半胱氨酸天冬氨酸蛋白酶在视网膜毛细血管周细胞凋亡中的活性及意义

目的探讨糖基化终产物（AGE）在糖尿病视网膜病变（DR）发生中的作用。方法培养的牛视网膜毛细血管周细胞分别与不同浓度（0．47、1．88、7．50μmol／L）的AGE共同培养4d后,分别检测细胞凋亡、半胱氨酸天冬氨酸蛋白酶（caspase-3）活性及caspase-3抑制剂Z—DEVD-fmk对周细胞凋亡及凋亡调节基因Bcl-2／Bax比率的影响。结果AGE能以剂量依赖的方式诱导培养的牛视网膜毛细血管周细胞凋亡（r=0．867,P〈0．01）、增加细胞内caspase-3的活性,而选择性caspase-3抑制剂Z—DEVD—fmk能明显抑制AGE作用下的周细胞凋亡及提高凋亡调节基因Bcl-2／Bax的比率。结论凋亡是DR中视网膜毛细血管周细胞选择性丧失的机制之一,caspase-3活性的增加是AGE诱导周细胞发生凋亡的关键因素。     

超氧化物歧化酶在视网膜毛细血管周细胞凋亡中的活性及意义

目的通过检测糖基化终产物（AGE）诱导培养的牛视网膜毛细血管周细胞凋亡及凋亡周细胞中超氧化物歧化酶（SOD）的活性及意义，进一步探讨糖尿病视网膜病变（DR）的发生机制。方法周细胞分别与不同浓度的AGE（0．47、1．88、7．5μmol／L）共同培养4d后，分别检测细胞凋亡、SOD活性，以及SOD对细胞凋亡及凋亡调节基因Bcl-2／Bax比率的影响。结果AGE能以浓度依赖的方式诱导周细胞凋亡（r=0．878，P〈0．01）、降低细胞内SOD的活性（r=-0．878，P〈0．01），而应用SOD能明显抑制AGE作用下的周细胞凋亡及提高凋亡调节基因Bcl-2／Bax的比率.结论凋亡及氧化应激的增加是DRP中周细胞选择性丧失的主要原因，SOD活性的下降是AGE诱导周细胞发生凋亡的关键因素。     

去势雄兔干眼病模型角膜上皮细胞凋亡及相关基因表达的研究

目的　探讨去势雄兔干眼病模型角膜上皮细胞凋亡及相关基因表达与组织损伤的关系。方法　制作 6只去势雄兔干眼病模型 ,采用原位末端标记及免疫组织化学方法 ,对模型兔及 6只对照兔角膜上皮细胞中的凋亡细胞及Fas、FasL、Bax和bcl 2等相关基因蛋白的表达进行检测。结果　干眼病模型兔角膜上皮细胞凋亡数为 ( 5 2 3± 2 67)个 /高倍视野 ;对照兔为 ( 0 5 8± 0 13 )个 /高倍视野 (P <0 0 1)。模型兔角膜上皮细胞中 ,Fas、FasL及Bax阳性表达的细胞数均明显高于对照组 (P <0 0 1) ,与凋亡细胞数呈正相关 (r=0 78,0 82 ,0 76,P <0 0 5 ) ;bcl 2阳性表达的细胞数明显低于对照组 (P <0 0 1) ,与凋亡细胞数呈负相关 (r =-0 72 ,P <0 0 5 )。结论　去势雄兔角膜组织中上皮细胞凋亡可能是导致角膜变薄、干燥 ,进而功能丧失的原因之一 ;Fas、FasL及Bax的增加及bcl 2的减少均与促进细胞凋亡有关。     

