Figure 4: Pharmacological inhibition of STAT3 stabilizes endothelial barrier integrity following VEGF stimulation in human endothelial cells.

(A) Human VEGF-165 recombinant protein (VEGF; 25 ng/ml) stimulation of HUVEC promotes ZO-1 (green) disorganization at endothelial cell junctions (top: DMSO vehicle control pretreatment for 1 hour prior to VEGF stimulation). ZO-1 organization is maintained upon pretreatment with 30 μM AQ for 4 hours (middle) or 10 μM PYR for 1 hour (bottom) prior to VEGF stimulation. VEGF-induced phosphorylation of STAT3 at Y705 (p-STAT3; red) was reduced upon AQ or PYR pretreatment. Nuclei: DAPI (blue). Insets: ZO-1 staining trace of 1 representative cell/field. (A’) Quantification of p-STAT3 (red). (B) Serum-starved HUVEC were pretreated with DMSO (vehicle control) for 1 hour, 30 μM AQ for 4 hours, or 10 μM PYR for 1 hour prior to VEGF (25 ng/ml) stimulation for 0, 2 or 5 minutes. Lysates were immunoblotted. Please see Supplemental Figure 2 for densitometry analysis. (C) Serum-starved HPAEC were pretreated with 10 μM PYR for 1 hour prior to VEGF (25 ng/ml) stimulation for 0, 5 or 30 minutes. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions. ZO-1 organization is maintained when HPAEC were pretreated with PYR. VEGF-induced p-STAT3 (red) was reduced upon PYR pretreatment. Nuclei were stained with DAPI (blue). Insets: trace of ZO-1 staining on 1 representative cell per field. (C’) Quantification of p-STAT3 (red). (D) VEGF (25 ng/ml) stimulation of HMVEC-L promotes ZO-1 (green) disorganization at endothelial cell junctions. ZO-1 organization is maintained upon pretreatment with 20 μM PYR for 6 hours prior to VEGF stimulation. VEGF-induced p-STAT3 (red) was reduced upon PYR pretreatment. Nuclei: DAPI (blue). Insets: ZO-1 staining trace of 1 representative cell/field. (D’) Quantification of p-STAT3 (red). ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, one-way ANOVA.