Marfan Aortic Embryologic Origin Influences Aneurysm Formation

项目总结 马凡综合征(MFS)是最常见的遗传性连接性疾病，由纤维蛋白-1基因突变引起 组织紊乱。患者通常会发展为主动脉根部动脉瘤，并随后发生主动脉夹层和破裂。 仍然是导致死亡的首要原因。如果不进行预防性手术，预期寿命将减少到40岁。 新的靶向药物疗法一直没有出现，可能是由于机制理解的局限性。因此， 迫切需要剖析参与MFS的主动脉壁细胞外基质的通路(S)。 破裂导致动脉瘤的形成。我们假设不同的SMC的胚胎起源 主动脉根部参与了主动脉根部特异性动脉瘤的病理生理过程。在最近的资助期，我们 建立并鉴定MFS患者的诱导多能干细胞(IPSCs)，并将其分化为 来自每个胚胎来源的SMC。我们了解到microRNA、miR-29b和相关的下游基因 依赖于SMC的胚胎起源。在应用高通量蛋白质组学之后，我们还发现 来自MFS患者的IPSC来源的SMC表达独特的、特定于谱系的蛋白质组特征，影响关键的 生物学过程包括细胞外基质的合成/退化和SMC的收缩/运动。利用单细胞RNA 测序，我们确定了一个独特的、疾病特异性的SMC簇，它会发生转录改变 在小鼠模型和人主动脉根部动脉瘤中均有局部研究。免费赠送MFS Fbn1C1041G/鼠标 在活体研究中，我们发现了几个区域性的上游转化生长因子-β依赖的触发因素，它们最终会增加 基质金属蛋白酶介导的细胞外基质重塑，包括：(A)RAS(GTPase)信号转导；(B)NADPH (C)雄激素对转化生长因子-β信号转导的增强作用 雄鼠。药物阻断每一条已发现的通路可减少动脉瘤的形成。 在本次续展申请中，我们严格扩展了我们的研究进展，提出了几个创新的 实验研究SMC谱系在局部ECM重塑中的相对贡献，调查 调节的SMC在动脉瘤形成过程中的上游触发和病理作用，并促进 翻译疾病特定的ipsc模型以发现新的途径。具体目标1寻求 MFS、SHF-1和神经脊-SMC之间的相互作用可诱导合成SMC表型和ECM重塑。 我们研究了还原的SHF-SMC甘露糖受体2(MRC2)对细胞外基质组成的新作用。 蛋白分解和SMC收缩功能。我们还将阐明ECM的生物力学特性对 谱系特异性的SMC反应，包括SMC转录调控和单细胞机械反应 属性。特异性目标2致力于确定调节的SMC群体对ECM重塑的影响 使用我们创新的MFS脱细胞基质支架进行体外培养。一种新的谱系追踪转基因小鼠模型 将被用来确定调节的SMC胚胎起源和在体内动脉瘤进展中的作用。

PROJECT SUMMARY Marfan syndrome (MFS), caused by mutations in the fibrillin-1 gene, is the most common inherited connective tissue disorder. Patients typically develop aortic root aneurysms with ensuing aortic dissection and rupture remaining the leading cause of death. Without prophylactic surgery, life-expectancy is reduced to age 40 years. Novel targeted drug therapies have been absent, likely due to limitations in mechanistic understanding. Thus, there is an urgent need to dissect the pathway(s) involved in MFS aortic wall extracellular matrix (ECM) breakdown resulting in aneurysm formation. We hypothesize that distinct embryonic origins of SMCs populating the aortic root contribute to aortic root-specific aneurysm pathophysiology. In the recent funding period, we created and characterized induced-pluripotent stem cells (iPSCs) from MFS patients and differentiated them into SMCs from each embryologic origin. We learned that the microRNA, miR-29b and related downstream genes were dependent on SMC embryologic origin. After applying high-throughput proteomics, we also discovered that iPSC-derived SMCs from MFS patients express distinct, lineage-specific proteomic profiles affecting critical biologic processes including ECM synthesis/degeneration and SMC contraction/motility. Utilizing single-cell RNA sequencing, we identified a distinct, disease-specific SMC cluster that develops transcriptomic alterations regionally in both murine models and human aortic root aneurysms. In complimentary MFS Fbn1C1041G/+ mouse in vivo studies, we discovered several regional upstream TGF-β-dependent triggers that ultimately increase matrix metalloproteinase-mediated ECM remodeling, including: (a) Ras (GTPase) signaling; (b) NADPH activation leading to reactive oxygen species production; and (c) androgen potentiation of TGF-β signaling in male mice. Drug blockade of each discovered pathway reduced aneurysm formation. In this renewal application, we rigorously expand our research progress by proposing several innovative experiments to study the relative contributions of SMC lineages to regional ECM remodeling, investigate the upstream triggers and pathologic role of modulated SMCs during aneurysm formation, and advance the translation of disease-specific iPSC modeling for novel pathway discovery. Specific Aim 1 seeks whether interactions between MFS SHF- and neural crest-SMCs induce synthetic SMC phenotype and ECM remodeling. We investigate the novel role of reduced SHF-SMC mannose receptor 2 (MRC2) on ECM composition, proteolysis and SMC contractile function. We will also elucidate the effect of ECM biomechanical properties on lineage-specific SMC responses, including SMC transcriptomic modulation and single cell mechanical properties. Specific Aim 2 strives to determine the effects of modulated SMC populations on ECM remodeling in vitro using our innovative MFS decellularized matrix scaffolds. Novel lineage-tracing transgenic mouse models will be utilized to identify modulated SMC embryologic origin and role in aneurysm progression in vivo.