肾上腺髓质素对血管紧张素II诱导的大鼠血管外膜成纤维细胞增殖、迁移、转化及胶原生成的影响
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摘要
研究背景和目的
血管重构在高血压、动脉粥样硬化和血管形成术后再狭窄等心血管疾病的发病中具有重要意义。血管重构广泛存在于血管的损伤应答过程中,并且涉及整个血管壁,包括血管内膜、中膜和外膜的代偿性和失代偿性改变。传统观念认为,血管损伤的主要反应在中膜,以平滑肌细胞(VSMC)增生、迁移为显著特征,因而对血管重构的研究多集中在中层平滑肌细胞上,而忽略了血管外膜改变在血管重构形成中的作用。近年的研究结果指出外膜在血管功能中并非只起支持及营养作用,它在维持血管正常功能和血管性疾病的发生中都起着重要作用。在动脉粥样硬化、高血压和血管损伤等疾病过程中,外膜成纤维细胞(AF)活化,其表型转变为肌成纤维细胞(MF),后者可向中膜和新生内膜迁移、增殖,同时胶原纤维合成增加,参与血管重塑过程。
基于血管重构是多种心血管疾病的病理生理基础,现已在治疗原发病如高血压、动脉硬化和糖尿病等的同时也注意对血管重构的防治,逆转血管重构的研究已成为血管生物学研究的热点之一。循环及血管局部的肾素-血管紧张素-醛固酮系统(RAAS)在血管病理性重构的发生发展中具有重要的作用。拮抗肾素-血管紧张素-醛固酮系统的作用将有利于改善血管病理性重构。肾上腺髓质素(ADM)是1993年从人的嗜铬细胞瘤组织中分离出的一种具有多种生物学作用的内源性活性肽,由52个氨基酸组成。ADM由二硫键形成一个分子内环状结构,与降钙素基因相关肽具有同源性,大量动物实验及临床研究显示,肾上腺髓质素在维持循环稳态和心血管疾病的防治中具有重要意义。ADM作为一种心脏保护肽,对冠心病发生、发展,高血压、心力衰竭进程,心肌梗死预后,及PTCA术后再狭窄的共同病理生理机制——心血管病理性重构起着一定的防治作用。研究表明,血管外膜不仅存在AngII受体,而且血管外膜也可表达分泌ADM,而旁分泌自分泌的ADM与循环及血管局部的肾素-血管紧张素-醛固酮之间存在相互调节的关系。AngII通过促进血管外膜成纤维细胞增殖、迁移、表型转化及细胞外基质沉积参与血管重构的发生。本研究旨在弄清心血管保护肽ADM是否以及如何通过上述几个环节来调节AngII促进的血管外膜重构的发生,为临床上预防和治疗血管重构提供新思路。
第一部分肾上腺髓质素对血管紧张素II诱导的血管外膜成纤维细胞增殖转化的影响
目的:观察肾上腺髓质素(ADM)对血管紧张素II的促成纤维细胞增殖及转化作用的影响。方法:原代培养大鼠胸主动脉外膜成纤维细胞,随机分为对照组、ADM组、AngII组,AngII加不同浓度ADM(10-9-10-7mol/L)组。用细胞计数、四氮唑蓝(MTT)比色法测定细胞增殖活性,流式细胞分析仪检测细胞周期,蛋白免疫印迹方法测定细胞内α-SM actin表达情况。结果:与对照组相比,AngII组S期和G2/M期细胞增多,MTT A值检测细胞增殖率为23.8%;ADM对基础水平的细胞周期中各期细胞构成比及细胞增殖率并无明显影响;与AngII组相比,AngII加不同浓度ADM(10-9-10-7mol/L)组S期细胞构成比明显减少,细胞增殖率分别为18.5%、14.1%、11.2%,均低于AngII组。成纤维细胞表达少量α-SM actin,经AngII (10-7mol/L)诱导后成纤维细胞转变为肌成纤维细胞,α-SM actin表达明显上调,与对照组相比差异有显著性(P<0.01)。ADM呈浓度依赖的抑制AngII诱导的α-SM actin表达。结论:ADM对血管外膜成纤维细胞的增殖及转化无明显影响,但ADM抑制AngII诱导的血管血管外膜成纤维细胞增殖及转化。
第二部分肾上腺髓质素对血管紧张素II诱导的血管外膜成纤维细胞迁移的影响及机制
目的:研究肾上腺髓质素(ADM)对血管紧张素II(AngII)诱导的大鼠胸主动脉外膜成纤维细胞迁移的影响及机制。方法:采用体外培养的大鼠胸主动脉外膜成纤维细胞,用刮伤试验测定AngII及不同浓度ADM对外膜成纤维细胞迁移的影响,用RT-PCR及Westernblotting分析不同浓度AngII及ADM干预后大鼠胸主动脉外膜成纤维细胞内骨桥蛋白mRNA及蛋白的表达。结果:在AngII (10-6 mol/L)趋化作用下,外膜成纤维细胞迁移活性较对照组显著增强,ADM可抑制AngII刺激的细胞迁移,迁移细胞数目在一定范围内随着ADM的浓度增加而减少;对照组细胞表达少量的骨桥蛋白,AngII在10-8-10-5mol/L浓度范围内均可诱导骨桥蛋白呈高表达,与对照组相比均有显著性差异。肾上腺髓质素在10-9-10-7mol/L的浓度范围内,呈剂量依赖性抑制由AngII(10-6 mol/L)刺激的骨桥蛋白表达,RT-PCR结果分析骨桥蛋白与β-actin mRNA比值,分别较AngII组降低了29%、40%、50%,差异有显著性(P<0.05);Western-blotting分析骨桥蛋白与β-actin条带光密度比值分别较AngII组降低了29%、39%、48% (P<0.05)。结论:ADM明显抑制AngII刺激的外膜成纤维细胞迁移,其机制可能与抑制AngII刺激的骨桥蛋白表达有关。
