High glucose, glucose fluctuation and carbonyl stress enhance brain microvascular endothelial barrier dysfunction: Implications for diabetic cerebral microvasculature

摘要

We previously demonstrated that in normal glucose (5 mM), methylglyoxal (MG, a model of carbonyl stress) induced brain microvascular endothelial cell (IHEC) dysfunction that was associated with occludin glycation and prevented by N-acetylcysteine (NAC). Herein, we investigated the impact of high glucose and low GSH, conditions that mimicked the diabetic state, on MG-induced {IHEC} dysfunction. MG-induced loss of transendothelial electrical resistance (TEER) was potentiated in {IHECs} cultured for 7 or 12?days in 25 mM glucose (hyperglycemia); moreover, barrier function remained disrupted 6 h after cell transfer to normal glucose media (acute glycemic fluctuation). Notably, basal occludin glycation was elevated under these glycemic states. {TEER} loss was exaggerated by inhibition of glutathione (GSH) synthesis and abrogated by NAC, which corresponded to {GSH} decreases and increases, respectively. Significantly, glyoxalase {II} activity was attenuated in hyperglycemic cells. Moreover, hyperglycemia and {GSH} inhibition increased {MG} accumulation, consistent with a compromised capacity for {MG} elimination. α-Oxoaldehydes (MG plus glyoxal) levels were elevated in streptozotocin-induced diabetic rat plasma. Immunohistochemistry revealed a prevalence of MG-positive, but fewer occludin-positive microvessels in the diabetic brain in vivo, and Western analysis confirmed an increase in MG–occludin adducts. These results provide the first evidence that hyperglycemia and acute glucose fluctuation promote MG–occludin formation and exacerbate brain microvascular endothelial dysfunction. Low occludin expression and high glycated-occludin contents in diabetic brain in vivo are factors that would contribute to the dysfunction of the cerebral microvasculature during diabetes.