Role of ciliary proteins in endothelial cells morphogenesis

描述(申请人提供)：在各种各样的细胞表面发现不活动的原生纤毛，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退化。携带纤毛基因突变的小鼠的一个显著特征是在发育早期就有致命的心血管缺陷。然而，目前尚不清楚纤毛蛋白是否在血管系统组装和重塑中发挥直接作用。研究人员最近发现，在95%的常染色体显性遗传性多囊肾病(ADPKD)患者中突变的多囊蛋白-1(PC1)与毛细血管形态发生基因-1产物(CMG-1)共定位于内皮初级纤毛中，CMG-1是CRIFT71的人类同源基因，是纤毛组装所需的鞭毛内运输(IFT)机制的组成部分。CMG-1和PC1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。携带IFT基因突变或多囊蛋白突变的小鼠在子宫中死亡，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患者的主要死亡原因是高血压、动脉瘤和发病机制不明的出血等心血管并发症。 这一应用解决了一个主要假设，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过程中，需要CMG-1和PC1等纤毛蛋白。在特定的目标1中，研究人员将测试CMG-1和PC1在体外系统中毛细血管样结构的EC组装过程中的作用。他们将通过用慢病毒RNAi载体转导HUVEC来击倒CMG-1和PC1，并分别从携带PKD1靶向突变的小鼠(Pkd1del17-21geo)和IFT88缺陷小鼠(Tg737orpk/orpk)获得EC系。后者的几种细胞类型中纤毛缩短或消融。原代培养的低水平表达CMG-1和PC1的EC和突变小鼠的EC将用于体外功能分析，如毛细血管形态发生、芽形成、细胞增殖、细胞黏附和细胞迁移。内皮细胞在血流动力学的作用下会发生显著的形态变化。在特定目标2中，研究人员将测试纤毛或PC1缺乏的EC在体外和体内系统中沿着层流方向极化的能力。纤毛蛋白在内皮细胞形态发生中所需的直接证据将有助于阐明ADPKD血管表现的发病机制，并为进一步研究发育中的心血管缺陷和血管疾病提供新的视角。 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维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，这将导致治疗心血管疾病的新疗法。

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.