Effect of beta-amyloid on endothelial cells: lack of direct toxicity, enhancement of MTT-induced cell death and intracellular accumulation.
Several cerebrovascular alterations have been described in Alzheimer's disease (AD) including an accumulation of beta-amyloid (betaA) on the vascular walls in the brain. To investigate the potential toxic activity of betaA on endothelial cells (EC), two endothelial murine cell lines derived from heart and brain were exposed to betaA1-42 and the biologically active fragment betaA25-35 in the range from 5nM to 50 microM. In a low concentration range (50 nM to 2.5 microM) both peptides significantly reduced the 3-(4,5-dimethylthiazol-2y1)-2-5-diphenyltetrazolium bromide (MTT) signal in the endothelial cell lines exposed for 24h. However, microscopic examination, lactate dehydrogenase (LDH) release determination and Neutral Red assay did not confirm any toxic effect associated with inhibition of MTT formazan reduction. The effect on MTT was not susceptible to anti-oxidant treatment and did not increase the sensitivity to oxidative stress. However, when the EC were exposed to betaA and MTT for 1h, cell viability, determined by LDH release, was strongly reduced, while in normal conditions MTT-induced cell death only after 2h. An inhibitor of lysosomal ATPase activity, bafilomycin A1, completely antagonized this effect. The morphological examination showed that the functional activation by betaA in EC enhanced the production of MTT formazan crystals. To verify the accumulation of betaA in the lysosomal compartment we analyzed the subcellular distribution of betaA1-42 at different exposure times of EC to the peptide. The peptide was found in several organelles and was absent in the cytoplasmic compartment; co-treatment with bafilomycin A1 did not reduce the intracellular presence of betaA1-42. In our condition, the exposure of EC to betaA induced an intracellular accumulation of the peptide and a vasoactive effect that did not appear associated with direct toxic activity.
Soriano
Francesc X
FX
Laboratory of Biology of Neurodegenerative Disorders, Istituto di Ricerche Fannacologiche Mario Negri, Via Eritrea 62, Milan 20157, Italy.
Galbete
José Luis
JL
Forloni
Gianluigi
G
D016229:Amyloid beta-Peptides / Q000378:metabolism / Q000633:toxicity*; D000818:Animals; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects*; D002460:Cell Line; D004727:Endothelium / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D013347:Subcellular Fractions / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism / Q000494:pharmacology*; D013844:Thiazoles / Q000378:metabolism / Q000494:pharmacology*
D016229:Amyloid beta-Peptides; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue