Role of ciliary proteins in endothelial cells morphogenesis

描述（由申请人提供）：初级纤毛不活动，存在于多种细胞表面，在发育和疾病中起关键作用，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发生致命性心血管缺陷。然而，纤毛蛋白是否在血管组装和重塑中起直接作用尚不清楚。研究人员最近发现，在95%的常染色体显性多囊肾病（ADPKD）患者中发生突变的多囊蛋白-1 （PC1）与毛细血管形态发生基因-1产物（CMG-1） （CrIFT71的人类同源基因）共定位于内皮初级纤毛，后者是纤毛组装所需的纤束内运输（IFT）机制的一个组成部分。CMG-1和PC1均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基因或多囊毒素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，但导致ADPKD患者死亡的主要原因是心血管并发症，如高血压、动脉瘤、发病机制尚不清楚的出血。

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.