Myeloid glycolysis in pathological ocular angiogenesis

病理性眼血管生成中的髓样糖酵解

• 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
• 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

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

项目摘要 这项建议旨在开发一种新的分子策略，以限制致盲中的病理性视网膜血管生成 眼疾。一个新的共识是巨噬细胞/小胶质细胞驱动异常的新血管形成。然而，在这方面， 所涉机制仍不明确。糖酵解是巨噬细胞的主要代谢过程。 PFKFB 3（6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase isoform 3，Pfkfb 3 for mice）是一种重要的果糖磷酸化酶 用于激活血管细胞和白细胞中的糖酵解。它催化果糖-2，6- 二磷酸，它是限速酶6-磷酸果糖-1-激酶的最有效的变构激活剂 糖酵解PFKFB 3对于巨噬细胞的炎症表型至关重要，并且已经显示糖酵解 以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，PFKFB 3在调节血管生成中的作用和潜在的信号传导途径完全不清楚。 未知我们的初步数据表明，在小鼠模型的视网膜髓样细胞的氧诱导 视网膜病变是超糖酵解的，如由高水平的糖酵解分子和调节剂/激活剂所证明的。 糖酵解包括Pfkfb 3。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活化的细胞表面标志物水平以及增加表达 促炎症和促血管生成因子的作用。这种巨噬细胞表型在小鼠中重现 用来自缺氧小鼠视网膜内皮细胞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- 相关的糖酵解大分子化合物（PAGM）。pfkfb 3在巨噬细胞中的敲低降低了它们的 促炎因子和促血管生成因子，并抑制缺氧视网膜中的新血管形成。这些数据 提示Pfkfb 3介导的髓样细胞糖酵解通过激活Hifs诱导血管生长的假设 和组蛋白乙酰化导致促炎因子和促血管生成因子的产生增加。测试 根据我们的假设，我们已经产生了骨髓细胞特异性Pfkfb 3缺陷小鼠。我们还建立 产生Pfkfb 3缺陷（低糖酵解）、Pfkfb 3过表达（高糖酵解）、Pfkfb 3过量表达（高糖酵解）的BMDM的方法， 糖酵解）或Pfkfb 3野生型以及用于敲低Hif 1a和Hif 2a。我们将研究调节 使用这些遗传工具以及在体内、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)糖酵解过度的PAGM调节细胞的发芽和增殖 视网膜或脉络膜内皮细胞通过产生和释放促炎因子和促血管生成因子。 2)Hifs和组蛋白乙酰化途径关键地参与由Hifs诱导的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)Pfkfb 3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展中起重要作用。 新生血管形成 1