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

摘要 主动脉瓣上狭窄(SVAS)的特征是主动脉局部狭窄，这增加了风险。 因为心源性猝死。SVAS是由弹性蛋白基因突变导致弹性蛋白减少引起的 目前尚无药物治疗方法。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，有助于 被动刚度(或弹性系数)，它决定在外加压力下主动脉将变形(或应变)的程度。 血液动力学压力。主动脉壁内的平滑肌细胞(SMC)的压力会影响分化， 扩散和迁徙。细胞跨膜通道，包括Piezo1/2，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。 激活Piezo通道导致细胞内钙离子增加，从而刺激核转位 YAP/TAZ是包括CTGF在内的靶基因的转录调控因子。CTGF是一种已知的调节因子 鼓励去分化、迁移和增殖的SMC表型-所有特征都受 可能导致SVAS的菌株。我们独特的SVAS小鼠模型(TaglnCre；Elnf/f)中的初步数据显示 主动脉弹性系数降低，可能增加SMC应变，增加SMC中Piezo2和CTGF的表达， 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以使主动脉壁变硬并防止增加时的机械转导 在发育过程中，SMC应变作为应力随着血压的增加而增加。SMC压力增加导致 Piezo2的过表达/激活，导致细胞内钙离子增加，核转位 YAP/TAZ，以及CTGF转录增加，导致SMC表型调节，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 SMC来源于SVAS患者诱导的多能干细胞。在目标1中，我们将测量全球 以及生理载荷条件下TaglnCre、Elnf/f主动脉和SMC应变的局部弹性模量。 不同的发育时间点(狭窄形成之前和之后)，并将这些结果与 单细胞RNA-Seq法检测SMC表型的变化。在目标2中，我们将对小鼠的主动脉施加应变 以及鼠和人的SMC，并测量Piezo2的表达和活性。我们会在化学和基因上 改变Piezo2的表达/活性并确定其对体外钙信号和体内狭窄的影响 严肃性。在目标3中，我们将从化学和基因上操纵Piezo2的表达/活性，YAP/TAZ CTGF在小鼠主动脉、小鼠和人血管内皮细胞中的定位和含量，并确定其对 SMC表型和狭窄程度。我们的结果将对发现新的药物很重要 预防弹性蛋白缺乏引起的SMC表型改变和治疗SVAS的策略。 。

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. .