Abstract
We conducted a randomized, double-blind, parallel-group study to assess the effect of 6 weeks treatment with low-dose (100 μg twice a day) or high-dose (500 μg twice a day) inhaled fluticasone propionate (FP) on the vascular component of airway remodeling in 30 patients with mild to moderate asthma. We also studied the effect on the inflammatory cells and the basement membrane thickness, and we compared findings from bronchial biopsies taken in patients with asthma with those in eight control subjects. Bronchial responsiveness to methacholine and asthma symptom score were measured before and after treatments. Eight patients in the low-dose FP group and eight patients in high-dose FP group completed the study. At baseline, patients with asthma showed an increase in the number of vessels and in vascular area as compared with control subjects. In the subjects with asthma, number of vessels correlated with vascular area (p < 0.01) and with number of mast cells (p < 0.01). Bronchial responsiveness to methacholine, asthma symptom score, and inflammatory cells decreased significantly after both low- and high-dose FP (p < 0.05). However, the number of vessels, the vascular area, and the basement membrane thickness decreased only after high-dose FP (p < 0.05). In conclusion, this study shows that in patients with mild to moderate asthma, high dose of inhaled FP given over 6 weeks can significantly affect airway remodeling by reducing both submucosal vascularity and basement membrane thickness.
The remodeling of airway wall is a distinctive feature of asthma, characterized by hypertrophy and hyperplasia of airway smooth muscle (1), increase in mucous glands (2), thickening of the reticular basement membrane (3), and qualitative and quantitative changes of airway blood vessels. Early studies on the pathology of asthma showed edematous bronchial mucosa with dilated and congested blood vessels in patients with fatal disease (4, 5). More recent in vivo quantitative studies found an increase in total number of vessels and in vascular area in patients with asthma when compared with control subjects (6, 7).
Mediators involved in asthmatic inflammation, such as histamine (8) and bradykinin (9) can induce vasodilatation, as well as some cytokines present in the airways could determine formation of new vessels (10). The functional significance of vascular changes in airway wall in asthma is not yet well known. However, the increase in the number and size of vessels can contribute to thickening of the airway wall, which in turn may lead to critical narrowing of bronchial lumen, when bronchial smooth muscle contraction occurs (11).
Inhaled corticosteroids are the most effective antiasthma drugs because they act on airway cellular inflammation (12) and may reduce the thickness of the basement membrane (13). However, to date, studies on the effect of inhaled corticosteroids on microvascular airway remodeling in patients with asthma are scant. In a cross-sectional study, Orsida and coworkers (14) found that patients with asthma who did not receive inhaled corticosteroids did not differ from those who received inhaled beclomethasone dipropionate in terms of number of vessels in the airway wall. However, patients treated with high dose of inhaled steroids tended to have a reduced number of vessels, and in the overall group of treated patients the percentage of vascular area was inversely related to the dosage of the drug. Recently, in a controlled longitudinal study, Hoshino and coworkers (15) showed that a 6-month treatment with a daily dose of 800 μg beclomethasone dipropionate significantly reduced the number of vessels and the vascular area in patients with asthma. None of these studies provided any information on inflammatory cells and their relationship with vascular remodeling.
The present study was specifically addressed to assess in a double-blind manner, the effect of short-term treatment with high-dose (500 μg twice a day) and low-dose (100 μg twice a day) inhaled fluticasone propionate (FP) on the vascular component of airway remodeling in patients with asthma. In addition, we studied the effect of high- and low-dose FP on the inflammatory cells and the basement membrane thickness as well as on bronchial responsiveness and symptoms. For comparison purposes we also examined bronchial biopsies obtained from healthy volunteers. Lastly, we evaluated the relationship between inflammatory cells and number of vessels and percentage of vascular area.
We conducted a double-blind, randomized, parallel-group study. Patients received FP at a dose of 500 μg twice daily or 100 μg twice daily via a metered dose inhaler with a spacer device. Treatments were administrated for 6 weeks, and patients attended the laboratory on five different days.
On Day 1, participants completed a clinical questionnaire, received a daily diary card, and underwent spirometry (16). On Day 2, after 3 weeks, diary cards were revised, the degree of asthma severity scored, and patients performed spirometry. On the same occasion, patients underwent methacholine challenge (17) and the provocative concentration producing a 20% decrease in FEV1 (PD20 M, in μmol) was measured. Three days after methacholine challenge (Day 3), the patients underwent fiberoptic bronchoscopy (3). After 6 weeks of treatment, diary cards were revised, the degree of asthma severity was scored, and FEV1 and PD20 M values were measured (Day 4). Three days later (Day 5), patients underwent a second bronchoscopy. Healthy volunteers underwent the same bronchoscopy procedures on a single occasion.
