STIM1/TRPC1复合体对EPCs修复损伤血管能力的影响
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摘要
1.背景与目的
血管内皮损伤和损伤后的不良修复是冠心病、高血压、经皮冠状动脉介入术(percutaneous coronary intervention ,PCI)后再狭窄等血管损伤性疾病发病的共同病理生理基础。如何尽早促进血管损伤后的再内皮化是防治这类疾病形成的关键策略。传统观点认为,内皮损伤的修复主要依靠临近成熟内皮的迁移和增殖,但其增殖能力有限。晚近的研究证实,骨髓和外周血中内皮祖细胞(endothelial progenitor cells ,EPCs)能归巢至损伤血管局部,分化为内皮细胞,促进血管的良性修复。EPCs的增殖、迁移等生物学行为是其修复内皮功能的基础,然而,目前调控EPCs生物学行为的机制,尤其是离子通道方面,仍不清楚。
钙离子是调节细胞生理功能比如增殖、分化等的重要物质基础。研究发现,钙离子参与调控EPCs的增殖、归巢及分化功能。钙离子对细胞功能的影响通过胞膜钙通道调控的钙内流来实现,在EPCs这种非兴奋性细胞,由于缺乏电压依赖性钙通道,主要以钙库操纵性钙通道为主(store-operated calcium channals ,SOCs), SOCs由基质交联分子1(stromal interacting molecule 1,STIM1)、Orai和瞬时受体电位通道(transient receptor potential canonical,TRPC)家族构成。STIM1在SOCs中发挥感受器作用,STIM1感受胞内钙库耗竭,激活Orai和TRPC通道,介导钙库操纵性钙内流(store-operated calcium entry ,SOCE),以补充胞内钙。
晚近的研究发现,基因沉默STIM1可通过抑制血管平滑肌细胞的增殖而抑制血管损伤后新生内膜的形成。TRPC1抗体也可抑制隐静脉平滑肌细胞增殖导致的新生内膜肥厚。而EPCs作为血管损伤修复中的一重要细胞来源,STIM1及TRPC1是否也通过影响EPCs的功能而参与血管损伤修复过程的调节,目前仍不清楚。我们的前期研究发现,EPCs上有STIM1和TRPC1分子的表达。因此,我们提出假设,STIM1和TRPC1可能参与EPCs增殖、迁移等生物理学形为的调节,从而影响其修复损伤血管的能力。
2.方法
2.1 STIM1对EPCs修复损伤血管能力的影响
2.1.1 STIM1对EPCs增殖、迁移的作用
2.1.1.1大鼠骨髓源EPCs的分离培养和鉴定
用密度梯度离心法分离大鼠骨髓源的单个核细胞,用含20%FBS的DMEM-L培养基培养。在普通显微镜下观察细胞的形态学特征,荧光双染实验鉴定对DiI-Ac-LDL-和UEA-1均染色阳性的细胞为正在分化的EPCs。用流式细胞术鉴定EPCs表面分子CD34、CD45、CD133、及VEGFR2的表达。
2.1.1.2 STIM1对EPCs增殖、迁移的作用
分离培养5-7天的大鼠骨髓源EPCs用于实验。将STIM1干扰腺病毒质粒(Ad-si/rSTIM1)、人源STIM1表达质粒(Ad-hSTIM1)及对应的非沉默作用的对照腺病毒(NSC)转染细胞,转染48小时后用于实验。采用细胞计数法和3~H-TdR掺入法检测EPCs的增殖情况,流式细胞仪检测细胞周期分布情况,较正的boyden小室检测细胞的迁移能力。
2.1.2观察STIM1对EPCs修复损伤血管能力的影响
复制大鼠颈动脉球囊损伤模型,用2%戊巴比妥钠麻醉后,以颈前正中线为切口,暴露左颈总动脉分叉,分离颈外动脉,分别在颈外动脉近、远两侧套线,结扎其远侧。用动脉夹暂时阻断颈内和颈总动脉血流,在颈外动脉结扎近侧穿刺,继之将2F Forgarty导管沿小口送入颈总动脉大约1-2cm,以1.5 atm-2 atm充盈球囊,阻断血流约30秒,然后缓慢来回抽动球囊,反复3次,退出导管,结扎颈外动脉,常规缝合伤口。术后立即经尾静脉注入事先转染Ad-si/rSTIM1、Ad-hSTIM1和NSC并且经Dil-LDL标记的EPCs(1×10~6)。术后用青霉素预防感染。伊文氏蓝染色观察损伤后7天、14天血管再内皮化。HE染色观察损伤后14天新生内膜的增生情况。
2.2观察STIM1与TRPC1相互作用调控EPCs的钙库操纵性钙内流
免疫荧光检测TRPC1在EPCs的定位,免疫共沉淀检测STIM1与TRPC1在EPCs的相互作用。RT-PCR和Western blot检测TRPC1的表达,ELISA方法检测STIM1质粒转染后上清中IP3蛋白的表达情况。激光共聚焦检测EPCs内钙变化情况。
2.3 TRPC1对EPCs的增殖和迁移的影响及其机制探讨
分离培养5-7天的大鼠骨髓源EPCs用于实验。将TRPC1的小RNA干扰质粒(siRNA-TRPC1)及对应的非沉默作用的siRNA对照质粒(siRNA-control)、TRPC1的发夹状RNA干扰质粒(shRNA-TRPC1)及对应的非沉默作用的shRNA对照质粒(shRNA-control)转染细胞,转染48～72小时后用于实验。采用细胞计数法和3~H-TdR掺入法检测EPCs的增殖情况,流式细胞仪检测细胞周期,较正的boyden小室检测细胞的迁移能力,激光共聚焦检测EPCs内钙变化情况。用细胞周期基因芯片筛选TRPC1干扰质粒转染EPCs 48小时后细胞周期基因的变化情况,筛选TRPC1作用的下游靶点。选择芯片中变化最大的基因激活和或阻断其功能,观察在沉默TRPC1的基础上EPCs的增殖和细胞周期的变化,探讨TRPC1的下游靶点。
3.结果
3.1 STIM1对EPCs修复损伤血管能力的影响
3.1.1 STIM1对EPCs增殖、迁移的影响
3.1.1.1大鼠骨髓源EPCs的分离培养和鉴定:成功分离培养大鼠骨髓源性的EPCs,呈梭形、卵圆形、多边形,典型的呈克隆样、线样、血管环样生长。用UEA-I和Dil-Ac-LDL进行染色鉴定,激光共聚焦下观察,摄取Dil-Ac-LDL表现红色荧光,摄取UEA-I表现为蓝色荧光,同时摄取Dil-Ac-LDL和UEA-I的细胞表现黄色荧光,即为双染阳性细胞,表示正在分化的EPCs(N>90%)。通过细胞流式仪检测CD34、CD133、CD45及VEGFR2分子在分离培养5-7天的EPCs上的表达,结果显示VEGFR2 91.24%、CD133 90.58%、CD34 77.32%,而CD45的表达仅4.5%。
3.1.1.2 STIM1对EPCs增殖、迁移的影响
通过RT-PCR及Western blot方法证实在EPCs上有STIM1的表达,免疫组化提示STIM1主要定位在细胞内。用不同感染复数NSC、Ad-hSTIM1及Ad-si/rSTIM1转染EPCs,并在转染后48小时通过RT-PCR及Western blot检测STIM1的表达,结果显示,Ad-si/rSTIM1转染组(感染复数分别为10MOI和20MOI)STIM1 mRNA和蛋白的表达均较NSC组明显下降(P<0.05),而共转染Ad-si/rSTIM1(10MOI)和Ad-hSTIM1(10MOI)组,STIM1的表达恢复到NSC转染组水平。STIM1对EPCs增殖的影响: Ad-si/rSTIM1转染组3~H-TdR掺入量较NSC组显著降低(P<0.05),共转染Ad-si/rSTIM1(10MOI)和Ad-hSTIM1(10MOI)组3~H-TdR掺入量恢复到NSC转染组水平。STIM1对EPCs迁移的影响:Ad-si/rSTIM1转染组细胞的迁移数目较NSC转染组显著减少(P<0.05),共转染Ad-si/rSTIM1(10MOI)和Ad-hSTIM1(10MOI)组细胞的迁移数目恢复到NSC转染组水平。STIM1对EPCs细胞周期的分布的影响: Ad-si/rSTIM1转染组,G1期分布的细胞数目较NSC转染组显著增多,S期分布的数目显著减少(P<0.05),而共转染组与NSC组相比无显著差异(P>0.05)。
3.1.2 STIM1对EPCs修复损伤血管能力的影响:用Ad-hSTIM1、Ad-si/rSTIM1及NSC转染EPCs后,并用Dil-Ac-LDL标记,将其移植到颈动脉球襄损伤的大鼠模型,分别在7天、14天,用Evans蓝染色检测损伤血管再内皮化率,结果显示Ad-si/rSTIM1转染组再内皮化率较NSC组显著降低(P<0.05),共转染Ad-si/rSTIM1与Ad-hSTIM1组与NSC组相比无明显差别(P>0.05)。HE染色检测新生内膜厚度,结果显示:在损伤后14天,Ad-si/rSTIM1转染组内膜/中膜比值与NSC组相比显著增加(P<0.05),而共转染组与NSC组相比无显著差别(P>0.05)。
3.2 STIM1与TRPC1对EPCs的SOCE的影响
3.2.1 STIM1对EPCs的SOCE的影响
分别用NSC、Ad-si/rSTIM1及Ad-hSTIM1转染EPCs,在转染后48小时,以TG耗竭细胞内钙后,再增加外钙浓度,观察细胞内钙离子浓度变化(以测量的钙离子探针的荧光密度值表示相对钙离子浓度),取增量值加以比较。结果发现在Ad-si/rSTIM1转染组钙离子浓度的增量值相对NSC组明显减少(P<0.05),而共转染组与NSC组相比无明显差别(P>0.05)。
3.2.2 TRPC1-SOCs参与STIM1对EPCs钙内流的调节
我们通过RT-PCR、Western blot证实EPCs上有TRPC1的表达,免疫组化提示TRPC1主要定位在细胞膜。此外,免疫共沉淀检测结果显示用STIM1与TRPC1在EPCs相互作用形成复合体,且在TG的剌激下相互作用增强。
