DDAH在糖尿病视网膜病变中的作用及机制研究

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes, and is also the importnat cause of blindness in the working age populatin around the world. The study about molecular mechanisms and corresponding therapeutic scheme has become the hot topic. The main pathological characteristics of diabetic retinopathy are blood-retinal barrier dysfunction and retinal neovascularization. Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme metabolisming endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine(ADMA), plays important role in the regulation of endotheilal function and angiogenesis by promoting the production of nitric oxide and vascular endothelial growth factor (VEGF) . The preliminary results of the present proposal showed that expression of DDAH1 decreased, while the expression of DDAH2 increased in retinal vascular endothelial cells pretreated with high glucose. Accordingly, this proposal will study the roles of DDAH in patients with diabetic retinopathy and diabetic animal model, explore the enzymatic and non-enzymatic roles of DDAH1 (DDAH1/ADMA/Cx43) and DDAH2 (DDAH2/Sp1/VEGF or DDAH2/Sp1/EphrinB2) in blood-retinal barrier dysfunction and pathological angiogenesis associated with diabetic retinopathy and elucidate the underlying molecular mechanisms by gene transfection technique and specific inhibitors in streptozotocin-induced diabetic rat model, high oxygen-induced angiogenesis mouse model and cultured retinal vascular cells. These proposed studies will not only contribute to understanding of the role of DDAH in diabetic retinopathy, but may also open a new direction to develop novel drugs for diabetic retinopathy.

糖尿病视网膜病变( DR）是糖尿病最常见的微血管并发症，是成人失明的重要病因，探明其发病机制、寻求有效防治方案是糖尿病领域的研究热点。血-视网膜屏障损伤和血管新生是DR的主要病理特征，病理机制尚未阐明。二甲基精氨酸-二甲胺水解酶（DDAH）是一种胞浆蛋白酶，能水解内源性NOS抑制物ADMA，促进NO和VEGF生成，在调节内皮功能和血管新生中起重要作用，可能参与DR。本项目预实验发现高糖处理视网膜内皮细胞时DDAH1表达降低，而DDAH2明显增加。据此，我们将结合动物实验和临床研究，确证DDAH在DR中的重要作用；在STZ诱导的糖尿病大鼠模型、高氧诱导血管新生的小鼠模型和视网膜血管细胞，通过基因转染技术和特异性抑制剂，从酶和非酶途径探讨DDAH1在DR早期血-视网膜屏障损伤（ADMA/Cx43）和DDAH2在晚期血管新生中(Sp1/VEGF和Sp1/EphrinB2)的作用及分子机制。

血-视网膜屏障损伤和血管新生是糖尿病视网膜病变的主要病理特征。本项目研究内容如下：建立不同病程的糖尿病大鼠模型，检测血-视网膜屏障损伤和血管新生、ADMA、DDAH、Cx43、EphA2和EphrinB2等指标，初步确定DDAH/ADMA、Cx43、EphA2和EphrinB2与DR的相关性；在原代培养的大鼠视网膜周细胞（RPCs）中进一步确证DDAH/ADMA在高糖所诱导的血-视网膜屏障损伤中的作用，采用siRNA方法观察内、外源性ADMA是否能模拟高糖所诱导的血-视网膜屏障损伤，并进一步探讨DDAH/ADMA对血-视网膜屏障损伤的影响是否涉及ADMA/Cx43/GJIC途径和/或ADMA/EphA2途径；在培养的视网膜内皮细胞，观察ADMA在低氧所诱导的血管新生中的作用，采用siRNA方法观察内、外源性ADMA是否能模拟低氧所诱导的血管新生，并进一步探讨ADMA对视网膜血管新生的影响是否涉及ADMA/EphrinB2途径。本项主要发现包括：①DDAH1/ADMA在糖尿病视网膜病变早期血-视网膜屏障损伤中发挥重要作用，并参与调节细胞间通讯功能，机制涉及DDAH1/ADMA/Cx43/GJIC和ADMA/EphA2信号通路；②ADMA促进糖尿病视网膜病变晚期血管新生，机制涉及PRMT-1/ADMA/EphrinB2信号通路。这些发现为糖尿病视网膜病变在不同时期的损伤机制提供了新的理论依据，为寻找防治DR药物提供了新思路。