The role of TWIST1 in smooth muscle cells during atherosclerosis

这一奖项的目的是为罗伯特·维尔卡博士提供受保护的时间，使他能够获得培训 有必要成为一名独立的研究人员，利用人类基因发现来定义新的机制 导致冠状动脉疾病(CAD)。职业发展活动旨在加强Wirka博士的 实现这一研究范式所需的三个关键领域的技能：一)将与疾病相关的变异联系起来 因果基因；ii)确定因果基因如何影响细胞和血管生物学；以及iii)定义 受致病基因影响的分子途径和网络。他的导师奎特莫斯博士将指导和 监督他走向独立的进程。在动脉粥样硬化过程中，血管平滑肌细胞(SMCs)去 分化、增殖和迁移到病变中的过程称为“表型调节”。在最近一次 Wirka博士在《自然医学》上发表的第一篇论文中，从疾病中产生了单细胞基因表达数据集 并表征了小鼠和人动脉SMC表型变化过程中的前所未有 细节。在目前的提案中，Wirka博士使用这些数据来确定转录因子Twist1是一种 潜在的SMC表型调节的新型调节剂。在小鼠和人类基因的通径分析中 表达数据显示，Twist1被强烈预测促进SMC的表型调节。与此一致 预测作用，Twist1基因在小鼠和小鼠SMC表型改变过程中表达增加 人类单细胞数据，初步研究表明Twist1抑制SMC分化的表达 记号笔。重要的是，Twist1还与包括冠心病在内的多种血管疾病密切相关 人类基因研究。拟议的研究将确定Twist1在血管SMC中的作用 疾病，并确定发生这种情况的细胞和分子机制。在目标1中，SMC- 特定的条件性Twist1基因敲除小鼠将被用来确定Twist1如何影响SMC在 动脉粥样硬化病变：i)单细胞基因表达的评估将用于确定 SMC特异性Twist1基因敲除对SMC在疾病和 绘制Twist1影响的分子路径图，以及ii)对病变动脉壁的原位研究将检查 SMC特异性Twist1基因敲除对典型病变表型的影响及其作用 到病变处的SMC。在目标2中，培养的人冠状动脉平滑肌细胞(HCASMCs)将被用来确定 Twist1的细胞和分子效应：I)Twist1扰动对SMC表型的影响 动脉粥样硬化，如分化、增殖、迁移、侵袭和细胞死亡，将被确定，并 II)Twist1抑制SMC分化标志物表达的详细分子机制 将探索ACTA2。这些研究将确定细胞和分子机制的CAD- 相关基因和潜在的新型SMC主控调节因子影响疾病风险。重要的是，这些研究 这也将为Wirka博士提供实现科学独立所需的最后技能。

The purpose of this award is to provide Dr. Robert Wirka protected time, allowing him to obtain the training necessary to become an independent investigator using human genetic findings to define novel mechanisms driving coronary artery disease (CAD). Career development activities are designed to strengthen Dr. Wirka's skills in three key areas necessary for achieving this research paradigm: i) Linking disease-associated variation to causal genes; ii) Determining how causal genes affect cellular and vascular biology; and iii) Defining the molecular pathways and networks affected by causal genes. His mentor, Dr. Quertermous, will guide and monitor his progress towards independence. During atherosclerosis, vascular smooth muscle cells (SMCs) de- differentiate, proliferate and migrate into the lesion in a process known as “phenotypic modulation”. In a recent first-author paper in Nature Medicine, Dr. Wirka generated single-cell gene expression datasets from diseased mouse and human arteries and characterized the process of SMC phenotypic modulation in unprecedented detail. In the current proposal, Dr. Wirka has used these data to identify the transcription factor TWIST1 as a potential novel regulator of SMC phenotypic modulation. In pathway analysis of the mouse and human gene expression data, TWIST1 is strongly predicted to promote SMC phenotypic modulation. Consistent with this predicted role, TWIST1 gene expression increases in SMCs during phenotypic modulation in the mouse and human single-cell data, and preliminary studies show that TWIST1 inhibits expression of SMC differentiation markers. Importantly, TWIST1 is also strongly associated with multiple vascular diseases, including CAD, in human genetic studies. The proposed studies will determine the role of TWIST1 in vascular SMCs during disease, and determine the cellular and molecular mechanisms by which this occurs. In Aim 1, SMC- specific conditional Twist1 knockout mice will be used to determine how Twist1 affects SMC phenotype within the atherosclerotic lesion: i) assessment of single-cell gene expression will be used to determine the effect of SMC-specific Twist1 knockout on the ability of SMCs to undergo phenotypic modulation during disease and to map the molecular pathways affected by Twist1, and ii) in situ studies of the diseased artery wall will examine the effect of SMC-specific Twist1 knockout on classical aspects of lesion phenotype and the contribution of SMCs to the lesion. In Aim 2, cultured human coronary artery SMCs (HCASMCs) will be used to determine the cellular and molecular effects of TWIST1: i) the effect of TWIST1 perturbation on SMC phenotypes relevant to atherosclerosis such as differentiation, proliferation, migration, invasion, and cell death will be determined, and ii) the detailed molecular mechanism by which TWIST1 inhibits expression of the SMC differentiation marker ACTA2 will be explored. These studies will determine the cellular and molecular mechanisms by which a CAD- associated gene and potential novel SMC master regulator influences disease risk. Importantly, these studies will also provide Dr. Wirka with the final skills necessary to achieve scientific independence.