Sirt1 In Vascular Smooth Muscle Cell Proliferation And Neointimal Formation In The Role And Molecular Mechanisms

Neointima formation, a process involved in vascular remodeling, is characterized by vascular smooth muscle cell proliferation, migration, apoptosis and extra cellular matrix deposition in the vascular intimal layer. Hyperplasia of vascular smooth muscle cell underlies the pathogenesis of major cardiovascular diseases, such as atherosclerosis, pulmonary hypertension, systemic hypertension, abdominal aneurysm and restenosis after angioplasty. The regulation of cell cycle mainly relies on three kinds of proteins including Cyclin-dependent kinase(CDK), Cyclin and Cyclin-dependent kinase inhibitor (CKI)。Among them, CyclinD1 is the earliest growth factor-induced gene. It forms complex with CDK4/CDK6 and determine transition of cells from G1 to S phase. The abundance of cyclin D1 is regulated transcriptionally and through the ubiquitin-proteasome pathway via protein degradation. The promoter region of the cyclin D1 gene contains multiple cis-acting elements, including binding sites for activator protein-1 (AP-1), for nuclear factorκB (NF-κB), for SP1 and so on. C-Fos and c-Jun, the activated immediate-early genes induced by serum, their roles on CyclinD1 regulation are well-established.SIRT1 (SIRTuin1), which belongs to classⅢhistone deacetylase (HDAC), is the closest homology to yeast Sir2 in the seven members of human classⅢHDAC. As a deacetylase, SIRT1 plays an essential role in embryonic development, differentiation, metabolic regulation, apoptosis, DNA damage repair and stress resistance through deacetylating a broad array of targets, including histones (H3,H4), many important transcription factors and transcription co-activators such as MyoD, p300, PGC-1α, PPARγ, NF-κB, Ku70, p53, FOXOs, E2F1 and so on. It is also found that SIRT1 could directly regulate cell cycle and cell growth in many kinds of cells. Thus, we hypothesize that SIRT1 may act as a proliferation suppressor in VSMCs, and in the way ameliorating vascular proliferate diseases.Using RT-PCR and Western blot, we examined the expression of SIRT1 in VSMCs incubated with serum and mouse ligated carotid arteries. Following that, SMC specific SIRT1 transgenic mouse was identified by RT-PCR, Southern blot, Western blot and immunohistochemistry. Neointima formation induced by carotid artery ligation was then observed by HE staining, immunohistochemistry, Masson staining and TUNEL assay. Furthermore, H3-TdR assay and FCM were used to detect the in vitro effects of SIRT1 on VSMCs proliferation. After over-expression and RNAi of SIRT1, expression of cell cycle proteins which functions in G1 state was examined by Western blot and RT-PCR. At last, the hypothesis whether SIRT1 fulfills its regulation on CyclinDl expression through c-Fos and c-Jun was confirmed by luciferase reporter assay and ChIP.We found that the expression of SIRT1 in VSMC increased first then decreased after serum stimulation. Similar results were also observed within neointima formation after carotid artery ligation. Moreover, neointima formation and ECM deposition were significantly suppressed in SMC-specific SIRT1 transgenic mice compared with wild type mice. However, there were no remarkable difference on VSMC apoptosis between this two kinds of mice. Furthermore, over-expression of SIRT1 inhibited VSMC proliferation and led to an arrest of the cells in phase G1. In addition, SIRT1 decreased the expression of CyclinD1, a G1 phase regulatory switch both in vitro and in vivo. The fact that SIRT1 decreased the enrichment of c-Fos and c-Jun on CyclinDl promoter might be an attractive explanation.To our knowledge, this report demonstrates for the first time that SIRT1 significantly decreases within neointima formation after carotid artery ligation. SIRT1 decreases CyclinD1 expression in ligated carotid artery, which might sustain the reason for remarkably suppression of neointima formation in SMC-SIRT1 Tg mice. SIRT1 decreases the enrichment of c-Fos and c-Jun on CyclinDl promoter, which leads to the reduce of CyclinD1 expression. Thus, SIRT1 makes G1 arrest in the cell cycle and then inhibits VSMC proliferation. In conclusion, SIRT1 might be a new target for ameliorating the impairment of inappropriate vascular proliferation diseases.