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Published ahead of print on March 30, 2006, doi:10.1165/rcmb.2005-0482OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp. 190-197, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0482OC

Hydrogen Peroxide Induces Vascular Permeability via Regulation of Vascular Endothelial Growth Factor

Kyung Sun Lee*, So Ri Kim*, Seoung Ju Park, Hee Sun Park, Kyung Hoon Min, Min Hee Lee, Sun Mi Jin, Gong Yong Jin, Wan Hee Yoo and Yong Chul Lee

Department of Internal Medicine, Airway Remodeling Laboratory, Research Center for Allergic Immune Diseases, and Department of Radiology, Chonbuk National University Medical School, Jeonju, South Korea

Correspondence and requests for reprints should be addressed to Yong Chul Lee, M.D., Ph.D., Department of Internal Medicine, Chonbuk National University Medical School, San 2-20 Geumam-dong, deokjin-gu, Jeonju, Jeonbuk 561-180, South Korea. E-mail: leeyc{at}chonbuk.ac.kr


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Oxidative stress plays critical roles in initiation and/or worsening of respiratory disease process. Although reactive oxygen species (ROS) are shown to cause vascular leakage, the mechanisms by which ROS induce an increase in vascular permeability are not clearly understood. In this study, we have used a murine model to evaluate the effect of hydrogen peroxide (H2O2) to examine roles of ROS and the molecular mechanism in vascular permeability. The results have revealed that ROS levels, vascular endothelial growth factor (VEGF) expression, hypoxia-inducible factor-1{alpha} protein level, airway hyperresponsiveness, and vascular permeability are increased after inhalation of H2O2. Administration of antioxidants markedly reduced plasma extravasation and VEGF levels in lungs treated with H2O2. These results indicate that ROS may modulate vascular permeability via upregulation of VEGF expression.

Key Words: airway • animal model • hydrogen peroxide • reactive oxygen species • vascular permeability • vascular endothelial growth factor


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Reactive oxygen species (ROS) is a collective term that includes a large variety of free oxygen radicals (e.g., superoxide anion and hydroxyl radicals) but also derivatives of oxygen that do not contain unpaired electrons (e.g., hydrogen peroxide [H2O2], hypochlorous acid, peroxynitrite, and ozone) (1). ROS play a crucial role in the pathogenesis of various lung disorders such as asthma, chronic obstructive pulmonary diseases, idiopathic pulmonary fibrosis, and acute respiratory distress syndrome (1, 2). When lung tissues are exposed to oxidative stress, enhanced levels of ROS can induce a variety of deleterious effects, thereby inducing pathophysiologic conditions. ROS can lead to endothelial barrier dysfunction with subsequent increased permeability to fluids, macromolecules, and inflammatory cells (1). However, the mechanisms by which ROS cause the increase in vascular permeability are not clearly understood. ROS are shown to induce vascular endothelial growth factor (VEGF) expression in vitro and in vivo (3). VEGF is an endothelial cell–specific mitogenic peptide and plays a key role in vasculogenesis and angiogenesis (4). VEGF also increases vascular permeability so that plasma proteins can leak into the extravascular space, which leads to edema and profound alterations in the extracellular matrix. VEGF is one of the major determinants of pulmonary inflammation, and thus the inhibition of VEGF receptor has been suggested to be a good therapeutic strategy (5, 6). Activation of hypoxia-responsive genes including VEGF is mediated by hypoxia-inducible factor-1 (HIF-1), a heterodimeric basic helix-loop-helix-PAS domain transcription factor (7, 8). HIF-1 is composed of two subunits, HIF-1{alpha} and HIF-1beta. Whereas the beta-subunit protein is constitutively expressed, the stability of the {alpha}-subunit and its transcriptional activity are precisely controlled by the intracellular oxygen concentration (9, 10).

This study investigates the effects of ROS, in particular H2O2, on the increase of vascular permeability in a murine model. The results have revealed that H2O2 enhances the vascular permeability by increasing VEGF protein expression through upregulation of HIF-1{alpha} protein expression.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals and Experimental Protocol
Female BALB/c mice, 8–10 wk of age and free of murine-specific pathogens, were obtained from the Korean Research Institute of Chemistry Technology (Daejon, Korea), were housed throughout the experiments in a laminar flow cabinet, and were maintained on standard laboratory chow ad libitum. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the Chonbuk National University Medical School. The care and use of laboratory animals in this study conformed to NIH guidelines (11). Mice were nebulized for 15 min with an aerosol of 1% or 3% (wt/vol) H2O2 (Junsei Chemical Co., Ltd., Tokyo, Japan) in saline (or with saline as a control) using an ultrasonic nebulizer (NE-U12; Omron Co., Tokyo, Japan). H2O2 was administered three times at 24-h intervals. Bronchoalveolar lavage (BAL) was performed 12 h after the last inhalation. At the time of lavage, the mice (8 mice in each group) were killed with an overdose of pentobarbital sodium (100 mg/kg body weight, administered intraperitoneally). The chest cavity was exposed to allow for expansion, after which the trachea was carefully intubated and the catheter secured with ligatures. Prewarmed 0.9% NaCl solution was slowly infused into the lungs and withdrawn. The collected solutions were pooled and then kept at 4°C. A part of each pool was then centrifuged and the supernatants were kept at –70°C until use.