第三部分肾上腺髓质素对血管紧张素II诱导的血管外膜成纤维细胞胶原生成的影响及机制
目的:探讨肾上腺髓质素(ADM)对血管紧张素II(AngII)诱导的血管外膜成纤维细胞胶原生成的影响及机制。方法:体外培养大鼠主动脉外膜成纤维细胞,采用酶联免疫吸附法(ELISA)测定培养上清中I、III型胶原蛋白含量,用RT-PCR及Western blot法检测转化生长因子β1(TGFβ1)和基质金属蛋白酶-2(MMP-2) mRNA及蛋白的表达。结果: AngII显著增加外膜成纤维细胞培养上清中I、III型胶原蛋白含量,ADM可抑制AngII刺激的I、III型胶原蛋白合成,呈剂量依赖趋势,ADM特异性受体拮抗剂ADM22-52与AngII共同孵育则增强了AngII的促胶原生成作用,与单独AngI(I10-6mol/L)组相比较,I、III型胶原合成分别增加了38%和43%。对照组和ADM组几乎检测不到TGFβ1mRNA及蛋白表达,在AngII诱导下,TGFβ1 mRNA及蛋白表达明显上调。ADM呈剂量依赖性抑制由AngII刺激的TGFβ1mRNA及蛋白表达,与AngII组相比, ADM(10-8mol/L)组中TGFβ1 mRNA及蛋白表达分别抑制了55%和45%( P<0.01)。AngII显著降低血管外膜成纤维细胞内MMP-2 mRNA及蛋白表达,较对照组分别降低了69%、64%(P<0.01)。ADM呈剂量依赖性上调由AngII抑制的MMP-2 mRNA及蛋白表达,其中10-8mol/L ADM增加了1.04倍和0.93倍(P<0.01),10-7mol/L ADM增加了1.5倍和1.3倍(P<0.01)。结论: ADM可能通过下调细胞内TGFβ1表达和上调MMP-2表达,抑制AngII刺激的I、III型胶原蛋白合成,从而发挥有效的抗血管重构作用。
Background & Objective
Vascular remodeling plays an important role in cardiovascular diseases suce as hypertension、atherosclerosis and restenosis after percutaneous transluminal coronary angioplasty. Vascular remodeling exists widely in arterial repair processes related to vascular injury and involves in the compensatory and discompensatory changes of intima、media and adventitia. It is a traditional concept that vascular injury response mainly happened in medial,which characterized by vascular smooth muscle cell (VSMC) proliferation and migration, so the most researches about vascular remodeling concentrated on VSMC while ignored the action of vascular adventitia. Recently researches found that vascular adventitia not only has the effect of support and nutrition to vascular wall but also plays an important role in maintaining normal vascular function and occurrence of vascular diseases. After blood vessel injury ,vascular adventitial fibroblasts changed to“active”state from“quiet”state and started a series of pathophysiological processes such as proliferation、migration and secretion of collagen which aggravate vascular remodeling.
