14d组为1.30±0.15,28d组为4.10±0.20较损伤7d组的0.28±0.02显著增加(分别为P<0.01,P=0.000)。
3.3.2 Id1在血管损伤局部血管壁的表达
免疫组化实验显示Id1在损伤血管的新生内膜、中膜以及外膜组织均有表达。Id1mRNA在正常血管组织低表达,血管损伤后表达迅速上升,114d即达到高峰,之后逐渐下降维持到血管损伤后第28天仍较对照组高。Western blot检测到Id1蛋白表达在血管损伤后第7d开始上调,14d时达到高峰,然后逐渐回落,在损伤后第28天仍有表达。
3.3.3移植过表达Id1的EPCs对损伤血管再内皮化的影响
损伤14d时Ad-Id1-EPCs转染组再内皮化率为68.36±4.51%,而Ad-GFP-EPCs转染组及未转染组再内皮化率分别为43.1±6.59%、40.5±7.82%,两者之间无统计学差异,但与Ad-Id1-EPCs转染组比较均存在明显差异(P<0.05),说明转染Id1过表达的EPCs可促进第14d时损伤血管再内皮化。
3.3.4移植过表达Id1的EPCs对损伤血管局部新生内膜增殖的影响
血管损伤后第14d,Ad-Id1-EPCs转染组小鼠损伤颈动脉内、中膜比值为1.08±0.15,结果与Ad-GFP-EPCs组(1.16±0.14)及未转染组(1.15±0.17)比较三组之间比较无明显差异(P>0.05),而三组中膜面积均无明显差异(P>0.05),提示转移植表达Id1的EPCs未减轻血管损伤后新生内膜的增殖程度。
4.结论:
4.1 Id1在静止状态的脾源性EPCs呈低表达,定位于细胞浆内;
4.2 Id1影响EPCs增殖、迁移功能:过表达Id1可促进EPCs的增殖、迁移,干扰Id1则抑制EPCs的增殖、迁移;
4.3 Id1在损伤血管局部呈动态表达,14d为其表达高峰期;
4.4移植过表达Id1的EPCs到颈动脉损伤动物模型,可促进14d后损伤血管再内皮化,但未明显抑制局部新生内膜的增殖。
1. Background and Objective:
Enhancement of reendothelialization is a critical therapeutic option to repair injured blood vessels. Regeneration of injured endothelium has been attributed to the migration and proliferation of neighboring endothelial cells (ECs). Mature ECs are terminally differentiated cells with a low proliferative potential, and their capacity to substitute damaged endothelium is limited. Increasing evidence suggests that circulating endothelial progenitor cells (EPCs), which can home to sites of tissue injury and differentiate into mature ECs and participate in re-endothelialization after vascular injury, may be an endogenous repair mechanism to maintain the integrity of the endothelial monolayer by replacing denuded parts of the artery. EPCs also secrete various cytoprotective or pro-angiogenic factors in a paracrine manner to promote the survival and proliferation of ECs. The physiological and therapeutic significance of EPCs in reendothelialization processes is currently the subject of intensive investigation.
Mobilization of EPCs by cytokines, growth factors or drugs; infusion ex-vivo expanded EPCs; and EPC-based gene therapy have been suggested to contribute to re-endothelialization and to inhibit intimal hyperplasia after vascular injury. A critical limitation for therapeutic application of post-natal EPCs is their low number in the circulation (which is even lower in patients with cardiovascular risk factors). The approaches mentioned above have the potential to overcome the problem. The mechanisms underlying reendothelialization by EPCs must be intensively investigated and these mechanisms understood before novel strategies for EPC therapy are translated into clinical applications.
