HGF/c-Met在血管损伤修复中促进内皮祖细胞增殖机制及归巢作用的研究
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摘要
1.背景与目的:
血管内皮受损是粥样硬化斑块形成的始动因素,也是血管损伤后不良修复反应的主要原因之一。长期以来,传统观念认为血管内膜损伤后内皮修复只有依靠损伤血管内膜边缘的内皮细胞(endothelial cell, EC)参与再生。近年来,实践证明邻近参与再生的ECs丧失再生能力或修复的范围有限,虽其远端细胞具有复制能力,但邻近局部内皮增生、迁移形成的再生内皮还会存在表型改变、功能不足或紊乱而不能发挥正常内皮功能,也难到达损伤位置而不能完成损伤血管内膜的修复。
细胞移植治疗近年来已成为治疗多种疾病的新策略,其目的是替代、修复或加强受损组织或器官的生物学功能。循环、骨髓及脾脏来源的内皮祖细胞(endothelial progenitor cell, EPC)被认为在血管损伤后的内皮修复中起到了越来越重要的作用。并且,研究发现EPCs易于在体外被基因修饰后再进行移植,这使得EPCs成为血管损伤后一种理想的治疗手段。但是,尽管目前研究证实机体自身动员的EPCs能够参与内皮功能不全及血管损伤的修复,但并不能完全修复损伤血管并阻止过度新生内膜形成及血管不良重构的发生。其认为一方面是机体自身动员不足、自身来源的EPCs具有不同程度的功能不全;另一方面是循环中EPCs不能充分的优势分布并粘附至血管靶部位。因此深入了解EPCs的功能调节机制,找到促进移植的EPCs向损伤血管优势分布的措施将会很大程度的提高修复血管损伤的疗效。
EPCs的功能能够被许多生长因子调节,其中包括血管内皮细胞生长因子(vascular endothelial cell growth factor, VEGF)、肝细胞生长因子(hepatocyte growth factor, HGF)、成纤维细胞生长因子(fibroblast growth factor, FGF)、雌激素(estrogen, ESG)等。新近,HGF对EPCs的调节作用逐渐引起重视。HGF被发现能够促进人类CD34+造血干细胞的增殖与生存,在肺部受到损伤后能够从骨髓动员EPCs至受损伤的局部。在高血压、急性心肌梗塞、糖尿病并发严重并发症、外周血管阻塞性疾病和动脉粥样硬化的病人血清中,HGF的浓度是升高的。并且,有许多证据表明,在血管损伤局部有HGF的表达,对促进局部内皮修复有重要的作用。
HGF促进细胞增殖主要是通过与其特异性的由原癌基因编码的受体c-Met相结合后开始的。HGF与其受体结合后,磷脂酶C-γ(phosphatidase c-γ, PLC-γ)被磷酸化,磷酸化的PLC-γ引起了三磷酸肌醇(inositol triphosphate, IP3)介导的内质网(endocytoplasmic reticulum, ER)钙库耗竭和接下来的细胞外钙离子通过细胞膜的内流。ER钙库耗竭引起的细胞膜钙离子内流通道被称为钙库操作性钙通道(store-operated Ca2+ channels, SOCCs)。在许多非兴奋性细胞,SOCCs被认为是广泛存在的调节钙离子内流的机制。Ca2+在细胞内充当第二信使的作用,参与细胞增殖、生存、分化、运动等多种功能。SOCCs的激活依赖于钙池的耗竭,因此ER必须把钙库耗竭的信息传递给细胞膜上的钙通道。一种名为间质交感分子1(stromal interaction molecule 1, STIM1)的跨膜蛋白被认为是激活SOCCs的重要分子。STIM1被认为是ER上钙库容量的感受器,细胞在静息状态时分布于ER膜表面,钙离子的耗竭导致了STIM1构象迅速发生改变并聚集到细胞膜附近。这种重新分布被认为起到了信号传递作用,将ER上钙离子耗竭的信号传递给了细胞膜上的钙离子通道,导致钙离子通道的开放。在HGF对EPCs的增殖调控中,STIM1及SOCCs是否参与其中,调控机制如何,目前国内外均无深入研究。
另外,国外及我们的研究证实静脉输注的EPCs能够优势分布至血管损伤部位参与损伤血管修复,但移植的EPCs定向优势分布的机制仍不清楚。一般认为,损伤局部的微环境和移植干细胞之间的相互作用促使移植的干细胞向损伤血管定向优势分布。损伤局部上调表达的炎性因子可能起到一定作用。除炎性因子外,其他一些相对特异的因子如基质细胞衍生因子1(stromal cell-derived factor 1, SDF-1)也发挥一定作用。近来研究发现,HGF也能介导细胞优势分布,在血管损伤局部存在HGF表达。因此,我们有理由推测HGF/c-Met能够通过趋化作用介导EPCs的优势分布,且局部的HGF能够促进移植的EPCs存活,提高细胞移植的效果。
本课题通过1、用RNA干扰STIM1的方法,研究了STIM1在HGF诱导的EPCs增殖效应中的作用。2、构建c-Met的腺病毒表达载体,转染至EPCs,使之高表达c-Met,通过静脉将之输注大鼠体内。利用血管损伤部位含有HGF的特点,以期达到其促进EPCs在移植体内存活并通过HGF/c-Met介导其向血管损伤部位优势分布的目的。从而为丰富干细胞研究的理论、进一步认识血管损伤修复机制及探索修复血管损伤的有效方法提供帮助。
2.方法:
2.1 EPCs的分离、培养、鉴定
密度梯度离心法获取大鼠骨髓及脾脏单个核细胞,在添加VEGF的DMEM培养液中常规培养,显微镜下观察细胞生长、形态学变化;UEA-I结合和DiI-LDL摄取实验观察EPCs是否具有内皮细胞功能特性;CD34、CD133、VEGFR-2流式鉴定是否具有内皮祖细胞表型。
2.2观察HGF对EPCs增殖功能的影响
胰酶消化EPCs后,接种至96孔板(1×105 cells/mL), EPCs经无血清培养同步化后,用四唑盐比色法(MTT)测定EPCs增殖变化。
2.3 RNA提取与半定量及实时定量PCR
用TRIzol试剂盒提取总RNA,根据M-MLV反转录试剂盒说明反转录CDNA,随后进行PCR检测,ABI PRISM 7000软件及荧光染料PCR法行定量PCR检测。
2.4 Westernblot检测STIM1蛋白表达。
2.5激光共聚焦检测内皮祖细胞细胞内钙离子荧光强度变化。
2.6 STIM1的RNA干扰载体由本实验室郭瑞威博士提供。
2.7重组腺病毒Ad-c-Met的构建
从美国ATCC购得目的基因c-Met片段,由北京宝赛生物公司通过同源重组系统构建c-Met的病毒过表达载体。
