摘要
研究背景
血管损伤性疾病如动脉粥样硬化、高血压及糖尿病血管并发症的发病率正逐年升高,是仅次肿瘤的的第二大致死因素,严重威胁着人类的健康。机械、炎性等损伤因素损伤血管内皮后可诱导炎性细胞浸入、血管平滑肌细胞增殖,最终导致粥样硬化、斑块破裂及血管再狭窄,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。内皮细胞损伤后,在生长因子、化学因子、切应力刺激及炎性介质等因素的作用下,血管平滑肌细胞由血管中层向内膜迁移、增殖、合成及分泌细胞外基质等是血管不良修复、新生内膜过度增生的病理生理学基础,也是动脉粥样硬化、再狭窄等血管损伤性疾病发生、发展的重要环节。抑制血管平滑肌的迁移、增殖是防治血管损伤性疾病的重要措施之一。因此,探讨血管平滑肌迁移、增殖的自身内在机制,从而寻找抑制血管平滑肌细胞迁移、增殖的措施,将会为血管损伤性疾病的防治提供新的策略。
维持血管壁自身稳定需要血管壁细胞成分(如血管平滑肌细胞和内皮细胞)间的复杂的相互作用。已经证实,促进损伤血管的再内皮化、恢复损伤血管的内皮完整性能够有效抑制血管平滑肌细胞迁移、增殖,可抑制新生内膜的过度增生,最终可减少血管损伤性疾病的发生及抑制其发展。既往认为,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参与再生。近年来,实践证明邻近参与再生的内皮细胞丧失再生能力或修复的范围有限,不能有效修复损伤内皮。因此,探讨影响内皮损伤修复的机制对血管损伤性疾病的防治具有重要意义。
损伤血管的再内皮化能够抑制血管平滑肌细胞的迁移及增殖,进而减少新生内膜形成、促进损伤血管的良性修复。但其作用的机制目前仍未完全清楚。既往认为主要机制是二者通过自分泌和旁分泌产生多种生物活性物质而相互调节。但在动物实验和体外实验中都发现,即使阻断这些作用途径,血管内皮和平滑肌细胞之间的相互调节作用并未完全消失,这提示血管内皮和平滑肌细胞之间还存在其它的联系通路。
缝隙连接在维持组织动态平衡、调节细胞生长、发育及分化中具有重要作用。近年来研究发现,Cx在血管损伤性疾病的发生、发展过程中具有重要作用。在体外培养的大隐静脉血管中出现新生内膜过度增生,其中伴有Cx43表达上调。在动脉粥样硬化的早期斑块中Cx表达发生改变。在颈动脉损伤形成新生内膜中Cx43表达升高,而抑制Cx43的表达可抑制新生内膜的过度增生。但是,在这些过程中,Cx是通过形成缝隙连接介导的细胞间直接通讯还是通过Cx独立于缝隙连接的生物学功能来发挥作用,目前仍不清楚。另外,缝隙连接传递的信号介质目前也不清楚。
研究目的
缝隙连接介导的细胞间直接通讯可能在血管损伤修复中具有重要作用。本研究拟观察缝隙连接在内皮损伤修复、血管平滑肌细胞增殖及表型转化中的作用,并观察缝隙连接在血管损伤后新生内膜过度增生中的作用。另外,通过建立血管内皮细胞及平滑肌细胞双层共培养模型以观察缝隙连接在内皮细胞与血管平滑肌细胞相互调节中的作用,并初步探讨在内皮细胞与血管平滑肌细胞相互调节的过程中通过缝隙连接传递的信号介质及可能的机制,从而为寻找血管损伤性疾病的防治新策略提供实验依据。
研究方法
1.为了探讨内皮缝隙连接在介导细胞间通讯及在损伤血管内皮修复中的作用,本实验采用植块法培养大鼠主动脉内皮细胞,并用抗vWF免疫荧光染色及透射电镜观察来鉴定培养的大鼠主动脉内皮细胞,Annexin V/PI染色及流式细胞检测技术来检测内皮细胞凋亡,用体外血管新生试剂盒分析内皮细胞体外成管能力,细胞免疫荧光染色法检测大鼠主动脉内皮细胞缝隙连接蛋白37、40及47的表达,荧光漂白后恢复技术检测缝隙连接介导的细胞间通讯。机械划痕建立内皮损伤模型,每24h摄像一次以定量分析内皮损伤修复速度,并观察缝隙连接特异性阻断剂18α-甘草次酸(18α-GA)对内皮损伤后修复的影响。
2.为探讨缝隙连接阻断剂对大鼠血管平滑肌细胞增殖及表型转化的影响,本实验采用贴块法培养大鼠主动脉平滑肌细胞,采用抗SMα-actin免疫荧光染色鉴定平滑肌细胞,细胞免疫荧光染色法及western blot检测大鼠主动脉平滑肌细胞缝隙连接蛋白43的表达,荧光漂白后恢复技术检测缝隙连接介导的细胞间通讯,四唑盐比色法测定细胞增殖能力以及采用逆转录聚合酶链式反应检测平滑肌α-肌动蛋白的表达,并观察缝隙连接特异性阻断剂观察18α-GA对上述指标的影响。
3.为了观察缝隙连接在调节血管张力及在血管损伤修复中的作用,本实验取大鼠主动脉制成血管环分别测量在18α-GA作用前后血管环对N E和Ach反应性的变化,建立大鼠颈动脉损伤模型,给予生胃酮3mg/kg/d腹腔注射,对照组给予生理盐水腹腔注射(2ml/d),14天后处死动物,取目标血管段,用HE及DAPI-伊文思蓝染色观察新生内膜厚度,细胞免疫荧光染色法及western blot检测靶血管缝隙连接蛋白43的表达。
4.为了探讨钙离子跨缝隙连接在内皮细胞与平滑肌细胞间的传递及其意义,本实验采用前述的植块法分别培养大鼠主动脉内皮及平滑肌细胞,建立内皮细胞与平滑肌细胞双层共培养模型,并用透射电镜观察两种细胞在transwell insert膜两侧生长情况,然后3H-TdR掺入法观察平滑肌细胞的增殖情况,将细胞负载Fluo-3后,在用ETA (10-5M)和/或ETB(10-5M)受体阻断剂处理一种细胞后用ET-1(10-7M)刺激另一种细胞在激光共聚焦显微镜测定未刺激细胞内荧光强度变化,并观察18α-GA及肝素对上述变化的影响。用RT-PCR测定在上述处理中刺激内皮细胞后平滑肌细胞内c-fos及c-jun mRNA表达的变化。
