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doi:10.1378/chest.06-2758
(Chest. 2007; 131:1035-1041)
© 2007 American College of Chest Physicians
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Roles of Angiopoietin-1 and Angiopoietin-2 on Airway Microvascular Permeability in Asthmatic Patients*

Hiroshi Kanazawa, MD, PhD; Saeko Nomura, MD, PhD and Kazuhisa Asai, MD, PhD

* From the Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan.

Correspondence to: Hiroshi Kanazawa, MD, PhD, Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, 1–4-3, Asahi-machi, Abenoku, Osaka, 545-8585, Japan; e-mail: kanazawa-h{at}med.osaka-cu.ac.jp

Abstract

Background: Vascular endothelial growth factor (VEGF) increases microvascular permeability. Recently, considerable attention has been devoted to the physiologic roles of angiopoietin-1 and angiopoietin-2 as regulatory factors of VEGF. This study was designed to examine the roles of angiopoietin-1 and angiopoietin-2 in controlling airway microvascular permeability in asthma.

Methods: Levels of these angiogenic factors and airway vascular permeability index were examined in 30 asthmatics and 12 control subjects. After 2-week run-in period, all asthmatics were randomly assigned to receive fluticasone propionate (400 µg/d) or montelukast (10 mg) for 12 weeks.

Results: VEGF, angiopoietin-1, and angiopoietin-2 levels in induced sputum were significantly higher in asthmatics than in control subjects. We found an inverse correlation between angiopoietin-1 level and vascular permeability index in asthmatics, while there was a positive correlation between angiopoietin-2 level and that index. VEGF and angiopoietin-1 levels were significantly decreased after fluticasone therapy, while VEGF and angiopoietin-2 levels were significantly decreased after montelukast therapy. Although VEGF levels after treatment were different between two groups, vascular permeability index in the montelukast group was the same level as that in the fluticasone group. Moreover, improvement in vascular permeability index after fluticasone therapy was inversely correlated with decrease in angiopoietin-1 level, while that after montelukast therapy was positively correlated with decrease in angiopoietin-2 level.

Conclusions: Angiopoietin-1 and angiopoietin-2 play complementary and coordinated roles in regulating microvascular permeability stimulated by VEGF in asthma. Combination of corticosteroids with leukotriene antagonists might effectively improve plasma leakage and provide a new strategy in treating bronchial asthma.

Key Words: airway remodeling • bronchial asthma • vascular endothelial growth factor

Asthma is a chronic airway inflammatory disease associated with airway wall remodeling. The structural alterations of airway walls in asthma include hypertrophy and hyperplasia of airway smooth muscle, mucous gland hyperplasia, thickening of the reticular basement membrane, and qualitative and quantitative changes of airway blood vessels.1 Indeed, it has been recognized that airway mucosa is edematous and contains dilated, congested blood vessels even in mild asthma.2 Previous studies34 reported that the airway wall of subjects with asthma is abnormally thick, a feature that has been confirmed by morphometric studies and CT. It was subsequently reported that both the total number of vessels and vascular area in biopsy specimens obtained from asthmatic patients were increased compared with those in normal control subjects.5 Angiogenesis and vascular enlargement, and consequent thickening of the airway wall mucosa, lead to narrowing of the airway lumen, which is associated with airway function in asthma.6 Thus, it has been emphasized that airway microcirculation has the potential to contribute to the pathophysiology of asthma.

Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation and increases microvascular permeability, so that plasma proteins can leak into the extravascular spaces, leading to mucosal edema and thereby narrow airway diameters, which could amplify the effect of airway smooth-muscle contraction.78 The possibility that VEGF plays an active role in angiogenesis and microvascular remodeling was recognized by Hoshino and coworkers,9 who found that VEGF expression is increased in the airways of subjects with asthma and correlates with mucosal vascularity. Moreover, we found that VEGF level in induced sputum from asthmatic patients is increased compared with that in normal control subjects, and that it is correlated with degree of airway obstruction.10 In fact, VEGF administration can initiate vessel formation but by itself promotes formation of only leaky, immature, and unstable vessels.11 Plasma leakage results from gaps between endothelial cells, as well as from increased vascular surface area in the newly formed and remodeled blood vessels. In addition to the elevated baseline leakage, the remodeled vessels in the airway mucosa are abnormally sensitive to various stimuli. Thus, the molecular mechanism of vascular leakage in asthma is of considerable interest.

