【作者】 吴光哲；

【导师】 韩雅玲； 闫承慧；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 研究背景和目的细胞凋亡是指细胞在一定的生理或病理条件下,遵循自身的程序,自我结束生命的死亡方式。凋亡是细胞在一系列凋亡诱导因素的作用下,相关的凋亡信号通路被激活,使凋亡调节基因的表达发生改变,进而在Ca2+、Mg2+的参与下激活相关酶而启动的。近年研究发现血管平滑肌细胞(VSMCs)作为构成血管壁的重要成分,其凋亡参与了动脉粥样硬化(AS)及经皮冠状动脉介入治疗(PCI)术后再狭窄(RS)、高血压等心血管疾病的发生发展过程。VSMCs凋亡是AS和血管成形术后RS的重要病理特征,提示细胞凋亡在AS的发生和发展过程中起到了重要作用。因此,有关VSMCs凋亡的研究已经成为心血管疾病防治研究的热点,深入探讨调控VSMCs凋亡的分子机制具有重要的临床意义,可能成为预防AS进展和PCI术后RS的有效策略。E1A激活基因阻遏子(cellular repressor of E1A-activated genes,CREG)是新近发现的一种分泌型糖蛋白,在组织和细胞发育中起重要作用。已有研究表明,CREG蛋白在成熟的组织和细胞中高表达,而在未成熟的细胞如干细胞、人畸胎瘤细胞及未分化的VSMCs中呈低表达,说明CREG对于维持机体细胞和组织的成熟和稳态具有重要的作用。我室前期研究发现CREG过表达能够对抗长期血清饥饿诱导的VSMCs凋亡,而抑制CREG表达后,细胞不耐受去血清培养而发生大量凋亡。本研究以逆转录病毒载体转染CREG过表达及表达抑制的人VSMCs和球囊损伤的人胸廓内动脉(ITA)为研究对象,深入探讨CREG在调控人VSMCs凋亡中的作用及相关分子机制,以期为PCI术后RS的防治提供新的思路和治疗方法。材料和方法我们首先应用逆转录病毒载体,分别将过表达hCREG的pLNCX2-CREG和能够沉默hCREG表达的pSM2-siCREG感染体外培养的人VSMCs。通过抗性药物筛选,获得能够稳定传代的hCREG过表达细胞株(hVSMCs-CREG)和hCREG表达抑制的细胞株(hVSMCs-siCREG)。Western blot鉴定稳定转染细胞株的CREG表达,为进一步探讨CREG调控VSMCs凋亡等生物学行为提供研究平台。应用已构建的CREG过表达和表达抑制的hVSMCs,分别给予凋亡诱导剂斯托斯普林(Staurosporine,STS)和依托泊苷(Etoposide,VP-16)诱导细胞凋亡,采用Western blot、流式细胞计数和TUNEL染色等实验方法检测细胞凋亡情况和相关信号通路变化。并通过细胞瞬时转染和添加有关信号通路抑制剂进一步明确CREG调控细胞凋亡的信号转导通路。在体外实验的基础上,选择患冠心病(CAD)并行冠脉搭桥手术病人的ITA建立球囊损伤模型,在球囊损伤和外源性添加CREG蛋白干预后的不同时间点收取受损动脉段。采用苏木精-曙红染色观察CREG和细胞凋亡标志物cleaved caspase-3变化情况。Western blotting检测CREG、VSMCs分化及凋亡指标和相关信号通路的变化。并给予特异性信号通路抑制剂,进一步明确在血管组织急性损伤的情况下CREG调控细胞凋亡的信号通路。研究结果1、CREG过表达促进VSMCs分化,抑制细胞凋亡逆转录病毒介导CREG基因表达前后三种hVSMCs的形态学发生变化:pLNCX(+)/GFP稳定感染细胞(Ctl cells)呈典型的波峰波谷样生长;CREG过表达细胞(CREG-SN cells)体积变小且更加细长,易于环绕排列生长呈管腔样结构,细胞呈现典型的分化表型生长;CREG表达抑制细胞(CREG-AS cells)体积明显增大,细胞失去极性呈无序样生长。采用Western blot检测CREG-SN、Ctl和CREG-AS三种细胞的CREG表达及分泌情况。发现CREG-SN细胞胞浆中CREG的表达与Ctl细胞无明显差别,而培养上清中表达较Ctl细胞明显增加。CREG-AS细胞与前两组细胞比较,培养上清及胞浆中CREG的表达明显降低(P <0.01)。分别选择不同浓度的STS(50、100、200nmol/L)、VP-16(20、50、100μmol/L)作用于CREG-AS细胞,在不同时间点(0、4、8、12、24 h)收集细胞。Annexin V/PI双染色流式细胞分析显示:不同浓度的STS和VP-16对细胞凋亡均有作用,且其诱导细胞凋亡的效应具有时间和浓度依赖性,即随浓度增加和时间延长,细胞凋亡的效应越明显。其中STS 200nmol/L和VP-16 100μmol/L诱导细胞凋亡的效果最为明显。CREG-SN、Ctl和CREG-AS细胞分别在有效浓度的STS和VP-16诱导下,通过Western blot检测CREG、细胞分化和凋亡指标表达情况。结果发现VSMCs分化标记物SMα-actin和SM MHC的表达趋势与CREG相似,而凋亡标志物cleaved caspase-3及细胞外基质纤维连接蛋白(Fibronectin,FN)和层粘连蛋白(Laminin-1,LN)与CREG表达趋势相反。同时发现随着药物作用时间延长,在CREG表达抑制组,cleaved caspase-3的增高更明显,SMα-actin和SM MHC的减少更为突出,而CREG过表达明显抑制了细胞凋亡标志物的表达,分化指标表达水平却相对较高。Annexin-V/PI双染流式分析和TUNEL染色也发现CREG过表达明显抑制细胞凋亡。以上结果提示,CREG过表达在维持hVSMCs分化状态的同时,抑制其凋亡。2、CREG通过p38/JNK信号通路调控VSMCs凋亡三种细胞在STS和VP-16作用下,在不同时间点,采用Western blot检测细胞凋亡有关的信号通路,结果发现磷酸化的p38、JNK和Akt均有增高趋势。然后在分别给予信号通路抑制剂SB203580、SP600125和LY294002预处理的基础上,诱导细胞凋亡。通过Annexin-V/PI流式分析,发现p38/JNK信号通路参与调控了VSMCs凋亡,而PI3K?Akt信号通路无明显作用。为进一步明确p38信号通路在CREG调控VSMCs凋亡中的作用,在CREG-AS细胞中瞬时转染p38表达抑制腺病毒质粒和给予特异性p38抑制剂,通过流式分析和Western blot分析进一步明确了p38在细胞凋亡中的作用,同时也发现在p38活化的同时,进一步激活了JNK,两者在调控VSMCs凋亡中存在协同作用。3、在急性损伤的动脉模型中,CREG促进VSMCs分化,同时通过p38/JNK信号通路抑制细胞凋亡体内研究发现,在人ITA球囊损伤修复过程中CREG表达与VSMCs分化状态相关。Western blot结果显示,CREG在AS组织中低度表达,在球囊损伤后7d,CREG表达下降,到第14d和28d表达略升高,始终维持在低表达水平。VSMCs分化标记物SMα-actin和SM MHC的表达趋势与CREG相似,而细胞凋亡标记物cleaved caspase-3与CREG表达趋势相反。在给予CREG蛋白干预后血管中CREG维持在高表达水平,SMα-actin和SM MHC也明显增加并维持在高度表达水平,而cleaved caspase-3表达明显下降。免疫组织化学结果显示cleaved caspase-3在球囊损伤后28d明显增高,而在给予CREG蛋白干预后,其表达水平明显下降。