主要结果
1.内皮缝隙连接在介导细胞间通讯及在损伤血管内皮修复中的作用:成功培养了大鼠主动脉内皮细胞,vWF免疫荧光染色阳性、透射电镜观察可见WP小体。在内皮细胞中缝隙连蛋白37、40及47中均有表达。荧光物质能够通过缝隙连接在相邻细胞间进行传递,孤立细胞荧光恢复率显著低于相邻细胞(5.7±0.63 % vs. 82.26±1.68%,P<0.01);而18α-GA能够抑制缝隙连接介导的细胞间通讯,18α-GA组的荧光恢复率显著低于对照组(33.58±1.73% vs. 82.26±1.68%, P<0.01)。18α-GA可抑制内皮细胞的体外成管能力,与正常对照组相比有显著性差异(4.12±0.41 vs. 2.32±0.58, P<0.01)。在50μM浓度下,流式细胞检测结果显示18α-GA并不增加内皮细胞凋亡。内皮划痕宽度在对照组与18α-GA干预组之间无显著性差异(396.57±25.32μm vs. 370.12±19.4μm, P>0.05),在内皮损伤24h后,18α-GA组划痕宽度显著大于对照组(237.38±20.40μm vs. 126.29±21.40μm, P<0.05)。内皮损伤完全愈合时间,18α-GA显著多于对照组(4.2±0.2d vs. 2.6±0.3d,P<0.05)。
2.缝隙连接阻断剂对大鼠血管平滑肌细胞增殖及表型转化的影响:成功培养了大鼠主动脉平滑肌细胞,SMα-actin免疫荧光染色阳性。大鼠血管平滑肌细胞体外培养3天后可表达缝隙连接蛋白43。荧光物质能够通过缝隙连接在相邻细胞间进行传递,孤立细胞荧光恢复率显著低于相邻细胞(7.3±0.58 % vs. 80.61±6.57 %,P<0.01);而18α-GA能够抑制缝隙连接介导的细胞间通讯,18α-GA组的荧光恢复率显著低于对照组(41.43±7.62% vs. 80.61±6.57%,P<0.05)。18α-GA并不诱导平滑肌细胞表达Cx43蛋白(0.85±0.06 vs 0.83±0.03,P>0.05),但18α-GA可抑制血管平滑肌细胞的增殖(与对照组相比,0.465±0.016 vs. 0.563±0.081,P<0.05),并且可促进平滑肌α-actin mRNA的表达(与对照组相比,1.384±0.13 vs.0.753±0.09,P<0.01)。
3.缝隙连接在调节血管张力及在血管损伤修复中的作用:单纯给予18α-GA处理血管环并未出现明显的收缩或舒张反应。在对照组中,给予去甲肾上腺素或乙酰胆碱后,血管环发生明显的收缩或舒张反应,而经18α-GA预处理后,去甲肾上腺素或乙酰胆碱引起的血管环收缩或舒张反应幅度显著降低(NE: 0.60±0.03 vs. 0.21±0.04; Ach: 0.15±0.01 vs. 0.62±0.03;P<0.05)。损伤2周生胃酮干预组血管血管新生内膜明显减轻,血管腔狭窄较单纯损伤组轻。用新生内膜细胞核计数的方法评价新生内膜形成情况显示,生胃酮干预组新生内膜细胞核数量显著低于对照组(89±28. 40 vs. 236±15.04,n=5,P<0.01)。在球囊损伤后2周后血管组织切片免疫荧光染色发现,在形成的新生内膜中免疫荧光染色发现缝隙连接蛋白Cx43表达丰富。Western blot结果显示,对照组缝隙连接蛋白Cx43的表达显著高于生胃酮干预组(0.93±0.06 vs. 0.38±0.11, n=3,P<0.01)。
4.钙离子跨缝隙连接在内皮细胞与平滑肌细胞间的传递:在共培养后第一天,EC/SMC组及SMC/SMC组平滑肌细胞3H-TdR掺入相比并无统计学差异(10900±1320 cpm/well 10430±1200, P>0.05)。第二天,EC/SMC组平滑肌细胞3H-TdR的掺入就明显小于SMC/SMC组(17200±1734 cpm/well vs. 20900±1659 cpm/well ),两者差异未达到统计学标准。到第三天这种差异更加明显,EC/SMC组平滑肌细胞3H-TdR的掺入为25800±1984 cpm/well,SMC/SMC组的则为33070±3569 cpm/well,两者相比有显著性差异(P<0.05)。在用18α-GA预处理24h后,EC/SMC共培养第三天后平滑肌细胞3H-TdR的掺入为明显高于未处理EC/SMC组,两者相比有显著性差异(30500±2650cpm/well vs. 25800±1984 cpm/well ,P<0.05)。在内皮细胞与平滑肌细胞双层共培养体系中,用ET-1刺激上层的内皮细胞,下层平滑肌细胞内游离钙浓度显著升高,与加药前相比有显著差异。加入18α-GA以阻断双层细胞间的肌内皮连接后,可抑制平滑肌细胞内游离钙浓度的升高(P<0.01)。而用肝素处理平滑肌细胞则对细胞内游离钙浓度无显著影响(P>0.05)。用ET-1刺激平滑肌细胞,下层内皮细胞内游离钙浓度显著升高,与加药前相比有显著差异。加入18α-GA以阻断双层细胞间的肌内皮连接后,可抑制平滑肌细胞内游离钙浓度的升高(P<0.01)。用肝素处理内皮细胞后可抑制细胞内游离钙浓度升高的幅度,但不能完全阻断其升高。在刺激内皮细胞后,平滑肌细胞内c-fos及c-jun mRNA表达显著增加,与未刺激内皮细胞相比有显著性差异(c-fos:0.957±0.051 vs. 0.682±0.038; c-jun:0.816±0.054 vs.0.467±0.032;P<0.01)。
