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. 2021 Feb 1;131(3):e140315. doi: 10.1172/JCI140315

Figure 1. M2 macrophages induce hypopermeability by downregulating VCAM1 in ECs (A) KEGG pathway analysis of DEGs in M1 macrophage–treated HUVECs versus M2 macrophage–treated HUVECs.

Figure 1

(B) Gene expression heatmap of differentially expressed CAM genes from A. (C) Expression of VCAM1 in HUVECs detected by Western blot in different cocultures (n = 3). (D) Localization of VCAM1 protein (red) by immunofluorescence analysis. DAPI stains cell nucleus. Scale bar: 20 μm. (E) p–VE-cad expression in M1 macrophage–cocultured HUVECs that were transiently transfected with VCAM1-specific shRNAs (shVCAM1-a, shVCAM1-b) or a control shRNA (shCtrl) (n = 3). (F) Immunofluorescence analysis of p–VE-cad in M1 macrophage–cocultured HUVECs with VCAM1 knocked down. Scale bar: 20 μm. (G) p–VE-cad expression in M2 macrophage–cocultured HUVECs that were transiently transfected with a VCAM1-specific vector (Ove-VCAM1) or a control vector (n = 3). (H) Immunofluorescence analysis of p–VE-cad in M2 macrophage–cocultured HUVECs with VCAM1 overexpressed. Scale bar: 20 μm. (I) TRITC-Dextran tracer fluorescence from M1 macrophage–cocultured HUVECs that were transfected with VCAM1 knockdown shRNAs (shVCAM1-a, shVCAM1-b) or a control shRNA (shCtrl; n = 5). (J) TRITC-dextran tracer fluorescence from M2 macrophage–cocultured HUVECs that were transfected with VCAM1-specific vector (Ove-VCAM1) or control vector (n = 5). Data represent 3 independent experiments. Results are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001, 1-way ANOVA (C, E, I) and Student’s t test (G and J).