Extracellular Matrix Biomechanical Properties Contribute to Aneurysm Formation in Marfan Syndrome

项目摘要/摘要 马凡综合征(MFS)是最常见的遗传性结缔组织疾病，由基因突变引起 纤维蛋白-1(FBN1)基因，每5,000人中就有1人受到影响。主动脉根部动脉瘤会导致主动脉夹层或破裂， 导致预期寿命缩短，除非进行预防性的主动脉手术。正常情况下，主动脉壁 动态平衡依赖于SMC感知和响应ECM机械力的过程，称为 机械转导。ECM维持功能障碍会导致主动脉壁僵硬，但 机械转导在动脉瘤形成中的作用仍然存在争议。此外，驱动焦点的机制 动脉瘤的发展仅限于主动脉根部(最靠近主动脉瓣的部分)，尽管 人们对FBN1突变的系统性影响知之甚少。填充在主动脉中的SMC来源于特定的 胚胎学起源，如主动脉根部起源于第二心区和升主动脉 节段起源于神经脊(NC)。我们已经开发了一种体外诱导多能干细胞系统来 胚胎学血管病理学模型。利用IPSC模型放弃对外科手术的依赖 组织标本，为个性化精准医学打开大门。我的初步工作表明， 生长在不同硬度平板上的IPSC来源的SMC显示出明显的胚胎反应 ECM硬度。建议的研究将促进我们目前对ECM-SMC机械转导的理解 在动脉瘤形成过程中使用两个互补的目标。目标1将评估ECM的转录效应 应用单细胞RNA测序技术研究两种胚胎来源的SMC的硬度和组成 细胞生长在不同的硬度和细胞外基质组成。电解加工材料的组成和力学性能 每个胚胎来源产生的SMC将与原子力显微镜和质量进行比较 光谱分析。Aim 2将利用IPSC来源的SMC研究胚胎依赖的ECM硬化 转导过表达甘露糖受体2(MRC2)，以研究细胞内胶原的体外循环。 转基因系谱追踪小鼠模型将被用来表征转化生长因子-b对MRC2诱导的ECM的影响 活体病理学。这些研究将使人们更好地理解细胞外基质的组成和变化 僵硬影响ECM-SMC机械转导，为预防疾病提供新的治疗靶点 动脉瘤的形成。拟议的研究培训计划以来自不同委员会的直接指导为特色 临床医生-科学家和使用最先进的设施和技术。这项计划包含了专业人员 发展和职业规划战略，利用心胸外科、 心血管内科和心血管研究所，以最大限度地发挥我的培训潜力。

Project Summary/Abstract Marfan syndrome (MFS) is the most common inherited connective tissue disorder, caused by mutations in the fibrillin-1 (FBN1) gene, affecting 1 in 5,000 individuals. Aortic root aneurysms lead to aortic dissection or rupture, resulting in reduced life expectancy unless preventative aortic surgery is performed. Normally, aortic wall homeostasis depends on SMC sensing and responding to ECM mechanical force in a process called mechanotransduction. Dysfunctional ECM maintenance results in aortic wall stiffening, but the role of mechanotransduction in aneurysm development remains controversial. Furthermore, mechanisms driving focal aneurysm development restricted to the aortic root (the segment most proximal to the aortic valve) despite systemic effects of FBN1 mutations are poorly understood. SMCs populating the aorta are derived from specific embryologic origins such that the aortic root is derived from the second heart field (SHF) and ascending aortic segments arise from neural crest (NC). We have developed an induced pluripotent stem cell in vitro system to model embryologic derived vascular pathology. Utilizing an iPSC model relinquishes the dependance for surgical tissue specimens and opens the door for personalized precision medicine. My preliminary work showed that iPSC-derived SMC grown on varying stiffness plates demonstrated a distinct embryologic response to increasing ECM stiffness. The proposed study will advance our current understanding in ECM-SMC mechanotransduction during aneurysm formation using two complimentary aims. Aim 1 will assess the transcriptomic effects of ECM stiffness and composition on SMCs from both embryologic origins by applying single cell RNA sequencing to cells grown on varying stiffness and ECM composition. The composition and mechanical properties of ECM produced by each embryologic origin SMC will be compared with atomic force microscopy and mass spectrometry. Aim 2 will investigate embryologic dependent ECM stiffening by utilizing iPSC-derived SMCs transduced to overexpress mannose receptor 2 (MRC2) to characterized intracellular collagen recycling in vitro. A transgenic lineage traced murine model will be used to characterize TGF-b effects on MRC2 induced ECM pathology in vivo. These studies will generate a greater understanding of how altered ECM composition and stiffness influences ECM-SMC mechanotransduction to provide insight into new therapeutic targets to prevent aneurysm formation. The proposed research training plan features direct mentorship from a diverse committee of clinician-scientist and access to state-of-the-art facilities and techniques. The plan incorporates professional development and career planning strategies, utilizing collaborative resources between Cardiothoracic Surgery, Cardiovascular Medicine, and the Cardiovascular Institute to maximize my training potential.