全文结论
1.内皮细胞间存在缝隙连接介导的细胞间通讯,而这种通讯能够调节内皮损伤后的修复过程。
2.缝隙连接介导的细胞间直接通讯能够调节平滑肌细胞的增殖能力以及平滑肌细胞由合成型向收缩型的转化。
3.缝隙连接在生理条件下参与维持及调节血管张力,在病理条件能够促进血管损伤后新生内膜的过度增生,在血管损伤性疾病的发生、发展过程中具有重要作用。
4.钙离子能跨缝隙连接在内皮细胞与平滑肌细胞间进行流动,是协调内皮细胞与平滑肌细胞功能的重要信号介质。钙离子跨缝隙连接由内皮细胞向平滑肌细胞流动的过程中不需IP3参与,而由平滑肌细胞向内皮细胞流动的过程中需IP3参与。钙离子由内皮细胞经缝隙连接进入平滑肌细胞后能促进平滑肌细胞中c-fos及c-jun的表达。
The prevalence of vascular injury diseases such as atherosclerosis, hypertension and the vascular complication of diabetes are increasing, and become second major cause leading to death after tumor. The injury and dysfunction of endothelial cells (ECs) by mechanical removal and inflammation,which induces a cascade of proinflammatory events resulting in infiltration of monocytic cells and smooth muscle cells(SMCs) proliferation, is a initial event of pathogenesis of atherosclerosis and a major cause of adverse repair response to vascular injury. After ECs injured, under the action of many factors such as growth factors, cytokines, chemical factors, inflammatory mediators and shear stress, vascular SMCs migrate from media to intima, proliferate, synthesize and secrete extracellular matrix. These processes are pathophysiological mechanism of vascular injury adverse repair and neointimal formation, and are also primary elements involving in that vascular injury diseases develop and progress. To inhibit the migration and proliferation of vascular SMCs is of improtance to treat vascular injury diseases. However, knowledge about its mechanism of these processes is still poor. Therefore, to explore itself internal mechanism of migrating and proliferating and to find a measure of inhibiting its migration and proliferation will provide a new strategy for preventing and treating vascular injury diseases.
The maintenance of vessel wall homeostasis requires cellular interaction and coordination of the behavior of individual cells. It is established that measures to promote the reendothelization of injured vessel could effectively inhibit the migration and proliferation of vascular smooth muscle, subsequently reduce neointimal formation, and finally prevent vascular injury diseases from developing or progressing. In the past, it had been thought that endothelial repair only depended on the regeneration and migration of ECs at the edge of injured endothelium. But recently, it was proved that the adjacent ECs could not regenerate or its regenerative ability was limited and could not effectively repair the injured endothelium. Hence, to study the mechanism of endothelial wound repair is very important for preventing and treating vascular injury diseases.