2.8观察c-Met在血管损伤修复过程中的作用
用1-2月龄的SD大鼠复制颈动脉球囊损伤模型,然后从尾静脉注入转染了c-Met的EPCs。观察过表达c-Met对损伤后第10天血管再内皮化以及第21天新生内膜增厚的影响。
3.结果:
3.1 EPCs分离、培养、鉴定:相差显微镜下可见分离后单个核细胞呈圆形悬浮于培养基中,胞体透亮,折光性好;细胞培养5d,可以观察到细胞增多增大并伸展呈椭圆形、短梭型细胞,有少量细胞突起,集落形成较多表明细胞增殖旺盛,有时可见到内皮祖细胞首尾相连,呈线状排列;培养7-10d,长梭型细胞较前明显增多,并呈簇状生长,有一定方向性,形成细胞团,簇状细胞中央细胞呈圆形,外周呈放射状;培养14d,有些培养瓶中可看到细胞首尾相连,具有成网状血管样趋势生长;继续培养至21d,细胞相互融合呈铺路石状,用acLDL-Dil和FITC-UEA-l对细胞染色后,通过荧光倒置显微镜鉴定,FITC-UEA-l结合阳性细胞呈现绿色荧光, acLDL-Dil摄取阳性细胞呈现红色荧光, UEA-l和DiLDL双染色阳性细胞呈现黄色荧光,这些双染的细胞被认为是正在分化的EPCs。利用细胞流式分析技术(FACS)分别检测CD133、CD34、VEFGR-2在分离培养7d左右细胞表面的表达,我们观察到CD133、CD34及VEGFR-2在培养细胞中的阳性率分别为85.28%、70.49%和96.68%。
3.2 HGF对EPCs增殖能力的影响
不同浓度梯度的HGF(2-20 ng/ml)刺激EPCs 48小时后发现,EPCs的增殖能力随着HGF刺激浓度的增加而增加。因此,我们的结果显示HGF能够促进EPCs的增殖。在本实验中其最大效应发生在10 ng/ml左右。
3.3 HGF刺激EPCs引起了经SOCCs的钙离子内流增加
使用HGF对EPCs刺激48小时再测定经钙库操纵性钙通道的钙内流(store-operated Ca2+ entry, SOCE),用共聚焦显微镜检测了单个细胞内ER清空前后钙离子浓度的变化。在细胞外钙缺乏的情况下,当加入2μM内质网钙离子泵拮抗剂毒胡萝卜素后(thapsigargin, TG),ER钙离子库被清空。当细胞外钙恢复以后(CaCl2; 5 mM)可以看到一个很快的钙离子内流,这是由于钙库清空后引起的SOCE而引起的,与对照组相比,SOCE的最大幅度约为原来的1.5倍。
3.4 SOCE对HGF诱导的EPC增殖能力的影响。
观察了SOCCs阻断剂对EPCs增殖效应的影响。在给予2-APB;100μM and BTP-2;10μM提前处理的EPCs组,同HGF刺激组比较,明显地抑制了EPCs的增殖效应。这些结果表明,SOCE参与了EPCs的增殖活动,而且在其中起到了重要的作用。
3.5 HGF作用于EPCs引起了STIM1表达的上调。
使用了荧光定量PCR及Westernblot检测了STIM1在HGF刺激的表达情况。STIM1 mRNA水平在刺激前的EPCs中处于非常低的水平,在HGF刺激12小时后增加了约2.5倍。24小时后增加幅度最大,约3.8倍。免疫印迹法分析也证实了相同的结果。这些结果证实了STIM1的表达在HGF刺激后发生了上调。
3.6 RNA干扰后STIM1表达后抑制了SOCE及HGF诱导的EPCs的增殖效应
用STIM1的腺病毒干扰载体,干扰STIM1的蛋白表达。转染48小时后,与对照组相比较STIM1蛋白表达减低了约70%。与对照组相比较,HGF刺激组STIM1的蛋白表达升高了约3倍。在siRNA+HGF组,STIM1蛋白表达与HGF组比较降低了约80%。而且,我们还研究了RNA干扰STIM1后对EPCs上SOCE的影响。与非干扰组比较,干扰组的SOCE上升最大幅度明显下降。MTT法测定了细胞的增殖能力,与对照组相比较,干扰组的增殖能力明显下降。这些结果表明STIM1在EPCs的增殖效应中起到了重要的作用。
3.7过表达c-Met对球囊损伤后血管再内皮化的影响
EPCs转染c-Met后,移植入球囊损伤颈动脉后的大鼠,伊文氏蓝染色来观察过表达c-Met对损伤血管第10天再内皮化的作用,选用再内皮化面积/动脉总面积这一比值作为评价指标。损伤10天时对照组(未移植组)、Ad-GFP-EPCs、Ad-c-Met-EPCs组再内皮化率分别是25.92±4.15%、43.21±7.24%、64.25±8.90%,说明c-Met过表达明显促进早期时损伤血管的再内皮化,提示c-Met转染EPCs后对损伤血管的再内皮化有促进作用
3.8过表达c-Met对球囊损伤后血管内膜增生的影响
在血管损伤后第21天,对照组(未移植组)、Ad-GFP-EPCs、Ad-c-Met-EPCs转染组大鼠损伤颈动脉内、中膜比值为1.28±0.23、0.63±0.13、0.29±0.06,c-Met转染组明显低于GFP及对照组大鼠损伤颈动脉内、中膜的比值,提示过表达c-Met抑制了球囊损伤后第21天大鼠颈动脉新生内膜的增厚。
4.结论:
1)、HGF促进EPCs的增殖;
2)、HGF刺激EPCs引起了经SOCCs的钙离子内流增加;
3)、SOCCs阻断剂明显地抑制了HGF诱导的EPCs的增殖效应;
4)、HGF作用于EPCs引起了STIM1表达的上调;
5)、RNA干扰STIM1表达后抑制了SOCE及HGF诱导的EPCs的增殖效应;
6)、过表达c-Met显著促进大鼠球囊损伤早期血管再内皮化;
7)、过表达c-Met抑制了大鼠球囊损伤后血管新生内膜的增厚。
1. Background and Objective:
Endothelial damage is a major contributing factor to atherosclerosis and post-angioplasty restenosis. Although endothelial cell (EC) repair mechanisms are considered to be mediated by adjacent mature endothelial cells, vascular endothelial cells only regenerate moderately in physiological conditions. If the endothelium persists under risk factors, local endothelial repair is incomplete.
