Cigarette smoke causes lung vascular barrier dysfunction via oxidative stress-mediated inhibition of RhoA and focal adhesion kinase

Abstract

Cigarette smoke (CS) is a major cause of chronic lung and cardiovascular diseases. Recent studies indicate that tobacco use is also a risk factor for acute lung injury (ALI) associated with blunt trauma. Increased endothelial cell (EC) permeability is a hallmark of ALI. CS increases EC permeability in vitro and in vivo; however, the underlying mechanism is not well understood. In this study, we found that only 6 h of exposure to CS impaired endothelial barrier function in vivo, an effect associated with increased oxidative stress in the lungs and attenuated by the antioxidant N-acetylcysteine (NAC). CS also exacerbated lipopolysaccharide (LPS)-induced increase in vascular permeability in vivo. Similar additive effects were also seen in cultured lung EC exposed to cigarette smoke extract (CSE) and LPS. We further demonstrated that CSE caused disruption of focal adhesion complexes (FAC), F-actin fibers, and adherens junctions (AJ) and decreased activities of RhoA and focal adhesion kinase (FAK) in cultured lung EC. CSE-induced inhibition of RhoA and FAK, endothelial barrier dysfunction, and disassembly of FAC, F-actin, and AJ were prevented by NAC. In addition, the deleterious effects of CSE on FAC, F-actin fibers, and AJ were blunted by overexpression of constitutively active RhoA and of FAK. Our data indicate that CS causes endothelial barrier dysfunction via oxidative stress-mediated inhibition of RhoA and FAK.

Fig. 1.

Cigarette smoke (CS) increased lung vascular permeability and exacerbated lipopolysaccharide (LPS)-induced lung edema. C57BL/6 mice were exposed to CS or room air (RA) for 6 h and then intratracheally given 2.5 mg/kg LPS or equal volume of 0.9% NaCl [vehicle (V), ∼50 μl]. At 24 h after LPS or vehicle challenge, the lungs were lavaged with 600 μl of saline and the protein content in bronchoalveolar lavage (BAL) fluid was assessed (A). Total cell counts in BAL fluid were also assessed (C). Parallel set of animals were used for assessment of lung wet-to-dry weight ratio (Wet/Dry) (B). Data are presented as means ± SE for 6 mice per group (n = 6) in each panel, *P < 0.05 vs. mice exposed to RA and treated with vehicle; ξP < 0.05 vs. mice exposed to RA and treated with LPS.

Fig. 2.

CS exposure caused lung edema via oxidative stress. A and B: C57BL/6 mice were exposed to CS or RA for 6 h and then intratracheally given 2.5 mg/kg LPS or equal volume of 0.9% NaCl (V, ∼50 μl). 24 h after LPS or vehicle challenge, lung tissue was collected for assessment of reactive oxygen species (ROS) levels by dichlorofluorescein assay (A) and by immunohistochemistry analysis of 8-oxo-7,8-dihydro-2′-deoxyguanisine levels (B). In A, data are presented as means ± SE. *P < 0.05 vs. mice exposed to RA and treated with vehicle. B: representative images. Scale bar represents 50 μm; 6 mice per group (n = 6) in A and B. C and D: C57BL/6 mice were pretreated with N-acetylcysteine (NAC; 150 mg/kg, ∼200 μl) or equal volume of vehicle (saline) via intraperitoneal injection 16 and 1 h prior to CS exposure (total suspended particles of 120 mg/m³) or RA exposure for 6 h. All animals were then intratracheally given 0.9% NaCl (50 μl), and 24 h later the lungs were lavaged with 750 μl of saline, and the protein content (C) and cell counts (D) in BAL fluid were assessed. Data are presented as means ± SE; 3 mice per group (n = 3). *P < 0.05 vs. mice exposed to RA and treated with vehicle, ξP < 0.05 vs. mice exposed to CS and treated with vehicle.

Fig. 3.

Cigarette smoke extract (CSE) exacerbated LPS-induced increase in endothelial monolayer permeability. Bovine pulmonary artery endothelial cells (PAEC) were treated with vehicle (5% sham PBS) or 5% CSE in the absence or presence of LPS (0.5 μg/ml) for indicated times. Endothelial monolayer permeability was assessed by measuring electrical resistance across monolayers over time by electrical cell impedance sensor (ECIS). Data are presented as means ± SE of the normalized electrical resistance relative to the time when agents were added, as indicated by an arrow; n = 6. *P < 0.05 vs. vehicle-treated cells; ξ <0.05 vs. LPS alone-treated cells.

