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Possible role of airway microvascular permeability on airway obstruction in patients with chronic obstructive pulmonary disease

Open ArchivePublished:December 21, 2018DOI:https://doi.org/10.1016/j.rmed.2018.12.007

      Abstract

      Background

      Airway microvascular system participates in the airway inflammation that is central to the pathophysiology of inflammatory lung disorders.

      Objective

      To examine the role of airway microvascular permeability on airway obstruction in patients with chronic obstructive pulmonary disease (COPD).

      Methods

      We measured the airway microvascular permeability index (AMPI) separately in the central or peripheral airways using a bronchoscopic microsampling technique in 9 non-smokers, 18 smokers without COPD (10 former smokers and 8 current smokers), and 26 smokers with COPD (12 former smokers and 14 current smokers).

      Results

      AMPI in the central airways was relatively low, and this index was comparable among the five groups. In contrast, AMPI in the peripheral airways was significantly higher in smokers with or without COPD compared with non-smokers. Moreover, AMPI in the peripheral airways was significantly higher in current smokers than in former smokers with COPD. Especially, AMPI in the peripheral airways, but not in the central airways, showed a significant correlation with the degree of airway obstruction in former or current smokers with COPD. However, AMPI in the peripheral airways was not correlated with the diffusing capacity of the lung in former or current smokers with COPD.

      Conclusion

      Airway microvascular permeability in the peripheral airways is increased in patients with COPD, and is associated with the severity of airway obstruction. We may need to consider this characteristic feature as a target in any therapeutic strategy for the treatment of the disease. (237 words).

      Keywords

      Abbreviations:

      AMPI (airway microvascular permeability index), COPD (chronic obstructive pulmonary disease), ELF (epithelial lining fluid), FEV1 (forced expiratory volume in 1 s), FVC (forced vital capacity), DLCO (diffusing capacity of the lung for carbon monoxide)

      1. Introduction

      Chronic obstructive pulmonary disease (COPD) is closely linked to the inhalation of noxious agents, especially cigarette smoke [
      • Rennard S.I.
      • Drummond M.B.
      Early chronic obstructive pulmonary disease: definition, assessment, and prevention.
      ]. It is associated with an abnormal inflammatory response of the lung, which results in progressive airway obstruction [
      • Decramer M.
      • Janssens W.
      • Miravitlles M.
      Chronic obstructive pulmonary disease.
      ]. Airway microvascular system participates in the airway inflammation that is central to the pathophysiology of inflammatory lung disorders. Airway inflammation induces the increased airway microvascular permeability, which is causally related to airway wall edema and swelling [
      • Goldie R.G.
      • Pedersen K.E.
      Mechanisms of increased airway microvascular permeability: role in airway inflammation and obstruction.
      ]. Eventually, in inflammatory conditions, airway microvascular hyper-permeability may contribute to, at least a part, airway obstruction via airway wall thickening.
      It is now recognized that airway obstruction in asthma could involve airway wall edema in addition to elevated airway smooth muscle contraction, mucus hypersecretion, and potentially airway wall remodeling [
      • Grainge C.L.
      • Lau L.C.K.
      • Ward J.A.
      • Dulay V.
      • Lahiff G.
      • Wilson S.
      • Holgate S.
      • Davies D.E.
      • Howarth P.H.
      Effect of bronchoconstriction on airway remodeling in asthma.
      ]. Several inflammatory mediators released into the asthmatic airways including histamine, leukotrienes and bradykinin are potent inducers of increased airway microvascular permeability, and are promoters of airway wall thickening and the resultant of reduction in the airway calibers [
      • Van Rensen E.L.J.
      • Hiemstra P.S.
      • Rabe K.F.
      • Sterk P.J.
      Assessment of microvascular leakage via sputum induction.
      ]. Moreover, the extravasation of plasma proteins may promote the production of viscous mucus and the formation of luminal mucus plugs [
      • Hogg J.C.
      • Chu F.
      • Utokaparch S.
      • Woods R.
      • Elliot W.M.
      • Buzatu L.
      • Cherniack R.M.
      • Rogers R.M.
      • Sciurba F.C.
      • Coxson H.O.
      • Pare P.D.
      The nature of small-airway obstruction in chronic obstructive pulmonary disease.
      ]. Thus, the leakage of plasma from airway microvessels into the airway wall and lumen may have significant effects on lung function. We have previously demonstrated that cigarette smoking augments airway microvascular permeability in the airways of patients with asthma [
      • Kanazawa H.
      • Asai K.
      • Tochino Y.
      • Kyoh S.
      • Kodama T.
      • Hirata K.
      Increased levels of angiopoietin-2 in induced sputum from smoking asthmatic patients.
      ]. Thus, smoking per se may contribute to airway narrowing via increased airway microvascular permeability in asthma.
      However, to our knowledge, the physiological roles of airway microvascular permeability on airway obstruction in COPD have not been determined. It is widely accepted that airway inflammation is relates to the airway obstruction in COPD [
      • Angelis N.
      • Porpodis K.
      • Zarogoulidis P.
      • Spyratos D.
      • Kioumis I.
      • Papaiwannou A.
      • Pitsiou G.
      • Tsakiridis K.
      • Mpakas A.
      • Arikas S.
      • Tsiouda T.
      • Katsikogiannis N.
      • Kougioumtzi I.
      • Machairiotis N.
      • Argyriou M.
      • Kessisis G.
      • Zarogoulidis K.
      Airway inflammation in chronic obstructive pulmonary disease.
      ]. Notably, a previous study reported that there is a strong correlation between the obstructive impairments in lung function and the degree of small airways inflammation in COPD [
      • Hogg J.C.
      • Paré P.D.
      • Hackett T.L.
      The contribution of small airway obstruction to the pathogenesis of chronic obstructive pulmonary disease.
      ]. On the basis of these findings, it is plausible that small airways inflammation may play a pivotal role on airway obstruction in COPD. Therefore, to elucidate the evidence for the involvement of airway microvascular system in airway obstruction in COPD, we would be required to discriminate the degree of airway microvascular permeability separately in central or peripheral airways. However, useful tools for assessment of peripheral airways are not well validated and remain difficult to use in clinical practice. In this respect, we have already established the method of measurement of biochemical constituents in epithelial lining fluid (ELF) samples separately obtained from the central or peripheral airways using a bronchoscopic microsampling technique [
      • Kodama T.
      • Kanazawa H.
      • Tochino Y.
      • Kyoh S.
      • Asai K.
      • Hirata K.
      A technological advance comparing epithelial lining fluid from different regions of the lung in smokers.
      ]. In previous studies, measurement of specific serum proteins in sputum samples has been used as an index of airway microvascular permeability [
      • Kanazawa H.
      • Asai K.
      • Hirata K.
      • Yoshikawa J.
      Vascular involvement in exercise-induced airway narrowing in patients with bronchial asthma.
      ,
      • Kanazawa H.
      • Nomura S.
      • Yoshikawa J.
      Role of microvascular permeability on physiologic differences in asthma and eosinophilic bronchitis.
      ]. In this study, we used the modified method which measures the airway microvascular permeability index (AMPI) using ELF samples. Using this method, we have attempted to describe the role of airway microvascular permeability in central or peripheral airways on airway obstruction in patients with COPD.

