Adenosine, Toll-Like Receptors and Angiogenesis

描述（申请人提供）：巨噬细胞在伤口愈合过程中介导炎症和血管生成。我们发现了一种新的途径，可以将巨噬细胞从M1（产生炎症细胞因子如TNF1）转换为m2样表型（产生VEGF）。该途径涉及tlr2、4、7或9与腺苷A2A受体（A2AR）信号的协同相互作用。TLR激动剂上调A2AR表达。然后A2AR激动剂上调HIF11-I。1 mRNA水平，导致VEGF表达的强烈转录上调。缺乏A2ARs的小鼠伤口愈合受损，缺乏MyD88的小鼠也是如此，MyD88是TLR信号的关键介质。MyD88-/-小鼠也对A2AR激动剂的刺激作用没有反应。在本应用中，我们将研究：1。TLR激动剂对A2AR表达的调控：TLR激动剂是否通过转录诱导A2AR表达将被研究。lps处理的巨噬细胞对A2AR激活的反应性增加是由于A2ARs水平的增加，还是由于抑制脱敏导致A2ARs的反应性增加，我们将进行测试。LPS诱导的A2AR mRNA特异性异构体也将被定义。2. 体内巨噬细胞表达A2ARs：为了确定体内A2AR表达是否上调，我们将在损伤后的不同时间点从愈合伤口中分离巨噬细胞，检测其A2ARs表达。我们将检测Ly-6Chi和Ly-6Clo巨噬亚群及其A2ARs的表达。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。我们将把A2AR-/-小鼠的骨髓移植到辐照的A2AR+/+小鼠体内（反之亦然），以确定髓系细胞A2AR表达在伤口愈合中的作用。为了避免辐照引起的潜在并发症，将使用Cre-Lox技术培育髓细胞中特异性缺失A2ARs的小鼠。内皮细胞A2ARs缺失的小鼠也将用Tie2-Cre小鼠繁殖。由于这些小鼠内皮细胞和造血细胞中都缺失了A2ARs，因此将使用野生型小鼠（UbC-GFP小鼠）的骨髓进行重构。4. 巨噬细胞HIF11-I的作用。1异构体在伤口愈合：HIF11-I。1是巨噬细胞中TLR- a2ar活化诱导的主要亚型。HIF11-I基因全基因敲除小鼠。将首先研究1异构体（HIF11-I.1-/-）。确定HIF11-I的作用。I由巨噬细胞表达，HIF11-I。然后将研究1-/-小鼠移植野生型骨髓，反之亦然。特异性敲除HIF11-I的小鼠。hf11 - 1在骨髓细胞中的表达，是通过给小鼠注射hf11 - 1获得的。然后将对LysM-Cre小鼠进行研究。这些研究将为巨噬细胞对A2AR表达的调控提供有价值的见解，并进一步阐明巨噬细胞表型中tlr -A2AR依赖性开关在体内调节炎症和伤口愈合中的作用。公共卫生相关性：巨噬细胞是在伤口愈合、炎性纤维增生性疾病和癌症中调节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（TLRs）和腺苷A2A受体之间的相互作用。这一途径将巨噬细胞从炎症型转变为血管生成型和伤口愈合型。在本次申请中，我们拟详细研究TLRs在调节A2ARs表达和功能中的作用，并研究A2AR信号传导的关键介质HIF11转录因子，特别是HIF11- i的作用。1)同种型。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控途径，将有助于确定这些过程的药理学调控的新靶点，从而有可能开发出针对这些过程发挥关键作用的疾病的新疗法。

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.