Molecular Mechanisms of MGP; Role in AVMs

项目概要： 血管系统由精细的网络组成，这些网络的发展与以下因素的密切调节相结合： 内皮细胞（EC）。了解这种调节对于开发新的治疗方法至关重要 针对血管畸形的策略，如动静脉畸形（AVM）和遗传性 出血性毛细血管扩张症（HHT），由激活素受体样激酶1（ALK 1）突变引起。 我们先前的研究表明，小鼠中基质Gla蛋白（MGP）的基因缺失， 形态发生蛋白（BMPs）导致多个器官中的AVM，类似于HHT。我们发现BMP 9/ALK 1 信号传导诱导EC中的MGP表达，其中MGP在分化中起重要作用。BMP 9/ALK 1 信号传导还以不同的诱导延迟诱导Crossveinless-2（CV 2），从而产生两种负性的 反馈回路MGP和CV 2通过一种以前未知的机制共同调节BMP 9信号传导。在 在培养的EC中，我们发现MGP和CV 2表达的振荡在时间上协调了向EC的过渡， 内皮细胞柄细胞表型。这也导致了茎细胞的标记物振荡，而尖端细胞的标记物则是 抑制Mgp的缺失消除了振荡行为。在体内，MGP和CV 2被视为“塑造 生长中的视网膜出现“波浪”或条纹，缺乏MGP扰乱了血管网络。 我们的假设是MGP和CV 2是BMP 9信号传导和血管形态发生的调节因子， 表达的波动或波动的产生。在目标1中，我们将描述MGP和CV 2如何协调 使用基因表达的振荡响应于BMP 9的EC分化。我们将BMP 9诱导的 柄细胞表型的振荡，并探索能够这种行为的EC的表达谱。我们将 通过使用已建立的shRNA技术在体外删除Mgp基因来破坏系统，并确定 抑制剂和柄细胞标记物对波的影响。我们还将调查MGP和 CV 2可以在正常血管系统中检测到，焦点在视网膜上。在目标2中，我们将获得关键信息 关于MGP在视网膜血管网络和AVM中的作用，通过删除Mgp，损害MGP蛋白功能， 并调节细胞起源。我们将使用Mgp-/-小鼠调节AVM治疗的潜在靶点。 作为AVM模型。我们将从CV 2的调节开始，并使用包括杂交育种的方法， 基因改变的小鼠和经乳腺免疫阻断，并随后筛选其他因素， BMP 9响应。我们的研究将有助于确定BMP 9反应系统中的靶点，这些靶点可能用于 设计脑动静脉畸形的治疗方法

PROJECT SUMMARY: The vascular system consists of elaborate networks that develop in combination with close regulation of endothelial cells (ECs). An understanding of such regulation is essential for the development of new treatment strategies aimed at vascular malformations, such as arteriovenous malformations (AVMs) and hereditary hemorrhagic telangiectasia (HHT), caused by mutations in activin receptor-like kinase 1 (ALK1). We previously showed that gene deletion in mice of matrix Gla protein (MGP), an inhibitor of bone morphogenetic proteins (BMPs), causes AVMs in multiple organs similar to HHT. We showed that BMP9/ALK1 signaling induces MGP expression in ECs, where MGP plays an important role in differentiation. BMP9/ALK1 signaling also induces Crossveinless-2 (CV2) with a different induction delay, thereby creating two negative feedback loops. Together, MGP and CV2 regulate BMP9 signaling by a previously unknown mechanism. In cultured ECs, we found oscillations of MGP and CV2 expression that temporally coordinated transition to EC stalk cell phenotype in ECs. This also caused markers of stalk cells to oscillate, whereas tip cell markers were suppressed. Deletion of Mgp abolished the oscillatory behavior. In vivo, MGP and CV2 were seen as “shaping waves” or stripes in the growing retina, and lack of MGP perturbed the vascular networks. Our hypothesis is that MGP and CV2 are regulators of BMP9 signaling and vascular morphogenesis through generation of oscillations or waves of expression. In Aim 1, we will characterize how MGP and CV2 orchestrate EC differentiation in response to BMP9 using oscillations of gene expression. We will relate BMP9-induced stalk cell phenotype to the oscillations, and explore expression profiles of ECs capable of this behavior. We will disrupt the system by deleting the Mgp gene in vitro using established techniques of shRNA, and determine the effect on the waves of inhibitors and stalk cell markers. We will also investigate whether waves of MGP and CV2 can be detected in normal vasculature, with focus on the retina. In Aim 2, we will obtain key information about the role of MGP in retinal vascular networks and AVMs by deleting Mgp, impairing MGP protein function, and modulating the cellular origin. We will modulate potential targets for AVM treatments using the Mgp-/- mice as an AVM model. We will start with modulation of CV2 and use approaches that include crossbreeding with genetically altered mice and transmammary immunoblocking, and subsequently screen other factors in the BMP9 response. Our studies will help identify targets in the BMP9 response system that might be used in designing treatments for AVMs.