Reendothelization of injured vessel could inhibit SMC migration and proliferation, subsequently reduce neointimal formation and promote the benign repair of injured vessel. But the mechanisms still remain unclear. It has been thought that ECs and SMCs could interact through bioactive substances in an autocrine and/or paracrine way. But it is found in vivo and in vitro that interaction between ECs and SMCs didn’t disappear after blocking the pathway of autocrine and/or paracrine. It suggested that other pathway exist between two type cells besides autocrine and paracrine.
Gap junctions play a key role in the maintenance of tissue homeostasis and the regulation of cellular growth, differentiation, and development. Recently, it was found that connexins is of very importance during the development and progression of vascular injury diseases. It was reported that upregulation of Cx43 protein was found in neointima of saphenous vein cultured in vitro and that the expression of Cx43 protein changed in the early stages of atherosclerosis. However, whether connexins act through the function dependent or independent on direct intercellular communication via gap junction remain unknown. And substances transferred between ECs and SMCs across gap junction are still unclear.
Objectives
Intercellular communication via gap junctions may play a key role on vascular injury repair. In this study, we attempted to explore the role of gap junctions on endothelial wound repair, the proliferation and phenotypic transition of SMCs and neointimal formation of vessel after balloon injury. Subsequently, we also tried to investigate if Ca2+ could transfer between ECs and SMCs across gap junctions during the processes of interaction of ECs and SMCs through a coculture system of ECs and SMCs. Lastly, we wanted to study a possible mechanism of Ca2+ transferred from ECs and SMCs across gap junctions would act on SMCs. And we hoped that this study could provide some experimental evidences for the strategy of preventing and treating vascular injury diseases.