维生素A缺乏干眼症兔泪腺凋亡及相关基因的表达

目的　探讨维生素A缺乏兔干眼症模型泪腺上皮细胞凋亡及相关基因表达与组织损伤的关系。方法　制作 6只维生素A缺乏兔干眼症模型 ,采用原位末端标记及免疫组织化学方法 ,对模型兔及 6只对照兔泪腺组织上皮细胞中的凋亡细胞及Fas、FasL、Bax和bcl 2等相关基因蛋白的表达进行检测。结果　干眼症模型兔泪腺上皮细胞凋亡数为每高倍视野 (18.17± 5 .33)个 ,对照兔为每高倍视野 (3.5 6± 1.18)个 ,P <0 .0 0 1。模型兔泪腺导管及腺泡上皮细胞中 ,Fas、FasL及Bax阳性表达的细胞数 [每 10 0 0个细胞(416 .33± 112 .0 9)个、(45 8.6 7± 12 0 .5 2 )个及 (32 8.74± 89.0 8)个 ]均明显高于对照组 [每10 0 0个细胞阳性数为 (98.83± 5 1.2 3)个、(12 6 .88± 5 4 .0 9)个及 (87.75± 4 3.33)个 ],P <0 .0 0 1,与凋亡细胞数呈正相关 (r =0 .86 ,0 .91,0 .88;P <0 .0 1)。bcl 2阳性表达的细胞数[每 10 0 0个细胞阳性数为 (113.37± 72 .4 5 )个 ],明显低于对照组 [每 10 0 0个细胞阳性数为 (396 .6 7± 98.73)个 ],P <0 .0 0 1,与凋亡细胞数呈负相关 (r =- 0 .73,P <0 .0 5 )。结论　维生素A缺乏兔泪腺组织中上皮细胞凋亡可能是导致腺体破坏进而功能丧失的原因之一 ;Fas、FasL及Bax增加及bcl 2的减少均与促进细胞     

干燥综合征泪腺组织中细胞凋亡及相关基因表达的研究

目的　探讨干燥综合征 (Sj gren′ssyndrome,SS)患者泪腺组织上皮细胞凋亡及其相关基因表达与SS组织损伤的关系。方法　采用原位末端标记及免疫组织化学的方法 ,对 12例SS患者及7例对照泪腺组织上皮细胞中的凋亡细胞及Fas、FasL、bcl 2和Bax等相关基因蛋白的表达进行检测。结果　 12例SS患者泪腺组织中上皮细胞凋亡数为 (8 83± 3 2 7)个 /高倍视野 ;对照为 (0 88± 0 5 2 )个 /高倍视野 (P =0 0 0 0 )。SS患者泪腺导管及腺泡上皮细胞中 ,Fas、FasL及Bax阳性表达的细胞数均明显高于对照组 (P =0 0 0 0 ) ,与凋亡细胞数呈正相关 (R =0 83,0 88,0 8;P =0 0 0 0 ) ;bcl 2阳性表达的细胞数明显低于对照组 ,与凋亡细胞数呈负相关 (R =- 0 6 9,P =0 0 0 1)。结论　SS患者泪腺组织中上皮细胞凋亡可能是导致腺体破坏进而功能丧失的原因之一 ;Fas、FasL及Bax增加以及bcl 2的减少皆与促进细胞凋亡有关     

大剂量甲基泼尼松龙对大鼠视神经挤压伤后RGC凋亡的影响

目的　观察大剂量甲基泼尼松龙 (MP)对大鼠视神经挤压伤后视网膜神经节细胞 (RGC)凋亡的影响。方法　大鼠 12 6只分为正常对照组 18只 3 6眼、损伤组 54只 54眼和MP治疗组 54只 54眼。伤后 4、7和 14d ,通过视网膜铺片和切片相结合的技术 ,进行细胞原位凋亡检测以及Bcl 2和Bax阳性细胞计数 (免疫组化SP法 )。结果　正常对照组有少量凋亡细胞 ,Bcl 2和Bax阳性细胞数少 ;伤后 4、7、14d凋亡细胞大量增加 ,治疗组凋亡细胞各时间点均低于损伤组 ,差异有显著性 (P <0 0 5) ;治疗组Bcl 2阳性细胞数各时间点均高于损伤组 ,Bax阳性细胞数均低于损伤组 ,差异有显著性 (P <0 0 5)。结论　大剂量MP能抑制视神经挫伤后RGC的凋亡 ,上调bcl 2基因的表达和下调bax基因的表达可能是MP治疗作用机制之一     

