Adenosine, Toll-Like Receptors and Angiogenesis

腺苷、Toll 样受体和血管生成

• 批准号: 7691357
• 负责人: Samuel Joseph Leibovich
• 金额: $ 37.83万
• 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 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7691357
• 关键词: ADRBK1 gene; Adenosine; Adenosine A2A Receptor; Agonist; Antibodies; Arrestins; Biological Models; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Breeding; CD14 gene; Cells; Disease; Dose; Endothelial Cells; Ensure; Granulation Tissue; Granuloma; Healed; Hematopoietic; Impaired wound healing; In Vitro; Inflammation; Inflammatory; Injury; Knock-out; Knockout Mice; Lead; Ligands; Light; Luciferases; Malignant Neoplasms; Mediating; Mediator of activation protein; Messenger RNA; Modeling; Mus; Myelogenous; Myeloid Cells; Pathway interactions; Phase; Phenotype; Plasmids; Play; Population; Porifera; Process; Production; Protein Isoforms; Proteins; Receptor Activation; Receptor Gene; Receptor Signaling; Regulation; Reporter; Resolution; Role; Signal Pathway; Signal Transduction; Skin; TIE-2 Receptor; TLR2 gene; Technology; Testing; Time; Toll-Like Receptor 2; Toll-like receptors; Transplantation; Universities; Up-Regulation; Vascular Endothelial Growth Factors; Wild Type Mouse; Wound Healing; angiogenesis; cytokine; desensitization; healing; in vivo; insight; irradiation; macrophage; novel; promoter; public health relevance; receptor; receptor expression; reconstitution; response; transcription factor; vector; wound

DESCRIPTION (provided by applicant): Macrophages mediate inflammation and angiogenesis in healing wounds. We have discovered a novel pathway that switches macrophages from an M1 (production of inflammatory cytokines such as TNF1) to an M2-like phenotype (production of VEGF). This pathway involves a synergistic interaction of TLR 2, 4, 7 or 9 and adenosine A2A receptor (A2AR) signaling. TLR agonists up-regulate A2AR expression. A2AR agonists then up-regulate HIF11-I.1 mRNA levels, resulting in strong transcriptional up-regulation of VEGF expression. Mice lacking A2ARs have impaired wound healing, as do mice lacking MyD88, a key mediator of TLR signaling. MyD88-/- mice also fail to respond to the stimulatory effects of A2AR agonists. In this application, we will study: 1. Regulation of A2AR expression by TLR agonists: whether TLR agonists induce A2AR expression transcriptionally will be studied. Whether increased responsiveness of LPS-treated macrophages to A2AR activation is due to increased levels of A2ARs or to increased responsiveness of A2ARs due to inhibition of desensitization will be tested. The specific isoform of A2AR mRNA induced by LPS will also be defined. 2. Expression of A2ARs by macrophages in vivo: To determine if A2AR expression is upregulated in vivo, macrophages will be isolated from healing wounds at various time points following injury, and their expression of A2ARs examined. Ly-6Chi and Ly-6Clo macrophagesub-populations and their expression of A2ARs will be examined. 3. The role of macrophage versus endothelial cell A2AR expression in wound healing: A2AR- /- mice heal poorly, with reduced granulation tissue formation. We will transplant bone marrow from A2AR-/- to irradiated A2AR+/+ mice (and vice versa) to determine the role of myeloid cell A2AR expression in wound healing. To avoid potential complications due to irradiation, mice with A2ARs deleted specifically in myeloid cells using Cre-Lox technology will be bred. Mice with an endothelial cell deletion of A2ARs bred using Tie2-Cre mice will also be studied. As these mice have A2ARs deleted in both endothelial and hematopoietic cells, reconstitution with bone marrow from wild type mice (UbC-GFP mice) will be performed. 4. The role of the macrophage HIF11-I.1 isoform in wound healing: HIF11-I.1 is the predominant isoform induced by TLR- A2ARactivation in macrophages. Mice with global knockout of HIF11-I.1 isoform (HIF11-I.1-/-) will first be studied. To determine the role of HIF11-I.I expressed by macrophages, HIF11-I.1-/- mice transplanted with wild-type bone marrow and vice versa will then be studied. Mice with specific knockout of HIF11-I.1 in myeloid cells, obtained by breeding mice with floxed HIF11-I.1 with LysM-Cre mice, will then be studied. These studies should provide valuable insights into the regulation of A2AR expression by macrophages, and throw further light on the role of the TLR-A2AR-dependent switch in macrophage phenotype in the regulation of inflammation and wound healing in vivo. PUBLIC HEALTH RELEVANCE:: Macrophages are key cells that regulate inflammation and angiogenesis in wound healing, inflammatory fibroproliferative diseases and cancer. We have discovered a novel pathway that regulates macrophage function that requires an interaction between two disparate receptor types, namely Toll-like receptors (TLRs) and adenosine A2A receptors. This pathway switches macrophages from an inflammatory to an angiogenic, wound healing phenotype. In this application, we propose to study in detail the role of TLRs in regulating the expression and function of A2ARs, and to study the role of a key mediator of A2AR signaling, namely the HIF11 transcription factor and in particular, the HIF11-I.1 isoform. These factors will be studied both in vitro, and in vivo in models of wound healing. Understanding the pathways by which wound healing and angiogenesis are regulated should lead to the identification of novel targets for the pharmacological regulation of these processes, and thus potentially to novel therapies for diseases where these processes play a key role.

