CLIC function in angiogenesis

CLIC 在血管生成中的功能

• 批准号: 8653256
• 负责人: Jan K. Kitajewski
• 金额: $ 39.68万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 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/8653256
• 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; 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; migration; mutant; postnatal; protein function; public health relevance; retina circulation disorder; retinal angiogenesis; sphingosine 1-phosphate; therapeutic target; trafficking; vasculogenesis

Project Summary (Abstract) 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.

项目摘要(摘要) CLIC家族由6个与线虫Exc4相关的人类蛋白(1-6)组成，Exc4对线虫的 管腔形成。我们研究了CLICs作为血管生成调节因子的作用，发现CLIC1和CLIC1 CLIC4在内皮细胞中表达，是血管内皮细胞增殖、迁移和管腔形成所必需的 培养内皮细胞。我们培育了具有异常视网膜血管生成的Clic4基因敲除小鼠。 Clic1/Clic4双突变胚胎在子宫内死亡，并且在血管发育方面存在缺陷，这表明Clic1 和Clic4在内皮功能上是多余的。我们假设小鼠的Clic1和Clic4 在发育和病理性血管生成中的作用。为了验证这一假设，我们将使用以下命令删除CLIC4 血管内皮细胞特异性驱动因子(Clic4ECKO)，在Clic1-/-基因背景下分析血管 胚胎、出生后视网膜和缺氧驱动的视网膜血管生成过程中的表型。尽管克里奇 编码可能的氯通道的基因，哺乳动物CLIC蛋白的分子作用很差 已定义。我们最近发现，鞘氨醇-1-磷酸(S1P)驱动的内皮细胞迁移， 粘连连接的形成和肌动蛋白应力纤维的形成需要CLIC1或CLIC4。按选择性CLIC 在内皮细胞中的敲除，我们记录了CLIC1重叠和不同的内皮功能 和CLIC4，突出S1P受体对CLICs的特异性活性。因此，我们提供了第一个证据 CLIC蛋白作为已知的血管生成途径、S1P信号和我们的功能研究的一部分发挥作用 将老生常谈与G蛋白偶联受体信号联系起来。我们建议使用体外内皮细胞研究来探索 CLICS与S1P通路之间的分子相互作用。我们将确定内皮细胞是否 细胞需要CLIC1或CLIC4来介导S1P驱动的增殖、存活、迁移、屏障形成、 胶原凝胶侵入，管腔形成。我们假设CLIC1和CLIC4促进S1P1信号转导 通过PI3K/RAC1和PI3K/Akt通路。我们假设CLIC1是S1P唯一需要的 激活S1P2-RhoA/ROCK途径，而S1P1-S1P激活需要CLIC4- RAS/ERK通路。我们将评估CLICS与S1P受体或其下游细胞内的结合 信令组件。S1P1在视网膜新生血管形成过程中发挥作用。内皮细胞特异性S1P1 丢失导致视网膜发芽增加，S1P1功能增强发芽减少。通过激活或 使用药物和遗传方法以及内皮细胞使小鼠的S1P1信号失活 丢失Clic4或Clic1-/-小鼠，我们将解决S1P信号需要内皮细胞克隆来 调节新生血管的萌发。