Since vascular remodeling is the pathophysiologic basis of many cardiovascular diseases, now care workers not only treat primary disease such as hypertension、atherosclorosis and diabetes mellitus but also pay attention to the prevention and therapy of vascular remodeling. How to reverse vascular remodeling have become one of the hot topic in the field of blood vessel biology. Circulating and local renin-angiotensin-aldosteron-system (RAAS)play an important role in the progression of vascular remodeling . Antagonizing the effect of RAAS will improve vascular remodeling. Adrenomedullin(ADM) is a multiple functional vasoactive peptide originally isolated from human pheochromocytoma tissue in 1993. This peptide, consisting of 52 amino acids, has one intracellular disulfide bond and shows homology with calcitonin gene-related peptide. A mass of experimental and clinical studies have shown that adrenomedullin plays an important role in maintaining circulation homeostasis and prevention and cure of cardiovascular diseases. ADM, acting as a cardiac protective peptide, has prevention and cure effect on cardiovascular negative remodeling which is the common pathophysiologic mechanism of onset and progression of coronary artery disease,the progress of hypertension, heart failure as well as restenosis after PTCA. Recent studies demonstrated that vascular adventitia not only has angiotensin II receptor but also expresses and secretes ADM. There were a interregulate relationship between paracrine/autocrine ADM and circulating and local renin-angiotensin-aldosteron-system. Angiotensin II induces vascular remodeling by stimulating fibroblast proliferation、migration、phenotypic transformation and secretion of collagen. The objectives of the current study were to determine whether ADM can depress the vascular remodeling effect of Angiotensin II by regulating fibroblast proliferation、migration、phenotypic transformation and secretion of collagen and supply a new research field for prevention and cure of vascular remodeling in clinic.
Part one Effects of adrenomedullin on angiotensin II- induced vascular adventitial fibroblast proliferation and phenotypic transformation in vitro
Objective To investigate the effects of adrenomedullin on AngII–induced proliferation and phenotypic transformation in cultured rat vascular adventitial fibroblasts. Methods Rat vascular adventitial fibroblasts were cultured in vitro and incubated with AngII、ADM or both.Cellular proliferation was determined by cell count and MTT.Cell cycle analysis was performed using flow cytomertry. The expression ofα-SM actin in adventitial fibroblasts stimualted in different conditions was measured by Western blotting. Results Compared with the control cells, AngII significantly increased the number of cells in S and G2/M phase ,and cell proliferation rate of MTT was 23.8%,wherea ADM had no such effect. Compared with the AngII group,the number of cells in S phase treated with both AngII and ADM(10-9、10-8、10-7mol/L) decreased,and the cell proliferation rate decreased by 18.5%、14.1%、11.2%,respectively. During the course of the phenotypic transformation to myofibroblast from adventitial fibroblasts induced by AngII (10-7mol/L),the expression ofα-SM actin was significantly upregulated and had a significant difference compared with the control group (P<0.01).ADM can inhibit AngII-induced expression ofα-SM actin of adventitial fibroblasts. Conclusion ADM did not influence the proliferation and phenotypic tranformation of adventitial fibroblasts alone ,but it markedly inhibited AngII-induced proliferation and phenotypic tranformation of adventitial fibroblasts.
Part two The effect and mechanism of Adrenomedullin on rat vascular adventitial fibroblasts Migration
Objective To investigate the effect and mechanism of adrenomedullin on angiotensin II–induced migration in cultured rat vascular adventitial fibroblasts. Methods Rat vascular adventitial fibroblasts were cultured in vitro.The adventitial fibroblasts migration assays were performed using the wounding assay.The expression of osteopontin(OPN) in adventitial fibroblasts stimualted in different conditions was measured by RT-PCR and Western blotting. Results Compared with the control group, AngII (10-6 mol/L) significantly stimulated adventitial fibroblasts migration, and adrenomedullin clearly inhibited AngII-induced migration of adventitial fibroblasts in a dose-dependent manner between 10-7and 10-9mol/L. There was a small expression of OPN in control group, and AngII significantly induced the expression of OPN in a dose-dependent manner between 10-7and 10-9mol/L and had a significant difference compared with the control group. ADM clearly inhibited AngII-induced the expression of OPN with dose-dependent between 10-7and 10-9mol/L,and the expression of OPN mRNA decreased by 29%、40%、50% respectively (all P<0.05) analyzed by semi-quantified RT-PCR and expression of OPN protein decreased by 29%、39%、48% respectively (all P<0.05) analyzed by Western-blot. Conclusion ADM inhibited Ang II–induced adventitial fibroblasts migration which may be resulted from decreasing the expression of osteopontin.