为了明确STIM1表达变化是否影响TRPC1的表达,我们检测各转染组TRPC1的表达,结果显示Ad-si/rSTIM1转染组TRPC1的表达相对NSC组下降(P<0.05)。而Ad-si/rSTIM1+ Ad-hSTIM1共转染组TRPC1的表达恢复到NSC组的水平。为了检测TRPC1的SOCs还是受体操纵性钙通道(ROC)参与STIM1对EPCs的SOCE的调节,我们用ELASA法检测各转染组IP3浓度(TRPC1-ROC受IP3激活),结果显示各转染(Ad-si/rSTIM1、Ad-si/rSTIM1+ Ad-hSTIM1及NSC)组无明显差别,间接提示TRPC1的SOCs参与STIM1对EPCs的SOCE的调节。
3.3. TRPC1对EPCs增殖、迁移的影响及机制。
3.3.1 TRPC1对EPCs增殖、迁移的影响
EPCs分别转染siRNA-TRPC1、shRNA-TRPC1及相应的阴性对照siRNA-control及shRNA-control。48小时后观察TRPC1的表达,结果显示两种不同转染质粒转染组TRPC1的表达较相应对照组均显著下降(P<0.05)。转染后各组细胞的增殖及迁移情况:在siRNA-TRPC1及shRNA-TRPC1转染组3~H-TdR掺入量较对照组显著下降(P<0.05),同时细胞计数也较对照组显著下降(P<0.05),而细胞迁移数目也较对照组显著下降(P<0.05)。我们通过流式细胞术检测TRPC1对EPCs的细胞周期的影响,结果显示:在siRNA-TRPC1及shRNA-TRPC1转染组细胞分布在G1期数目较对照组显著增多(P<0.05),而分布在S期的细胞数目较对照组显著减少(P<0.05)。转染后各组细胞的SOCE情况:在siRNA-TRPC1及shRNA-TRPC1转染组均明显抑制了EPCs的SOCE。
3.3.2 TRPC1对EPCs的细胞周期基因表达的影响
通过基因芯片检测TRPC1对EPCs的细胞周期基因表达的影响,结果发现在siRNA-TRPC1转染组相对对照组有9个基因上调,4个基因下调,上调基因包括Ak1、Brca2、Camk2b、p21、Ddit3、Inha、Slfn1、Mdm2、Prm1。下调基因包括Bcl2, Mki67, Pmp22, Ppp2r3a。其中变化最明显的基因是Slfn1(上调9.4倍)。
3.3.3 Slfn1在TRPC1对EPCs增殖中的作用
由于Slfn1是变化最明显的基因,我们通过PCR和Western blot方法也证实Slfn1在siRNA-TRPC1转染组相对对照组上调9倍左右。因此我们对Slfn1进行下一步的功能实验,在TRPC1沉默的基础上,通过Slfn1封闭肽阻断Slfn1的功能,观察对EPCs增殖和细胞周期的影响。结果发现:在TRPC1沉默的基础上,用Slfn1封闭肽阻断Slfn1功能组, 3~H-TdR掺入量及细胞计数均恢复到空白对照组水平,此外,细胞周期分布也部分恢复到空白对照组水平。由此说明Slfn1是TRPC1对EPCs作用的下游靶点。
4.结论
本研究主要结论:
4.1基因沉默STIM1负性调控EPCs修复损伤血管的能力。
4.2 STIM1与TRPC1在EPCs相互作用形成复合体,共同调节EPCs的SOCE。
4.2基因沉默TRPC1负性调控EPCs修复损伤血管的能力。
1.Background and Objective:
Endothelial cell (EC) damage is an important pathophysiological step of atherosclerosis,hypertension and restenosis following percutaneous coronary intervention (PCI) such as angioplasty and stenting. Accelerated reendothelialization effectively inhibits smooth muscle cell (SMC) migration, proliferation, and resulting neointimal formation, and is therefore of special interest with regard to prevention of the early stages of atherosclerosis and restenosis. The traditional paradigm of vascular repair is based on the proliferation and migration of pre-existing mature endothelial cells from the adjacent vasculature. Recently, there is a growing understanding that endothelial progenitor cells also contribute to this process. EPCs, which can be mobilized and recruited into injured vessels, differentionate EC, replace dysfunctional endothelium,are increasingly recognized to play a key role in the maintenance of vascular integrity and to act as“repair”cells in response to vascular injury. During re-endothelialization, the key steps regulated by various mechanisms are migration and proliferation of EPCs.However; the mechanisms by which the biological properties of EPCs are regulated remain unclear, especially with respect to those ion channels.
Calcium (Ca~(2+)) signaling is essential for a variety of cell functions, such as the regulation of proliferation, differentiation and so on. Previous study suggested that Ca~(2+) was involved to regulate the proliferation、differentiation and homing of EPCs. The effects of Ca~(2+) on cell function depend on Ca~(2+) entry induced by Ca~(2+)channels on the plasma membrane. A major Ca~(2+) entry pathway component in non-excitable cells, including EPCs,are store-operated Ca~(2+) channels (SOCs). Major components of SOCs are stromal interaction molecule 1 (STIM1), Orai and transient receptor potential canonical (TRPC) protein families. STIM1 is a sensor of SOCs that aggregates and relocates into clusters at the ER-plasma membrane junctions, where it functionally interacts with and activates plasma membrane TRPC and Orai channels, and then mediate store-operated Ca~(2+) entery (SOCE) under store depletion.
Recently, our study demonstrated that knockdown of STIM1 significantly suppressed neointimal hyperplasia by inhibiting vascular smooth cell (SMC) proliferation after vascular injury. In addition, It has been reported that an antibody to TRPC1 decreased neointimal hyperplasia after vascular injury by inhibiting SMC proliferation. Moreover, EPCs also play a crucial role on the process of re-endothelialization by reducing neointima formation after vascular injury. However, it remains unclear whether STIM1 and TRPC1 effect on EPCs function. Our previous study found that STIM1 and TRPC1 were expressed in EPCs. Therefore, we hypothesized that STIM1 and TRPC1 affect the biological properties and re-endothelialization of EPCs.
