Western blotting analysis showing VEGF-stimulated (20 ng/ml for 0 to 30 min) eNOS phosphorylation at serine 1179 in BAECs cultured for 24 h in the presence (black bars) or absence (control, white bars) of 10 μM Aβ25–35
(A) and 5 μM Aβ1–42
(B). Blots were subjected to densitometric analysis and the obtained data were analyzed for statistical significance (*P < 0.01 versus 0 min control group, n = 3). (C-D) Western blotting analysis demonstrating VEGF-stimulated eNOS phosphorylation at serine 1179 in BAECs cultured for 24 h in the presence of reversed inactive 10 μM Aβ35–25
(C) and 5 μM Aβ42–1
(D). (E) Endothelial nitric oxide release. cGMP formation in BAECs cultured for 24 h in the presence (black bars) or absence (white bars) of 10 μM Aβ25–35 and 5 μM Aβ1–42 and following stimulation with 20 ng/ml VEGF for 30 min. cGMP formation, which directly reflects the nitric oxide release, was determined by reporter cell assay by measuring picomoles of cGMP generated in rat aortic smooth muscle cells after 3 min of exposure of the cells to media of BAECs after the different treatments, as indicated above and as explained in the text. To assess the assay specificity, some experiments were conducted with BAECs pre-stimulated with 100 μM of the nitric oxide synthase inhibitor L-NAME (LN). (*P < 0.05 versus control, n = 4, #P < 0.05 versus VEGF, n = 4). VEGF, vascular endothelial growth factor; eNOS, endothelial nitric oxide synthase; min, minutes.