Regulation of blood-retina barrier by placental growth factor

项目摘要 糖尿病视网膜病变（DR）是糖尿病常见的并发症，可导致视力下降。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 导致DR发生和发展的致病因素。本建议将完成两个目标：（1） 阐明胎盘生长因子（PlGF）如何通过抑制葡萄糖6 磷酸脱氢酶（G6 PD）活性和氧化戊糖磷酸途径（oxPPP）通量。(2)到 确定G6 PD的抗氧化功能通过oxPPP在视网膜细胞中对糖尿病的保护作用- 诱导氧化损伤。更具体地说，参与调节的PlGF信号通路分子 G6 PD表达和活性将被揭示。PlGF介导糖尿病的新机制- 诱导视网膜内皮细胞屏障功能障碍和其他DR相关的病理通过代谢 从PPP到糖酵解和线粒体功能障碍的转变将在细胞培养物和小鼠中阐明。 模型G6 PD在DR中的保护作用将使用细胞/组织特异性G6 PD条件来确定 敲除（cKO）和条件性过表达（cOE）小鼠，以及基于AAV的G6 PD表达 载体和小分子G6 PD激活剂。总的来说，这项研究旨在阐明 以及“糖尿病-PlGF-G6 PD-氧化应激”信号级联在DR发病机制中的作用。的 结果将给出一般的DR发病机制和特别是PlGF功能的机制性见解。 因此，PlGF已成为治疗DR和糖尿病性黄斑水肿的有希望的靶分子 (DEM)因此，了解其内在机制具有重要意义。鉴于G6 PD门控的核心作用， oxPPP在调节氧化还原稳态和应激条件下细胞存活方面的作用，该项目将填补 关于G6 PD通过oxPPP的抗氧化功能是否在 保护视网膜细胞免受糖尿病诱导的氧化损伤和受损的G6 PD活性， 有限的oxPPP通量有助于DR发病机制。为了检测代谢通量和线粒体功能， 将应用定量测量，包括稳定标记示踪剂[1，2 - 13 C2]葡萄糖、液体 色谱/质谱，靶向代谢组学，耗氧率/细胞外 酸化率将采用最先进的方法来确定G6 PD的保护作用，例如 如基因沉默（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。总之，这个项目是创新的，因为它 将阐明PlGF通过调节葡萄糖介导DR相关病理的新机制 并提供新的见解的问题（S）“为什么或如何视网膜细胞变得脆弱 糖尿病引起的氧化应激？”它还将探索预防或延迟DR的新方法， 通过提高抗氧化能力开发DME。

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.