Abstract
Hypervascularity in the bronchial wall is part of airway remodeling, but has remained an ill-defined process in asthma pathogenesis. Previous morphologic assessment has been limited to biopsy specimens, and therefore a high-magnification bronchovideoscope (side-viewing type) was developed for less invasive examination of subepithelial vessels. We evaluated vascularity in the lower trachea, using this novel scope in 12 normal control subjects, 13 patients with chronic obstructive pulmonary disease, and 24 subjects with stable asthma; 8 were steroid naive with newly diagnosed asthma (Group A) and 16 had been treated with inhaled corticosteroids for more than 5 years (Group B). The redness of bronchial mucosa in patients with asthma observed by conventional fiberoptic bronchoscopy proved to be due to a fine vascular network. Morphometric measurements of subepithelial vessels showed that both vessel area density and vessel length density were significantly (p < 0.0001) increased in subjects with asthma as compared with control subjects and patients with chronic obstructive pulmonary disease. The degree of increase in vessels did not differ between Group A and Group B. The increase in subepithelial vessels of the airway is present even in newly diagnosed asthma. This novel bronchovideoscope is useful for assessment of vessel network in the surface of the airway lumen in vivo.
Asthma is a chronic airway inflammatory disease associated with reversible airflow limitation and bronchial hyperresponsiveness. Pathologically, the bronchial wall shows epithelial shedding, goblet cell metaplasia, subepithelial collagen deposition, angiogenesis, eosinophil dominant inflammatory cell infiltration, smooth muscle hyperplasia and hypertrophy, and mucus gland hyperplasia, which together cause persistent thickening of the airway wall (1–3). However, hypervascularity within the bronchial wall remains an ill-defined process in the pathogenesis of asthma and airway chronic inflammation, such as chronic obstructive pulmonary disease (COPD) (3, 4). It has been proposed that a small increase in airway wall thickness, resulting from mucosal edema and vascular engorgement, might lead to excessive narrowing of the airway lumen in asthma (5). The number of vessels in the bronchial wall and airflow obstruction were reported to be reduced in patients taking high doses of inhaled corticosteroid or inhaled long-acting β2-agonist (salmeterol), as assessed by bronchial biopsy (6–8).
Previous assessments of vascularity in the airway have employed histologic methods (6–11), which are invasive and difficult to perform repeatedly. All commercial fiberoptic bronchoscopes are of a front-viewing type and have no magnification. The characteristic bronchoscopic findings in patients with asthma are of a red edematous mucosa, but there has been no previous detailed examination of the subepithelial vessels. We have developed a high-magnification bronchovideoscope (side-viewing type) in cooperation with Olympus (Tokyo, Japan) (12). This new bronchovideoscope permits a front view of the bronchial wall from the inner surface (Figure 1)
Figure 1. High-magnification bronchovideoscope (side-viewing type) in the lower trachea and details of its distal end. This novel scope possesses two systems: video observation for high magnification (side viewing) and fiber observation of forward viewing for orientation. The side-viewing images are reconstructed by an EVIS CV-240 video processor (Olympus Optical), which stores their digital data on a magneto-optical (MO) disk. CCD = charge-coupled device.
