Imatinib attenuates inflammation and vascular leak in a clinically relevant two-hit model of acute lung injury

Abstract

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS), an illness characterized by life-threatening vascular leak, is a significant cause of morbidity and mortality in critically ill patients. Recent preclinical studies and clinical observations have suggested a potential role for the chemotherapeutic agent imatinib in restoring vascular integrity. Our prior work demonstrates differential effects of imatinib in mouse models of ALI, namely attenuation of LPS-induced lung injury but exacerbation of ventilator-induced lung injury (VILI). Because of the critical role of mechanical ventilation in the care of patients with ARDS, in the present study we pursued an assessment of the effectiveness of imatinib in a "two-hit" model of ALI caused by combined LPS and VILI. Imatinib significantly decreased bronchoalveolar lavage protein, total cells, neutrophils, and TNF-α levels in mice exposed to LPS plus VILI, indicating that it attenuates ALI in this clinically relevant model. In subsequent experiments focusing on its protective role in LPS-induced lung injury, imatinib attenuated ALI when given 4 h after LPS, suggesting potential therapeutic effectiveness when given after the onset of injury. Mechanistic studies in mouse lung tissue and human lung endothelial cells revealed that imatinib inhibits LPS-induced NF-κB expression and activation. Overall, these results further characterize the therapeutic potential of imatinib against inflammatory vascular leak.

Keywords: NF-κB; acute lung injury; acute respiratory distress syndrome; endothelium; imatinib; lipopolysaccharide; mechanical ventilation.

Fig. 1.

Imatinib attenuates vascular leak in mice challenged with a 2-hit model of LPS and mechanical ventilation (MV). Mice were challenged with LPS [0.5 mg/kg, intratracheally (it)] (t = 0 h) and MV (respiratory rate 75, tidal volume 30 ml/kg, positive end-expiratory pressure 0 cm H₂O) (t = 20–24 h) as outlined in A. Spontaneously breathing (SB) control mice received PBS (vs. LPS) and were intubated for 4 h without MV. Bronchoalveolar lavage (BAL) fluid, plasma, and lungs were harvested from the animals immediately after MV. Imatinib (IM) (75 mg/kg, ip) or vehicle was administered 0.5 h before LPS administration and before the initiation of MV. Lung permeability was assessed by BAL protein content (B), BAL fluid albumin (C), and lung tissue albumin (D). Additionally, in separate animals, Evans Blue dye (EBD) was injected (30 mg/kg, iv) 1 h before harvest, and representative extravasation into harvested lung tissue is shown (E) and quantified in multiple samples (F). The left lung of each of these animals was used for calculation of lung wet:dry ratio (G). SB (n = 3), SB + imatinib (n = 3), LPS + ventilator-induced lung injury (VILI) (n = 3–11) and LPS + VILI + imatinib (n = 3–6). *P < 0.05 compared with SB controls and #P < 0.05 compared with untreated animals.

Fig. 2.

Imatinib decreases lung inflammation in mice challenged with LPS and MV. Mice were subjected to the 2-hit lung injury model (LPS + VILI), and lung inflammation was quantified by BAL total cell counts (A) and BAL neutrophil counts (B). Representative hematoxylin and eosin (H and E)-stained lung sections are shown (C). Each H and E image was obtained from a different animal. SB (n = 3), SB + imatinib (n = 3), LPS + VILI (n = 3–11), and LPS + VILI + imatinib (n = 3–6). *P < 0.05 compared with SB controls and #P < 0.05 compared with untreated animals.

Fig. 3.

Imatinib decreases LPS-induced vascular leak when given after injury onset. Mice were challenged with LPS (1.0 mg/kg, it) (vs. PBS) and then received imatinib (75 mg/kg, ip) (vs. vehicle) 4 h later. Samples were harvested 18 h after LPS administration. Pulmonary vascular permeability was quantified in these animals by measuring BAL protein (A), BAL albumin (B), and lung tissue albumin (C). In separate animals, EBD was injected (30 mg/kg, iv) 1 h before harvest, and representative extravasation into harvested lung tissue is shown (D) and quantified in multiple samples (E). The left lung of each of these animals was used for calculation of lung wet:dry ratio (F). SB (n = 3), SB + imatinib (n = 3), LPS (n = 3–6), and LPS + imatinib (n = 3–6). *P < 0.05 compared with SB controls and #P < 0.05 compared with untreated animals.

Fig. 4.

Imatinib decreases LPS-induced inflammation when given after injury onset. Mice were challenged with LPS (1.0 mg/kg, it) (vs. PBS) and then received imatinib (75 mg/kg, ip) (vs. vehicle) 4 h later. Lung inflammation was then quantified by BAL total cell counts (A) and BAL neutrophil counts (B). Representative H and E-stained lung sections are shown (C). Each H and E image was obtained from a different animal. SB (n = 3), SB + imatinib (n = 3), LPS (n = 3–6), and LPS + imatinib (n = 3–6). *P < 0.05 compared with SB controls and #P < 0.05 compared with untreated animals.

Fig. 5.

Imatinib decreases BAL TNF-α levels in a clinically relevant mouse model of acute lung injury (ALI). Inflammatory cytokines implicated in ALI (TNF-α, IL-6) were measured in the BAL fluid of mice challenged with the 2-hit model (LPS + VILI) (A and B) or the LPS-only postinjury model (C and D). 2-hit injury data represent SB (n = 3), SB + imatinib (n = 3), LPS + MV (n = 6), and LPS + MV + imatinib (n = 7). Post-LPS data represent SB (n = 3), SB + imatinib (n = 3), LPS (n = 3–6), and LPS + imatinib (n = 3–6). #P < 0.05.

Fig. 6.

Imatinib inhibits LPS-induced NF-κB phosphorylation and nuclear translocation in vitro. Human pulmonary artery endothelial cells (HPAEC) were treated with imatinib (40 μM, 60 min) and then challenged with LPS (1 μg/ml, 0–60 min). Lysates were harvested for Western blots for phosphorylated NF-κB p65 (S536), total NF-κB p65 protein, IκB, and actin and quantified by densitometry (A and B). HPAEC plated on glass coverslips were subjected to identical conditions, and immunofluorescence microscopy was conducted to determine the localization of total NF-κB p65 protein (red). 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used to stain the nuclei (white arrows) (C). Data are representative of 3 independent experiments. *P < 0.05 compared with nonchallenged samples.

Fig. 7.

Imatinib decreases NF-κB expression in mouse lungs after LPS. Mice were challenged with LPS (1.0 mg/kg, it) (vs. PBS) and then received imatinib (75 mg/kg, ip) (vs. vehicle) 4 h later. Lung tissue homogenates were collected, subjected to Western blotting for total NF-κB p65 protein, and quantified by densitometry (A and B). Lanes in A represent lung homogenates from individual animals. Representative immunohistochemistry images are shown for NF-κB p65 taken by an individual blinded to experimental condition (C). *P < 0.05.

Fig. 8.

Potential protective effects of imatinib in lung endothelial inflammation induced by LPS plus VILI. In this proposed schema, stimulation of lung endothelial cells (EC) by LPS + VILI results in activation of the Abl kinases and subsequent downstream signaling that includes increased NF-κB activity, upregulation of VCAM-1, release of cytokines (TNF-α, IL-6), and increased pulmonary neutrophil (PMN) recruitment. Imatinib inhibits Abl family kinases to attenuate these effects in vitro and in vivo. Nuc, nucleus.