Regulation of blood-retina barrier by placental growth factor

胎盘生长因子对血视网膜屏障的调节

• 批准号: 10530910
• 负责人: Hu Huang
• 金额: $ 44.22万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10530910
• 关键词: Address; Antioxidants; Apoptosis; Apoptotic; Area; Automobile Driving; Belief; Blindness; Blood-Retinal Barrier; CRISPR/Cas technology; Cell Culture Techniques; Cell Survival; Cells; Cessation of life; Clinical; Complications of Diabetes Mellitus; Development; Diabetes Mellitus; Diabetic Retinopathy; Diabetic mouse; Endothelial Cells; Erythrocytes; Eye; FOXO1A gene; Functional disorder; Gene Expression Profile; Gene Silencing; Gene Transfer; Genetic Models; Glucose; Glucosephosphate Dehydrogenase; Glucosephosphate Dehydrogenase Deficiency; Glycolysis; Goals; HDAC9 gene; Homeostasis; Human; Impairment; In Vitro; Intervention; Knock-out; Knockout Mice; Knowledge; Label; Lentivirus; Ligands; LoxP-flanked allele; MAP Kinase Gene; Measurement; Measures; Mediating; Metabolic; Mitochondria; Mononuclear; Muller' s cell; Mus; NADP; Natural regeneration; Neurons; Outcome; Oxidation-Reduction; Oxidative Stress; Oxides; Oxygen Consumption; PGF gene; PI3K/AKT; Pathogenesis; Pathology; Pentosephosphate Pathway; Pharmacologic Substance; Phenotype; Phosphotransferases; Play; Predisposition; Reaction; Reduced Glutathione; Regulation; Reporting; Research; Retina; Rod; Role; STAT1 gene; Signal Pathway; Signal Transduction; Stable Isotope Labeling; Streptozocin; Stress; Testing; Tissues; Tracer; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors; Virulence Factors; Virus; Vision; antioxidant enzyme; base; conditional knockout; dehydroepiandrosterone; design; diabetic; experimental study; expression vector; extracellular; gain of function; genome editing; glucose metabolism; in vivo; innovation; insight; kidney cell; knock-down; liquid chromatography mass spectrometry; macular edema; metabolomics; mitochondrial dysfunction; mouse model; novel; novel strategies; overexpression; oxidative damage; prevent; protective effect; receptor; retinal damage; retinal rods; small hairpin RNA; small molecule; stem cells; transcription factor; translational potential; vector

Project Abstract Diabetic retinopathy (DR) is a common complication of diabetes causing vision loss. Diabetes- induced metabolic abnormality and mitochondrial dysfunction resulting in oxidative stress is a primary pathogenic factor driving DR onset and development. This proposal will complete the two Aims: (1) To elucidate how placental growth factor (PlGF) mediates DR-related pathology by suppressing glucose 6 phosphate dehydrogenase (G6PD) activity and oxidative pentose phosphate pathway (oxPPP) flux. (2) To determine the protective role of G6PD’s antioxidant function via oxPPP in retinal cells against diabetes- induced oxidative injury. More specifically, the PlGF signaling pathway molecules involved in regulating G6PD expression and activity will be uncovered. The new mechanisms by which PlGF mediates diabetes- induced retinal endothelial cell barrier dysfunction and other DR-related pathologies through the metabolic shift from PPP to glycolysis and mitochondrial dysfunction will be elucidated in cell cultures and murine models. G6PD’s protective role in DR will be determined using cell-/tissue-specific G6PD conditional knockout (cKO) and conditional overexpression (cOE) mice, as well as AAV-based G6PD expression vector and small-molecule G6PD activator. Overall, the research is designed to elucidate the regulation and role of the “diabetes-PlGF-G6PD-oxidative stress” signaling cascades in DR pathogenesis. The outcomes will give mechanistic insight into DR pathogenesis in general and PlGF function in particular. Since that PlGF has emerged as a promising target molecule to treat DR and diabetic macular edema (DEM), understanding the underlying mechanism is significant. Given the central role of G6PD-gated oxPPP in regulating redox homeostasis and cell survival under stress conditions, this project will fill a critical knowledge gap regarding whether G6PD’s antioxidant function via oxPPP plays a pivotal role in protecting retinal cells from diabetes-induced oxidative damage and compromised G6PD activity and limited oxPPP flux contribute to DR pathogenesis. To examine metabolic flux and mitochondrial function, quantitative measurements will be applied, including stable labeling tracer [1,2-13C2]glucose, liquid chromatography/mass spectrometry, targeted metabolomics, oxygen consumption rate/extracellular acidification rate. Cutting-edge approaches will be employed to determine G6PD’s protective effect, such as gene silence (shRNA), genetic models (cKO and cOE mouse), CRISPR-Cas9 genome editing, virus- based gene transfer, and pharmaceutical compounds. In summary, this project is innovative because it will elucidate a novel mechanism for PlGF to mediate DR-related pathology by regulating glucose metabolism and provide new insights into the question(s) “why or how are retinal cells become vulnerable to diabetes-induced oxidative stress?”. It will also explore the new approaches to prevent or delay DR and DME development by enhancing antioxidant capacity.

