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Published ahead of print on March 20, 2008, doi:10.1164/rccm.200708-1141OC
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American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 1314-1321, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200708-1141OC

Original Article

Angiopoietin-1 Protects against Airway Inflammation and Hyperreactivity in Asthma

Davina C. M. Simoes1, Theodoros Vassilakopoulos1, Dimitrios Toumpanakis1, Kalomira Petrochilou1, Charis Roussos1 and Andreas Papapetropoulos1,2

1 "G. P. Livanos and M. Simou" Laboratories, Department of Critical Care and Pulmonary Services, Evangelismos Hospital, University of Athens School of Medicine, Athens, Greece; and 2 Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras, Greece

Correspondence and requests for reprints should be addressed to Andreas Papapetropoulos, Ph.D., "G. P. Livanos and M. Simou" Laboratories, Evangelismos Hospital, Department of Critical Care and Pulmonary Services, University of Athens School of Medicine, Athens, Greece 10675. E-mail: apapapet{at}upatras.gr


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: The angiopoietins (Ang) comprise a family of growth factors mainly known for their role in blood vessel formation and remodeling. The best-studied member, Ang-1, exhibits antiapoptotic and antiinflammatory effects. Although the involvement of Ang-1 in angiogenesis is well recognized, little information exists about its role in respiratory physiology and disease. On the basis of its ability to inhibit vascular permeability, adhesion molecule expression, and cytokine production, we hypothesized that Ang-1 administration might exert a protective role in asthma.

Objectives: To determine changes in the expression of Ang and to assess the ability of Ang-1 to prevent the histologic, biochemical, and functional changes observed in an animal model of asthma.

Methods: To test our hypothesis, a model of allergic airway disease that develops after ovalbumin (OVA) sensitization and challenge was used.

Measurements and Main Results: Ang-1 expression was reduced at the mRNA and protein levels in lung tissue of mice sensitized and challenged with OVA, leading to reduced Tie2 phosphorylation. Intranasal Ang-1 treatment prevented the OVA-induced eosinophilic lung infiltration, attenuated the increase in IL-5 and IL-13, and reduced eotaxin and vascular cell adhesion molecule 1 expression. These antiinflammatory actions of Ang-1 coincided with higher levels of I{kappa}B and decreased nuclear factor-{kappa}B binding activity. More importantly, Ang-1 reversed the OVA-induced increase in tissue resistance and elastance, improving lung function.

Conclusions: We conclude that Ang-1 levels are decreased in asthma and that administration of Ang-1 might be of therapeutic value because it prevents the increased responsiveness of the airways to constrictors and ameliorates inflammation.

Key Words: airway resistance • interleukin 5 • eotaxin • VCAM-1 • nuclear factor-{kappa}B



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Angiopoietin (Ang)-1 exhibits antiinflammatory properties by inhibiting leukocyte transendothelial migration, cytokine production, and vascular permeability. Ang-1 administration ameliorates endotoxin-induced lung injury and improves survival.

What This Study Adds to the Field
Ang-1 levels are reduced in the lungs of animals with experimental asthma. Local Ang-1 administration prevents the biochemical and functional changes observed in asthma, suggesting that Tie2 agonists might have therapeutic potential in this disease.

 
Asthma is a chronic inflammatory lung disease with exaggerated T-helper (Th2) cell inflammation characterized by elevated serum IgE levels, mucus hypersecretion, and airway hyperresponsiveness (1, 2). Th2 cells, together with other inflammatory cells, have been proposed to play a critical role in the initiation, development, and chronicity of asthma (3). During the course of the disease, structural changes also occur in the airways, known as remodeling; these include subepithelial fibrosis, increase in the airway smooth muscle mass, and increased vascularization (4). Enhanced expression of angiogenic factors has been observed in the lungs of patients with asthma and in animal models of asthma (5–9). Remodeling of the microvasculature in the airways of individuals with bronchial asthma is believed to contribute to airway wall thickness, luminal narrowing, and airway hyperreactivity (10). In some studies, vessel number and vascularity have been positively correlated with the severity of asthma (11, 12).