More recently, circulating, bone marrow or spleen-derived endothelial progenitor cells (EPCs) have been found to present several advantages for endothelial regeneration after vascular injury. EPCs can be mobilized to the sites of injury and are amenable to ex vivo genetic engineering with viral vectors, making them ideal vehicles for delivery of therapeutic genes to sites of injury. Although rescent studies identified that automobilized EPCs can be involved in the progress of revasculation, the endothelium repair is ineffective. The reasonable explanation is that the founction of automobilized EPCs is defective and the number of EPCs homing to the injury site is insufficient. Thus, understanding the regulation mechanism of EPCs and how to improve the efficience of the EPCs homing is becoming the hot spot.
Hepatocyte growth factor (HGF), initially identified as a powerful stimulatory agent for primary cultured hepatocytes, is a multifunctional cytokine that regulates growth, motility, and morphogenesis of various cell types. Serum HGF levels are elevated in response to hypertension, acute myocardial infarction, diabetes mellitus with hypertensive complications, peripheral arterial occlusive diseases and carotid atherosclerosis . Several lines of evidence have implied that HGF is expressed locally in the arterial wall after injury and promotes the survival and proliferation of EPCs and ECs. Furthermore, HGF has also been demonstrated to promote the proliferation and survival of human CD34+ hematopoietic progenitors and induce angiogenesis in injured lungs through mobilizing EPCs from bone marrow, suggesting that HGF is involved in arterial repair and atherogenesis.
HGF promotes cell growth by stimulating the tyrosine kinase activity of the HGF-specific receptor encoded by the c-Met proto-oncogene. Following HGF activation of c-Met, phospholipase c-γ(PLC-γ) is phosphorylated. Phosphorylation of PLC-γresults in inositol triphosphate (IP3) mediated Ca2+ store depletion and subsequent Ca2+ influx across the plasma membrane (PM). Endoplasmic reticulum (ER) Ca2+ depletion activates PM-localized Ca2+ influx channels known as store-operated Ca2+ channels (SOCCs). Ca2+ signaling through SOCCs activates the expression of specific genes necessary for the regulation of cell growth and cell division.
SOCC activation relies exclusively on store depletion. Thus, the ER must communicate with SOCCs within the plasma to signal Ca2+ store depletion. After years of research, a membrane-spanning protein named stromal interaction molecule 1 (STIM1) was revealed as the key molecule required for the activation of SOCCs. Whereas STIM1 is likely the“sensor”of Ca2+ within ER Ca2+ stores and disperses on the ER membrane in quiescence, Ca2+ store depletion results in the rapid translocation of STIM1 into puncta close to the PM. This relocalization of the protein is thought to act as the store depletion signal to the SOCCs in the PM, subsequently leading to the opening of SOCCs.
However, it is not known whether STIM1 is involved in HGF-induced EPC growth, which is an important event for the vascular repair process, and whether HGF/c-Met can accelerate the re-endothelialization after ballon injury in rat model. Therefore, in this study, we siRNAed STIM1 and overexpressed c-Met to study the mechanism and effects of STIM1 and HGF/c-Met on EPC proliferation and recovery of balloon-injured artery.
2. Methods:
2.1 EPCs isolation and characterization
Total bone marrow or spleen-derived mononuclear cells (MNCs) were isolated by density gradient centrifugation (Lymphoprep 1.083) at 400×g for 20 min. After three washes, cells were plated on cell culture flasks and propagated in low glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FCS), 10 ng/ml recombinant human vascular endothelial growth factor (VEGF), 100 IU/mL penicillin, and 100μg/mL streptomycin. Cells were maintained at 37°C in a humidified atmosphere (95% air and 5% CO2) condition. Twenty-four hours later, nonadherent cells were transferred to a new flask to remove adherent hematopoietic cells and mature endothelial cells. Media were refreshed every 3 days. For characterization, differentiating cells were incubated with acLDL-Dil (10 mg/ml) for 4 h, fixed with 4% paraformaldehyde and then incubated with FITC-labeled lectin (UEA-1) for 1 h. Additionally, flow cytometry (FACS) analysis was performed using antibodies against rat CD133, CD34, VEGFR-2 and the corresponding isotype control antibodies.
2.2 Cell proliferation studies
EPCs were trypsinized and replaced into fibronectin-coated 96-well plates (1×105 cells/mL). Mitochondrial dehydrogenase activity was measured by the cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO) to purple formazan as an index of cell viability.
2.3 RNA isolation and quantitative real-time polymerase chain reaction (RT-PCR) Total RNA was extracted from EPCs using the TRIzol reagent. cDNA was synthesized using oligo (dT) and M-MLV reverse transcriptase according to the manufacturer’s protocol. For quantitative RT-PCR analyses, the ABI PRISM 7000 Sequence Detection System and SYBR Green PCR Master Mix were used.
2.4 Western blot analysis
After treatment, cells were lysed in lysis buffer. Protein concentrations of cell extracts were measured using the Bradford method. Equal amounts of protein (100μg) were separated by SDS-PAGE (10% polyacrylamide gel) and electrophoretically transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% nonfat milk solution in TBS with 0.5% Tween- 20 and incubated in STIM1 primary antibody purchased from BD, at 4 ?C for 4 h. Finally, membranes were incubated with anti-rabbit horseradish peroxidase-conjugated IgG for 1 h. Protein bands were visualized by chemiluminescent detection and quantified by a gel image analysis system. Anti-GAPDH monoclonal antibody was used to test for equal protein loading.
2.5 Fluorescence Cell Imaging
EPCs placed in a special chamber were loaded with the Ca2+ indicator Fluo-3/AM and continuously cultured at 37 ?C for 30 min in a 5% CO2 incubator. After loading with the fluorescence probe, changes in intracellular Ca2+ infux ([Ca2+]i) in individual cells were measured using a digital imaging system equipped with a laser confocal scanning microscope with an excitation wavelength of 488 nm.
