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. 2018 Feb 19;128(3):1106–1124. doi: 10.1172/JCI93025

Figure 4. M(Hb) macrophages promote vascular permeability via VEGF-A/VEGFR2 signaling.

Figure 4

(A) TEER measurements after treatment of scramble siRNA– (Scr) or VEGFR2 siRNA–transfected HAECs with supernatant from control macrophages [M(con)] or HH-differentiated [M(Hb)] macrophages. The relative TEER compared with control is shown (n = 4 per group). (B) FITC-dextran permeability in scramble siRNA– or VEGFR2 siRNA–transfected HAECs treated with control or HH-differentiated macrophage supernatants. Percentage change of FITC-dextran compared with control (n = 4 per group). (C) Immunofluorescence imaging of scramble siRNA– or VEGFR2 siRNA–transfected HAECs treated with control or HH-differentiated macrophage supernatants for VE-cadherin (green) and DAPI (blue) (original magnification, ×60). Scale bar: 50 μm. Note the loss of plasma membrane VE-cadherin in endothelial cells treated with M(Hb) supernatants versus M(con) supernatants and the restoration of membrane VE-cadherin in endothelial cells transfected with VEGFR2 siRNA and treated with M(Hb) supernatants. (D) Quantitation of plasma membrane VE-cadherin in the experiment shown in C (n = 10 per group). (E) Immunoblot of the membrane fraction from scramble siRNA– or VEGFR2 siRNA–transfected HAECs treated with control or HH-differentiated macrophage supernatants, with quantitation of densitometry for VE-cadherin (n = 4 per group). (F) Quantitation of plasma membrane VE-cadherin in the experiment shown in E. All error bars indicate the mean ± SEM. *P < 0.05 versus other groups. For multiple group comparisons, a 1-way ANOVA was applied. If the variance ratio test (F test) was significant, a more detailed post-hoc analysis of differences between groups was done using a Tukey-Kramer honest significant difference test.