Understanding the Molecular Mechanisms of Fibromuscular Dysplasia

项目摘要 纤维肌性发育不良（FMD）是一个研究不足，有时是致命的医学之谜，可导致动脉粥样硬化， 纤维化、狭窄、夹层、迂曲、动脉瘤和闭塞。诊断时的平均年龄为50岁- 55岁，94%是女性。虽然它在女性中的患病率高达5%，但没有具体的治疗方法， 关于其病因学知之甚少。在新闻界，这种缺乏知识，低估的流行， 有时致命的结果导致FMD被称为“不是罕见的疾病”（WSJ，2009年6月27日）。 我们的团队是FMD领域的世界领导者，我们对其临床特征的了解不断加深。为了解决缺乏 为了了解其原因，我们在2013年启动了DEFINE-FMD研究-一项针对FMD的大型功能性组学研究。 FMD的遗传和分子基础。DEFINE-FMD已经帮助提供了重要的见解， FMD，表明它有一个复杂的（非孟德尔）遗传基础。在这里，我们提出了详细的功能和 机制研究，以了解在DEFINE-FMD研究中确定的FMD的主要因果候选者- a 关键调控基因网络（RGN），我们称之为“FMD-RGN”。使用不同的方法，我们有 反复验证了该RGN与FMD的相关性，P值始终小于1 x 10-16。此外，本发明还提供了一种方法， 我们已经确定FMD-RGN的最重要的驱动因子之一是UBR 4（泛素蛋白连接酶E3组分n-1）。 recognin 4）. UBR 4是FMD的一个强有力的因果候选者，我们已经证实它发挥了强大的影响 调节FMD-RGN中其他基因的表达水平。作为我们的总体目标，我们旨在确定 FMD-RGN对血管细胞和动脉表型的特异性作用，并了解UBR 4在 控制FMD-RGN和引起FMD。·在具体目标1中，我们将详细分析 UBR 4和FMD-RGN对细胞表型的影响。我们将进行一系列体外研究， 人成纤维细胞与UBR 4的敲低和过表达，以了解该基因和FMD的作用， FMD中的RGN。·在具体目标2中，我们将表征细胞特异性Ubr 4的体内心血管作用。 删除。我们将在小鼠中进行一系列的体内研究，包括内皮细胞、平滑肌细胞和成纤维细胞， 特异性Ubr 4缺失。我们将提供这些小鼠的心血管表型的详细表征， 线，包括组织病理学，原子力显微镜的生物力学特性，和蛋白质组学使用液体 色谱串联质谱法。·在具体目标3中，我们将进行进一步的研究，以了解 表达UBR 4的血管细胞的体内命运和功能。我们将应用单细胞RNA测序， 其他尖端技术，以新鲜获得的小鼠和人类动脉样本，以提供决定性的体内 表达人UBR 4的血管细胞的表征，以及Ubr 4缺失的细胞特异性表型效应 对小鼠总的来说，使用这些集成但独立的方法，这个R 01将完全剖析分子 UBR 4和FMD-RGN的机制，以建立FMD的血管病理生物学的整体功能图。 作为1938年首次报道的一种疾病，我们认为这些关于口蹄疫的研究是必要的，而且早就应该进行了。

PROJECT SUMMARY Fibromuscular dysplasia (FMD) is an understudied and sometimes fatal medical enigma that can cause arterial fibrosis, stenosis, dissection, tortuosity, aneurysm and occlusion, throughout the body. Mean age at diagnosis is 50- 55 yrs and 94% are female. Although it has a prevalence of up to 5% in females, there is no specific treatment, and very little is known about its etiology. In the press, this lack of knowledge, underappreciated prevalence and sometimes fatal outcomes have led to FMD being called “The Rare Disease That Isn’t” (WSJ, June 27, 2009). Our team, world leaders in FMD, have advanced our knowledge of its clinical features. To address the lack of understanding about its cause, in 2013 we initiated the DEFINE-FMD study - a large, functional ‘omics study of the genetic and molecular basis of FMD. Already, DEFINE-FMD has helped provide important insights into the cause of FMD, showing that it has a complex (non-Mendelian) genetic basis. Here, we propose detailed functional and mechanistic studies to understand a top causal candidate for FMD that was identified in the DEFINE-FMD study – a critical regulatory gene network (RGN) we refer to as the “FMD-RGN.” Using differing approaches, we have repeatedly validated the association of this RGN with FMD, with P values consistently less than 1 x 10-16. In addition, we have identified that one of the top key drivers of the FMD-RGN is UBR4 (ubiquitin protein ligase E3 component n- recognin 4). UBR4 is a strong causal candidate for FMD, and we have already confirmed that it exerts strong effects on modulating the expression levels of other genes in the FMD-RGN. As our overall goals we aim to determine the specific effects of the FMD-RGN on the vascular cell and arterial phenotypes, and to understand the role of UBR4 in governing the FMD-RGN and in causing FMD. • In Specific Aim 1 we will undertake detailed analyses of the impact of UBR4 and the FMD-RGN on the cellular phenotype. We will perform a series of in vitro studies using human fibroblasts with knockdown and overexpression of UBR4 to understand the role of this gene and the FMD- RGN in FMD. • In Specific Aim 2 we will characterize the in vivo cardiovascular effects of cell-specific Ubr4 deletion. We will perform a series of in vivo studies in mice with endothelial-, smooth muscle cell-, and fibroblast- specific Ubr4 deletion. We will provide a detailed characterization of the cardiovascular phenotypes of these mouse lines, including histopathology, biomechanical properties by atomic force microscopy, and proteomics using liquid chromatography tandem mass spectrometry. • In Specific Aim 3 we will perform further studies to understand the in vivo fate and function of vascular cells expressing UBR4. We will apply single cell RNA sequencing and other cutting edge techniques to freshly obtained mouse and human artery samples to provide a decisive in vivo characterization of human UBR4-expressing vascular cells, and the cell-specific phenotypic effects of Ubr4 deletion in mice. Collectively, using these integrated but independent approaches, this R01 will fully dissect the molecular mechanisms of UBR4 and the FMD-RGN, to build a holistic functional picture of the vascular pathobiology of FMD. As a disease first reported in 1938, we believe these proposed studies on FMD are imperative, and long overdue.