Marfan Aortic Embryologic Origin Influences Aneurysm Formation

项目概要 马凡综合征 (MFS) 由原纤维蛋白 1 基因突变引起，是最常见的遗传性结缔组织病 组织紊乱。患者通常会出现主动脉根部动脉瘤并随后发生主动脉夹层和破裂 仍然是主要的死亡原因。如果不进行预防性手术，预期寿命将缩短至 40 岁。 新型靶向药物疗法一直不存在，可能是由于机制理解的局限性。因此， 迫切需要剖析参与 MFS 主动脉壁细胞外基质 (ECM) 的通路 破裂导致动脉瘤形成。我们假设 SMC 的不同胚胎起源 主动脉根部有助于主动脉根部特异性动脉瘤的病理生理学。在最近的资助期内，我们 从 MFS 患者中创建并表征诱导多能干细胞 (iPSC)，并将其分化为 来自每个胚胎学起源的 SMC。我们了解到microRNA、miR-29b及相关下游基因 依赖于 SMC 胚胎学起源。应用高通量蛋白质组学后，我们还发现 来自 MFS 患者的 iPSC 衍生的 SMC 表达不同的、谱系特异性的蛋白质组谱，影响关键 生物过程，包括 ECM 合成/变性和 SMC 收缩/运动。利用单细胞 RNA 通过测序，我们发现了一个独特的、疾病特异性的 SMC 簇，它会发生转录组改变 在小鼠模型和人类主动脉根部动脉瘤中都有区域性的结果。附赠 MFS Fbn1C1041G/鼠标 在体内研究中，我们发现了几个区域上游 TGF-β 依赖性触发器，最终增加 基质金属蛋白酶介导的 ECM 重塑，包括： (a) Ras (GTPase) 信号传导； (b) NADPH 活化导致活性氧的产生； (c) TGF-β信号传导的雄激素增强作用 雄性小鼠。对每条发现的通路进行药物阻断可减少动脉瘤的形成。 在这次更新申请中，我们通过提出几项创新方案来严格扩展我们的研究进展 实验研究 SMC 谱系对区域 ECM 重塑的相对贡献，调查 调节 SMC 在动脉瘤形成过程中的上游触发因素和病理作用，并推进 将疾病特异性 iPSC 模型转化为新途径发现。具体目标 1 寻求是否 MFS SHF-和神经嵴-SMC 之间的相互作用诱导合成 SMC 表型和 ECM 重塑。 我们研究了减少的 SHF-SMC 甘露糖受体 2 (MRC2) 对 ECM 成分的新作用， 蛋白水解和 SMC 收缩功能。我们还将阐明 ECM 生物力学特性对 谱系特异性 SMC 反应，包括 SMC 转录组调节和单细胞机械 属性。具体目标 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实验。新型谱系追踪转基因小鼠模型 将用于鉴定调节的 SMC 胚胎学起源及其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.