Angiopoietins (Ang) are ligands for the endothelial receptor tyrosine kinase Tie2 with important roles in angiogenesis (13). Ang-1 phosphorylates and activates Tie2; it is perceived as a stabilizing factor that promotes structural and functional integrity of the vasculature (14, 15). In contrast, Ang-2 exerts context-dependent effects and is believed to prime vessel plasticity, allowing for other angiogenic factors to drive the angiogenic response (13, 16). Initially, Tie2 expression was believed to be restricted to the vascular endothelium. However, recent observations have provided evidence for the existence of functional Tie2 receptors on leukocytes (neutrophils and eosinophils) (17, 18). It should be noted that, apart from their role in angiogenesis, the angiopoietins have been shown to modify the inflammatory response in vivo (15, 19–21). We and others have shown that Ang-1 inhibits vascular permeability, leukocyte adhesion to the endothelium, cytokine production, and adhesion molecule expression in response to a variety of inflammatory mediators (15, 22, 23). Because mediators that are elevated in asthma, such as transforming growth factor (TGF)-β1, thrombin, and cytokines, down-regulate Ang-1 expression (16, 24–26), we hypothesized that Ang-1 expression is reduced in the lung in the course of allergic airway inflammation.

Vascular endothelial growth factor (VEGF), a growth factor that plays a key role in angiogenesis (27), has been shown to enhance the Th2 response in animal models of asthma and to be increased in the lungs of patients with asthma (7, 8). Because Ang-1 has been shown to counteract many effects of VEGF linked to inflammation (15, 22), we also hypothesized that Ang-1 exerts a protective effect in asthma. To this end, the effects of local Ang-1 administration were evaluated in a murine model of allergic airway inflammation. Some of the results of these studies have been previously reported in the form of abstracts (28, 29)


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Protocol
Male BALB/c mice (8–12 wk of age) were sensitized using ovalbumin (OVA) (0.01 mg/mouse) intraperitoneally on Days 0 and 12. Control mice received the same volume of alum. Animals were challenged daily with 5% OVA (aerosolized) from Days 18 to 21. Administration of Ang-1 (1 µg, intranasally) or vehicle (saline) was performed under light anesthesia (ketamine/xylazine) 30 minutes before each OVA challenge. All measurements were made 24 hours after the final OVA challenge.

Airway Hyperresponsiveness
Mice were anesthetized, tracheostomized, paralyzed, and ventilated with Flexivent (SCIREQ Scientific Respiratory Equipment, Inc., Montreal, PQ, Canada) (see the online supplement). After baseline measurements of impedance (Zrs), methacholine (3 or 10 mg/ml) or saline were delivered (Aeroneb; SCIREQ) for 10 seconds. Afterwards, a 2-second forced oscillation perturbation (1–20 Hz) was performed every 10 seconds for 3 minutes. Before measurements and before every aerosol delivery, the volume history of the lung was established with two 6-second deep inflations to a pressure limit of 30 cm H2O. Measurements of Zrs were fit with the constant phase model (30), where Rn is the Newtonian resistance of the airways, G represents tissue resistance, and H tissue elasticity. After each dose of methacholine, model parameters were expressed as % ratio of the baseline (see the online supplement).

Bronchoalveolar Lavage Measurements and Lung Histology
Airways of mice were lavaged, the resulting bronchoalveolar lavage (BAL) fluid was immediately centrifuged (700 x g, 5 min at 4°C), and supernatants collected. Differential cell counts were performed on Wright-Giemsa–stained cytospins. To evaluate the extent of inflammation and mucus production, lung sections were stained with hematoxylin and eosin and periodic acid Schiff (PAS), respectively.

Inflammatory Mediators and Serum Antibody Concentration
Lung tissue was homogenized in Hanks' balanced salt solution containing protease inhibitors, centrifuged (1,900 rpm for 10 min), and the supernatant collected. Levels of cytokines in the lung tissue and serum levels of total and specific anti-OVA IgE were determined by ELISA (see the online supplement).

Nuclear Factor-{kappa}B Binding Assay
Active nuclear factor (NF)-{kappa}B contained in 20 µg of lung nuclear extracts was bound to an oligonucleotide coating the plate surface of the NF-{kappa}B TransAM kit (Active Motif, Carlsbad, CA). An antibody directed against the p65 subunit was used to determine the presence of this NF-{kappa}B subunit. Optical density at 450 nm was determined using a horseradish peroxidase–conjugated secondary antibody.

Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from the lungs, quantified, and treated with DNase I. After cDNA synthesis, the levels of Ang-1 and Ang-2 were assessed in a Light Cycler System (Bio-Rad, Hercules, CA) and analyzed. To calculate the relative quantity of the respective subunit, the {Delta}{Delta}CT method was used, employing glyceraldehyde-3-phosphate dehydrogenase for normalization.

Western Blot Analysis
One lobe was homogenized in 10 vol (wt/vol) of a lysis buffer (see the online supplement). Samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by blotting with appropriate primary and secondary antibodies. Immunoblots were visualized using chemiluminescence.

Statistical Analysis
Results are presented as means ± SEM. Comparisons were made using the t test or analysis of variance (ANOVA) followed by the Tukey's post hoc or Kruskal-Wallis ANOVA and Mann-Whitney U test, where appropriate (SPSS Version 13.0 software; SPSS, Inc., Chicago, IL). Differences were considered significant when P values were less than 0.05.


   RESULTS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ang-1 mRNA and Protein Levels Are Reduced in Allergen-induced Airway Inflammation
To determine whether asthmalike airway inflammation leads to changes in angiopoietin levels, mice were sensitized and challenged with OVA and Ang-1 and Ang-2 mRNA levels were determined. Ang-1 mRNA levels were reduced by approximately 50% in OVA mice, whereas the levels of Ang-2 were not altered (Figure 1A). Exposing mice to a greater number of OVA challenges (6 instead of 4) led to a more pronounced decrease in Ang-1 levels (data not shown). To test if the changes observed at the mRNA level also translated into changes in protein expression, angiopoietin content in lung homogenates was determined by Western blotting (Figure 1B). In these experiments, we observed that Ang-1 was reduced to 20.8 ± 8.2% of control in OVA-sensitized mice. Ang-2 protein levels were also reduced in the OVA mice; however, this reduction did not reach statistical significance (P = 0.06; Figure 1C). Tie2 immunoprecipitation experiments revealed that smaller amounts of Ang-1 were bound to its receptor in mice sensitized and challenged with OVA (Figures 2A and 2B). In addition, Tie2 phosphorylation in OVA-treated mice was reduced compared with control animals (Figures 2A and 2C). Upon administration of exogenous Ang-1, more of this growth factor was found to be bound to Tie2, leading to an increase in phospho-Tie2/total Tie2 ratio (Figures 2A and 2C).


Figure 1
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  		Figure 1. Angiopoietin (Ang)-1 is reduced in mice with asthmalike airway inflammation. (A) Angiopoietin mRNA levels in lung homogenates were analyzed with real-time reverse transcriptase–polymerase chain reaction as described in METHODS. (B) Representative Western blots depicting Ang-1 and Ang-2 levels in lung homogenates of control (CTL) and ovalbumin-treated (OVA) mice. (C) Quantitation of band intensity from blots shown in (B) by densitometry. Results are presented as means ± SEM; relative expression of Ang-1 or Ang2 was set at 100% for control mice after normalization against either GAPDH or actin for mRNA and protein measurements, respectively. n = 8–10 mice per group; *P < 0.05 compared with control.

