Fig. 1: Importance of the AT1R-RAGE axis in Ang II-induced endothelial hyperpermeability in HUVECs.

a Changes in the phosphorylation of VE-cadherin (Y731), Src (Tyr416), and β-catenin (Ser552) in HUVECs following transfection with RAGE siRNA and treatment with Ang II. Expression values were normalized to those of VE-cadherin, Src, and β-catenin (n = 4 for each lane). b Immunocytochemistry of VE-cadherin (green) and DAPI (nuclei, blue), as examined under a confocal microscope (white scale bars: 50 μm in merged images and 20 μm in magnified images; ×400 magnification). The main images were selected from representative regions. c HUVECs transfected with siRNA targeting RAGE or with scrambled sequences were incubated for 72 h and were then stimulated with Ang II for 6 h. TEER was measured every 2 h. ***p < 0.001 vs. control, ^###p < 0.001 vs. Ang II (n = 3 for each lane). d, e Protein expression in HUVECs treated with Ang II and losartan (10 μM) alone or in combination for 4 h, as determined by western blotting. The relative values of AT1R, RAGE, and mDia1 expression values were normalized to that of GAPDH (n = 4 for each lane). The relative values of phospho-Src, phospho-β-catenin, and phospho-VE-cadherin expression were normalized to those of Src, β-catenin, and VE-cadherin, respectively (n = 3 for each lane). f Changes in AT1R protein levels in HUVECs following transfection with RAGE siRNA, as determined by western blotting. Expression was normalized to that of GAPDH (top). Changes in AT1R mRNA expression in HUVECs following transfection with RAGE siRNA, as determined by RT-PCR (bottom). Expression was normalized to that of the 18S rRNA gene (n = 4 for each lane). The values are presented as the means ± SEMs. *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test