Developmental basis for vascular smooth muscle cell dysfunction in Marfan syndrome aortic aneurysm

项目摘要/摘要 马凡综合征(MFS)是最常见的遗传性疾病，由纤维蛋白1(FBN1)基因突变引起 结缔组织疾病，每5,000人中就有1人患病。主动脉根部动脉瘤导致预期寿命缩短 由于动脉瘤的夹层或破裂，除非进行预防性的主动脉手术。正常情况下，血管 平滑肌细胞(SMC)通过动态收缩/松弛来维持主动脉内的动态平衡。 细胞外基质的产生，然而，这些细胞保持显著的可塑性，以改变其表型在 对伤害、生长因子或其他刺激的反应。已知功能失调的SMC表型调节 有助于MFS的动脉瘤发展。转化生长因子-β信号转导失调 也会导致主动脉瘤，尽管这一通路的确切作用仍存在争议。此外， 推动主动脉根部(主动脉瓣最近端的部分)发展的机制 尽管FBN1突变对全身有影响，但局灶性动脉瘤仍鲜为人知。独特的胚胎起源 主动脉根部(来自第二心区，SHF)和升主动脉段(来自神经)的SMC Crest(北卡罗来纳州CREST)被认为与主动脉根部特异性病理有关，但目前尚不清楚这些因素是如何起作用的 胚胎起源会影响动脉瘤的发展倾向。最近，单细胞RNA测序(scRNA- SMC(SEQ：行情)提供了对单个SMC基因表达的高分辨率分析。我的前期工作适用于 ScRNA-seq以小鼠为模型的MFS已经鉴定出具有严重调制的、病理的SMC亚群 表型。拟议的研究将促进我们目前对SMC发育和功能障碍的理解 在MFS主动脉瘤中使用两个互补的目标。在目标1中，我将定义胸廓的不同表型 应用单细胞RNA技术从第二心野和神经脊来源的主动脉平滑肌细胞 胚胎谱系追踪小鼠模型的测序及转化生长因子-β调控SHF的体外研究 和NC来源的SMC表型。在目标2中，我将描述调制的来源和病理效应 小鼠MFS模型中早期SMC的谱系追踪及其在MFS主动脉瘤中的应用 计算转录分析工具对scRNAseq数据进行分析，以确定驱动它们的分子机制 表型变化。表型调控的SMC与健康的主动脉SMC共培养实验 体外模拟主动脉瘤病理。这些研究将产生重要的数据，有助于准确定位 推动MFS和其他遗传性动脉瘤疾病向新的方向发展的分子机制 治疗的发展。拟议的研究培训计划的特点是由一个委员会直接指导 有经验的临床医生-科学家和获得最先进的设施和技术。这个计划也是 结合专业发展和职业规划策略，采用独特的协作精神 在斯坦福大学，心血管外科和医学之间的合作旨在最大限度地发挥培训潜力。

Project Summary/Abstract Marfan syndrome (MFS), caused by mutations in the fibrillin-1 (FBN1) gene, is the most common inherited connective tissue disorder, affecting 1 in 5,000 individuals. Aortic root aneurysm leads to reduced life expectancy due to dissection or rupture of the aneurysm unless preventative aortic surgery is performed. Normally, vascular smooth muscle cells (SMCs) maintain homeostasis within the aorta via dynamic contraction/relaxation and extracellular matrix production, however these cells retain significant plasticity to alter their phenotype in response to injury, growth factors, or other stimuli. Dysfunctional SMC phenotype modulation is known to contribute to aneurysm development in MFS. Dysregulated transforming growth factor-beta (TGF-b) signaling also contributes to aortic aneurysm, though the precise role of this pathway remains controversial. Furthermore, mechanisms driving the tendency of the aortic root (the segment most proximal to the aortic valve) to develop focal aneurysm despite systemic effects of FBN1 mutations are poorly understood. Distinct embryonic origins of SMCs populating the aortic root (from the second heart field, SHF) and ascending aortic segments (from neural crest, NC) is hypothesized to contribute to aortic root-specific pathology, however it remains unclear how these embryonic origins affect propensity for aneurysm development. Recently, single-cell RNA sequencing (scRNA- seq) has permitted high-resolution analysis of individual SMC gene expression. My preliminary work applying scRNA-seq to a mouse model of MFS has identified a subset of SMCs with a severely modulated, pathologic phenotype. The proposed study will advance our current understanding of SMC development and dysfunction in MFS aortic aneurysm using two complementary aims. In Aim 1 I will define the distinct phenotypes of thoracic aortic SMCs derived from the second heart field and neural crest lineages by applying single-cell RNA sequencing to an embryonic lineage-tracing mouse model and in vitro studies of TGF-b dysregulation on SHF and NC-derived SMC phenotype. In Aim 2 I will characterize the source and pathologic effects of modulated SMCs in MFS aortic root aneurysm by lineage-tracing early SMCs in a murine MFS model and applying computational transcriptomic analysis tools to scRNAseq data to determine molecular mechanisms driving their phenotype changes. Co-culture experiments of phenotypically modulated SMCs with healthy aortic SMCs will model aortic aneurysm pathology in vitro. These studies will generate important data that will help pinpoint molecular mechanisms driving aortic pathology in MFS and other hereditary aneurysm disorders toward new therapy development. The proposed research training plan features direct mentorship from a committee of accomplished clinician-scientists and access to state-of-the art facilities and techniques. This plan also incorporates professional development and career planning strategies, employing the unique collaborative spirit between Cardiovascular Surgery and Medicine at Stanford University intended to maximize training potential.