Molecular Mechanisms of MGP; Role in AVMs

项目总结： 血管系统由精细的网络组成，这些网络与密切的调节 内皮细胞(ECs)。对这种规则的理解对于开发新的治疗方法是至关重要的 针对血管畸形(如动静脉畸形)和遗传性血管畸形的策略 出血性毛细血管扩张(HHT)，由激活素受体样激酶1(ALK1)突变引起。 我们之前在小鼠中发现了骨抑制因子基质GLA蛋白(MGP)的基因缺失 形态发生蛋白(BMPs)，可引起类似于HHT的多个器官的动静脉曲张。我们发现BMP9/ALK1 信号诱导内皮细胞表达Mgp，其中Mgp在细胞分化中起重要作用。BMP9/ALK1 信号也以不同的诱导延迟诱导无交叉-2(CV2)，从而产生两个负值 反馈循环。MGP和CV2共同通过一种以前未知的机制来调节BMP9信号。在……里面 培养的内皮细胞，我们发现Mgp和CV2的表达振荡，并在时间上协调过渡到内皮细胞 内皮细胞中的柄细胞表型。这也导致茎细胞的标记振荡，而茎细胞的标记 被压制了。Mgp基因的缺失消除了这种振荡行为。在活体内，MGP和CV2被视为“成形” 生长中的视网膜上的“波”或条纹，以及缺乏MGP扰乱了血管网络。 我们的假设是MGP和CV2是BMP9信号和血管形态发生的调节者 表达的振荡或波动的产生。在目标1中，我们将描述MGP和CV2如何协调 通过基因表达的振荡反应BMP9对EC的分化。我们将把BMP9诱导的 茎细胞的表型与振荡有关，并探索具有这种行为的内皮细胞的表达谱。我们会 利用已建立的shRNA技术，通过在体外删除MGP基因来扰乱系统，并确定 抑制物和茎细胞标记物对波的影响。我们还将调查MGP和MGP的浪潮 CV2可以在正常的血管系统中检测到，主要集中在视网膜。在目标2中，我们将获得关键信息 关于MGP通过缺失MGP，损害MGP蛋白功能，在视网膜血管网络和动静脉畸形中的作用， 以及调节细胞起源。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CV2的调制开始，并使用包括与 转基因小鼠和经乳腺免疫阻断，并随后筛选其他因素在 BMP9反应。我们的研究将有助于确定BMP9反应系统中可能用于 设计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.