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. Author manuscript; available in PMC: 2018 Jul 25.
Published in final edited form as: Circulation. 2017 Apr 27;136(4):388–403. doi: 10.1161/CIRCULATIONAHA.117.027646

Figure 4. The CXCL12/CXCR4 axis sustains endothelial barrier function through WNT/β-catenin signaling regulating VE-cadherin.

Figure 4

(A) Experimental scheme. (B) WNT activity in hAoECs as determined in a Gaussia luciferase WNT reporter assay, after stimulation with CXCL12 (100 ng/ml) for indicated periods. Cells were pretreated by the CXCR4 antagonist AMD3100 (1 µg/ml) or CXCR4 knock-down was performed using siRNA (CXCR4-kd). WNT activation was blocked by the WNT inhibitor endo-IWR1 (10 µM) (n=3; 1-way ANOVA with Sidak’s multiple comparison test). (C) β-Catenin protein levels in cytoplasmic and nuclear extracts of hAoECs after stimulation with CXCL12 (100 ng/ml) for 24 h. Cells were pretreated with endo-IWR1 (10 µM). Silencing of CXCR4 was performed using siRNA (CXCR4-kd). β-Catenin levels were normalized to cytoplasmic (tubulin) and nuclear (lamin β1) loading controls, respectively, and presented relative to untreated controls, as quantified by densitometry (n=3; 1-way ANOVA with Tukey’s multiple comparison test). A representative western blot and densitometry quantification is shown (left panel). (D) Permeability of hAoECs to FITC-dextran after stimulation with WNT3a (200 ng/ml) or the WNT activator LiCl (30 mM) for 24 h (n=5-10 wells from 4 independent experiments; Kruskal-Wallis test with Dunn’s post-test). (E) Permeability of hAoECs to FITC-dextran after stimulation with WNT3a (200 ng/ml) or CXCL12 (100 ng/ml) for 24 h and pre-treatment with endo-IWR1 (1 µM) for 1 h, as indicated (n=7-9 wells from 3 independent experiments; 1-way ANOVA with Tukey’s multiple comparison test). (F) VE-cadherin protein levels in cytoplasmic extracts of hAoECs after stimulation with CXCL12 (100 ng/ml) for 24 h and treatment with endo-IWR1 (10 µM), as indicated. Knock-down of CXCR4 was performed using siRNA (CXCR4-kd). VE-cadherin expression was normalized to tubulin and presented relative to untreated, as quantified by densitometry (n=3; 1-way ANOVA with Tukey’s multiple comparison test). A representative Western blot and densitometry quantification is shown. Controls received control siRNA (B,C,F). (G) Quantification of VE-cadherin expression in carotid artery lysates of BmxCre+ vs BmxCre-Cxcr4fl/flApoe−/− mice (n=3; Mann-Whitney test). (H) Permeability of hAoECs to FITC-dextran after stimulation with CXCL12 (100 ng/ml) or WNT3a (200 ng/ml) for 24 h and blockade of SHP2 (5 µM) or PTP1B (25 µM) for 1 h, as indicated (n=8-12 wells from 4 independent experiments; Kruskal-Wallis test with Dunn’s post-test). (I-K) Vascular permeability to Evans blue induced by histamine (10 µg i.v., 10 min) in VE-PTP-FRB+/VE-cadherin-FKBP C57/Bl6 knock-in mice pretreated with the CXCR4 antagonist AMD3465 (125 µg) for 12 h and/or Rapalog (250 µg) for 4 h or vehicle, as indicated (n=8-16; 2-way ANOVA with Bonferroni post-test). (A-K) Data present mean±SD (A-J) or mean±SEM (K). *P<0.05; **P<0.01; ***P<0.001.