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防止再狭窄，使冠状动脉病变的血运重建发生了革命性变化。重要的是，新出现的长期安全数据表明，雷帕霉素洗脱支架会增加晚期血栓形成的风险，这可能是由于内皮未完全愈合所致。因此，理想的支架药物将选择性地抑制血管平滑肌细胞(VSMC)的增殖和促进其分化，而不会抑制再内皮化。在我们对血管生成、动脉粥样硬化和再狭窄所必需的VSMC表型改变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促进VSMC的分化。我们发现，雷帕霉素抑制mTOR效应器S6K1，并由此激活Akt2是这一效应所必需的。令人惊讶的是，Akt1抑制了VSMC的分化。我们还发现雷帕霉素能激活VSMC转录因子GATA-6，并且该因子是雷帕霉素诱导分化所必需的。这种转录因子是VSMC特有的，在血管内皮细胞中没有发现。我们发现雷帕霉素也能诱导转录辅活化子myocardin的表达，从而促进VSMC的分化。众所周知，mTOR途径调节蛋白质的合成。值得注意的是，我们发现VSMC特异性转录的调节是这一途径的一种新功能。我们推测雷帕霉素通过Akt2调节转录因子是该药物抑制VSMC增殖和促进VSMC分化的关键机制。我们的目的是了解雷帕霉素调节VSMC中前分化转录因子的机制。(1)在解决这一假设时，我们的目标是确定Akt2的雷帕霉素激活如何调节GATA-6。我们假设雷帕霉素诱导GATA-6的磷酸化，从而导致其核转位和激活。我们将使用siRNA、Akt1或Akt2基因敲除小鼠的VSMC和GATA-6磷酸化位点突变体来确定哪个激酶使GATA-6磷酸化，以及磷酸化如何影响GATA-6的活性。我们推测，一个模拟磷酸的突变体GATA-6可能具有结构性活性，因此可能是一种潜在的VSMC特异性前分化治疗药物。(2)利用siRNA方法研究myocardin在雷帕霉素诱导表达中的作用。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肌钙蛋白的表达和/或活性。(3)我们将使用野生型、Akt1或Akt2基因敲除小鼠的损伤模型，确定Akt2缺失是否加剧，Akt1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Akt2。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改进支架治疗的开发提供关键靶点。 与公共卫生相关心血管疾病是西方世界发病率和死亡率的主要原因。虽然涂层了药物雷帕霉素的支架极大地降低了冠状动脉血管成形术后再狭窄(血管再次堵塞)的风险，但最近的研究发现，它们具有微小但重大的心脏病发作或死亡风险。该项目旨在了解雷帕霉素对血管平滑肌细胞有益的抗再狭窄反应的分子机制，因为这一知识可能使我们能够定制未来的治疗方法来特异性地抑制这些细胞，避免对其他类型的细胞产生有害的副作用，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.