Eye and Vision Health, College of Medicine and Public Health, Flinders University, Adelaide, South Australia, Australia.
Diabetic Retinopathy, Department of Diabetes, Monash University, Melbourne, Victoria, Australia.
Clin Exp Ophthalmol. 2018 Aug;46(6):652-660. doi: 10.1111/ceo.13155. Epub 2018 Feb 23.
Over-production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS-generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans.
Differential Nox enzyme expression was studied by real-time-quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2',7'-dichlorofluorescein. DMOG-stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production.
Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG-induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth.
Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases.
活性氧（ROS）的过度产生和由此产生的氧化应激导致包括糖尿病性视网膜病变、早产儿视网膜病变和主要视网膜血管闭塞在内的血管疾病中的视网膜损伤。NADPH 氧化酶（Nox）蛋白是专业的 ROS 生成酶，在这些疾病中的治疗靶向具有很强的吸引力。Nox4 的药理学抑制可减轻实验性视网膜血管病变的严重程度。我们研究了这种药物方法在人类中的潜在应用。
通过实时定量聚合酶链反应在原代人视网膜内皮细胞分离物和特征明确的人视网膜内皮细胞系中研究差异 Nox 酶表达。通过用二甲基草酰甘氨酸（DMOG；100μM）化学诱导内皮细胞中的氧化应激，测量 Nox4 和血管内皮生长因子（VEGFA）转录物；并通过 2'，7'-二氯荧光素检测 ROS 的产生。用两种 Nox1/Nox4 抑制剂 GKT136901 和 GKT137831 处理 DMOG 刺激的内皮细胞；通过 DNA 定量监测细胞生长，以及 VEGFA 转录物和 ROS 产生。
Nox4（Nox4A 同工型）是人视网膜内皮细胞表达的主要 Nox 酶。用 DMOG 处理可在 72 小时内显著增加内皮细胞中 Nox4 的表达，伴随 ROS 产生和 VEGFA 表达增加。用 GKT136901 或 GKT137831 处理可显著降低 DMOG 诱导的内皮细胞中 ROS 的产生和 VEGFA 表达，并抑制 DMOG 对细胞生长的作用。
我们在激活的人视网膜内皮细胞实验中的发现为视网膜血管病变实验模型中的研究提供了转化佐证，并支持 GKT136901 和 GKT137831 通过抑制 Nox4 在视网膜血管疾病患者中的治疗应用。
