Toll-Like Receptors, Adenosine and Angiogenesis

Toll 样受体、腺苷和血管生成

• 批准号: 7006488
• 负责人: Samuel Joseph Leibovich
• 金额: $ 2.36万
• 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 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7006488
• 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起关键作用。巨噬细胞对它们的微环境非常敏感，细胞因子和生长因子的产生受到严格的调节。我们已经在巨噬细胞中发现了一种新的信号通路，其导致VEGF的强烈上调和TNF α和IL-12表达的下调，构成了血管生成开关。该途径涉及Toll样受体（TLR）-2，4和9激动剂与腺苷A2 A受体（A2 AR）激动剂之间的协同相互作用。该基金将研究这些不同信号通路之间协同作用的基础。目的1：免疫共沉淀技术将用于确定A2 AR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。目标二：将使用基因敲除小鼠并将显性负性转录物转染到RAW264.7A2A细胞中，研究TLR途径的下游信号传导组分（MyD 88、IRAK-1、IRAK-4、IRAK-M）的作用。目的3：研究A2 AR通路的下游信号成分。将研究G蛋白亚单位（Gsalpha和Gi）以及腺苷酸环化酶活化和cAMP产生在协同相互作用中的作用。目的4：研究A2 ARs与TLRs之间的相互作用是否对VEGF以外基因的表达有调节作用。 使用Affyssin基因芯片的初步研究表明，该途径调节一小群基因。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，以分析受这种协同途径差异调节的蛋白质。目的5将检查TLR激动剂处理的巨噬细胞中A2 AR激动剂下调TNF α的机制。将研究调节水平（转录、转录后、mRNA稳定性）和NF-κ B活化的作用。目的6研究A2 ARS与TLRs之间的协同作用在体内创伤愈合和血管生成中的作用。将分析A2 AR激动剂和拮抗剂对MyD 88小鼠（TLR信号传导缺陷）中切除伤口的影响。这些实验应有助于阐明这种协同作用在体内的意义。