Essential functions of Fibulin proteins in outflow tract morphogenesis.

项目总结 先天性心脏病(CHD)是人类和圆锥动脉干患者最常见的出生缺陷 缺陷占这一人群的20%。脑室远端畸形，漏斗畸形， 大动脉的近端导致圆锥干畸形。具体的例子包括 大血管转位，右心室双出口，法洛四联症。此外，相比之下， 对于其他类型的先天性心脏病，圆锥干缺陷更多地与遗传和综合征有关 异常现象。在这一患者队列中，17%的死亡率表明我们的 对早期发育线索的理解引导这些流出道(OFT)形态发生的异常。 这个应用程序的目的是揭示造成干扰的分子和细胞机制 在OFT的发展和人类圆锥干先天性心脏病的基础上。我们鉴定出纤维蛋白(Fbln)为新的蛋白质 OFT形态发生所必需的细胞外基质(ECM)的调节剂。我们的初步数据显示 Fbln是OFT中的平滑肌细胞和分化晚期的转化生长因子-β信号转导所必需的 对动脉极有贡献的祖细胞。在目标1中，我将剖析细胞和分子机制。 在OFT生长过程中由fbln基因介导。我将评估前SHF祖细胞群体的增殖 在Fbln功能丧失模型中评估心肌细胞的分化和增殖，内皮细胞 细胞，以及平滑肌细胞对动脉极部SHF来源的前谱系的贡献。我会雇佣埃杜， 细胞凋亡和发育计时分析来剖析这些细胞类型特有的Fbln基因功能。在AIM 2、我将探讨pSmad3在心肌细胞、内皮细胞和平滑肌细胞类型中的特异性表达。 通常是Fbln功能丧失的胚胎。此外，我还将进行功能增益和功能损失实验 具有结构性活性的Alk5转基因和Smad3磷酸化的小分子抑制物， 分别进行了分析。这些策略将阐明Fbln蛋白如何通过转化生长因子-β信号介导性肥胖症的发生。 从这些拟议研究中获得的见解将有助于阐明动脉粥样硬化的机制。 动脉极的顺应性和弹性变形及其对涉及动脉狭窄的疾病的影响 主动脉瓣和肺动脉瓣。此外，探索Fbln蛋白的细胞类型特异性作用将增强我们的 识别组织工程组织工程的新靶点和方案的能力 阀门。

PROJECT SUMMARY Congenital heart disease (CHD) is the most common birth defect in humans and patients with conotruncal defects comprise 20% of this population. Malformations of the distal aspect of the ventricle, the infundibulum, and the proximal aspect of the great arteries lead to conotruncal anomalies. Specific examples include transposition of the great vessels, double outlet right ventricle, and Tetralogy of Fallot. Moreover, in comparison to other types of CHDs, conotruncal defects are more frequently associated with genetic and syndromic abnormalities. Among this patient cohort, the mortality rate of 17% indicates a dire need for improvement in our understanding of the early developmental cues guiding these aberrations in outflow tract (OFT) morphogenesis. The purpose of this application is to uncover the molecular and cellular mechanisms that account for disruptions in OFT development and underlie human conotruncal CHDs. We identified Fibulin (Fbln) proteins as novel regulators of the extracellular matrix (ECM) essential for OFT morphogenesis. Our preliminary data demonstrate that Fblns are required for smooth muscle addition to the OFT and for TGF-β signaling in the late-differentiating progenitors that contribute to the arterial pole. In Aim 1, I will dissect the cellular and molecular mechanisms mediated by fbln genes during OFT growth. I will assess proliferation of the anterior SHF progenitor population in the Fbln loss-of-function model and evaluate differentiation and proliferation of the cardiomyocyte, endothelial cell, and smooth muscle cell contribution to anterior SHF-derived lineages at the arterial pole. I will employ EdU, apoptosis, and developmental timing assays to dissect these cell type-specific functions of Fbln genes. In Aim 2, I will probe cardiomyocyte, endothelial cell, and smooth muscle cell-type specific expression of pSmad3 in the OFT of Fbln loss-of-function embryos. Moreover, I will perform gain-of-function and loss-of-function experiments with a constitutively active Alk5 transgene and a small molecular inhibitor of Smad3 phosphorylation, respectively. These strategies will illuminate how Fbln proteins mediate OFT development via TGF-β signaling. Insights gained from these proposed studies will shed light on the mechanisms responsible for arterial compliance and elastic deformation at the arterial pole with implications for diseases involving stenosis of the aortic and pulmonary valves. Furthermore, probing the cell type-specific roles of Fbln proteins will augment our ability to identify novel therapeutic targets and protocols for tissue engineering of OFT conduits and artificial valves.