There are currently two known members of Tie receptors, Tie-1 and Tie-2.12 Since Tie-2 messenger RNA and protein are most abundant in the lung, it appears that the lung is uniquely dependent on Tie-2 signaling.13 The two of the ligands for Tie-2 receptor are angiopoietin-1 and angiopoietin-2,14 both of which bind to Tie-2 receptor with equal affinity but result in distinct effects. Angiopoietin-1 is known to stabilize nascent vessels and make them leak resistant.15 Indeed, angiopoietin-1 has been shown to protect the adult vasculature against plasma leakage induced by VEGF. In contrast, angiopoietin-2 is an antagonist of angiopoietin-1 that competes for Tie-2 receptor, and subsequently reduces vascular integrity. Therefore, we hypothesized that angiopoietins and VEGF apparently act in a complementary and coordinated manner in the process of airway microvascular permeability. In this study, we examined the roles of angiopoietin-1 and angiopoietin-2 in controlling airway microvascular permeability in asthma. We also clarified the differences in microvascular effects of angiopoietin-1 and angiopoietin-2 by examining the changes in levels of these angiogenic factors following standard asthma therapy.

Methods and Materials

Study Subjects
Thirty patients with asthma and 12 age-matched control subjects were included in the study. All control subjects were healthy, nonsmoking volunteers who had no history of lung disease. All patients with asthma were recruited from respiratory outpatient clinics at our institution; they were nonsmokers and satisfied American Thoracic Society criteria for asthma.16 Methacholine inhalation challenge testing was performed for all study subjects as we previously described.17 All subjects with asthma in this study demonstrated bronchial hyperreactivity to methacholine. Exhaled nitric oxide (NO) was measured for all subjects with a chemiluminescence analyzer (CLM-500; Shimazu; Kyoto, Japan) in accordance with American Thoracic Society standards.18 The mean value of three expiratory NO concentrations was calculated for each subject and expressed as parts per billion. At study entry, regular medication in asthmatic patients consisted of short-acting ß2-agonists on demand. No patients were receiving oral or inhaled corticosteroids. All patients with asthma were clinically stable, and none had a history of respiratory infection for at least the 4-week period preceding the study. All subjects gave their written informed consent for participation in the study, which was approved by the ethics committee of Osaka City University.

Sputum Induction and Processing
Sputum induction was performed as we previously described.19 The volume of sputum samples was measured, and the sample was divided into two portions. One portion was diluted with phosphate-buffered saline solution containing dithiothreitol (DTT) [a final concentration of 1 mmol/L] (WAKO Pure Chemical Industries; Osaka, Japan) was then centrifuged at 400g for 10 min, and the cell pellet was resuspended. The slides were prepared using a cytospin (Cytospin 3: Shandon; Tokyo, Japan) and stained with May-Grunwald-Giemsa stain for differential cell counts. The other portion of sputum samples, for assay of angiopoietin-1 and angiopoietin-2, was diluted with phosphate-buffered saline solution without DTT because we had preliminarily detected a detrimental effect of DTT on measurement of angiopoietin-1 and angiopoietin-2 levels in induced sputum. The supernatant was stored at – 70°C for subsequent assay of eosinophil cationic protein (ECP), VEGF, angiopoietin-1, angiopoietin-2, and albumin. VEGF, angiopoietin-1, and angiopoietin-2 concentrations were measured by quantitative sandwich enzyme immunoassays (Quantikine; R&D Systems; Minneapolis, MN). Samples were analyzed in duplicate. ECP concentration was measured using a radioimmunoassay kit (Pharmacia Diagnostics; Uppsala, Sweden). The limits of detection for VEGF, angiopoietin-1, and, angiopoietin-2 were 9 pg/mL, 62.5 pg/mL, and 8.29 pg/mL, respectively. Albumin concentration was measured by laser nephelometry, and then we calculated the airway vascular permeability index (the ratio of albumin concentrations in induced sputum and serum).20 All subjects produced an adequate specimen of sputum; a sample was considered adequate if the patient was able to expectorate at least 2 mL of sputum and if on differential cell counting the slides contained < 10% squamous cells.

