Toll-Like Receptors, Adenosine and Angiogenesis

Toll 样受体、腺苷和血管生成

• 批准号: 7067191
• 负责人: Samuel Joseph Leibovich
• 金额: $ 36.3万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7067191
• 关键词: G protein; adenosine; adenylate cyclase; alpha adrenergic agent; angiogenesis; biological signal transduction; cyclic AMP; gene expression; gene induction /repression; gene targeting; genetically modified animals; immunoprecipitation; laboratory mouse; ligands; macrophage; matrix assisted laser desorption ionization; microarray technology; protein protein interaction; proteomics; purinergic receptor; toll like receptor; tumor necrosis factor alpha; two dimensional gel electrophoresis; vascular endothelial growth factors; wound healing

DESCRIPTION (provided by applicant): Macrophages play a key role in wound repair and fibroproliferation by producing cytokines and growth factors that regulate both inflammation and angiogenesis. Macrophages are exquisitely sensitive to their micro-environment, and production of cytokines and growth factors is tightly regulated. We have discovered a novel signaling pathway in macrophages that results in the strong up-regulation of VEGF and downregulation of TNFalpha and IL-12 expression, constituting an angiogenic switch. This pathway involves a synergistic interaction between Toll-like receptor (TLR)-2, 4 and 9 agonists and adenosine A2A receptor (A2AR) agonists. This grant will study the basis for the synergistic interaction between these disparate signaling pathways. Aim 1: Co-immunoprecipitation techniques will be used to determine whether physical interaction occurs between A2ARs and TLRs. Primary macrophages and RAW264.7 cells transfected with epitope-tagged expression constructs will be used. Aim 2: The role of down-stream signaling components of the TLR pathway (MyD88, IRAK-1, IRAK-4, IRAK-M) will be studied, using knockout mice, and transfection of dominant negative transcripts into RAW264.7A2A cells. Aim 3: Down-stream signaling components of the A2AR pathway will be studied. The role of G-protein sub-units (Gsalpha and Gi), and adenylyl cyclase activation and cAMP production in the synergistic interaction will be studied. Aim 4 will determine whether the expression of genes other than VEGF is regulated by the interaction between A2ARs and TLRs. Initial studies using Affymetrix gene chips indicate that this pathway regulates a small group of genes. We will extend and confirm the gene chip analysis, and then perform proteomic analysis using 2-D Difference Gel Electrophoresis and MALDI-TOF MS, to analyze proteins that are differentially regulated by this synergistic pathway. Aim 5 will examine the mechanism of down-regulation of TNFalpha by A2AR agonists in TLR-agonist-treated macrophages. The level of regulation (transcriptional, post-trancriptional, mRNA stability), and the role of NF-KappaB activation will be studied. Aim 6 will study the role of the synergistic interaction between A2ARS and TLRs in regulating wound healing and angiogenesis in vivo. The effects of A2AR agonists and antagonists on excisional wounds in MyD88 mice (deficient in TLR signaling) will be analyzed. These experiments should help to clarify the significance of this synergistic interaction in vivo.

描述（由申请人提供）： 巨噬细胞通过产生调节炎症和血管生成的细胞因子和生长因子，在伤口修复和纤维增殖中发挥关键作用。巨噬细胞对其微环境极其敏感，细胞因子和生长因子的产生受到严格调节。我们在巨噬细胞中发现了一条新的信号通路，导致 VEGF 强烈上调，TNFα 和 IL-12 表达下调，构成血管生成开关。该途径涉及 Toll 样受体 (TLR)-2、4 和 9 激动剂与腺苷 A2A 受体 (A2AR) 激动剂之间的协同相互作用。这笔赠款将研究这些不同信号通路之间协同相互作用的基础。目标 1：免疫共沉淀技术将用于确定 A2AR 和 TLR 之间是否发生物理相互作用。将使用用表位标记的表达构建体转染的原代巨噬细胞和RAW264.7细胞。目标 2：将使用基因敲除小鼠以及将显性失活转录物转染至 RAW264.7A2A 细胞中来研究 TLR 通路下游信号成分（MyD88、IRAK-1、IRAK-4、IRAK-M）的作用。目标 3：将研究 A2AR 通路的下游信号传导成分。将研究 G 蛋白亚基（Gsalpha 和 Gi）以及腺苷酸环化酶激活和 cAMP 产生在协同相互作用中的作用。目标 4 将确定 VEGF 以外的基因表达是否受到 A2AR 和 TLR 之间的相互作用的调节。 使用 Affymetrix 基因芯片的初步研究表明，该途径调节一小部分基因。我们将扩展并确认基因芯片分析，然后使用二维差异凝胶电泳和MALDI-TOF MS进行蛋白质组分析，以分析受该协同途径差异调节的蛋白质。目标 5 将研究 TLR 激动剂处理的巨噬细胞中 A2AR 激动剂下调 TNFα 的机制。将研究调节水平（转录、转录后、mRNA 稳定性）以及 NF-KappaB 激活的作用。目标 6 将研究 A2ARS 和 TLR 之间的协同相互作用在调节体内伤口愈合和血管生成中的作用。将分析 A2AR 激动剂和拮抗剂对 MyD88 小鼠（TLR 信号传导缺陷）切除伤口的影响。这些实验应该有助于阐明这种体内协同相互作用的重要性。