Li W, Liu X, He Z, Yanoff M, Jian B, Ye X
Department of Ophthalmology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102, USA.
Invest Ophthalmol Vis Sci. 1998 Aug;39(9):1535-43.
Retinal capillary pericytes underwent apoptosis in vitro after they had been exposed long-term to high levels of glucose followed by a rapid glucose reduction. The present work was designed to study the expression of bcl-2 family members and apoptosis regulatory genes and to determine the status of oxidative stress induced by high concentrations of glucose in this in vitro apoptosis model.
Pericytes were grown in normal or high glucose concentrations (5, 20, 30, and 40 mM) for 10 days and then exposed to a rapid reduction of glucose to 0.5 mM or 5 mM. Pericyte cell death was evaluated by determining the loss of cell viability and the fragmentation of DNA using agarose gel electrophoresis. In parallel, the quantitative reverse transcription-polymerase chain reaction technique was used to determine the expression of bcl-2, bax, p53, and glutathione peroxidase (GSH-Px) genes. The intracellular level of glutathione (GSH) and the DNA fragmentation were determined simultaneously for pericytes treated with or without exogenous GSH monoethylester. Retinal capillary endothelial cells, experiencing the same glucose variation, were studied as a comparison.
For pericytes, downregulation of bcl-2 was observed as early as 24 hours after rapid glucose reduction, whereas DNA fragmentation was not detectable at that time. After 72 hours, a decreased protein ratio of Bcl-2 to Bax was concomitant to evident loss of pericyte viability. During the period of high glucose and the following glucose reduction, p53 expression essentially was unchanged. Decreased levels of GSH induced by high concentrations of glucose (>30 mM) became further depleted when the glucose levels were rapidly reduced. Addition of GSH monoethylester to the medium restored the level of GSH in pericytes and prevented pericyte apoptosis induced by glucose variation. Moreover, the mRNA levels of GSH-Px were significantly elevated. By contrast, with the same glucose reduction endothelial cells did not undergo apoptosis. Their mRNA levels of bcl-2, bax, and GSH-Px essentially were unchanged.
High levels of glucose and rapid reduction of glucose modulate the expression of bcl-2 family genes in retinal pericytes. Upregulation of GSH-Px and depletion of GSH indicate a reparative process of accelerated elimination of reactive oxygen species following rapid glucose reduction. These findings indicate that the aggravated oxidative stress and the weakened antioxidant defense induced by the combined effects of high levels of glucose and subsequent rapid glucose reduction cause pericyte apoptosis. Prevention of DNA fragmentation of pericytes by exogenous GSH further supports this notion. Because endothelial cells did not show similar pathologic changes, this proposed mechanism seems to be specific to pericytes.
视网膜毛细血管周细胞在长期暴露于高糖环境后再迅速降低葡萄糖浓度时，会在体外发生凋亡。本研究旨在探讨bcl-2家族成员及凋亡调控基因的表达情况，并确定在该体外凋亡模型中高浓度葡萄糖诱导的氧化应激状态。
将周细胞分别培养于正常或高糖浓度（5、20、30和40 mM）环境中10天，然后迅速将葡萄糖浓度降至0.5 mM或5 mM。通过测定细胞活力丧失及利用琼脂糖凝胶电泳检测DNA片段化来评估周细胞死亡情况。同时，采用定量逆转录-聚合酶链反应技术检测bcl-2、bax、p53和谷胱甘肽过氧化物酶（GSH-Px）基因的表达。对添加或未添加外源性谷胱甘肽单乙酯处理的周细胞，同时测定细胞内谷胱甘肽（GSH）水平和DNA片段化情况。作为对照，研究经历相同葡萄糖浓度变化的视网膜毛细血管内皮细胞。
对于周细胞，在迅速降低葡萄糖浓度后24小时即观察到bcl-2下调，而此时DNA片段化尚未检测到。72小时后，Bcl-2与Bax的蛋白比例下降，同时周细胞活力明显丧失。在高糖期及随后的葡萄糖降低过程中，p53表达基本未变。高浓度葡萄糖（>30 mM）诱导的GSH水平降低在葡萄糖迅速降低时进一步加剧。向培养基中添加谷胱甘肽单乙酯可恢复周细胞内GSH水平，并防止葡萄糖变化诱导的周细胞凋亡。此外，GSH-Px的mRNA水平显著升高。相比之下，相同的葡萄糖降低过程中内皮细胞未发生凋亡。它们的bcl-2、bax和GSH-Px的mRNA水平基本未变。
高糖及葡萄糖迅速降低可调节视网膜周细胞中bcl-2家族基因的表达。GSH-Px上调及GSH耗竭表明在葡萄糖迅速降低后加速清除活性氧的修复过程。这些发现表明，高糖及随后葡萄糖迅速降低的联合作用诱导的氧化应激加剧及抗氧化防御减弱导致周细胞凋亡。外源性GSH预防周细胞DNA片段化进一步支持了这一观点。由于内皮细胞未表现出类似的病理变化，该机制似乎是周细胞特有的。