Administration of L-2-Oxothiazolidine-4-Carboxylic Acid, {alpha}-Lipoic Acid, SU5614, CBO-P11, Wortmannin, LY-294002, or 2-Methoxyestradiol
L-2-Oxothiazolidine-4-Carboxylic Acid (OTC, 160 mg/kg body weight/d; Sigma-Aldrich, St. Louis, MO) was freshly prepared by dissolving the chemical in PBS and adjusting the pH to 7.2 with 3 N NaOH as described elsewhere (12), and was administered intraperitoneally three times at 24-h intervals, beginning 1 h before the first inhalation. {alpha}-Lipoic acid (100 mg/kg body weight/d; Sigma-Aldrich), which is a nonenzymatic antioxidant, was administered four times by oral gavage at 24-h intervals, beginning 1 d before the first inhalation. An inhibitor of VEGF receptor tyrosine kinase, SU5614 (Flk-1; IC50 = 1.2 µM, 5-Chloro-3-[(3,5-dimethylpyrrol-2-yl)methylene]-2-indolinone; Calbiochem, San Diego, CA) and cyclopeptidic vascular endothelial growth inhibitor, CBO-P11 (Flt-1; IC50 = 700nM, Flk-1/KDR; IC50 = 1.3 µM, D-Phe-Pro[79–93], Calbiochem) were used to inhibit VEGF activity. SU5614 (2.5 mg/kg body weight/d) dissolved in 0.05% DMSO diluted with 0.9% NaCl and CBO-P11 (2 mg/kg body weight/d) administered intraperitoneally three times at 24-h intervals, beginning at 1 h before the first inhalation (13). Wortmannin (100 µg/kg body weight/d; Calbiochem) or LY-294002 (1.5 mg/kg body weight/d; BIOMOL Research Laboratories Inc., Plymouth Meeting, PA) dissolved in 0.05% DMSO and diluted with 0.9% NaCl was administered in a volume of 50 µl, as described previously (14, 15). Wortmannin or LY-294002 was administered intratracheally two times to each treated animal, once at 1 h before the first inhalation with H2O2 and the second time at 1 h before the last inhalation with H2O2. The vehicle was 0.9% NaCl containing DMSO. An HIF-1{alpha} inhibitor, 2-methoxyestradiol (2ME2, 100 mg/kg body weight/d; Sigma-Aldrich) was suspended in 0.5% carboxymethylcellulose (Sigma-Aldrich), and administered by oral gavage five times at 24-h interval, beginning 2 d before the first challenge (16, 17).

Measurement of Intracellular ROS
ROS were measured by a method previously described, with a modification (18, 19). BAL cells were washed with PBS. To measure intracellular ROS, cells were incubated for 10 min at room temperature with PBS containing 3.3 µM 2',7'-dichlorofluorescein diacetate (Molecular Probes, Eugene, OR), to label intracellular ROS. The cells were then immediately observed under fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY) and FACS analysis (Partec, Münster, Germany). The numbers of ROS-positive cells stained by dichlorofluorescein were counted. Data were presented as numbers of ROS-positive cells divided by numbers of total cells in each group.

Measurement of Plasma Exudation
To assess lung permeability, Evans blue dye (EBD) was dissolved in 0.9% saline at a final concentration of 5 mg/ml. Animals were weighed and injected with 20 mg/kg EBD in the tail vein. After 30 min, the animals were killed and their chests were opened. Normal saline containing 5 mM EDTA was perfused through the aorta until all venous fluid returning to the opened right atrium was clear. The lungs were removed and weighed wet. EBD was extracted in 2 ml formamide kept in a water bath at 60°C for 3 h, and the absorption of light at 620 nm was measured using a spectrophotometer (Spectra Max Plus Microplate Spectrophotometer; Molecular Devices, Sunnyvale, CA). The dye extracted was quantified by interpolation against a standard curve of dye concentration in the range of 0.01–10 µg/ml and is expressed as ng of dye/mg of wet lung.

