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家族由与C. Elegans Exc 4，这对于 管腔形成。我们研究了CLIC作为血管生成调节因子的作用，发现CLIC 1和 CLIC 4在内皮细胞中表达，是内皮细胞增殖、迁移和管腔形成所必需的。 培养的内皮细胞。我们开发了具有异常视网膜血管生成的Clic 4敲除小鼠。 Clic 1/Clic 4双突变胚胎在子宫内死亡，并且具有血管发育缺陷，表明Clic 1 和Clic 4在内皮中是功能冗余的。我们假设小鼠Clic 1和Clic 4 在发育和病理性血管生成中的功能。为了检验这个假设，我们将删除Clic 4， 内皮特异性驱动因子（Clic 4 ECKO），在Clic 1-/-基因型的背景下，并分析血管 表型在胚胎、出生后视网膜和缺氧驱动的视网膜血管生成过程中。虽然克利奇 基因编码假定的氯离子通道，哺乳动物CLIC蛋白的分子作用很差， 定义了我们最近发现鞘氨醇-1-磷酸（S1 P）驱动的内皮细胞迁移， 粘附连接的形成和肌动蛋白应力纤维的形成需要CLIC 1或CLIC 4。通过选择性CLIC 敲低内皮细胞，我们记录了CLIC 1的重叠和不同的内皮功能 和CLIC 4，突出了CLIC的S1 P受体特异性活性。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S1 P信号传导的一部分发挥作用，我们的功能研究 将CLIC与G蛋白偶联受体信号传导连接。我们建议使用体外内皮研究来探索 CLIC和S1 P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CLIC 1或CLIC 4来介导S1 P驱动的增殖、存活、迁移、屏障形成 胶原凝胶侵入和管腔形成。我们假设CLIC 1和CLIC 4促进S1 P1信号传导 通过PI 3 K/RAC 1和PI 3 K/Akt途径。我们假设CLIC 1是S1 P唯一需要的 S1 P2-RhoA/ROCK通路的激活，而CLIC 4是S1 P1-RhoA/ROCK通路的S1 P激活所必需的。 RAS/ERK通路。我们将评估CLIC与S1 P受体或其下游细胞内 信号组件。S1 P1在视网膜的萌芽血管生成过程中起作用。内皮细胞特异性S1 P1 缺失导致视网膜出芽增加，而S1 P1功能获得性减少出芽。通过激活或 使用药理学和遗传学方法灭活小鼠中的S1 P1信号传导，沿着内皮细胞 Clic 4或Clic 1-/-小鼠的缺失，我们将阐述S1 P信号传导需要内皮CLIC来 调节血管新生发芽。