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

通过调节视网膜血管细胞和神经元凋亡来开发糖尿病视网膜病变治疗剂的新战略方法

• 批准号: 14571656
• 负责人: TAKAMURA Hiroshi
• 金额: $ 2.24万
• 依托单位: Yamagata University Faculty of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571656/
• 关键词: diabetic retinopathy; oxidative stress; 8-OHdG; NOx; VEGF; diabetes mellitus(DM) model rats; PKC; DGK; 細胞障害; VEGP; 可溶性VEGP受容体; IL-6; プロテインキナーゼCβ

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

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）的凋亡由包括氧化应激在内的各种损伤引起。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）和氮氧化物（NOx）在人糖尿病视网膜病变中的表达。NOx是NO产生的标志物，8-OHdG是氧化应激引起的DNA损伤的生物标志物。8-用ELISA法测定人玻璃体标本中的OHdG和NOx水平。在获得受试者的书面许可后，在玻璃体手术期间从患有增殖性糖尿病视网膜病变（PDR）、视网膜分支静脉阻塞（BRVO）或黄斑裂孔和/或视网膜前膜（MH/ERM）的眼睛获得玻璃体标本。本研究已获得山形大学医学部伦理委员会的批准。平均玻璃体浓度 ...更多信息 PDR组的NO_x和8-OHdG浓度明显高于BRVO组和MH/ERM组。免疫组化法观察正常大鼠和糖尿病模型大鼠视网膜中8-OHdG的表达。8-OHdG主要在STZ大鼠眼神经节细胞层表达。总之，糖尿病眼的氧化损伤比BRVO或MH/ERM眼更显著。这些结果表明，抗氧化剂是非常好的候选药物治疗糖尿病视网膜病变。我们调查的其他途径，以开发新的治疗药物的糖尿病视网膜病变。在糖尿病视网膜病变中，甘油二酯（DG）的产生增加并激活蛋白激酶C（PKC），其参与来自各种生长因子的信号转导途径。二酰基甘油激酶（DGK）磷酸化DG，降解为磷脂酸（PA），并通过调节DG水平和肌醇-磷脂转换来调节胞质内信号转导。观察DGK亚型和细胞因子在糖尿病视网膜病变中的表达，探讨DGK在糖尿病视网膜病变发病中的临床意义。在来自链脲佐菌素诱导的大鼠（STZ）和自发性糖尿病Torii（SDT）大鼠（由Torii Pharmaceutical Co.，有限公司、Tokyo，Japan），免疫组化和Western印迹法观察DGK亚型和细胞因子（VEGF、IL-6、Angiotensin II、TGF-1、2和PEDF）的表达。糖尿病模型大鼠视网膜中DGK、VEGF、IL-6和血管紧张素-II的表达模式和表达水平与正常对照组相比发生了变化。糖尿病大鼠视网膜中DGK和IL-6主要表达于血管，血管紧张素-II主要表达于血管和内核层。VEGF在糖尿病视网膜全层表达增加。DGK和VEGF可能参与了DMR的早期发病过程，是开发新的治疗药物的良好靶点。少

项目成果

S.Sato, Y.Katagiri, K.Goto, M.Igarashi, H.Yamashita: "Immunohistochemical Observation of Diacylglycerol Kinase (DGK) in Normal and the Early Stage of Diabetic Rat."Invest Ophthalmol Vis Sci(75th ARVO 2003.5.4-8). 44. (2003)

S.Sato、Y.Katagiri、K.Goto、M.Igarashi、H.Yamashita：“正常和糖尿病大鼠早期阶段二酰基甘油激酶 (DGK) 的免疫组织化学观察”Invest Ophasemol Vis Sci（第 75 届 ARVO 2003.5.4）

Sato H, Kawasaki R< Yamashita H.: "Observation of Idiopathic Full-Thickness Macular Hole Closure in Early Postoperative Period as Evaluated by Optical Coherence Tomography."Am J Ophthalmol. 136. 185-187 (2003)

Sato H、Kawasaki R< Yamashita H.：“通过光学相干断层扫描评估术后早期特发性全层黄斑裂孔闭合的观察”Am J Ophamol。

寺島和人, 高村浩, 山下英俊: "TGF-βと眼疾患"眼科. 45. 31-38 (2003)

Kazuto Terashima、Hiroshi Takamura、Hidetoshi Yamashita：“TGF-β 和眼部疾病” 眼科学 45. 31-38 (2003)。

H.Sato, R.Kawasaki, T.Yamamoto, T.Yamashita, H.Yamashita: "Evaluation of Cell Damage by Measuring Vitreous Level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in Retinal Diseases."Invest Ophthalmol Vis Sci(75th ARVO 2003.5.4-8). 44. (2003)

H.Sato、R.Kawasaki、T.Yamamoto、T.Yamashita、H.Yamashita：“通过测量视网膜疾病中 8-羟基-2-脱氧鸟苷 (8-OHdG) 的玻璃体水平来评估细胞损伤。”Invest Ophasemol

Yamane K, Minamoto A, Mishima HK, Yamashita H, Takamura H, Yoshizato K: "Proteome analysis of normal human vitreous fluid"Invest Ophthalmol Vis Sci(74th ARVO 2002.5.5-9). 43. (2002)

Yamane K、Minamoto A、Mishima HK、Yamashita H、Takamura H、Yoshizato K：“正常人玻璃体液的蛋白质组分析”Invest Ophasemol Vis Sci（第 74 届 ARVO 2002.5.5-9）。