Histology and Immunocytochemistry
At 12 h after the last inhalation, lungs were removed from the mice after killing. Before the lungs were removed, the lungs and trachea were filled intratracheally with a fixative (0.8% formalin, 4% acetic acid) using a ligature around the trachea. Lung tissues were fixed with 10% (vol/vol) neutral buffered formalin. The specimens were dehydrated and embedded in paraffin. For histologic examination, 4-µm sections of fixed embedded tissues were cut on a Leica model 2165 rotary microtome (Leica, Nussloch, Germany), placed on glass slides, deparaffinized, and stained sequentially with hematoxylin 2 and eosin-Y (Richard-Allan Scientific, Kalamazoo, MI). For immunocytochemistry of VEGF, the cytocentrifuge preparations of BAL cells were incubated sequentially in accordance with the instructions of the R. T. U. Vectastain Universal Quick kit from Vector Laboratories Inc. (Burlingame, CA). Briefly, the slides were incubated in Endo/Blocker (Biomeda Corp., Foster City, CA) for 5 min and in pepsin solution for 4 min at 40°C. The slides were incubated in normal horse serum for 15 min at room temperature. The slides were then probed with antibody against VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, and were incubated with prediluted biotinylated pan-specific IgG for 10 min. The slides were incubated in streptavidin/peroxidase complex reagent for 5 min, and then in 3-amino-9-ethylcarbazole substrate kit for 12 min. Controls consisted of BAL cells from mice that were incubated without the primary antibody. After immunostaining, the slides were counterstained for 1 min with Gill's hematoxylin in 20% ethylene glycol and then mounted with Aqueous Mounting Medium (InnoGenex, San Ramon, CA) and photomicrographed (Vanox T; Olympus, Tokyo, Japan).

Measurements of VEGF in BAL Fluids
Levels of VEGF in BAL fluids were quantified by an enzyme immunoassay according to the manufacturer's protocol (R&D Systems Inc., Minneapolis, MN). The minimum detectable level of mouse VEGF is < 3.0 pg/ml.

Western Blot Analysis
Lung tissues were homogenized in the presence of protease inhibitors to obtain extracts of lung proteins. Protein concentrations were determined using Bradford reagent (Bio-Rad Inc., Hercules, CA). Samples (30 µg protein per lane) were loaded on a 10% SDS-PAGE gel. After electrophoresis, separated proteins were transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ) by the wet transfer method (250 mA, 90 min). Nonspecific sites were blocked with 5% nonfat milk in Tris-buffered saline Tween 20 (TBST; 25 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 2 h, and the blots were then incubated with an anti-VEGF antibody (Santa Cruz Biotechnology), anti-Akt antibody (Cell Signaling Technology Inc., Beverly, MA), or anti-phosphorylated Akt (p-Akt) antibody (Cell Signaling Technology Inc.) overnight at 4°C. Anti-rabbit horseradish peroxidase–conjugated IgG was used to detect binding of the antibodies. The binding of the specific antibody was visualized by exposing to photographic film after treating with enhanced chemiluminescence system reagents (Amersham Pharmacia Biotech).

Cytosolic or Nuclear Protein Extractions for Analysis of HIF-1{alpha} and HIF-1beta
Lungs were removed and homogenized in 2 vols of buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 0.5 mM DTT, 5 mM MgCl2, and 1 mM PMSF) containing protease inhibitor cocktails. The homogenates were centrifuged at 1,000 x g for 15 min at 4°C. The supernatant fraction was incubated on ice for 10 min and centrifuged at 100,000 x g for 1 h at 4°C to obtain cytosolic proteins for analysis of HIF-1{alpha} and HIF-1beta. The pellets were washed twice in buffer A and resuspended in buffer B (1.3 M sucrose, 1.0 mM MgCl2, and 10 mM potassium phosphate buffer, pH 6.8) and pelleted at 1,000 x g for 15 min. The pellets were suspended in buffer B with a final sucrose concentration of 2.2 M and centrifuged at 100,000 x g for 1 h. The resulting pellets were washed once with a solution containing 0.25 M sucrose, 0.5 mM MgCl2, and 20 mM Tris-HCl, pH 7.2, and centrifuged at 1,000 x g for 10 min. The pellets were solubilized with a solution containing 50 mM Tris-HCl (pH 7.2), 0.3 M sucrose, 150 mM NaCl, 2 mM EDTA, 20% glycerol, 2% Triton X-100, 2 mM PMSF, and protease inhibitor cocktails. The mixture was kept on ice for 1 h with gentle stirring and centrifuged at 12,000 x g for 30 min. The resulting supernatant was used as soluble nuclear proteins for analysis of HIF-1{alpha} and HIF-1beta. The levels of these proteins were analyzed by Western blotting using antibody against HIF-1{alpha} (Novus Biologicals Inc., Littleton, CO) or HIF-1beta (Novus Biologicals Inc.) as described above.