Part three The effect and mechanism of adrenomedullin on angiotensin II- induced collagen synthesis in vascular adventitial fibroblasts
Objective To investigate the effect and mechanism of adrenomedullin on AngII–induced collagen synthesis in cultured rat vascular adventitial fibroblasts.Methods Rat vascular adventitial fibroblasts were cultured in vitro. Type I、III collagen synthesis in adventitia cells was studied by ELISA,and the expression of TGFβ1 and MMP-2 was measured by RT-PCR and Western blotting. Results AngII significantly stimulated type I、III collagen synthesis in adventitial fibroblasts,and adrenomedullin clearly reduced the AngII-induced type I、III collagen synthesis of adventitial fibroblasts in a dose-dependent manner, wereas antagonist of specific receptor of ADM potentiated the effect of type I、III collagen synthesis induced by AngII by 38% and 43%. AngII significantly induced the expression of TGFβ1 mRNA and protein compared with the control group. ADM alone had no effect on the expression of TGFβ1,wereas ADM abolished the effect of AngII in a dose-dependent manner, reducing the expression of TGFβ1 mRNA and protein by 55% and 45% (P<0.01) in adrenomedullin(10-8mol/L)group compared with the AngII group. MMP-2 mRNA and protein expression in AngII group decreased by 69% and 64% (P<0.01) respectively compared with the control group , compared with AngII group, MMP-2 mRNA and protein expression upregulated by 1.04 and 0.9 folds (P<0.01) in adrenomedullin(10-8mol/L)group, and by 1.5 and 1.3 folds (P<0.01) in adrenomedullin(10-7mol/L)group. Conclusion ADM inhibited AngII-induced type I、III collagen synthesis of adventitial fibroblasts which may be though reducing the expression of TGFβ1 and stimulating the expression of MMP-2. ADM may play an important role in vascular remodeling as an antifibrotic factor.
血管重构在高血压、动脉粥样硬化和血管形成术后再狭窄等心血管疾病的发病中具有重要意义。血管重构广泛存在于血管的损伤应答过程中,并且涉及整个血管壁,包括血管内膜、中膜和外膜的代偿性和失代偿性改变。传统观念认为,血管损伤的主要反应在中膜,以平滑肌细胞(VSMC)增生、迁移为显著特征,因而对血管重构的研究多集中在中层平滑肌细胞上,而忽略了血管外膜改变在血管重构形成中的作用。近年的研究结果指出外膜在血管功能中并非只起支持及营养作用,它在维持血管正常功能和血管性疾病的发生中都起着重要作用。在动脉粥样硬化、高血压和血管损伤等疾病过程中,外膜成纤维细胞(AF)活化,其表型转变为肌成纤维细胞(MF),后者可向中膜和新生内膜迁移、增殖,同时胶原纤维合成增加,参与血管重塑过程。
基于血管重构是多种心血管疾病的病理生理基础,现已在治疗原发病如高血压、动脉硬化和糖尿病等的同时也注意对血管重构的防治,逆转血管重构的研究已成为血管生物学研究的热点之一。循环及血管局部的肾素-血管紧张素-醛固酮系统(RAAS)在血管病理性重构的发生发展中具有重要的作用。拮抗肾素-血管紧张素-醛固酮系统的作用将有利于改善血管病理性重构。肾上腺髓质素(ADM)是1993年从人的嗜铬细胞瘤组织中分离出的一种具有多种生物学作用的内源性活性肽,由52个氨基酸组成。ADM由二硫键形成一个分子内环状结构,与降钙素基因相关肽具有同源性,大量动物实验及临床研究显示,肾上腺髓质素在维持循环稳态和心血管疾病的防治中具有重要意义。ADM作为一种心脏保护肽,对冠心病发生、发展,高血压、心力衰竭进程,心肌梗死预后,及PTCA术后再狭窄的共同病理生理机制——心血管病理性重构起着一定的防治作用。研究表明,血管外膜不仅存在AngII受体,而且血管外膜也可表达分泌ADM,而旁分泌自分泌的ADM与循环及血管局部的肾素-血管紧张素-醛固酮之间存在相互调节的关系。AngII通过促进血管外膜成纤维细胞增殖、迁移、表型转化及细胞外基质沉积参与血管重构的发生。本研究旨在弄清心血管保护肽ADM是否以及如何通过上述几个环节来调节AngII促进的血管外膜重构的发生,为临床上预防和治疗血管重构提供新思路。
