Figure 3.

Loss of Piezo1 in ECs augments Src-dependent phosphorylation of VE-Cad. (A) Western blot analysis of Src autophosphorylation at Y416 and paxillin phosphorylation at Y118 in lungs of Piezo1^fl/fl and Piezo1^iEC−/− mice after mechanical ventilation with tidal volume of 40 ml/kg for 30 minutes. Piezo1^iEC−/− mice show increased phosphorylation of Src and paxillin, indicative of Src activation. Mean ± SEM; n = 3. ***P < 0.001 and ****P < 0.0001 by ANOVA. (B and C) Western blot analysis of Src phosphorylation in HLMVEC monolayers transfected with control or Piezo1 siRNA (B) or treated with 500 nM GsmTx-4 (C) after exposure to 18% CS for 30 minutes. Genetic depletion of Piezo1 (B) or pharmacological inhibition of Piezo1 (C) augmented Src phosphorylation. Mean ± SEM; n = 3. *P < 0.05 by two-tailed t test. (D) Pulmonary EBA permeability in Piezo1^fl/fl and Piezo1^iEC−/− mice mechanically ventilated with tidal volume of 40 ml/kg for 2 hours. Piezo1^iEC−/− mice received intraperitoneal injection of PP2, an Src inhibitor, at 1 mg/kg body weight before HVMV. Inhibition of Src with PP2 prevents lung microvascular leakage in Piezo1^iEC−/− mice. Mean ± SEM. Data points depict individual mice. *P < 0.05 by ANOVA.