Li Jun, Wang Peipei, Yu Songping, Zheng Zhi, Xu Xun
Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, PR China.
Mol Vis. 2012;18:2371-9. Epub 2012 Sep 20.
Hyperglycemia-induced vascular cell apoptosis is a seminal early event in diabetic retinopathy. Prolonged hyperglycemia is known to increase intracellular cytosolic free calcium ([Ca(2+)]i) in retinal vascular endothelial cells (RECs), suggesting that [Ca(2+)]i is a critical trigger for microvascular degeneration. This study aims to elucidate Ca(2+)-dependent signaling mechanisms that mediate hyperglycemia-induced apoptosis in RECs.
A cultured macaque choroid-retinal endothelial cell line (RF/6A) was incubated in normal glucose (NG), NG plus the Ca(2+) entry blocker 2-aminoethoxydiphenyl borate (2-APB), high glucose (HG), or HG plus either 2-APB, the c-jun N-terminal kinase (JNK) inhibitor SP600125, or the calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93. Changes in [Ca(2+)]i evoked by adenosine 5'-triphosphate (ATP) were measured in fluo-3/AM-loaded RF/6A cells by confocal microscopy. The mitochondrial membrane potential (ΔΨm) and apoptosis were assessed by flow cytometry. Expression levels of CaMKII, phosphorylated CaMKII (p-CaMKII), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), the death receptor (Fas), and cytochrome c were detected by western blotting analysis.
Prolonged exposure to HG (96 h) potentiated ATP-evoked Ca(2+) entry as well as CaMKII phosphorylation and RF/6A cell apoptosis. Enhanced apoptosis was blocked by 2-APB and KN93. Furthermore, HG increased JNK phosphorylation and Fas expression, and both responses were partially blocked by 2-APB and KN93, while the JNK inhibitor SP600125 partially reduced HG-induced Fas expression. In addition, HG depolarized the ΔΨm and triggered the release of mitochondrial cytochrome c. These early signs of mitochondria-dependent apoptosis were partially reversed by 2-APB and KN93.
HG-induced apoptosis in RF/6A cells depends on Ca(2+) entry and CaMKII activation, leading to the activation of both Fas-dependent and mitochondria-dependent apoptosis pathways. The CaMKII-JNK-Fas pathway is involved in HG-evoked apoptosis of RECs.
高血糖诱导的血管细胞凋亡是糖尿病视网膜病变早期的一个关键事件。已知长期高血糖会增加视网膜血管内皮细胞（RECs）内的细胞溶质游离钙（[Ca(2+)]i），这表明[Ca(2+)]i是微血管退变的关键触发因素。本研究旨在阐明介导高血糖诱导RECs凋亡的钙依赖信号机制。
将培养的猕猴脉络膜视网膜内皮细胞系（RF/6A）分别置于正常葡萄糖（NG）、NG加钙内流阻滞剂2-氨基乙氧基二苯基硼酸（2-APB）、高葡萄糖（HG）或HG加2-APB、c-jun氨基末端激酶（JNK）抑制剂SP600125或钙/钙调蛋白依赖性蛋白激酶II（CaMKII）抑制剂KN93中孵育。通过共聚焦显微镜测量用fluo-3/AM负载的RF/6A细胞中由三磷酸腺苷（ATP）诱发的[Ca(2+)]i变化。通过流式细胞术评估线粒体膜电位（ΔΨm）和细胞凋亡情况。通过蛋白质印迹分析检测CaMKII、磷酸化CaMKII（p-CaMKII）、c-Jun氨基末端激酶（JNK）、磷酸化JNK（p-JNK）、死亡受体（Fas）和细胞色素c的表达水平。
长时间暴露于HG（96小时）会增强ATP诱发的钙内流以及CaMKII磷酸化和RF/6A细胞凋亡。2-APB和KN93可阻断增强的细胞凋亡。此外，HG会增加JNK磷酸化和Fas表达，这两种反应均被2-APB和KN93部分阻断，而JNK抑制剂SP600125可部分降低HG诱导的Fas表达。此外，HG使ΔΨm去极化并触发线粒体细胞色素c的释放。这些线粒体依赖性细胞凋亡的早期迹象被2-APB和KN93部分逆转。
HG诱导的RF/6A细胞凋亡依赖于钙内流和CaMKII激活，导致Fas依赖性和线粒体依赖性凋亡途径均被激活。CaMKII-JNK-Fas途径参与了HG诱发的RECs凋亡。