 

Figure 2
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  		Figure 2. Tie2 phosphorylation in mice sensitized and challenged with ovalbumin (OVA). (A) The Tie2 receptor was immunoprecipitated and samples were processed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Membranes were then blotted using an antibody that recognizes both human and mouse Ang-1 (top panel), a phosphotyrosine antibody (middle panel) or total Tie2 (lower panel). (B, C) Densitometric analysis of blots shown in (A). OVA challenge mice (CTL), mice sensitized and challenged with OVA (OVA), and mice treated with Ang-1 (OVA+Ang-1) before each OVA challenge. Results are presented as means ± SEM, n = 5–6; *P < 0.05 from control, #P < 0.05 compared with OVA.

 
Administration of Ang-1 Reduces Lung Inflammation and Prevents Airway Hyperresponsiveness
We next sought to investigate whether increasing Ang-1 levels through local administration of recombinant protein could prevent the changes observed in the airways of mice with an asthmalike disease. OVA mice exhibited an increase in BAL cellularity; differential cell counts revealed that this was due primarily to infiltration of eosinophils, and to a smaller extent macrophages and lymphocytes (Figure 3A). Ang-1 administration significantly reduced total cell and eosinophil counts, whereas it did not affect the number of macrophages, lymphocytes, and neutrophils present in the BAL after OVA sensitization and challenge (Figure 3A). It should be noted that administration of Ang-1 in the absence of OVA sensitization had no effect on BAL cell numbers (data not shown). The extent of inflammation in the lungs of OVA mice was also evaluated in histologic sections stained with hematoxylin and eosin (Figure 3B). Large numbers of infiltrating inflammatory cells were present in OVA mice and Ang-1 treatment ameliorated this inflammatory response. In contrast, production of mucus by goblet cells was not different between the OVA and OVA+Ang-1 mice, as suggested by the similar PAS scores obtained in the two groups of animals (Figure 3C).


Figure 3
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  		Figure 3. Lung inflammation is reduced in ovalbumin (OVA)-mice treated with Ang-1. (A) Differential cell counts in the bronchoalveolar lavage (BAL) of control animals only challenged with OVA (CTL), mice sensitized and challenged with OVA (OVA), and mice treated with Ang-1 before the OVA challenge (OVA+Ang-1). Animals were killed 24 hours after the final OVA challenge and BAL cells were isolated and counted. In (B) and (C) lungs were prepared for histology, stained with hematoxylin and eosin (B) or periodic acid Schiff (C) and scored by a blinded observer. Values are expressed as means ± SEM, n = 10; *P < 0.05 compared with control, #P < 0.05 compared with OVA. Eos = eosinophils; LMs = lymphocytes; Macs = macrophages; Neuts = neutrophils.

 
To determine the effects of Ang-1 on lung function, airway (Newtonian) resistance, tissue resistance (G), and tissue elastance (H) were next determined. Neither OVA immunization nor Ang-1 treatment had any effect on baseline lung function, because neither airway resistance nor tissue mechanics differed among the different groups (see the online supplement). In OVA-treated mice, all three parameters were increased in a dose-dependent manner after exposure to methacholine (Figure 4). Intranasal treatment with Ang-1, in addition to ameliorating inflammation, significantly decreased tissue resistance and elastance (Figures 4B and 4C), without affecting Newtonian resistance (Figure 4A).


Figure 4
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  		Figure 4. Ang-1 treatment attenuates the ovalbumin (OVA)-induced increase in resistance and elastance. Airway reactivity to increasing amounts of methacholine was measured 24 hours after the final OVA challenge in control mice (CTL), mice receiving Ang-1 only (CTL+Ang-1), mice sensitized and challenged with OVA (OVA), and mice treated with Ang-1 before each OVA challenge (OVA+Ang-1). Newtonian resistance (A), tissue resistance (B), and tissue elastance (C) were determined as described in METHODS. Data are presented as means ± SEM; n = 9–11; *P < 0.05 compared with control, #P < 0.05 compared with OVA.