      2. Materials and methods

      2.1 Subjects

      This study enrolled 9 non-smokers, 18 smokers without COPD (10 former smokers and 8 current smokers) and 26 smokers with COPD (12 former smokers and 14 current smokers) from the outpatient clinic of Osaka City University Hospital. All former smokers had stopped smoking for at least more than 1 year. Study subjects who agreed to undergo ELF samplings were consecutively selected, and were subjected to bronchoscopic examination to identify the cause of persistent cough or small peripheral nodules. All study subjects underwent pulmonary function tests by using a CHESTAC-8800 unit (Chest, Tokyo, Japan). The forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were examined, and the best of three consecutive attempts of spirometric measurements were recorded [
      • Miller M.R.
      • Hankinson J.
      • Brusasco V.
      • Burgos F.
      • Casaburi R.
      • Coates A.
      • Crapo R.
      • Enright P.
      • van der Grinten C.P.M.
      • Gustafsson P.
      • Jensen R.
      • Johnson D.C.
      • MacIntyre N.
      • McKay R.
      • Navajas D.
      • Pedersen O.F.
      • Pellegrino R.
      • Viegi G.
      ]. In addition, the diffusing capacity of the lung for carbon monoxide (DLCO) was also measured in smokers with COPD [
      • Pellegrino R.
      • Viegi G.
      • Brusasco V.
      • Crapo R.O.
      • Burgos F.
      • Casaburi R.
      • Coates A.
      • van der Grinten C.P.M.
      • Gustafsson P.
      • Hankinson J.
      • Jensen R.
      • Johnson D.C.
      • MacIntyre N.
      • McKay R.
      • Miller M.R.
      • Navajas D.
      • Pedersen O.F.
      J. Wanger. Interpretative strategies for lung function tests.
      ]. In all subjects, chest computed tomographic scans showed absence of abnormal diffuse interstitial infiltrates, and results of arterial blood gas analyses were normal. All COPD patients satisfied the Global Initiative for Chronic Obstructive Lung Disease criteria for the diagnosis [
      • Vogelmeier F.C.
      • Criner G.J.
      • Martinez J.F.
      • Anzueto A.
      • Barnes P.J.
      • Bourbeau J.
      • Celli B.R.
      • Chen R.
      • Decramer M.
      • Fabbri L.M.
      • Frith P.
      • Halpin D.M.G.
      • López Varela M.V.
      • Nishimura M.
      • Roche N.
      • Rodriguez-Roisin R.
      • Sin D.D.
      • Singh D.
      • Stockley R.
      • Vestbo J.
      • Wedzicha J.A.
      • Agustí A.
      Global strategy for the diagnosis, management, and prevention of chronic obstructive lung disease 2017 report. GOLD executive summary.
      ]. All data in COPD patients were collected at the time of diagnosis before administration of any medications. No COPD patients suffered from a recent respiratory infection or an exacerbation for at least 4 weeks prior to the study. COPD patients with main comorbidities such as endocrine disorders, hypertension, cardiovascular disorders, and renal impairment were excluded in this study. All subjects gave their written informed consent for participation in the study, which was approved by the Ethics Committee of Osaka City University (IRB number #685).