糖尿病视网膜毛细血管周细胞凋亡分子生物学机制的研究进展

随着糖尿病视网膜病变研究的进展,已证实糖尿病视网膜毛细血管周细胞的丧失是由细胞凋亡所引起的。糖尿病患者体内血糖水平的增加使视网膜毛细血管周细胞内氧自由基产生增加,导致DNA修复酶聚ADP核糖多聚酶(poly ADP-ribose polymerase,PARP)激活、线粒体内细胞色素C(cytochrome C,CytC)的释放激活半胱天冬酶(caspase)家族、周细胞内Ca2 浓度增加、激活核转录因子-κB(the nuclear factor-κB,NF-κB),从而引起细胞凋亡。糖尿病患者由于糖化血红蛋白的增多等原因,视网膜长期处于低氧状态。低氧则引起视网膜毛细血管周细胞DNA损伤、氧自由基的产生增加、细胞凋亡抑制基因Bcl-2的低表达,导致细胞凋亡。糖尿病患者体内高血糖水平和继发形成的糖基化终末产物(advancedglycation end-products,AGEs)都能够使视网膜毛细血管周细胞的Bax基因高表达与Bcl-2基因的表达减少,引起促凋亡、抗凋亡基因比例失衡,导致细胞凋亡。色素上皮衍生因子(pig-ment epithelium-derivedfactor,PDEF)可以增加视网膜毛细血管周细胞内谷胱苷肽过氧化物酶(glutathione peroxidase,GPx)mRNA转录水平,阻止高糖引起的视网膜毛细血管周细胞内氧自由基的增加,因而具有细胞凋亡作用。糖尿病患者体内PDEF水平的降低,导致了视网膜毛细血管周细胞凋亡的发生。     

高糖诱导视网膜血管周细胞凋亡中线粒体细胞色素C的变化

目的:研究高糖诱导牛视网膜血管周细胞凋亡中线粒体细胞色素C(Cyt-C)的变化.方法:以在体外培养3代近融合的牛视网膜毛细血管周细胞为对象,分别在对照组(5.5mmol/L)与高糖各组(15,25,35mmol/L)中孵育6d,透射电镜观察周细胞超微结构改变,TUNEL法检测培养后周细胞的凋亡情况,分光光度计检测周细胞胞质Cyt-C变化.结果:高糖各组周细胞出现明显凋亡改变,凋亡率明显高于对照组(t1=57.450,t2=83.754,t3=136.403,P＜0.01);高糖各组胞质Cyt-C浓度明显高于对照组(t1=17.361,t2=17.866,t3=24.072,P＜0.01),且Cyt-C浓度与周细胞凋亡率明显正相关(r=0.964,P＜0.01).结论:高糖呈剂量依赖性诱导牛视网膜血管周细胞凋亡,线粒体Cyt-C释放可能参与周细胞凋亡过程.     

