"Myeloid PFKFB3 in subretinal fibrosis"

修订后的项目摘要 视网膜下纤维化是在新生血管性老年性黄斑变性(NAMD)过程中由脉络膜新生血管(CNV)发展而来的终末期纤维性斑块/盘状瘢痕。视网膜下纤维化损害了组织严密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转的视力损害。目前对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势在必行。 多种细胞类型，包括内皮细胞、视网膜色素上皮细胞、巨噬细胞和神经胶质细胞，通过分化为间充质样细胞和进一步分化为α-平滑肌肌动蛋白阳性的肌成纤维细胞和/或产生促纤维化和促炎因子而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，视网膜下纤维化区的细胞是高糖酵解的，高水平的糖酵解酶和糖酵解调节/激活剂，包括6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶异构体3(PFKFB3)，是在各种高增殖细胞中激活糖酵解的关键酶。PFKFB3催化合成2，6-二磷酸果糖(F2，6P2)，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C57BL/6J小鼠和极低密度脂蛋白受体缺陷(Vldlr-/-)小鼠中，从激光诱导的自发性视网膜下纤维化中分离出的RPE/脉络膜复合体中存在包括PFKFB3在内的高水平的糖酵解酶，并且在PFKFB3-/+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，髓系细胞中PFKFB3/PFKFB3的缺失抑制了其向肌成纤维细胞的转化，并减少了其促炎和促纤维化因子的产生。我们推测，PFKFB3介导的髓系细胞糖酵解诱导髓系细胞向肌成纤维细胞转化，并通过激活HIFs通路诱导其产生促纤维化和促炎因子，最终导致视网膜下纤维化的发生。为了验证我们的假设，我们建立了各种遗传小鼠，并用激光诱导的CNV和Vldlr-/-小鼠的自发性CNV建立了小鼠视网膜下纤维化模型。我们将采用体内和体外模型相结合的方法，利用特定的遗传学和药理学工具，研究PFKFB3缺乏或抑制髓系细胞对视网膜下纤维化的影响。我们的研究将确定PFKFB3介导的髓系细胞代谢在视网膜下纤维化发展中的作用，并验证抑制髓系PFKFB3作为治疗视网膜下纤维化的新策略。

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.