Abstract
Airway-wall remodeling leading to thickening of the bronchial wall in asthma has been invoked to account for airflow obstruction and increased bronchial reactivity to provocative stimuli. Bronchial-wall changes characteristic of asthma are thought to include increased vascularity with vasodilatation. The contention that inflammatory mediators cause bronchial vasodilatation and that growth factors may induce increased vascularity is based on little structural evidence. We took bronchoscopic biopsies from the major airways of 12 subjects with mild asthma and 11 control subjects, and evaluated bronchial vessel numbers and size, using computerized image analysis after staining for type IV collagen in vessel walls. The airways of asthmatic subjects were significantly more vascular (17.2 ± 4.2 versus 10.3 ± 1.9%, p < 0.001), with more vessels (738 ± 150 versus 539 ± 276 vessels/mm2 [mean ± SD], p < 0.05) than those of the controls. There were significantly more asthmatic bronchial than control vessels with a cross-sectional area greater than 300 μ m2 (19.4 versus 12.7%, p < 0.05). These findings provide the first confirmatory evidence that bronchial biopsies from patients with mild asthma are more vascular than those of normal controls, that there are more vessels in asthmatic airways, and that asthmatic bronchial vessels are larger than controls.
Airway-wall remodeling in asthma, leading to thickening of the bronchial wall, has been attributed to inflammatory cell infiltration, the deposition of scar-type collagen below the epithelium, and smooth-muscle hyperplasia/hypertrophy and increased vascularity within the submucosa (1-3). Airway-wall remodeling has been invoked to account for fixed airflow obstruction in severe asthma (4) and reduced distensibility of the airway wall in mild asthma (5).
The contribution of the vascular bed to airway-wall remodeling has not been fully elucidated. The bronchial circulation arises from the aorta and forms a dense peribronchial plexus of interconnecting vessels. Branches then penetrate the muscular layer to form a second complex in the submucosa (6). The bronchial and pulmonary circulations anastomose freely with each other throughout the tracheobronchial tree. Capillary engorgement and/or leakage in this circulatory bed could directly alter airway-wall thickness. Capillary engorgement has been described in models of anaphylactic reactions in both the rat (7) and the guinea pig (8). Inflammatory mediators, known to be released in asthma (1), may contribute to bronchial vascular dilatation. Potential candidates include histamine (9), bradykinin (10, 11), sulfidopeptide leukotrienes (12), and mediators released by autonomic sensory nerves (13). Platelet activating factor (PAF) may also increase bronchial vascular blood flow (14, 15). There are, hence, several inflammatory mediators, each having known agonists, capable of causing bronchial vascular dilatation. It has been demonstrated in sheep that the microvascular volume fraction of the airway subepithelial tissue may be as high as 16% and can double with pulmonary hypertension and vasodilation, resulting in a narrowed bronchiolar lumen (16). When blood or saline is used to induce engorgement of canine airways in vivo, there is subsequent bronchial thickening and luminal narrowing (17). Currently, it is uncertain whether increased bronchial vascular congestion directly contributes to airway resistance (18). A model of airway-wall thickening proposed by Moreno and colleagues (19) suggests that a small increase in thickness, such as that caused by edema or vascular engorgement, might account for the increased airway resistance seen with bronchial provocation (20). In humans, it has also been hypothesized that the vascular engorgement of these vessels, and consequent thickening of the airway mucosa, leads to narrowing of the lumen of the bronchi, an increase in airway resistance, and a decrease in forced expiratory flow rates (21). Supporting this notion is the observation that patients with impaired left-ventricular function have heightened sensitivity to methacholine within the asthmatic range (22). Furthermore, normal subjects may develop similar changes in airway responsiveness upon the rapid infusion of intravenous fluids (23). Airflow obstruction induced by exercise and isocapnic hyperventilation may be attributable to vascular hyperemia without a significant contribution by airway smooth muscle (24, 25). Among patients with chronic asthma, Dunnill described dilatation of the capillary blood vessels as a striking feature of the bronchial mucosa in 20 cases of fatal asthma (26). A study of small-airway vascularity (vessels as the percentage of submucosal area in specimen sections) in resected lung and postmortem specimens found a vascularity of 3.3% in asthmatic individuals and 0.6% in airways of control subjects (27). These studies suggest that the bronchial circulation may play a role in the pathophysiology of chronic asthma. The vascularity of the proximal airways in bronchial biopsies from subjects with mild asthma has not previously been quantified with vessel-specific stains. The aim of the present study was to examine the large airways of volunteers with mild asthma for evidence of the possible significance of vascular changes in airway-wall remodeling.
