Mechanisms of outflow tract morphogenesis regulated by extracellular matrix

项目摘要 先天性心脏病（CHD）是人类最常见的出生缺陷， 缺陷占总数的30%。脑室远端畸形，漏斗， 和大动脉近端导致圆锥动脉干异常。具体实例包括 大血管转位、右心室双出口和法洛四联症。在该患者队列中， 17%的死亡率表明我们迫切需要提高对早期发育的理解， 在流出道（OFT）形态发生中引导这些畸变的信号。本申请的目的是 揭示导致OFT发育中断的分子、细胞和生物力学机制 并构成人类圆锥动脉干CHD的基础。细胞外基质（ECM）在心脏病中的作用 发育和疾病的研究表明，这种专门的非细胞蛋白质网络是塑造细胞的关键因素。 圆锥干然而，我们对OFT过程中ECM的个体功能了解有限 阵我们鉴定了Fibulin（Fbln）蛋白作为OFT生长所必需的ECM的新调节剂， 扩张.我们的初步数据表明，需要Fblns来建立OFT的适当大小。 Fblns是通过Smad 3依赖性TGF-β信号积累内皮细胞（EC）所必需的。此外，本发明还 Fblns刺激平滑肌细胞（SMC）分化和弹性蛋白组装以建立OFT口径。 重要的是，该辅助腔室的组织硬度升高;这种降低的弹性有助于改变组织硬度。 流动剖面总而言之，我们的初步数据强调， fbln功能丧失模型中OFT生长受损是多因素的，代表了以下因素的复杂相互作用： EC积累、SMC分化、组织硬度和流动相关因素的缺陷。我们提出了 总体假设，Fbln基因是必不可少的，以建立OFT的尺寸和遵守，通过调节 TGF-β在EC中信号传导，并通过SMC促进ECM沉积，最终产生剪切力， 进一步扩大。我们将研究这个模型，目的如下： Fbln 2介导的EC积聚和OFT生长（目的1），研究Fbln 5在调节EC积聚和OFT生长中的作用。 弹性蛋白组装，刚度和OFT扩张（目标2），并确定剪切应力和机械生物学 在EC和SMC中通过Piezo 1在Fbln蛋白下游操作的机制（Aim 3）。从以下方面获得的见解： 这些拟议的研究将阐明负责顺应性和弹性的生物力学机制。 动脉极点的变形与圆锥动脉干区域狭窄相关的疾病有关。 此外，探索Fbln蛋白的细胞类型特异性功能将增强我们鉴定新的细胞因子的能力。 用于OFT导管和人工瓣膜的组织工程的治疗靶点和方案。

PROJECT SUMMARY Congenital heart disease (CHD) is the most common birth defect in humans and patients with conotruncal defects comprise 30% 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. Among this patient cohort, the mortality rate of 17% indicates a dire need for improvement in our understanding of the early developmental signals guiding these aberrations in outflow tract (OFT) morphogenesis. The purpose of this application is to uncover the molecular, cellular, and biomechanical mechanisms that account for disruptions in OFT development and underlie human conotruncal CHDs. The emerging focus on extracellular matrix (ECM) in cardiac development and disease points to this specialized, non-cellular protein network as a key player in sculpting the conotruncus. Yet, we have limited appreciation of the individual ECM complements functioning during OFT formation. We identified Fibulin (Fbln) proteins as novel regulators of the ECM essential for OFT growth and expansion. Our preliminary data demonstrate that Fblns are required to establish the proper size of the OFT. Fblns are required for accumulation of endothelial cells (ECs) through Smad3-dependent TGF-β cues. Further, Fblns stimulate smooth muscle cell (SMC) differentiation and elastin assembly to establish the OFT caliber. Importantly, tissue stiffness of this auxiliary chamber is elevated; this decreased elasticity contributes to altered flow profiles. Altogether, our preliminary data highlight that the underlying mechanisms responsible for the impaired OFT growth in the fbln loss-of-function model are multifactorial, representing a complex interplay of defects in EC accumulation, SMC differentiation, tissue stiffness, and flow-related factors. We put forth the overarching hypothesis that Fbln genes are essential to establish OFT dimensions and compliance by regulating TGF-β signals in ECs and by promoting ECM deposition by SMCs, ultimately generating shear forces that propagate further expansion. We will examine this model with the following aims: dissect the mechanisms mediated by fbln2 in EC accumulation and OFT growth (Aim 1), investigate the function of Fbln5 in regulating elastin assembly, stiffness, and OFT expansion (Aim 2), and determine shear stress and mechanobiological mechanisms operating downstream of Fbln proteins via Piezo1 in ECs and SMCs (Aim 3). Insights gained from these proposed studies will shed light on the biomechanical mechanisms responsible for compliance and elastic deformation at the arterial pole with implications for diseases involving stenosis of the conotruncal region. Furthermore, probing the cell type-specific functions of Fbln proteins will augment our ability to identify novel therapeutic targets and protocols for tissue engineering of OFT conduits and artificial valves.