Figure 4. TNFα-stimulated venous barrier permeability required Panx1.

(A) Effect of spironolactone (Spiro, 80 μM) on TRPV4 sparklets induced by TNFα (10 ng/mL) in en face mesenteric veins of C57BL/6 mice (n = 4 veins for each treatment). (B,C) Effect of spironolactone (Spiro, 80 μM) on TNFα-induced permeability of veins from C57BL/6 mice (B) or from human adipose tissue biopsy samples (C) (n = 5 veins for each treatment). (D) Effect of the TRPV4 inhibitor GSK2193784 (GSK219) on TRPV4 sparklets induced by TNFα (10 ng/mL) in second order mesenteric veins from Cdh5-CreER^T2+/Panx1^fl/fl mice injected with tamoxifen (+TMX) to induce Panx1 deficiency or veins from control mice (−TMX) (n = 4 veins for each treatment). (E) TNFα-induced permeability of mesenteric veins from Cdh5-CreER^T2+/Panx1^fl/fl mice injected with tamoxifen (+TMX) or veins isolated from mice treated with vehicle control alone (−TMX, n = 4 veins for each treatment). (F,G) Representative microscopic images of TNFα-treated (10 ng/mL), en face second order mesenteric veins either pretreated with spironolactone (15 min, Spiro, 10 μM) from C57BL/6 mice or from Cdh5-CreER^T2+/Panx1^fl/fl mice injected with tamoxifen. Cldn11 was immunostained with a specific antibody (red) and nuclei were stained with DAPI (blue). The amount of Cldn11 localized to endothelial tight junctions was measured by the presence of junctional discontinuities (G). Scale bar, 100 μm. n = 3-4 fields imaged of vessel preparations from 3 mice for each treatment (H, I) Representative immunoblot (H) and quantitative analysis (I) of whole cell lysates of either spironolactone-pretreated and TNFα-treated (10 ng/mL) second order mesenteric veins from C57BL/6 mice or of second order mesenteric veins from Cdh5-CreER^T2+/Panx1^fl/fl mice injected with tamoxifen or vehicle control and treated with TNFα (10 ng/mL). Total protein expression was used as loading control. n = 3 protein isolates from biological replicates. *** P = 0.0004 (A); ** P = 0.0071 (B); ** P = 0.0026 (C); ** P = 0.0012 (E) by unpaired two-tailed t-test. *** P = 0.001, ** P = 0.0097 (D); * P = 0.0336, 0.0165 (G); ** P = 0.0054, # P = 0.119 (vs +Spiro) (I) by One-way ANOVA with Fisher’s LSD test. Graphs represent mean ± SD.