2. Methods
2.1 Effect of STIM1 on EPCs proliferation, migration and involved in the repair process after vascular injury
2.1.1 Effect of STIM1 on EPCs proliferation, migration
2.1.1.1 Isolation and characterization of rat bone marrow derived EPCs
Rat bone marrow derived (BM-derived) EPCs were isolated using density-gradient centrifugation at 1500×g for 20 min. Following purification with three washing steps, cells were resuspended in low-glucose Dulbecco’s Modified Eagle’s Medium (L-DMEM) supplemented with 10 ng/mL vascular endothelial growth factor (VEGF),20% fetal calf serum. To confirm the phenotype of EPCs, first, cells were incubated with Dil–Ac-LDL for 4 h, in addition, cells were fixed with 4% paraformaldehyde, last, and cells were incubated with FITC-labeled lectin (UEA-1) for 1 h and examined under a laser confocal scanning microscope (LCSM). Cells that stained positive for both acLDL-DiI and UEA-1 were identified as EPCs, nearly all adherent cells (90%) were positive for both markers. Additionally, antibodies against rat CD34, CD45, VEGFR-2, CD133 and the corresponding isotype control antibodies were examined by flow cytometry analysis.
2.1.1.2 Effect of STIM1 on EPCs proliferation and migration
Rat BM-derived EPCs were isolated and cultured, and EPCs between days 5 and 7 were used in vitro experiments.EPCs were transduced with Ad-si/rSTIM1, Ad-hSTIM1, non silence control Adenovirus (NSC) for 48 h and used in experiments.STIM1 mRNA and protein levels were examined by PCR and Western blot. The proliferation of EPCs was measured by [3~H]-Thymidine incorporation and cell counting. EPCs migration was determined using a modified Boyden chamber assay. Cell-cycle distribution was analyzed using fluorescence-activated cell sorting (FACS).
2.1.2 Effect of STIM1 on EPCs reendothelialization: to determin the effect of EPCs induced by STIM1 during vascular repair in vivo, angioplasty of the rat left carotid artery was constructed by using a balloon embolectomy catheter. Firstly, animals were subjected to anesthesia and surgical procedure. Secondly, we transduced Ad-si/rSTIM1, Ad-hSTIM1 and NSC into EPCs, and then EPCs were labelled with acLDL-DiI. Last, these transfected EPCs were transplanted by intravenous tail vein after induction of carotid arterial injury. Evans Blue dye was performed to measured reendothelialization at 7 and 14 day after carotid arterial injury. In addition, the neointimal formation was evaluated by staining with hematoxylin and eosin at 14 day after injury.
2.2 The interaction between STIM1 and TRPC1 regulate SOCE of EPCs
Changes in intracellular Ca~(2+) in individual cells were examined by an Anquacosmos system.The interaction between STIM1 and TRPC1 were examined by Co-immunoprecipitation. Protein levels of inositol 1, 4, 5-trisphosphate (IP3) in the cell supernatants were determined with an ELISA kit.
2.3 The role and mechanism of TRPC1 on EPCs proliferation and migration
Rat BM-derived EPCs were isolated and cultured, and EPCs between days 5 and 7 was used in vitro experiments. EPCs were transduced with siRNA-TRPC1,shRNA-TRPC1, siRNA-control and shRNA-control respectively for 48～72 h and used in experiments. TRPC1 mRNA and protein levels were examined by realtime PCR and Western blott. The proliferation of EPCs was measured by [3~H]-Thymidine incorporation and cell counting. EPCs migration was determined using a modified Boyden chamber assay. Cell-cycle distribution was analyzed using fluorescence- activated cell sorting. Changes in intracellular Ca~(2+) in individual cells were examined by an Anquacosmos system. Cell cycle gene was measured by a Cell Cycle PCR Array.
3.Result
3.1.Effect of STIM1 on EPCs proliferation、migration and reendothelialization during vascular repair process after vascular injury
3.1.1 Effect of STIM1 on EPCs proliferation、migration.
3.1.1.1 Rat BM-derived EPCs isolation and characterization
Representative BM-derived EPCs exhibited a cord-like, cluster-like, or tubular-like appearance. After 5–7 days in culture, attached EPCs were examined by flow cytometry analysis and immunofluorescence. LSCM showed that the majority of cells were dual-stained cells (N>90%), positive for both acLDL-DiI and UEA-1.Flow cytometry analysis demonstrated that the cells expressed endothelial /stem cell markers, including VEGFR-2, CD34, and CD133, but not CD45.
3.1.1.2 Effect of STIM1 on EPCs proliferation and migration
STIM1 was expressed in EPCs by semi-quantitative RT-PCR and Western blotting. Immunocytochemistry was used to further investigate the cellular location of STIM1 in EPCs, and STIM1 was found to be localized in the cytoplasm of EPCs.
Rat bone-marrow-derived EPCs were cultured for in vitro experiments. Adenovirus constructs expressing NSC, Ad-hSTIM1, and Ad-si/rSTIM1 were transfected into those EPCs. Transduction of EPCs with Ad-si/rSTIM1 at multiplicities of infection (MOI) of 10 and 20 plaque forming units (pfu)/cell effectively decreased STIM1 mRNA and protein expression at 48 h post-transduction. However, the co-transfection of Ad-hSTIM1 (MOI 10 pfu/cell) with Ad-si/rSTIM1 (MOI 10 pfu/cell) restored the expression of STIM1 both at mRNA level and protein level. Transfection of EPCs with Ad-si/rSTIM1 decreased the uptake of [3~H]-thymidine at 48 h after infection when compared with NSC. The co-transfection of Ad-hSTIM1 (MOI 10 pfu/cell) reversed the effects of STIM1 knockdown on [3~H]-thymidine uptake. In addition, EPCs were first serum starved for 24 h to obtain synchronization in G_0 phase, and then transduced with Ad-si/rSTIM1, Ad-hSTIM1 and NSC for 48 h. FACS was used to measure the cell cycle distribution. Approximately 12.8% of EPCs infected with the NSC progressed into S phase. EPCs infected with Ad-si/rSTIM1 were distributed mainly in G1 phase, and only 0.967% of cells progressed into S phase. EPCs co-infected with Ad-si/rSTIM1 and Ad-hSTIM1 approximately 10.7% of cells progressed into S phase.Last, we used the modified Boyden’s chambers to assess the effects of STIM1 on EPC migration. transfection of EPCs with Ad-si/rSTIM1 decreased the number of migrating cells significantly 48 h after infection compared with NSC-transfected cells In the si/rSTIM1 (MOI 10 pfu/cell) knockdown background, Ad-hSTIM1 (MOI 10 pfu/cell) re-expression reversed the effects of STIM1 knockdown in a number of migrating cells.
3.1.2. Effect of STIM1 on EPCs function during vascular repair process
Evans Blue dye was performed to measured reendothelialization at 7 and 14 day after vascular injury. Blue represented nonendothelialized lesions at injured vessels, white represented the reendothelialized area at uninjured vessels. At 7 and 14 day, the reendothelialized area in the Ad-si/rSTIM1-EPCs infected arteries was obviously less than that in NSC-infected groups (P<0.05). Interestingly, the reendothelialized area in the Ad-si/rSTIM1+ Ad-hSTIM1-EPCs infected arteries restored the levels of NSC-transduced groups (P> 0.05).
The neointimal formation was evaluated by staining with hematoxylin and eosin at 14 day after injury. A marked increase in the neointimal area and I/M ratio(0.50±0.01 vs 0.21±0.02, P<0.05)was shown in Ad-si/rSTIM1-EPCs group compared with NSC-transduced groups at 14 day. I/M ratio in the Ad-si/rSTIM1+ Ad-hSTIM1-EPCs transduced arteries restored the levels of NSC-transduced groups (P >0.05).