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Forty-nine subjects were recruited from Sapporo Medical University Hospital: 24 with asthma, 13 with COPD, and 12 normal control subjects. The basic characteristics of this study population are shown in Table 1
Subjects with Asthma | |||||
|---|---|---|---|---|---|
| Control Subjects
(n = 12) | Subjects with Chronic Obstructive
Pulmonary Disease
(n = 13) | Newly Diagnosed
(n = 8) | More Than
5 Years
(n = 16) | ||
| Age, yr | |||||
| Median | 59 | 65 | 55 | 59 | |
| Range | 28–76 | 58–72 | 26–71 | 25–73 | |
| Male sex, n | 9 | 11 | 6 | 10 | |
| Smoker, n | 0 | 13 | 0 | 0 | |
| Pack-years | 51 ± 25 | ||||
| VC, % predicted | 93.8 ± 8.5 | 97.2 ± 7.3 | 101 ± 6.3 | 98 ± 5.3 | |
| FEV1/FVC, % | 84.0 ± 2.4 | 50.2 ± 2.6 | 69.0 ± 4.3 | 67.0 ± 6.2 | |
| FEV1, % predicted | 101 ± 9 | 56 ± 16 | 71 ± 16 | 74 ± 18 | |
| V̇50, % predicted | 87.0 ± 13.0 | 24.6 ± 10.1 | 51.2 ± 10.7 | 50.3 ± 11.2 | |
| V̇25, % predicted | 62.0 ± 7.2 | 20.2 ± 3.4 | 40.0 ± 9.1 | 38.9 ± 10.2 | |
| Duration of asthma, yr | — | — | 0.7 ± 0.3 | 9.8 ± 3.7 | |
| Treatment | |||||
| Inhaled corticosteroids | — | — | — | 16 | |
| Inhaled β2-agonists | — | 2 | — | 10 | |
| Inhaled anticholinergics | — | 6 | — | — | |
COPD was diagnosed on the basis of the Global Initiative for Chronic Obstructive Lung Diseases in 2001, National Heart, Lung, and Blood Institute/World Health Organization workshop report (17): FEV1/forced vital capacity × 100 < 70% and with a lack of 15% or more response in FEV1 after inhalation of a β2-agonist. All patients with COPD had undergone computed tomography scans and there were many low-attenuation areas; therefore they were of the emphysema predominant type, and had little sputum. The severity of COPD according to guidelines (17) was as follows: 9 were moderate (FEV1% predicted was between 40 and 60%) and 4 were mild (FEV1% predicted was more than 60%), and no hypoxia was associated (PaO2 was not under 60 torr even after a 6-minute walk). Seven patients were current smokers, and six were ex-smokers (average secession term was 4 years). No patients with COPD had received long-acting β2-agonist.
All subjects gave written informed consent and the study protocol was approved by the ethics committee of Sapporo Medical University School of Medicine.
The high-magnification bronchovideoscope (side-viewing type), XBF 200HM3, was developed on the basis of the BF type 200, a commercial front-viewing flexible bronchovideoscope (Olympus Optical). The new bronchovideoscope combines a video observation system for high magnification (side viewing) with a fiber observation system for orientation of the bronchoscope end (Figure 1). The signals from the XBF 200HM3 were reconstructed as visual images by an EVIS CV-240 video processor (Olympus Optical) and projected on a 14-inch video monitor. As this bronchovideoscope is flexible and we can easily bend the end portion, we are able to attach the side surface to the tracheal wall when taking pictures, and consequently the actual focal distance (from side lens to tracheal surface) is about 1 mm and magnification is about 105-fold. One bronchoscopist performed the procedure and took all images without knowledge of the medical condition of the subjects.
Bronchovideoscopy was performed 12 hours after the cessation of smoking and treatment with anticholinergics and short-acting β2-agonists. Only patients in Group B received inhaled corticosteroid 3 hours before this evaluation. During the procedure ambient air temperature was kept at 25°C by room air conditioner. Subjects underwent bronchovideoscopy with local anesthesia (2% lidocaine solution). Two milliliters of this local anesthesia was nebulized 15 min before the examination, and for patients with asthma, bronchodilator (2 mg of salbutamol) was added to this solution. We gave inhaled bronchodilators or oxygen during or after the procedure if required and observed the subjects until they had fully recovered from any respiratory discomfort.
For the evaluation of subepithelial blood vessels of the bronchial mucosa we measured vessel area density and vessel length density. Vessel area density was represented as the number of vessel-overlaid pixels relative to the total number of picture area pixels, expressed as a percentage. The vessel density was calculated with Adobe Photoshop software on a Macintosh computer. Vessel length density was represented as the length (in millimeters) of blood vessels in a given area (1 mm2) of tissue and reflected the total length of vessels independent of vessel size. Each image was graded by three pulmonologists without knowledge of the clinical data or the results of pulmonary function tests, and we used the mean value. The mean coefficient of variation for repeated measurements was 7% for the vessel area density and 8% for the vessel length density.
Vessel area density and vessel length density are expressed as means ± standard deviation. Differences in the mean of vascular findings were assessed by analysis of variance and Scheffé test. p values less than 0.05 were considered statistically significant.
We safely performed this new bronchovideoscopy in all patients with asthma and none suffered an asthma exacerbation either during or after the examination. No patients needed O2 inhalation during the procedure. The redness of the bronchial mucosa in patients with asthma, observed by conventional fiberoptic bronchoscopy, proved to be due to increased fine subepithelial vascular networks, as revealed with this novel bronchoscope. Airway vascularity was dense in intercartilage areas, but sparse in cartilage or membranous areas of the trachea. Vascularity was similar between the trachea and main bronchi. Therefore, we assessed in each subject three images from the intercartilage area of the lower trachea: each image was taken in the space between the first and second cartilaginous rings from the carina or that between the second and third cartilaginous rings. The blood microvessels on bronchial mucosa were increased in patients with asthma (both newly diagnosed and steroid treated) as compared with subjects with COPD and control subjects (Figure 2)
Figure 2. Side-viewing and high-magnification bronchovideoscopic images of the lower trachea of a control subject (A); a subject with COPD (B); a steroid-naive patient with newly diagnosed, stable asthma (C); and a patient who has had stable asthma for more than 5 years, treated with inhaled corticosteroid (D). Fine vessel networks are prominently increased in the patients with asthma as compared with the control subject and the patient with COPD.