2.6 Knockdown of STIM1 by siRNA
Ad-si/STIM1 and non-silencing control (NSC) were a gift from Dr. Guo. The selected siRNA duplex sequences specifically targeted rat STIM1 (rSTIM1, NM_001108496), and showed no homology to any other sequences by a blast search. The two sequences used in this study are (i) Start nucleotide 935, GCAUGGAAGGCAUCAGAAGUGUAUA; and (ii) start nucleotide 970, GGAUGAGGUGAUACAGUGGCUGAUU. A non-silencing control (NSC) sequence was designed according to the sequence of a negative control.
2.7 Construction of recombinant adenoviral vectors
Adenoviral vector expressing c-Met was generated using the AdEasy system. Briefly, full-length rat C-Met cDNA was buyed from Baosai company. The shuttle vector was used to generate recombinant adenoviruses according to the manufacturer’s protocol. An adenovirus encoding green fluorescent protein (GFP; Ad-GFP) was used as control.
2.8 Vascular injury model
To evaluate the role of HGF/Met in vascular repair in vivo, we used balloon-injured rat carotid artery model. Evans Blue dye was administered to evaluate reendothelialization after 10 days injury, and the neointimal formation was assessed at 21 days following vascular injury.
3. Results:
3.1 EPCs isolation and characterization
Freshly plated bone and spleen derived MNCs possessed a rounded shape. After 3 to 5 days in culture, adherent cells formed cluster- or cord-like structures. The number of EPCs continually increased and the cells elongated to possess a spindle shape over the next 7 days. EPC colony-forming units became obvious after 10 days in culture. EPCs cultured 14 days formed tubular-like appearance and grew to confluence with a cobblestone shape at 21 days. For further characterization, after 7 days in culture, attached cells were analyzed with immunofluorescence and FACS. Cells took up Dil-Ac-LDL, bound lectin, and expressed endothelial/stem cell markers, including CD133, CD34 and VEGFR-2 were EPCs.
3.2 Effects of HGF on EPC proliferation
The effect of HGF on EPC proliferation was examined using the MTT assay 48 h after exposure to different quantities of HGF (range 2-20 ng/ml). The proliferation effect was strongly dose-dependent and increased in HGF-treated EPCs but not in control cells. Thus, our results suggest that HGF accelerates EPC proliferation. Because the maximum proliferation effect occurred even at 10 ng/ml, we used 10 ng/ml HGF as the proliferation stimulus in subsequent experiments.
3.3 Increased SOCE in HGF-treated EPCs
In order to test whether SOCE plays a role in HGF-induced EPC proliferation, the long-term effect of HGF on SOCCs activation in EPCs was assessed after treated with HGF for 48 h. Changes in [Ca2+]i in individual cells were estimated and compared before and after store depletion between control and HGF-treated cells using fluorescence microscopy. Ca2+ stores were initially depleted by inhibition of ER Ca-ATPase activity with 2μM thapsigargin (TG) in the absence of extracellular Ca2+. After addition of TG, transient cytosolic Ca2+ release from the ER occurred, confirming the emptying of ER Ca2+ stores. Following restoration of extracellular Ca2+ (CaCl2; 5 mM), a rapid increase of Ca2+ influx was observed, which was due to SOCCs activation. The maximum amplitude in [Ca2+]i caused by SOCE was greatly increased in HGF-treated EPCs compared to control cells (140 % versus control).
To assess further the dependence of SOCE on HGF-induced EPC proliferation, we investigated the effects of SOCC inhibitors on EPC proliferation. As shown in Fig. 3b, this inhibition was evaluated by the MTT assay after 12 h of pre-incubation with SOCC inhibitors (2-APB;100μM and BTP-2;10μM). Both BTP-2 and 2-APB, compared to the HGF treated group, inhibited the proliferation response significantly. These common effects clearly suggested that SOCE is needed for HGF-induced EPCs proliferation.
3.4 Expression of STIM1 in HGF-treated EPCs
To test whether HGF affects the expression of STIM1, quantitative real-time PCR and western blotting were performed after HGF stimulation. STIM1 mRNA levels were very low at resting states, but greatly increased 12 h after HGF treatment and reached a maximum of 4-fold greater than baseline levels after 24 h of HGF treatment. Western blot analysis indicated that the STIM1 protein level increased significantly after 24 and 48 h treatment with HGF. These results indicated that STIM1 expression is upregulated in EPCs after HGF stimulation.
3.5 Knockdown of STIM1 expression attenuates SOCE and HGF-induced EPC proliferation
To determine whether endogenous STIM1 affects SOCE and HGF-induced EPC proliferation, we delivered adenovirus constructs expressing si/rSTIM1 to knockdown STIM1 protein levels. Cells treated with HGF but transfected with a random sequence were defined as the non-silencing group (nsRNA) and used as a control to monitor non-sequence-specific effects. Forty-eight hours after transfection, the level of STIM1 protein decreased by 76% compared to the control. HGF stimulation for 48 h increased the STIM1 protein level to 340% compared to the control group. In the siRNA+HGF group, STIM1 protein expression decreased about by 81% compare to the HGF group. Furthermore, When we measured the effect of an siRNA targeted against rSTIM1 on SOCE, the maximum increase in [Ca2+]i caused by SOCE robustly decreased in siRNA-treated EPCs compared to the nsRNA group. Cell proferation measured by MTT in the siRNA group was significantly lower compared to the nsRNA group. Taken together, these results indicate that STIM1 molecules play a key role in the proliferation response of EPCs.
3.6 Effect of Ad-Met on vascular reendothelialization
Evans Blue dye was administered to evaluate reendothelialization after balloon injury. Nonendothelialized lesions were marked blue at injured vessels, whereas the reendothelialized area appeared white at uninjured vessels. The reendothelialized area in the Ad-Met-infected EPCs transplantation group was significantly larger than that in Ad-GFP-infected EPCs transplantation and control groups.
3.7 Effect of Ad-Met on neointimal formation
A marked decrease in the neointimal area and I/M ratio was shown in Ad-Met–EPCs treated rats compared with that of Ad-GFP-EPCs treated and control group at day 21.
4. Conclusions:
4.1 HGF accelerated EPC proliferation.
4.2 Store-operated Ca2+ entry (SOCE) was elevated in HGF-treated EPCs.
4.3 STIM1 mRNA and protein expression levels were increased in response to HGF stimulation.
4.4 Knockdown of STMI1 decreased SOCE and prevented HGF-induced EPC proliferation.
4.5 Ad-Met-infected EPCs promoted reendothelialization and inhibited neointimal formation than those in Ad-GFP-infected EPCs transplantation and control groups.