Measurement of Phosphatidylinositol 3-Kinase Enzyme Activity in Lung Tissues
Lung tissues were homogenized in the presence of protease inhibitors to obtain extracts of lung proteins. Protein concentrations were determined using Bradford reagent (Bio-Rad). The amount of phosphatidyl inositol-3,4,5-triphosphate (PIP3) produced was quantified by PIP3 competition enzyme immunoassays according to the manufacturer's protocol (Echelon, Inc., Salt Lake City, UT). The enzyme activity was expressed as pmol PIP3 produced by 1 ml of lung tissue extracts containing equal amounts of total protein.

Determination of Airway Responsiveness to Methacholine
Airway responsiveness was assessed as a change in airway function after challenge with aerosolized methacholine via airways, as described elsewhere (20). Anesthesia was achieved with 80 mg/kg of pentobarbital sodium injected intraperitoneally. The trachea was then exposed through midcervical incision, tracheostomized, and an 18-gauge metal needle was inserted. Mice were connected to a computer-controlled small animal ventilator (flexiVent; SCIREQ, Montreal, PQ, Canada). The mouse was quasi-sinusoidally ventilated with nominal tidal vol- ume of 10 ml/kg at a frequency of 150 breaths/min and a positive end-expiratory pressure of 2 cm H2O to achieve a mean lung volume close to that during spontaneous breathing. This was achieved by connecting the expiratory port of the ventilator to a water column. Before methacholine challenge, an aerosol of saline was given to obtain baseline of airway responsiveness in each group. Methacholine aerosol was generated with an in-line nebulizer and administered directly through the ventilator. To determine the differences in airway response to methacholine, each mouse was challenged with methacholine aerosol in increasing concentrations (2.5–50 mg/ml in saline). After each methacholine challenge, the data of airway resistance (RL) was continuously collected. Maximum values of RL were selected to express changes in airway function, which were represented as a percentage change from baseline after saline aerosol.

Densitometric Analyses and Statistics
All immunoreactive and phosphorylative signals were analyzed by densitometric scanning (Gel Doc XR; Bio-Rad Laboratories Inc.). Data are expressed as mean ± SEM. Statistical comparisons were performed using one-way ANOVA followed by the Scheffe's test. Pearson's correlation was calculated to assess the correlation between data. Significant differences between groups were determined using t test. Statistical significance was set at P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effect of H2O2 on ROS Levels in BAL Cells
ROS levels in BAL cells were increased dose-dependently at 12 h after the last inhalation of H2O2. H2O2-induced ROS levels were increased 2.1-fold at 1% H2O2 and 3.1-fold at 3% H2O2 administration compared with the levels after saline inhalation (Figure 1).


Figure 1
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  		Figure 1. Effect of H2O2 on ROS levels in BAL cells. Sampling was performed at 12 h after the last inhalation of 1% H2O2 or 3% H2O2. Dichlorofluorescein fluorescence intensity is presented as the relative ratio of ROS levels. The ROS levels in the BAL cells of control mice are arbitrarily presented as 100. Bars represent mean ± SEM from eight mice per group. #P < 0.05 versus control; *P < 0.05 versus 1% H2O2.

 
Effect of H2O2 on Airway Responsiveness
Airway responsiveness was assessed as a percent increase of RL in response to increasing doses of methacholine. In mice given inhalations of 1% H2O2 or 3% H2O2, the dose–response curve of RL shifted to the left compared with that of control mice (see Figure E1 in the online supplement). In addition, the RL produced by methacholine administration (at doses from 2.5–50 mg/ml) increased significantly in the mice given inhalations of 1% H2O2 or 3% H2O2 compared with the controls. These results indicate that H2O2 administration induces airway hyperresponsiveness.

Effect of H2O2 on VEGF Protein Levels in BAL Fluids and in Lung Tissues
Western blot analysis revealed that levels of VEGF protein in lung tissues were significantly increased dose-dependently at 12 h after the last inhalation of H2O2 (Figures 2A and 2B). The increase in VEGF level at 12 h after the last inhalation of H2O2 was 2.2- and 4.2-fold at 1% and 3% H2O2 administration, respectively, as compared with the levels after saline inhalation. Consistent with the results obtained from the Western blot analysis, enzyme immunoassay revealed that levels of VEGF protein in BAL fluids were significantly increased at 12 h after the last inhalation of H2O2 (Figure 2C). The increase in VEGF level at 12 h after the last inhalation of H2O2 was 2.3- and 3.7-fold at 1% and 3% H2O2 administration, respectively, as compared with the levels after saline inhalation.