[More] [Minimize]Vessel area density was 15.1 ± 5.1% (control subjects), 15.4 ± 6.1% (COPD), 24.1 ± 5.2% (asthma, Group A), and 21.9 ± 6.9% (asthma, Group B). Vessel length density was 5.2 ± 1.1 mm/mm2 (control subjects), 4.5 ± 1.2 mm/mm2 (COPD), 8.9 ± 1.3 mm/mm2 (asthma, Group A), 9.1 ± 1.5 mm/mm2 (asthma, Group B). As shown in Figures 3 and 4
Figure 3. Vessel area density in the lower tracheal mucosa. Density is significantly (p < 0.0001) different between subjects with asthma and control subjects. The degree of vascularity in subjects treated with inhaled corticosteroid (ICS) for more than 5 years had a trend to decrease compared with steroid-naive patients with newly diagnosed asthma, but the difference was not significant. There is no difference between patients with COPD and control subjects.
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Figure 4. Vessel length density in the lower tracheal mucosa. Density is significantly (p < 0.0001) different between subjects with asthma and control subjects. The degree of vascularity in subjects treated with inhaled corticosteroid (ICS) for more than 5 years had a trend to decrease compared with steroid-naive patients with newly diagnosed asthma, but the difference was not significant. There is no difference between patients with COPD and control subjects.
[More] [Minimize]This study demonstrates that steroid-naive patients with newly diagnosed asthma have increased vessel networks in the airway mucosa as compared with control subjects and subjects with COPD, and the degree of increase in newly diagnosed asthma is similar to that of subjects with asthma who had received long-term inhaled corticosteroid treatment. We were surprised that increased vessels in the bronchial mucosa seem extensive even in the early phases of chronic adult asthma. There has been no previous study of when the airway hypervessel network is completed, and we are therefore the first to address this issue. However, five of the eight subjects with newly diagnosed asthma were of moderate severity in the present study, and therefore there remains the possibility of a longstanding subclinical course or unrecognized disease in these five cases. Pathologically, the number and percentage of vessels in airways taken from subjects with mild asthma have been reported to be higher than in control subjects (9, 10). Because mast cell–derived mediators such as histamine and leukotrienes may cause not only airway smooth muscle contraction but also airway edema due to plasma leakage from mucosal capillary vessels, it is suggested that hypervascularity of the bronchial mucosa makes a critical contribution to acute asthma exacerbations even in the onset phase of asthma.
This new method can visualize fine vessel structure of the lumenal surface of airways, which was undetectable by previous technology. This is the first direct observation of submucosal vessels on the trachea in vivo in patients with asthma, and employs a simple repeatable technology. Pathologically, more than 50% of the subepithelial vessels had a diameter of less than 20 μm in patients with asthma, which we calculated on the basis of data concerning the cross-sectional area of vessels in a previous report (10). This novel bronchovideoscope cannot detect these smaller vessels, because its limit of detection is 20 μm. A second limitation is that this technique cannot distinguish new vessels from vascular engorgement or vasodilation. Vessels visualized during bronchoscopy were assumed to represent both the actual density of vessels resulting from neovascularization (sprouting) and vessel enlargement due to increased bronchial blood flow. We also recognized that vessel enlargement implies endothelial cell proliferation resulting in increased vessel diameter. Another difference from previous studies is the site examined. We evaluated the tracheal wall, whereas previous reports used bifurcation specimens from lobar or segmental bronchi (6–11, 18). As the degree of submucosal vascularity was similar between the trachea and main bronchi in our preliminary study, it suggests that vascular remodeling occurred not only in the large airway but also in the trachea. This new assessment of vessel network in the trachea could replace more invasive bronchial biopsy and seems preferable for patients with increased airway hyperresponsiveness.
