Apelin inhibits VEGF-mediated hyperpermeability. (A) Effect of apelin on VE-cadherin–mediated endothelial junctions. VE-cadherin expression was studied in HUVEC monolayers. HUVECs cultured with or without pretreatment with apelin for 10 minutes were stimulated with VEGF (20 ng/mL) for 10 minutes. F-actin (red) and VE-cadherin (VE-cad.; green) staining is shown. Arrowheads indicate the gaps between adjacent ECs and lack of VE-cadherin. Nuclei were labeled with TOPRO-3 (TOP.; blue). (B) Suppression of VEGF-mediated VE-cadherin internalization by apelin. HUVEC cell surface VE-cadherin labeled by anti–VE-cadherin antibody and stimulated with VEGF (20 ng/mL) in the presence (apelin) or absence (control) of apelin (100 ng/mL). VE-cadherin internalization was visualized in fixed cells using secondary fluorescent antibodies (green). To confirm the internalization of VE-cadherin, cell surface bound VE-cadherin antibodies were removed by a mild acid wash before fixation (right 2 lines of images). Nuclei were labeled with TOPRO-3 (blue). (C) The internalization of endogenous VE-cadherin (in the right 2 lines of panel B) was quantified by the fluorescence intensity of secondary antibodies per cell. *P < .01. (D) Western blot analysis of cell surface and cytoplasmic VE-cadherin protein on HUVECs that had been stimulated with VEGF (20 ng/mL) and/or apelin (100 ng/mL) for 20 minutes. The purity of cytoplasmic protein was confirmed by the lack of expression of the cell surface protein N-cadherin. GAPDH expression was analyzed as a control for an unrelated intracellular protein.