Myeloid glycolysis in pathological ocular angiogenesis

病理性眼血管生成中的髓样糖酵解

基本信息

批准号：
10456819
负责人：
Ruth B Caldwell
金额：
$ 43.92万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10456819
关键词：
6-Phosphofructo-2-kinase 6-Phosphofructokinase Adult Age Age related macular degeneration Angiogenic Factor Area Biological Assay Blindness Blood Vessels Bone Marrow Cell surface Cells Choroid Choroidal Neovascularization Conditioned Culture Media Consensus Critical Pathways Data Development Elderly Endothelial Cells Endothelium Enzyme Activation Enzymes Exhibits Eye diseases Fructose Fructose-2,6-bisphosphatase Genetic Glycolysis Growth Histone Acetylation Hypoxia Hypoxia Inducible Factor In Vitro Individual Inflammatory Laser injury Lesion Leukocytes Measures Mediating Messenger RNA Metabolism Methods Microglia Molecular Mus Myelogenous Myeloid Cells Newborn Infant Oxygen Pathologic Pathologic Neovascularization Pathway interactions Peripheral Phenotype Play Production Protein Isoforms Proteins Retina Retinal Diseases Retinal Neovascularization Retinopathy of Prematurity Role Sampling Signal Pathway Signal Transduction Testing Transgenic Organisms Vascular Endothelial Growth Factors angiogenesis cell type chromatin immunoprecipitation cytokine design in vitro Model in vivo knock-down macrophage middle age mouse model neovascular neovascularization ocular angiogenesis overexpression proliferative diabetic retinopathy promoter retinal angiogenesis tool

项目摘要

PROJECT SUMMARY This proposal seeks to develop a new molecular strategy to limit pathological retinal angiogenesis in blinding eye disease. An emerging consensus is that macrophages/microglia drive aberrant neovascularization. However, the mechanisms involved remain poorly defined. Glycolysis is a major metabolic process in macrophages. PFKFB3 (6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase isoform 3, Pfkfb3 for mice) is a critical enzyme for activation of glycolysis in vascular cells and leukocytes. It catalyzes the synthesis of fructose-2, 6- bisphosphate, which is the most potent allosteric activator of 6-phosphofructo-1-kinase, a rate-limiting enzyme for glycolysis. PFKFB3 is critical for the inflammatory phenotype of macrophages, and glycolysis has been shown to promote activation of hypoxia-inducible factor (HIF) or histone acetylation signaling in some cell types. However the role of PFKFB3 in regulating angiogenesis and the underlying signaling pathways are completely unknown. Our preliminary data show that myeloid cells in the retinas of the mouse model of oxygen-induced retinopathy are hyper-glycolytic, as evidenced by high levels of glycolytic molecules and regulators/activators of glycolysis including Pfkfb3. The majority of these retinal macrophages exhibit a mixed phenotype characterized by increased levels of cell surface markers for both classical and alternative activation and increased expression of both pro-inflammatory and pro-angiogenic factors. This macrophage phenotype is recapitulated in mouse bone marrow derived macrophages treated with conditioned medium from hypoxic mouse retinal endothelial cells or the glycolytic metabolite lactate. We term these unique macrophages as Pathological Angiogenesis- associated Glycolytic Macrophages (PAGMs). Pfkfb3 knockdown in macrophages reduces their expression of pro-inflammatory and pro-angiogenic factors and suppresses neovascularization in hypoxic retinas. These data inform the hypothesis that Pfkfb3-mediated glycolysis in myeloid cells induces vascular growth by activating Hifs and histone acetylation leading to increased production of pro-inflammatory and pro-angiogenic factors. To test our hypothesis, we have generated myeloid cell specific Pfkfb3-deficient mice. We have also established methods for generating BMDMs that are Pfkfb3 deficient (hypo-glycolytic), Pfkfb3 overexpressing (hyper- glycolytic) or Pfkfb3 wild type and for knockdown of Hif1a and Hif2a. We will investigate the effect of modulating glycolysis in PAGMs on pathological angiogenesis using these genetic tools and in vivo, ex vivo, and in vitro models. We propose three specific aims. 1) Hyper-glycolytic PAGMs regulate the sprouting and proliferation of retinal or choroidal endothelial cells by production and release of pro-inflammatory and pro-angiogenic factors. 2) Hifs and histone acetylation pathways are critically involved in PAGM polarization and activation induced by endogenous glycolysis or endothelial derived glycolytic metabolites. 3) Pfkfb3-mediated glycolysis in macrophages/microglia plays an important role in the development of pathological choroidal and intraretinal neovascularization. 1

项目概要该提案旨在开发一种新的分子策略来限制致盲时的病理性视网膜血管生成眼部疾病。一个新兴的共识是巨噬细胞/小胶质细胞驱动异常的新生血管形成。然而，所涉及的机制仍然不明确。糖酵解是巨噬细胞的主要代谢过程。 PFKFB3（6-磷酸果糖-2-激酶/果糖-2、6-双磷酸酶亚型 3、小鼠 Pfkfb3）是一种关键酶用于激活血管细胞和白细胞中的糖酵解。催化果糖-2, 6-的合成二磷酸盐，是 6-磷酸果糖-1-激酶（一种限速酶）最有效的变构激活剂用于糖酵解。 PFKFB3 对于巨噬细胞的炎症表型至关重要，并且糖酵解已被证明促进某些细胞类型中缺氧诱导因子 (HIF) 或组蛋白乙酰化信号的激活。然而PFKFB3在调节血管生成中的作用及其潜在的信号通路是完全未知的。未知。我们的初步数据表明，氧诱导小鼠模型视网膜中的骨髓细胞视网膜病变是糖酵解过度的，高水平的糖酵解分子和调节剂/激活剂就证明了这一点糖酵解包括 Pfkfb3。这些视网膜巨噬细胞中的大多数表现出混合表型，其特征在于通过增加经典和替代激活的细胞表面标志物水平以及增加表达促炎因子和促血管生成因子。这种巨噬细胞表型在小鼠身上得到了重现用来自缺氧小鼠视网膜内皮的条件培养基处理的骨髓源性巨噬细胞细胞或糖酵解代谢物乳酸。我们将这些独特的巨噬细胞称为病理性血管生成- 相关的糖酵解巨噬细胞（PAGM）。巨噬细胞中 Pfkfb3 的敲低会降低其表达促炎和促血管生成因子并抑制缺氧视网膜中的新血管形成。这些数据提出以下假设：Pfkfb3 介导的骨髓细胞糖酵解通过激活 Hifs 诱导血管生长组蛋白乙酰化导致促炎和促血管生成因子的产生增加。测试我们的假设是，我们已经培育出了骨髓细胞特异性 Pfkfb3 缺陷小鼠。我们还设立了生成 Pfkfb3 缺陷（低糖酵解）、Pfkfb3 过表达（高糖酵解）的 BMDM 的方法糖酵解）或 Pfkfb3 野生型以及用于敲低 Hif1a 和 Hif2a。我们将研究调节的效果使用这些遗传工具在体内、离体和体外研究 PAGM 中的糖酵解对病理性血管生成的影响模型。我们提出三个具体目标。 1）高糖酵解PAGMs调节发芽和增殖视网膜或脉络膜内皮细胞通过产生和释放促炎和促血管生成因子。 2) Hifs和组蛋白乙酰化途径关键参与PAGM极化和激活内源性糖酵解或内皮衍生的糖酵解代谢物。 3）Pfkfb3介导的糖酵解巨噬细胞/小胶质细胞在病理性脉络膜和视网膜内的发育中发挥重要作用新血管形成。 1