 
Ang-1 Treatment Reduces the Production of Inflammatory Mediators
To investigate the mechanisms associated with the protective action of Ang-1, we measured the levels of Th2 cytokines implicated in the pathogenesis of asthma, namely IL-4, IL-5, and IL-13. As expected, lung homogenates of mice sensitized and challenged with OVA exhibited increased levels of all the above-mentioned cytokines compared with controls. Interestingly, administration of Ang-1 significantly reduced the levels of IL-5 and IL-13, but not IL-4 (Figures 5A–5C). In addition, Ang-1 prevented the OVA-induced increase in eotaxin expression (Figure 5D), suggesting that reduced eosinophil infiltration could be due to the reduction in this major chemoattractant.


Figure 5
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  		Figure 5. Ang-1 treatment prevents the increase in inflammatory mediators of asthma. Levels of IL-5 (A), IL-13 (B), eotaxin (C), and IL-4 (D) were measured in total lung homogenates by ELISA. The groups of mice used are the same as in Figure 3. Data are expressed as means ± SEM, n = 11–15; *P < 0.05 compared with control, #P < 0.05 compared with OVA.

 
VCAM-1 is an adhesion molecule of major importance for the recruitment of eosinophils into tissues (31). In agreement with current knowledge (24), VCAM-1 was up-regulated in the lungs of OVA-treated mice (Figures 6A and 6B). Ang-1 administration abolished the increase in VCAM-1 expression in mice sensitized and challenged with OVA (Figure 6B).


Figure 6
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  		Figure 6. Ovalbumin (OVA)-induced VCAM-1 levels are decreased in mice treated with Ang-1. VCAM-1 protein levels were determined by Western blotting in total lung homogenates of control animals (CTL), mice sensitized and challenged with OVA (OVA), and mice treated with Ang-1 (OVA+Ang-1) before each OVA challenge. (A) Representative Western blot depicting VCAM-1 expression; (B) densitometric quantitation of blots shown in (A). Relative expression of VCAM-1 was set at 100% for control mice after normalization to actin expression. Results are presented as means ± SEM; n = 7, *P < 0.05 compared with control; #P < 0.05 compared with OVA.

 
Apart from its local effects in the lung, intranasal Ang-1 administration exerted systemic effects. Circulating total IgE increased several fold to 18.8 ± 1.7 µg/ml in OVA mice; treatment with Ang-1 reduced these levels to 9.7 ± 0.98 µg/ml (Figure 7A). Similarly, Ang-1 was able to attenuate the production of OVA-specific IgE (639.2 ± 80.8 vs. 389 ± 38 U/ml in OVA vs. OVA+Ang-1 mice, respectively) (Figure 7B). Ang-1 administration also inhibited the number of eosinophils in peripheral blood of mice sensitized and challenged with OVA, suggesting this therapeutic intervention can reduce leukocyte trafficking from the bone marrow (control, 21.5 ± 6.0 x 103; OVA, 81.7 ± 13.6 x 103; OVA+Ang-1, 49.2 ± 7.8 x 103 cells/ml; n = 14–17, P < 0.05). It should, however, be noted that when human Ang-1 was measured in plasma at various time points (15 min to 24 h) after intranasal administration of the growth factor, its levels were undetectable.


Figure 7
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  		Figure 7. Serum IgE levels in mice sensitized and challenged with ovalbumin (OVA) are reduced after Ang-1 administration. Levels of total IgE (A) and OVA-specific IgE (B) were measured by ELISA in serum samples 24 hours after the final OVA challenge. Specific IgE antibody titters were then related to pooled standards generated in the laboratory and assigned arbitrary units. Data are expressed as means ± SEM, n = 11–15; *P < 0.05 compared with control, #P < 0.05 compared with OVA.