Thirty nonsmoking patients with mild to moderate asthma were recruited from the outpatient clinic of the Department of Clinical Sciences of the Parma University. All had a well-documented history of asthma (18), and baseline FEV1 had to be 70% or more of predicted. All patients were free from asthma attacks in the previous 2 months and controlled their symptoms with inhaled salbutamol only. They did not receive corticosteroids during the 6 months before the study and were free from respiratory infections in the 4 weeks preceding the study. The degree of asthma severity was assessed according to an asthma severity score (3). Atopy was assessed by skin prick tests with a battery of 10 common inhalant allergens. Eight healthy volunteers were enrolled as the control group. The study was approved by the University of Parma ethical committee, and subjects gave written informed consent.
Mucosal biopsies were immediately transferred into ice-cold acetone containing the protease inhibitor iodoacetamide (20 mM) and phenylmethylsulfonylfluoride (2 mM) for fixation; then they were stored at −20°C for 24 hours and processed into the water-soluble resin, glycolmethacrylate (Polysciences, Northampton, UK), for embedding (19). Sections of 2 μm were cut and stained using monoclonal antibodies against Type IV collagen to outline the endothelial basement membrane (Novocastra Lab., Newcastle, UK), tryptase (DAKO, Glostrup, Denmark), and EG2 (Pharmacia and Upjohn Diagnostics AB, Uppsala, Sweden) to identify mast cells and eosinophils (19).
Light microscopy was performed with a Leica microscope (Leica DMLB, Wetzlar, Germany) at magnification ×1000. Briefly, the number of vessels, mast cells, and eosinophils were counted in all nonoverlapping high power fields in the lamina propria (defined as a zone 100 μm deep to the epithelial basement membrane) and expressed as number of vessels and cells per square millimeter of lamina propria. In each section, all available nonoverlapping high power fields with intact tissue covered by intact basement membrane were examined. Vascular area was expressed as percentage of the area of the assessed lamina propria. The mean size of vessels was estimated by dividing the total vascular area by the total number of vessels. Basement membrane thickness was measured as described previously (20). Final values represent the mean of at least two sections from two different biopsies in each subject. A single observer who had no knowledge of patient characteristics evaluated tissue sections using an image analysis system (Leica Q500C; Leica Cambridge Ltd., Cambridge, UK). An average of eight high power fields was examined for each subject included in the study. The mean coefficient of variation for repeated measurements was 9% for number of vessels, 11% for vascular area, 5% for mast cells, 7% for eosinophils, and 11% for basement membrane thickness.
Values are presented as mean ± SD. The Mann–Whitney and χ2 tests were used for comparison between patients with asthma and control subjects, when appropriate. Changes in functional data and morphologic data before and after treatment were calculated using the Wilcoxon matched pairs test. Relationships were estimated by the Spearman's rank correlation coefficient (rs). A p value less than or equal to 0.05 was taken as significant.
In patients with asthma (six females, age range 18–56 years), baseline FEV1 values ranged from 70 to 148% and the PD20 M values ranged from 0.056 to 14.26 μmol. According to the asthma severity score, patients with asthma had mild persistent to moderately severe asthma (score range 3–11, median value 6). In addition, the duration of disease ranged from 1 to 27 years. In healthy volunteers (four females, age range 22–29 years), baseline FEV1 values ranged from 93 to 117%.
