Diabetes Mellitus Aggravates Hemorrhagic Transformation after Ischemic Stroke via Mitochondrial Defects Leading to Endothelial Apoptosis

Diabetes is a crucial risk factor for stroke and is associated with increased frequency and poor prognosis. Although endothelial dysfunction is a known contributor of stroke, the underlying mechanisms have not been elucidated. The aim of this study was to elucidate the mechanism by which chronic hyperglycemia may contribute to the worsened prognosis following stroke, especially focusing on mitochondrial alterations. We examined the effect of hyperglycemia on hemorrhagic transformation at 24 hours after middle cerebral artery occlusion (MCAO) in streptozotocin (STZ) -induced diabetic mice. We also examined the effects of high-glucose exposure for 6 days on cell death, mitochondrial functions and morphology in human brain microvascular endothelial cells (HBMVECs) or human endothelial cells derived from induced pluripotent stem cells (iCell endothelial cells). Hyperglycemia aggravated hemorrhagic transformation, but not infarction following stroke. High-glucose exposure increased apoptosis, capase-3 activity, and release of apoptosis inducing factor (AIF) and cytochrome c in HBMVECs as well as affected mitochondrial functions (decreased cell proliferation, ATP contents, mitochondrial membrane potential, and increased matrix metalloproteinase (MMP)-9 activity, but not reactive oxygen species production). Furthermore, morphological aberration of mitochondria was observed in diabetic cells (a great deal of fragmentation, vacuolation, and cristae disruption). A similar phenomena were seen also in iCell endothelial cells. In conclusion, chronic hyperglycemia aggravated hemorrhagic transformation after stroke through mitochondrial dysfunction and morphological alteration, partially via MMP-9 activation, leading to caspase-dependent apoptosis of endothelial cells of diabetic mice. Mitochondria-targeting therapy may be a clinically innovative therapeutic strategy for diabetic complications in the future.

糖尿病是脑卒中的重要危险因素，且与脑卒中发病频率升高及预后不良密切相关。尽管已知内皮功能障碍是脑卒中的致病因素之一，但其具体潜在机制尚未阐明。本研究旨在阐明慢性高血糖加重脑卒中后不良预后的潜在机制，重点关注线粒体结构与功能的异常改变。本研究以链脲佐菌素（streptozotocin, STZ）诱导的糖尿病小鼠为模型，观察了高血糖对大脑中动脉闭塞（middle cerebral artery occlusion, MCAO）术后24小时出血性转化的影响。此外，本研究还观察了6天高糖暴露对人脑微血管内皮细胞（human brain microvascular endothelial cells, HBMVECs）及诱导多能干细胞来源的人内皮细胞（iCell内皮细胞）的细胞死亡、线粒体功能与形态的影响。实验结果显示，高血糖可加重脑卒中后的出血性转化，但对脑梗死体积无显著影响。高糖暴露可上调人脑微血管内皮细胞的凋亡水平、半胱天冬氨酸蛋白酶3（caspase-3）活性，促进凋亡诱导因子（apoptosis inducing factor, AIF）及细胞色素c的释放；同时损伤线粒体功能：表现为细胞增殖能力下降、三磷酸腺苷（ATP）含量降低、线粒体膜电位受损，且升高基质金属蛋白酶-9（matrix metalloproteinase, MMP-9）活性，但未显著影响活性氧（reactive oxygen species）的生成。此外，糖尿病模型小鼠的内皮细胞中可见线粒体形态异常，表现为大量碎片化、空泡化及嵴结构断裂。iCell内皮细胞中也观察到了类似的线粒体形态异常现象。综上，慢性高血糖可通过诱导线粒体功能障碍与形态异常，部分经由基质金属蛋白酶-9活化通路，加重糖尿病小鼠脑卒中后的出血性转化，最终引发内皮细胞的半胱天冬氨酸蛋白酶依赖型凋亡。未来，线粒体靶向治疗有望成为糖尿病相关并发症的创新性临床治疗策略。