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

基本信息

批准号：
10684755
负责人：
Hu Huang
金额：
$ 42.74万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
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/10684755
关键词：
Address Antioxidants Apoptosis Apoptotic Area Automobile Driving Belief Blindness Blood-Retinal Barrier Breeding 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&apos s cell Mus NADP Natural regeneration Neurons Outcome Oxidation-Reduction Oxidative Stress Oxidative Stress Induction 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 System Testing Tissues Tracer Transfection VEGFA gene Vascular Endothelial Growth Factor Receptor-1 Virulence Factors Virus Vision antioxidant enzyme 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 protective pathway receptor retinal damage retinal rods small hairpin RNA small molecule stem cells transcription factor translational potential vector

项目摘要

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.

项目摘要糖尿病性视网膜病（DR）是导致视力丧失的常见并发症。糖尿病- 诱导的代谢绝对和线粒体功能障碍导致氧化应激是主要的驱动DR发作和发育的致病因素。该建议将完成两个目标：（1）阐明位置生长因子（PLGF）如何通过抑制葡萄糖6 磷酸脱氢酶（G6PD）活性和氧化五糖磷酸途径（OXPPP）通量。（2）至确定G6PD通过OXPPP在残留细胞中针对糖尿病的抗氧化功能的保护作用 - 诱导氧化损伤。更具体地，参与调节的PLGF信号通路分子 G6PD表达和活性将被发现。 PLGF介导糖尿病的新机制 - 通过代谢引起的视网膜内皮细胞屏障功能障碍和其他与DR相关的病理从PPP转向糖酵解和线粒体功能障碍将在细胞培养物和鼠中阐明型号。 G6PD在DR中的保护作用将使用细胞/组织特异性G6PD确定敲除（CKO）和有条件的过表达（COE）小鼠以及基于AAV的G6PD表达式向量和小分子G6PD激活剂。总体而言，该研究旨在阐明调节 “糖尿病-PLGF-G6PD氧化应激”在DR发病机理中的作用。这结果将使一般的DR发病机理，特别是PLGF功能提供机械洞察力。由于该PLGF已成为有望治疗DR和糖尿病性黄斑水肿的承诺靶分子（DEM），了解潜在机制是重要的。鉴于G6PD门控的核心作用 OXPPP在调节氧化还原稳态和在压力条件下的细胞存活方面，该项目将填充关于G6PD是否通过OXPPP的抗氧化功能是否起关键作用的关键知识差距在保护视网膜细胞免受糖尿病诱导的氧化损伤和损害的G6PD活性和有限的OXPPP通量有助于DR发病机理。要检查代谢通量和线粒体功能，将应用定量测量值，包括稳定的标记示踪剂[1,2-13C2]葡萄糖，液体色谱/质谱法，靶向代谢组学，氧气消耗率/细胞外酸化率。将采用尖端的方法来确定G6PD的保护效果，例如作为基因静音（SHRNA），遗传模型（CKO和COE小鼠），CRISPR-CAS9基因组编辑，病毒 - 基因转移和药物化合物。总而言之，这个项目具有创新性，因为它将通过调节葡萄糖来阐明PLGF介导与DR相关的病理的新型机制代谢并提供了对问题的新见解：“为什么或如何成为视网膜细胞变得脆弱为糖尿病引起的氧化压力吗？”。它还将探索预防或延迟DR的新方法通过增强抗氧化能力来开发DME。