Effects Of Vascular Endothelial Growth Factor In Cultured Brain Microvascular Endothelial Cells During Hypoglycemia

Hypoglycemia is a common and serious complication among patients with diabetes receiving intensive treatment with insulin. Clinical studies have shown that hypoglycemic brain edema initiated the process of hypoglycemic brain injury. Brain endomicrovascular cell is an important component of blood-brain barrier. Tight junctions(TJs) are located between adjacent endothelial cells and maintain the stability of the internal environment by limiting the small molecules. However, whether hypoglycemia can destroy TJs and, in turn, affect the brain microvascular endothelial cells function as result ofbrain edema, which has so far failed to be studied. Vascular endothelial growth factor(VEGF), as a potential factor of regulating blood vessel function, plays a protective role in cerebral ischemia, which has been confirmed. It also can promote the growth of neurons and synapses of survival. This prompted us to explore the protective effect of VEGF on the hypoglycemic cerebral endothelial cell function.We adopt immortalized mouse brain endomicrovascular cells as the research object, mimicking in vitro the blood-brain barrier. First, we investigated the effect of low glucose conditions on brain endomicrovascularcells. We detected the transendothelial cells permeability with fluorescein sodium(Na-F), and tested the expression and distribution of TJs(claudin-5, ZO-1 and occludin) with Western blot and cell immunofluorescence(IF). The effect of low glucose on cell toxicity also has been detected with Lactate dehydrogenase(LDH) experiment. The results showed that, compared with the normal glucose group, the transendothelial cells permeability in groups of low glucose(0, 0.5, 1 mM) were significantly increased and the expression of the protein claudin-5 also dropped accordingly. But when cells exposed to a long time(24 h) and lower glucose concentration(0, 0.5 mM) induced higher cell toxicity.Furthermore, we detected the VEGF secretion of DMEM under the condition of low glucose with ELISA for 24 h. We found the VEGF content of low glucose group was higher than normal glucose group. Finally, we detected transendothelial cells permeability, the expression and distribution of TJs(claudin-5, ZO-1 and occludin) and cell toxicity to explore the protective effect of exogenous VEGF under hypoglycemic condition on the cerebral endothelial cell function. In addition, we also tested the the expression changes of Glucose transporter-1(Glut-1) and apoptosis regulator Bcl-2. The results showed that, compared with low sugar group, the transendothelial cells permeability under the condition of low glucose in presence of VEGF decreased, the expression of claudin-5 also increased accordingly. Moreover, the endothelial cell toxicity has dropped significantly. In addition, the Glut-1 and the Bcl-2 protein expression level were significantly upregulated.To sum up, the study showed that hypoglycemia can significantly increase transendothelial permeability by downregulating claudin-5 expression. We further showed that exogenous VEGF protected brain endothelial cells against hypoglycemia.