Bak和bcl-xl在增生性玻璃体视网膜病变玻璃体内细胞和视网膜前膜中的表达

目的　观察增生性玻璃体视网膜病变 (PVR)玻璃体内和视网膜前膜 (ERM)中细胞凋亡、bak和bcl xl基因表达、以及细胞凋亡和基因表达之间的关系。方法　对 12例PVRB级玻璃体切割液离心收集物 ,18例C级以上PVR的ERM进行研究 ,末端标记 (TUNEL)染色法检测凋亡细胞 ,免疫组织化学染色法检测bak和bcl xl基因的表达。结果　B级玻璃体切割液标本中bak和bcl xl阳性细胞百分数分别为 73 0 %和 65 1% ,TUNEL阳性细胞百分率平均为 2 7 2 %。C级以上PVR的ERM标本中bak和bcl xl的阳性细胞比例分别为 61 4%和 77 0 % ,TUNEL阳性细胞百分率平均为 18 6%。PVRB级的凋亡细胞百分数及bak的表达与C级以上标本之间相差明显 (P <0 0 5 ,P <0 0 1) ,而bcl xl的表达在两者间没有差别 (P >0 0 5 )。凋亡细胞bak表达与凋亡细胞百分数相关系数r值为 0 86(P <0 0 1) ,与bcl xl的r值为 0 3 2 (P >0 0 5 )。结论　PVR眼增殖细胞中可见bak和bcl xl的表达和细胞凋亡的存在。bak的高表达可能与PVR中增生细胞凋亡有关。     

糖尿病大鼠视网膜毛细血管细胞凋亡及其凋亡相关基因的表达

目的 确定早期糖尿病大鼠视网膜毛细血管细胞凋亡并观测凋亡相关基因（Bax、bcl-2）在早期糖尿病大鼠视网膜的表达。方法 选择健康成年Wistar大鼠40只,随机分成正常对照组（contrast group,CON）和糖尿病（diabetes mellitus)1个月（DM1）、3个月（DM3）及6个月（DM6）组。腹腔内注射链脲佐菌素（streptozotocin,STZ)诱发大鼠糖尿病。制备大鼠视网膜血管辅片及切片,分别行TUNEL法和免疫组化ABC法染色,并对ABC法染色结果进行计算机图像分析。结果 TUNEL法标记的阳性周细胞核出现于DM3和DM6组,而内皮细胞见于DM6组,CON和DM1组未见阳性反应。TUNEL阳性反应细胞核染色质分布不均,表现为环形核、新月形核等典型凋亡细胞特征。ABC法染色：于CON及DM各组,Bax和bcl-2二基因的蛋白均在视网膜血管表达。随病程进展,其阳性反应强度逐渐增加。此外,bcl-2阳性反应在DM3组于色素上皮细胞出现。在DM6组进一步扩大到视杆内节和节细胞。而Bax在DM6组亦出现于细胞。结论 早期糖尿病大鼠视网膜毛细血管周细胞丧失的性质为凋亡,内皮细胞亦存在凋亡;Bax和bcl-2在早期糖尿病大鼠视网膜的表达增加。     

糖基化终产物对牛视网膜毛细血管周细胞内抗氧化能力的影响

目的 通过测量糖基化终产物对培养的牛视网膜毛细血管周细胞抗氧化物酶和脂质过氧化物含量的影响, 以探讨氧化应激在糖尿病视网膜病变进程中的作用。 方法 不同浓度的糖基化终产物（0，8，32，125，500，2 000 μg/ml）与周细胞作用4 d后，以分光光度法测量细胞内过氧化氢酶的活性及过氧化脂质丙二醛的含量。 结果 糖基化终产物能以剂量依赖的方式降低周细胞内过氧化氢酶的活性(r=-0.714, P＜0.01)，增加丙二醛的含量(r=0.748, P＜0.01)，与对照组相比，当糖基化终产物浓度达到32 μg/ml时，两者比较差异有显著性的意义（P＜0.01）。 结论 氧化应激的增加可能是早期糖尿病视网膜病变中周细胞丧失的原因之一。 (中华眼底病杂志, 2002, 18: 143-145)     

视网膜微血管周细胞ET-1的分泌及影响

目的观察糖基化终产物(Advanced glycation end products,AGE)及低氧对培养的牛视网膜微血管周细胞(Bovine retinal microvascular pericytes,BRPs)内内皮素-1(Endothelin-1,ET-1)产生的影响.方法培养的BRPs分别与不同浓度的AGE(8,32,125μg/ml)共同培养4 d;低氧(10%O2,5%CO2,85%N2)条件下培养12、24、48 h;与不同浓度的AGE作用2 d后,再低氧培养48 h;培养结束后,分别收集培养液,离心,取上清液测定ET-1的含量.结果低氧能以时间依赖的方式促进BRPs分泌ET-1(γ=0.943,P＜0.01).AGE虽能使BRPs分泌ET-1轻微增加,但与对照组相比,差异无显著性(P＞0.05),而AGE能明显增强低氧对BRPs内ET-1分泌的诱导作用,两者具有协同效应.结论AGE和低氧能影响培养的BRPs内ET-1的产生,ET-1的产生异常可能是糖尿病视网膜病变中视网膜微循环血流动力学异常的原因之一.     