During reendothelialization, migration and proliferation of EPCs are the key steps regulated by various mechanisms and signals. The inhibitor of DNA binding 1 (Id1) proteins, an important subfamily member of helix-loop-helix (HLH) transcriptional factors, has been implicated in regulating the growth, proliferation, migration and differentiation of cells. Several studies have focused on the Id1 transcription factor because Id1 knockout mice (Id1+/– Id3–/–) exhibit impaired tumor growth associated with impaired recruitment of EPCs. Restraint of the expression of the cyclin-dependent kinase inhibitor p21 by Id1 is one key element of its activity in facilitating EPC generation in the bone marrow. Recent studies also demonstrated tumor-induced expression of Id1 in EPCs, and conditional Id1 suppression resulted in impaired mobilization of EPCs. These studies shed light on the relationship between Id1 and the functional regulation of EPCs, including recruitment, population and mobilization of EPCs.
Based on the available data concerning Id1 on EPC behavior, Id1 may exert potential effects on the migration and proliferation of EPCs, contributing to the neovascularization and vascular repair process after injury. In this study, we overexpressed Id1 and si-RNA to evaluate the possible role of Id1 on EPCs proliferation, migration, and participation in vascular regeneration. Our findings provide a novel insight into understanding of biological function of Id1 and the molecular mechanisms behind EPCs-mediated vascular regeneration.
2. Methods:
2.1 Construction of recombinant adenoviral vectors
Adenoviral vectors repectively expressing Id1 were generated using the AdEasy system. Briefly, full-length rat Id1 cDNA were generated by RT-PCR using total RNA from Sprague–Dawley (SD) rat heart and spleen. The cDNA was first TA-cloned into pMD19-T simple vector and then subcloned into pAdTrack-CMV, resulting in pAdTrack-Id1. The shuttle vectors were used to generate recombinant adenoviruses according to the manufacturer’s protocol. All PCR-amplified fragments and cloning junctions were verified by DNA sequencing and enzymatic digestion. An adenovirus encoding green fluorescent protein (GFP; Ad-GFP) was used as control.
2.2 Effecs of Id1 on spleen derived EPCs proliferation and migration in vitro
spleen derived 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 mouse Scal-1 and VEGFR-2. To investigate the effect of Id1 on spleen derived EPCs proliferation and migaration in vitro, we transduced Ad- Id1 and pGenesil- Id1 into EPCs that were cultured in serum- and VEGF- free medium.
2.3 Expression and function of Id1 during vascular repair following mice carotid artery injured
To evaluate the role of Id1 in vascular repair in vivo, firstly, we examined the expression of Id1 in injured mice carotid artery, using PCR and Westen blot analysis. Secondly, we transduced Ad- Id1 and pGenesil- Id1 into EPCs. Thirdly, these transduced EPCs were injected by intravenous tail vein after induction of arterial injury. The injured segments were isolated 14 days after EPCs transplantation. Overexpression of Id1 was confirmed by immunohistochemistry. No observable adverse side effect (mortality or any other clinical signs of distress/morbidity) was found in experimental animals. Evans Blue dye was administered to evaluate reendothelialization at 14 day after injury, and the neointimal formation was assessed at 14 day following vascular injury.
3. Results:
3.1 Recombinant adenoviral vectors expressing Id1
Full length cDNA encoding Id1 was amplified by RT-PCR using total RNA from Sprague–Dawley (SD) rat heart or spleen. The cDNA was first TA-cloned into pMD19-T simple vector and then subcloned into adenoviral shuttle vector pAdTrack-CMV. Recombinant adenovirus Ad-Id1 were generated and purified according to the manufacturer’s protocol. The adenovirus virus titer was about 1.2×1010-2.8×1011 plaque-forming units per millilitre (pfu /ml), as determined by plaque assay.
3.2 Effecs of Id1 on spleen derived EPCs proliferation and migration in vitro
3.2.1 spleen derived EPCs isolation and characterization
After 4-7 days of culture, adherent EPCs were characterized by immunofluorescence and flow cytometry analysis (FACS). The majority of cells (>90%) stained positive for DiI-AcLDL and lectin, and expressed endothelial/stem cell markers, including Scal-1 (83.5%) and VEGFR-2 (57.6 %) confirming the cell type of EPCs.
3.2.2 Expression and location of Id1 in EPCs
Id1 was present at fairly low levels in quiescent EPCs, but was rapidly upregulated upon stimulation with serum and VEGF (a strong growth factor of EPCs) and was detected via mRNA in RT-PCR or by protein in western blotting. To analyze the subcellular localization of Id1, EPCs were fixed and subjected to DAB staining with anti-Id1 multiclonal antibody by immunocytochemical staining: Id1 was localized predominantly in the cytoplasm.