 
Protective Effects of Ang-1 Coincide with Reduced NF-{kappa}B Activation
Ang-1 has been proposed to inhibit NF-{kappa}B activation in vitro (32). To determine whether it also exerts a negative effect on NF-{kappa}B in vivo, we measured the levels of the NF-{kappa}B inhibitor protein I{kappa}B-{alpha}. As shown in Figures 8A and 8B, Ang-1 prevented the OVA-induced reduction in I{kappa}B-{alpha}. In line with this observation, binding activity of the p65 subunit present in nuclear extracts isolated from lung homogenates of OVA mice was decreased to control levels after Ang-1 administration (Figure 8C), suggesting that Ang-1 might reduce inflammatory gene expression in vivo by inhibiting NF-{kappa}B.


Figure 8
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  		Figure 8. Ang-1 treatment reduces nuclear factor (NF)-{kappa}B activation in the lung of mice sensitized and challenged with ovalbumin (OVA). (A) Representative Western blot of inhibitor of nuclear factor {kappa}B-{alpha} (I{kappa}B-{alpha}) in lung homogenates from control animals, and animals with inflammatory airway disease in the presence or absence of Ang-1 administration. (B) Densitometric analysis of blots shown in (A). I{kappa}B-{alpha} protein expression was set at 100% for control mice after normalization for actin content. (C) Graph depicts the DNA binding of the p65 subunit of NF-{kappa}B in lung cell nuclei in control animals challenged with OVA (CTL), mice sensitized and challenged with OVA (OVA), and mice treated with Ang-1 (OVA+Ang-1) before each OVA challenge. Results are presented as means ± SEM, n = 8–10; *P < 0.05 compared with control, #P < 0.05 compared with OVA.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ang-1 is known to exert a protective role in the vasculature because it promotes vascular integrity, reduces endothelial transmigration of leukocytes, and reinforces barrier function (13, 15, 23, 33). We hypothesized that, during allergic inflammation of the airways, the levels of this protective growth factor would be reduced. Indeed, mice sensitized and challenged with OVA exhibited reduced mRNA and protein levels of Ang-1, raising the possibility that Ang-1 down-regulation contributes to the pathogenesis of asthma. Under the same conditions, the levels of the context-dependent antagonist of the Tie2 receptor Ang-2 remained unaltered. As the balance between Ang-1 and Ang-2 in the asthmatic lung changes in favor of the latter, the protective action of Tie2 is diminished, as reflected by the reduction in Tie2 phosphorylation. Although the mechanisms responsible for the reduced expression of Ang-1 in asthma were not identified in the present study, several mediators and factors that are increased in asthma could contribute to the inhibition of Ang-1 expression. These include TGF-β1, thrombin, cytokines, and nitric oxide (NO) (16, 25, 26, 34). Interestingly, the combination of tumor necrosis factor and IFN-{gamma} down-regulates the expression of Ang-1 in an NF-{kappa}B and NO-dependent manner (through up-regulation of inducible NO synthase [iNOS]) (25). This is very similar to the milieu in the asthmatic lung, in which NF-{kappa}B activation coexists with increased production of NO via iNOS up-regulation (3, 24, 35).

Having established that Ang-1 protein levels are reduced in the lungs of mice with asthma, we asked whether administering Ang-1 would prevent the changes seen in animals with allergen-induced airway inflammation. Indeed, administration of Ang-1 reduced the OVA-induced increase in the number of eosinophils present in the BAL and ameliorated tissue inflammation in lung sections. In agreement with our observations, McCarter and colleagues (20) showed that Ang-1 improved morphologic and biochemical indices of inflammation in a model of LPS-induced acute lung injury. In another study, adenoviral delivery of Ang-1 improved the histologic appearance of the lung and restricted leukocyte infiltration in mice given endotoxin intraperitoneally (36). Our results showing reduced eosinophil infiltration in OVA mice treated with Ang-1 are in line with a recent report showing that Ang-1, although chemotactic for eosinophils, inhibited VEGF-induced migration (17).

