Mechanisms of outflow tract morphogenesis regulated by extracellular matrix

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30%。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。在这个患者队列中， 17%的死亡率表明我们迫切需要改善对早期发育的理解 在流出道(OFT)形态发生中引导这些异常的信号。此应用程序的目的是 揭示导致OFT发育中断的分子、细胞和生物力学机制 是人类圆锥干先天性心脏病的基础。细胞外基质(ECM)在心脏中的研究进展 发展和疾病表明，这种专门的、非细胞蛋白质网络是塑造 圆锥树干。然而，我们对OFT过程中发挥作用的单个ECM补充物的了解有限 队形。我们发现纤维蛋白(Fbln)是OFT生长和生长所必需的ECM的新调节因子。 扩张。我们的初步数据表明，FBLN是建立OFT的适当大小所必需的。 Fbln是内皮细胞(ECs)通过Smad3依赖的转化生长因子-β信号积累所必需的。此外， FBLN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，这个辅助室的组织硬度升高；这种弹性的降低有助于改变 流动剖面。总而言之，我们的初步数据突显了导致 FBLN功能丧失模型中的OFT增长受损是多因素的，代表着复杂的 内皮细胞积聚、SMC分化、组织僵硬和血流相关因素存在缺陷。我们提出了 最重要的假设是Fbln基因对建立OFT维度和通过调控顺应性至关重要 转化生长因子-β信号通过促进内皮细胞的细胞外基质沉积，最终产生剪切力， 传播进一步的扩张。我们将从以下几个方面考察这一模型：剖析其机制 Fbln2介导的EC积累和OFT生长(目标1)，研究Fbln5在调控中的作用 弹性蛋白装配、硬度和OFT扩张(目标2)，并确定剪应力和机械生物学 在ECs和SMC中通过Piezo1作用于Fbln蛋白下游的机制(目标3)。从以下方面获得的见解 这些拟议的研究将阐明导致顺应性和弹性的生物力学机制。 动脉极的变形与涉及圆锥干区狭窄的疾病有关。 此外，探索Fbln蛋白的细胞类型特定功能将增强我们识别新事物的能力 组织工程OFT管道和人工瓣膜的治疗靶点和方案。

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.