CLIC function in angiogenesis

CLIC 在血管生成中的功能

• 批准号: 9181450
• 负责人: Jan K. Kitajewski
• 金额: $ 39.98万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 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 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9181450
• 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; Dimensions; 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; Pathologic Neovascularization; Pathway interactions; Pharmacology; Phenotype; Process; Proteins; Regulatory Pathway; Retina; Retinal; Role; Signal Pathway; Signal Transduction; Sphingosine-1-Phosphate Receptor; Stress Fibers; Testing; Tumor Angiogenesis; angiogenesis; cadherin 5; cell behavior; cell motility; cell type; design; gain of function; in utero; in vitro Assay; in vivo; knock-down; migration; mutant; postnatal; protein function; public health relevance; retina circulation disorder; retinal angiogenesis; sphingosine 1-phosphate; therapeutic target; trafficking; vasculogenesis

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 家族由与线虫 Exc4 相关的六种人类蛋白 (1-6) 组成，对于管腔形成至关重要。我们研究了 CLIC 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，并且是培养内皮细胞的增殖、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 基因敲除小鼠。 Clic1/Clic4 双突变体胚胎会在子宫内死亡，并且血管发育存在缺陷，这表明 Clic1 和 Clic4 在内皮细胞中存在功能冗余。我们假设小鼠 Clic1 和 Clic4 在发育和病理性血管生成中发挥作用。为了检验这一假设，我们将在 Clic1-/- 基因型背景下使用内皮特异性驱动程序 (Clic4ECKO) 删除 Clic4，并分析胚胎、出生后视网膜和缺氧驱动的视网膜血管生成过程中的血管表型。尽管 Clic 基因编码假定的氯离子通道，但哺乳动物 CLIC 蛋白的分子作用尚不清楚。我们最近发现，1-磷酸鞘氨醇 (S1P) 驱动的内皮细胞迁移、粘附连接形成和肌动蛋白应力纤维形成需要 CLIC1 或 CLIC4。通过在内皮细胞中选择性敲除 CLIC，我们记录了 CLIC1 和 CLIC4 的重叠和不同的内皮功能，突出了 CLIC 的 S1P 受体特异性活性。因此，我们提供了第一个证据，证明 CLIC 蛋白作为已知血管生成途径、S1P 信号传导的一部分起作用，并且我们的功能研究将 CLIC 与 G 蛋白偶联受体信号传导联系起来。我们建议利用体外内皮研究来探索 CLIC 和 S1P 通路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、胶原凝胶侵入和管腔形成。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 通路的 S1P 激活所必需的，而 CLIC4 是 S1P1-RAS/ERK 通路的 S1P 激活所必需的。我们将评估 CLIC 与 S1P 受体或其下游细胞内信号传导成分的结合。 S1P1 在视网膜新生血管生成过程中发挥作用。内皮特异性 S1P1 缺失导致视网膜萌芽增加，而 S1P1 功能获得则导致视网膜萌芽减少。通过使用药理学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-/- 小鼠的内皮细胞损失，我们将解决 S1P 信号传导需要内皮 CLIC 来调节血管生成萌芽的假设。