Effect of dicarbonyl modification of fibronectin on retinal capillary pericytes

PURPOSE: To determine effects of alpha-dicarbonyl modification of an extracellular matrix protein on retinal capillary pericyte attachment and viability. METHODS: Primary cultures of bovine retinal pericytes (BRPs) were seeded on either normal fibronectin (FN) or FN modified by methylglyoxal (MGO) and glyoxal (GO). Apoptosis was measured by flow cytometry along with caspase-3 activity. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and Akt/PKB were evaluated by Western blot analysis. Cellular glutathione and reactive oxygen species were measured. alphaB-crystallin was measured by Western blot analysis and, to determine its role in apoptosis, experiments were conducted using BRPs that were transiently transfected with alphaB-crystallin. RESULTS: Cultures seeded on MGO- or GO-modified FN showed a significant reduction in the number of viable cells, an increase in the number of apoptotic cells, and increased caspase-3 activity, which correlated with the extent of FN modification. Pericytes seeded on either type of modified FN showed phosphorylation of p38 MAPK and dephosphorylation of Akt/PKB. Cultures seeded on dicarbonyl-modified FN had reduced glutathione and increased levels of reactive oxygen species compared with those on a normal matrix. Cells on the altered matrices had reduced alphaB-crystallin levels as well. Transient transfection of rat alphaB-crystallin into BRPs significantly reduced the apoptosis triggered by alpha-dicarbonyl-modified FN. CONCLUSIONS: These observations indicate that modification of FN by alpha-dicarbonyl compounds triggers apoptosis through a combination of increased oxidative stress and reduction of alphaB-crystallin. This mechanism may contribute to loss of pericytes in diabetic retinopathy and contribute to the resultant vascular lesions.     

恒定性及波动性高糖对牛视网膜周细胞凋亡的影响

目的探讨恒定性高糖及不同频率的波动性高糖对原代培养的牛视网膜周细胞增生、细胞周期及凋亡的影响。方法以体外培养3代近融合的牛视网膜毛细血管周细胞为对象，分别在对照组（5．5mmol／L）、恒定性高糖组（25mmol／L）及不同波动频率的高糖组（5．5mmol／L与25mmol／L交替，频率分别为6h和24h）中孵育6d，采用MTT比色法观察周细胞增生情况；流式细胞术观察周细胞周期情况；TUNEL法检测培养后周细胞的凋亡率。结果周细胞增生受抑制，周期阻滞，出现典型的细胞凋亡特征。恒定性高糖组的作用高于波动性高糖组（P〈0．05）；波动性高糖组内波动频率越高作用越明显（P〈0．05）。结论恒定性高糖较波动性高糖可能具有更强的抑制周细胞增生、周期阻滞及诱导凋亡作用，波动组间波动频率高者比频率低者作用更明显。     

High glucose alters connexin 43 expression and gap junction intercellular communication activity in retinal pericytes

