title: Effects of beta-amyloid on rat neuromicrovascular endothelial cells cultured in vitro.
abstract: Several studies have shown that beta-amyloid (beta A) deposits are associated with damage of cerebral vessels and that in Alzheimer's disease (AD) beta A peptides are cytotoxic for cerebral endothelial cells (ECs). However, little is known about the mechanisms underlying these effects of beta A peptides. Hence, we have investigated the effects of beta A(1-40) and beta A(1-42) on rat neuromicrovascular ECs (NECs) cultured in vitro. NECs were isolated, plated (1.5x10(4) cells/cm2) on collagen/fibronectin-coated Petri dishes and cultured in EC growth medium MV2. After 24 h of culture, NECs were incubated with beta A(1-40) and beta A(1-42) (10(-9) or 10(-7) M) and cultured for another 3, 24 or 48 h. Cell viability was assayed by either trypan blue exclusion or by measuring redox activity (MTS assay). Cell proliferation was assessed by measuring the incorporation of 5'-bromo-2'-deoxyuridine into DNA, cell apoptosis by TUNEL assay and cell necrosis by evaluating the release of lactate dehydrogenase. The morphology of cultured NECs was examined by transmission electron microscopy. Other NECs were plated (2.5x10(4) cells/cm2) on Matrigel coated-wells and incubated for 18 h in the presence or absence of beta A peptides for in vitro angiogenesis assay. Beta A peptides significantly decreased NEC viability and enhanced cell apoptosis and necrosis rates. NEC proliferation was not significantly affected. The effects were seen after an incubation period of 3 h (and also 24 h in the case of the redox activity) but not 48 h and beta A(1-42) was much more effective in its toxic effects than beta A(1-40). NECs incubated for 24 h with beta A peptides displayed ultrastructural signs of cell degeneration. beta A peptides prevented NECs cultured on Matrigel to form a capillary-like network and image analysis showed that the downloading of dimensional and topological parameters of the meshwork was significant only in the case of the incubation with beta A(1-42). Collectively our findings allow us to conclude that i) beta A peptides exert a marked toxic effect on cultured NECs, which conceivably reduces their in vitro angiogenic activity; ii) beta A(1-42) is the more toxic form, which could suggest its main role in the pathogenesis of cerebral vessel lesions in AD and iii) the maximum toxic action occurs after a short incubation period, which could be explained by assuming that beta A peptides are unable to accumulate in NECs due to their rapid degradation, thereby allowing NECs to fully recover within 48 h.
authors.affiliation: Department of Biology, University of Padua, Padua I-35121, Italy.
authors.forename: Marcella
authors.initials: M
authors.lastname: Folin
authors.forename: Silvia
authors.initials: S
authors.lastname: Baiguera
authors.forename: Mara
authors.initials: M
authors.lastname: Tommasini
authors.forename: Diego
authors.initials: D
authors.lastname: Guidolin
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Conconi
authors.forename: Eugenio
authors.initials: E
authors.lastname: De Carlo
authors.forename: Gastone G
authors.initials: GG
authors.lastname: Nussdorfer
authors.forename: Pier Paolo
authors.initials: PP
authors.lastname: Parnigotto
mesh_terms: D016229:Amyloid beta-Peptides / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001921:Brain / Q000098:blood supply; D001973:Bromodeoxyuridine / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D009336:Necrosis / Q000473:pathology; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
chemical_list: D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); D007770:L-Lactate Dehydrogenase; D001973:Bromodeoxyuridine