血管内皮受损是粥样硬化斑块形成的始动因素,也是血管损伤后不良修复反应的主要原因之一。长期以来,传统观念认为血管内膜损伤后内皮修复只有依靠损伤血管内膜边缘的内皮细胞(endothelial cell, EC)参与再生。近年来,实践证明邻近参与再生的ECs丧失再生能力或修复的范围有限,虽其远端细胞具有复制能力,但邻近局部内皮增生、迁移形成的再生内皮还会存在表型改变、功能不足或紊乱而不能发挥正常内皮功能,也难到达损伤位置而不能完成损伤血管内膜的修复。
细胞移植治疗近年来已成为治疗多种疾病的新策略,其目的是替代、修复或加强受损组织或器官的生物学功能。循环、骨髓及脾脏来源的内皮祖细胞(endothelial progenitor cell, EPC)被认为在血管损伤后的内皮修复中起到了越来越重要的作用。并且,研究发现EPCs易于在体外被基因修饰后再进行移植,这使得EPCs成为血管损伤后一种理想的治疗手段。但是,尽管目前研究证实机体自身动员的EPCs能够参与内皮功能不全及血管损伤的修复,但并不能完全修复损伤血管并阻止过度新生内膜形成及血管不良重构的发生。其认为一方面是机体自身动员不足、自身来源的EPCs具有不同程度的功能不全;另一方面是循环中EPCs不能充分的优势分布并粘附至血管靶部位。因此深入了解EPCs的功能调节机制,找到促进移植的EPCs向损伤血管优势分布的措施将会很大程度的提高修复血管损伤的疗效。
EPCs的功能能够被许多生长因子调节,其中包括血管内皮细胞生长因子(vascular endothelial cell growth factor, VEGF)、肝细胞生长因子(hepatocyte growth factor, HGF)、成纤维细胞生长因子(fibroblast growth factor, FGF)、雌激素(estrogen, ESG)等。新近,HGF对EPCs的调节作用逐渐引起重视。HGF被发现能够促进人类CD34+造血干细胞的增殖与生存,在肺部受到损伤后能够从骨髓动员EPCs至受损伤的局部。在高血压、急性心肌梗塞、糖尿病并发严重并发症、外周血管阻塞性疾病和动脉粥样硬化的病人血清中,HGF的浓度是升高的。并且,有许多证据表明,在血管损伤局部有HGF的表达,对促进局部内皮修复有重要的作用。
HGF促进细胞增殖主要是通过与其特异性的由原癌基因编码的受体c-Met相结合后开始的。HGF与其受体结合后,磷脂酶C-γ(phosphatidase c-γ, PLC-γ)被磷酸化,磷酸化的PLC-γ引起了三磷酸肌醇(inositol triphosphate, IP3)介导的内质网(endocytoplasmic reticulum, ER)钙库耗竭和接下来的细胞外钙离子通过细胞膜的内流。ER钙库耗竭引起的细胞膜钙离子内流通道被称为钙库操作性钙通道(store-operated Ca2+ channels, SOCCs)。在许多非兴奋性细胞,SOCCs被认为是广泛存在的调节钙离子内流的机制。Ca2+在细胞内充当第二信使的作用,参与细胞增殖、生存、分化、运动等多种功能。SOCCs的激活依赖于钙池的耗竭,因此ER必须把钙库耗竭的信息传递给细胞膜上的钙通道。一种名为间质交感分子1(stromal interaction molecule 1, STIM1)的跨膜蛋白被认为是激活SOCCs的重要分子。STIM1被认为是ER上钙库容量的感受器,细胞在静息状态时分布于ER膜表面,钙离子的耗竭导致了STIM1构象迅速发生改变并聚集到细胞膜附近。这种重新分布被认为起到了信号传递作用,将ER上钙离子耗竭的信号传递给了细胞膜上的钙离子通道,导致钙离子通道的开放。在HGF对EPCs的增殖调控中,STIM1及SOCCs是否参与其中,调控机制如何,目前国内外均无深入研究。
另外,国外及我们的研究证实静脉输注的EPCs能够优势分布至血管损伤部位参与损伤血管修复,但移植的EPCs定向优势分布的机制仍不清楚。一般认为,损伤局部的微环境和移植干细胞之间的相互作用促使移植的干细胞向损伤血管定向优势分布。损伤局部上调表达的炎性因子可能起到一定作用。除炎性因子外,其他一些相对特异的因子如基质细胞衍生因子1(stromal cell-derived factor 1, SDF-1)也发挥一定作用。近来研究发现,HGF也能介导细胞优势分布,在血管损伤局部存在HGF表达。因此,我们有理由推测HGF/c-Met能够通过趋化作用介导EPCs的优势分布,且局部的HGF能够促进移植的EPCs存活,提高细胞移植的效果。
本课题通过1、用RNA干扰STIM1的方法,研究了STIM1在HGF诱导的EPCs增殖效应中的作用。2、构建c-Met的腺病毒表达载体,转染至EPCs,使之高表达c-Met,通过静脉将之输注大鼠体内。利用血管损伤部位含有HGF的特点,以期达到其促进EPCs在移植体内存活并通过HGF/c-Met介导其向血管损伤部位优势分布的目的。从而为丰富干细胞研究的理论、进一步认识血管损伤修复机制及探索修复血管损伤的有效方法提供帮助。
2.方法:
2.1 EPCs的分离、培养、鉴定
密度梯度离心法获取大鼠骨髓及脾脏单个核细胞,在添加VEGF的DMEM培养液中常规培养,显微镜下观察细胞生长、形态学变化;UEA-I结合和DiI-LDL摄取实验观察EPCs是否具有内皮细胞功能特性;CD34、CD133、VEGFR-2流式鉴定是否具有内皮祖细胞表型。
2.2观察HGF对EPCs增殖功能的影响
胰酶消化EPCs后,接种至96孔板(1×105 cells/mL), EPCs经无血清培养同步化后,用四唑盐比色法(MTT)测定EPCs增殖变化。
2.3 RNA提取与半定量及实时定量PCR
用TRIzol试剂盒提取总RNA,根据M-MLV反转录试剂盒说明反转录CDNA,随后进行PCR检测,ABI PRISM 7000软件及荧光染料PCR法行定量PCR检测。
2.4 Westernblot检测STIM1蛋白表达。
2.5激光共聚焦检测内皮祖细胞细胞内钙离子荧光强度变化。
2.6 STIM1的RNA干扰载体由本实验室郭瑞威博士提供。
2.7重组腺病毒Ad-c-Met的构建
从美国ATCC购得目的基因c-Met片段,由北京宝赛生物公司通过同源重组系统构建c-Met的病毒过表达载体。
2.8观察c-Met在血管损伤修复过程中的作用