Study Design
A randomized study protocol was used. At the end of 2-week run-in period, all asthmatic patients were randomly assigned to receive fluticasone propionate (200 µg bid) or montelukast capsule (10 mg at night) for 12 weeks. Pulmonary function testing, methacholine provocation testing, determination of exhaled NO level, and sputum induction were performed on the last day of the run-in and treatment periods.

Statistical Analysis
All values are presented as mean ± SD. Multiple comparisons were performed by one-way analysis of variance, followed by the Bonferroni test. The significance of correlations was evaluated by determining Spearman rank correlation coefficients. A p value < 0.05 was considered significant.

Results

The clinical characteristics of the 30 asthmatic patients and 12 normal control subjects are shown in Table 1 . VEGF level in induced sputum was significantly higher in asthmatic patients (4,310 ± 860 pg/mL) than in control subjects (910 ± 590 pg/mL, p < 0.001). Similarly, angiopoietin-1 and angiopoietin-2 levels in induced sputum were also significantly higher in asthmatic patients (angiopoietin-1, 8.5 ± 3.1 ng/mL; angiopoietin-2, 660 ± 240 pg/mL) than in control subjects (angiopoietin-1, 4.9 ± 2.8 ng/mL, p < 0.001; angiopoietin-2, 250 ± 40 pg/mL, p < 0.001). VEGF level was significantly correlated with airway vascular permeability index in asthmatic patients (r = 0.81, p < 0.001). Moreover, we found an inverse correlation between angiopoietin-1 level and airway vascular permeability index (r = – 0.38, p = 0.003) [Fig 1 , left, A]. In contrast, there was a positive correlation between angiopoietin-2 level and airway vascular permeability index (r = 0.52, p < 0.001) [Fig 1, right, B].


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  		Table 1. Clinical Characteristics of Study Subjects*

 

Figure 1
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  		Figure 1. Correlation between angiopoietin-1 (left, A), and angiopoietin-2 (right, B) levels in induced sputum and airway vascular permeability index before treatment in asthmatic patients.

 
After 12 weeks of inhaled fluticasone therapy, airway obstruction and hyperreactivity to methacholine were markedly improved. Moreover, eosinophilic airway inflammation and exhaled NO levels had decreased to the same levels as in normal control subjects. VEGF and angiopoietin-1 levels were significantly decreased in the fluticasone group (VEGF: before, 4,310 ± 770 pg/mL; after, 2,300 ± 720 pg/mL, p < 0.001; angiopoietin-1: before, 8.7 ± 3.9 ng/mL; after, 5.7 ± 2.7 ng/mL, p = 0.02) [Fig 2 , top left, A, and top right, B]. In contrast, the angiopoietin-2 level did not differ from before to after fluticasone therapy (before, 650 ± 260 pg/mL; after, 630 ± 220 pg/mL) [Fig 2, bottom left, C]. In the montelukast group, airway obstruction did not change, but airway hyperreactivity to methacholine was significantly improved. In addition, eosinophilic airway inflammation and exhaled NO level had also decreased but were still higher compared with normal control levels. VEGF and angiopoietin-2 levels in the montelukast group were significantly decreased (VEGF: before, 4,320 ± 960 pg/mL; after, 3,720 ± 790 pg/mL, p = 0.04; angiopoietin-2: before, 670 ± 230 pg/mL; after, 480 ± 170 pg/mL, p = 0.01), but the angiopoietin-1 level was not (before, 8.3 ± 3.9 ng/mL; after, 9.1 ± 4.0 ng/mL). In addition, the VEGF level in the montelukast group was significantly higher than that in the fluticasone group (p < 0.001). Nevertheless, airway vascular permeability index in the montelukast group was significantly decreased, to the same level as that in the fluticasone group (montelukast group: before, 0.035 ± 0.009; after, 0.024 ± 0.005; fluticasone group: before, 0.034 ± 0.010; after, 0.028 ± 0.007) [Fig 2, bottom right, D]. Moreover, decrease in angiopoietin-1 level after fluticasone therapy was inversely correlated with improvement in airway vascular permeability index (r = – 0.53, p = 0.04) [Fig 3 , left, A]. In contrast, decrease in angiopoietin-2 level after montelukast therapy was positively correlated with improvement in airway vascular permeability index (r = 0.59, p = 0.03) [Fig 3, right, B].