3.2.3 EPCs transfection
Adenovirus-mediated Id1 expression was confirmed by fluorescence, RT-PCR, and Western blot analysis. The transfection efficiency was 60% at 24h post-transfection and 80% at 48-72h post-transfection. Four days after introduction of pGenesil1-Id1, an approximate 60% of Id1 expression loss was shown, as measured by Western blot and RT-PCR.
3.2.4 Effect of Ad- Id1 on EPCs proliferation
The proliferation of EPCs was not ehanced significantly by Ad- Id1. Despite a decrease observed in cells transfected with Ad- Id1 as compared with Ad-GFP (p<0.05), it has significant meaning in compare with untransfected control (p<0.05).
3.2.5 Effect of Ad- Id1 on EPCs migration
Over expression of exogenous Id1 extensively improved the migration of EPCs, and in fact, the average migrated cell number of the EPCs increased by approximately 3-fold, from 7.1±1.8 to 6.1±2.8 (* P<0.01) compared to that of the control cells.
3.2.6 Role of siRNA- Id1
After introduction of si-RNA- Id1 EPCs exhibited a decrease in cell proliferation and migration when compared with negative control siRNA transfected cells or untransfected cells (* p< 0.05).
3.3 Expression and function of Id1 during vascular repair following mice carotid artery injured
3.3.1 Mice carotid injury model
Histological analysis and H&E staining demonstrated that neointimal formation was initiated at 7days and obviously developed at 28 day after vascular injury in control mouse. In addition, after transfection with Ad- Id1 or Ad-GFP, green fluorescence was detected with vascular tissues under a fluorescence microscope at 48 h post-vascular injury, indicating the efficiency of adenovirus transfection in mice carotid arteries.
3.3.2 Expression of Id1 during vascular repair process
Id1 mRNA expression was detected at low levels in normal uninjured control arteries, whereas following vascular injury Id1 mRNA level was rapidly enhanced with a peak at 14 d which gradually declined thereafter in 14 days and remained elevated for up to at least 28days. Id1 protein expression was assessed by Western blotting and was consistently found to be up-regulated. Further, immunohistochemistry showed that Id1 was detected in the intima and media of local vessels.
3.3.3 Effect of Ad- Id1-EPCs transplantation on vascular reendothelialization
Evans Blue dye was administered to evaluate reendothelialization at 14 days after injury. Nonendothelialized lesions were marked blue about 100% at injured vessels, whereas the reendothelialized area appeared white at uninjured vessels. The reendothelialized area in the Ad- Id1-EPCs infected arteries was significantly larger than that in Ad-GFP-EPCs, and non-infected groups (reendothelialized area /totle vessel injured area ratio was 68.36±4.51, 43.1±6.59 and 40.5±7.82, respectively, p<0.05), suggesting that transplantation of Ad-Id1-EPCs effectively promoted reendothelialization after vascular injury.
3.3.4 Effect of Ad- Id1-EPCs transplantation on neointimal formation
No marked decrease in the I/M ratio was shown in Ad- Id1-EPCs group compared with Ad-GFP-EPCs or non-infected group at day 14 (1.08±0.1, 1.16±0.14 and 1.15±0.17, respectively, p>0.05).
4. Conclusions:
4.1 Id1 was present at fairly low levels in quiescent EPCs and was localized mainly in the cytoplasm. Id1 was rapidly upregulated upon stimulation with serum and VEGF;
4.2 Overexpression of Id1 stimulated spleen derived EPCs proliferation and migration, and that was reversed by si-RNA-mediated silencing of Id1 expression;
4.3 Id1 was dynamically expressed in vascular lesions, and attained the peak expression in14 day after injure.
4.4 Ad-Id1-EPCs transplantation could home into the vascular injury site and accelerate reendothelialization of denuded vessel, howere, could not inhibit the neointima formation at the same period after vascular injury.
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