第一部分肾上腺髓质素对血管紧张素II诱导的血管外膜成纤维细胞增殖转化的影响
目的:观察肾上腺髓质素(ADM)对血管紧张素II的促成纤维细胞增殖及转化作用的影响。方法:原代培养大鼠胸主动脉外膜成纤维细胞,随机分为对照组、ADM组、AngII组,AngII加不同浓度ADM(10-9-10-7mol/L)组。用细胞计数、四氮唑蓝(MTT)比色法测定细胞增殖活性,流式细胞分析仪检测细胞周期,蛋白免疫印迹方法测定细胞内α-SM actin表达情况。结果:与对照组相比,AngII组S期和G2/M期细胞增多,MTT A值检测细胞增殖率为23.8%;ADM对基础水平的细胞周期中各期细胞构成比及细胞增殖率并无明显影响;与AngII组相比,AngII加不同浓度ADM(10-9-10-7mol/L)组S期细胞构成比明显减少,细胞增殖率分别为18.5%、14.1%、11.2%,均低于AngII组。成纤维细胞表达少量α-SM actin,经AngII (10-7mol/L)诱导后成纤维细胞转变为肌成纤维细胞,α-SM actin表达明显上调,与对照组相比差异有显著性(P<0.01)。ADM呈浓度依赖的抑制AngII诱导的α-SM actin表达。结论:ADM对血管外膜成纤维细胞的增殖及转化无明显影响,但ADM抑制AngII诱导的血管血管外膜成纤维细胞增殖及转化。
第二部分肾上腺髓质素对血管紧张素II诱导的血管外膜成纤维细胞迁移的影响及机制
目的:研究肾上腺髓质素(ADM)对血管紧张素II(AngII)诱导的大鼠胸主动脉外膜成纤维细胞迁移的影响及机制。方法:采用体外培养的大鼠胸主动脉外膜成纤维细胞,用刮伤试验测定AngII及不同浓度ADM对外膜成纤维细胞迁移的影响,用RT-PCR及Westernblotting分析不同浓度AngII及ADM干预后大鼠胸主动脉外膜成纤维细胞内骨桥蛋白mRNA及蛋白的表达。结果:在AngII (10-6 mol/L)趋化作用下,外膜成纤维细胞迁移活性较对照组显著增强,ADM可抑制AngII刺激的细胞迁移,迁移细胞数目在一定范围内随着ADM的浓度增加而减少;对照组细胞表达少量的骨桥蛋白,AngII在10-8-10-5mol/L浓度范围内均可诱导骨桥蛋白呈高表达,与对照组相比均有显著性差异。肾上腺髓质素在10-9-10-7mol/L的浓度范围内,呈剂量依赖性抑制由AngII(10-6 mol/L)刺激的骨桥蛋白表达,RT-PCR结果分析骨桥蛋白与β-actin mRNA比值,分别较AngII组降低了29%、40%、50%,差异有显著性(P<0.05);Western-blotting分析骨桥蛋白与β-actin条带光密度比值分别较AngII组降低了29%、39%、48% (P<0.05)。结论:ADM明显抑制AngII刺激的外膜成纤维细胞迁移,其机制可能与抑制AngII刺激的骨桥蛋白表达有关。
第三部分肾上腺髓质素对血管紧张素II诱导的血管外膜成纤维细胞胶原生成的影响及机制
目的:探讨肾上腺髓质素(ADM)对血管紧张素II(AngII)诱导的血管外膜成纤维细胞胶原生成的影响及机制。方法:体外培养大鼠主动脉外膜成纤维细胞,采用酶联免疫吸附法(ELISA)测定培养上清中I、III型胶原蛋白含量,用RT-PCR及Western blot法检测转化生长因子β1(TGFβ1)和基质金属蛋白酶-2(MMP-2) mRNA及蛋白的表达。结果: AngII显著增加外膜成纤维细胞培养上清中I、III型胶原蛋白含量,ADM可抑制AngII刺激的I、III型胶原蛋白合成,呈剂量依赖趋势,ADM特异性受体拮抗剂ADM22-52与AngII共同孵育则增强了AngII的促胶原生成作用,与单独AngI(I10-6mol/L)组相比较,I、III型胶原合成分别增加了38%和43%。对照组和ADM组几乎检测不到TGFβ1mRNA及蛋白表达,在AngII诱导下,TGFβ1 mRNA及蛋白表达明显上调。ADM呈剂量依赖性抑制由AngII刺激的TGFβ1mRNA及蛋白表达,与AngII组相比, ADM(10-8mol/L)组中TGFβ1 mRNA及蛋白表达分别抑制了55%和45%( P<0.01)。AngII显著降低血管外膜成纤维细胞内MMP-2 mRNA及蛋白表达,较对照组分别降低了69%、64%(P<0.01)。ADM呈剂量依赖性上调由AngII抑制的MMP-2 mRNA及蛋白表达,其中10-8mol/L ADM增加了1.04倍和0.93倍(P<0.01),10-7mol/L ADM增加了1.5倍和1.3倍(P<0.01)。结论: ADM可能通过下调细胞内TGFβ1表达和上调MMP-2表达,抑制AngII刺激的I、III型胶原蛋白合成,从而发挥有效的抗血管重构作用。
Background & Objective
Vascular remodeling plays an important role in cardiovascular diseases suce as hypertension、atherosclerosis and restenosis after percutaneous transluminal coronary angioplasty. Vascular remodeling exists widely in arterial repair processes related to vascular injury and involves in the compensatory and discompensatory changes of intima、media and adventitia. It is a traditional concept that vascular injury response mainly happened in medial,which characterized by vascular smooth muscle cell (VSMC) proliferation and