There is a double capillary network in the wall of the bronchial tree; one part is under the epithelium, another is in a deeper peribronchial part (outside the airway smooth muscle). These two systems are connected by vessels that pass through and distribute around the smooth muscle. We performed a preliminary bronchoscopic–pathological correlation, using human bronchial specimens from patients undergoing surgery for lung cancer: vessels 40 μm in diameter existing 250 μm beneath the epithelial surface could be visualized with this novel bronchovideoscope (data not shown). We then speculated that the shallow capillary network 200–300 μm under the epithelium could be observed.
Inhaled corticosteroids are the most effective antiinflammatory treatment for asthma. Three reports suggest that inhaled corticosteroids downregulate the increased vascularity in human asthmatic airways as observed in pathological specimens (6, 8, 18). Tumor necrosis factor-α, tissue growth factor-β, basic fibroblast growth factor, and vascular endothelial growth factor can potentially induce angiogenesis, and these factors may be released in the airway wall as part of airway inflammation or the remodeling response (19–23). Corticosteroids inhibit vascular endothelial growth factor, according to one in vitro study (24). Other angiogenic factors, such as matrix metalloproteinase-9, interleukin-8, and interleukin-18, are elevated during asthma exacerbations and are suppressed by methylprednisolone (25, 26). Although our data showed a trend for subepithelial vascular networks to decrease in patients with asthma receiving inhaled corticosteroid, the trend was not significant. Previous reports (6–8) showed successful treatment with beclomethasone dipropionate at more than 500 μg/day, but failure with beclomethasone dipropionate at 200–500 μg/day. Chetta and coworkers (8) reported that fluticasone (1,000 μg/day) for 6 weeks significantly reduced airway vascularity but did not completely reverse it to control levels. The smaller dose of inhaled corticosteroid used may explain why subepithelial vessels were not reduced in the present study. On the other hand, Orsida and coworkers (7) reported that 3 months of treatment with long-acting β2-agonist (salmeterol) caused a marked fall in the number of vessels per millimeter squared of lamina propria, which was not seen with treatment with low-dose inhaled corticosteroids. In our study none of the patients had used salmeterol. The therapeutic implications of alterations of increased subepithelial microvessels are just beginning to be evaluated. As the present study was cross-sectional, further interventional studies of the therapeutic efficacy of high-dose inhaled corticosteroid or salmeterol on airway angiogenesis should be performed with this novel bronchovideoscope.
In conclusion, increased subepithelial microvessels in the tracheal mucosa were found even in steroid-naive patients with newly diagnosed asthma, suggesting increased airway vascularity may be completed when asthma symptoms begin. A newly developed high-magnification bronchovideoscope (side-viewing type) might be a useful tool in evaluating mucosal vascular networks and could replace more invasive bronchial biopsy.
H.T. has no declared conflict of interest; G.Y. has no declared conflict of interest; T.S. has no declared conflict of interest; M.H. has no declared conflict of interest; S.T. has no declared conflict of interest; K.S. has no declared conflict of interest; M.J. has no declared conflict of interest; H.T. has no declared conflict of interest; S.A. has no declared conflict of interest.
The authors thank Yuichi Morizane, Takeshi Takigawa, and Toshiyuki Kubonoya (Olympus Optical, Tokyo, Japan) for work in developing the high-magnification bronchovideoscope (side-viewing type).