HE染色也显示在球囊损伤后28d新生内膜明显增厚,而CREG蛋白干预后,明显抑制了损伤后新生内膜的形成。以上结果提示在血管组织急性损伤修复过程中CREG可能在促进VSMCs分化,抑制细胞凋亡方面发挥重要作用。Western blot也显示,在急性损伤后,尽管总的p38、JNK和Akt表达没有明显改变,但在损伤后14d磷酸化的p38、JNK和Akt表达明显增加。在外源性添加不同浓度的CREG蛋白干预后,p38、JNK和Akt活化形式的表达量明显减低。与此同时,分别给予p38、JNK和Akt信号通路抑制剂,结果显示在p38抑制剂组中磷酸化的p38和JNK表达均明显下降,在JNK抑制剂组中磷酸化形式的JNK明显减少,而p38的活化亦有所减低。同时发现p38和JNK抑制剂组中细胞凋亡指标cleaved caspase-3表达明显减少,而Akt抑制剂组无明显改变。进一步证实了p38和JNK信号通路共同参与调控急性血管损伤后的细胞凋亡,p38与JNK信号转导通路之间具有协同作用。结论体外和离体实验均证实了CREG通过p38/JNK丝裂原活化蛋白激酶信号通路调控血管平滑肌细胞凋亡,进一步阐明CREG对于临床PCI术后RS的预防可能有重要的价值。更多还原

【Abstract】 BackgroundApoptosis is a physiological cell death process involved in many pathological conditions. VSMC apoptosis is observed in the advanced atherosclerotic lesion as well as the injured blood vessel. VSMC apoptosis is a key pathologic feature of atherosclerosis and angioplasty-induced restenosis and plays an important role in determining the course of atherogenesis. Accordingly, modulating VSMC apoptosis might have applicational perspective in attenuating the progression of atherosclerotic plaques and arterial restenosis after balloon angioplasty.CREG is a recently established secreted glycoprotein that has been shown to antagonize transcription activation and cellular transformation induced by the adenovirus E1A oncoprotein. We and other studies have shown that the secreted CREG can inhibit several kinds of cellular proliferation such as VSMCs, NIH3T3 and human embryonal carcinoma cells in vitro. Previous studies also have shown that CREG is significantly upregulated at both the mRNA and protein level during the phenotypic conversion of proliferative VSMCs to quiescent differentiated ones in vitro. Furthermore, CREG is downregulated in the vascular media after balloon injury to the rat carotid artery. Adenovirus-mediated CREG overexpression in balloon-injured rabbit carotid arteries inhibits VSMCs proliferation and attenuates neointimal hyperplasia. Since CREG is highly expressed in normal adult tissues and keeps cells in a homeostatic state, the findings support the hypothesis that CREG may play a key role in protecting VSMCs from apoptosis. In this study, we used cultured human VSMCs and balloon-injuried human internal thoracic artery model to examine the effect of CREG on the apoptosis of VSMCs.Materials and methodsVSMCs were isolated from human internal thoracic artery medial smooth muscle cells. The retrovirus was added to cells with MOI (multiplicity of infection) of ~20 pfu/cell in complete medium plus polybrene (8μg/mL) for 72h (viral titer based on pLNCX-GFP). Stable cell lines were established by selection with G418 (500μg/mL) for 2 weeks. To generate CREG knockdown VSMCs, cells were infected with retrovirus expressing CREG shRNAs and selected with 6μg/mL puromycin. Cell lysates and medium were harvested and CREG expression was evaluated by Western analysis. We assessed the role of CREG on cellular apoptosis in stable VSMC lines.Human internal thoracic arteries were obtained at surgery from subjects with coronary artery disease (CAD) who underwent coronary artery bypass grafting. Vascular tissue pieces were grown in DMEM (Invitrogen) with 4 mM glutamine supplemented with 10 % fetal calf serum (FCS) and 100 U/mL penicillin and 100 U/mL streptomycin at 37℃in a humidified atmosphere of 5 % CO2 and 95 % air. The arteries were injured by a 6F Fogarty balloon embolectomy catheter (Baxter). CREG protein was continually infused into the injured artery segments. The tissues were extracted at 7, 14, 28 and 42 days after balloon injury and CREG protein treatment. The uninjured thoracic artery served as control.ResultsCREG overexpression inhibits cultured VSMCs apoptosis and enhances cellular differentiationRetroviral vector pLNCX-hCREG or pLNCX-hCREG/shRNA was used to infect cultured human VSMCs and to generate stable clones that overexpressing or knocking-down the expression of CREG (named CREG-SN cells or CREG-AS cells respectively). CREG in VSMC lysates displays different bands, possibly due to the levels of glycosylation. Meanwhile, the cells overexpressing CREG converted to a spindle shape, a characteristic of the mature VSMC phenotype when cultured in 10 %FCS. In contrast, the GFP-transduced cells (named Ctl cells) exhibited a classic“hill-and-valley”growth pattern at and those CREG knockdown cells assumed spread morphology. Western blotting revealed that cell lysates from CREG-SN was detected no obviously increase in CREG expression compared to Ctl cells, whereas the CREG protein secreted to cell medium was detected ~6-fold increase in CREG-SN cells than in Ctl cells. Meanwhile, the expression of CREG was detected a siginaficant reduction of CREG protein expression by ~80% compared to the Ctl cells both in cell medium and in cellular lysates from CREG-AS cells (P<0.01).The CREG-AS cells were treated with different concentrations of STS (50, 100 and 200 nmol/L) or VP-16 (20, 50 and 100μmol/L) for various periods (0, 4, 8, 12 and 24h). The results indicated that treatment with STS (200 nmol/L) and VP-16 (100μmol/L) for 24h greatly increased the number of apoptotic cells by flow cytometry. To observe the effect of CREG on VSMC apoptosis, three groups of cells were stimulated with STS (200 nmol/L) or VP-16 (100μmol/L) for 0,8, 12 and 24h, respectively. The amount of CREG and cleaved caspase-3 was quantified at designated time points by Western blotting. The results showed that CREG knockdown upregulated cleaved caspase-3 content, whereas CREG overexpression lowered the levels of cleaved caspase-3. We also observed that the number of cells undergoing apoptosis remarkably increased in response to STS or VP-16 provocation at different time points (0, 8 and 12h) in CREG-AS cells compared to in the Ctl cells, whereas apoptotic cells significantly reduced in CREG-SN cells compared to in the Ctl cells by annexin V/PI dual-color flow cytometry. Furthermore, the percentage of TUNEL-positive cells in CREG-AS cells with STS or VP-16 at 12h markedly increased compared to in CREG-SN cells. Western analysis also revealed that CREG overexpression increased VSMC differentiation markers SMα-actin and SM myosin heavy chains (SM MHC) content and reduced cell-associated Fibronectin(FN)and Laminin-1(LN). After STS or VP-16 provocation, the expression of CREG was relative to VSMC differentiation markers, and inversely in parallel with