      2.2 Bronchoscopic microsampling technique

      ELF was obtained as we have previously described [
      • Kanazawa H.
      • Tochino Y.
      • Asai K.
      • Hirata K.
      Simultaneous assessment of HGF and VEGF in epithelial lining fluid from patients with COPD.
      ,
      • Kanazawa H.
      • Tochino Y.
      • Asai K.
      • Ichimaru Y.
      • Watanabe T.
      • Hirata K.
      Validity of HMGB1 measurement in epithelial lining fluid in patients with COPD.
      ]. In all study subjects, bronchial microsampling from the second bronchus was performed using the microsampling probe (BC 402C, Olympus, Tokyo, Japan) (central airway sample). Following this, a thin, flexible fiberoptic bronchoscope was inserted into the lung, and the ELF from the seventh or eighth lower lobe bronchioles was collected under direct observation using an ultrafine probe (BC 401C, Olympus, Tokyo, Japan) (peripheral airway sample).

      2.3 Measurement of airway microvascular permeability index

      In this study, we measured the concentrations of albumin in serum and ELF samples. The degree of airway microvascular permeability was evaluated by the AMPI as measured by the albumin levels in ELF/albumin levels in serum.

      2.4 Statistical analysis

      All values are presented as the median [interquartile range (IQR)]. When multiple comparisons of nonparametric data between groups were performed, significant intergroup variability was first established with use of the Kruskal-Wallis test. The Mann-Whitney U test was then used for intergroup comparisons. The Wilcoxon signed-rank test was also used for comparisons of variables between the central and peripheral airways. The significance of correlations was evaluated by determining Spearman's rank-correlation coefficients. In all statistical analysis, p value of less than 0.05 was considered significant.

