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.
Macrophages are key cells that regulate inflammation and angiogenesis in woundhealing; inflammatory fibroproliferative diseases and cancer. We have discovered a novelpathway that regulates macrophage function that requires an interaction between twodisparate receptor types; namely Toll-like receptors (TLRs) and adenosine A2A receptors.This pathway switches macrophages from an inflammatory to an angiogenic; woundhealing phenotype. In this application; we propose to study in detail the role of TLRs inregulating the expression and function of A2ARs; and to study the role of a key mediatorof A2AR signaling; namely the HIF1 transcription factor and in particular; the HIF1 -I.1isoform. These factors will be studied both in vitro; and in vivo in models of woundhealing. Understanding the pathways by which wound healing and angiogenesis areregulated should lead to the identification of novel targets for the pharmacologicalregulation of these processes; and thus potentially to novel therapies for diseases wherethese processes play a key role.
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