BET Bromodomain proteins as Novel Epigenetic Targets for prevention of Intimal Hyperplasia after Vascular Surgery

项目摘要： 尽管技术进步，动脉血运重建，特别是外周血管成形术和旁路移植术， 通常由于内膜增生（IH）而失败。IH的主要病理是血管平滑肌细胞的转化， 肌细胞（SMC）从静止状态到激活状态，具有多种促IH表型。 目前，IH的预防主要集中在抗增殖剂上，成功率有限。此外，缺乏洞察力 存在于支配所有主要SMC致病表型的主近端调节因子中，不仅 增殖/迁移，还有去分化和炎症。查明并有效地针对 主调节剂将代表抗IH治疗的重大进展。最近，我们的团队发现， 被称为表观遗传阅读器的蛋白质的溴和末端外（BET）家族可能独特地适合于这一主人 作用最近的突破性研究表明，BET家族的蛋白质可以改变表型 通过将两个乙酰基结合溴结构域与乙酰化关键转录因子偶联， 从而激活所选择的基因子集的转录。我们发现了一个令人兴奋的现象 第一类BET抑制剂（JQ 1）阻止SMC致病性转化并减轻IH。这导致 我们的中心假设，与下游个体途径不同，BET 蛋白质作为节点转录因子（如STAT 3）的上游驱动因子， 下游致病性SMC表型。由于JQ 1在所有三个BET中均全面阻断两个溴结构域， 蛋白质，在这个建议中，我们将区分各种BET蛋白质和溴结构域的功能， SMC转换。在Aim 1中，我们将确定哪些BET蛋白决定SMC致病表型 和IH。我们已经观察到两种BET蛋白在动脉损伤后显著增加。在AIM 2中，我们将 进一步描述两个溴结构域中的哪一个负责BET致病功能。的理由 是基于我们的数据，用各自的阻断两个溴结构域（在所有三种BET蛋白中的每一种）， 抑制剂对SMC增殖和炎症产生不同的作用。在Aim 3中，我们将确定密钥 由BET蛋白控制的转录因子，从而介导SMC上的BET功能 转型我们已经初步确定STAT 3为主要候选者。我们的最终目标是阐明 特异性BET蛋白或溴结构域是预防内膜增生的最合适靶点， 增生为此，我们将利用革命性的CRISPR/Cas9技术来研究BET蛋白质。 敲除和溴结构域突变。该提案是新颖的，因为我们将确定一个 控制SMC致病性转化的关键表观遗传开关。这项研究意义重大，因为我们将 建立BET蛋白或溴结构域作为开发优化治疗方法的新靶点， 治疗IH和复发性血管疾病。

Project Summary: Despite technological advances, arterial revascularization  especially peripheral angioplasty and bypass  often fail due to intimal hyperplasia (IH). The primary pathology of IH is the transformation of vascular smooth muscle cells (SMCs) from a quiescent state to an activated state, with multiple IH-promoting phenotypes. Currently, prevention of IH focuses on anti-proliferative agents with limited success. Moreover, a lack of insight exists into master proximal regulators that govern all of the major SMC pathogenic phenotypes, not only proliferation/migration, but also de-differentiation and inflammation. Identification and effective targeting of such master regulators would represent a major advance in anti-IH therapy. Recently, our team has identified that the bromo and extraterminal (BET) family of proteins, termed epigenetic readers, may uniquely fit this master role. Recent groundbreaking studies have revealed that the BET family of proteins can change the phenotype of different cell types, by coupling two acetyl-binding bromo-domains with acetylated key transcription factors consequently activating the transcription of a select subset of genes. We have made the exciting observation that the first-in-class BET inhibitor (JQ1) halts SMC pathogenic transformation and mitigates IH. This leads to our central hypothesis, that unlike downstream individual pathways that are subject to redundancy, BET proteins act as upstream drivers of nodal transcription factors (such as STAT3) which in turn determine downstream pathogenic SMC phenotypes. Since JQ1 globally blocks both bromo-domains in all three BET proteins, in this proposal we will differentiate the functions of the various BET proteins and bromo-domains in SMC transformation. In Aim1 we will determine which of the BET proteins dictate SMC pathogenic phenotypes and IH. We have observed that two BET proteins dramatically increase after arterial injury. In Aim2 we will further delineate which of the two bromo-domains is responsible for the BET pathogenic function. The rationale is based on our data that blocking the two bromo-domains (each in all three BET proteins) with respective inhibitors produces differential effects on SMC proliferation and inflammation. In Aim3 we will identify the key transcription factors that are governed by BET proteins, thereby mediating the BET function on SMC transformation. We have preliminarily identified STAT3 as a leading candidate. Our ultimate goal is to elucidate the specific BET protein or bromo-domain that is the most opportune target in the prevention of intimal hyperplasia. To this end, we will make use of the revolutionary CRISPR/Cas9 technology for BET protein knockout and bromo-domain mutation in the mouse genome. The proposal is novel because we will identify a key epigenetic switch that controls SMC pathogenic transformation. The research is significant because we will establish a BET protein or bromo-domain as a novel target for developing optimized therapeutic methods to treat IH and recurrent vascular disease.