Chen Wei, Yang Jie, Chen Shuhua, Xiang Hong, Liu Hengdao, Lin Dan, Zhao Shaoli, Peng Hui, Chen Pan, Chen Alex F, Lu Hongwei
1 Center for Experimental Medical Research, The Third Xiangya Hospital of Central South University, Changsha, Hunan, P.R. China.
2 Department of Cardiology, The Third Xiangya Hospital of Central South University, Changsha, Hunan, P.R. China.
Diab Vasc Dis Res. 2017 Nov;14(6):494-501. doi: 10.1177/1479164117723270. Epub 2017 Aug 4.
Mitochondrial Ca overload is implicated in hyperglycaemia-induced endothelial cell dysfunction, but the key molecular events responsible remain unclear. We examined the involvement of mitochondrial calcium uniporter, which mediates mitochondrial Ca uptake, in endothelial cell dysfunction resulting from high-glucose treatment.
Human umbilical vein endothelial cells were exposed to various glucose concentrations and to high glucose (30 mM) following mitochondrial calcium uniporter inhibition or activation with ruthenium red and spermine, respectively. Subsequently, mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA and protein expression was measured by real-time polymerase chain reaction and western blotting. Ca concentrations were analysed by laser confocal microscopy, and cytoplasmic and mitochondrial oxidative stress was detected using 2',7'-dichlorofluorescein diacetate and MitoSOX Red, respectively. Apoptosis was assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and a wound-healing assay was performed using an in vitro model.
High glucose markedly upregulated mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA expression, as well as protein production, in a dose- and time-dependent manner with a maximum effect demonstrated at 72 h and 30 mM glucose concentration. Moreover, high-glucose treatment significantly raised both mitochondrial and cytoplasmic Ca and reactive oxygen species levels, increased apoptosis and compromised wound healing (all p < 0.05). These effects were enhanced by spermine and completely negated by ruthenium red, which are known to activate and inhibit mitochondrial calcium uniporter, respectively.
Mitochondrial calcium uniporter plays an important role in hyperglycaemia-induced endothelial cell dysfunction and may constitute a therapeutic target to reduce vascular complications in diabetes.
线粒体钙超载与高血糖诱导的内皮细胞功能障碍有关，但具体的关键分子事件仍不清楚。我们研究了介导线粒体钙摄取的线粒体钙单向转运体在高糖处理导致的内皮细胞功能障碍中的作用。
分别用钌红和精胺抑制或激活线粒体钙单向转运体后，将人脐静脉内皮细胞暴露于不同葡萄糖浓度及高糖（30 mM）环境中。随后，通过实时聚合酶链反应和蛋白质印迹法检测线粒体钙单向转运体及线粒体钙单向转运体调节因子1信使核糖核酸和蛋白质的表达。用激光共聚焦显微镜分析钙浓度，分别用2',7'-二氯荧光素二乙酸酯和MitoSOX Red检测细胞质和线粒体的氧化应激。通过膜联蛋白V-异硫氰酸荧光素/碘化丙啶染色评估细胞凋亡，并使用体外模型进行伤口愈合试验。
高糖以剂量和时间依赖性方式显著上调线粒体钙单向转运体及线粒体钙单向转运体调节因子1信使核糖核酸的表达以及蛋白质生成，在72小时和30 mM葡萄糖浓度时达到最大效应。此外，高糖处理显著提高了线粒体和细胞质中的钙及活性氧水平，增加了细胞凋亡并损害了伤口愈合（所有p < 0.05）。已知分别激活和抑制线粒体钙单向转运体的精胺增强了这些效应，而钌红则完全消除了这些效应。
线粒体钙单向转运体在高血糖诱导的内皮细胞功能障碍中起重要作用，可能构成减少糖尿病血管并发症的治疗靶点。