Figure 2
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  		Figure 2. Effect of H2O2 on plasma exudation and VEGF protein expression in BAL fluids and lung tissues. Sampling was performed at 12 h after the last inhalation of 1% H2O2 or 3% H2O2. (A) Western blotting of VEGF in lung tissues. (B) Densitometric analyses are presented as the relative ratio of VEGF to actin. The relative ratio of VEGF in the lung tissues of control mice is arbitrarily presented as 1. (C) Enzyme immunoassay of VEGF in BAL fluids. (D–F) Localization of immunoreactive VEGF in BAL fluids. Sampling was performed at 12 h after the last inhalation of saline (D), 1% H2O2 (E), or 3% H2O2 (F). The brown color indicates VEGF-positive cells. Bars indicate scale of 50 µm. (G) EBD assay in lung tissues. Bars represent mean ± SEM from eight mice per group. #P < 0.05 versus control; *P < 0.05 versus 1% H2O2.

 
Localization of Immunoreactive VEGF in BAL Fluids
Immunocytologic analyses of BAL fluids showed the localization of immunoreactive VEGF in the precipitated cells from the mice inhaled with 1% H2O2 or 3% H2O2 (Figures 2E and 2F). The expression of immunoreactive VEGF in BAL cells from H2O2-treated mice were higher than those in BAL cells from the control mice (Figure 2D).

Effect of H2O2 on Plasma Extravasation
The EBD assay showed that plasma extravasation was significantly increased in a H2O2 dose-dependent manner at 12 h after the last inhalation of H2O2 (Figure 2G). H2O2-mediated increase of EBD levels was 3.7-fold at 1% H2O2 and 5.3-fold at 3% H2O2 administration as compared with the levels after saline inhalation.

Effect of H2O2 on p-Akt and Akt Protein Levels and PI3K Enzyme Activity in Lung Tissues
Levels of p-Akt protein in the lung tissues were increased significantly at 12 h after the last inhalation of H2O2 compared with the levels in the control animals (Figures 3A and 3B). However, no significant changes in total Akt protein levels were observed in any of the groups tested. The increased p-Akt but not Akt protein levels in lung tissues at 12 h after the last inhalation of H2O2 was 2.0- and 4.3-fold at 1% and 3% H2O2 administration, respectively, as compared with the enzyme activity after saline inhalation.


Figure 3
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  		Figure 3. Effect of H2O2 on p-Akt and Akt protein levels and PI3K enzyme activity in lung tissues. (A) p-Akt and Akt protein levels in lung tissues were measured at 12 h after the last inhalation of 1% H2O2 or 3% H2O2. (B) Densitometric analyses are presented as the relative ratio of VEGF to actin. The relative ratio of p-Akt in the lung tissues of control mice is arbitrarily presented as 1. (C) PIP3 generation by PI3Ks in lung tissue extracts. Bars represent mean ± SEM from eight mice per group. #P < 0.05 versus control; *P < 0.05 versus 1% H2O2.

 
PI3K activity in the lung tissues was increased significantly at 12 h after the last inhalation of 1% H2O2 compared with the control mice (Figure 3C). The increased PI3K activity in lung tissues at 12 h after the last inhalation of H2O2 was 2.9- and 4.5-fold at 1% and 3% H2O2 administration, respectively, as compared with the levels after saline inhalation.

Effect of H2O2 on HIF-1{alpha} Levels in Nuclear Protein Extracts from Lung Tissues
Western blot analysis revealed that levels of HIF-1{alpha} protein in nuclear protein extracts from lung tissues were increased significantly at 12 h after the last inhalation of H2O2 compared with the levels in the control animals (Figures 4A and 4B). The increased HIF-1{alpha} levels in nuclear protein extracts from lung tissues at 12 h after the last inhalation of H2O2 was 1.7- and 2.4-fold at 1% and 3% H2O2 administration, respectively, as compared with the levels after saline inhalation.


