Comprehensive Analysis of Polycystin Related Endothelial Cell Signaling Pathways

描述（由申请人提供）：常染色体显性遗传性多囊肾病（ADPKD）是遗传性肾衰竭的最常见形式，由PKD 1和PKD 2两种基因突变引起。基于动脉瘤和ADPKD的相关性，推测多囊蛋白（PKD蛋白）在维持血管完整性方面发挥重要作用。由于Pkd无效动物表现出水肿和出血，因此该表型涉及内皮细胞缺陷。尽管这个问题的相关性，有很少知道的作用，多囊蛋白发挥内皮细胞。我们最近使用Cre-Lox技术诱导内皮细胞特异性敲除小鼠的Pkd 1或Pkd 2。我们发现，内皮细胞中任一基因的选择性失活产生了上述血管缺陷的一个子集。 在Pkd无效胚胎中，包括约30-40%的围产期死亡率、偶尔出血、羊水过多和胎盘异常。值得注意的是，这些胚胎缺乏水肿，这是所有靶向Pkd无效等位基因的普遍特征。这些研究证明了多囊蛋白在内皮细胞区室中的功能作用，并促使我们假设功能性多囊蛋白的丧失导致内皮细胞相关信号通路的失调。在本申请中，我们提出了三个具体的目标，寻求在细胞和整个动物水平上发展对多囊蛋白在内皮细胞中发挥的功能作用的机制理解。在Aim 1中，我们将使用Pkd无效和条件等位基因来探索Pkd 1/Pkd 2突变动物中水肿形成的基础。目的2是基于我们的观察，即多囊蛋白耗尽的内皮细胞表现出有缺陷的细胞迁移。我们将深入调查 使用延时成像和生物化学方法表征这种表型。此外，我们将使用新的细胞生物传感器来探测与细胞迁移有关的明确的信号通路。在目标3中，我们将采用从不同妊娠时间点的胚胎中分离的内皮细胞的全面微阵列研究来推断在体内Pkd突变内皮细胞中被破坏的调节网络。在这些研究的结论，我们将获得新的见解，多囊蛋白如何适应血管发育的背景。希望这些基本范式将对多囊蛋白功能的定义产生更广泛的影响，尽管多年的深入研究，这个主题仍然模糊不清。

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of inherited renal failure and is caused by mutations two genes, PKD1 and PKD2. Based on the association of aneurysms and ADPKD it has been speculated that polycystins (PKD proteins) play an important role in maintaining blood vessel integrity. An endothelial cell defect has been implicated in this phenotype since Pkd null animals exhibit edema and hemorrhage. Despite the relevance of this problem, there is little known about the role that polycystins play in endothelial cells. We recently used Cre-Lox technology to induce an endothelial cell specific knock out of Pkd1 or Pkd2 in mice. We found that selective inactivation of either gene in endothelial cells yielded a subset of the vascular defects described in Pkd null embryos including ~30-40% perinatal lethality, occasional hemorrhage, polyhydramnios and placental abnormalities. Remarkably, these embryos lacked edema, which is a universal feature of all targeted Pkd null alleles. These studies demonstrated a functional role for polycystins in the endothelial cell compartment and prompted us to hypothesize that loss of functional polycystins results in dysregulation of endothelial cell related signaling pathways. n this application, we propose three specific aims that seek to develop a mechanistic understanding of the functional role that polycystins play in endothelial cells at both the cellula and whole animal level. In Aim1 we will use Pkd null and conditional alleles to explore the basis of edema formation in Pkd1/Pkd2 mutant animals. Aim 2 is based on our observation that polycystin depleted endothelial cells exhibit defective cell migration. We will conduct an in depth characterization of this phenotype using time-lapse imaging and biochemical methods. In addition we will use novel cellular biosensors to probe well-defined signaling pathways implicated in cell migration. In Aim 3 we will employ comprehensive microarray studies of endothelial cells isolated from embryos at different gestational time points to infer the regulator networks that are disrupted in Pkd mutant endothelial cells in vivo. At the conclusion of these studies, we will have gained novel insights into how polycystins fit into the context of vascular development. The hope is that these fundamental paradigms will have broader implications for defining polycystin function, a subject that remains vaguely understood despite many years of intense investigation.