3.2 Effect of STIM1 and TRPC1 on EPCs SOCE
3.2.1 Effect of STIM1 on EPCs SOCE
We investigated the effect of STIM1 on EPCs SOCE, to activate SOCE, we depleted intracellular Ca~(2+) stores by treating cells using 1 mMol/L thapsigargin (TG) without extracellular Ca~(2+) and then adding extracellular Ca~(2+) to 2 mM. The TG-mediated SOCE may be attributed to the release of Ca~(2+) from the sarcoplasmic reticulum. The transfection of Ad-si/rSTIM1 at 48 h resulted in a marked decrease in SOCE compared to NSC group (P<0.05). However, in the Ad-si/rSTIM1 knockdown background, the co-transfection of cells with Ad-hSTIM1 reversed the effects of STIM1 knockdown on intracellular Ca~(2+) in EPCs.
3.2.2 TRPC1-SOCs participate with STIM1 in mediating SOCE of EPCs
Our results demonstrated that TRPC1 was expressed in EPCs by semi-quantitative PCR and Western blotting. At the same time, TRPC1 was localized to the plasma membrane and at intracellular sites in EPCs as determined by immunocytochemistry. Additionally, semi-quantitative RT-PCR and Western blotting were used to address whether the knockdown or re-expression of STIM1 affected the expression of TRPC1. The results demonstrated that TRPC1 mRNA and protein levels were greatly decreased 48 h after si/rSTIM1 transfection. However, Ad-hSTIM1 re-expression reversed the effects of STIM1 knockdown on TRPC1. To determine if STIM1 was associated with the TRPC1 channel in rat EPCs, a co-immunopreci -pitation study was performed. The results demonstrated that STIM1 co-precipitates with TRPC1, indicating a molecular complex had formed between STIM1 proteins and TRPC1 channels in rat EPCs. To further investigate how TRPC1 was regulated by STIM1, we tested the levels of IP3 by ELISA 48 h after NSC, Ad-hSTIM1 and Ad-si/rSTIM1 transfection. The results were not significantly different when the NSC and si/r STIM1 treatments were compared, nor were they significantly different when the si/r STIM1 and si/rSTIM1 + hSTIM1 groups were compared (P > 0.05). This indicated that the function of TRPC1-SOCs incorporated the regulation of SOCE in EPCs via STIM1.
3.3. The role and mechanism of TRPC1 on EPCs proliferation and migration
3.3.1 The role of TRPC1 on EPCs proliferation and migration
First, siRNA-TRPC1, shRNA-TRPC1, siRNA-control, and shRNA- control were transfected into the EPCs. After 48 h of transfection, TRPC1 levels were assessed by real-time RT-PCR and Western blotting 48 h post-transduction. Compared with the controls, transfection with siRNA-TRPC1 or shRNA-TRPC1 attenuated TRPC1 mRNA and protein expression significantly (P<0.05). Next, EPC proliferation was analyzed by a [3~H]-thymidine incorporation assay. The results indicated that transfection of EPCs with siRNA-TRPC1 decreased the uptake of [3~H]-thymidine post-infection when compared with the siRNA-control (877.67±13.74 vs. 1925.67±24.36, n =3, P< 0.05). Transfection of EPCs with shRNA-TRPC1 also decreased the uptake of [3~H]-thymidine compared with the shRNA-control (941.00±4.04 vs. 2058.67±52.42, P < 0.05). At the same time, we performed cell counting to further determine the proliferation of EPCs. Transfection of EPCs with either siRNA-TRPC1 or shRNA-TRPC1 significantly inhibited EPCs proliferation. We also investigated the effects of TRPC1 on EPCs migration using the modified Boyden chamber assay. We observed a notable inhibition of EPCs migration in the siRNA-TRPC1 group and shRNA-TRPC1 at 48 h post-infection compared with the control (P < 0.05). FACS was used to measure the cell cycle distribution. Approximately 16.14% of EPCs infected with the siRNA-control progressed into S phase. EPCs infected with siRNA-TRPC1 or shRNA-TRPC1 was distributed mainly in G1 phase. Approximately 2.15% of the siRNA-TRPC1 group and 2.67% of the shRNA-TRPC1 progressed into S phase.
Lastly, we measured whether silencing of TRPC1 inhibits SOCE. both the siRNA-TRPC1 group and the shRNA-TRPC1 group showed a significant downregulation in SOCE at 48 h after infection compared with the siRNA-control or shRNA-control (siRNA-TRPC1 vs. siRNA-control: 33.88±1.81 vs. 97.33±4.10; shRNA-TRPC1 vs. shRNA-control: 27.93±1.60 vs. 101.33±3.28, n=3, P < 0.05).
3.3.2 Effects of TRPC1 on EPC cell cycle gene expression
We analyzed the expression of cell cycle genes using a Cell Cycle PCR gene chips Array. Genes that were either upregulated or downregulated by at least 2-fold in siRNA-TRPC1-infected EPCs compared with the siRNA-control group (P<0.05), of these genes, 9 genes were upregulated in samples from siRNA-TRPC1-infected EPCs as compared with the siRNA-control. The upregulated genes were as follows: Ak1, Brca2, Camk2b, p21, Ddit3, Inha, Slfn1, Mdm2, and Prm1. Four genes were downregulated in siRNA-TRPC1-infected EPCs compared with the siRNA-control group. The downregulated genes were as follows: Bcl2, Mki67, Pmp22, and Ppp2r3a.
3.3.3 Impact of Slfn1 on EPC proliferation following TRPC1 silencing
To further confirm the increased expression of Slfn1 in response to TRPC1 silencing. We performed realtime RT-PCR and Western blot analysis. A 9.1-fold increase in Slfn1 mRNA levels and an 8.6-fold increase in Slfn1 protein levels were detected in siRNA-TRPC1 when compared with the siRNA-control. Next, we tested the impact of Slfn1 inhibition combined with TRPC1 silencing on EPC proliferation. To inhibit Slfn1 expression, EPCs were transfected with either siRNA-TRPC1 alone or with siRNA-TRPC1 and a Slfn1-blocking peptide. Interestingly, we found that the G1 arrest induced by TRPC1 silencing was partially reversed in the presence of the Slfn1-blocking peptide. The S-phase population was increased when EPCs is being stimulated in the presence of siRNA-TRPC1 and the Slfn1- blocking peptide,whereas the control antibody did not affect the cell cycle distribution. In addition, the proliferation of EPCs under siRNA-TRPC1 and Slfn1-blocking peptide stimulation was partially restored compared with the siRNA-TRPC1 group (n=3, P < 0.05). Lastly, the reduced uptake of [3~H]-thymidine by EPCs following TRPC1 silencing was also partially reversed by the Slfn1-blocking peptide.
4. Conclusions
4.1 knockdown of endogenous STIM1 significantly inhibited EPCs reendothelialization.
4.2 STIM1 co-precipitates with TRPC1, indicating a molecular complex, which regulate SOCE, had formed between STIM1 proteins and TRPC1 channels in rat EPCs.
4.3 knockdown of endogenous TRPC1 significantly suppressed EPCs reendothelialization.