描述（由申请人提供）：巨噬细胞在伤口愈合中介导炎症和血管生成。我们发现了一种新的途径，可将巨噬细胞从 M1（产生炎症细胞因子，如 TNF1）转变为 M2 样表型（产生 VEGF）。该通路涉及 TLR 2、4、7 或 9 与腺苷 A2A 受体 (A2AR) 信号传导的协同相互作用。 TLR 激动剂上调 A2AR 表达。 A2AR 激动剂随后上调 HIF11-I.1 mRNA 水平，导致 VEGF 表达的转录上调。缺乏 A2AR 的小鼠会损害伤口愈合，缺乏 MyD88（TLR 信号转导的关键介质）的小鼠也是如此。 MyD88-/- 小鼠也无法对 A2AR 激动剂的刺激作用做出反应。在本申请中，我们将研究： 1. TLR激动剂对A2AR表达的调节：将研究TLR激动剂是否在转录上诱导A2AR表达。将测试 LPS 处理的巨噬细胞对 A2AR 激活的反应性增加是由于 A2AR 水平增加还是由于抑制脱敏而导致 A2AR 反应性增加。 LPS 诱导的 A2AR mRNA 的特定亚型也将被定义。 2.体内巨噬细胞的A2AR表达：为了确定体内A2AR表达是否上调，将从损伤后不同时间点愈合的伤口中分离巨噬细胞，并检查它们的A2AR表达。将检查 Ly-6Chi 和 Ly-6Clo 巨噬细胞亚群及其 A2AR 表达。 3.巨噬细胞与内皮细胞A2AR表达在伤口愈合中的作用：A2AR-/-小鼠愈合不良，肉芽组织形成减少。我们将 A2AR-/- 的骨髓移植到受辐射的 A2AR+/+ 小鼠（反之亦然），以确定骨髓细胞 A2AR 表达在伤口愈合中的作用。为了避免因辐射引起的潜在并发症，将培育使用 Cre-Lox 技术在骨髓细胞中特异性删除 A2AR 的小鼠。还将研究使用 Tie2-Cre 小鼠培育的 A2AR 内皮细胞缺失的小鼠。由于这些小鼠的内皮细胞和造血细胞中的 A2AR 均被删除，因此将使用野生型小鼠（UbC-GFP 小鼠）的骨髓进行重建。 4.巨噬细胞HIF11-I.1亚型在伤口愈合中的作用：HIF11-I.1是巨噬细胞中TLR-A2AR激活诱导的主要亚型。首先将研究 HIF11-I.1 同种型 (HIF11-I.1-/-) 整体敲除的小鼠。为了确定巨噬细胞表达的 HIF11-I.I 的作用，将研究移植野生型骨髓的 HIF11-I.1-/- 小鼠，反之亦然。然后将研究骨髓细胞中 HIF11-I.1 特异性敲除的小鼠，这些小鼠是通过将 HIF11-I.1 缺失的小鼠与 LysM-Cre 小鼠交配而获得的。这些研究应该为巨噬细胞调节 A2AR 表达提供有价值的见解，并进一步阐明巨噬细胞表型中 TLR-A2AR 依赖性开关在调节体内炎症和伤口愈合中的作用。公共卫生相关性：巨噬细胞是调节伤口愈合、炎症性纤维增殖性疾病和癌症中炎症和血管生成的关键细胞。我们发现了一种调节巨噬细胞功能的新途径，该途径需要两种不同受体类型（即 Toll 样受体 (TLR) 和腺苷 A2A 受体）之间的相互作用。该途径将巨噬细胞从炎症表型转变为血管生成、伤口愈合表型。在本申请中，我们建议详细研究 TLR 在调节 A2AR 表达和功能中的作用，并研究 A2AR 信号传导的关键介质（即 HIF11 转录因子，特别是 HIF11-I.1 亚型）的作用。这些因素将在体外和体内伤口愈合模型中进行研究。了解伤口愈合和血管生成的调节途径应该有助于识别这些过程的药理学调节的新靶标，从而有可能为这些过程发挥关键作用的疾病提供新的治疗方法。