Of the 30 selected patients with asthma, 6 withdrew from the study after Day 3 of the protocol; 4 of them withdrew because they did not satisfactorily complete the symptoms diary card at Day 4 and the remaining 2 patients withdrew because they had a respiratory tract infection during the treatment period. In addition, 6 out of 30 patients refused to undergo the second bronchoscopy. Eighteen out of 30 patients completed the study protocol, and adequate paired biopsy material for immunostaining was obtained in 16 patients, 8 in the group who received 500 μg FP twice a day and 8 in the group who received 100 μg FP twice a day. The two groups of patients who completed the study, were not different in terms of age, sex, atopy, duration of disease, asthma severity score, baseline FEV1, and PD20 M values (Table 1)
Subjects | Age (yr) | Sex | Atopy | Duration of Asthma (yr) | Baseline FEV1 (% predicted) | Asthma Severity Score | PD20 (μmol) |
|---|---|---|---|---|---|---|---|
| 100 μg fluticasone twice a day | |||||||
| 1 | 18 | M | Y | 15 | 117 | 6 | 3.02 |
| 2 | 42 | M | Y | 15 | 105 | 10 | 0.12 |
| 3 | 18 | M | Y | 4 | 126 | 6 | 2.39 |
| 4 | 25 | M | Y | 10 | 105 | 7 | 0.21 |
| 5 | 32 | M | Y | 25 | 83 | 6 | 2.34 |
| 6 | 32 | M | Y | 11 | 86 | 6 | 0.11 |
| 7 | 23 | F | Y | 2 | 100 | 6 | 0.77 |
| 8 | 31 | M | Y | 10 | 74 | 9 | 5.33 |
| Mean | 28 | 11 | 100 | 7 | 0.81 | ||
| SD | 8 | 7 | 18 | 2 | 1.7 | ||
| 500 μg fluticasone twice a day | |||||||
| 1 | 24 | M | Y | 23 | 86 | 11 | 0.056 |
| 2 | 24 | M | Y | 10 | 108 | 6 | 0.75 |
| 3 | 18 | M | Y | 14 | 97 | 5 | 4.45 |
| 4 | 26 | M | Y | 7 | 130 | 6 | 0.72 |
| 5 | 43 | F | Y | 3 | 114 | 5 | 14.26 |
| 6 | 21 | F | Y | 6 | 112 | 5 | 4.74 |
| 7 | 18 | M | Y | 17 | 148 | 5 | 2.74 |
| 8 | 31 | M | Y | 27 | 84 | 8 | 0.18 |
| Mean | 26 | 13 | 110 | 6 | 0.71 | ||
| SD | 8 | 9 | 22 | 2 | 1.7 |
At baseline, patients with asthma significantly differed from healthy volunteers in number of vessels (207 ± 49 versus 153 ± 23 vessels/mm2, p < 0.01) and in vascular area (5.1 ± 1.6 versus 3.1 ± 0.7%, p < 0.01). However, the distribution of vessels and vascular area in patients with asthma and in healthy volunteers largely overlapped. In addition, there was no significant difference in mean vessel size (220 ± 53 versus 203 ± 31 μm2) in patients with asthma compared with control subjects. Duration of asthma, asthma symptom score, baseline FEV1, and PD20 M values were not significantly correlated with the number of vessels or percent vascularity. Lastly, when compared with healthy volunteers, patients with asthma had significantly higher values in number of mast cells (66 ± 26 versus 35 ± 9 cells/mm2, p < 0.01), in number of activated eosinophils (105 ± 76 versus 31 ± 22 cells/mm2, p < 0.01), and in basement membrane thickness (9.5 ± 1.1 versus 8.1 ± 0.4 μm, p < 0.01).
In patients with asthma, the number of vessels significantly correlated with vascular area (rs = 0.58, p < 0.01) and with the number of mast cells (rs = 0.67, p < 0.01) (Figure 1)
Figure 1. Relationship between number of vessels and number of mast cells in patients with asthma. rs = Spearman rank correlation coefficient.
[More] [Minimize]Patients who received 100 μg FP twice a day had a significant increase in PD20 M (0.81 ± 1.7 versus 3.77 ± 1.6 μmol, p < 0.05) (Figure 2)
Figure 2. Individual PD20 M values measured in patients with asthma before and after 6 weeks of treatment with 100 μg fluticasone propionate (FP) twice a day and 500 μg FP twice a day. Open squares indicate mean values.
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Figure 3. Individual values of mast cells measured in patients with asthma before and after 6 weeks of treatment with 100 μg FP twice a day and 500 μg FP twice a day. Open squares indicate mean values.
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Figure 4. Individual values of activated eosinophils measured in patients with asthma before and after 6 weeks of treatment with 100 μg FP twice a day and 500 μg FP twice a day. Open squares indicate mean values.
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Figure 5. Individual values of basement membrane thickness measured in patients with asthma before and after 6 weeks of treatment with 100 μg FP twice a day and 500 FP μg twice a day. Open squares indicate mean values.
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Figure 6. Individual values of number of vessels measured before and after 6 weeks of treatment with 100 μg FP twice a day and 500 μg FP twice a day. Open squares indicate mean values.
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Figure 7. Individual values of vascular area measured in patients with asthma before and after 6 weeks of treatment with 100 μg FP twice a day and 500 μg FP twice a day. Open squares indicate mean values.