血管内皮损伤和损伤后的不良修复是冠心病、高血压、经皮冠状动脉介入术(percutaneous coronary intervention ,PCI)后再狭窄等血管损伤性疾病发病的共同病理生理基础。如何尽早促进血管损伤后的再内皮化是防治这类疾病形成的关键策略。传统观点认为,内皮损伤的修复主要依靠临近成熟内皮的迁移和增殖,但其增殖能力有限。晚近的研究证实,骨髓和外周血中内皮祖细胞(endothelial progenitor cells ,EPCs)能归巢至损伤血管局部,分化为内皮细胞,促进血管的良性修复。EPCs的增殖、迁移等生物学行为是其修复内皮功能的基础,然而,目前调控EPCs生物学行为的机制,尤其是离子通道方面,仍不清楚。
钙离子是调节细胞生理功能比如增殖、分化等的重要物质基础。研究发现,钙离子参与调控EPCs的增殖、归巢及分化功能。钙离子对细胞功能的影响通过胞膜钙通道调控的钙内流来实现,在EPCs这种非兴奋性细胞,由于缺乏电压依赖性钙通道,主要以钙库操纵性钙通道为主(store-operated calcium channals ,SOCs), SOCs由基质交联分子1(stromal interacting molecule 1,STIM1)、Orai和瞬时受体电位通道(transient receptor potential canonical,TRPC)家族构成。STIM1在SOCs中发挥感受器作用,STIM1感受胞内钙库耗竭,激活Orai和TRPC通道,介导钙库操纵性钙内流(store-operated calcium entry ,SOCE),以补充胞内钙。
晚近的研究发现,基因沉默STIM1可通过抑制血管平滑肌细胞的增殖而抑制血管损伤后新生内膜的形成。TRPC1抗体也可抑制隐静脉平滑肌细胞增殖导致的新生内膜肥厚。而EPCs作为血管损伤修复中的一重要细胞来源,STIM1及TRPC1是否也通过影响EPCs的功能而参与血管损伤修复过程的调节,目前仍不清楚。我们的前期研究发现,EPCs上有STIM1和TRPC1分子的表达。因此,我们提出假设,STIM1和TRPC1可能参与EPCs增殖、迁移等生物理学形为的调节,从而影响其修复损伤血管的能力。
2.方法
2.1 STIM1对EPCs修复损伤血管能力的影响
2.1.1 STIM1对EPCs增殖、迁移的作用
2.1.1.1大鼠骨髓源EPCs的分离培养和鉴定
用密度梯度离心法分离大鼠骨髓源的单个核细胞,用含20%FBS的DMEM-L培养基培养。在普通显微镜下观察细胞的形态学特征,荧光双染实验鉴定对DiI-Ac-LDL-和UEA-1均染色阳性的细胞为正在分化的EPCs。用流式细胞术鉴定EPCs表面分子CD34、CD45、CD133、及VEGFR2的表达。
2.1.1.2 STIM1对EPCs增殖、迁移的作用
分离培养5-7天的大鼠骨髓源EPCs用于实验。将STIM1干扰腺病毒质粒(Ad-si/rSTIM1)、人源STIM1表达质粒(Ad-hSTIM1)及对应的非沉默作用的对照腺病毒(NSC)转染细胞,转染48小时后用于实验。采用细胞计数法和3~H-TdR掺入法检测EPCs的增殖情况,流式细胞仪检测细胞周期分布情况,较正的boyden小室检测细胞的迁移能力。
2.1.2观察STIM1对EPCs修复损伤血管能力的影响
复制大鼠颈动脉球囊损伤模型,用2%戊巴比妥钠麻醉后,以颈前正中线为切口,暴露左颈总动脉分叉,分离颈外动脉,分别在颈外动脉近、远两侧套线,结扎其远侧。用动脉夹暂时阻断颈内和颈总动脉血流,在颈外动脉结扎近侧穿刺,继之将2F Forgarty导管沿小口送入颈总动脉大约1-2cm,以1.5 atm-2 atm充盈球囊,阻断血流约30秒,然后缓慢来回抽动球囊,反复3次,退出导管,结扎颈外动脉,常规缝合伤口。术后立即经尾静脉注入事先转染Ad-si/rSTIM1、Ad-hSTIM1和NSC并且经Dil-LDL标记的EPCs(1×10~6)。术后用青霉素预防感染。伊文氏蓝染色观察损伤后7天、14天血管再内皮化。HE染色观察损伤后14天新生内膜的增生情况。
2.2观察STIM1与TRPC1相互作用调控EPCs的钙库操纵性钙内流
免疫荧光检测TRPC1在EPCs的定位,免疫共沉淀检测STIM1与TRPC1在EPCs的相互作用。RT-PCR和Western blot检测TRPC1的表达,ELISA方法检测STIM1质粒转染后上清中IP3蛋白的表达情况。激光共聚焦检测EPCs内钙变化情况。
2.3 TRPC1对EPCs的增殖和迁移的影响及其机制探讨
分离培养5-7天的大鼠骨髓源EPCs用于实验。将TRPC1的小RNA干扰质粒(siRNA-TRPC1)及对应的非沉默作用的siRNA对照质粒(siRNA-control)、TRPC1的发夹状RNA干扰质粒(shRNA-TRPC1)及对应的非沉默作用的shRNA对照质粒(shRNA-control)转染细胞,转染48～72小时后用于实验。采用细胞计数法和3~H-TdR掺入法检测EPCs的增殖情况,流式细胞仪检测细胞周期,较正的boyden小室检测细胞的迁移能力,激光共聚焦检测EPCs内钙变化情况。用细胞周期基因芯片筛选TRPC1干扰质粒转染EPCs 48小时后细胞周期基因的变化情况,筛选TRPC1作用的下游靶点。选择芯片中变化最大的基因激活和或阻断其功能,观察在沉默TRPC1的基础上EPCs的增殖和细胞周期的变化,探讨TRPC1的下游靶点。
3.结果
3.1 STIM1对EPCs修复损伤血管能力的影响
3.1.1 STIM1对EPCs增殖、迁移的影响
3.1.1.1大鼠骨髓源EPCs的分离培养和鉴定:成功分离培养大鼠骨髓源性的EPCs,呈梭形、卵圆形、多边形,典型的呈克隆样、线样、血管环样生长。用UEA-I和Dil-Ac-LDL进行染色鉴定,激光共聚焦下观察,摄取Dil-Ac-LDL表现红色荧光,摄取UEA-I表现为蓝色荧光,同时摄取Dil-Ac-LDL和UEA-I的细胞表现黄色荧光,即为双染阳性细胞,表示正在分化的EPCs(N>90%)。通过细胞流式仪检测CD34、CD133、CD45及VEGFR2分子在分离培养5-7天的EPCs上的表达,结果显示VEGFR2 91.24%、CD133 90.58%、CD34 77.32%,而CD45的表达仅4.5%。
3.1.1.2 STIM1对EPCs增殖、迁移的影响
通过RT-PCR及Western blot方法证实在EPCs上有STIM1的表达,免疫组化提示STIM1主要定位在细胞内。用不同感染复数NSC、Ad-hSTIM1及Ad-si/rSTIM1转染EPCs,并在转染后48小时通过RT-PCR及Western blot检测STIM1的表达,结果显示,Ad-si/rSTIM1转染组(感染复数分别为10MOI和20MOI)STIM1 mRNA和蛋白的表达均较NSC组明显下降(P<0.05),而共转染Ad-si/rSTIM1(10MOI)和Ad-hSTIM1(10MOI)组,STIM1的表达恢复到NSC转染组水平。STIM1对EPCs增殖的影响: Ad-si/rSTIM1转染组3~H-TdR掺入量较NSC组显著降低(P<0.05),共转染Ad-si/rSTIM1(10MOI)和Ad-hSTIM1(10MOI)组3~H-TdR掺入量恢复到NSC转染组水平。STIM1对EPCs迁移的影响:Ad-si/rSTIM1转染组细胞的迁移数目较NSC转染组显著减少(P<0.05),共转染Ad-si/rSTIM1(10MOI)和Ad-hSTIM1(10MOI)组细胞的迁移数目恢复到NSC转染组水平。STIM1对EPCs细胞周期的分布的影响: Ad-si/rSTIM1转染组,G1期分布的细胞数目较NSC转染组显著增多,S期分布的数目显著减少(P<0.05),而共转染组与NSC组相比无显著差异(P>0.05)。
3.1.2 STIM1对EPCs修复损伤血管能力的影响:用Ad-hSTIM1、Ad-si/rSTIM1及NSC转染EPCs后,并用Dil-Ac-LDL标记,将其移植到颈动脉球襄损伤的大鼠模型,分别在7天、14天,用Evans蓝染色检测损伤血管再内皮化率,结果显示Ad-si/rSTIM1转染组再内皮化率较NSC组显著降低(P<0.05),共转染Ad-si/rSTIM1与Ad-hSTIM1组与NSC组相比无明显差别(P>0.05)。HE染色检测新生内膜厚度,结果显示:在损伤后14天,Ad-si/rSTIM1转染组内膜/中膜比值与NSC组相比显著增加(P<0.05),而共转染组与NSC组相比无显著差别(P>0.05)。
3.2 STIM1与TRPC1对EPCs的SOCE的影响
3.2.1 STIM1对EPCs的SOCE的影响
分别用NSC、Ad-si/rSTIM1及Ad-hSTIM1转染EPCs,在转染后48小时,以TG耗竭细胞内钙后,再增加外钙浓度,观察细胞内钙离子浓度变化(以测量的钙离子探针的荧光密度值表示相对钙离子浓度),取增量值加以比较。结果发现在Ad-si/rSTIM1转染组钙离子浓度的增量值相对NSC组明显减少(P<0.05),而共转染组与NSC组相比无明显差别(P>0.05)。
3.2.2 TRPC1-SOCs参与STIM1对EPCs钙内流的调节
我们通过RT-PCR、Western blot证实EPCs上有TRPC1的表达,免疫组化提示TRPC1主要定位在细胞膜。此外,免疫共沉淀检测结果显示用STIM1与TRPC1在EPCs相互作用形成复合体,且在TG的剌激下相互作用增强。
为了明确STIM1表达变化是否影响TRPC1的表达,我们检测各转染组TRPC1的表达,结果显示Ad-si/rSTIM1转染组TRPC1的表达相对NSC组下降(P<0.05)。而Ad-si/rSTIM1+ Ad-hSTIM1共转染组TRPC1的表达恢复到NSC组的水平。为了检测TRPC1的SOCs还是受体操纵性钙通道(ROC)参与STIM1对EPCs的SOCE的调节,我们用ELASA法检测各转染组IP3浓度(TRPC1-ROC受IP3激活),结果显示各转染(Ad-si/rSTIM1、Ad-si/rSTIM1+ Ad-hSTIM1及NSC)组无明显差别,间接提示TRPC1的SOCs参与STIM1对EPCs的SOCE的调节。
3.3. TRPC1对EPCs增殖、迁移的影响及机制。
3.3.1 TRPC1对EPCs增殖、迁移的影响
EPCs分别转染siRNA-TRPC1、shRNA-TRPC1及相应的阴性对照siRNA-control及shRNA-control。48小时后观察TRPC1的表达,结果显示两种不同转染质粒转染组TRPC1的表达较相应对照组均显著下降(P<0.05)。转染后各组细胞的增殖及迁移情况:在siRNA-TRPC1及shRNA-TRPC1转染组3~H-TdR掺入量较对照组显著下降(P<0.05),同时细胞计数也较对照组显著下降(P<0.05),而细胞迁移数目也较对照组显著下降(P<0.05)。我们通过流式细胞术检测TRPC1对EPCs的细胞周期的影响,结果显示:在siRNA-TRPC1及shRNA-TRPC1转染组细胞分布在G1期数目较对照组显著增多(P<0.05),而分布在S期的细胞数目较对照组显著减少(P<0.05)。转染后各组细胞的SOCE情况:在siRNA-TRPC1及shRNA-TRPC1转染组均明显抑制了EPCs的SOCE。
