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

研究平滑肌细胞机械传导改变导致瓣膜上主动脉瓣狭窄的原因

• 批准号: 10568580
• 负责人: Jessica Wagenseil
• 金额: $ 39.17万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10568580
• 关键词: Affect; Antibodies; Aorta; Aortic Valve Stenosis; Biological; Blood Pressure; Calcium; Calcium Signaling; Cardiovascular Diseases; Characteristics; Chemicals; Child; Collagen; DNA Sequence Alteration; Data; Deposition; Development; Elasticity; Elastin; Extracellular Matrix; Extracellular Matrix Proteins; Genes; Genetic Transcription; Heart failure; Human; In Vitro; Ion Channel; Measures; Mechanics; Modulus; Mus; Mutation; Nuclear Translocation; Operative Surgical Procedures; Patients; Pharmacologic Substance; Phenotype; Physiological; Piezo 1 ion channel; Piezo 2 ion channel; Piezo ion channels; Proliferating; Property; Proteins; Repeat Surgery; Risk; Role; Severities; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Stenosis; Stress; Stretching; Supravalvular aortic stenosis; Thoracic Aortic Aneurysm; Time; Vascular Diseases; cell dedifferentiation; cell type; genetic association; genetic manipulation; hemodynamics; in vivo; induced pluripotent stem cell; mechanotransduction; migration; mouse model; novel therapeutic intervention; overexpression; prevent; public health relevance; response; single-cell RNA sequencing; sudden cardiac death

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. .

抽象的 上主动脉狭窄（SVA）的特征是主动脉的局灶性变窄，增加了风险 突然心脏死亡。 SVA是由弹性蛋白基因突变引起的，导致弹性蛋白降低 数量，目前没有药物治疗。弹性蛋白不足的机制 因为SVA并不理解。弹性蛋白是主动脉的关键机械成分，有助于 被动刚度（或模量）确定主动脉在应用下将变形（或应变）的数量 血液动力学应力。在主动脉壁内部的平滑肌细胞（SMC）会影响分化， 扩散和迁移。细胞跨膜通道，包括压电1/2，是机械敏感的 将机械变化（例如应变）转化为生物学作用（例如分化）的分子。 压电通道的激活导致细胞内钙的增加，可以刺激核易位 YAP/TAZ，是包括CTGF在内的靶基因的转录调节剂。 CTGF是已知的调节器 鼓励去分化，迁移和扩散的SMC表型 - 所有特征受 可能导致SVA的压力。在我们独特的SVAS鼠标模型（Taglncre; elnf/f）中显示的初步数据显示 主动脉模量降低，可能会增加SMC菌株，在主动脉SMC中增加压电2和CTGF表达， 以及去分化的主动脉SMC表型。我们假设SVA是由SMC改变引起的 机械转导足够的弹性蛋白未放置以使主动脉壁加强并防止增加 SMC应变随着发育过程中的压力随血压的增加而增加。增加SMC应变原因 压电2的过表达/激活，导致细胞内钙增加，核易位 YAP/TAZ，并增加CTGF转录，导致SMC表型调节导致狭窄。 我们将通过使用taglncre; elnf/f小鼠和人来解决我们的假设 来自SVAS患者的诱导多能干细胞的SMC。在AIM 1中，我们将衡量全球 在生理载荷条件下，taglncre的局部弹性模量； ELNF/F主动脉和SMC菌株 不同的发育时间点（狭窄形成之前和之后），并将这些结果与 通过单细胞RNA-seq测量的SMC表型的变化。在AIM 2中，我们将应对小鼠主动脉施加应变 小鼠和人类SMC，并测量压电2的表达和活性。我们将通过化学和遗传 改变压电2的表达/活性，并确定对体外钙信号传导和体内狭窄的影响 严重程度。在AIM 3中，我们将通过化学和遗传操纵压电2的表达/活性，YAP/TAZ 本地化，CTGF在小鼠主动脉和小鼠和人类SMC中的含量，并确定对 SMC表型和狭窄的严重程度。我们的结果对于识别新药物非常重要 可能会阻止SMC表型因弹性蛋白不足和治疗SVA而发生变化的策略。 。