New strategic approach to develop therapeutic agent for diabetic retinopathy by regulation of apoptosis of retinal vascular cells and neurons

我们研究了糖尿病视网膜病变的病理生理学方面，重点是细胞损伤。视网膜细胞成分(视网膜神经元和视网膜血管细胞)的凋亡是由包括氧化应激在内的各种损伤引起的。为了评价氧化应激对细胞的损伤，我们观察了8-羟基脱氧鸟苷(8-OHdG)和NOx在人糖尿病视网膜病变中的表达。NOx是NO产生的标志，8-OHdG是氧化应激造成DNA损伤的生物标志。用双抗体夹心法测定人玻璃体标本中8-OHdG和NOx的含量。玻璃体标本取自增殖性糖尿病视网膜病变(PDR)、视网膜分支静脉阻塞(BRVO)、黄斑裂孔和/或视网膜前膜(MH/ERM)患者玻璃体手术中，并征得受试者的书面许可。这项研究已经得到山形大学医学部伦理委员会的批准。平均玻璃体…PDR眼的NOx和8-OHdG浓度明显高于BRVO和MH/ERM眼。用免疫组织化学方法观察正常大鼠和DM模型大鼠视网膜中8-OHdG的表达。8-OHdG主要在STZ大鼠眼部神经节细胞层表达。总而言之，糖尿病眼的氧化损伤比BRVO或MH/ERM眼更明显。这些结果表明抗氧化剂是非常好的糖尿病视网膜病变治疗药物的候选药物。我们探索了另一种方法来开发糖尿病视网膜病变的新治疗药物。在糖尿病视网膜病变中，二酰甘油(DG)的产生增加并激活蛋白激酶C(PKC)，PKC参与了各种生长因子的信号转导途径。甘油二酯激酶(DGK)通过磷酸化DG，使DG降解为磷脂酸(PA)，通过调节DG水平和肌醇-磷脂代谢来调节胞质内的信号转导。我们观察了DGK亚型和细胞因子的表达模式，以探讨DGK在糖尿病视网膜病变发病机制中的临床意义。用免疫组织化学和Western blotting方法观察了链脲佐菌素诱导的大鼠(STZ)和自发性糖尿病大鼠(SDT)视网膜组织中DGK亚型和细胞因子(血管内皮生长因子、IL-6、血管紧张素Ⅱ、转化生长因子1、2和PEDF)的表达。糖尿病模型大鼠视网膜DGK、VEGF、IL-6和Ang-II的表达模式及表达水平与正常对照组比较，DGK和IL-6主要在视网膜血管表达，Ang-II在血管和内核层均有表达。糖尿病视网膜全层血管内皮生长因子表达增强。DGK和VEGF可能参与了糖尿病视网膜病变早期的发病机制，是开发糖尿病视网膜病变新的治疗药物的良好靶点。较少

We investigated the pathophysiological aspects of diabetic retinopathy, focusing on the cellular damage. The apoptosis of retinal cell components (retinal neurons and retinal vascular cells) is caused by various insults including oxidative stress. To evaluate the cell damage by oxidative stress, we observed the expression of 8-hydroxydeoxyguanosine (8-OHdG) and NOx in the eyes with diabetic retinopathy in human eyes. NOx is a marker for NO production, and 8-OHdG is a biological marker of DNA damage due to oxidative stress. 8-OHdG and NOx levels in the human vitreous specimens were measured by ELISA. The vitreous specimens were obtained during the vitreous surgeries from the eyes with proliferative diabetic retinopathy (PDR), branch retinal vein occulusion (BRVO), or macular hole and/or epiretinal membrane (MH/ERM)), after securing the written permission from the subjects. This study has been approved by Ethical Committee of Yamagata University Faculty of Medicine. The mean vitreal conc … More entration of NOx and 8-OHdG in the eyes with PDR was significantly higher than those with BRVO and MH/ERM. The expression of 8-OHdG was observed immunohistochemically in the retinas of the noromal rats and the DM model rats. The expression of 8-OHdG was detected mainly in ganglion cell layer of the STZ rat eyes. Taken together, oxidative damage was more significant in the diabetic eyes than in the BRVO or the MH/ERM eyes. These results suggest that anti-oxidant agents are very good candidates for therapeutic agents for diabetic retinopathy.We investigated the other approach to develop new therapeutic agents for diabetic retinopathy. In diabetic retinopathy, the production of diacylglycerol (DG) is increased and activates protein kinase C(PKC), which is involved in the signal transduction pathways from various growth factors. Diacylglycerol kinase (DGK) phosphorylates DG, which is degraded into phosphatidic acid (PA), and regulates the intracytoplasmic signal transduction by regulating DG level and inositol-phospholipid turnover. We observed the expression patterns of DGK isoforms and cytokines to investigate the clinical significance of DGK during the pathogenesis of diabetic retinopathy. In the retinal specimens from Streptozotocin induced rats (STZ) and Spontaneously Diabetic Torii (SDT) rats (supplied by the courtesy of Torii Pharmaceutical Co., Ltd., Tokyo, Japan), the expression of DGK isoforms and cytokines (VEGF, IL-6, Angiotensin II, TGF_1,2, and PEDF) was observed immunohistochemically and by Western blotting. The expression patterns and expression levels of DGK, VEGF, IL-6 and angiotensin-II changes in DM model rat retina in comparison with the normal control The expression of DGK and IL-6 was mainly detected in the retinal vessels, and angiotensin-II was observed in the vessels and the inner nuclear layer in the diabetic retina. VEGF expression increased in whole layere of the diabetic retina. It is possible that DGK and VEGF are involved in the pathogenesis in the early stage of DMR, and are good targets to develop new therapeutic agents for diabetic retinopathy. Less