3.3.2 TRPC1对EPCs的细胞周期基因表达的影响
通过基因芯片检测TRPC1对EPCs的细胞周期基因表达的影响,结果发现在siRNA-TRPC1转染组相对对照组有9个基因上调,4个基因下调,上调基因包括Ak1、Brca2、Camk2b、p21、Ddit3、Inha、Slfn1、Mdm2、Prm1。下调基因包括Bcl2, Mki67, Pmp22, Ppp2r3a。其中变化最明显的基因是Slfn1(上调9.4倍)。
3.3.3 Slfn1在TRPC1对EPCs增殖中的作用
由于Slfn1是变化最明显的基因,我们通过PCR和Western blot方法也证实Slfn1在siRNA-TRPC1转染组相对对照组上调9倍左右。因此我们对Slfn1进行下一步的功能实验,在TRPC1沉默的基础上,通过Slfn1封闭肽阻断Slfn1的功能,观察对EPCs增殖和细胞周期的影响。结果发现:在TRPC1沉默的基础上,用Slfn1封闭肽阻断Slfn1功能组, 3~H-TdR掺入量及细胞计数均恢复到空白对照组水平,此外,细胞周期分布也部分恢复到空白对照组水平。由此说明Slfn1是TRPC1对EPCs作用的下游靶点。
4.结论
本研究主要结论:
4.1基因沉默STIM1负性调控EPCs修复损伤血管的能力。
4.2 STIM1与TRPC1在EPCs相互作用形成复合体,共同调节EPCs的SOCE。
4.2基因沉默TRPC1负性调控EPCs修复损伤血管的能力。
1.Background and Objective:
Endothelial cell (EC) damage is an important pathophysiological step of atherosclerosis,hypertension and restenosis following percutaneous coronary intervention (PCI) such as angioplasty and stenting. Accelerated reendothelialization effectively inhibits smooth muscle cell (SMC) migration, proliferation, and resulting neointimal formation, and is therefore of special interest with regard to prevention of the early stages of atherosclerosis and restenosis. The traditional paradigm of vascular repair is based on the proliferation and migration of pre-existing mature endothelial cells from the adjacent vasculature. Recently, there is a growing understanding that endothelial progenitor cells also contribute to this process. EPCs, which can be mobilized and recruited into injured vessels, differentionate EC, replace dysfunctional endothelium,are increasingly recognized to play a key role in the maintenance of vascular integrity and to act as“repair”cells in response to vascular injury. During re-endothelialization, the key steps regulated by various mechanisms are migration and proliferation of EPCs.However; the mechanisms by which the biological properties of EPCs are regulated remain unclear, especially with respect to those ion channels.
Calcium (Ca~(2+)) signaling is essential for a variety of cell functions, such as the regulation of proliferation, differentiation and so on. Previous study suggested that Ca~(2+) was involved to regulate the proliferation、differentiation and homing of EPCs. The effects of Ca~(2+) on cell function depend on Ca~(2+) entry induced by Ca~(2+)channels on the plasma membrane. A major Ca~(2+) entry pathway component in non-excitable cells, including EPCs,are store-operated Ca~(2+) channels (SOCs). Major components of SOCs are stromal interaction molecule 1 (STIM1), Orai and transient receptor potential canonical (TRPC) protein families. STIM1 is a sensor of SOCs that aggregates and relocates into clusters at the ER-plasma membrane junctions, where it functionally interacts with and activates plasma membrane TRPC and Orai channels, and then mediate store-operated Ca~(2+) entery (SOCE) under store depletion.
Recently, our study demonstrated that knockdown of STIM1 significantly suppressed neointimal hyperplasia by inhibiting vascular smooth cell (SMC) proliferation after vascular injury. In addition, It has been reported that an antibody to TRPC1 decreased neointimal hyperplasia after vascular injury by inhibiting SMC proliferation. Moreover, EPCs also play a crucial role on the process of re-endothelialization by reducing neointima formation after vascular injury. However, it remains unclear whether STIM1 and TRPC1 effect on EPCs function. Our previous study found that STIM1 and TRPC1 were expressed in EPCs. Therefore, we hypothesized that STIM1 and TRPC1 affect the biological properties and re-endothelialization of EPCs.
2. Methods
2.1 Effect of STIM1 on EPCs proliferation, migration and involved in the repair process after vascular injury
2.1.1 Effect of STIM1 on EPCs proliferation, migration
2.1.1.1 Isolation and characterization of rat bone marrow derived EPCs
Rat bone marrow derived (BM-derived) EPCs were isolated using density-gradient centrifugation at 1500×g for 20 min. Following purification with three washing steps, cells were resuspended in low-glucose Dulbecco’s Modified Eagle’s Medium (L-DMEM) supplemented with 10 ng/mL vascular endothelial growth factor (VEGF),20% fetal calf serum. To confirm the phenotype of EPCs, first, cells were incubated with Dil–Ac-LDL for 4 h, in addition, cells were fixed with 4% paraformaldehyde, last, and cells were incubated with FITC-labeled lectin (UEA-1) for 1 h and examined under a laser confocal scanning microscope (LCSM). Cells that stained positive for both acLDL-DiI and UEA-1 were identified as EPCs, nearly all adherent cells (90%) were positive for both markers. Additionally, antibodies against rat CD34, CD45, VEGFR-2, CD133 and the corresponding isotype control antibodies were examined by flow cytometry analysis.
