RPE PFKFB3 in subretinal fibrosis

视网膜下纤维化中的 RPE PFKFB3

• 批准号: 10739396
• 负责人: Qiuhua Yang
• 金额: $ 11.55万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10739396
• 关键词: 6-Phosphofructo-2-kinase; 6-Phosphofructokinase; Actins; Anatomy; Area; Cells; Choroid; Choroidal Neovascularization; Cicatrix; Complex; Data; Development; Endothelial Cells; Endothelium; Enzyme Activation; Enzymes; Exudative age-related macular degeneration; Eye diseases; Fibrosis; Fructose; Fructose-2,6-bisphosphatase; Genetic; Glycolysis; HIF1A gene; Hypoxia; In Vitro; Inflammatory; Knock-out; Laser injury; Lasers; Lesion; Macrophage; Mediating; Mentors; Mesenchymal; Metabolic; Metabolic Pathway; Metabolism; Modeling; Molecular; Mus; Myeloid Cells; Myofibroblast; Neuroglia; Oxygen; Pathway interactions; Patients; Phosphotransferases; Production; Profibrotic signal; Protein Isoforms; Pyruvate; Retina; Retinal Diseases; Rodent Model; Role; Smooth Muscle; Structure of retinal pigment epithelium; Testing; Therapeutic; Transforming Growth Factor beta; VLDL receptor; Vision; Visual impairment; Work; cell type; design; epithelial to mesenchymal transition; in vitro Model; in vivo; knock-down; migration; novel strategies; overexpression; retinal angiogenesis; tool; treatment strategy

PROJECT SUMMARY Subretinal fibrosis, an end-stage fibrous plaque/disciform scar that progresses from choroidal neovascularization (CNV) of neovascular age-related macular degeneration (nAMD), compromises highly organized anatomical layers and tightly coordinated cellular interactions, inevitably leading to irreversible visual impairment. Current treatment for subretinal fibrosis is limited; thus, therapeutic strategies for the inhibition of subretinal fibrosis are essential. Many types of cells, including retinal pigment epithelium (RPE) cells, contribute to subretinal fibrosis by differentiating into mesenchymal-like cells, α-smooth muscle actin-positive myofibroblasts and/or producing profibrotic and proinflammatory factors. However, the underlying metabolic mechanisms for these cellular and molecular activities are not well defined. Glycolysis is a metabolic pathway utilized by many proliferative cells. my preliminary data show that cells in subretinal fibrotic areas are hyper-glycolytic, as evidenced by increased expression 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. I have found that high levels of glycolytic enzymes including PFKFB3 are present in the RPE/choroid complex isolated from laser-induced subretinal fibrosis in C57BL/6j mice, spontaneous lesion in very low–density lipoprotein receptor deficient (Vldlr-/-) mice and RPE layer of nAMD patients. Furthermore, I have also observed that the area of subretinal fibrosis is markedly decreased in Pfkfb3+/- mice. My in vitro studies have shown that PFKFB3 knockdown in RPE cells inhibits their transition to mesenchymal and reduces their production of proinflammatory and profibrotic factors. I hypothesize that PFKFB3-mediated glycolysis in RPE cells induces their transition to mesenchymal cells and their production of profibrotic and proinflammatory factors by activating HIFs pathways, eventually leading to the development of subretinal fibrosis. To test my hypothesis, I have generated RPE- specific Pfkfb3-deficient mice and established mouse subretinal fibrosis models with laser-induced CNV and spontaneous CNV in Vldlr-/- mice. I will investigate the effect of Pfkfb3 deficiency in RPE cells on subretinal fibrosis using specific genetic tools with an integrated approach of in vivo and in vitro models. My study will define the role of PFKFB3-mediated metabolism in RPE cells in the development of subretinal fibrosis and demonstrate PFKFB3 inhibition as a novel strategy for the treatment of subretinal fibrosis.

项目摘要 视网膜下纤维化，一种从脉络膜新生血管发展而来的终末期纤维斑块/瘢痕 (CNV)新生血管性年龄相关性黄斑变性（nAMD）， 层和紧密协调的细胞相互作用，不可避免地导致不可逆转的视觉障碍。电流 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 具有本质意义 许多类型的细胞，包括视网膜色素上皮（RPE）细胞，通过以下方式促成视网膜下纤维化： 分化为间充质样细胞、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。然而，这些细胞和代谢的潜在机制， 分子活性没有很好地定义。糖酵解是许多增殖细胞利用的代谢途径。 我的初步数据显示，视网膜下纤维化区域的细胞是糖酵解过度的， 糖酵解酶和糖酵解调节剂/激活剂（包括6-磷酸果糖-2-）的表达 激酶/果糖-2，6-二磷酸酶同种型3（PFKFB 3），一种激活各种糖酵解的关键酶， 高度增殖的细胞PFKFB 3催化果糖-2，6-二磷酸（F2，6P 2）的合成， 6-磷酸果糖-1-激酶（PFK-1）最有效的变构激活剂，是糖酵解的限速酶。我有 发现在分离的RPE/脉络膜复合体中存在高水平的糖酵解酶，包括PFKFB 3 C57 BL/6 j小鼠激光诱导的视网膜下纤维化，极低密度脂蛋白 受体缺陷（Vldlr-/-）小鼠和nAMD患者的RPE层。此外，我还注意到， 在Pfkfb 3 +/-小鼠中视网膜下纤维化的面积显著减少。我的体外研究表明PFKFB 3 RPE细胞中的敲低抑制它们向间充质的转化并减少它们的促炎性细胞因子的产生。 和促纤维化因子。我假设PFKFB 3介导的RPE细胞糖酵解诱导它们向糖酵解的转变。 间充质细胞及其通过激活HIF途径产生促纤维化和促炎因子， 最终导致视网膜下纤维化的发展。为了验证我的假设，我生成了视网膜色素上皮细胞- 特异性Pfkfb 3缺陷小鼠和建立的小鼠视网膜下纤维化模型， Vldlr-/-小鼠中的自发CNV。我将研究RPE细胞中Pfkfb 3缺陷对视网膜下细胞的影响。 使用特定的遗传工具，结合体内和体外模型的综合方法，研究纤维化。我的研究将定义 PFKFB 3介导的RPE细胞代谢在视网膜下纤维化发展中的作用，并证实 PFKFB 3抑制作为治疗视网膜下纤维化的新策略。