[More] [Minimize]Patients who received 500 μg FP twice a day had a significant increase in PD20 M (0.71 ± 1.7 versus 3.35 ± 1.7 μmol, p < 0.05) (Figure 2) and a significant decrease in asthma symptom score (6 ± 2 versus 0, p < 0.05) but not in FEV1 when compared with baseline values. Moreover, these patients had a significant decrease in the number of mast cells (79 ± 19 versus 31 ± 13 cells/mm2, p < 0.01) (Figure 3), in the number of activated eosinophils (105 ± 64 versus 34 ± 10 cells/mm2, p < 0.05) (Figure 4), in the basement membrane thickness (9.7 ± 1.3 versus 8.4 ± 0.7 μm, p < 0.05) (Figure 5), in the number of vessels (204 ± 42 versus 163 ± 42 vessels/mm2, p < 0.05) (Figure 6), in the vascular area (4.8 ± 1.5 versus 3.0 ± 0.9%, p < 0.05) (Figure 7), and in the mean vessel size (236 ± 45 versus 207 ± 14 μm2, p < 0.05). The number of inflammatory cells, the thickness of basement membrane, the number of vessels and the vascular area, measured after treatment with a high FP dose, were not different from those found in healthy volunteers. Figure 8
Figure 8. Microphotograph showing immunostaining for collagen IV to identify vessels in the lamina propria of: a patient with asthma (a); a healthy volunteer (b); a patient with asthma before (c) and after (d) treatment with 100 μg FP twice a day; a patient with asthma before (e) and after (f) treatment with 500 μg FP twice a day.
[More] [Minimize]This controlled study provides the first evidence that short-term treatment with high-dose FP significantly reduce the vascular component of airway remodeling in patients with mild to moderate asthma. This effect of high-dose FP was also observed on basement membrane thickness. Conversely, both high- and low-dose FP reduce the number of inflammatory cells in the bronchial mucosa, the degree of bronchial responsiveness to methacholine, and symptom score. In addition, the study shows that when compared with healthy control subjects, airway vascularity was significantly increased in patients with asthma and was correlated with the number of mast cells.
Blood vessels number and percentage of vascular area observed in patients with asthma in our study were similar to those reported by Salvato (7), but they were smaller (6, 14) or greater (21) than those reported by others. Differences in selection criteria of patients or in methods and in sampling sites can explain these different results. In addition, the vessel distribution could be uneven in the bronchial wall specimens because edema or fixation of the tissue can induce scattering or clustering of the vessels. However, our results are consistent with previous papers reporting an increased vascularity of the bronchial wall in patients with asthma when compared with control subjects (6, 7, 14, 15, 21). Taken together, these findings support the view that angiogenesis and microvascular remodeling of the airway wall can be one of the main aspects characterizing the chronic inflammatory changes in bronchial asthma (22). Whether the vascular component of airway remodeling is clinically relevant is not yet clear. In our study, bronchial vascularity was not related to clinical and functional grading of disease in agreement with the results of Li and Wilson (6), who did not find any relationship between clinical variables and vascularity in patients with mild asthma. However, other studies (7, 21), which included patients with a wider range of severity of disease, showed that patients with severe asthma had more vessels than patients with mild to moderate asthma, suggesting a possible relation between vascular remodeling of the airway wall and asthma severity.
The mechanism responsible for the formation of new vessels and the remodeling of the existing ones is unknown. Mediators and inflammatory cells can be involved in different ways. Among endothelial cell-specific growth factors, the vascular endothelial growth factor, whose expression is increased in the airways of subjects with asthma and is correlated with vascularity (23), has shown potent and clinically relevant effects on the microvasculature (24). Mast cells can also play a role in inducing the neovascularization process through the release of proangiogenic factors. Histamine, the major preformed mast cell mediator, stimulates new vessel growth by acting through both H1 and H2 receptors (25), and heparin, the main glycosaminoglycan constituent of mast cells granules, possess a proangiogenic activity (26). Our study, by providing evidence that mast cells are positively related to the number of vessels in patients with asthma, supports these observations. Moreover, mast cells produce and secrete vascular endothelial growth factor (27), which has been shown to stimulate mast cell migration at sites of angiogenesis (28). However, it must be taken in account that vascular endothelial growth factor may also cause increased vascular permeability, vessel leak, edema, and tissue swelling, thereby reducing the density of vessels (29).