用1-2月龄的SD大鼠复制颈动脉球囊损伤模型,然后从尾静脉注入转染了c-Met的EPCs。观察过表达c-Met对损伤后第10天血管再内皮化以及第21天新生内膜增厚的影响。
3.结果:
3.1 EPCs分离、培养、鉴定:相差显微镜下可见分离后单个核细胞呈圆形悬浮于培养基中,胞体透亮,折光性好;细胞培养5d,可以观察到细胞增多增大并伸展呈椭圆形、短梭型细胞,有少量细胞突起,集落形成较多表明细胞增殖旺盛,有时可见到内皮祖细胞首尾相连,呈线状排列;培养7-10d,长梭型细胞较前明显增多,并呈簇状生长,有一定方向性,形成细胞团,簇状细胞中央细胞呈圆形,外周呈放射状;培养14d,有些培养瓶中可看到细胞首尾相连,具有成网状血管样趋势生长;继续培养至21d,细胞相互融合呈铺路石状,用acLDL-Dil和FITC-UEA-l对细胞染色后,通过荧光倒置显微镜鉴定,FITC-UEA-l结合阳性细胞呈现绿色荧光, acLDL-Dil摄取阳性细胞呈现红色荧光, UEA-l和DiLDL双染色阳性细胞呈现黄色荧光,这些双染的细胞被认为是正在分化的EPCs。利用细胞流式分析技术(FACS)分别检测CD133、CD34、VEFGR-2在分离培养7d左右细胞表面的表达,我们观察到CD133、CD34及VEGFR-2在培养细胞中的阳性率分别为85.28%、70.49%和96.68%。
3.2 HGF对EPCs增殖能力的影响
不同浓度梯度的HGF(2-20 ng/ml)刺激EPCs 48小时后发现,EPCs的增殖能力随着HGF刺激浓度的增加而增加。因此,我们的结果显示HGF能够促进EPCs的增殖。在本实验中其最大效应发生在10 ng/ml左右。
3.3 HGF刺激EPCs引起了经SOCCs的钙离子内流增加
使用HGF对EPCs刺激48小时再测定经钙库操纵性钙通道的钙内流(store-operated Ca2+ entry, SOCE),用共聚焦显微镜检测了单个细胞内ER清空前后钙离子浓度的变化。在细胞外钙缺乏的情况下,当加入2μM内质网钙离子泵拮抗剂毒胡萝卜素后(thapsigargin, TG),ER钙离子库被清空。当细胞外钙恢复以后(CaCl2; 5 mM)可以看到一个很快的钙离子内流,这是由于钙库清空后引起的SOCE而引起的,与对照组相比,SOCE的最大幅度约为原来的1.5倍。
3.4 SOCE对HGF诱导的EPC增殖能力的影响。
观察了SOCCs阻断剂对EPCs增殖效应的影响。在给予2-APB;100μM and BTP-2;10μM提前处理的EPCs组,同HGF刺激组比较,明显地抑制了EPCs的增殖效应。这些结果表明,SOCE参与了EPCs的增殖活动,而且在其中起到了重要的作用。
3.5 HGF作用于EPCs引起了STIM1表达的上调。
使用了荧光定量PCR及Westernblot检测了STIM1在HGF刺激的表达情况。STIM1 mRNA水平在刺激前的EPCs中处于非常低的水平,在HGF刺激12小时后增加了约2.5倍。24小时后增加幅度最大,约3.8倍。免疫印迹法分析也证实了相同的结果。这些结果证实了STIM1的表达在HGF刺激后发生了上调。
3.6 RNA干扰后STIM1表达后抑制了SOCE及HGF诱导的EPCs的增殖效应
用STIM1的腺病毒干扰载体,干扰STIM1的蛋白表达。转染48小时后,与对照组相比较STIM1蛋白表达减低了约70%。与对照组相比较,HGF刺激组STIM1的蛋白表达升高了约3倍。在siRNA+HGF组,STIM1蛋白表达与HGF组比较降低了约80%。而且,我们还研究了RNA干扰STIM1后对EPCs上SOCE的影响。与非干扰组比较,干扰组的SOCE上升最大幅度明显下降。MTT法测定了细胞的增殖能力,与对照组相比较,干扰组的增殖能力明显下降。这些结果表明STIM1在EPCs的增殖效应中起到了重要的作用。
3.7过表达c-Met对球囊损伤后血管再内皮化的影响
EPCs转染c-Met后,移植入球囊损伤颈动脉后的大鼠,伊文氏蓝染色来观察过表达c-Met对损伤血管第10天再内皮化的作用,选用再内皮化面积/动脉总面积这一比值作为评价指标。损伤10天时对照组(未移植组)、Ad-GFP-EPCs、Ad-c-Met-EPCs组再内皮化率分别是25.92±4.15%、43.21±7.24%、64.25±8.90%,说明c-Met过表达明显促进早期时损伤血管的再内皮化,提示c-Met转染EPCs后对损伤血管的再内皮化有促进作用
3.8过表达c-Met对球囊损伤后血管内膜增生的影响
在血管损伤后第21天,对照组(未移植组)、Ad-GFP-EPCs、Ad-c-Met-EPCs转染组大鼠损伤颈动脉内、中膜比值为1.28±0.23、0.63±0.13、0.29±0.06,c-Met转染组明显低于GFP及对照组大鼠损伤颈动脉内、中膜的比值,提示过表达c-Met抑制了球囊损伤后第21天大鼠颈动脉新生内膜的增厚。
4.结论:
1)、HGF促进EPCs的增殖;
2)、HGF刺激EPCs引起了经SOCCs的钙离子内流增加;
3)、SOCCs阻断剂明显地抑制了HGF诱导的EPCs的增殖效应;
4)、HGF作用于EPCs引起了STIM1表达的上调;
5)、RNA干扰STIM1表达后抑制了SOCE及HGF诱导的EPCs的增殖效应;
6)、过表达c-Met显著促进大鼠球囊损伤早期血管再内皮化;
7)、过表达c-Met抑制了大鼠球囊损伤后血管新生内膜的增厚。
1. Background and Objective:
Endothelial damage is a major contributing factor to atherosclerosis and post-angioplasty restenosis. Although endothelial cell (EC) repair mechanisms are considered to be mediated by adjacent mature endothelial cells, vascular endothelial cells only regenerate moderately in physiological conditions. If the endothelium persists under risk factors, local endothelial repair is incomplete.
