Abstract
There is evidence that the receptor for advanced glycation endproducts (RAGE) is a binding site for amyloid β peptide (Aβ). RAGE is a multiligand cell surface receptor which is normally expressed in brain endothelium and, at low levels, in microglia and neurons. However, in Alzheimer's disease (AD) brains RAGE expression is increased by several-fold in cerebral endothelial cells (CECs), astrocytes, microglia, and neurons. Recent studies have demonstrated that RAGE is an important therapeutic target in AD. Therefore, there is a need to investigate new promising Aβ/RAGE axis blockers. It has been shown that azelnidipine (a highly lipid-soluble dihydropyridine-based calcium channel blocker) had a strong positive effect in RAGE-associated blood vessel damage in nondiabetic patients with stage I or II chronic kidney disease. In this study, we examined the effects of azelnidipine on A β 42 -induced activation of NADPH oxidase and consequent reactive oxygen species (ROS) generation, activation of NF-κB, ERK1/2 phosphorylation in CECs, and expression of P-selectin on the surface of the cells. In this study, immortalized CECs (bEnd3)were applied. To confirm azelnidipine -RAGE binding, we examined the quantitative immunofluorescence microscopy (QIM) of cellular surface RAGE for bEnd3 cells pretreated with azelnidipine and stained with antiRAGE primary antibody (Ab RAGE). To quantify the ROS production, we applied fluorescence microscopy of dihydroethidium (DHE) in the cells. Immunofluorescent microscopy was used to quantify the surface P-selectin expression and NF-κB activation. Confocal immunofluorescence microscopy of double immunofluorescent labeled gp91-phox and p47-phox subunits was performed to characterize NADPH oxidase complex assembly. Western blot analysis was applied to characterize phosphorylation of ERK1/2. We report that azelnidipine competed with the anti-RAGE antibody (Ab RAGE) to bind to RAGE on the surfaces of CECs. In addition, azelnidipine abrogates A β 42 -induced ROS production and co-localization between the cytosolic (p47-phox) and membrane (gp91-phox) subunits of NADPH oxidase. Moreover, azelnidipine suppressed A β 42 induced NF-κB activation, ERK1/2 phosphorylation in CECs, and accumulation of P-selectin on the surface of the cells. This study demonstrated that pretreatment of CECs with azelnidipine can, at least partially, neutralize toxic effect of Aβ on the CECs in vitro.