2.1.1.2 Effect of STIM1 on EPCs proliferation and migration
Rat BM-derived EPCs were isolated and cultured, and EPCs between days 5 and 7 were used in vitro experiments.EPCs were transduced with Ad-si/rSTIM1, Ad-hSTIM1, non silence control Adenovirus (NSC) for 48 h and used in experiments.STIM1 mRNA and protein levels were examined by PCR and Western blot. The proliferation of EPCs was measured by [3~H]-Thymidine incorporation and cell counting. EPCs migration was determined using a modified Boyden chamber assay. Cell-cycle distribution was analyzed using fluorescence-activated cell sorting (FACS).
2.1.2 Effect of STIM1 on EPCs reendothelialization: to determin the effect of EPCs induced by STIM1 during vascular repair in vivo, angioplasty of the rat left carotid artery was constructed by using a balloon embolectomy catheter. Firstly, animals were subjected to anesthesia and surgical procedure. Secondly, we transduced Ad-si/rSTIM1, Ad-hSTIM1 and NSC into EPCs, and then EPCs were labelled with acLDL-DiI. Last, these transfected EPCs were transplanted by intravenous tail vein after induction of carotid arterial injury. Evans Blue dye was performed to measured reendothelialization at 7 and 14 day after carotid arterial injury. In addition, the neointimal formation was evaluated by staining with hematoxylin and eosin at 14 day after injury.
2.2 The interaction between STIM1 and TRPC1 regulate SOCE of EPCs
Changes in intracellular Ca~(2+) in individual cells were examined by an Anquacosmos system.The interaction between STIM1 and TRPC1 were examined by Co-immunoprecipitation. Protein levels of inositol 1, 4, 5-trisphosphate (IP3) in the cell supernatants were determined with an ELISA kit.
2.3 The role and mechanism of TRPC1 on EPCs proliferation and migration
Rat BM-derived EPCs were isolated and cultured, and EPCs between days 5 and 7 was used in vitro experiments. EPCs were transduced with siRNA-TRPC1,shRNA-TRPC1, siRNA-control and shRNA-control respectively for 48～72 h and used in experiments. TRPC1 mRNA and protein levels were examined by realtime PCR and Western blott. The proliferation of EPCs was measured by [3~H]-Thymidine incorporation and cell counting. EPCs migration was determined using a modified Boyden chamber assay. Cell-cycle distribution was analyzed using fluorescence- activated cell sorting. Changes in intracellular Ca~(2+) in individual cells were examined by an Anquacosmos system. Cell cycle gene was measured by a Cell Cycle PCR Array.
3.Result
3.1.Effect of STIM1 on EPCs proliferation、migration and reendothelialization during vascular repair process after vascular injury
3.1.1 Effect of STIM1 on EPCs proliferation、migration.
3.1.1.1 Rat BM-derived EPCs isolation and characterization
Representative BM-derived EPCs exhibited a cord-like, cluster-like, or tubular-like appearance. After 5–7 days in culture, attached EPCs were examined by flow cytometry analysis and immunofluorescence. LSCM showed that the majority of cells were dual-stained cells (N>90%), positive for both acLDL-DiI and UEA-1.Flow cytometry analysis demonstrated that the cells expressed endothelial /stem cell markers, including VEGFR-2, CD34, and CD133, but not CD45.
3.1.1.2 Effect of STIM1 on EPCs proliferation and migration
STIM1 was expressed in EPCs by semi-quantitative RT-PCR and Western blotting. Immunocytochemistry was used to further investigate the cellular location of STIM1 in EPCs, and STIM1 was found to be localized in the cytoplasm of EPCs.
Rat bone-marrow-derived EPCs were cultured for in vitro experiments. Adenovirus constructs expressing NSC, Ad-hSTIM1, and Ad-si/rSTIM1 were transfected into those EPCs. Transduction of EPCs with Ad-si/rSTIM1 at multiplicities of infection (MOI) of 10 and 20 plaque forming units (pfu)/cell effectively decreased STIM1 mRNA and protein expression at 48 h post-transduction. However, the co-transfection of Ad-hSTIM1 (MOI 10 pfu/cell) with Ad-si/rSTIM1 (MOI 10 pfu/cell) restored the expression of STIM1 both at mRNA level and protein level. Transfection of EPCs with Ad-si/rSTIM1 decreased the uptake of [3~H]-thymidine at 48 h after infection when compared with NSC. The co-transfection of Ad-hSTIM1 (MOI 10 pfu/cell) reversed the effects of STIM1 knockdown on [3~H]-thymidine uptake. In addition, EPCs were first serum starved for 24 h to obtain synchronization in G_0 phase, and then transduced with Ad-si/rSTIM1, Ad-hSTIM1 and NSC for 48 h. FACS was used to measure the cell cycle distribution. Approximately 12.8% of EPCs infected with the NSC progressed into S phase. EPCs infected with Ad-si/rSTIM1 were distributed mainly in G1 phase, and only 0.967% of cells progressed into S phase. EPCs co-infected with Ad-si/rSTIM1 and Ad-hSTIM1 approximately 10.7% of cells progressed into S phase.Last, we used the modified Boyden’s chambers to assess the effects of STIM1 on EPC migration. transfection of EPCs with Ad-si/rSTIM1 decreased the number of migrating cells significantly 48 h after infection compared with NSC-transfected cells In the si/rSTIM1 (MOI 10 pfu/cell) knockdown background, Ad-hSTIM1 (MOI 10 pfu/cell) re-expression reversed the effects of STIM1 knockdown in a number of migrating cells.
3.1.2. Effect of STIM1 on EPCs function during vascular repair process
Evans Blue dye was performed to measured reendothelialization at 7 and 14 day after vascular injury. Blue represented nonendothelialized lesions at injured vessels, white represented the reendothelialized area at uninjured vessels. At 7 and 14 day, the reendothelialized area in the Ad-si/rSTIM1-EPCs infected arteries was obviously less than that in NSC-infected groups (P<0.05). Interestingly, the reendothelialized area in the Ad-si/rSTIM1+ Ad-hSTIM1-EPCs infected arteries restored the levels of NSC-transduced groups (P> 0.05).
The neointimal formation was evaluated by staining with hematoxylin and eosin at 14 day after injury. A marked increase in the neointimal area and I/M ratio(0.50±0.01 vs 0.21±0.02, P<0.05)was shown in Ad-si/rSTIM1-EPCs group compared with NSC-transduced groups at 14 day. I/M ratio in the Ad-si/rSTIM1+ Ad-hSTIM1-EPCs transduced arteries restored the levels of NSC-transduced groups (P >0.05).
3.2 Effect of STIM1 and TRPC1 on EPCs SOCE
3.2.1 Effect of STIM1 on EPCs SOCE
We investigated the effect of STIM1 on EPCs SOCE, to activate SOCE, we depleted intracellular Ca~(2+) stores by treating cells using 1 mMol/L thapsigargin (TG) without extracellular Ca~(2+) and then adding extracellular Ca~(2+) to 2 mM. The TG-mediated SOCE may be attributed to the release of Ca~(2+) from the sarcoplasmic reticulum. The transfection of Ad-si/rSTIM1 at 48 h resulted in a marked decrease in SOCE compared to NSC group (P<0.05). However, in the Ad-si/rSTIM1 knockdown background, the co-transfection of cells with Ad-hSTIM1 reversed the effects of STIM1 knockdown on intracellular Ca~(2+) in EPCs.
