Role of ciliary proteins in endothelial cells morphogenesis

描述（由申请人提供）：在多种细胞类型的表面上发现了非运动的初级纤毛，并在发育和疾病中发挥关键作用，包括多囊肾病、脑积水、综合征性肥胖和视网膜变性。 携带纤毛基因突变的小鼠的一个突出特征是在发育早期致命的心血管缺陷的叛乱。 然而，目前还不清楚纤毛蛋白是否在血管组装和重塑中发挥直接作用。 研究人员最近表明，多囊蛋白-1（PC 1）在95%的常染色体显性遗传性多囊肾病（ADPKD）患者中发生突变，与毛细血管形态发生基因-1产物（CMG-1）一起共定位于内皮初级纤毛中，CMG-1是CrIFT 71的人类直系同源物，是纤毛组装所需的鞭毛内转运（IFT）机制的一个组成部分。 CMG-1和PC 1都在胚胎心血管系统中表达，并在毛细血管形态发生过程中上调。 IFT基因或多囊蛋白突变的小鼠在子宫内死亡，主要是因为心血管和肾脏发育异常。 此外，虽然肾脏表现导致肾功能逐渐下降，但ADPKD患者死亡的主要原因是心血管并发症，如高血压、动脉瘤和发病机制不明的出血。 本申请解决了主要假设，即在血管发育和重塑所必需的内皮细胞（EC）的正常形态发生变化期间需要纤毛蛋白如CMG-1和PC 1。 在具体目标1中，研究人员将测试CMG-1和PC 1在体外系统中毛细管样结构的EC组装过程中的作用。 他们将通过用慢病毒RNAi载体转导HUVEC来敲低CMG-1和PC 1，并分别从携带Pkd 1靶向突变的敲除小鼠（Pkd 1del 17 - 21 geo）和IFFT 88缺陷小鼠（Tg 737 orpk/orpk）中获得EC系。 在后者的几种细胞类型中报告了纤毛缩短或消融。 表达低水平CMG-1和PC 1的EC的原代培养物以及来自突变小鼠的EC将用于进行体外功能测定，例如毛细血管形态发生、芽形成、细胞增殖、细胞粘附和细胞迁移。 EC暴露于血流动力学的力量发生显着的形态发生变化。 在特定目标2中，研究者将测试纤毛或PC 1缺陷EC在体外和体内系统中沿层流方向沿着流动的能力。 纤毛蛋白是EC形态发生所必需的直接证据，将阐明ADPKD血管表现的发病机制，并为未来的研究提供了一个新的视角，以了解发育过程中的心血管缺陷和血管疾病。 公共卫生相关性：血管并发症是许多疾病发病率和死亡率的主要原因。 这个项目将阐明初级纤毛在血管树的组装和重塑中所起的作用。 如果研究人员的假设是正确的，这将导致新的治疗方法来治疗心血管疾病。

DESCRIPTION (provided by applicant): Non-motile, primary cilia are found on the surface of a strikingly diverse range of cell types and play critical roles in development and disease, including polycystic kidney disease, hydrocephalus, syndromic obesity, and retinal degeneration. A prominent feature of mice carrying mutations in ciliary genes is the insurgency of lethal cardiovascular defects early in development. It is unclear however, whether ciliary proteins play a direct role in vasculature assembly and remodeling. The investigators have recently shown that polycystin-1 (PC1), which is mutated in 95% of patients affected by autosomal dominant polycystic kidney disease (ADPKD), co-localizes in the endothelial primary cilium together with the capillary morphogenesis gene-1 product (CMG-1), the human ortholog of CrIFT71, a component of the intraflagellar transport (IFT) machinery required for ciliary assembly. Both, CMG-1 and PC1 are expressed in the embryonic cardiovascular system and are upregulated during capillary morphogenesis. Mice with mutations in IFT genes or in polycystins die in utero, primarily because of abnormal cardiovascular and renal development. In addition, although the kidney manifestation results in a gradual decline in renal function, the leading cause of death in patients with ADPKD is due to cardiovascular complications such as hypertension, aneurysm, and hemorrhage of unclear pathogenesis. This application addresses the main hypothesis that ciliary proteins such as CMG-1 and PC1 are required during normal morphogenetic changes of endothelial cells (EC) essential for vasculature development and remodeling. In Specific Aim 1, the investigators will test the role of CMG-1 and PC1 during EC assembly of capillary-like structures in in vitro systems. They will knock-down CMG-1 and PC1 by transducing HUVEC with lentiviral RNAi vectors and obtain EC lines from knock-out mice carrying a targeted mutation in Pkd1 (Pkd1del17-21geo) and mice defective in IFT88 (Tg737orpk/orpk), respectively. Shortened or ablated cilia were reported in several cell types of the latter. Primary cultures of EC expressing low levels of CMG-1 and PC1, and EC from mutant mice will be utilized to perform in vitro functional assays such as capillary morphogenesis, sprout formation, cell proliferation, cell adhesion, and cell migration. EC exposed to hemodynamic forces undergo significant morphogenetic changes. In Specific Aim 2, the investigators will test the ability of cilia- or PC1-deficient EC to polarize along the direction of a laminar flow in in vitro and in vivo systems. Direct evidence that ciliary proteins are required for morphogenesis of the EC would shed light on the pathogenesis of the vascular manifestation of ADPKD, and provide a novel perspective for future studies to understand cardiovascular defects during development and vascular diseases. PUBLIC HEALTH RELEVANCE: Vascular complications are a leading cause of morbidity and mortality in a number of diseases. This project will elucidate the role that primary cilia play in the assembly and remodeling of the vascular tree. If the investigators' hypothesis is correct, this will lead to novel therapeutic approaches to the treatment of cardiovascular diseases.