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. 2009 Mar;33(3):612-24.
doi: 10.1183/09031936.00014808. Epub 2008 Nov 14.

Amifostine reduces lung vascular permeability via suppression of inflammatory signalling

P Fu  1 , A A Birukova, J Xing, S Sammani, J S Murley, J G N Garcia, D J Grdina, K G Birukov
Affiliations

Amifostine reduces lung vascular permeability via suppression of inflammatory signalling

P Fu et al. Eur Respir J. 2009 Mar.

Abstract

Despite an encouraging outcome of antioxidant therapy in animal models of acute lung injury, effective antioxidant agents for clinical application remain to be developed. The present study investigated the effect of pre-treatment with amifostine, a thiol antioxidant compound, on lung endothelial barrier dysfunction induced by Gram-negative bacteria wall-lipopolysaccharide (LPS). Endothelial permeability was monitored by changes in transendothelial electrical resistance. Cytoskeletal remodelling and reactive oxygen species (ROS) production was examined by immunofluorescence. Cell signalling was assessed by Western blot. Measurements of Evans blue extravasation, cell count and protein content in bronchoalveolar lavage fluid were used as in vivo parameters of lung vascular permeability. Hydrogen peroxide, LPS and interleukin-6 caused cytoskeletal reorganisation and increased permeability in the pulmonary endothelial cells, reflecting endothelial barrier dysfunction. These disruptive effects were inhibited by pre-treatment with amifostine and linked to the amifostine-mediated abrogation of ROS production and redox-sensitive signalling cascades, including p38, extracellular signal regulated kinase 1/2, mitogen-activated protein kinases and the nuclear factor-kappaB pathway. In vivo, concurrent amifostine administration inhibited LPS-induced oxidative stress and p38 mitogen-activated protein kinase activation, which was associated with reduced vascular leak and neutrophil recruitment to the lungs. The present study demonstrates, for the first time, protective effects of amifostine against lipopolysaccharide-induced lung vascular leak in vitro and in animal models of lipopolysaccharide-induced acute lung injury.

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Figures

Figure 1
Figure 1. Effect of amifostine on H2O2, LPS and IL-6-induced endothelial barrier dysfunction
Human pulmonary EC were grown on golden microelectrodes. At the time point indicated by first arrow, cells were pretreated with WR-1065 (0.4 mM, 1 mM or 4 mM, 30 min) followed by stimulation with 250 mM H2O2 (Panel A). EC were pretreated with 4 mM WR-1065 for 30 min followed by 200 ng/ml LPS stimulation with LPS (200 ng/ml) (Panel B), or combination of IL-6 (25 ng/ml) and its soluble co-receptor IL-6-SR (10 U/ml) (Panel C) marked by second arrow, and TER reflecting EC permeability changes was monitored over time. Shown are pooled data from three independent experiments.
Figure 2
Figure 2. Amifostine prevents agonist-induced lung EC cytoskeletal remodeling and adherens junction disruption
EC were grown on glass cover-slips pretreated with amifostine (4 mM, 30 min) or not, followed by H2O2 (250 mM, 15 min), LPS (200 ng/ml, 6 hrs), or IL-6 + IL-6-SR (25 ng/ml and 10 U/ml, 6 hrs) stimulation. Analysis of actin cytoskeletal remodeling was performed by immunofluorescent staining with Texas Red phalloidin. Paracellular gap formation is shown by arrows (Panel A). VE-Cadherin staining was performed, to visualize LPS-induced disruption of adherens junctions (shown by arrows) (Panel B). The panels are representative of the entire cell monolayer. Shown are results of three independent experiments.
Figure 3
Figure 3. Amifostine inhibits ROS production induced by LPS
HPAEC were pretreated with vehicle (top row), NAC (5 mM, 1 h) (middle row) or WR-1065 (4 mM, 30 min) (low row) followed by LPS stimulation (100 ng/ml, 200 ng/ml, 500 ng/ml). ROS was detected by DCFDA fluorescent assay described in “Material and Methods”. The results are representative of the entire cell monolayer and have been reproduced in three independent experiments.
Figure 4
Figure 4. Amifostine attenuates intracellular signaling activated by inflammatory agonists
HPAEC were pretreated with amifostine (4 mM, 30 min) or vehicle followed by stimulation with H2O2 (250 mM, 15 min) (Panel A), IL-6 + IL-6-SR (25 ng/ml and 10 U/ml, 2 hrs) (Panel B), or LPS (200 ng/ml, 2 hrs) (Panel C). Phosphorylation of p38, MEK1/2, Erk1/2, IκBα, NκB-p65, MLC and Hsp27 was detected with corresponding phospho-specific antibodies. Results are representative of three independent experiments. In vivo, five animals per each experimental group were treated with LPS (0.7 mg/kg) with or without concurrent administration of WR-2721 (200 mg/kg), or with WR-2721 (200 mg/kg) alone.
Figure 5
Figure 5. Amifostine attenuates LPS-induced neutrophil accumulation in BAL, increased MPO activity and lung barrier dysfunction
C57BL/6J mice were subjected to treatment of LPS (0.7 mg/kg), with or without concurrent treatment with WR-2721 (200 mg/kg). Control animals were treated with vehicle or WR-2721 (200 mg/kg) alone. After 18 hrs of stimulation, cell count (Panel A), MPO activity (Panel B) and protein concentration (Panel C) were measured in bronchoalveolar lavage fluid taken from control and experimental animals. Results are represented as mean + SE; *p < 0.001 vs. control; **p < 0.001 vs. LPS; n=6–9 per group.
Figure 6
Figure 6. Effects of amifostine on LPS-induced acute lung injury
Lung specimens were obtained from control mice (A), mice treated with amifostine (B), LPS (C) and LPS + amifostine (D). At the end of experiment, lungs were excised, fixed in 4% paraformaldehyde, embedded in paraffin, and used for histochemical analysis after H&E staining. Images are representative of 6–9 lung specimens for each condition. Original magnification: X200.
Figure 7
Figure 7. Effects of amifostine on LPS-induced p38 activation and nitrotyrosine accumulation in mouse lungs
Mouse lungs were harvested after 2 hrs and 6 hrs of LPS instillation, and tissue samples were prepared for western blot analysis of p38 MAP kinase phosphorylation and accumulation of tyrosine nitrated proteins. Results are representative of three independent experiments.
Figure 7
Figure 7. Effects of amifostine on LPS-induced p38 activation and nitrotyrosine accumulation in mouse lungs
Mouse lungs were harvested after 2 hrs and 6 hrs of LPS instillation, and tissue samples were prepared for western blot analysis of p38 MAP kinase phosphorylation and accumulation of tyrosine nitrated proteins. Results are representative of three independent experiments.
Figure 8
Figure 8. Inhibition of LPS-induced inflammation and endothelial barrier dysfunction by amifostine
Lung exposure to LPS stimulates production of reactive oxygen and nitrogen species (ROS and RNS) by activating NADPH oxidase, NO-synthase, xanthine oxidoreductase and other enzymes, which leads to cellular oxidative stress. As a result, activation of redox-sensitive signaling cascades including NFkB, Erk-1,2 and p38 MAP kinases triggers cellular responses to LPS such as elevated expression of pro-inflammatory cytokines, cytoskeletal remodeling and disruption of endothelial monolayer integrity leading to acute lung injury. Amifostine reduces LPS-induced inflammatory signaling via inactivation of ROS and RNS and blunting the redox-sensitive inflammatory signaling.
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