Figure 4
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  		Figure 4. Effect of H2O2 on HIF-1{alpha} and HIF-1beta levels in nuclear protein extracts from lung tissues. (A) HIF-1{alpha} and HIF-1beta expression in nuclear protein extracts from lung tissues. HIF-1{alpha} and HIF-1beta levels in lung tissues were measured at 12 h after the last inhalation of 1% H2O2 or 3% H2O2. (B) Densitometric analyses are presented as the relative ratio of HIF-1{alpha} to HIF-1beta. The relative ratio of HIF-1{alpha} in the lung tissues of control mice is arbitrarily presented as 1. Bars represent mean ± SEM from eight mice per group. #P < 0.05 versus control; *P < 0.05 versus 1% H2O2.

 
VEGF Protein in BAL Fluids and Plasma Exudation
In 3% H2O2–exposed mice, levels of VEGF protein had increased ~ 1.4-, 1.7-, 1.8-, 2.1-, 2.8-, and 2.1-fold at 0.5, 1, 4, 6, 12, and 24 h after 3% H2O2 inhalation, respectively, compared with before the first 3% H2O2 inhalation (Figure 5A). In 3% H2O2-exposed mice, plasma extravasation had also increased ~ 2.0-, 2.6-, 3.1-, 3.3-, 3.9-, and 3.1-fold at 0.5, 1, 4, 6, 12, and 24 h after 3% H2O2 inhalation, respectively, compared with before the first 3% H2O2 inhalation (Figure 5B). In addition, the levels of VEGF protein in BAL fluids correlated significantly with the levels of plasma exudation in 3% H2O2-exposed mice (r = 0.685; P < 0.05) (Figure 5C).


Figure 5
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  		Figure 5. The kinetics and correlation of VEGF protein expression in BAL fluids and plasma exudation of H2O2-inhaled mice. (A) Enzyme immunoassay of VEGF protein in BAL fluids. Sampling was performed in BAL fluids after the last inhalation of 3% H2O2. (B) EBD assay in lung tissues. Data represent mean ± SEM from eight mice per group; 0.5, 1, 4, 6, 12, and 24 h are time periods after the last inhalation of 3% H2O2. Pre, before the first 3% H2O2 inhalation; #P < 0.05 versus Pre. (C) Correlation between VEGF levels in BAL fluids and plasma exudation.

 
Effect of OTC, {alpha}-Lipoic Acid, SU5614, or CBO-P11 on VEGF Protein in Lungs and Plasma Exudation
Western blot analysis revealed that levels of VEGF protein in lung tissues were increased significantly at 12 h after the last inhalation of 3% H2O2 compared with the levels in the control mice (Figures 6A and 6B). The increased VEGF levels at 12 h after the last inhalation of 3% H2O2 were decreased significantly by the administration of OTC, {alpha}-lipoic acid, SU5614, or CBO-P11. Consistent with the results obtained from the Western blot analysis, enzyme immunoassay revealed that levels of VEGF protein in BAL fluids were increased at 12 h after the last inhalation of 3% H2O2 compared with the levels in the control mice (Figure 6C). The increased VEGF levels at 12 h after the last inhalation of 3% H2O2 were decreased significantly by the administration of OTC, {alpha}-lipoic acid, SU5614, or CBO-P11. However, the increased VEGF levels in lung tissues and in BAL fluids at 12 h after the last inhalation of 3% H2O2 were not changed significantly by the administration of DMSO alone.


Figure 6
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  		Figure 6. Effect of OTC, {alpha}-lipoic acid, SU5614, or CBO-P11 on plasma exudation and VEGF levels in lung tissues and BAL fluids. Sampling was performed at 12 h after the last inhalation in mice administered saline after saline inhalation (SAL+SAL), mice administered saline after 3% H2O2 inhalation (3% H2O2+SAL), mice administered drug vehicle (PBS) after 3% H2O2 inhalation (3% H2O2+VEH), mice administered OTC after 3% H2O2 inhalation (3% H2O2+OTC), mice administered {alpha}-lipoic acid after 3% H2O2 inhalation (3% H2O2+{alpha}-lipoic acid), mice administered SU5614 after 3% H2O2 inhalation (3% H2O2+SU5614), mice administered CBO-P11 after 3% H2O2 inhalation (3% H2O2+CBO-P11), and mice administered 0.05% DMSO after 3% H2O2 inhalation (3% H2O2+DMSO). (A) Western blot analysis of VEGF. (B) Densitometric analyses are presented as the relative ratio of VEGF to actin. The relative ratio of VEGF in the lung tissues of SAL+SAL is arbitrarily presented as 1. (C) Enzyme immunoassay of VEGF in BAL fluids. (D) EBD assay in lung tissues. Data represent mean ± SEM from eight mice per group. #P < 0.05 versus SAL+SAL; *P < 0.05 versus 3% H2O2+SAL.