Twelve nonsmoking atopic asthmatic subjects who satisfied American Thoracic Society (ATS) criteria for asthma (28) and who were using β-agonists alone were recruited from the hospital outpatient department. All responded to one or more common aeroallergens (Dermatophagoides pteronyssinus, Alternaria, animal dander, feathers, rye grass and mixed grass; Holister-Stier, Spokane, WA) on skin-prick testing with a wheal ≥ 3 mm, and responded to bronchoprovocative methacholine challenge using the method of Chai and colleagues (29). Normal volunteer subjects had no history of airway symptoms and did not respond to methacholine challenge or skin prick testing.
Fiberoptic bronchoscopy was performed transnasally after administration of 2% lidocaine to the upper airway and nares as previously described (14). Six mucosal biopsies were taken from the subsegmental carinae of the right lower lobe. After fixation in 100% ethanol and processing in paraffin, one of the best blocks was sectioned to provide two sections at 40-μm intervals. Well-oriented 4-μm sections were stained with goat monoclonal antihuman collagen type IV antibodies diluted 1:150 (Southern Biotechnology Associates, Inc., Birmingham, AL). Staining was amplified with horseradish peroxidase-conjugated streptavidin and third-stage biotinylated rabbit antigoat antibody (Dako Corporation, Carpinteria, CA). Stained sections were washed, counterstained with hematoxylin, and mounted under coverslips. Vessels were identified by typical staining of type IV collagen in the true basement membrane supporting the endothelium (Figure 1a and b). Coded samples to which a single observer was blinded were assessed. Continuous analysis of all lamina propria to a depth of 150 μm below the basement membrane was done with a computerized image analyzer (Video Pro; Leading Edge Pty. Ltd., Adelaide, Australia) at a final magnification of ×500. Smooth muscle and glands were excluded from areas used to evaluate vessel density. All structures internal to the vessel endothelial basement membrane were evaluated as vascular area. For each subject, the total vascular area was divided by the biopsy area examined in order to determine the percent vascularity. The total number of vessels was divided by the biopsy area examined to determine the number of vessels per square millimeter of lamina propria to a depth of 150 μm below the basement membrane. The mean size of vessels was estimated by dividing the total vascular area by the total number of vessels for each subject. The frequency distribution of all vessels according to their area was used for further analysis of vessel size in the two study groups.
Fig. 1. Bronchial biopsies stained with anticollagen type IV (magnification ×400), indicating vessels below the true basement membrane in controls (a) and asthmatic subjects (b).
[More] [Minimize]Comparisons between groups were made with the Mann–Whitney U-test, and comparisons between asthmatic and normal subjects' large and small vessels were made with the chi-square test.
Asthmatic subjects had a mean (± SD) predicted FEV1 of 80.3 ± 21.3%, with a geometric mean (± SD) provocative concentration of methacholine causing a 20% decrease in FEV1 (PD20) of 1.78 ± 3.67 mg/ml.
Section areas were approximately 0.25 mm2 in all biopsies. The coefficient of variation (CV) used for analysis of variation of vascularity between sections of 1.0% in the control group and 10.5% in the asthmatic group. The CV for numbers of vessels in different sections was 4.2% in controls and 10.0% in asthmatics. The total area of lamina propria examined per patient was the same for the two groups (Table 1). The percentage of biopsy area covered with vessels was greater in asthmatics than in control subjects (17.2 ± 4.2% versus 10.3 ± 1.9%, p < 0.001 (Figure 2). There was an increase in density of vessels in asthmatic subjects as compared with controls (738 ± 150/mm2 versus 539 ± 276/mm2, p < 0.05) (Figure 3). There was no significant correlation between any of the variables of duration of asthma, medication use, FEV1, or PC20 and either percent vascularity or number of vessels for asthmatic biopsies.
