| Background & ObjectiveVascular 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 vitroObjective 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 MigrationObjective 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 fibroblastsObjective 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.
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