Ferrochelatase as a mediator of ocular angiogenesis

新生血管性眼病包括早产儿视网膜病变、增殖性糖尿病视网膜病变和新生血管性视网膜病变。 年龄相关性黄斑变性是终生失明的主要原因。并非所有患者都有反应 因此，迫切需要找到新的细胞成分，可以靶向 阻断这些疾病特有的病理性血管生成。铁螯合酶（FECH）是一种 这些成分是体外和体内内皮细胞增殖所必需的。FECH负责 亚铁离子插入原卟啉IX，血红素生物合成的最后一步。上一个赠款期 FECH在小鼠和人类脉络膜和视网膜新生血管中上调， FECH抑制导致内皮细胞中血红素蛋白的消耗，并且FECH损失阻断血红素- 依赖于内皮细胞的氧化磷酸化。降低FECH活性也令人惊讶地阻断了 糖酵解此外，开发了第一种药物样FECH抑制剂SH-17023，并显示其 在体外和体内抗血管生成。在这项工作的基础上，目前的目标是确定如何 通过FECH和糖酵解的血红素合成影响内皮细胞生物学和新血管形成。的 这项研究的基本原理是FECH是血管生成的重要介质，是潜在的治疗靶点， 以及糖酵解酶的表达和功能的以前未被认识的调节剂。整体 假设是FECH通过控制血红素的可用性，是多种血红素的综合主调节剂， 促血管生成途径，包括糖酵解。在强有力的初步数据的指导下， 通过两个具体目标：1.阐明FECH影响糖酵解和血管生成的机制。的 内皮细胞中血红素合成抑制引起糖酵解酶失调及其血红素依赖性 将在内皮细胞中评估转录调节。糖酵解功能和相关代谢 将通过Seahorse、靶向代谢组学和稳定同位素示踪评估这些途径。的影响 这种血红素依赖性糖酵解对内皮细胞功能的调节将随着细胞的变化而沿着确定， 这种效应的类型特异性。2.评估第一个眼部类药物小分子FECH抑制剂 新生血管形成将量化SH-17023的药代动力学和毒性。那么，这部小说 将测试分子在氧诱导的视网膜病变、Vldlr-/-和JR 5558视网膜、视网膜下 和脉络膜新血管形成模型，加上与抗VEGF治疗的协同作用。总的来说，这项工作是 创新，因为它是血红素合成，糖酵解和后循环之间联系的第一个机制研究。 眼部血管生成，以及用药物样小分子直接抑制FECH的第一个特征， 视网膜和脉络膜新生血管形成。这项工作意义重大，因为它将全面 阐明血红素代谢和糖酵解过程之间的联系，以前没有连接， 建立FECH阻断作为一种可行的治疗方法，从而开发出预防失明的新方法。

The neovascular eye diseases retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular age-related macular degeneration are major causes of blindness through the lifespan. Not all patients respond to existing therapies, so there is thus a critical need to find novel cellular components that could be targeted to block the pathological angiogenesis that is characteristic of these diseases. Ferrochelatase (FECH) is one such component, necessary for proliferation of endothelial cells in vitro and in vivo. FECH is responsible for inserting ferrous ion into protoporphyrin IX, the final step in heme biosynthesis. The previous grant period yielded findings that FECH is upregulated in murine and human choroidal and retinal neovascularization, that FECH inhibition leads to depletion of hemoproteins in endothelial cells, and that FECH loss blocks heme- dependent oxidative phosphorylation in endothelial cells. Reduced FECH activity also surprisingly blocks glycolysis. In addition, the first drug-like FECH inhibitor, SH-17023 was developed and shown to be antiangiogenic in vitro and in vivo. Building on this work, the current goal is to define the mechanism of how heme synthesis through FECH and glycolysis impacts endothelial cell biology and neovascularization. The rationale for this research is that FECH is a significant mediator of angiogenesis, a potential therapeutic target, and a previously unappreciated regulator of the expression and function of glycolytic enzymes. The overall hypothesis is that FECH, via controlling heme availability, is an integrated master regulator of multiple proangiogenic pathways, including glycolysis. Guided by strong preliminary data, the hypothesis will be tested via two specific aims: 1. Delineate the mechanism of FECH’s influence on glycolysis and angiogenesis. The glycolysis enzymes dysregulated by heme synthesis inhibition in endothelial cells and their heme-dependent transcriptional regulation will be assessed in endothelial cells. Glycolytic function and related metabolic pathways will be assessed by Seahorse, targeted metabolomics, and stable isotope tracing. The influence of this heme-dependent glycolysis regulation on endothelial cell function will be determined, along with the cell- type specificity of this effect. 2. Evaluate the first drug-like small molecule FECH inhibitor in ocular neovascularization. The pharmacokinetics and toxicity of SH-17023 will be quantified. Then, this novel molecule will be tested for efficacy in the oxygen-induced retinopathy, Vldlr-/-, and JR5558 retinal, subretinal, and choroidal neovascularization models, plus synergy with anti-VEGF therapy. Overall, this work is innovative, as it is the first mechanistic study of the links between heme synthesis, glycolysis, and posterior ocular angiogenesis, and the first characterization of direct FECH inhibition with a drug-like small molecule for retinal and choroidal neovascularization. The work is highly significant because it will comprehensively elucidate linkages between heme metabolism and the process of glycolysis, not previously connected, and establish FECH blockade as a viable therapy, leading to development of new ways to prevent blindness.