The Role And Mechanism Of Autophagy In Diabetic Stroke Vascular Endothelial Cells Injury

Background:As the fact that type2diabetic patients hold2-4folds higher risk for cerebrovascular disease and stroke, but the mechanism how diabetes associated with ischemia is still not very clear by now. Accumulating evidences show that methylglyoxal (MGO) plays a critical role in the progression of diabetic vascular complications as one of the highly reactive intermediate carbonyl groups which holds200～50000folds higher glycosylation activity than glucose. The cytotoxicity of MGO is mediated by the modification of DNA and activation of apoptosis. Studies show that autophagy is associated with diabetes mellitus by removing its increased fissile mitochondria in the endothelium which is known as a process by which cells recycle cytoplasm and remove organelles with defective structures. However, it is now widely accepted that autophagy is activated as a survival mechanism by removal of toxic subjects and damaged organelles when the injury is under a certain threshold, but when damage exceeds this warning line, autophagy leads to cell death. But in cerebrovascular diseases with diabetes, the evidence that whether autography in involved in the mechanism of pathogenesis is still not very clear. Previous studies have reported that cancer cells can survive when the the focal adhesion kinase (FAK) pathway is severely perturbed, and this mechanism is strongly associated to the enhanced expression of autophagy signaling as a protective procedure, and interestingly, FAK activity can be inhibited by induction of autophagy. But whether FAK pathway is associated with autophagy in normal cells has not reported yet. This study is devoted to confirm the role of antophagy in diabetic stroke vascular endothelial cells injury and find the possible mechanism of it.Methods:1、MGO was used as a trigger factor of hyperglycemic condition that induced cell injury in cultured human umbilical vein vascular endothelial cells (HUVECs) in vitro. Cell viability was detected after MGO administration and autophagy activity was investigated during the treatment.2、Rapamycin or3-MA was added as the inducer or inhibitor of autophagy to investigate the autophagy’s role in MGO-induced cell injury. Cell viability was detected after MGO administration and autophagy activity was investigated during the treatment.3、PF573228was added into MGO-induced cell injury to block the FAK pathway. The autophagy activity was investigated and cell viability was also detected.4、An oxygen and glucose deprivation (OGD) experiment was followed after MGO administration to simulate ischemia. Rapamycin,3-MA or PF573228was added into the system and the cell viability was detected.5、A permanent middle cerebral artery occlusion (pMCAO) surgery was performed in type I diabetic male SD rats. The infarct volume and neurobehavioral test were assessed to verify the exacerbation of ischemic injury in diabetic rats. Then, BBB permeability and the expression of autophagy in cerebrovascular endothelial cells was evaluated.6、Before the pMCAO surgery, a dose of rapamycin was injected into the right lateral ventricle. After surgery, BBB permeability was evaluated also.Results:1. MGO injured HUVECs viability with a dose-dependent manner. MGO-induced injury was associated with transient elevation of autophagy prompted by the up-regulated LC3-II/LC3-I expression ratio in cultured HUVECs.2. Rapamycin up-regulated the autophagy expression prompted by the up-regulated LC3-II/LC3-I ratio and improved the cell viability on MGO-induced injury in cultured HUVECs.3. PF573228also blocked autophagy pathway and made the cell viability in MGO-induced injury even worse in cultured HUVECs.4. MGO administration exacerbated OGD-induced injury in cultured HUVECs. Rapamycin improved the cell viability, while3-MA and PF573228worsened it on MGO enhancing OGD-induced injury in cultured HUVECs.5. Type I diabetic rats showed a significant increased infarct volume, a reduced neurobehavioral test score, and a higher BBB permeability after pMCAO onset. The autophagy related protein Beclin-1in cerebrovascular endothelial cells was higher in diabetic rats.6. Enhancing of autophagy by right lateral ventricle injection of rapamycin improved the BBB permeability on diabetes enhancing ischemia injury in SD Rats.Conclusions:In the present study, we evaluated the LC3-II/LC3-I expression ratio to investigated autophagy activity in HUVECs. Data showed that autophagy was activated in the early stage of MGO-induced injury. The MGO-induced transient elevation of autophagy played a protective effect in cultured HUVECs. Induction of autophagy also provided a better outcome on MGO enhancing OGD-induced cell injury. And this effect may associated with FAK phosphorylation.Furthermore, in type I diabetic male SD rats, the ischemic injury was worse than normoglycemic ones. The transient elevation of autophagy related protein Beclin-1was also verified in diabetic rats’ cerebrovascular endothelial cells early after a pMCAO surgery onset and played as a protective role on cerebrovascular endothelial function.All above proved that autophagy plays a protective role in diabetic stroke vascular endothelial cells injury and may through FAK phosphorylation.