Differential and opposing effects of imatinib on LPS- and ventilator-induced lung injury

Abstract

Endothelial dysfunction underlies the pathophysiology of vascular disorders such as acute lung injury (ALI) syndromes. Recent work has identified the Abl family kinases (c-Abl and Arg) as important regulators of endothelial cell (EC) barrier function and suggests that their inhibition by currently available pharmaceutical agents such as imatinib may be EC protective. Here we describe novel and differential effects of imatinib in regulating lung pathophysiology in two clinically relevant experimental models of ALI. Imatinib attenuates endotoxin (LPS)-induced vascular leak and lung inflammation in mice but exacerbates these features in a mouse model of ventilator-induced lung injury (VILI). We next explored these discrepant observations in vitro through investigation of the roles for Abl kinases in cultured lung EC. Imatinib attenuates LPS-induced lung EC permeability, restores VE-cadherin junctions, and reduces inflammation by suppressing VCAM-1 expression and inflammatory cytokine (IL-8 and IL-6) secretion. Conversely, in EC exposed to pathological 18% cyclic stretch (CS) (in vitro model of VILI), imatinib decreases VE-cadherin expression, disrupts cell-cell junctions, and increases IL-8 levels. Downregulation of c-Abl expression with siRNA attenuates LPS-induced VCAM-1 expression, whereas specific reduction of Arg reduces VE-cadherin expression in 18% CS-challenged ECs to mimic the imatinib effects. In summary, imatinib exhibits pulmonary barrier-protective and anti-inflammatory effects in LPS-injured mice and lung EC; however, imatinib exacerbates VILI as well as dysfunction in 18% CS-EC. These findings identify the Abl family kinases as important modulators of EC function and potential therapeutic targets in lung injury syndromes.

Keywords: ARDS; Arg; LPS; VILI; c-Abl; endothelium; imatinib; permeability; stretch.

Copyright © 2015 the American Physiological Society.

Figures

Fig. 1.

Effects of imatinib on lung vascular leak in LPS- and ventilator-induced lung injury (VILI)-treated mice. C57BL/6J mice were injected with imatinib (Im) (75 mg/kg ip) or water [vehicle (Veh)] and then exposed to either LPS (1 mg/kg, 18 h) (vs. PBS) or VILI (30 ml/kg, 4 h) [vs. spontaneously breathing (SB)]. Lung permeability was assessed by measuring the bronchoalveolar lavage (BAL) protein content (A and B) and the albumin concentration in lung homogenates (C and D). Data represent means ± SE (n = 3–8 mice in each group). *P < 0.05, Tukey's post hoc test (A and B); t-test (C and D).

Fig. 2.

Effects of imatinib on lung histopathology and inflammation in LPS- and VILI-treated mice. A and B: hematoxylin and eosin staining of lung tissue sections. A: vehicle (Veh) or imatinib (Im) (75 mg/kg) after PBS or LPS (1 mg/kg, 18 h). B: vehicle or imatinib (75 mg/kg) in spontaneously breathing (SB) or VILI (30 ml/kg, 4 h) exposed mice. Images shown are representative lung tissue sections from multiple animals (original magnification, ×100). C and D: BAL total cell counts. E and F: BAL neutrophil cell counts. Data represent means ± SE (n = 3–8 mice in each group). *P < 0.05, Tukey's post hoc test.

Fig. 3.

Effects of imatinib on inflammatory cytokine production by LPS or VILI in mice. Mice were injected with imatinib (75 mg/kg ip) and then challenged with LPS (1 mg/kg, 18 h) or VILI (30 ml/kg, 4 h). The levels (pg/ml) of IL-6 (A and B) and KC (C and D) were assayed in the BAL. Data represent means ± SE (n = 3–7 mice per group), *P < 0.05 compared with treated group (LPS or VILI). E: absolute values of cytokines for all experimental groups (means ± SD).

Fig. 4.

Imatinib prevents LPS-induced endothelial dysfunction. A: representative transendothelial electrical resistance (TER) tracing. Human pulmonary artery endothelial cells (HPAEC) were plated on gold microelectrodes and at the time indicated by arrows were pretreated with imatinib (10, 20, or 40 μM) (Im 10, Im 20, Im 40) followed by stimulation with LPS (1 μg/ml). TER values were normalized to the baseline and then measured over time. Decreased resistance values reflect increases in permeability. B: normalized TER values were pooled from each independent experiment at the time point at which LPS induced the maximum decrease. Data values are means ± SE from 3–5 independent experiments (*P < 0.05 compared with LPS, Tukey's post hoc test). C: immunofluorescence images for VE-cadherin in HPAEC pretreated for 1 h with imatinib (20 μM), followed by LPS challenge (5 h). LPS induced loss of VE-cadherin from the junctions as shown by arrows. Representative images from 3 independent experiments are shown. D: HPAEC were treated with imatinib for 1 h followed by LPS for 3 h. VCAM-1 and β-actin expression were detected by immunoblotting. A representative Western blot is shown demonstrating an upregulation of VCAM-1 expression after LPS challenge that is decreased by imatinib in a dose-dependent manner.

Fig. 5.

Effects of imatinib on 18% cyclic stretch (CS)-induced endothelial dysfunction. HPAEC were treated with imatinib as indicated for 1 h and then subjected to cyclic stretch (18% CS, 24 h). A: representative Western blot for VE-cadherin. B: densitometry of VE-cadherin normalized to β-actin. Data values are means ± SE from 3 independent experiments. (*P < 0.05 compared with control, Dunnett's post hoc test). C: immunofluorescence staining for VE-cadherin. 18% CS in combination with imatinib induced a loss of VE-cadherin from the junctions as indicated by arrows. Representative images from multiple independent experiments are shown.

Fig. 6.

Effects of imatinib on inflammatory cytokine production by LPS or 18% CS in HPAEC. HPAEC were treated with imatinib as indicated for 1 h followed by challenge with LPS (1 μg/ml, 3 h) or 18% CS (24 h). Cell supernatants were then collected and assayed for IL-8 (A and B) and IL-6 (C and D) by ELISA. Data represent means ± SE from 3 or more independent experiments. *P < 0.05 compared with LPS or 18% CS group, Dunnett's post hoc test.

Fig. 7.

Effects of c-Abl or Arg siRNA on LPS and 18% CS-induced endothelial dysfunction. HPAEC were transfected with control, c-Abl-, or Arg-specific siRNA for 48 h and then stimulated with LPS (1 μg/ml, 3 h) or 18% CS (24 h). Cell lysates were then collected and analyzed by immunoblotting. Representative Western blots of at least 3 experiments depict the specific downregulation of c-Abl (A) or Arg (B) protein expression by siRNA, VCAM-1 expression, and the corresponding densitometry (P < 0.05, *compared with si-Ctr and #compared with si-Ctr/LPS and si-c-Abl/LPS), Tukey's post hoc test (C) and VE-cadherin expression and the corresponding densitometry (D). Data represent means ± SE from 3 or more independent experiments. *P < 0.05, compared with si-Ctr, si-Ctr/CS, and si-c-Abl/CS, Tukey's post hoc test.