Inhaled corticosteroids are able to downregulate several airway inflammatory cytokines (30), to reduce cell infiltration of bronchial wall (12), and may reverse the basement membrane thickening (13). They may also have some in vitro antiangiogenic properties because they directly inhibit the expression of the vascular endothelial growth factor gene (31). Moreover, inhaled corticosteroids can act on airway mucosal blood flow by causing vasoconstriction and reducing edema, therefore increasing vessel density. Indeed, in subjects with asthma and normal control subjects, inhaled FP can induce a vasoconstrictive action in the airway mucosa, with 880 μg being the lowest effective dose (32). In this study, we provided evidence that 6 weeks treatment with 500 μg FP twice a day is able to reduce the vascular component of airway remodeling in patients with mild to moderate asthma. After treatment with high-dose FP, the total number of vessels and the vascular area in patients with asthma were similar to those in healthy control subjects. However, from this observation we cannot infer that high-dose FP can completely reverse the airway wall vascularity to the values of control subjects. In fact, given the small number of subjects who completed the study, the lack of difference observed may in part be due to this small number and reduced power to detect a persistent difference.
Our results are consistent with the previous findings by Hoshino and coworkers (15), who showed that 800 μg beclomethasone dipropionate reduced airway wall vascularity in patients with asthma. Interestingly, the duration of our study protocol was shorter than that of Hoshino and coworkers (6 weeks versus 6 months). This suggests that a short-term treatment with a more potent inhaled steroid, given at a high dose, can be enough to reduce the airway vascular remodeling in asthma. Orsida and coworkers (14) showed an effect of inhaled steroids on airway wall vascularity only more than 500 μg of beclomethasone dipropionate. Moreover, another study by Orsida and coworkers (33) failed to demonstrate any effect of 3 months treatment with a dose of 100 μg FP twice a day plus a background dose of inhaled steroids (200–500 μg/day of beclometasone or 200–400 μg/day of budesonide) on vascularity in patients with asthma. We believe that our study, although it does not establish the lowest dose and duration of FP that may be required to treat airway remodeling, adds a new finding to this debate by showing that short-term treatment with 500 μg FP twice a day can reduce the airway vascular remodeling in patients with asthma.
In this study, we found an effect of high-dose FP not only on the vascular component of airway remodeling but also on another component, i.e., the basement membrane thickening. In patients receiving 500 μg FP twice a day, the basement membrane thickness was significantly reduced, whereas in patients treated with 100 μg FP twice a day, the basement membrane thickness was unchanged. Our data are consistent with previous studies that showed that high doses of inhaled steroids, such as 250 μg FP twice a day (13) or 750 μg FP twice a day (34) and 400 μg beclometasone twice a day (15) or 500 μg beclometasone twice a day (35), can be effective in reducing the basement membrane thickness of patients with asthma. On the other hand, lower doses of inhaled steroids, such as 200 μg budesonide twice a day, did not demonstrate any effect on the thickening of basement membrane, despite a significant change in airway mucosal inflammation (36). Similarly, in this study, we found that the low dose was able to reduce the number of inflammatory cells in the bronchial mucosa as well as the bronchial responsiveness to methacholine and asthma symptoms. This latter observation is in agreement with the findings of the meta-analysis of Holt and coworkers (37), who showed that most of the therapeutic benefit of inhaled fluticasone is achieved with a total daily dose of 250 μg.
The results of a recent report by Sont and coworkers (38) are of relevance for our study. The authors compared a treatment strategy aimed at reducing bronchial hyperresponsiveness with a standard treatment (aimed at reducing symptoms and improving lung function). The treatment aimed at reducing bronchial hyperresponsiveness was achieved with an increase in the dose of inhaled steroids (the median difference in dose of inhaled steroids between the two treatment strategies was 400 μg/day) and resulted in more effective asthma control. Both the standard treatment and the treatment aimed at reducing bronchial hyperresponsiveness decreased the number of airway inflammatory cells, but only the latter one had direct effects on remodeling, as it reduced the thickness of basement membrane. Our results are in line with those of Sont and coworkers by showing that both basement membrane thickness and the vascular component of airway remodeling were improved only after treatment with high doses of inhaled corticosteroids.
In summary, we demonstrated that in patients with mild to moderate asthma, short-term treatment with high doses of inhaled FP can significantly reduce not only inflammatory cells but also the vascular component of airway remodeling and basement membrane thickening, suggesting a potential reversibility of both inflammatory and structural changes in asthma.
The authors thank Ms. Iris Spanevello of the Department of Clinical Sciences of the University of Parma for performing bronchial biopsies processing and staining procedure. The authors are also indebted to Ms. Sabrina Muti from the Department of Clinical Sciences of the University of Parma for expert secretarial help.
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