"Myeloid PFKFB3 in subretinal fibrosis"

“视网膜下纤维化中的骨髓 PFKFB3”

• 批准号: 10584490
• 负责人: Ruth B Caldwell
• 金额: $ 40.03万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10584490
• 关键词: 6-Phosphofructo-2-kinase; 6-Phosphofructokinase; Actins; Anatomy; Area; Bone Marrow; Cells; Choroid; Choroidal Neovascularization; Cicatrix; Citric Acid Cycle; Complex; Data; Development; Endothelial Cells; Enzyme Activation; Enzymes; Epithelial Cells; Exudative age-related macular degeneration; Eye diseases; Fibrosis; Fructose; Fructose-2,6-bisphosphatase; Genetic; Glycolysis; In Vitro; Inflammatory; Lasers; Macrophage; Measures; Mediating; Mesenchymal; Metabolic; Metabolic Pathway; Metabolism; Modeling; Molecular; Mus; Myelogenous; Myeloid Cells; Myofibroblast; Neuroglia; Oxygen; Pathway interactions; Phosphotransferases; Production; Profibrotic signal; Protein Isoforms; Retinal Diseases; Role; Smooth Muscle; Structure of retinal pigment epithelium; Testing; Therapeutic; VLDL receptor; Visual impairment; cell type; design; epithelial to mesenchymal transition; in vitro Model; in vivo; inhibitor; knock-down; novel strategies; overexpression; pharmacologic; retinal angiogenesis; tool; treatment strategy

REVISED PROJECT SUMMARY Subretinal fibrosis is an end-stage fibrous plaque/disciform scar that progresses from choroidal neovascularization (CNV) during neovascular age-related macular degeneration (nAMD). Subretinal fibrosis compromises highly organized anatomical layers and tightly coordinated cellular interactions, inevitably leading to irreversible visual impairment. Current treatment for subretinal fibrosis is limited and thus, therapeutic strategies for the inhibition of subretinal fibrosis are imperative. Multiple cell types, including endothelial cells (ECs), retinal pigment epithelium (RPE) cells, macrophages, and glial cells, contribute to subretinal fibrosis by either differentiating into mesenchymal-like cells and further differentiating into α-smooth muscle actin-positive myofibroblasts and/or producing profibrotic and proinflammatory factors. However, the underlying metabolic mechanisms for these cellular and molecular activities remain poorly defined. Glycolysis is a metabolic pathway utilized by many proliferative cells. Our preliminary data show that cells in subretinal fibrotic areas are hyper-glycolytic, as evidenced by high levels of glycolytic enzymes and glycolytic regulators/activators including 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase isoform 3 (Pfkfb3), a critical enzyme for activation of glycolysis in various highly proliferative cells. Pfkfb3 catalyzes the synthesis of fructose-2,6-bisphosphate (F2, 6P2), which is the most potent allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme for glycolysis. We have demonstrated that high levels of glycolytic enzymes including Pfkfb3 are present in the RPE/choroid complex isolated from laser-induced and spontaneous subretinal fibrosis in C57BL/6j mice and very low–density lipoprotein receptor deficient (Vldlr-/-) mice and that the area of subretinal fibrosis is markedly decreased in Pfkfb3-/+ mice. Our in vitro studies have also shown that PFKFB3/Pfkfb3 deletion in myeloid cells inhibits their transition to myofibroblast cells as well as reducing their production of proinflammatory and profibrotic factors. We hypothesize that Pfkfb3-mediated glycolysis in myeloid cells induces their transition to myofibroblasts and induces their production of profibrotic and proinflammatory factors by activating HIFs pathways, eventually leading to the development of subretinal fibrosis. To test our hypothesis, we have generated a variety of genetic mice and established mouse subretinal fibrosis models with laser-induced CNV and spontaneous CNV in Vldlr-/- mice. We will investigate the effect on subretinal fibrosis of Pfkfb3 deficiency or inhibition in myeloid cells using specific genetic and pharmacological tools with an integrated approach of in vivo and in vitro models. Our study will define the role of PFKFB3-mediated metabolism in myeloid cells in the development of subretinal fibrosis and validate inhibition of myeloid PFKFB3 as a novel strategy for the treatment of subretinal fibrosis.

修订项目总结 视网膜下纤维化是在新生血管性年龄相关性黄斑变性（nAMD）期间从脉络膜新生血管（CNV）进展的终末期纤维斑块/带状瘢痕。视网膜下纤维化损害高度组织化的解剖层和紧密协调的细胞相互作用，不可避免地导致不可逆的视力损害。目前视网膜下纤维化的治疗是有限的，因此，抑制视网膜下纤维化的治疗策略是必要的。 多种细胞类型，包括内皮细胞（EC）、视网膜色素上皮（RPE）细胞、巨噬细胞和神经胶质细胞，通过分化为间充质样细胞并进一步分化为α-平滑肌肌动蛋白阳性肌成纤维细胞和/或产生促纤维化和促炎因子，导致视网膜下纤维化。然而，这些细胞和分子活动的潜在代谢机制仍然不清楚。糖酵解是许多增殖细胞利用的代谢途径。我们的初步数据显示，视网膜下纤维化区域中的细胞是糖酵解过度的，如通过高水平的糖酵解酶和糖酵解调节剂/激活剂所证明的，所述糖酵解调节剂/激活剂包括6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶同种型3（Pfkfb 3），其是在各种高度增殖细胞中激活糖酵解的关键酶。Pfkfb 3催化果糖-2，6-二磷酸（F2，6P 2）的合成，果糖-2，6-二磷酸是糖酵解限速酶6-磷酸果糖-1-激酶（PFK-1）最有效的变构激活剂。我们已经证明，在C57 BL/6 j小鼠和极低密度脂蛋白受体缺陷（Vldlr-/-）小鼠中，从激光诱导和自发性视网膜下纤维化分离的RPE/脉络膜复合物中存在高水平的糖酵解酶（包括Pfkfb 3），并且在Pfkfb 3-/+小鼠中视网膜下纤维化的面积显著减少。我们的体外研究还表明，骨髓细胞中的PFKFB 3/Pfkfb 3缺失抑制了它们向肌成纤维细胞的转变，并减少了它们促炎因子和促纤维化因子的产生。我们假设，Pfkfb 3介导的糖酵解在髓样细胞诱导其过渡到肌成纤维细胞，并诱导其生产的促纤维化和促炎因子通过激活HIF途径，最终导致视网膜下纤维化的发展。为了验证我们的假设，我们已经产生了各种遗传小鼠，并建立了小鼠视网膜下纤维化模型与激光诱导的CNV和自发性CNV的Vldlr-/-小鼠。我们将使用特定的遗传和药理学工具，采用体内和体外模型的综合方法，研究骨髓细胞中Pfkfb 3缺乏或抑制对视网膜下纤维化的影响。我们的研究将确定髓样细胞中PFKFB 3介导的代谢在视网膜下纤维化发展中的作用，并验证抑制髓样PFKFB 3作为治疗视网膜下纤维化的新策略。