More recently, circulating, bone marrow or spleen-derived endothelial progenitor cells (EPCs) have been found to present several advantages for endothelial regeneration after vascular injury. EPCs can be mobilized to the sites of injury and are amenable to ex vivo genetic engineering with viral vectors, making them ideal vehicles for delivery of therapeutic genes to sites of injury. Although rescent studies identified that automobilized EPCs can be involved in the progress of revasculation, the endothelium repair is ineffective. The reasonable explanation is that the founction of automobilized EPCs is defective and the number of EPCs homing to the injury site is insufficient. Thus, understanding the regulation mechanism of EPCs and how to improve the efficience of the EPCs homing is becoming the hot spot.
Hepatocyte growth factor (HGF), initially identified as a powerful stimulatory agent for primary cultured hepatocytes, is a multifunctional cytokine that regulates growth, motility, and morphogenesis of various cell types. Serum HGF levels are elevated in response to hypertension, acute myocardial infarction, diabetes mellitus with hypertensive complications, peripheral arterial occlusive diseases and carotid atherosclerosis . Several lines of evidence have implied that HGF is expressed locally in the arterial wall after injury and promotes the survival and proliferation of EPCs and ECs. Furthermore, HGF has also been demonstrated to promote the proliferation and survival of human CD34+ hematopoietic progenitors and induce angiogenesis in injured lungs through mobilizing EPCs from bone marrow, suggesting that HGF is involved in arterial repair and atherogenesis.
HGF promotes cell growth by stimulating the tyrosine kinase activity of the HGF-specific receptor encoded by the c-Met proto-oncogene. Following HGF activation of c-Met, phospholipase c-γ(PLC-γ) is phosphorylated. Phosphorylation of PLC-γresults in inositol triphosphate (IP3) mediated Ca2+ store depletion and subsequent Ca2+ influx across the plasma membrane (PM). Endoplasmic reticulum (ER) Ca2+ depletion activates PM-localized Ca2+ influx channels known as store-operated Ca2+ channels (SOCCs). Ca2+ signaling through SOCCs activates the expression of specific genes necessary for the regulation of cell growth and cell division.
SOCC activation relies exclusively on store depletion. Thus, the ER must communicate with SOCCs within the plasma to signal Ca2+ store depletion. After years of research, a membrane-spanning protein named stromal interaction molecule 1 (STIM1) was revealed as the key molecule required for the activation of SOCCs. Whereas STIM1 is likely the“sensor”of Ca2+ within ER Ca2+ stores and disperses on the ER membrane in quiescence, Ca2+ store depletion results in the rapid translocation of STIM1 into puncta close to the PM. This relocalization of the protein is thought to act as the store depletion signal to the SOCCs in the PM, subsequently leading to the opening of SOCCs.
However, it is not known whether STIM1 is involved in HGF-induced EPC growth, which is an important event for the vascular repair process, and whether HGF/c-Met can accelerate the re-endothelialization after ballon injury in rat model. Therefore, in this study, we siRNAed STIM1 and overexpressed c-Met to study the mechanism and effects of STIM1 and HGF/c-Met on EPC proliferation and recovery of balloon-injured artery.
2. Methods:
2.1 EPCs isolation and characterization
Total bone marrow or spleen-derived mononuclear cells (MNCs) were isolated by density gradient centrifugation (Lymphoprep 1.083) at 400×g for 20 min. After three washes, cells were plated on cell culture flasks and propagated in low glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FCS), 10 ng/ml recombinant human vascular endothelial growth factor (VEGF), 100 IU/mL penicillin, and 100μg/mL streptomycin. Cells were maintained at 37°C in a humidified atmosphere (95% air and 5% CO2) condition. Twenty-four hours later, nonadherent cells were transferred to a new flask to remove adherent hematopoietic cells and mature endothelial cells. Media were refreshed every 3 days. For characterization, differentiating cells were incubated with acLDL-Dil (10 mg/ml) for 4 h, fixed with 4% paraformaldehyde and then incubated with FITC-labeled lectin (UEA-1) for 1 h. Additionally, flow cytometry (FACS) analysis was performed using antibodies against rat CD133, CD34, VEGFR-2 and the corresponding isotype control antibodies.
2.2 Cell proliferation studies
EPCs were trypsinized and replaced into fibronectin-coated 96-well plates (1×105 cells/mL). Mitochondrial dehydrogenase activity was measured by the cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO) to purple formazan as an index of cell viability.
2.3 RNA isolation and quantitative real-time polymerase chain reaction (RT-PCR) Total RNA was extracted from EPCs using the TRIzol reagent. cDNA was synthesized using oligo (dT) and M-MLV reverse transcriptase according to the manufacturer’s protocol. For quantitative RT-PCR analyses, the ABI PRISM 7000 Sequence Detection System and SYBR Green PCR Master Mix were used.
2.4 Western blot analysis
After treatment, cells were lysed in lysis buffer. Protein concentrations of cell extracts were measured using the Bradford method. Equal amounts of protein (100μg) were separated by SDS-PAGE (10% polyacrylamide gel) and electrophoretically transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% nonfat milk solution in TBS with 0.5% Tween- 20 and incubated in STIM1 primary antibody purchased from BD, at 4 ?C for 4 h. Finally, membranes were incubated with anti-rabbit horseradish peroxidase-conjugated IgG for 1 h. Protein bands were visualized by chemiluminescent detection and quantified by a gel image analysis system. Anti-GAPDH monoclonal antibody was used to test for equal protein loading.
2.5 Fluorescence Cell Imaging
EPCs placed in a special chamber were loaded with the Ca2+ indicator Fluo-3/AM and continuously cultured at 37 ?C for 30 min in a 5% CO2 incubator. After loading with the fluorescence probe, changes in intracellular Ca2+ infux ([Ca2+]i) in individual cells were measured using a digital imaging system equipped with a laser confocal scanning microscope with an excitation wavelength of 488 nm.
