Restoration of normal paracellular permeability in Cav-1^−/− endothelial monolayers and vessels by inhibition of either RhoA or eNOS. (a) Immunofluorescent staining of MLVECs isolated from Cav-1^−/− and Cav-1/eNOS double knockout (DKO) mice for β-catenin (green), F-actin (red), and nuclei (blue). Cav-1^−/− cells were treated with Rho inhibitor C3 transferase and AP-CSD peptide. Bar, 10 µm. (b) β-catenin accumulation at AJs as in Fig. 1 e; *, P < 0.01 as compared with Wt control; **, P < 0.01 as compared with Cav-1^−/−; n = 10. (c) Endothelial permeability to EBA; mean and SEM are as in Fig. 1 e; *, P < 0.01 as compared with Wt control; **, P < 0.05 as compared with Cav-1^−/−; n = 4. (d) Lung weight changes after a step increase in transvascular oncotic pressure gradient. Recordings were smoothed by averaging successive groups of five points. Lungs isolated from Wt and Cav-1^−/− mice were perfused with 0% BSA for 10 min, with 10% BSA for 30 min, and with 0% BSA for 10 min; an additional Cav-1^−/− group received AP-CSD peptide starting at 10 min of BSA profusion. AP-CSD peptide reversed lung weight loss during high albumin perfusion in Cav-1^−/− lungs and largely restored the transvascular fluid filtration rate between 40 and 50 min. (e) The filtration rate was calculated from the initial slope of slow exponential component of lung weight gain; mean and SEM are as in Fig. 1 e; *, P < 0.05 as compared with Wt control; n = 5–9.