proliferating cell associated protein. The results suggest that CREG participates in regulating VSMC apoptosis and the maintaining differentiated VSMC phenotype.CREG attenuates the induction of apoptosis through p38/JNK MAPK pathways in cultured VSMCsAfter STS or VP-16 stimulation, although there was no obvious change in the total p38, JNK and Akt amount, the phosphorylation of p38, JNK and Akt in CREG-AS cells significantly increased by Western blotting. The data in CREG-SN cells revealed a little increase in the amount of phosphorylated p38, JNK and Akt, which were maintained at a very low level.In order to further elucidate the effect of CREG on the involvement of signal transduction pathways in VSMC apoptosis, the specific inhibitor SB203580, SP600125 and LY294002 were used. CREG-AS cells were pretreated for 30 ~ 60 min with SB203580 (10, 20 and 50μmol/L) or SP600125 (10, 20 and 50μmol/L) or LY294002 (10, 20 and 50μmol/L), then the cells were stimulated with STS or VP-16 for 12h respectively. Using flow cytometry to estimate the number of apoptotic cells, treatment with SB203580 (10μmol/L) and SP600125 (10μmol/L) significantly lowered the number of STS-induced apoptotic cells from 41.33±3.8 % to 20.39±1.5 % and 29.05±2.3 % (P<0.01), and similarly reduced in VP-16-induced apoptotic cells from 53.45±5.36 % to 26.78±3.1 % and 38.05±3.3 % (P<0.01). But pretreatment with LY294002 was not associated with an increased apoptotic index in CREG-AS cells.We transfected CREG-AS cells with the plasmids p38αCA and p38αAGF (named p38αCA-CREG-AS cells or p38AGF-CREG-AS cells,respectively). After transfection was carried out for 48h, cell lysates were harvested and phosphorylated p38 expression was evaluated by Western analysis. Furthermore, CREG-AS cells were divided into three groups according to the transfection and p38 inhibitor pretreatment or not (CREG-AS cells, p38 AGF-CREG-AS cells and SB203580-CREG-AS cells respectively). After SB203580-CREG-AS cells were treated with the specific p38 inhibitor SB203580 for 30 min, these groups were provocated by STS at designed times. As shown in these blots, p38 activation diminished in parallel with decrease in the amount of cleaved caspases-3 in p38AGF-CREG-AS cells and SB203580-CREG-AS cells. Flow cytometric analysis showed that cellular apoptosis was substantially inhibited in p38AGF-CREG-AS cells and SB203580-CREG-AS cells from 36.23±5.8 % to 17.7±3.6 % and 24.39±2.7 % at 12h (P<0.01). We also found that the AGF mutant of p38 and the specific p38 inhibitor decreased the level of phosphorylated JNK, especially in p38AGF-CREG-AS cells. These results indicate that these p38 fusion proteins are functionally active in regulating JNK activity and induced JNK phosphorylation contributes positively to VSMC apoptosis.CREG enhances VSMC differentiation and inhibits cellular apoptosis via p38/JNK MAPK pathways in balloon-injured arteryIn the present study, we observed that CREG expression closely correlated with VSMC apoptosis and cellular differentiation in vitro. To detect whether CREG might participate in cellular apoptosis and whether its expression might involved in phenotypic modulation of VSMCs