Lung endothelial barrier protection by iloprost in the 2-hit models of ventilator-induced lung injury (VILI) involves inhibition of Rho signaling

Abstract

Mechanical ventilation at high tidal volume (HTV) may cause pulmonary capillary leakage and acute lung inflammation culminating in ventilator-induced lung injury. Iloprost is a stable, synthetic analog of prostaglandin I(2) used to treat pulmonary hypertension, which also showed endothelium-dependent antiedemagenic effects in the models of lung injury. To test the hypothesis that iloprost may attenuate lung inflammation and lung endothelial barrier disruption caused by pathologic lung distension and coagulation system component thrombin, we used cell and animal 2-hit models of ventilator-induced lung injury. Mice received a triple injection of iloprost (2 microg/kg, intravenous instillation) at 0, 40, and 80 min after the onset of HTV mechanical ventilation (30 mL/kg, 4h), combined with the administration of a thrombin receptor-activating peptide 6 (TRAP6, 3 x 10(-7)mol/mouse, intratracheal instillation). After 4h of ventilation, bronchoalveolar lavage (BAL), histologic analysis, and measurements of Evans blue accumulation in the lung tissue were performed. The effects of iloprost on endothelial barrier dysfunction were subsequently assessed in pulmonary endothelial cells (ECs) exposed to thrombin and pathologic (18%) cyclic stretch. The combination of HTV and TRAP6 enhanced the accumulation of neutrophils in BAL fluid and lung parenchyma, as well as increased the BAL protein content and endothelial permeability judged by Evans blue extravasation in the lung tissue. These effects were markedly attenuated by iloprost. The application of 18% cyclic stretch to pulmonary ECs enhanced the thrombin-induced EC paracellular gap formation and Rho-GTPase-mediated phosphorylation of regulatory myosin light chains and myosin phosphatase. Iloprost markedly inhibited the Rho-kinase-mediated site-specific phosphorylation of myosin phosphatase, and it prevented cyclic stretch- and thrombin-induced endothelial monolayer disruption. This study characterizes for the first time the protective effects of iloprost in the in vitro and in vivo 2-hit models of VILI and supports consideration of iloprost as a new therapeutic treatment of VILI.

Figure 1. Effects of iloprost on BAL cell count and protein content in the two-hit VILI model

Mice were subjected to mechanical ventilation at high tidal volume (HTV, 30 ml/kg, 4 hr) with or without TRAP6 injection (3×10⁻⁷ mol/mouse i/t), or left spontaneously ventilated. Animals were treated with iloprost (2 μg/kg, i/v) or sterile saline at three time points (0, 40, and 80 min) during mechanical ventilation. Cell count (A) and measurements of protein concentration (B) were performed in BAL fluid taken from control and experimental animals; n=6–10 per condition; *p<0.03, **p<0.005.

Figure 2. Histological assessment of iloprost effect in the VILI model

Histological assessment of lung injury was performed in lung tissue samples from control and TRAP6/HTV treated mice with or without iloprost instillation. Whole lungs were fixed in formaldehyde, embedded in paraffin, and used for histological evaluation by hematoxylin and eosin staining; n=4–6 per condition; magnification × 40.

Figure 3. Effects of iloprost on lung vascular leak in the VILI model

Effects of iloprost (2 μg/kg, i/v) on the TRAP6/HTV-induced vascular leak were evaluated visually by Evans blue accumulation in the lung tissue samples (A). In addition, quantitative analysis of Evans blue labeled albumin extravasation was performed by spectrophotometric analysis of Evans blue extracted from the lung tissue samples (B); n=4–8 per condition; *p<0.01 for TRAP6/HTV vs. non-treated controls; **p<0.05 for iloprost + TRAP6/HTV vs. TRAP6/HTV.

Figure 4. Effects of iloprost on EC monolayer integrity in the in vitro model of VILI

EC monolayers grown on Flexcell plates were preconditioned at pathologic (18%) levels of CS for 2.5 hr and stimulated with thrombin (0.1 U/ml, 15 min) with or without iloprost (200 ng/ml, 15 min) pretreatment. F-actin was visualized by immunofluorescence staining with Texas-Red phalloidin. Paracellular gaps are marked by arrows. Results are representative of four independent experiments.

Figure 5. Effects of iloprost on MLC and MYPT phosphorylation in the in vitro model of VILI

Pulmonary EC grown to confluence on Flexcell plates were pre-treated with iloprost (200 ng/ml, 15 min) and exposed to 18% CS for indicated time periods. Phosphorylation of MLC (A) and MYPT (B) was detected by western blot with phospho-specific antibodies. HPAEC were further stimulated with thrombin (0.1 U/ml, 15 or 30 min) with or without iloprost (200 ng/ml, 15 min) pretreatment. Phosphorylated MLC (C) and MYPT (D) were detected by immunoblotting with specific antibodies. Results are representative of three to six independent experiments.

Figure 5. Effects of iloprost on MLC and MYPT phosphorylation in the in vitro model of VILI

Pulmonary EC grown to confluence on Flexcell plates were pre-treated with iloprost (200 ng/ml, 15 min) and exposed to 18% CS for indicated time periods. Phosphorylation of MLC (A) and MYPT (B) was detected by western blot with phospho-specific antibodies. HPAEC were further stimulated with thrombin (0.1 U/ml, 15 or 30 min) with or without iloprost (200 ng/ml, 15 min) pretreatment. Phosphorylated MLC (C) and MYPT (D) were detected by immunoblotting with specific antibodies. Results are representative of three to six independent experiments.