Fig. 4.

CSE increased endothelial monolayer permeability via oxidative stress. PAEC were treated with vehicle (20% sham PBS) or varying concentrations of CSE (10, 20%) for indicated times (A), or preincubated with vehicle (HEPES) or 25 mM NAC for 40 min and then treated with vehicle (20% sham PBS) or 20% CSE in the absence or presence of 25 mM NAC for indicated times (B). Endothelial monolayer permeability was assessed by ECIS. Data are presented as means ± SE of the normalized electrical resistance relative to the time when agents were added (at 1 h in A; at 30 min in B). Arrows indicate the times for addition of treatments. A, n = 10, *P < 0.05 vs. vehicle-treated cells. B, n = 6, *P < 0.05 vs. vehicle-treated cells; ξP < 0.05 vs. vehicle, NAC, and NAC+CSE-treated cells.

Fig. 5.

CSE disrupted focal adhesion complexes (FAC), F-actin fibers, and adherens junctions (AJ) via oxidative stress. PAEC (A) and primary cultured fetal mouse alveolar type II epithelial cells (B) were preincubated with vehicle or 25 mM NAC for 1 h and then exposed to vehicle (10% sham PBS) or 10% CSE in the absence or presence of 25 mM NAC for 4 h. FAC and AJ were assessed by immunofluorescence staining of vinculin and β-catenin, respectively, and visualized by fluorescence microscopy. F-actin fibers were assessed by phalloidin staining of F-actin. Arrows indicate vinculin, F-actin, and β-catenin staining. Asterisks indicate intercellular gaps. Scale bar = 25 μm. Representative images from 4 independent experiments for each panel are shown.

Fig. 6.

CSE caused endothelial barrier dysfunction via inhibition of focal adhesion kinase (FAK). A: PAEC were treated with vehicle (20% sham PBS) or 20% CSE (S) for indicated times. Lysates were collected for assessment of protein levels of FAK phosphorylation at tyrosine-397 (FAK∼P^Y397), total FAK, paxillin, vinculin, VE-cadherin, β-catenin, and actin by immunoblot analysis. B: PAEC were preincubated with vehicle or 25 mM NAC (N) for 1 h and then exposed to vehicle (20% sham PBS) or 20% CSE in the absence or presence of 25 mM NAC for 4 h. FAK∼P^Y397 and total FAK were assessed. C: PAEC were transfected with cDNA coding for GFP or GFP-conjugated FAK (GFP-FAK) for 24 h and then exposed to vehicle (10% sham PBS) or 10% CSE for 4 h. FAC formation was detected by assessing localization of vinculin-containing structures. F-actin fibers were assessed by phalloidin staining of F-actin. Adherens junctions were assessed by localization of β-catenin. GFP- and GFP-FAK-overexpressing cells were visualized by fluorescence microscopy (green). Arrows indicate vinculin, F-actin fibers, and β-catenin staining (red). Asterisks indicate intercellular gaps. Scale bars represent 25 μm. Representative images from 3 independent experiments for each panel are shown.

Fig. 7.

CSE caused endothelial barrier dysfunction via inhibition of RhoA. PAEC (A) and fetal mouse alveolar type II epithelial cells (B) were exposed to vehicle (20% sham PBS) or 20% CSE for indicated times. C: PAEC were preincubated with vehicle or 25 mM NAC for 1 h and then exposed to vehicle (20% sham PBS) or 20% CSE in the absence or presence of 25 mM NAC for indicated times. GTP-RhoA was assessed by pull-down assay using GST-RBD beads. Total RhoA was also assessed in lysates. D: PAEC were transfected with cDNA coding for GFP or GFP-conjugated constitutively active RhoA [GFP-RhoA(Q63L)] for 24 h and then exposed to vehicle (10% sham PBS) or 10% CSE for 4 h. FAC formation was detected by assessing localization of vinculin-containing structures. F-actin fibers were assessed by phalloidin staining of F-actin. Adherens junctions were assessed by localization of β-catenin. GFP- or GFP-RhoA(Q63L)-overexpressing cells were visualized by fluorescence microscopy (green). Arrows indicate vinculin, F-actin fibers, and β-catenin staining (red). Asterisks indicate intercellular gaps. Scale bars represent 25 μm. Representative images from 3 independent experiments for each panel are shown.