Transcriptional Regulation by Angiotensin II in Vascular Smooth Muscle Cells

摘要：血管壁中的血管平滑肌细胞(VSMCs)在心血管疾病中起着关键作用。 疾病(心血管疾病；例如高血压)，影响到约48%的美国成年人，而且 糖尿病(DCVD)。当受到糖尿病和生长因子血管紧张素II(AngII)等因素的刺激时，成熟 VSMC通过“表型转换”(Phe-Sw)通过“收缩”和“合成”的失调而去分化 基因，导致VSMC增殖、迁移、炎症和细胞外基质(ECM)增加 制作。在之前的融资周期中，我们解开了AngII调控的Long的第一个功能角色 VSMC中的非编码RNA和(超级)增强子(一个关键的表观遗传调节层)。我们的总体目标是 这一更新是为了阐明调控糖尿病和血管紧张素Ⅱ诱导的VSMC Phe-Sw的机制 表观基因组和单细胞(Sc)水平以及这些机制如何建立血管代谢记忆(其中 既往高血糖/血管紧张素Ⅱ暴露会导致持续的长期DCVD，尽管随后会出现血糖 正规化)。我们将使用最先进的多重组学和sc测序(Seq)方法来破译VSMC。 DCVD的行为和代谢记忆，并确定新的药物靶点。我们的假设是糖尿病 Conditions和AngII协调重新编程VSMC转录组和表观基因组，这导致了持久性 促进Phe-Sw的基因异常调节到导致VSMC功能障碍和加速的独特细胞状态 DCVD。这一假设得到了大量新的初步数据的支持，这些数据表明：i)糖尿病状态增加 Angii作用并促进VSMC增殖和Phe-Sw基因谱，即使在葡萄糖作用下也会持续存在 正常化；ii)DNA甲基化减少，染色质可及性增加 糖尿病小鼠VSMCs的ECM和炎症基因，即使在正常葡萄糖培养后也是如此；iii)新细胞 在血管紧张素Ⅱ注射的小鼠的主动脉中出现了指示Phe-Sw的簇，用整合的scRNA-和 糖尿病刺激诱导血管细胞染色质3D改变，使用染色质 构象分析。我们将在3个具体目标上验证我们的假设：1)定义糖尿病诱导的转录 和Phe-Sw相关基因VSMCs[Angii(In)依赖性]的表观基因组变化及其记忆 体外葡萄糖正常化后的持续性失调；2)阐明去分化的VSMC亚型 以及它们在糖尿病和血管紧张素Ⅱ诱导的动脉Phe-SW中的作用，以及它们在葡萄糖后的持久性 体内标准化，使用scRNA-seq和scatac-seq；以及3)确定 靶向介导糖尿病和血管紧张素Ⅱ诱导的候选基因/位点逆转DCVD和血管记忆 VSMC PHE-软件。这项创新性的研究，使用尖端技术和体内功能模型，将提供 对糖尿病血管病变的VSMC调节网络和表观遗传记忆的新见解。这一知识 有可能为急需的新疗法的开发提供信息，特别是对无反应的患者 对目前可用的糖尿病药物和血管紧张素受体阻滞剂有良好的疗效，对DCVD具有深远的临床意义。

SUMMARY: Vascular smooth muscle cells (VSMCs) in the blood vessel wall play pivotal roles in cardiovascular disease (CVD; e.g., hypertension), which affects ~48% of US adults, and which is significantly accelerated by diabetes (DCVD). When stimulated by factors like diabetes and the growth factor Angiotensin II (AngII), mature VSMCs de-differentiate through “phenotypic switching” (Phe-sw) via dysregulation of "contractile" and "synthetic” genes, resulting in increased VSMC proliferation, migration, inflammation, and extracellular matrix (ECM) production. In the previous funding cycles, we unraveled the first functional roles for AngII-regulated long noncoding RNAs and (super-)enhancers (a key epigenetic regulatory layer) in VSMCs. Our overall objective in this renewal is to elucidate the mechanisms regulating diabetes- and AngII-induced VSMC Phe-sw at the epigenome and single-cell (sc) level and how these mechanisms establish vascular metabolic memory (in which prior hyperglycemia/AngII exposure leads to persistent long-term DCVD despite subsequent glucose normalization). We will use state-of-the-art multi-omics and sc-sequencing (seq) approaches to decipher VSMC behavior in DCVD and metabolic memory and identify new drug targets. Our hypothesis is that diabetic conditions and AngII coordinately re-program the VSMC transcriptome and epigenome, which lead to persistent dysregulation of genes promoting Phe-sw to unique cellular states underlying VSMC dysfunction and accelerated DCVD. This hypothesis is supported by extensive new preliminary data that show: i) a diabetic state augments AngII actions and promotes VSMC proliferation and Phe-sw gene profiles, which persist even after glucose normalization; ii) DNA methylation is decreased, and chromatin accessibility is increased at key upregulated ECM and inflammatory genes in VSMCs from diabetic mice, even after culture in normal glucose; iii) new cell clusters indicative of Phe-sw occur in aortas of Ang II-infused mice, identified using integrated scRNA- and scATAC-seq; iv) diabetic stimuli induce 3D chromatin changes in vascular cells, seen using chromatin conformation assays. We will test our hypothesis in 3 specific aims: 1) Define diabetes-induced transcriptomic and epigenomic changes [AngII-(in)dependent] in VSMCs at Phe-sw-related genes, and the memory of their persistent dysregulation after glucose normalization in vitro; 2) Elucidate the de-differentiated VSMC subtypes and their functions in diabetes- and AngII-induced Phe-sw in arteries, and their persistence after glucose normalization in vivo, using scRNA-seq and scATAC-seq; and 3) Determine the translational potential for reversing DCVD and vascular memory by targeting candidate genes/loci mediating diabetes- and AngII-induced VSMC Phe-sw. This innovative study, using cutting-edge technologies and functional in vivo models, will provide novel insights into VSMC regulatory networks and epigenetic memory of diabetic vasculopathy. This knowledge has the potential to inform development of much-needed new therapies, especially for patients not responding well to currently available diabetes drugs and AngII blockers, with far-reaching clinical implications for DCVD.