Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University, 4717 St. Antoine, Detroit, MI, 48201, USA.
Pharmaceutical Sciences, Wayne State University, Detroit, MI, USA.
Mol Neurobiol. 2019 Dec;56(12):8643-8655. doi: 10.1007/s12035-019-01696-5. Epub 2019 Jul 13.
Early activation of cytosolic NADPH oxidase-2 (Nox2) in diabetes increases retinal ROS production, damaging their mitochondria. The assembly of Nox2 holoenzyme requires activation of a small molecular weight G protein Rac1. Rac1 activation is regulated by guanine exchange factors and guanine nucleotide-dissociation inhibitors, and post-translational modifications assist in its association with exchange factors and dissociation inhibitors. The goal of this study is to investigate the mechanisms of Rac1 activation in the development of diabetic retinopathy.
The levels of the dissociation inhibitor, prenylating enzyme (farnesyltransferase, FNTA), and exchange factor Vav2 were quantified in human retinal endothelial cells, incubated in normal or high glucose for 96 h. The roles of prenylation and Vav2 in Rac1-Nox2-ROS mitochondrial damage were confirmed in FNTA-siRNA-transfected cells and using the Vav2 inhibitor EHop, respectively. Retinal histopathology and functional changes associated with diabetic retinopathy were analyzed in diabetic mice receiving EHop for 6 months. Key parameters of Rac1 activation were confirmed in the retinal microvasculature from human donors with diabetic retinopathy.
In HRECs, glucose increased FNTA and Vav2 and decreased the dissociation inhibitor. FNTA-siRNA and EHop inhibited glucose-induced activation of Rac1-Nox2-ROS signaling. In diabetic mice, EHop ameliorated the development of retinopathy and functional/structural abnormalities and attenuated Rac1-Nox2-mitochondrial damage. Similar alterations in Rac1 regulators were observed in retinal microvasculature from human donors with diabetic retinopathy. In diabetes, Rac1 prenylation and its interactions with Vav2 contribute to Nox2-ROS-mitochondrial damage, and the pharmacological inhibitors to attenuate Rac1 interactions with its regulators could have the potential to halt/inhibit the development of diabetic retinopathy. Graphical Abstract Activation of prenylating enzyme farnesyltransferase (FNTA) in diabetes, prenylates Rac1. The binding of Rac1 with guanine nucleotide-dissociation inhibitor (GDI) is decreased, but its association with the guanine exchange factor, Vav2, is increased, resulting in Rac1 activation. Active Rac1 helps in the assembly of Nox2 holoenzyme, and Nox2 activation increases cytosolic ROS production, damaging the mitochondria. Damaged mitochondria accelerate capillary cell apoptosis, and ultimately, results in the development of diabetic retinopathy.
糖尿病早期激活胞浆型 NADPH 氧化酶-2（Nox2）会增加视网膜 ROS 的产生，损害其线粒体。Nox2 全酶的组装需要小分子 G 蛋白 Rac1 的激活。Rac1 的激活受鸟嘌呤核苷酸交换因子和鸟嘌呤核苷酸解离抑制剂调节，并且翻译后修饰有助于其与交换因子和解离抑制剂结合。本研究的目的是探讨糖尿病性视网膜病变发展过程中 Rac1 激活的机制。
在正常或高糖孵育 96 小时的人视网膜内皮细胞中定量测定分离抑制剂、 prenylating 酶（法呢基转移酶，FNTA）和交换因子 Vav2 的水平。在用 FNTA-siRNA 转染的细胞中和使用 Vav2 抑制剂 EHop 分别证实 prenylation 和 Vav2 在 Rac1-Nox2-ROS 线粒体损伤中的作用。用 EHop 治疗 6 个月的糖尿病小鼠分析与糖尿病性视网膜病变相关的视网膜组织病理学和功能变化。在患有糖尿病性视网膜病变的人类供体的视网膜微血管中证实 Rac1 激活的关键参数。
在 HRECs 中，葡萄糖增加了 FNTA 和 Vav2，降低了分离抑制剂。FNTA-siRNA 和 EHop 抑制了葡萄糖诱导的 Rac1-Nox2-ROS 信号转导的激活。在糖尿病小鼠中，EHop 改善了视网膜病变的发展以及功能/结构异常，并减轻了 Rac1-Nox2-线粒体损伤。在患有糖尿病性视网膜病变的人类供体的视网膜微血管中观察到类似的 Rac1 调节因子改变。在糖尿病中，Rac1 的 prenylation 及其与 Vav2 的相互作用导致 Nox2-ROS-线粒体损伤，而药理学抑制剂减弱 Rac1 与其调节剂的相互作用可能具有阻止/抑制糖尿病性视网膜病变发展的潜力。
糖尿病中，prenylating 酶法尼基转移酶（FNTA）的激活，使 Rac1 发生 prenylation。Rac1 与鸟嘌呤核苷酸解离抑制剂（GDI）的结合减少，但与鸟嘌呤核苷酸交换因子 Vav2 的结合增加，导致 Rac1 激活。活性 Rac1 有助于 Nox2 全酶的组装，Nox2 的激活增加了胞浆 ROS 的产生，损害了线粒体。受损的线粒体加速毛细血管细胞凋亡，最终导致糖尿病性视网膜病变的发生。