| 1. | Bousquet J, Jeffery PK, Busse WW, Johnson M, Vignola AM. Asthma: from bronchoconstriction to airway inflammation and remodeling. Am J Respir Crit Care Med 2000;161:1720–1745. |
| 2. | Davies DE, Wicks J, Powell RM, Puddicombe SM, Holgate ST. Airway remodeling in asthma: new insights. J Allergy Clin Immunol 2003;111:215–225. |
| 3. | McDonald DM. Angiogenesis and remodeling of airway vasculature in chronic inflammation. Am J Respir Crit Care Med 2001;164:S39–S45. |
| 4. | Jeffery PK. Remodeling in asthma and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001;164:S28–S38. |
| 5. | Hogg JC, Pare PD, Moreno R. The effect of submucosal edema on airways resistance. Am Rev Respir Dis 1987;135:S54–S56. |
| 6. | Orsida BE, Li X, Hickey B, Thien F, Wilson JW, Walters EH. Vascularity in asthmatic airways: relation to inhaled steroid dose. Thorax 1999;54:289–295. |
| 7. | Orsida BE, Ward C, Li R, Bish R, Wilson JW, Thien F, Walters EH. Effect of a long-acting β2-agonist over three months on airway wall vascular remodeling in asthma. Am J Respir Crit Care Med 2001;164:117–121. |
| 8. | Chetta A, Zanini A, Foresi A, Del Donno M, Castagnaro A, d'Ippolito R, Baraldo S, Testi R, Saetta M, Olivieri D. Vascular compartment of airway remodeling in asthma reduced by high dose of fluticasone. Am J Respir Crit Care Med 2003;167:751–757. |
| 9. | Kuwano K, Bosken K, Pare PD, Bai TR, Wiggs BR, Hogg JC. Small airway dimensions in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1993;148:1220–1225. |
| 10. | Li X, Wilson JW. Increased vascularity of the bronchial mucosa in mild asthma. Am J Respir Crit Care Med 1997;156:229–233. |
| 11. | Carroll NG, Cooke C, James AL. Bronchial blood vessel dimensions in asthma. Am J Respir Crit Care Med 1997;155:689–695. |
| 12. | Takahasi H, Itoh T, Shiratori M, Oouchi H, Nishino M, Yamada G, Tanaka H, Koba H, Abe S. Observation of the structure of trachea and bronchial lumens in healthy volunteers by using a newly developed high-magnification bronchovideoscope. In: Yoshimura H, Kida A, Arai T, editors. Bronchology and bronchoesophagology. London: Elsevier; 2001. p. 568–569. |
| 13. | Tanaka H, Yamada G, Itoh T, Hashimoto M, Suzuki K, Tanaka S, Oashi K, Saikai T, Teramoto S, Takahashi H, et al. Airway hypervascularity in asthmatic patients assessed by side-viewing high-magnified bronchoscopy: a new point of view [abstract]. Am J Respir Crit Care Med 2001;163:A787. |
| 14. | Tanaka H, Yamada G, Saikai T, Hashimoto M, Suzuki K, Fujii M, Takahashi H, Abe S. Fine vessels of the airway mucosa are increased in asthma but not in COPD, observed by a novel high-magnified bronchovideoscope (side-viewing type) [abstract]. Am J Respir Crit Care Med 2003;167:A454. |
| 15. | American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 1987;136:225–244. |
| 16. | National Institutes of Health: National Heart, Lung, and Blood Institute. Guidelines for the diagnosis and management of asthma. Publication no. 97–4051. Washington, DC: National Institutes of Health; 1997. |
| 17. | National Institutes of Health: National Heart, Lung, and Blood Institute. Global initiative for chronic obstructive lung disease. Washington, DC: National Institutes of Health; 2001. p. 6–9. |
| 18. | Hoshino M, Takahashi M, Takai Y, Sim JJ, Aoike N. Inhaled corticosteroids decrease vascularity of the bronchial mucosa in patients with asthma. Clin Exp Allergy 2000;31:722–730. |
| 19. | Leibovich SJ, Polverini PJ, Shepard HM, Wiseman DM, Shively V, Nuseir N. Macrophage-induced angiogenesis is mediated by tumour necrosis factor-α. Nature 1987;329:630–632. |
| 20. | Wilson J. The bronchial microcirculation in asthma. Clin Exp Allergy 2000;30:51–53. |
| 21. | D'Amore P. Mechanisms of endothelial growth control. Am J Respir Cell Mol Biol 1992;6:1–8. |
| 22. | Connolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL, Delfino JJ, Siegel NR, Leimgruber RM, Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 1989;84:1470–1478. |
| 23. | Hoshino M, Takahashi M, Aoike N. Expression of vascular endothelial growth factors, basic fibroblast growth factor, and angiogenin immunoreactivity in asthmatic airways and its relationship to angiogenesis. J Allergy Clin Immunol 2001;107:295–301. |
| 24. | Nauck M, Roth M, Tamm M, Eickelberg O, Wieland H, Stulz P, Perruchoud AP. Induction of vascular endothelial growth factor by platelet-activating factor and platelet-derived growth factors is downregulated by corticosteroid. Am J Respir Cell Mol Biol 1997;16:398–406. |
| 25. | Tanaka H, Miyazaki N, Oashi K, Tanaka S, Ohmichi M, Abe S. Sputum matrix metalloproteinase-9: tissue inhibitor of metalloproteinase-1 ratio in acute asthma. J Allergy Clin Immunol 2000;105:900–905. |
| 26. | Tanaka H, Miyazaki N, Oashi K, Teramoto S, Shiratori M, Hashimoto M, Ohmichi M, Abe S. IL-18 might reflect disease activity in mild and moderate asthma exacerbation. J Allergy Clin Immunol 2001;107:331–336. |