PURPOSE: To investigate the role of the gap junction protein, connexin-43 (Cx43) in the maintenance of retinal vascular homeostasis in diabetic retinopathy. METHODS: In human retinal pericytes (HRPs) and bovine retinal pericytes (BRPs) grown for 7 days in normal (5 mM) or high (30 mM)-glucose medium, the Cx43 protein level was determined by Western blot analysis. Parallel experiments were performed in HRPs to determine the Cx43 mRNA level by RT-PCR, the distribution and localization of Cx43 protein by immunostaining, and gap junction intercellular communication (GJIC) activity by a scrape-loading dye transfer technique. Distribution and localization of Cx43 protein was also determined in pericyte-endothelial cell cocultures. RESULTS: Western blot analysis of the Cx43 protein level in HRPs and BRPs indicated reduced Cx43 expression in the high-glucose condition (69.1% +/- 17% of control, P = 0.004; 62.3% +/- 19% of control, P = 0.001, respectively). The Cx43 mRNA level in HRPs grown in high-glucose medium also showed significant reduction (71.4% +/- 16.8% of control, P = 0.02). The relative number of Cx43 plaques indicative of Cx43 localization at specific sites of contact between adjacent cells showed significant reduction in the high-glucose condition (61% +/- 10% of control, P = 0.002); similarly, a significant reduction in the number of plaques was observed in cocultures grown in high-glucose medium compared with those in normal medium (59.4% +/- 29% of control, P = 0.001). Cells with reduced Cx43 expression showed significantly reduced transfer of lucifer yellow (61% +/- 13% of control, P = 0.001; r = 0.9). CONCLUSIONS: High-glucose-induced downregulation of Cx43 expression and inhibition of GJIC in retinal pericytes may play a role in the disruption of vascular homeostasis in diabetic retinopathy.     

Reactive oxygen species mediates a metabolic memory of high glucose stress signaling in bovine retinal pericytes

AIM: To investigate the role of reactive oxygen species (ROS) and antioxidant mechanism underlying the metabolic memory of bovine retinal pericytes (BRPs) induced by high glucose. METHODS: Effects of high glucose levels and culture time on BRPs viability were evaluated by CCK-8. BRPs were grown in high-glucose media (30 mmol/L) for 4d followed by culture in normal glucose condition (5.6 mmol/L) for 4d in an experimental group. In contrast, in negative and positive control groups, BRPs were grown in either normal-glucose media or high-glucose media for 8d, respectively. The ROS levels, apoptosis, the expression and activity of manganese superoxide dismutase (MnSOD) in BRPs, as well as the protective effect of adeno-associated viral (AAV)-mediated over expression of MnSOD were determined separately by DCHFA, ELISA and Western blot. RESULTS: Comparing the result of cells apoptosis, activity and protein expression of MnSOD and caspase-3, the cell culture system that exposed in sequence in 30 mmol/L and normal glucose for 4d was demonstrated as a suitable model of metabolic memory. Furthermore, delivery of antioxidant gene MnSOD can decrease BRPs apoptosis, reduce activated caspase-3, and reverse hyperglycemic memory by reducing the ROS of mitochondria. CONCLUSION: Increased ROS levels and decreased MnSOD levels may play important roles in pericyte loss of diabetic retinopathy. BRPs cultured in high glucose for 4d followed by normal glucose for 4d could be an appropriate model of metabolic memory. rAAV-MnSOD gene therapy provides a promising strategy to inhibit this blinding disease.     

体外孵育的非酶糖基化终产物诱导正常大鼠类糖尿病性视网膜血管病变研究

目的　探讨外源性非酶糖基化终产物 (advancednon enzymaticglycationendproducts ,AGE)在血管壁的沉积与糖尿病性视网膜血管病变之间的关系。方法　将 16只健康两月龄SD大鼠 ,随机分为 4组 ,每组 4只鼠。正常对照组不做任何处理 ;糖尿病对照组应用AGE制作糖尿病模型 ;血清白蛋白 (ratserumalbumin ,RSA)组每日自鼠尾静脉注射RSA(4 0mg kg体重 ) ,连续注射 2周 ;AGE RSA组注射AGE RSA ,注射方法、剂量与RSA组相同。 2周后观察各组鼠视网膜毛细血管周细胞密度变化。结果　注射RSA组鼠视网膜毛细血管周细胞数平均为 (5 80± 0 4 8)个 每油镜视野 ,注射AGE组为(4 31± 0 34)个 每油镜视野 ,显示实验组毛细血管周细胞密度低于对照组 ,差异有显著意义 (F =7 16 4 ,P <0 0 1)。结论　外源性AGE注入正常大鼠会引起类糖尿病性视网膜血管病变 ,AGE可能是糖尿病性视网膜血管病变的独立致病因素之一。     

