CLIC function in angiogenesis

CLIC 在血管生成中的功能

基本信息

批准号：
9295237
负责人：
Jan K. Kitajewski
金额：
$ 32.58万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-12-20 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9295237
关键词：
Actins Address Adhesions Agonist Apoptosis Binding Biology Blood Vessels CLIC4 gene Caenorhabditis elegans Cell Mobility Cell Polarity Cell Proliferation Cell surface Cells Chloride Channels Collagen Data Defect Development Disease Embryo Embryonic Development Endothelial Cells Endothelium Family G Protein-Coupled Receptor Signaling Gel Genes Genetic Genotype Growth Health Human Hypoxia In Vitro Inflammatory Knockout Mice Knowledge Link Mediating Methods Modeling Molecular Mus Pathologic Neovascularization Pathway interactions Phenotype Process Proteins Regulatory Pathway Research Design Retina Retinal Role Signal Pathway Signal Transduction Sphingosine-1-Phosphate Receptor Staging Stress Fibers Testing Tumor Angiogenesis angiogenesis cadherin 5 cell behavior cell motility cell type gain of function in utero in vitro Assay in vivo knock-down migration mutant postnatal protein function retina circulation disorder retinal angiogenesis sphingosine 1-phosphate therapeutic target trafficking vasculogenesis

项目摘要

DESCRIPTION (provided by applicant): The CLIC family consists of six human proteins (1-6) related to C. elegans Exc4, which is essential for lumen formation. We investigated the roles of CLICs as angiogenic regulators and found that CLIC1 and CLIC4 are expressed in endothelial cells and are required for proliferation, migration and lumen formation of cultured endothelial cells. We developed Clic4 knockout mice that had aberrant retinal angiogenesis. Clic1/Clic4 double mutant embryos die in utero and have defects in vascular development, suggesting Clic1 and Clic4 are functionally redundant in the endothelium. We hypothesize that murine Clic1 and Clic4 function in developmental and pathological angiogenesis. To test this hypothesis, we will delete Clic4 using endothelial specific drivers (Clic4ECKO), in the background of the Clic1-/- genotype and analyze vascular phenotypes in the embryo, postnatal retina and during hypoxia-driven retinal angiogenesis. Although Clic genes encode putative chloride channels, the molecular action of mammalian CLIC proteins is poorly defined. We recently discovered that sphingosine-1-phosphate (S1P)-driven endothelial cell migration, adhesion junction formation, and actin stress fiber formation requires CLIC1 or CLIC4. By selective CLIC knockdowns in endothelial cells, we document both overlapping and distinct endothelial functions for CLIC1 and CLIC4, highlighting S1P receptor specific activities for CLICs. Thus, we provide the first evidence that CLIC proteins function as part of a known angiogenic pathway, S1P signaling, and our functional studies link CLICs to G protein coupled receptor signaling. We propose to use in vitro endothelial studies to explore the molecular interactions between CLICs and the S1P pathway. We will determine whether endothelial cells require CLIC1 or CLIC4 to mediate S1P-driven proliferation, survival, migration, barrier formation, collagen gel invasion, and lumen formation. We hypothesize that CLIC1 and CLIC4 promote S1P1 signaling via the PI3K/RAC1 and PI3K/Akt pathways. We hypothesize that CLIC1 is uniquely required for S1P activation of the S1P2-RhoA/ROCK pathway, while CLIC4 is required for S1P activation of the S1P1- RAS/ERK pathway. We will assess binding of CLICs to S1P receptors or their downstream intracellular signaling components. S1P1 functions during sprouting angiogenesis of the retina. Endothelial specific S1P1 loss caused increased retinal sprouting and S1P1 gain-of-function decreased sprouting. By activating or inactivating S1P1 signaling in mice, using both pharmacologic and genetic methods, along with endothelial loss of Clic4 or Clic1-/- mice, we will address the hypothesis that S1P signaling requires endothelial CLICs to regulate angiogenic sprouting.

描述（由申请人提供）：CLIC家族由六种与秀丽隐杆线虫Exc4相关的人类蛋白质（1-6）组成，这对于管腔形成至关重要。我们研究了库作为血管生成调节剂的作用，发现CLIC1和CLIC4在内皮细胞中表达，是培养的内皮细胞的增殖，迁移和管腔形成所必需的。我们开发了具有异常视网膜血管生成的Clic4敲除小鼠。 CLIC1/CLIC4双突变胚胎在子宫内死亡，并在血管发育中存在缺陷，这表明CLIC1和CLIC4在内皮中功能上是多余的。我们假设鼠Clic1和Clic4在发育和病理血管生成中的功能。为了检验该假设，我们将在Clic1 - / - 基因型的背景下使用内皮特异性驱动因素（Clic4ecko）删除CLIC4，并分析胚胎，产后视网膜和低氧驱动视网膜血管生成期间的血管表型。尽管CLIC基因编码推定的氯化物通道，但哺乳动物Clic蛋白的分子作用却很差。我们最近发现，鞘氨醇1-磷酸（S1P）驱动的内皮细胞迁移，粘附连接形成和肌动蛋白应激纤维形成需要CLIC1或CLIC4。通过在内皮细胞中的选择性CLIC敲低，我们记录了CLIC1和CLIC4的重叠和不同的内皮功能，突出了CLICS的S1P受体特定活性。因此，我们提供了第一个证据，表明Clic蛋白作为已知的血管生成途径，S1P信号传导的一部分，我们的功能研究将CLIC与G蛋白偶联受体信号连接起来。我们建议使用体外内皮研究来探索CLICS与S1P途径之间的分子相互作用。我们将确定内皮细胞是否需要CLIC1或CLIC4来介导S1P驱动的增殖，存活，迁移，屏障形成，胶原蛋白凝胶侵袭和管腔形成。我们假设CLIC1和CLIC4通过PI3K/RAC1和PI3K/AKT途径促进S1P1信号传导。我们假设CLIC1是S1P2-RHOA/ROCK途径的S1P激活所必需的，而S1P激活S1P1- RAS/ERK途径需要CLIC4。我们将评估CLIC与S1P受体或其下游细胞内信号传导分量的结合。 S1P1在视网膜发芽血管生成过程中起作用。内皮特异性S1P1损失导致视网膜发芽增加，而S1P1功能获得降低了发芽。通过使用药物和遗传学方法激活或灭活小鼠中的S1P1信号传导，以及CLIC4或CLIC1 - / - 小鼠的内皮损失，我们将解决S1P信号传导需要内皮层来调节血管疾病发育的假设。