      3. Results

      The clinical characteristics of study subjects are shown in Table 1. All study subjects were male, and the five groups were well matched for age. Although the smoking index in the four smoking groups were similar levels, FEV1 and FEV1/FVC were significantly lower in former and current smokers with COPD. Though the DLCO value was similar levels in former and current smokers with COPD, FEV1 and FEV/FVC were significantly lower in current smokers than in former smokers with COPD.
      Table 1Clinical characteristics of study subjects.
      Non-smokersSmokers without COPDSmokers with COPD
      formercurrentformercurrent
      Subject No.91081214
      Age (year)61 [60–64]66 [63–69]61 [60–64]62 [57–69]64 [60–69]
      Smoking index (pack-year)054 [45–60]58 [49–62]65 [62–73]63 [45–75]
      FEV1 (%predicted)88 [83–95]86 [74–94]79 [76–80]64 [61–68]#58 [54–61]†*
      FEV1/FVC (%)80 [78–82]76 [74–79]74 [71–76]56 [47–62]#48 [44–50]†*
      DLCO (%)N.D.N.D.N.D.58 [53–65]57 [55–61]
      All values are presented as median [IQR].
      Definition of abbreviations: COPD = chronic obstructive pulmonary disease, FEV1 = forced expiratory volume in 1 s, FVC = forced vital capacity, DLco = diffusing capacity of carbon monoxide, N.D. = not determined.
      *p < 0.05, compared with former smokers with COPD.
      #p < 0.01, compared with former smokers without COPD.
      †p < 0.01, compared with current smokers without COPD.
      We successfully measured the AMPI separately in central or peripheral airways in all study subjects. Our sampling method had no detrimental effects on baseline lung function and oxygenation during the procedure. AMPI was relatively low in ELF obtained from central airways (AMPI in the central airways: non-smokers: 0 [0–0.04]; without COPD: former smokers: 0 [0–0.09]; current smokes: 0.08 [0.03–0.15]; with COPD: former smokers: 0.07 [0–0.15]; current smokers: 0.08 [0.03–0.24]) (Fig. 1). The index in the central airways was not significantly different between these five groups. However, the index was markedly higher in the peripheral airways than in the central airways (AMPI in the peripheral airways: non-smokers: 0 [0–0.10], p = 0.07; without COPD: former smokers: 0.19 [0–0.33], p = 0.08; current smokes: 0.23 [0.18–0.30], p = 0.02; with COPD: former smokers: 0.33 [0.24–0.48], p = 0.003; current smokers: 0.64 [0.38–0.79], p = 0.001). Moreover, AMPI in the central airways did not differ between former and current smokers with or without COPD. In contrast, although there was no significant difference in AMPI in the peripheral airways between former and current smokers without COPD, the index was significantly higher in current smokers than in former smokers with COPD (p = 0.03).
      Fig. 1
      Fig. 1Comparisons of airway microvascular permeability index from central or peripheral airways in non-smokers, smokers without COPD, and smokers with COPD. Each bar represents the median value. *p < 0.01 compared with current smokers without COPD. †p < 0.05 compared with former smokers with COPD.
      Subsequently, we evaluated the correlations between AMPI in the central or peripheral airways and the pulmonary function parameters. AMPI in the central airways was not significantly correlated with FEV1 and FEV1/FVC both in former and current smokers with or without COPD. Moreover, the index in the peripheral airways from former or current smokers without COPD was not also significantly correlated with FEV1 and FEV1/FVC. However, the index in the peripheral airways showed a significant correlation with FEV1 (r = −0.74, p = 0.01) and FEV1/FVC (r = −0.70, p = 0.02) in former smokers with COPD (Fig. 2). In addition, in current smokers with COPD, the index in the peripheral airways was also significantly correlated with FEV1 (r = −0.68, p = 0.01) and FEV1/FVC (r = −0.88, p = 0.001) (Fig. 3). In contrast, AMPI in the central or peripheral airways was not significantly correlated with DLco both in former and current smokers with or without COPD.
      Fig. 2
      Fig. 2Correlations between airway microvascular permeability index and degree of airway obstruction (FEV1 (% predicted) and FEV1/FVC) in former smokers with COPD.
      Fig. 3
      Fig. 3Correlations between airway microvascular permeability index and degree of airway obstruction (FEV1 (% predicted) and FEV1/FVC) in current smokers with COPD.

