Investigating altered smooth muscle cell mechanotransduction as a cause of supravalvular aortic stenosis

摘要 主动脉瓣上狭窄（SVAS）的特征是主动脉局灶性狭窄， 心脏性猝死SVAS是由弹性蛋白基因突变导致弹性蛋白减少引起的 量，目前没有药物治疗。弹性蛋白不足的机制 因为SVAS还没有被很好地理解。弹性蛋白是主动脉的关键机械成分， 被动刚度（或模量），其确定在施加的情况下主动脉将变形（或应变）多少 血流动力学应力主动脉壁内平滑肌细胞（SMC）的应变影响分化， 扩散和迁移。细胞跨膜通道，包括Piezo 1/2，是机械敏感的 将机械变化（如应变）转化为生物效应（如分化）的分子。 Piezo通道的激活导致细胞内钙的增加，这可以刺激核转位 雅普/TAZ是Ctgf等靶基因的转录调控因子。CTGF是已知的 SMC表型促进去分化、迁移和增殖-所有特征均受 可能导致SVAS菌株。我们独特的SVAS小鼠模型（TaglnCre; Elnf/f）的初步数据显示， 降低的主动脉模量可能增加SMC应变，增加主动脉SMC中的Piezo2和Ctgf表达， 和去分化的主动脉SMC表型。我们假设SVAS是由SMC改变引起的 当没有足够的弹性蛋白铺设到主动脉壁并防止增加时， 在发育过程中，随着血压的升高，SMC应变随着应力的增加而增加。SMC应变增加导致 Piezo2的过度表达/激活，导致细胞内钙增加， 雅普/TAZ和Ctgf转录增加，导致SMC表型调节，从而导致狭窄。 我们将通过使用TaglnCre; Elnf/f小鼠和人类的三个互补目标来解决我们的假设。 来源于SVAS患者的诱导多能干细胞的SMC。在目标1中，我们将衡量全球 在生理负荷条件下TagInCre的局部弹性模量; Elnf/f主动脉和SMC应变， 不同的发育时间点（狭窄形成前后），并将这些结果与 通过单细胞RNA-Seq测量的SMC表型变化。在目标2中，我们将对小鼠主动脉施加应变 以及小鼠和人SMC，并测量Piezo2表达和活性。我们将从化学和基因上 改变Piezo2表达/活性并确定对体外钙信号传导和体内狭窄的影响 严重性。在目标3中，我们将化学和遗传操纵Piezo2表达/活性，雅普/TAZ 定位和小鼠主动脉以及小鼠和人SMC中的Ctgf量，并确定对 SMC表型和狭窄严重程度。我们的研究结果将是重要的，以确定新的药物 这些策略可以预防SMC表型变化对弹性蛋白不足的反应并治疗SVAS。 .

ABSTRACT Supravalvular aortic stenosis (SVAS) is characterized by focal narrowing of the aorta that increases the risk for sudden cardiac death. SVAS is caused by mutations in the elastin gene that lead to decreased elastin amounts and there are currently no pharmaceutical treatments. The mechanisms by which elastin insufficiency cause SVAS are not well understood. Elastin is a critical mechanical component of the aorta and contributes to the passive stiffness (or modulus) that determines how much the aorta will deform (or strain) under applied hemodynamic stresses. Strain on smooth muscle cells (SMCs) within the aortic wall affects differentiation, proliferation, and migration. Cellular transmembrane channels, including Piezo1/2, are mechanosensitive molecules that transduce mechanical changes (such as strain) into biological effects (such as differentiation). Activation of Piezo channels leads to increases in intracellular calcium that can stimulate nuclear translocation of YAP/TAZ, which are transcriptional regulators of target genes including Ctgf. Ctgf is a known modulator of SMC phenotype that encourages dedifferentiation, migration, and proliferation - all characteristics affected by strain that may contribute to SVAS. Preliminary data in our unique SVAS mouse model (TaglnCre;Elnf/f) show a reduced aortic modulus that may increase SMC strain, increased Piezo2 and Ctgf expression in aortic SMCs, and a dedifferentiated aortic SMC phenotype. We hypothesize that SVAS is caused by altered SMC mechanotransduction when enough elastin is not laid down to stiffen the aortic wall and prevent increased SMC strain as stress increases with blood pressure during development. Increased SMC strain causes overexpression/activation of Piezo2, leading to increased intracellular calcium, nuclear translocation of YAP/TAZ, and increased Ctgf transcription that causes SMC phenotype modulation contributing to stenosis. We will address our hypothesis through three complementary aims using TaglnCre;Elnf/f mice and human SMCs derived from induced pluripotent stem cells from SVAS patients. In Aim 1, we will measure the global and local elastic modulus of TaglnCre;Elnf/f aorta and SMC strain under physiologic loading conditions at different developmental time points (before and after stenosis formation) and correlate these results with changes in SMC phenotype as measured by single cell RNA-Seq. In Aim 2, we will apply strain to mouse aorta and mouse and human SMCs and measure Piezo2 expression and activity. We will chemically and genetically alter Piezo2 expression/activity and determine effects on in vitro calcium signaling and in vivo stenosis severity. In Aim 3, we will chemically and genetically manipulate Piezo2 expression/activity, YAP/TAZ localization, and Ctgf amounts in mouse aorta and mouse and human SMCs and determine the effects on SMC phenotype and stenosis severity. Our results will be important for identifying new pharmaceutical strategies that may prevent SMC phenotype changes in response to elastin insufficiency and treat SVAS. .