Define the mechanisms of aortopathy in bicuspid aortic valve patients

胸主动脉瘤发生在大约50-70%的二叶式主动脉瓣（BAV）患者中， 影响1-2%的总人口。迫切需要发展证据，以确定 手术干预和开发治疗，将防止或扭转BAV患者的动脉瘤。胸 BAV中的主动脉瘤经常累及近端主动脉，平滑肌细胞（SMC）起源于 神经嵴干细胞（NCSC）然而，它保留了降胸主动脉，其中有SMC， 起源于近轴中胚层。我们研究的长期目标是确定 负责与BAV相关的动脉病变的机制，并制定主动脉瘤的治疗策略 BAV动脉瘤。本研究的目的是使用来源于BAV的SMC在体外模拟BAV血管病变。 BAV诱导的多能干细胞（iPSC）和体内使用由这些细胞产生的组织工程化血管 SMC。模型将用于探索BAV中主动脉瘤的治疗方法。我们的中心假设是BAV 主动脉平滑肌细胞在转化生长因子β 1依赖性分化中有缺陷，而非轴旁中胚层 并且该缺陷导致这些细胞的收缩功能和细胞外基质分泌降低， 导致BAV近端主动脉的动脉病和随后的动脉瘤。目标1：我们将描述 体外BAV/动脉瘤病例中血管SMC分化缺陷。我们将区分BAV和对照 iPSC转化为NCSC和近轴中胚层，然后是SMC。我们将确定分化，收缩力， BAV和对照SMC的细胞外基质。我们将定义导致缺陷的关键信号通路， BAV NCSC-SMC的分化，重点是典型的TGF-β信号传导和心肌蛋白转录， 在我们的试点研究中发现。我们还将进行RNA-seq以无偏见地识别其他潜在途径，导致 SMC与NCSC的分化缺陷。我们将挽救BAV SMC的分化缺陷， NCSC谱系通过独立于TGF-β途径的途径，如雷帕霉素，其促进 SMC分化通过抑制哺乳动物雷帕霉素靶点和激活Akt 2。目标2：我们将确定 BAV构建的工程化主动脉在裸鼠体内形成动脉瘤的机制 患者的iPSC。我们将创建填充有BAV NCSC-SMC的组织工程化血管，以取代BAV NCSC-SMC。 家兔腹主动脉。我们将确定工程血管的生物力学和动脉瘤形成 在兔中，我们将定义SMC的分化和成熟以及TGF-β 1信号传导。 工程船。最后，我们将在支架中加入雷帕霉素，以防止动脉瘤在工程中形成。 船舶.我们的人iPSC体外和体内模型是研究胸主动脉粥样硬化机制的创新。 BAV中的动脉瘤和筛选治疗剂。这些研究将产生关于脊椎病的新数据， 阐明BAV患者主动脉瘤形成的机制，指导外科干预，并提供 为BAV主动脉瘤的未来医学治疗奠定了基础。

Thoracic aortic aneurysm occurs in approximately 50–70% of patients with bicuspid aortic valve (BAV), which affects 1–2% of the general population. There is a critical need to develop evidence to determine criteria for surgical intervention and to develop treatment that will prevent or reverse aneurysms for BAV patients. Thoracic aortic aneurysm in BAV frequently involves the proximal aorta, with smooth muscle cells (SMCs) that originate from neural crest stem cells (NCSCs). However, it spares the descending thoracic aorta, which has SMCs that originate from the paraxial mesoderm. The long-term goal of our research is to determine the molecular mechanisms responsible for the aortopathy associated with BAV and develop therapeutic strategies for aortic aneurysm in BAV. The objective of this research is to model BAV aortopathy in vitro using SMCs derived from BAV induced pluripotent stem cells (iPSCs) and in vivo using tissue-engineered vessels generated from these SMCs. Models will be used to explore treatments for aortic aneurysm in BAV. Our central hypothesis is that BAV aortic SMCs are defective in TGF--dependent differentiation of SMCs from NCSCs but not paraxial mesoderm and this defect causes decreased contractile function and secretion of extracellular matrix from these cells, resulting in aortopathy and subsequent aneurysm at the proximal aorta in BAV. Aim 1: We will characterize defects in vascular SMC differentiation in BAV/aneurysm cases in vitro. We will differentiate BAV and control iPSCs into NCSCs and paraxial mesoderm, then SMCs. We will determine the differentiation, contractility, and extracellular matrix of the BAV- and control-SMCs. We will define the key signaling pathway leading to defective differentiation in BAV NCSC-SMCs with a focus on canonical TGF-β signaling and myocardin transcription as identified in our pilot study. We will also perform RNA-seq to unbiasedly identify other potential pathways causing the defective differentiation of SMCs from NCSCs. We will rescue the defective differentiation of BAV SMCs from the NCSC lineage through pathways independent of the TGF- pathway, such as rapamycin, which promotes SMC differentiation by inhibiting mammalian target of rapamycin and activating Akt2. Aim 2: We will determine the mechanism of aneurysm formation in vivo using nude rabbits that host engineered aorta generated from BAV patients’ iPSCs. We will create tissue-engineered vessels populated with BAV NCSC-SMCs to replace the rabbits’ abdominal aorta. We will determine the biomechanics and aneurysm formation of the engineered vessels in rabbits, and we will define differentiation and maturation of the SMCs and the TGF- signaling of the engineered vessel. Finally, we will incorporate rapamycin into the scaffold to prevent the aneurysm in engineered vessels. Our human iPSC in vitro and in vivo model is innovative for studying the mechanisms of thoracic aortic aneurysm in BAV and for screening therapeutic agents. These studies will produce novel data on aortopathy, clarify mechanisms of aortic aneurysm formation in BAV patients, guide surgical intervention, and provide a foundation for future medical treatment of aortic aneurysm in BAV.