Increased Susceptibility to Amyloid-β Toxicity in Rat Brain Microvascular Endothelial Cells under Hyperglycemic Conditions

Abstract

We hypothesized that hyperglycemia-induced mitochondrial dysfunction and oxidative stress are closely associated with amyloid-β peptide (Aβ) toxicity in endothelial cells. Brain microvascular endothelial cells from rat (RBMEC) and mice (MBMEC) were isolated from adult Sprague-Dawley rats and homozygous db/db (Lepr^db/Lepr^db) and heterozygous (Dock7^m/Lepr^db) mice, and cultured under normo- and hyperglycemic conditions for 7 d followed by 24 h exposure to Aβ_1-40. Some experiments were also performed with two mitochondrial superoxide (O₂^•−) scavengers, MitoTempo and Peg-SOD. Cell viability was measured by the Alamar blue assay and mitochondrial membrane potential (ΔΨm) by confocal microscopy. Mitochondrial O₂^•− and hydrogen peroxide (H₂O₂) production was assessed by fluorescence microscopy and H₂O₂ production was confirmed by microplate reader. Hyperglycemia or Aβ_1-40 alone did not affect cell viability in RBMEC. However, the simultaneous presence of high glucose and Aβ_1-40 reduced cell viability and ΔΨm, and enhanced mitochondrial O₂^•− and H₂O₂ production. MitoTempo and PEG-SOD prevented Aβ_1-40 toxicity. Interestingly, MBMEC presented a similar pattern of alterations with db/db cultures presenting higher susceptibility to Aβ_1-40. Overall, our results show that high glucose levels increase the susceptibility of brain microvascular endothelial cells to Aβ toxicity supporting the idea that hyperglycemia is a major risk factor for vascular injury associated with AD.