The angiopoietin/Tie-2 system regulates pericyte survival and recruitment in diabetic retinopathy

PURPOSE: The angiopoietin (Ang) system plays an important role in vascular stabilization and pathologic neovascularization. The hypothesis for the study was that, in addition to modulating endothelial cell behavior, the angiopoietin/Tie-2 system also regulates the pericyte apoptosis and/or the vessel maturation associated with diabetic retinopathy. METHODS: Tie-2 expression in cultured retinal pericytes was analyzed by using ELISA, Western Blot analysis, and flow cytometry. CD13 (aminopeptidase N) expression in pericytes was determined by Western blot analysis and Ang effects verified with Tie-2 antisense treatment. Cell proliferation was monitored by crystal violet uptake, and pericyte migration was assessed in a scrape wound. Annexin V-FITC flow cytometry was used to quantify pericyte apoptosis. RESULTS: Pericytes expressed a functionally active Tie-2 receptor upregulated by both Ang-1 and -2 (P < 0.05). In pericytes undergoing apoptosis induced by either TNF-alpha or high glucose Ang-1 increased survival (P < 0.05 for TNF-alpha; P < 0.01 for high glucose), whereas Ang-2 increased apoptosis. Ang-1 enhanced CD13 expression in a dose-dependent manner (P < 0.05) and stimulated pericyte migration in a synthetic matrix wound-healing assay that was associated with a change in F-actin organization. Addition of Tie-2 antisense confirmed that angiopoietins act through Tie-2. CONCLUSIONS: These findings demonstrate that Tie-2 is functional in pericytes and may play an important role in the progression of diabetic retinopathy, by regulating pericyte loss and influencing the activation state and recruitment of pericytes.     

Diabetes-induced mitochondrial dysfunction in the retina

PURPOSE: Oxidative stress is increased in the retina in diabetes, and antioxidants inhibit activation of caspase-3 and the development of retinopathy. The purpose of this study was to investigate the effect of diabetes on the release of cytochrome c from mitochondria and translocation of Bax into mitochondria in the rat retina and in the isolated retinal capillary cells. METHODS: Mitochondria and cytosol fractions were prepared from retina of rats with streptozotocin-induced diabetes and from the isolated retinal endothelial cells and pericytes incubated in 5 or 20 mM glucose medium for up to 10 days in the presence of superoxide dismutase (SOD) or a synthetic mimetic of SOD (MnTBAP). The release of cytochrome c into the cytosol and translocation of the proapoptotic protein Bax into the mitochondria were determined by the Western blot technique and cell death by caspase-3 activity and ELISA assay. RESULTS: Diabetes of 8 months' duration in rats increased the release of cytochrome c into the cytosol and Bax into the mitochondria prepared from the retina, and this phenomenon was not observed at 2 months of diabetes. Incubation of isolated retinal capillary cells with 20 mM glucose increased cytochrome c content in the cytosol and Bax in the mitochondria, and these abnormalities were accompanied by increased cell apoptosis. Inclusion of SOD or its mimetic inhibited glucose-induced release of cytochrome c, translocation of Bax, and apoptosis. CONCLUSIONS: Retinal mitochondria become leaky when the duration of diabetes is such that capillary cell apoptosis can be observed; cytochrome c starts to accumulate in the cytosol and Bax into the mitochondria. Inhibition of superoxides inhibits glucose-induced release of cytochrome c and Bax and inhibits apoptosis in both endothelial cells and pericytes. Identifying the mechanism by which retinal capillary cells undergo apoptosis may reveal novel therapies to inhibit the development of retinopathy in diabetes.