in vivo, we introduced balloon injury to the human internal thoracic artery and harvested the injured arteries on days 7, 14, 28 and 42. We tested the effects of CREG on cellular apoptosis and VSMC differentiation at designed time points. Western analysis revealed that CREG was markedly downregulated in the human atherosclerotic artery. Although CREG revealed a little increase, its expression maintained at a very low level inversely correlated with cellular apoptosis as revealed by cleaved caspase-3 after vascular injury. This is also reflected by the reduction of VSMC differentiation markers SMα-actin and SM MHC in parallel with the expression of CREG after vascular injure. These data strongly suggest that CREG is associated with quiescent mature VSMCs and that CREG downregulation may facilitate cellular apoptosis and dedifferentiation. To further clarify the effects of CREG on cellular apoptosis and phenotypic modulation, treatment with CREG protein (600 ng/mL and 1200 ng/mL) significantly lowered the amount of cleaved caspase-3 and increased in the expression of SMα-actin and SM MHC in the injured artery. Immunohistochemistry also revealed that cleaved caspase-3 was markedly upregulated at the fourth week after balloon injury. Addition of CREG protein in medium abrogated the expression of cleaved caspase-3 in injured artery wall. Meanwhile, HE staining also indentified that the neointimal area was increased at the fourth week after injury. Inversely, treatment with CREG protein markedly reduced neointimal area after balloon injury. These data strongly suggest that CREG expression in the injury artery may atteuate VSMC apotosis and inhibit the neointimal formation associated with maintainence of the quiescent mature VSMCs.To test whether CREG might regulate cellular apoptosis by these signaling pathways in the injured artery, we performed Western analysis using antibodies that specifically reacts with active, phosphorylated ERK1/2, p38, JNK, Akt and so on. After 2 weeks, balloon injury to the internal thoracic artery induced significant activation of p38, JNK and Akt, not ERK1/2. Treatment with CREG protein markedly reduced the amount of phosphorylated p38, JNK and Akt as compared to the injured artery, especially in the group of treatment with CREG protein (600 ng/mL). Furthermore, the specific inhibitor SB203580 (10μmol/L), SP600125 (10μmol/L) and LY294002 (50μmol/L) were used in the balloon-injured artery for 7, 14 and 28d,respectively. Western blot showed that treatment with SB203580 and SP600125 in the injured artery significantly lowered the levels of cleaved caspase-3, and that stimulation with LY294002 did not increase the activation of caspase-3. The blots also showed that the inactivation of p38 decreased JNK phosphorylation. These results suggest that CREG-mediated inhibition of p38 and JNK activation and cooperative activities among p38 and JNK pathways are involved in mediating cellular apoptosis in the injured arterial wall.ConclusionThese results demonstrate for the first time that CREG plays a key role in modulating VSMC apoptosis by p38/JNK MAPK signaling transduction pathways in vitro and in vivo.更多还原