Figure 2
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  		Figure 2. Comparison of VEGF (top left, A), angiopoietin-1 (top right, B), angiopoietin-2 (bottom left, C), and airway vascular permeability index (bottom right, D) before and after treatment in normal control subjects and asthmatic patients. *p < 0.05 and **p < 0.01 compared with pretreatment. {dagger}p < 0.05 and {ddagger}p < 0.01 compared with postfluticasone group.

 

Figure 3
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  		Figure 3. Correlation between improvement of airway vascular permeability index and decrease in angiopoietin-1 level after fluticasone therapy (left, A) and angiopoietin-2 level (right, B) after montelukast therapy in asthmatic patients.

 
Discussion

In the present study, we found that levels of three angiogenic factors—VEGF, angiopoietin-1, and angiopoietin-2—in induced sputum from asthmatic patients were higher than those from normal control subjects. We also found an inverse correlation between angiopoietin-1 level and airway vascular permeability index in asthmatic airways. In contrast, a positive correlation between angiopoietin-2 and airway vascular permeability index was observed. Angiopoietin-1 promotes recruitment of the pericytes and smooth-muscle cells, thereby playing a role in establishing and maintaining the vascular integrity and quiescence.21 The transgenic mice overexpressing angiopoietin-1 displayed increased vascularization and decreased adult vasculature leakage. Thus, angiopoietin-1 appears to stabilize nascent vessels and make them leak resistant.22 On the basis of these findings, the inhibitory mechanism of vascular leak by angiopoietin-1 is clear. A second angiopoietin, angiopoietin-2, antagonizes the effects of angiopoietin-1 on the Tie-2 receptor, and in some contexts acts as a natural inhibitor of angiopoietin-1.23 Whereas angiopoietin-1 is widely expressed in normal adult tissues, angiopoietin-2 is expressed mainly at sites of vascular remodeling such as chronic inflammation.24 As an antagonist of angiopoietin-1, angiopoietin-2 loosens the interactions between endothelial cells, perivascular support cells, and extracellular matrix, leading to the increase in airway microvascular permeability. Thus, the roles of angiopoietin-2 are very hot issues and, somehow, still remain to be controversial. For example, Bhandari et al25 reported that angiopoietin-2 in plasma and alveolar edema fluid is increased in adults with acute lung injury, and that angiopoietin-2 is thus a mediator of epithelial necrosis with an important role in hyperoxic lung injury and pulmonary edema. In contrast, Daly et al26 reported that angiopoietin-2 might act as an autocrine Tie-2 agonist and protective factor. Moreover, the ratio of angiopoietin-1/angiopoietin-2 may be more important than the absolute angiopoietin-2 levels for determining the effect of Tie-2 signaling on endothelial cell permeability. However, since we attempted to clarify the opposite effects of angiopoietin-1 and angiopoietin-2 on airway microvascular permeability, we focused on the separate roles of both substances in the current study.

Although the importance of VEGF in the pathophysiology of asthma has been established, angiopoietins have remained largely unexplored. Recently, Feltis et al27 determined that VEGF and angiopoietin-1 levels were elevated in the airway of subjects with asthma. We have already found that VEGF and angiopoietin-2 levels in induced sputum were significantly higher in asthmatics than in normal control subjects.28 Moreover, angiopoietin-2 levels were significantly correlated with VEGF levels, suggesting that interaction between VEGF and angiopoietin-2 in asthmatic airways may exist. A expression-profiling study29 showed that the main sources of angiopoietin-1 and angiopoietin-2 are pericytes and endothelial cells, respectively. Angiopoietin-2 levels can be transcriptionally and posttranscriptionally regulated by hypoxia or exposure to growth factors, such as VEGF.30 Coexpression of VEGF, angiopoietin-1, and angiopoietin-2 is critical to vascularization and vessel stability, and appears to play complementary and coordinated roles in the process of airway microvascular permeability.31 In the presence of high levels of VEGF, angiopoietin-2 promotes rapid increase in capillary diameter, remodeling of the basal lamina, proliferation and migration of endothelial cells, and stimulates sprouting of new blood vessels, leading to airway hyperpermeability. Thus, the coexistence of VEGF and angiopoietin-2 stimulates vascular leakage in asthmatic airways. A previous study32 have also presented evidence that VEGF and angiopoietin-2 play an ongoing role in regulation of vascular permeability. Our findings also suggested that local regulation of VEGF and angiopoietin-2 could be involved in the control of vascular permeability in asthmatic airways. Given these findings, high levels of VEGF and angiopoietin-2 in asthmatic patients indicate that blood vessels in asthmatic airways are in a hypervascularized, destabilized state, and that this contributes to upregulation of airway vascular hyperpermeability. In contrast, angiopoietin-1 appears to counterregulate airway vascular permeability stimulated by VEGF and angiopoietin-2.

