Fig. 4: Extracellular interactions with VE-cadherin are necessary for APEL induction by Aβ aggregates.
From: Endothelial leakiness elicited by amyloid protein aggregation
a APEL arising from Aβo or Aβs (20 µM, 30 min) was inhibited by an EC1 domain antibody (EC1a) pre-treatment (EC1a dilutions 1:100 and 1:500, 1 h prior to amyloid protein treatments), which specifically blocked the extracellular domain of VE-cadherin (n = 3 biologically independent experiments, representative micrographs are presented). The post-addition of EC1a after 0.5 h-incubation of amyloid proteins did not negate the EL occurrence induced by Aβo/Aβs (Supplementary Fig. 7). Gap distributions were obtained from captured confocal images through trainable Weka segmentation plugin in ImageJ software. Scale bars: 20 μm. Black dots in the images represent holes in the HMVECs monolayer. Red: VE-cadherin. Y and X axes revealed the combination of pre-treatment (Media/EC1a) and Aβo/Aβs (−/+) employed. b, c Gap area percentages were analyzed by ImageJ according to the images from panel a and Supplementary Fig. 7. Data are shown as mean ± SD (n = 3 images belongs to three biologically independent samples), analyzed via two-tailed Student’s t tests. The derived P values compared between groups are shown. d Co-localization of ThT-labeled Aβs and VE-cadherin was observed in HMVECs near their cell junctions, revealing association between them (n = 3 biologically independent experiments). Red: VE-cadherin, green: ThT-labeled Aβs. Scale bars: 20 μm. e Co-immunoprecipitation assay pulled down with an anti-APP antibody, for probing the interactions between VE-cadherin (VEC) and the four forms of Aβ following HMVECs exposure to Aβo (0, 20, and 40 µM, 30 min). A representative blot is displayed, out of three biologically independent experiments.
