Toll-Like Receptors, Adenosine and Angiogenesis

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非常敏感，细胞因子和生长因子的产生受到严格调控。我们在巨噬细胞中发现了一条新的信号通路，导致血管内皮生长因子的强烈上调和肿瘤坏死因子α和IL-12的表达下调，构成了血管生成的开关。该通路涉及Toll样受体(TLR)-2、4和9激动剂与腺苷A2a受体(A2AR)激动剂的协同作用。这笔赠款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A2ARs和TLRs之间是否发生物理作用。将使用原代巨噬细胞和RAW264.7细胞，其中RAW264.7细胞带有表位标记的表达构建体。目的：利用基因敲除小鼠，将显性负性转录本导入RAW264.7A2A细胞，研究TLR通路下游信号成分(MyD88、IRAK-1、IRAK-4、IRAK-M)的作用。目的3：研究A2AR途径的下游信号成分。将研究G蛋白亚基(Gsalpha和Gi)以及腺苷环化酶激活和cAMP产生在协同作用中的作用。目的4将确定除血管内皮生长因子外的其他基因的表达是否受A2ARs和TLRs之间相互作用的调节。使用Affymetrix基因芯片的初步研究表明，这一途径调节着一小群基因。我们将扩展和确认基因芯片分析，然后使用二维差异凝胶电泳和MALDI-TOF MS进行蛋白质组学分析，以分析受这种协同作用途径差异调控的蛋白质。目的5将研究在TLR激动剂处理的巨噬细胞中A2AR激动剂下调肿瘤坏死因子α的机制。将研究调节水平(转录、转录后、mRNA稳定性)以及核因子-kappaB激活的作用。目的6研究A2ARS和TLRs之间的协同作用在体内调节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信号缺失)切除伤口的影响。这些实验应该有助于阐明体内这种协同作用的意义。

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.