| Subject | Total Area (mm2 ×10− 2) | Vessel Area (mm2 ×10− 2) | Percent Vascularity | Total Vessels | Vessels per mm2 | Vessel Size (μm 2) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | ||||||||||||
| 1 | 5.36 | 0.55 | 10.22 | 40 | 745.77 | 137.50 | ||||||
| 2 | 11.23 | 0.82 | 7.29 | 26 | 231.56 | 314.62 | ||||||
| 3 | 9.92 | 1.01 | 10.16 | 50 | 503.90 | 201.56 | ||||||
| 4 | 18.98 | 2.18 | 11.49 | 124 | 653.35 | 175.79 | ||||||
| 5 | 15.48 | 1.48 | 9.59 | 88 | 568.31 | 168.70 | ||||||
| 6 | 12.10 | 1.11 | 9.20 | 74 | 611.34 | 150.54 | ||||||
| 7 | 39.29 | 5.32 | 13.53 | 158 | 402.14 | 336.40 | ||||||
| 8 | 33.62 | 4.46 | 13.28 | 180 | 535.45 | 248.02 | ||||||
| 9 | 2.50 | 0.28 | 11.25 | 30 | 1,198.27 | 93.87 | ||||||
| 10 | 9.81 | 0.91 | 9.32 | 24 | 244.70 | 380.83 | ||||||
| 11 | 5.43 | 0.44 | 8.13 | 26 | 479.19 | 169.69 | ||||||
| Mean | 14.88 | 1.69 | 10.31 | 74.55 | 561.27 | 216.09 | ||||||
| SD | 11.70 | 1.68 | 1.95 | 56.36 | 263.78 | 91.69 | ||||||
| Asthma | ||||||||||||
| 1 | 3.35 | 0.27 | 7.99 | 36 | 1,075.33 | 74.32 | ||||||
| 2 | 4.63 | 0.57 | 12.29 | 40 | 864.38 | 142.20 | ||||||
| 3 | 9.67 | 1.66 | 17.13 | 62 | 641.00 | 267.16 | ||||||
| 4 | 10.11 | 1.72 | 17.01 | 72 | 712.00 | 238.86 | ||||||
| 5 | 14.84 | 2.10 | 14.13 | 132 | 889.67 | 158.82 | ||||||
| 6 | 43.00 | 10.46 | 24.32 | 226 | 525.63 | 462.62 | ||||||
| 7 | 7.50 | 1.17 | 15.60 | 46 | 613.73 | 254.17 | ||||||
| 8 | 5.26 | 0.91 | 17.34 | 36 | 684.88 | 253.17 | ||||||
| 9 | 26.44 | 4.79 | 18.14 | 216 | 817.07 | 221.95 | ||||||
| 10 | 18.98 | 4.12 | 21.68 | 134 | 705.98 | 307.10 | ||||||
| 11 | 9.01 | 1.50 | 16.63 | 56 | 621.63 | 267.54 | ||||||
| 12 | 1.95 | 0.39 | 19.88 | 14 | 719.20 | 276.43 | ||||||
| Mean | 12.89 | 2.47 | 16.84 | 89.17 | 739.21 | 243.70 | ||||||
| SD | 11.79 | 2.88 | 4.25 | 71.59 | 149.96 | 95.83 | ||||||
| p Value | 0.42 | 0.5 | 0.001 | 0.58 | 0.018 | 0.49 | ||||||
Fig. 2. Comparison of percent vascularity of bronchial biopsies from asthmatic subjects and controls.
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Fig. 3. Comparison of vessel numbers in bronchial biopsies from asthmatic subjects and controls.
[More] [Minimize]Vessels from biopsies obtained from asthmatic and control subjects were compared for distribution of vessel size (Figure 4). With chi-square analysis, asthmatic vessels were more likely than control vessels to be over 300 μm2 in cross-sectional area (p < 0.05). When the theoretical mean area for vessels in each biopsy was calculated (total area of vasculature divided by total number of vessels), there was no apparent increase in mean area for asthmatic vessels as compared with controls (243.7 ± 95.8 μm2 versus 235 ± 91.7 μm2, p = 0.48).
Fig. 4. Histogram indicating size distributions of vessels in asthmatic and control bronchial biopsies.
[More] [Minimize]This study indicates that the vascularity of the airways as assessed by identification of the true basement membrane within vessel walls in the major bronchi is increased in asthmatic subjects as compared with controls. The use of image-analysis techniques allowed the counting of pixels within vessels as well as the accurate measurement of submucosal area.
