Supplementary MaterialsSupplemental data jciinsight-4-124329-s096. epithelial-endothelial paracrine signaling induced endothelial barrier failure, as detected by microvascular dextran leakage and lung water quantification. Remarkably, endothelial mitochondria regulated the barrier failure by activating uncoupling protein 2 (UCP2), thereby inducing transient mitochondrial depolarization that led to cofilin-induced actin depolymerization. Knockdown, or endothelium-targeted deletion of UCP2 expression, blocked these responses, including pulmonary edema. To our knowledge, these findings are the first to mechanistically implicate endothelial mitochondria in acid-induced barrier deterioration and pulmonary edema. Pparg We suggest endothelial UCP2 may be a therapeutic target for acid-induced acute lung injury. = 5 lungs. Scale bar: 20 m. (B) Fluorescence of CG, CR, and tetramethylrhodamine ethyl ester (TMRE, intravascularly, 2 M) at indicated time points before (baseline) and after alveolar HCl injection. Images at 30 minutes were obtained after repeat injections of TAS-114 CG and TMRE. Scale bars: 20 m. (C) Bars are quantifications in cytosol (calcein) and mitochondria (TMRE) for the indicated cell TAS-114 types following alveolar injections of PBS or HCl. White, PBS; orange, 10 minutes after HCl; blue, 30 minutes after HCl. * 0.05 versus PBS. (D) Effects of indicated treatments on endothelial TMRE following alveolar HCl or microvascular H2O2 injection. EV, intranasal empty vector; CAT, alveolar catalase transfection; NAC, intravascular 0.05 versus EV. Data are shown as mean SEM for the number of injections indicated by dots. = 4 lungs. Scale bar: 20 m. (BCE) Data are for analyses carried out 48 hours after tail vein injection of indicated siRNA. (B) Gel and bars show immunoblotting (IB) and densitometry of mitochondria isolated from lung homogenates. The antibodies were UCP2 mouse monoclonal antibody and voltage-dependent anion channel (VDAC) rabbit polyclonal antibody. Lanes were run on the same gel. Vertical line indicates the lanes are not contiguous. Results were identical for IB using UCP2 goat polyclonal antibody (data not shown). SI, siUCP2; SC, scRNA. * 0.05 versus scRNA. (C) Bars show effects of indicated treatments following alveolar HCl or microvascular H2O2 injections. KO, endothelial cellCspecific 0.05 versus left bar. (D) Confocal images show dextran distribution at indicated locations. TAS-114 The endothelial cytosol was loaded with CR. FITC-D70 was infused in vessels at baseline and then again after injection of alveolar HCl. Scale bar: 20 m. (E) Bars quantify FITC-D70Cfilled alveoli (edematous alveoli) following the indicated alveolar injections. Equal numbers of alveoli were injected in each group. Alveoli with greater than 50% luminal area filled with FITC-D70 were defined as edematous. UN, untreated. * 0.05 versus PBS. Data are shown as mean SEM for the number of injections indicated by dots. 0.05), was considerably lower than that of untreated controls. Importantly, EVLW did not increase significantly. These findings mechanistically implicated UCP2 activation in the HCl-induced increases of BAL cell count, protein content, and EVLW. We conclude TAS-114 that alveolar HClCinduced UCP2 activation caused global loss of endothelial barrier function in the lung, resulting in pulmonary edema. Open in a separate window Figure 3 Effects of endothelial UCP2 activation on intranasal HClCinduced lung injury.Data are for responses obtained 2 days after the indicated treatments. The analyses of the BAL (A and B) and the EVLW (C) were obtained 2 hours after intranasal instillation of PBS or HCl, as indicated. TAS-114 Data are shown as mean SEM. 0.05 versus PBS by ANOVA with post hoc Bonferronis correction. Actin depolymerization underlies alveolar acidCinduced hyperpermeability. Because the HCl-induced endothelial barrier loss was rapid, we considered that alveolar HCl might induce an endothelial signaling pathway through actin depolymerization, thereby destabilizing the barrier (17). Endothelial actin depolymerization could occur by known alveolar HClCinduced endothelial Ca2+ increases (1), activating the Ca2+-dependent phosphatase, calcineurin. Calcineurin dephosphorylates cofilin, causing actin depolymerization (18, 19). The alveolar HClCinduced FITC-D70 hyperpermeability was absent in microvessels pretreated with the calcineurin inhibitor, tacrolimus, implicating calcineurin in the barrier effect. Subsequently, we transfected the lung capillary endothelium to express constitutively inactive cofilin (inCFLN, Figure 4A), which cannot depolymerize actin (20). Alveolar HClCinduced FITC-D70 hyperpermeability was absent following inCFLN transfection (Figure 4B). To rule out nonspecific effects, we confirmed that transfection of WT cofilin had no effect on the HCl-induced hyperpermeability (data not shown). We then assessed the actin response in lung capillaries. Alveolar HCl injection decreased endothelial F-actin within 20 minutes, as indicated by loss of rhodamine phalloidin fluorescence (Figure 4, C and D). This effect was absent in inCFLN-expressing (Figure.