migration, so the most researches about vascular remodeling concentrated on VSMC while ignored the action of vascular adventitia. Recently researches found that vascular adventitia not only has the effect of support and nutrition to vascular wall but also plays an important role in maintaining normal vascular function and occurrence of vascular diseases. After blood vessel injury ,vascular adventitial fibroblasts changed to“active”state from“quiet”state and started a series of pathophysiological processes such as proliferation、migration and secretion of collagen which aggravate vascular remodeling.
Since vascular remodeling is the pathophysiologic basis of many cardiovascular diseases, now care workers not only treat primary disease such as hypertension、atherosclorosis and diabetes mellitus but also pay attention to the prevention and therapy of vascular remodeling. How to reverse vascular remodeling have become one of the hot topic in the field of blood vessel biology. Circulating and local renin-angiotensin-aldosteron-system (RAAS)play an important role in the progression of vascular remodeling . Antagonizing the effect of RAAS will improve vascular remodeling. Adrenomedullin(ADM) is a multiple functional vasoactive peptide originally isolated from human pheochromocytoma tissue in 1993. This peptide, consisting of 52 amino acids, has one intracellular disulfide bond and shows homology with calcitonin gene-related peptide. A mass of experimental and clinical studies have shown that adrenomedullin plays an important role in maintaining circulation homeostasis and prevention and cure of cardiovascular diseases. ADM, acting as a cardiac protective peptide, has prevention and cure effect on cardiovascular negative remodeling which is the common pathophysiologic mechanism of onset and progression of coronary artery disease,the progress of hypertension, heart failure as well as restenosis after PTCA. Recent studies demonstrated that vascular adventitia not only has angiotensin II receptor but also expresses and secretes ADM. There were a interregulate relationship between paracrine/autocrine ADM and circulating and local renin-angiotensin-aldosteron-system. Angiotensin II induces vascular remodeling by stimulating fibroblast proliferation、migration、phenotypic transformation and secretion of collagen. The objectives of the current study were to determine whether ADM can depress the vascular remodeling effect of Angiotensin II by regulating fibroblast proliferation、migration、phenotypic transformation and secretion of collagen and supply a new research field for prevention and cure of vascular remodeling in clinic.