 
The EBD assay showed that plasma extravasation was significantly increased at 12 h after the last inhalation of 3% H2O2 compared with the levels in the control mice (Figure 6D). The increase in plasma extravasation at 12 h after the last inhalation of 3% H2O2 was decreased significantly by the administration of OTC, {alpha}-lipoic acid, SU5614, or CBO-P11. The increase in plasma extravasation at 12 h after the last inhalation of 3% H2O2 was not changed significantly by the administration of DMSO alone.

Effect of Antioxidants on Pathologic Changes
Histologic analyses revealed that numerous inflammatory cells infiltrated around the bronchioles and mucus and debris had accumulated in the lumen of bronchioles at 12 h after the last inhalation of 3% H2O2 (Figure 7B) as compared with the control (Figure 7A). Mice treated with OTC (Figure 7C) and {alpha}-lipoic acid (Figure 7D) showed marked reductions in the infiltration of inflammatory cells in the peribronchiolar region and in the amount of mucus and debris in the airway lumen.


Figure 7
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  		Figure 7. Effect of OTC and {alpha}-lipoic acid on pathologic changes in lung tissues. Sampling was performed at 12 h after the last inhalation in mice administered saline after saline inhalation (A), mice administered saline after 3% H2O2 inhalation (B), mice administered OTC after 3% H2O2 inhalation (C), and mice administered {alpha}-lipoic acid after 3% H2O2 inhalation (D). Bars indicate scale of 50 µm.

 
Effect of LY-294002 or Wortmannin on HIF-1{alpha} Protein Levels in Lung Tissues of 3% H2O2–Exposed Mice
Western blot analysis revealed that levels of HIF-1{alpha} protein in nuclear protein extracts from lung tissues were increased significantly at 12 h after H2O2 inhalation compared with the levels in the control mice (Figure E2). The increased HIF-1{alpha} levels in nuclear protein extracts from lung tissues at 12 h after H2O2 inhalation were decreased significantly by the administration of LY-294002 or wortmannin.

Effect of 2ME2 on VEGF Protein Levels in Lung Tissues and in BAL Fluids and Plasma Exudation of 3% H2O2–Exposed Mice
Western blot analysis showed that VEGF protein levels in lung tissues were increased significantly at 12 h after 3% H2O2 inhalation compared with the levels after saline inhalation. The increased VEGF levels were significantly reduced by the administration of 2ME2 (Figures E3A and E3B). Consistent with the results obtained from the Western blot analysis, an enzyme immunoassay revealed that levels of VEGF in BAL fluids were also increased significantly at 12 h after 3% H2O2 inhalation compared with the levels after saline inhalation (Figure E3C). The increased VEGF levels were significantly reduced by the administration of 2ME2. The EBD assay showed that plasma extravasation was significantly increased at 12 h after the last inhalation of 3% H2O2 compared with the levels in the control mice (Figure E3D). The increase in plasma extravasation at 12 h after the last inhalation of 3% H2O2 was decreased significantly by the administration of 2ME2.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In this study, we have used a murine model challenged with H2O2 to examine roles of ROS and the molecular mechanism in vascular permeability. The results have revealed that ROS level, VEGF expression, HIF-1{alpha} protein level, and airway hyperresponsiveness, as well as vascular permeability, are increased after inhalation of H2O2. Administration of antioxidants or VEGF receptor inhibitor markedly reduced the increased vascular permeability and VEGF levels in ROS-induced lungs. These results suggest that ROS modulate vascular permeability via upregulation of VEGF expression.

ROS are involved in airway smooth muscle contraction, increased mucus production, decreased numbers and function of epithelial cilia, altered release of inflammatory mediators, influx of inflammatory cells, and increased vascular permeability (1). Previous studies have demonstrated that ROS can lead to endothelial barrier dysfunction with subsequent increased permeability to fluid, macromolecules, and inflammatory cells (21, 22). Although the lung has a well-developed antioxidant system (23), overproduction of ROS or depression of the protective system results in epithelial cell damage, cell shedding, and bronchial hyperreactivity (24, 25). Studies with animal models have indicated that ROS contribute to airway hyperresponsiveness by increasing vagal tone due to damage of oxidant-sensitive beta-adrenergic receptors as well as decreasing mucociliary clearance (26). Consistent with these observations, our results indicate that plasma extravasation, airway inflammation, and bronchial hyperresponsiveness caused by increased vascular permeability were elevated after H2O2 inhalation.