Current guidelines for asthma recommend use of inhaled corticosteroids as a first-line control therapy. Inhaled corticosteroids are able to downregulate several airway inflammatory cytokines, and to reduce cellular infiltration of bronchial walls.33 Moreover, corticosteroids have also been reported to reduce airway vascularity and to inhibit vascular permeability.3435 After 12 weeks of fluticasone therapy, VEGF and angiopoietin-1 levels in induced sputum were significantly decreased, whereas angiopoietin-2 levels were not. Indeed, a previous study36 showed that transcription of VEGF messenger RNA and VEGF protein were downregulated in the presence of corticosteroids. However, there are no reports on the effect of any corticosteroid on angiopoietin-1 proteins or messenger RNA expression in asthmatic airways. For the first time, in the present study we found that inhaled fluticasone therapy reduced angiopoietin-1 level in the asthmatic airways. Although inhaled fluticasone therapy markedly improved airway obstruction, hyperreactivity, eosinophilic inflammation, and NO production to the same levels as in normal control subjects, airway vascular permeability index was still high even after inhaled fluticasone therapy. Therefore, it is possible that reduction of angiopoietin-1 level after inhaled fluticasone therapy is responsible for sustained high levels of airway microvascular permeability. The cysteinyl leukotrienes induce bronchoconstriction, mucus hypersecretion, mucosal edema, and enhance airway hyperreactivity. Therefore, it is not surprising that leukotriene receptor antagonists improve lung function, attenuate bronchial hyperresponsiveness, and reduce the number of exacerbations in patients with mild-to-moderate asthma. Moreover, addition of leukotriene receptor antagonists to inhaled corticosteroids results in better control of asthma and can decrease the requirement for inhaled corticosteroids.3738 We previously found that pranlukast, a selective leukotriene receptor antagonist, decreases airway VEGF level in asthmatic patients.39 Our findings indicate that leukotriene antagonists may reduce airway microvascular permeability via reduction in airway VEGF level. In this study, we found that VEGF and angiopoietin-2 levels were decreased following montelukast therapy, but the angiopoietin-1 level was not. Although montelukast therapy was inferior to inhaled fluticasone therapy in reduction of VEGF level, montelukast equaled inhaled fluticasone in capacity to reduce airway microvascular permeability. These findings suggest that VEGF is not solely essential for controlling of airway microvascular permeability, and that angiopoietin-1 and angiopoietin-2 may act as cofactors in regulating the effects of VEGF.

In summary, we determined the potential roles of three angiogenic factors—VEGF, angiopoietin-1, and angiopoietin-2—on airway microvascular permeability. Measurement of not only VEGF but also of angiopoietin-1 and angiopoietin-2 levels in induced sputum is useful for evaluating the pathophysiology of asthma, in particular airway microvascular permeability. Inhaled corticosteroid therapy decreased VEGF and angiopoietin-1 levels, while leukotriene receptor antagonists decreased VEGF and angiopoietin-2 levels. These results suggest that combination of inhaled corticosteroid with leukotriene receptor antagonists can effectively improve vascular remodeling and plasma leakage in asthmatic airways, and might provide a new strategy in treating bronchial asthma. Our study raises many questions and makes it clear that more work is needed to understand the complexity of control of microvascular permeability under pathologic conditions such as asthma.

Acknowledgements

We thank Miss Yukari Matsuyama for her help in the preparation and editing of the manuscript.

Footnotes

Abbreviations: DTT = dithiothreitol; ECP = eosinophil cationic protein; NO = nitric oxide; VEGF = vascular endothelial growth factor

This work was supported by Grant-in-Aid for Scientific Research (No. 17590800) from the Japan Society for the Promotion of Science.

The authors have no conflicts of interest to disclose.

Received for publication November 13, 2006. Accepted for publication December 15, 2006.

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