Methods
1. Role of endothelial gap junctions in intercellular communication and vascular endothelial wound repair: Rat aortic ECs (RAECs) were cultured by explanted rat aortic wall tissue and were identified with cell immunofluorescence staining for vWF and transmission electron microscope. Cell immunofluorescence staining was applied to detect the expressions of connexin (Cx) 37, Cx40 and Cx47 in RAECs. Fluorescence redistribution after photobleaching (FRAP) was used to measure the communications between cells via gap junctions. Cell apoptosis was detected by Annexin V/PI staining combined with flow cytometry (FCM). The monolayer of cultured RAECs was scraped by a mechanical method and the endothelial wound healing rate was quantified by an analysis of the photographs taken every 24h after endothelial cell layers were wounded. Meanwhile, 18α-glycyrrhetinic acid (18α-GA), a specific blocker of gap junction, was administered to observe its effect on endothelial wound repair.
2. Effect of the blocker of gap junction on phenotypic transition in cultured rat vascular SMCs: Rat aortic SMCs (RASMCs) were cultured by explanted rat aortic wall tissue and identified with cell immunofluorescence staining for SMα-actin. Cell immunofluorescence staining was applied to detect the expressions of connexin (Cx) 43 in RASMCs. FRAP was used to measure the communications between cells via gap junctions. MTT and RT-PCR were used to measure the proliferative capability of RASMCs and the expression of smooth muscle (SM)α-actin respectively. Meanwhile, 18α-glycyrrhetinic acid (18α-GA), a specific blocker of gap junction, was administered to observe its effect on the contents above.
3. Role of gap junctions on the regulation of vascular tone and the repair of vascular wound: vascular rings from rat Carotid artery were made and used to compare to the changes of vascular response to NE or Ach with or without 18α-GA. The model of vascular injury was established with rat carotid balloon injury. And animals were administrated with intraperitoneal injections of carbenoxolone (3mg/kg.d) in carbenoxolone group or saline (2ml/d) in control group for 2weeks after carotid balloon injury. After 2 weeks, HE staining and DAPI-Evens blue double staining were applied to evaluate the neointimal formation of targeted vessels. And cell immunofluorescence staining and western blot were used to detect protein Cx43 expression on targeted vessels.
4. To explore the transfer of Ca2+ between endothelia cells and SMCs across gap junctions and its implication: ECs and SMCs were cultured according to the protocols above. A coculture system was made by seeding ECs and SMCs on the both side of transwell insert with millipores respectively and observed with transmission electron microscope. Then, the proliferation of SMCs was evaluated by 3H-TdR incorporation. After loading both type cells with fluo-3 and pretreating ECs with ETB receptor blocker or SMCs with ETA(10-5M) and ETB (10-5M)receptor blocker, one type cells was stimulated with ET-1 and laser confocal scanning microscope was used to observe the change of immuofluorescence intensity in unstimulated other type cells. The effect of 18α-GA or heparin on above was also observed. After stimulating ECs according to above, the expression of c-fos and c-jun mRNA in unstimulated SMCs was detected with RT-PCR.
Results
1. Role of endothelial gap junctions in intercellular communication and vascular endothelial wound repair: In cultured RAECs, immunofluorescence staining for vWF was positive and Weibel-Palade bodies were found. Cx37, Cx40 and Cx47 were all expressed in RAECs. Fluorescent dye could only be transferred between conjugated cells, and mean fluorescence recovery rate in isolated cells were significantly lower compared with that in conjugated cells (5.7±0.63 % vs. 82.26±1.68%,P<0.01). Compared with in control group, mean fluorescence recovery rate in 18α-GA group were significant lower (33.58±1.73% vs. 82.26±1.68%, P<0.01). Therefore, 18α-GA could inhibit dye transfer between conjugated cells. FCS also showed that 18α-GA at a concentration of 50μM could not increase the apoptosis of RAECs (2.044 vs. 2.156, P>0.05). The scrape widths of endothelium were similar in two groups at the time of wound. However, at 24h after endothelial wound, the scrape widths in 18α-GA group were significantly bigger than those in control group(237.38±20.40μm vs. 126.29±21.40μm, P<0.05)。The times of the wound need to reach complete recover in 18α-GA group were significantly more than those in control group(4.2±0.2d vs.2.6±0.3d,P<0.05).