3.2.2 TRPC1-SOCs participate with STIM1 in mediating SOCE of EPCs
Our results demonstrated that TRPC1 was expressed in EPCs by semi-quantitative PCR and Western blotting. At the same time, TRPC1 was localized to the plasma membrane and at intracellular sites in EPCs as determined by immunocytochemistry. Additionally, semi-quantitative RT-PCR and Western blotting were used to address whether the knockdown or re-expression of STIM1 affected the expression of TRPC1. The results demonstrated that TRPC1 mRNA and protein levels were greatly decreased 48 h after si/rSTIM1 transfection. However, Ad-hSTIM1 re-expression reversed the effects of STIM1 knockdown on TRPC1. To determine if STIM1 was associated with the TRPC1 channel in rat EPCs, a co-immunopreci -pitation study was performed. The results demonstrated that STIM1 co-precipitates with TRPC1, indicating a molecular complex had formed between STIM1 proteins and TRPC1 channels in rat EPCs. To further investigate how TRPC1 was regulated by STIM1, we tested the levels of IP3 by ELISA 48 h after NSC, Ad-hSTIM1 and Ad-si/rSTIM1 transfection. The results were not significantly different when the NSC and si/r STIM1 treatments were compared, nor were they significantly different when the si/r STIM1 and si/rSTIM1 + hSTIM1 groups were compared (P > 0.05). This indicated that the function of TRPC1-SOCs incorporated the regulation of SOCE in EPCs via STIM1.
3.3. The role and mechanism of TRPC1 on EPCs proliferation and migration
3.3.1 The role of TRPC1 on EPCs proliferation and migration
First, siRNA-TRPC1, shRNA-TRPC1, siRNA-control, and shRNA- control were transfected into the EPCs. After 48 h of transfection, TRPC1 levels were assessed by real-time RT-PCR and Western blotting 48 h post-transduction. Compared with the controls, transfection with siRNA-TRPC1 or shRNA-TRPC1 attenuated TRPC1 mRNA and protein expression significantly (P<0.05). Next, EPC proliferation was analyzed by a [3~H]-thymidine incorporation assay. The results indicated that transfection of EPCs with siRNA-TRPC1 decreased the uptake of [3~H]-thymidine post-infection when compared with the siRNA-control (877.67±13.74 vs. 1925.67±24.36, n =3, P< 0.05). Transfection of EPCs with shRNA-TRPC1 also decreased the uptake of [3~H]-thymidine compared with the shRNA-control (941.00±4.04 vs. 2058.67±52.42, P < 0.05). At the same time, we performed cell counting to further determine the proliferation of EPCs. Transfection of EPCs with either siRNA-TRPC1 or shRNA-TRPC1 significantly inhibited EPCs proliferation. We also investigated the effects of TRPC1 on EPCs migration using the modified Boyden chamber assay. We observed a notable inhibition of EPCs migration in the siRNA-TRPC1 group and shRNA-TRPC1 at 48 h post-infection compared with the control (P < 0.05). FACS was used to measure the cell cycle distribution. Approximately 16.14% of EPCs infected with the siRNA-control progressed into S phase. EPCs infected with siRNA-TRPC1 or shRNA-TRPC1 was distributed mainly in G1 phase. Approximately 2.15% of the siRNA-TRPC1 group and 2.67% of the shRNA-TRPC1 progressed into S phase.
Lastly, we measured whether silencing of TRPC1 inhibits SOCE. both the siRNA-TRPC1 group and the shRNA-TRPC1 group showed a significant downregulation in SOCE at 48 h after infection compared with the siRNA-control or shRNA-control (siRNA-TRPC1 vs. siRNA-control: 33.88±1.81 vs. 97.33±4.10; shRNA-TRPC1 vs. shRNA-control: 27.93±1.60 vs. 101.33±3.28, n=3, P < 0.05).
3.3.2 Effects of TRPC1 on EPC cell cycle gene expression
We analyzed the expression of cell cycle genes using a Cell Cycle PCR gene chips Array. Genes that were either upregulated or downregulated by at least 2-fold in siRNA-TRPC1-infected EPCs compared with the siRNA-control group (P<0.05), of these genes, 9 genes were upregulated in samples from siRNA-TRPC1-infected EPCs as compared with the siRNA-control. The upregulated genes were as follows: Ak1, Brca2, Camk2b, p21, Ddit3, Inha, Slfn1, Mdm2, and Prm1. Four genes were downregulated in siRNA-TRPC1-infected EPCs compared with the siRNA-control group. The downregulated genes were as follows: Bcl2, Mki67, Pmp22, and Ppp2r3a.
3.3.3 Impact of Slfn1 on EPC proliferation following TRPC1 silencing
To further confirm the increased expression of Slfn1 in response to TRPC1 silencing. We performed realtime RT-PCR and Western blot analysis. A 9.1-fold increase in Slfn1 mRNA levels and an 8.6-fold increase in Slfn1 protein levels were detected in siRNA-TRPC1 when compared with the siRNA-control. Next, we tested the impact of Slfn1 inhibition combined with TRPC1 silencing on EPC proliferation. To inhibit Slfn1 expression, EPCs were transfected with either siRNA-TRPC1 alone or with siRNA-TRPC1 and a Slfn1-blocking peptide. Interestingly, we found that the G1 arrest induced by TRPC1 silencing was partially reversed in the presence of the Slfn1-blocking peptide. The S-phase population was increased when EPCs is being stimulated in the presence of siRNA-TRPC1 and the Slfn1- blocking peptide,whereas the control antibody did not affect the cell cycle distribution. In addition, the proliferation of EPCs under siRNA-TRPC1 and Slfn1-blocking peptide stimulation was partially restored compared with the siRNA-TRPC1 group (n=3, P < 0.05). Lastly, the reduced uptake of [3~H]-thymidine by EPCs following TRPC1 silencing was also partially reversed by the Slfn1-blocking peptide.
4. Conclusions
4.1 knockdown of endogenous STIM1 significantly inhibited EPCs reendothelialization.
4.2 STIM1 co-precipitates with TRPC1, indicating a molecular complex, which regulate SOCE, had formed between STIM1 proteins and TRPC1 channels in rat EPCs.
4.3 knockdown of endogenous TRPC1 significantly suppressed EPCs reendothelialization.
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3. Alicia S, Angelica Z, Carlos S, et al.STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: moving TRPC1 in and out of lipid rafts. Cell Calcium, 2008, 44:479-491.
4. Zeng W, Yuan JP, Kim MS, et al.STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction. Mol Cell, 2008, 32:439-448.
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1. Ramsey IS, Delling M, Clapham DE.An introduction to TRP channels. Annual Review of Physiology, 2006, 68:619-647.
2. Pani B, Ong HL, Brazer SC, et al.Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1.Proc Natl Acad Sci U S A,2009,106:20087-20092.
3. Alicia S, Angelica Z, Carlos S, et al.STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: moving TRPC1 in and out of lipid rafts. Cell Calcium, 2008, 44:479-491.
4. Zeng W, Yuan JP, Kim MS, et al.STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction. Mol Cell, 2008, 32:439-448.
5. Birnbaumer L.The TRPC class of ion channels: a critical review of their roles in slow, sustained increases in intracellμlar Ca (2+) concentrations. Annu Rev Pharmacol Toxicol, 2009, 49:395-426.
6. Maroto R, Raso A, Wood TG, et al.TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nat Cell Biol, 2005, 7:179-185.
7. Kumar B, Dreja K, Shah SS, et al.Upregμlated TRPC1 channel in vascμlar injury in vivo and its role in human neointimal hyperplasia.Circ Res,2006,98:557-563.
8. Takahashi Y, Watanabe H, Murakami M, et al.Involvement of transient receptor potential canonical 1 (TRPC1) in angiotensin II-induced vascμlar smooth muscle cell hypertrophy.Atherosclerosis,2007,195:287-296.
9. Ingueneau C, Huynh UD, Marcheix B, et al.TRPC1 is regμlated by caveolin-1 and is involved in oxidized LDL-induced apoptosis of vascμlar smooth muscle cells.J Cell Mol Med,2009,13:1620-1631.
10. Ambudkar IS, Ong HL, Liu X, et al.TRPC1: the link between functionally distinct store-operated calcium channels.Cell Calcium, 2007, 42:213-223.
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