2.6 Knockdown of STIM1 by siRNA
Ad-si/STIM1 and non-silencing control (NSC) were a gift from Dr. Guo. The selected siRNA duplex sequences specifically targeted rat STIM1 (rSTIM1, NM_001108496), and showed no homology to any other sequences by a blast search. The two sequences used in this study are (i) Start nucleotide 935, GCAUGGAAGGCAUCAGAAGUGUAUA; and (ii) start nucleotide 970, GGAUGAGGUGAUACAGUGGCUGAUU. A non-silencing control (NSC) sequence was designed according to the sequence of a negative control.
2.7 Construction of recombinant adenoviral vectors
Adenoviral vector expressing c-Met was generated using the AdEasy system. Briefly, full-length rat C-Met cDNA was buyed from Baosai company. The shuttle vector was used to generate recombinant adenoviruses according to the manufacturer’s protocol. An adenovirus encoding green fluorescent protein (GFP; Ad-GFP) was used as control.
2.8 Vascular injury model
To evaluate the role of HGF/Met in vascular repair in vivo, we used balloon-injured rat carotid artery model. Evans Blue dye was administered to evaluate reendothelialization after 10 days injury, and the neointimal formation was assessed at 21 days following vascular injury.
3. Results:
3.1 EPCs isolation and characterization
Freshly plated bone and spleen derived MNCs possessed a rounded shape. After 3 to 5 days in culture, adherent cells formed cluster- or cord-like structures. The number of EPCs continually increased and the cells elongated to possess a spindle shape over the next 7 days. EPC colony-forming units became obvious after 10 days in culture. EPCs cultured 14 days formed tubular-like appearance and grew to confluence with a cobblestone shape at 21 days. For further characterization, after 7 days in culture, attached cells were analyzed with immunofluorescence and FACS. Cells took up Dil-Ac-LDL, bound lectin, and expressed endothelial/stem cell markers, including CD133, CD34 and VEGFR-2 were EPCs.
3.2 Effects of HGF on EPC proliferation
The effect of HGF on EPC proliferation was examined using the MTT assay 48 h after exposure to different quantities of HGF (range 2-20 ng/ml). The proliferation effect was strongly dose-dependent and increased in HGF-treated EPCs but not in control cells. Thus, our results suggest that HGF accelerates EPC proliferation. Because the maximum proliferation effect occurred even at 10 ng/ml, we used 10 ng/ml HGF as the proliferation stimulus in subsequent experiments.
3.3 Increased SOCE in HGF-treated EPCs
In order to test whether SOCE plays a role in HGF-induced EPC proliferation, the long-term effect of HGF on SOCCs activation in EPCs was assessed after treated with HGF for 48 h. Changes in [Ca2+]i in individual cells were estimated and compared before and after store depletion between control and HGF-treated cells using fluorescence microscopy. Ca2+ stores were initially depleted by inhibition of ER Ca-ATPase activity with 2μM thapsigargin (TG) in the absence of extracellular Ca2+. After addition of TG, transient cytosolic Ca2+ release from the ER occurred, confirming the emptying of ER Ca2+ stores. Following restoration of extracellular Ca2+ (CaCl2; 5 mM), a rapid increase of Ca2+ influx was observed, which was due to SOCCs activation. The maximum amplitude in [Ca2+]i caused by SOCE was greatly increased in HGF-treated EPCs compared to control cells (140 % versus control).
To assess further the dependence of SOCE on HGF-induced EPC proliferation, we investigated the effects of SOCC inhibitors on EPC proliferation. As shown in Fig. 3b, this inhibition was evaluated by the MTT assay after 12 h of pre-incubation with SOCC inhibitors (2-APB;100μM and BTP-2;10μM). Both BTP-2 and 2-APB, compared to the HGF treated group, inhibited the proliferation response significantly. These common effects clearly suggested that SOCE is needed for HGF-induced EPCs proliferation.
3.4 Expression of STIM1 in HGF-treated EPCs
To test whether HGF affects the expression of STIM1, quantitative real-time PCR and western blotting were performed after HGF stimulation. STIM1 mRNA levels were very low at resting states, but greatly increased 12 h after HGF treatment and reached a maximum of 4-fold greater than baseline levels after 24 h of HGF treatment. Western blot analysis indicated that the STIM1 protein level increased significantly after 24 and 48 h treatment with HGF. These results indicated that STIM1 expression is upregulated in EPCs after HGF stimulation.
3.5 Knockdown of STIM1 expression attenuates SOCE and HGF-induced EPC proliferation
To determine whether endogenous STIM1 affects SOCE and HGF-induced EPC proliferation, we delivered adenovirus constructs expressing si/rSTIM1 to knockdown STIM1 protein levels. Cells treated with HGF but transfected with a random sequence were defined as the non-silencing group (nsRNA) and used as a control to monitor non-sequence-specific effects. Forty-eight hours after transfection, the level of STIM1 protein decreased by 76% compared to the control. HGF stimulation for 48 h increased the STIM1 protein level to 340% compared to the control group. In the siRNA+HGF group, STIM1 protein expression decreased about by 81% compare to the HGF group. Furthermore, When we measured the effect of an siRNA targeted against rSTIM1 on SOCE, the maximum increase in [Ca2+]i caused by SOCE robustly decreased in siRNA-treated EPCs compared to the nsRNA group. Cell proferation measured by MTT in the siRNA group was significantly lower compared to the nsRNA group. Taken together, these results indicate that STIM1 molecules play a key role in the proliferation response of EPCs.
3.6 Effect of Ad-Met on vascular reendothelialization
Evans Blue dye was administered to evaluate reendothelialization after balloon injury. Nonendothelialized lesions were marked blue at injured vessels, whereas the reendothelialized area appeared white at uninjured vessels. The reendothelialized area in the Ad-Met-infected EPCs transplantation group was significantly larger than that in Ad-GFP-infected EPCs transplantation and control groups.
3.7 Effect of Ad-Met on neointimal formation
A marked decrease in the neointimal area and I/M ratio was shown in Ad-Met–EPCs treated rats compared with that of Ad-GFP-EPCs treated and control group at day 21.
4. Conclusions:
4.1 HGF accelerated EPC proliferation.
4.2 Store-operated Ca2+ entry (SOCE) was elevated in HGF-treated EPCs.
4.3 STIM1 mRNA and protein expression levels were increased in response to HGF stimulation.
4.4 Knockdown of STMI1 decreased SOCE and prevented HGF-induced EPC proliferation.
4.5 Ad-Met-infected EPCs promoted reendothelialization and inhibited neointimal formation than those in Ad-GFP-infected EPCs transplantation and control groups.
引文
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