2. Effect of the blocker of gap junction on phenotypic transition in cultured rat vascular SMCs: RASCMCs were cultured successfully with positive immunofluorescence staining for SMα-actin. At 3rd day after cultured, Cx43 was expressed in RASMCs. Fluorescent dye could only be transferred between conjugated cells, and mean fluorescence recovery rate in isolated cells were significantly lower compared with that in conjugated cells[(7.3±0.58 )% vs. ( 80.61±6.57 )%,P<0.01]. Compared to control group, mean fluorescence recovery rate in 18α-GA group were significant lower(41.43±7.62% vs. 80.61±6.57 % , P < 0.05). Therefore, 18α-GA could inhibit dye transfer between conjugate3.d cells. In this experiment, we found that 18α-GA couldn’t induce the expression of Cx43 protein in RASMCs(0.85±0.06 vs 0.83±0.03,P>0.05), but 18α-GA could inhibit the proliferation of RASMCs(0.465±0.016 vs. 0.563±0.081,P<0.05 vs. control group) and promote the expression of SMα-actin in cultured RASMCs(1.384±0.13 vs.0.753±0.09,P<0.01 vs. control group).
3. Role of gap junctions on the regulation of vascular tone and the repair of vascular wound: 18α-GA alone didn’t cause significantly the response of vascular rings to systole or diastole. In control group, NE or Ach could induce vascular rings to contract or relax, but 18α-GA could inhibit the contraction or relaxation of vascular rings triggered by NE or Ach(NE: 0.60±0.03 vs. 0.21±0.04; Ach: 0.15±0.01 vs. 0.62±0.03; P<0.05). 2 weeks after carotid balloon injury, carbenoxolone could significantly reduce the neointimal formation and the stenosis of blood vessel lumen. When nuclei number of neointima was used to evaluate the neointimal formation, result suggested that nuclei number of neointima in carbenoxolone group was significantly lower than that in control group((89±28. 40 vs. 236±15.04,n=5,P<0.01). The expression of Cx43 protein in neointima was abundant. Western blot result also suggested that the expression of Cx 43 in carbenoxolone group was significantly lower than that in control group(0.93±0.06 vs. 0.38±0.11, n=3,P<0.01).
4. To explore the transfer of Ca2+ between endothelia cells and SMCs across gap junctions and its implication: At first day after coculture, there was no significantly difference of 3H-TdR incorporation between EC/SMC and SMC/SMC(10900±1320 10430±1200, P>0.05). At second day, 3H-TdR incorporation in EC/SMC and SMC/SMC was 17200±1734 and 20900±1659 respectively. Although there were some difference, it didn’t reach statistical standard. At third day, there was significantly difference on 3H-TdR incorporation between EC/SMC and SMC/SMC (25800±1984 vs. 33070±3569, P<0.05). after pretreating EC/SMC cocultured for 3d with 18α-GA for 24h, 3H-TdR incorporation was significantly higher compared to EC/SMC which was not pretreated((30500±2650l vs. 25800±1984,P<0.05). In the coculture system, [Ca2+]i in SMCs increased following stimulation of endothelia cells with ET-1, and 18α-GA could inhibit the increase of [Ca2+], but heparin not. [Ca2+]i in ECs also increased following stimulation of SMCs with ET-1, and 18α-GA inhibit the increase of [Ca2+]i and heparin could inhibit the increase of [Ca2+]i partly. The expression of c-fos and c-jun mRNA in the unstimulated SMCs increased following stimulation of endothelia cells with ET-1, compared with no stimulation(c-fos:0.957±0.051 vs. 0.682±0.038; c-jun:0.816±0.054 vs.0.467±0.032;P<0.01)。.
Conclusions
1. There are intercellular communications via gap junctions among conjugated RAECs and this kind of intercellular communication might regulate the process of endothelial wound repair.
2. Intercellular communications via gap junctions could influence smooth muscle cell proliferation and phenotypic transition of SMCs from synthetic to contractile state.
3. Gap junctions participate in maintaining and regulating vascular tone under the physiological condition, and promote the neointimal formation after vascular injury under the pathological condition. Hence, gap junctions play a key role on the development and progression of vascular injury diseases.
4. Ca2+ can flow between ECs and SMCs across gap junction and is an important signal mediator coordinates the behavior of ECs and SMCs. Ca2+ flow from SMCs to ECs need the involvement of IP3, but which don’t involve in Ca2+ flow from ECs to SMCs. Ca2+ flowing into SMCs from ECs via gap junctions increase the expression of c-fos and c-jun in SMCs.
引文
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