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，重点关注氧化应激等各种损伤引起的视网膜细胞成分（视网膜神经元和视网膜血管细胞）的凋亡。糖尿病视网膜病变患者眼中 8-羟基脱氧鸟苷 (8-OHdG) 和 NOx 的表达是 NO 产生的标志物，并且8-OHdG 是氧化应激引起的 DNA 损伤的生物标志物，通过 ELISA 测量玻璃体标本中的增殖性糖尿病视网膜病变 (PDR) 眼睛。 、视网膜分支静脉阻塞（BRVO）或黄斑裂孔和/或视网膜前膜（MH/ERM）），在固定视网膜后本研究已获得山形大学医学院伦理委员会的批准。PDR 眼中 NOx 和 8-OHdG 的平均浓度高于 BRVO 和 MH/ERM 患者。免疫组化观察正常大鼠和DM模型大鼠视网膜中8-OHdG的表达，主要在神经节中检测到8-OHdG的表达。总而言之，糖尿病眼中的氧化损伤比 BRVO 或 MH/ERM 眼中的氧化损伤更显着。这些结果表明，抗氧化剂是糖尿病视网膜病变的非常好的治疗剂。我们研究了开发糖尿病视网膜病变新治疗药物的另一种方法。在糖尿病视网膜病变中，二酰基甘油 (DG) 的产生增加并激活蛋白激酶。 C(PKC)参与多种生长因子的信号转导途径，使DG磷酸化，降解为磷脂酸(PA)，并通过调节DG水平和肌醇磷脂来调节胞质内信号转导。我们观察了 DGK 亚型和细胞因子的表达模式，以研究 DGK 在糖尿病发病过程中的临床意义。在链脲佐菌素诱导的大鼠 (STZ) 和自发性糖尿病 Torii (SDT) 大鼠（由日本东京 Torii Pharmaceutical Co., Ltd. 提供）的视网膜标本中，DGK 同种型和细胞因子 (VEGF、通过免疫组织化学和蛋白质印迹法观察IL-6、血管紧张素II、TGF_1,2和PEDF的表达模式和结果。 DM 模型大鼠视网膜中 DGK、VEGF、IL-6、血管紧张素-II 表达水平与正常对照相比的变化 DGK、IL-6 主要在视网膜血管中表达，血管紧张素-II 在视网膜血管中表达。糖尿病视网膜血管和内核层VEGF表达在糖尿病视网膜全层增加，DGK和VEGF可能参与了早期的发病机制。 DMR，并且是开发糖尿病视网膜病变新治疗药物的良好目标。

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）

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)。

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。

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）。

佐藤さくら, 佐藤浩章, 川崎良, 高村浩, 山下英俊: "眼疾患とRAS"Angiotensin Research (Journal of Angiotensin Research). 1. 73-77 (2004)

Sakura Sato、Hiroaki Sato、Ryo Kawasaki、Hiroshi Takamura、Hidetoshi Yamashita：“眼部疾病和 RAS”血管紧张素研究（血管紧张素研究杂志）1. 73-77 (2004)。