Inhibition of lung eosinophilia by Ang-1 could result either from reduced infiltration or mobilization of eosinophils from the bone marrow. Local treatment with Ang-1 significantly reduced the increase in the number of circulating eosinophils triggered by OVA sensitization and challenge and inhibited the levels of eotaxin, a chemokine that induces eosinophil transendothelial migration of eosinophils in vivo and in vitro (31, 37). Moreover, Ang-1 blocked the OVA-stimulated expression of vascular cell adhesion molecule 1 (VCAM-1), another molecule that aids in the recruitment of eosinophils into the asthmatic lung, suggesting that Ang-1 inhibits the presence of eosinophils in the lung by acting at multiple levels. In line with our data on the ability of Ang-1 to inhibit VCAM-1 expression, Kim and coworkers (22) reported that Ang-1 reduces the VEGF-stimulated increase in intercellular adhesion molecule (ICAM)-1, VCAM-1, and E-selectin steady-state mRNA levels. Interestingly, Ang-1 attenuates adhesion molecule expression in vivo in the lungs of animals challenged with LPS (20, 36).

To further study the ability of Ang-1 to restrict inflammation in the asthmatic lung, we measured the levels of Th2 cytokines with well-established roles in the pathogenesis of asthma. IL-5 is the major stimulus for the differentiation, recruitment, and survival of eosinophils, which plays a key role in the induction of allergen-induced eosinophilic airway inflammation (3, 24). In line with the ability of Ang-1 to inhibit eosinophil infiltration, this growth factor abolished the IL-5 increase seen in OVA mice. Another key mediator in asthma is IL-13, a cytokine that promotes B-cell differentiation, capable of inducing isotype switching leading to increased IgE production; IL-13 has also been shown to contribute to airway hyperresponsiveness (2, 24). Intranasal Ang-1 treatment inhibited the OVA-stimulated increase in IL-13 production. This finding is in line with the ability of Ang-1 to reduce IgE levels and to attenuate airway hyperresponsiveness. Unlike what was observed with IL-5 and IL-13, Ang-1 was ineffective in reducing IL-4, a cytokine that promotes Th2 cell differentiation, mucin gene expression, and goblet cell metaplasia (2, 3, 24). The lack of effect of Ang-1 on IL-4 levels may also explain the fact that mucus production was not reduced in Ang-1–treated mice, as suggested by the similar PAS scores of OVA and OVA+Ang-1 mice. It should be kept in mind that the cellular sources contributing to inflammatory cytokine production were not identified in the course of our experiments; infiltrating leukocytes might contribute to inflammatory cytokine production together with resident lung cells.

It has been previously reported that Ang-1 inhibits NF-{kappa}B–driven gene expression in a heterologous system (32). Because NF-{kappa}B regulates the expression of several cytokines, chemokines, and cell adhesion molecules involved in the pathogenesis of asthma, we hypothesized that blockade of this transcription factor might contribute to the protective mechanism of Ang-1. In our experiments, we observed that Ang-1 prevented the OVA-induced reduction in I{kappa}B-{alpha} levels in lung homogenates, indicating that in mice treated with Ang-1 more NF-{kappa}B would be retained in the cytosol, in its inactive form. To prove that in animals exposed to Ang-1 smaller amounts of NF-{kappa}B translocate to the nucleus, nuclear proteins were extracted from lung homogenates and the p65 binding to a NF-{kappa}B consensus sequence was measured. Indeed, Ang-1 treatment reduced the amount of active NF-{kappa}B in mice sensitized and challenged with OVA.