Numbers of blood vessels identified in our study were greater than those described by Beasley and colleagues (2), who found 123 vessels/mm2 in controls and 142 vessels/mm2 in subjects with mild asthma with an Azure II–methylene blue– basic fuchsin method, which may not have specifically identified vessels. Using a more sensitive method, but sampling similar airways by fiberoptic bronchoscopy, we found greater numbers of vessels in each group. By comparison, the number of vessels identified in our study was significantly greater than that reported by Kuwano and coworkers (27), who found 80 vessels/mm2 in asthmatic subjects and 28 vessels/mm2 in control subjects with a trichrome stain. With an antibody to Factor VII, Kuwano and coworkers found considerably more vessels in both asthmatic (465 ± 149/mm2) and control airways (320 ± 148/mm2). They also found a considerably lower percentage of airway vasculature, of 3.3% in asthmatic subjects and 0.6% in controls. The size of the airways used to determine these values from postmortem and lung-resection specimens was not stated, but may have been smaller than that of the proximal airways sampled in our study. Although derived from few subjects (n = 3 in each group), our results (n = 11 controls, n = 12 asthmatic subjects) support the observations of Kuwano and coworkers. Higher counts and sensitivity might also have been achieved with our technique, which excluded nonvascular structures from the counting field.
Kuwano and coworkers suggest that their estimate of airway vascularity may well be an underestimate of the true value, since the majority of asthmatic samples were from postmortem lungs, in which blood may have drained from the tissues. Unlike subjects in Kuwano and coworkers' study, our volunteers were not subject to hypercapnia, hypoxia, or acidosis. It is therefore unlikely that these factors, present in terminal asthma, can be invoked to explain vascular differences in the airways of the mildly asthmatic subjects described here. A further factor, the use of β-agonists in asthma, may be associated with vascular dilatation of large-airway vessels, such as has been observed in dogs (12). The asthmatic subjects in our study were premedicated with albuterol prior to bronchoscopy (14). Although unlikely to be a major factor, it is possible that premedication contributed to airway-vessel dilatation in the asthmatic subjects but not to an increase in vessel numbers.
The finding of increased numbers of vessels in the asthmatic submucosa in this study and others (2, 27) suggests that angiogenesis is a component of the chronic inflammatory response even in mild-to-moderate asthma. The number of vessels seen in sections of the airway may also be increased by vessel elongation, with folding of vessels in and out of the plane of section. In either case, a vascular proliferative response must occur to account for the increase in the density of vessels. Possible factors responsible for angiogenesis in airway inflammation include basic fibroblast growth factor (bFGF) (30), tumor necrosis factor-α (TNF-α) (31), and vascular endothelial growth factor (VEGF) (32). Any release of angiogenic growth factors in the bronchial wall may be either secondary to an established inflammatory response or part of the remodeling response to intramural mechanical forces (33, 34).
Antiinflammatory treatment with inhaled corticosteroids may be expected to reduce the production of growth factors and lead to a reduction in airway vascularity. This postulate is in keeping with the observation that the introduction of inhaled steroids early in the course of mild asthma leads to better airflow and reduced bronchial reactivity (35).
The contribution of increased vascularity, ranging from 10.3 to 17.2% in mild asthma, is associated with increased bronchial reactivity. A model of airway-wall thickening proposed by Moreno and associates (19) suggests that a small increase, such as that caused by edema or engorgement, might account for the increased airway resistance seen with bronchial provocation (20). Mitzner and colleagues have used blood or saline engorgement of canine airways in vivo to demonstrate a correlation between wall thickening and luminal narrowing (17). In addition, the indirect effect of edema associated with vessel proliferation may contribute to both the airway narrowing and subepithelial injury typical of asthma. Increased engorgement may also contribute to the loss of airway distensibility seen in asthma (5). The relative contribution of each of vessel size and vessel number to airway resistance is difficult to estimate from the current study. More large vessels (> 300 μm2) have been identified in asthmatic than in normal airways. Rapid responsiveness of vessel diameter following allergen or exercise challenge might become significant when numbers of vessels are increased. Bronchodilators such as albuterol can potentially abrogate bronchospasm associated with either of these stimuli, but their bronchial vascular actions in humans are yet to be established.
This study has shown that with fiberoptic bronchoscopic biopsies, the identification of type IV collagen in the true basement membrane of blood vessels reveals increased vascularity in the major airways of asthmatic subjects as compared with controls. This finding suggests that significantly more airway-wall thickening may be attributable to vascular proliferation and dilatation than was previously thought. Future strategies to control airflow obstructions in the known forms of asthma may need to account for increased vascularity of the bronchial submucosa.
The authors wish to thank Dr. Michael Pain of the Royal Melbourne Hospital for providing bronchoscopy facilities, and Mr. Ian Birchall of the University of Melbourne for assistance with tissue processing. Dr. Alistair Stewart of the Bernard O'Brien Institute kindly reviewed the manuscript.
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