Transcriptional Regulation by Angiotensin II in Vascular Smooth Muscle Cells

血管平滑肌细胞（VSMC）在心血管系统中起着重要的作用。 疾病（CVD;例如，高血压），影响约48%的美国成年人，并显著加速 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， VSMC通过“收缩”和“合成”的调节异常通过“表型转换”（Phe-sw）去分化 基因，导致VSMC增殖、迁移、炎症和细胞外基质（ECM）增加 生产在之前的融资周期中，我们揭示了AngII监管的长期融资的第一个功能角色， 非编码RNA和（超级）增强子（一个关键的表观遗传调控层）。我们的总体目标是 这一更新旨在阐明糖尿病和AngII诱导的VSMC Phe-sw的调节机制， 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/AngII暴露导致持续性长期DCVD，尽管随后血糖 标准化）。我们将使用最先进的多组学和sc测序（seq）方法来破译VSMC DCVD和代谢记忆中的行为，并确定新的药物靶点。我们假设糖尿病患者 条件和AngII协调地重新编程VSMC转录组和表观基因组，这导致持续的 促进Phe-sw进入独特细胞状态的基因失调，是VSMC功能障碍的基础， DCVD。这一假设得到了广泛的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 正常化; ii）DNA甲基化降低，并且在关键上调位点染色质可及性增加。 来自糖尿病小鼠的VSMC中的ECM和炎症基因，即使在正常葡萄糖中培养后; iii）新细胞 指示Phe-sw的簇出现在血管紧张素II输注小鼠的动脉中，使用整合的scRNA和 scATAC-seq; iv）糖尿病刺激诱导血管细胞中的3D染色质变化，使用染色质 构象分析我们将在3个具体目标中测试我们的假设：1）定义糖尿病诱导的转录组学 和VSMCs中Phe-sw相关基因的表观基因组变化[AngII-（in）依赖性]，以及它们的记忆 体外血糖正常化后持续失调; 2）阐明去分化VSMC亚型 以及它们在糖尿病和AngII诱导的动脉中Phe-sw的功能，以及它们在葡萄糖 使用scRNA-seq和scATAC-seq进行体内标准化;以及3）确定 通过靶向介导糖尿病和AngII诱导的DCVD和血管记忆的候选基因/位点来逆转DCVD和血管记忆 VSMC Phe-sw。这项创新性研究，使用尖端技术和功能性体内模型，将提供 对VSMC调控网络和糖尿病血管病变的表观遗传记忆的新见解。这些知识 有可能为急需的新疗法的开发提供信息，特别是对于没有反应的患者 以及目前可用的糖尿病药物和血管紧张素II受体阻滞剂，具有深远的临床意义的DCVD。

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.