Myeloid glycolysis in pathological ocular angiogenesis

病理性眼血管生成中的髓样糖酵解

• 批准号: 10673058
• 负责人: Ruth B Caldwell
• 金额: $ 45.28万
• 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/10673058
• 关键词: 6-Phosphofructo-2-kinase; 6-Phosphofructokinase; Adult; Age; Age related macular degeneration; Angiogenic Factor; Aorta; Area; Biological Assay; Blindness; Blood Vessels; Bone Marrow; Breeding; Cell surface; Cells; Choroid; Coculture Techniques; 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; HIF1A gene; Histone Acetylation; Hypoxia; Hypoxia Inducible Factor; In Vitro; Individual; Inflammatory; Laser injury; Lesion; Leukocytes; Macrophage; Measures; Mediating; Messenger RNA; Metabolism; Methods; Microglia; Molecular; Mus; Myelogenous; Myeloid Cells; Newborn Infant; Oxygen; Pathologic; Pathologic Neovascularization; Pathway interactions; Peripheral; Phenotype; Phosphotransferases; Play; Production; Proliferating; 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; 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

项目总结 这项提议试图开发一种新的分子策略来限制致盲的病理性视网膜血管生成。 眼疾。一个新出现的共识是巨噬细胞/小胶质细胞驱动异常新生血管。然而， 所涉及的机制仍不明确。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-2，6-二磷酸酶异构体3，对小鼠来说，PFKFB3)是一种关键酶 用于激活血管细胞和白细胞的糖酵解。它催化合成果糖-2，6- 二磷酸，它是限速酶6-磷酸果糖-1-激酶最有效的变构激活剂 用于糖酵解。PFKFB3对巨噬细胞的炎症表型至关重要，糖酵解已被证明 在某些细胞类型中促进低氧诱导因子(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和潜在的信号通路是完全 未知。我们的初步数据显示，小鼠视网膜中的髓细胞在氧气诱导的模型中 视网膜病变是高糖酵解，表现为高水平的糖酵解分子和调节/激活因子 糖酵解，包括PFKFB3。大多数视网膜巨噬细胞表现出混合的表型特征 通过增加经典和替代激活的细胞表面标志物水平和增加表达 促炎因子和促血管生成因子。这种巨噬细胞表型在小鼠中重现。 低氧小鼠视网膜内皮细胞条件培养液对骨髓来源巨噬细胞的作用 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- 相关糖酵解巨噬细胞(PAGM)。在巨噬细胞中敲除PFKFB3可降低其表达 促炎症和促血管生成因子和抑制缺氧性视网膜新生血管。这些数据 PFKFB3介导的髓系细胞糖酵解通过激活HIF诱导血管生长的假说 组蛋白乙酰化导致促炎和促血管生成因子的产生增加。为了测试 我们的假设是，我们已经产生了髓系细胞特异性的PFKFB3缺陷小鼠。我们还建立了 用于产生PFKFB3缺乏(低糖酵解)、PFKFB3过表达(高表达)的BMDM的方法 糖酵解)或PFKFB3野生型，以及HIF1a和HIF2a的敲除。我们将研究调制的效果 利用这些基因工具以及体内、体外和体外研究PAGMS中的糖酵解对病理性血管生成的影响 模特们。我们提出了三个具体目标。1)高糖酵解PAGMS调控植物的萌发和增殖 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在PAGM极化和激活中起关键作用。 内源性糖酵解或内皮源性糖酵解代谢产物。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展中起重要作用 新生血管。 1

项目成果

Gene-dosage effect of Pfkfb3 on monocyte/macrophage biology in atherosclerosis.

• DOI: 10.1111/bph.15926
• 发表时间: 2022-11
• 期刊: British journal of pharmacology
• 影响因子: 7.3

Adenosine kinase promotes post-infarction cardiac repair by epigenetically maintaining reparative macrophage phenotype.

• DOI: 10.1016/j.yjmcc.2022.11.007
• 发表时间: 2023-01
• 期刊: JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
• 影响因子: 5
• 作者: Zhang, Min;Wang, Caiping;Wang, Rongning;Xu, Jiean;Wang, Zhefeng;Yan, Jianlong;Cai, Yongfeng;Li, Liangping;Huo, Yuqing;Dong, Shaohong
• 通讯作者: Dong, Shaohong