项目摘要 糖尿病视网膜病变(DR)是糖尿病导致视力丧失的常见并发症。糖尿病- 诱导的代谢异常和线粒体功能障碍导致的氧化应激是主要的 推动糖尿病视网膜病变发生发展的致病因素。这项建议将实现两个目标：(1) 阐明胎盘生长因子(PlGF)如何通过抑制葡萄糖6而介导DR相关病理 磷酸脱氢酶(G6PD)活性和磷酸戊糖氧化途径(OxPPP)通量。(2)至 通过oxPPP在视网膜细胞中确定G6PD的抗氧化功能对糖尿病的保护作用 导致氧化损伤。更具体地说，PlGF信号通路分子参与调控 G6PD的表达和活性将被揭开。PlGF介导糖尿病的新机制-- 通过代谢诱导视网膜内皮细胞屏障功能障碍和其他DR相关病理 从PPP到糖酵解的转变和线粒体功能障碍将在细胞培养和小鼠中阐明 模特们。G6PD在DR中的保护作用将通过细胞/组织特异性G6PD条件 基因敲除(CKO)和条件性过度表达(COE)小鼠，以及基于AAV的G6PD表达 载体和小分子G6PD激活剂。总体而言，这项研究旨在阐明这一规定 糖尿病-PlGF-G6PD-氧化应激信号通路在DR发病机制中的作用。这个 这些结果将使我们从机制上深入了解DR的发病机制，特别是PlGF的功能。 由于PlGF已成为治疗糖尿病视网膜病变和糖尿病黄斑水肿很有希望的靶分子 (DEM)，了解潜在机制具有重要意义。鉴于G6PD-GATED的核心作用 OxPPP在调节氧化还原动态平衡和应激条件下的细胞存活方面，该项目将填补 关于G6PD通过oxPPP的抗氧化功能是否在 保护视网膜细胞免受糖尿病引起的氧化损伤和G6PD活性的影响 有限的oxPPP通量参与了DR的发病。为了检测代谢流量和线粒体功能， 将应用定量测量，包括稳定的标记示踪剂[1，2-13C2]葡萄糖，液体 色质联用、靶向代谢组学、耗氧率/胞外 酸化速度。将使用尖端方法来确定G6PD的保护作用，如 AS基因沉默(ShRNA)，遗传模型(CKO和COE小鼠)，CRISPR-Cas9基因组编辑，病毒- 基于基因转移和药物化合物。总而言之，这个项目是创新的，因为它 将阐明PlGF通过调节血糖来介导DR相关病理的新机制 新陈代谢并提供了对问题(S)的新见解：视网膜细胞为什么或如何变得脆弱 糖尿病引起的氧化应激？“它还将探索预防或延迟DR和 通过提高抗氧化能力来开发二甲醚。