Airway hyperresponsiveness is a hallmark of asthma, but the exact mechanisms underlying it are still unclear. Similar to other studies (38, 39), our OVA mice presented increased Rn, G, and H compared with control mice after methacholine challenge. This hyperresponsiveness of BALB/c mice with allergically inflamed airways can be explained by thickening of the airway mucosa, allowing for excessive narrowing of the airway lumen with some airways proceeding to full closure even when the degree of airway smooth muscle shortening is normal (40, 41). Closure is likely exacerbated by disruption of surfactant function, possibly due to fibrin deposition, which would increase the propensity for the formation of liquid bridges across the lumen of small airways (41) and by the presence of a critical volume of intraluminal liquid due to plasma exudation (42). The greater proportional increase in G relative to H (Figure 3) suggests that this derangement in mechanical function exhibits regional heterogeneity (43). Ang-1 administration, apart from its protective effects limiting inflammation, normalized G and H. The reduction of inflammation would reduce airway mucosal thickness as well as the volume of extravasated plasma into the airway lumen. Furthermore, Ang-1 has direct antileakage action in the airway microvasculature (10, 44), which would prevent airway wall edema and intraluminal fluid accumulation, thus reversing the majority of derangements of mechanical lung function induced by allergic airway inflammation. Ang-1 treatment had no significant effect on the peak value of Rn in response to methacholine. However, the area under the curve of the Rn response to methacholine over time tended to normalize in Ang-1–treated OVA mice (P = 0.06 vs. OVA-treated mice, P = not significant vs. controls; see the online supplement), suggesting that Ang-1 treatment had some effect on airway smooth muscle contractility or on the clearance of methacholine from the lung, apart from the effect of diminished airway closure on Rn.

Clinical Relevance
Our finding of the protective effect of Ang-1 treatment would be clinically relevant if the animal model used reflected not only molecular mechanisms but also lung function derangements of human asthma. Accordingly, there is substantial evidence to support the importance of airway closure for the airway hyperresponsiveness in humans with asthma. Single-photon emission computed tomography with Technegas has revealed that patients with asthma have increased range of closing pressures and a patchy distribution of airway closure (45). Positron emission tomography has shown that bronchoconsticted patients with asthma have large ventilation defects in their lungs, with patchy distribution (46, 47) and clusters of underventilated areas adjacent to normally ventilated areas that can be attributed to clusters of small airway closures (48). Furthermore, the increased amount of air trapped in the asthmatic lung correlates with the reversibility of small airway obstruction (49). Thus, our animal model of allergic airway inflammation reflects significant aspects of deranged mechanical lung function of human asthma.

In a recent study, changes in the number of Ang-1–positive vessels in biopsies from patients with asthma were reported to increase, without, however, quantifying the expression of this growth factor (5). Moreover, Ang-1 was found to be increased in induced sputum of patients with asthma (50), whereas Ang-1 levels were inversely correlated with airway vascular permeability index (which lends credence to the protective role of Ang-1 in asthma). Clearly, additional experimentation is needed to comprehend the reasons for the divergent results between the present study and the results published so far with subjects with asthma. One might speculate that initially in the course of asthma development, Ang-1 levels are reduced, allowing for greater vessel plasticity, as has been described for other vascular beds (16). The concomitant rise in VEGF expression at the onset of asthma promotes new blood vessel formation (13) Later on, Ang-1 levels recover and might even be increased as part of a homeostatic response to limit vascular permeability (13).

In summary, we have demonstrated that local administration of recombinant Ang-1 protects against the histologic, biochemical, and functional changes observed in allergic asthma. Ang-1 inhibits NF-{kappa}B activation and reduces inflammatory cytokine production. In addition, Ang-1 inhibits eosinophil infiltration by reducing adhesion molecule expression and chemoattractant production, and by inhibiting eosinophil trafficking. More importantly, Ang-1 improves lung function in the context of asthmatic inflammation. This later beneficial effect of Ang-1 might be related to the barrier-promoting properties of this growth factor. Our results reinforce the notion that Ang-1 exerts a protective action in the setting of inflammation and suggest that exogenous Ang-1 administration could be of therapeutic benefit in asthma.


   FOOTNOTES
 
Supported by the Thorax Foundation (Athens, Greece) and by funds from the Greek Ministry of Education and the Greek Secretariat of Research and Technology.

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: 10.1164/rccm.200708-1141OC on March 20, 2008

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 August 1, 2007; accepted in final form March 19, 2008


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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