Several reports have shown that overproduction of VEGF is associated with increased vascular permeability and plasma exudation in a murine model of pulmonary inflammation, which is associated with an increased release of ROS in the airways (6, 27, 28). In this study, we have found that VEGF protein expression was upregulated and that increased levels of VEGF closely correlated with increased vascular permeability in an ROS-induced animal model. Interestingly, administration of OTC, {alpha}-lipoic acid, SU5614, or CBO-P11 reduced the increased VEGF levels in the lungs. These results suggest that oxidative stress is associated with the regulation of VEGF expression and that treatment of the antioxidant may decrease the vascular permeability by inhibiting upregulation of VEGF expression. The major role of VEGF in airway inflammation appears to be the enhancement of vascular permeability, resulting in leakage of plasma proteins into the extravascular space (4, 6). This may cause edema and profound alterations in the extracellular matrix. The mechanism of VEGF-mediated induction of the vascular permeability seems to be the enhanced functional activity of vesicular–vacuolar organelles (4). VEGF expression is regulated through HIF-1{alpha} expression (7, 8). Consistent with these reports, this present study has shown that the increased VEGF levels after H2O2 inhalation were decreased significantly by the administration of HIF-1{alpha} inhibitor, 2ME2. HIF-1 is a transcriptional activator that mediates changes in gene expression in response to changes in cellular oxygen concentrations (29). Moreover, recent studies have demonstrated that HIF-1 plays a central role in stress responses beyond hypoxia. A number of peptide and nonpeptide mediators of inflammation can activate HIF-1 even under normoxic conditions (30, 31). Previous reports have demonstrated that HIF-1{alpha} plays a critical role in immune and inflammatory responses (32, 33). Determination of HIF-1{alpha} protein level in nuclear extracts has revealed that this protein level was substantially increased in our present H2O2-inhaled model, suggesting that HIF-1{alpha} was activated. Several reports have shown that increase of PI3K/Akt activity can activate the HIF pathway (34, 35). Li and coworkers have also reported that activation of Akt turns on HIF-1{alpha} independently of hypoxia (36). In addition, ROS have been shown to stabilize HIF-1{alpha} during hypoxia and/or normoxia (37, 38). Our results have revealed that levels of p-Akt protein in the lung tissues and PI3K activitiy were increased after H2O2 inhalation. In addition, HIF-1{alpha} protein levels were also increased significantly after H2O2 inhalation, and the increased HIF-1{alpha} protein levels were decreased significantly by the administration of PI3K inhibitors. Taken together, we suggest that ROS regulate HIF-1{alpha} action through a PI3K/Akt pathway, resulting in increased VEGF expression in a murine model.

Recently, {alpha}-lipoic acid and OTC, which act as antioxidants, have been shown to reduce airway inflammation and hyperresponsiveness in a murine model of pulmonary inflammation (27, 39). In this study, we have found that plasma extravasation caused by increased vascular permeability was elevated after H2O2 inhalation and that administration of OTC or {alpha}-lipoic acid significantly reduced the increased plasma extravasation at 12 h after H2O2 inhalation. Although the pathogenesis of airway inflammation induced by plasma extravasation is not clearly defined, plasma protein leakage has been implicated to play a role in the induction of a thickened, engorged, and edematous airway wall, resulting in the airway lumen narrowing. Exudation of plasma proteins into the airways correlates with bronchial hyperreactivity (40). It is also possible that the plasma exudate may readily pass the inflamed mucosa and reach the airway lumen through leaky epithelium, thus compromising epithelial integrity and reducing ciliary function and mucus clearance (41).

In summary, we have examined the role of the ROS in a murine model in the increase of vascular permeability. By using an exogenous ROS, H2O2, we have shown the important role of ROS in airway inflammation, bronchial hyperresponsiveness, and increased vascular permeability, including expression of VEGF and HIF-1{alpha}. By examining the effects of the antioxidants OTC or {alpha}-lipoic acid on H2O2-induced plasma exudation and VEGF expression, we suggest that ROS increase vascular permeability through enhancing VEGF expression. Our study also provides evidence that oxidative stress is one of the important determinants of lung disorders and that antioxidant treatment may be a recommendable therapeutic strategy.


   Acknowledgments
 
The authors thank professor Mie-Jae Im for critical readings of the manuscript.


   Footnotes
 
* These authors contributed equally to this work. Back

This work was supported by Korean Research Foundation Grant funded by Korea Government (MOEHRD, Basic Research Promotion Fund) (KRF-2005–201-E00014) and grant from the National Research Laboratory Program of the Korea Science and Engineering Foundation, Republic of Korea.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0482OC on March 30, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form December 27, 2005

Accepted in final form February 24, 2006


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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