| Global aging has become a looming catastrophe during the21st century. Age is one of the most powerful determinants of cardiovascular risk and is associated with a number of deleterious changes in the cardiovascular system. As the first barrier of blood vessels, the endothelium plays an extremely important role in maintaining vessel homeostasis. Endothelial dysfunction, indicated as reduction in endothel ium-dependent vasorelaxation in response to vasodilators, is increasingly accepted as an early characteristic feature of cardiovascular diseases (CVDs). It is implicated in vast majority of CVDs such as primary hypertension, atherosclerosis, stroke and heart failure. Endothelial cell senescence is an important cause for endothelial dysfunction, and contributes to vascular aging. Senescent endothelial cells have been detected in human atherosclerotic tissues and in diabetic vasculopathy, suggesting that senescence of vascular cells contributes to the pathophysiology of age-related vascular diseases.Mammalian sirtuin1(SIRT1), the closest homolog of yeast sir2, functions as a NAD+-dependent class III histone deacetylase. It mediates life span extension in response to caloric restriction (CR) in many organisms, including yeast, worms, flies and possibly mammals. It is reported that SIRT1can inhibit oxidative stress-induced and high glucose-induced endothelial cell senescence in vitro. However, whether SIRT1has a protective role against age-related vascular aging in vivo remains unknown. PAI-1is a rapid and specific inhibitor of both tissue-type and urokinase-type plasminogen activators (t-PA and u-PA) and is the major inhibitor of fibrinolysis in vivo. The expression of PAI-1is not only elevated in the elderly but also significantly induced in a variety of pathologies associated with the process of aging. PAI-1has been regarded as an important marker for cardiovascular risk and a senescence-associated biomarker both in vitro and in vivo. It may play a key role in the progression of cardiovascular aging. So, we developed our hypothesis that overexpression of SIRT1in endothelium could inhibit endothelial cell senescence and ameliorate age-related vascular aging in vivo. The downregulation of PAI-1by SIRT1, at least in part, contributed to its anti-aging effect.We previously reported that SIRT expression in aortas was decreased by a high-fat diet and hyperglycemia, but increased in mice with a CR diet, implying that SIRT1might play a protective role in vascular aging. In the present study, to determine the role of SIRT1in endothel ial cell senescence and age-related vascular aging, we constructed the in vitro model of HUVEC replicative senescence and the in vivo naturally aging mouse model with wild-type (WT) mice and endothel ium-specific SIRT1transgenic (Tg-VE-STRT1) mice. Firstly we detected the expression profiles of SIRT1and senescence-associated biomarkers in senescent HUVECs and in the aortas of old WT mice with western blot and realtime RT-PCR. Results showed that SIRT1expression was dramatically decreased while senescence-associated biomarkers (PAI-1,p66Shc,Acp53,p16,I CAM-1) were increased in senescent HUVECs compared with young HUVECs. Furthermore, in the aortas of old WT mice, the expression of SIRT1was downregulated while PAI-1, p66Shc, p53and p21expressions were greatly upregulated. Using SA-β-gal staining, we detected the role of SIRT1in endothelial senescence. SIRT1RNAi in young HUVFCs promoted cell senescence. By contrast, in senescent HUVECs, SIRT1overexpression inhibited cell senescence. In naturally aging mouse model, endothelium-specific SIRT1overexpression ameliorated vascular aging as indicated by the downregulation of senescence-associated biomarkers, PAI-1, p66Shc, p53and p21in lysed aortas. Plasma PAI-1activity was also decreased in old Tg-VE-SIRT1mice compared with old WT mice. And what's more, endothelium-specifie SIRT1overexpression improved the endothelium-dependent vasorelaxation using isometric tension studies in thoracic aortic rings. In addition, by the left common carotid artery (LCCA) PWV analysis, we found that arterial stiffness was ameliorated in old Tg-VE-SIRT1mice. Then to explore the mechanism by which SIRT1exerted its protective role against vascular aging, we elucidated whether SIRT1could downregulate PAI-lexpression. Results showed that sirtuin inhibitors (NAM, suramin and sirtinol) induced PAI-1mRNA expression in293A, and SIRT1-specifie inhibitor, EX527, also induced PAI-1expression at both mRNA and protein level in HUVECs. Furthermore, SIRT1RNA1in young HUVECs increased PAI-1expression, while SIRT1overexpression in senescent HUVECs decrease PAI-1expression at both mRNA and protein levels. ChIP analysis in HUVECs showed that SIRT1bound to the specific region of PAI-1promoter (-572bp/-454bp) and EX527treatment decreased the binding of SIRT1to the PAI-1promoter. In addition, in293A cells after treatment with NAM, the binding of SIRT1to the PAI-1promoter was dramatically decreased, and the acetylation of H4K16on the PAI-1promoter was greatly increased. In contrast, the acetylation of H3K9, H3K9/K14and H4K12on the PAI-1promoter were not affected by NAM treatment.In this study, our findings indicate for the first time that endothelium-specific SIRT1overexpression ameliorates age-related vascular aging and prevents against aging-associated endothelial dysfunction and arterial stiffness, and that the downregulation of PAI-1by SIRT1through epigenetic modification, at least in part, contributes to the anti-aging effect of SIRT1.
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