Novel targets of rapamycin and Akt in vascular smooth muscle cell differentiation

描述（申请人提供）：药物洗脱支架通过在很大程度上预防再狭窄，彻底改变了冠状动脉病变的血运重建。重要的是，新出现的长期安全性数据表明，雷帕霉素洗脱支架造成晚期血栓形成的风险升高，可能是由于内皮愈合不完全。因此，理想的支架药物将选择性地抑制血管平滑肌细胞（VSMC）的增殖并促进其分化，而不抑制再内皮化。在我们对VSMC表型调节（血管生成、动脉粥样硬化和再狭窄所必需的过程）的分子机制的研究中，我们发现mTOR抑制剂雷帕霉素通过诱导包括收缩蛋白在内的新的基因表达程序来促进VSMC分化。我们已经发现，雷帕霉素抑制mTOR效应子S6 K1，以及由此产生的Akt 2激活是这种效应所必需的。令人惊讶的是，Akt 1抑制VSMC分化。我们还发现雷帕霉素激活VSMC转录因子加塔-6，并且该因子是雷帕霉素诱导分化所必需的。该转录因子对VSMC具有特异性，在内皮细胞中未发现。我们发现，雷帕霉素也诱导表达的主调节转录辅激活因子myocardin，促进VSMC分化。众所周知，mTOR通路调节蛋白质合成。值得注意的是，我们确定了VSMC特异性转录的调节作为该途径的新功能。我们推测雷帕霉素通过Akt 2调节转录因子是一种重要的机制，通过这种机制，该药物抑制增殖并促进VSMC分化。我们的目的是了解雷帕霉素调节VSMC中促分化转录因子的机制。(1)在解决这一假设，我们的目标是确定雷帕霉素激活Akt 2调节加塔-6。我们假设雷帕霉素诱导加塔-6磷酸化，导致其核转位和激活。我们将使用siRNA、来自Akt 1或Akt 2敲除小鼠的VSMC和加塔-6磷酸化位点突变体来确定哪种激酶磷酸化加塔-6，以及磷酸化如何影响加塔-6活性。我们假设磷酸模拟突变体加塔-6可能具有组成性活性，因此可能是一种潜在的VSMC特异性促分化治疗剂。(2)我们的目的是使用siRNA方法来确定myocardin在雷帕霉素诱导的表达中的作用。我们将使用DNA结合、染色质免疫沉淀和启动子报告方法来确定雷帕霉素是否以及如何促进myocardin的表达和/或活性。(3)我们将使用野生型、Akt 1或Akt 2敲除小鼠的损伤模型，确定Akt 2缺失是否加重内膜增生，Akt 1缺失是否减少内膜增生，以及对雷帕霉素的治疗反应是否需要Akt 2。我们认为，了解雷帕霉素的这些下游靶点及其调控的分子机制，将为开发改进的支架疗法提供关键靶点。 心血管疾病是西方世界发病率和死亡率的主要原因。虽然涂有药物雷帕霉素的支架大大降低了冠状动脉血管成形术后再狭窄（血管再阻塞）的风险，但最近的研究结果表明，它们会带来很小但很大的心脏病发作或死亡风险。该项目旨在了解血管平滑肌细胞对雷帕霉素的有益抗再狭窄反应的分子机制，因为这一知识可以使我们能够定制未来的治疗方法来特异性抑制这些细胞，避免对其他细胞类型的有害副作用，从而导致危险的并发症。

DESCRIPTION (provided by applicant): Drug-eluting stents have revolutionized revascularization of coronary artery lesions by largely preventing restenosis. Importantly, emerging long term safety data suggests that rapamycin-eluting stents pose an elevated risk of late thrombosis, likely due to incomplete healing of the endothelium. An ideal stent drug would, therefore, selectively inhibit proliferation and promote differentiation of vascular smooth muscle cells (VSMC), without inhibiting re-endothelialization. In our studies of the molecular mechanisms underlying VSMC phenotypic modulation, a process necessary for angiogenesis, atherosclerosis, and restenosis, we have discovered that the mTOR inhibitor rapamycin promotes VSMC differentiation by inducing a new program of gene expression, including contractile proteins. We have found that rapamycin inhibition of the mTOR effector S6K1, and the resulting activation of Akt2 is necessary for this effect. Surprisingly, Akt1 inhibited VSMC differentiation. We have also made the exciting discovery that rapamycin activates a VSMC transcription factor, GATA-6, and that this factor is necessary for rapamycin-induced differentiation. This transcription factor is specific to VSMC and not found in endothelial cells. We find that rapamycin also induces expression of the master regulatory transcriptional coactivator myocardin that promotes VSMC differentiation. The mTOR pathway is well known to regulate protein synthesis. Notably, we identify regulation of VSMC-specific transcription as a novel function for this pathway. We hypothesize that rapamycin regulation of transcription factors via Akt2 is a critical mechanism by which this drug inhibits proliferation and promotes differentiation in VSMC. We aim to understand the mechanisms by which rapamycin regulates prodifferentiation transcription factors in VSMC. (1) In addressing this hypothesis, we aim to determine how rapamycin activation of Akt2 regulates GATA-6. We hypothesize that rapamycin induces phosphorylation of GATA-6 that leads to its nuclear translocation and activation. We will use siRNA, VSMC from Akt1 or Akt2 knockout mice, and GATA-6 phosphorylation site mutants to determine which kinase phosphorylates GATA-6, and how phosphorylation influences GATA-6 activity. We hypothesize that a phospho-mimetic mutant GATA-6 may be constitutively active, and therefore a potential VSMC-specific prodifferentiation therapeutic. (2) We aim to use siRNA methods to determine the role of myocardin in rapamycin-induced expression. We will determine whether and how rapamycin promotes myocardin expression and/or activity using DNA binding, chromatin immunoprecipitation, and promoter reporter methods. (3) We will determine whether Akt2 deletion exacerbates, and Akt1 deletion diminishes intimal hyperplasia, and whether the therapeutic response to rapamycin requires Akt2, using an injury model in wild type, Akt1 or Akt2 knockout mice. We propose that understanding these downstream targets of rapamycin and the molecular mechanisms by which they are regulated will provide key targets for development of improved stent therapeutics. PUBLIC HEALTH RELEVANCE Cardiovascular disease is a major cause of morbidity and mortality in the western world. While stents coated with the drug rapamycin have greatly reduced the risk of restenosis (re-blockage of the vessel) after coronary artery angioplasty, recent findings have revealed that they confer a small but significant risk of heart attack or death. This project aims to understand the molecular mechanisms underlying the beneficial anti-restenotic response of vascular smooth muscle cells to rapamycin, as this knowledge may allow us to tailor future therapeutics to inhibit these cells specifically, avoiding detrimental side effects on other cell types that can cause the dangerous complications.