Part one Effects of adrenomedullin on angiotensin II- induced vascular adventitial fibroblast proliferation and phenotypic transformation in vitro
Objective To investigate the effects of adrenomedullin on AngII–induced proliferation and phenotypic transformation in cultured rat vascular adventitial fibroblasts. Methods Rat vascular adventitial fibroblasts were cultured in vitro and incubated with AngII、ADM or both.Cellular proliferation was determined by cell count and MTT.Cell cycle analysis was performed using flow cytomertry. The expression ofα-SM actin in adventitial fibroblasts stimualted in different conditions was measured by Western blotting. Results Compared with the control cells, AngII significantly increased the number of cells in S and G2/M phase ,and cell proliferation rate of MTT was 23.8%,wherea ADM had no such effect. Compared with the AngII group,the number of cells in S phase treated with both AngII and ADM(10-9、10-8、10-7mol/L) decreased,and the cell proliferation rate decreased by 18.5%、14.1%、11.2%,respectively. During the course of the phenotypic transformation to myofibroblast from adventitial fibroblasts induced by AngII (10-7mol/L),the expression ofα-SM actin was significantly upregulated and had a significant difference compared with the control group (P<0.01).ADM can inhibit AngII-induced expression ofα-SM actin of adventitial fibroblasts. Conclusion ADM did not influence the proliferation and phenotypic tranformation of adventitial fibroblasts alone ,but it markedly inhibited AngII-induced proliferation and phenotypic tranformation of adventitial fibroblasts.
Part two The effect and mechanism of Adrenomedullin on rat vascular adventitial fibroblasts Migration
Objective To investigate the effect and mechanism of adrenomedullin on angiotensin II–induced migration in cultured rat vascular adventitial fibroblasts. Methods Rat vascular adventitial fibroblasts were cultured in vitro.The adventitial fibroblasts migration assays were performed using the wounding assay.The expression of osteopontin(OPN) in adventitial fibroblasts stimualted in different conditions was measured by RT-PCR and Western blotting. Results Compared with the control group, AngII (10-6 mol/L) significantly stimulated adventitial fibroblasts migration, and adrenomedullin clearly inhibited AngII-induced migration of adventitial fibroblasts in a dose-dependent manner between 10-7and 10-9mol/L. There was a small expression of OPN in control group, and AngII significantly induced the expression of OPN in a dose-dependent manner between 10-7and 10-9mol/L and had a significant difference compared with the control group. ADM clearly inhibited AngII-induced the expression of OPN with dose-dependent between 10-7and 10-9mol/L,and the expression of OPN mRNA decreased by 29%、40%、50% respectively (all P<0.05) analyzed by semi-quantified RT-PCR and expression of OPN protein decreased by 29%、39%、48% respectively (all P<0.05) analyzed by Western-blot. Conclusion ADM inhibited Ang II–induced adventitial fibroblasts migration which may be resulted from decreasing the expression of osteopontin.
Part three The effect and mechanism of adrenomedullin on angiotensin II- induced collagen synthesis in vascular adventitial fibroblasts
Objective To investigate the effect and mechanism of adrenomedullin on AngII–induced collagen synthesis in cultured rat vascular adventitial fibroblasts.Methods Rat vascular adventitial fibroblasts were cultured in vitro. Type I、III collagen synthesis in adventitia cells was studied by ELISA,and the expression of TGFβ1 and MMP-2 was measured by RT-PCR and Western blotting. Results AngII significantly stimulated type I、III collagen synthesis in adventitial fibroblasts,and adrenomedullin clearly reduced the AngII-induced type I、III collagen synthesis of adventitial fibroblasts in a dose-dependent manner, wereas antagonist of specific receptor of ADM potentiated the effect of type I、III collagen synthesis induced by AngII by 38% and 43%. AngII significantly induced the expression of TGFβ1 mRNA and protein compared with the control group. ADM alone had no effect on the expression of TGFβ1,wereas ADM abolished the effect of AngII in a dose-dependent manner, reducing the expression of TGFβ1 mRNA and protein by 55% and 45% (P<0.01) in adrenomedullin(10-8mol/L)group compared with the AngII group. MMP-2 mRNA and protein expression in AngII group decreased by 69% and 64% (P<0.01) respectively compared with the control group , compared with AngII group, MMP-2 mRNA and protein expression upregulated by 1.04 and 0.9 folds (P<0.01) in adrenomedullin(10-8mol/L)group, and by 1.5 and 1.3 folds (P<0.01) in adrenomedullin(10-7mol/L)group. Conclusion ADM inhibited AngII-induced type I、III collagen synthesis of adventitial fibroblasts which may be though reducing the expression of TGFβ1 and stimulating the expression of MMP-2. ADM may play an important role in vascular remodeling as an antifibrotic factor.
引文
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