      4. Discussion

      In the present study, we found that AMPI in the central airways was relatively low, and that this index was comparable among the five groups. Compared with the central airways, the index in the peripheral airways was significantly higher in smokers with or without COPD. A previous study reported that smoking-induced airway inflammation mainly originates in the peripheral airways [
      • Mahmood M.Q.
      • Sohal S.S.
      • Shukla S.D.
      • Ward C.
      • Hardikar A.
      • Noor W.D.
      • Muller H.K.
      • Knight D.A.
      • Walters E.H.
      Epithelial mesenchymal transition in smokers: large versus small airways and relation to airflow obstruction.
      ]. We have also found that levels of biomarkers reflecting airway inflammation and oxidative stress in ELF from smokers with or without COPD were significantly higher in the peripheral airways than in the central airways [
      • Kanazawa H.
      • Yoshikawa J J.
      Elevated oxidative stress and reciprocal reduction of vascular endothelial growth factor levels with severity of chronic obstructive pulmonary disease.
      ]. Therefore, it is plausible that the peripheral airways are sites of intense airway microvascular permeability as a consequence of smoking. Moreover, an important pathological feature of COPD is chronic airway inflammation in the peripheral airways [
      • Berg K.
      • Joanne L.
      • Wright J.L.
      The pathology of chronic obstructive pulmonary disease.
      ]. Actually, AMPI in the peripheral airways was markedly increased in smokers with COPD. From the viewpoint, it appears that cigarette smoking may cause excessive oxidative stress and induce inflammatory mediators in the peripheral airways of COPD, thereby leading to up-regulation of airway microvascular permeability. However, in this study, the markers of disease activity (i.e. symptom scores, exacerbation history, rate of lung function decline) were not evaluated in patients with COPD. Further studies will be required to examine the association between the markers of disease activity and AMPI.
      The second novel finding is that there was no significant difference in AMPI in the peripheral airways between former and current smokers without COPD. However, this index in the peripheral airways was significantly higher in current smokers than in former smokers with COPD. Thus, current cigarette smoking augments airway microvascular permeability in the peripheral airways of COPD per se. Despite its clinical importance, there were few published data on the effects of current cigarette smoking on airway microvascular system in various airway disorders. However, the microvascular system in the airway mucosa of COPD patients have recently aroused much interest for pulmonary investigators [
      • Hueper K.
      • Vogel-Claussen J.
      • Parikh M.A.
      • Austin J.H.M.
      • Bluemke D.A.
      • Carr J.
      • Choi J.
      • Goldstein T.A.
      • Gomes A.S.
      • Hoffman E.A.
      • Kawut S.M.
      • Limal J.
      • Michos E.D.
      • Post W.S.
      • Po1 M.J.
      • Prince M.R.
      • Liu K.
      • Rabinowitz D.
      • Skrok J.
      • Smith B.M.
      • Watson K.
      • Yin Y.
      • Zambeli-Ljepovic A.M.
      • Barr R.G.
      Pulmonary microvascular blood flow in mild chronic obstructive pulmonary disease and emphysema: the MESA COPD study.
      ]. Our findings in this study clearly show that current smoking has undesirable effects on airway microvascular system in COPD.
      Another novel finding in this study is that AMPI in the peripheral airways, but not in the central airways, showed a significant correlation with FEV1 and FEV1/FVC both in former and current smokers with COPD. It is widely accepted that cigarette smoking has detrimental effects on lung function in COPD. For example, cigarette smoking accelerated the decline in lung function [
      • Bhatt S.P.
      • Soler X.
      • Wang X.
      • Murray S.
      • Anzueto A.R.
      • Beaty T.H.
      • Boriek A.M.
      • Casaburi R.
      • Criner G.J.
      • Diaz A.A.
      • Dransfield M.T.
      • Curran-Everett D.
      • Galba´n C.J.
      • Hoffman E.A.
      • Hogg J.C.
      • Kazerooni E.A.
      • Kim V.
      • Kinney G.L.
      • Lagstein A.
      • Lynch d.A.
      • Make B.J.
      • Martinez F.J.
      • Ramsdell J.W.
      • Reddy R.
      • Ross B.D.
      • Rossiter H.B.
      • Steiner R.M.
      • Strand M.J.
      • van Beek E.J.R.
      • Wan E.S.
      • Washko G.R.
      • Wells J.M.
      • Wendt C.H.
      • Wise R.A.
      • Silverman E.K.
      • Crapo J.D.
      • Bowler R.P.
      • Han M.K.
      For the COPD Gene Investigators. Association between functional small airway disease and FEV1 decline in chronic obstructive pulmonary disease.
      ]. However, there is limited information on the effects of airway microvascular permeability on airway function in COPD. In this study, the DLCO value was similar in former and current smokers with COPD, suggesting that these two groups have similar level in alveolar destruction. However, FEV1 and FEV/FVC were significantly lower in current smokers than in former smokers with COPD. In addition, AMPI was also significantly higher in current smokers with COPD. These findings suggest the possibility that increased airway microvascular permeability may cause progressive airway obstruction in COPD. Moreover, the difference in AMPI between emphysematous and bronchitis phenotypes of COPD may be important for the treatment of this disease. In particular, increased airway microvascular permeability in the peripheral airways may lead to high risk of a frequent-exacerbation phenotype of COPD [
      • Hurst J.R.
      • Vestbo J.
      • Anzueto A.
      • Locantore N.
      • Müllerova H.
      • Tal-Singer R.
      • Miller B.
      • Lomas D.A.
      • Agusti A.
      • MacNee W.
      • Calverley P.
      • Rennard S.
      • Wouters E.F.M.
      • Wedzicha J.A.
      For the evaluation of COPD longitudinally to identify predictive surrogate endpoints (ECLIPSE) investigators. Susceptibility to exacerbation in chronic obstructive pulmonary disease.
      ].
      Cigarette smoking is the main risk factor for persistent airway inflammation in COPD, which contributes to irreversible pathological changes in the lung, such as airway remodeling. Moreover, it is well known that smoking-induced airway remodeling mainly originate in the peripheral airways [
      • Koo H.-K.
      • Vasilescu D.M.
      • Booth S.
      • Hsieh A.
      • Katsamenis O.L.
      • Fishbane N.
      • Elliott W.M.
      • Kirby M.
      • Lackie P.
      • Sinclair I.
      • Warner J.A.
      • Cooper J.D.
      • Coxson H.O.
      • Paré P.D.
      • Hogg J.C.
      • Hackett T.-L.
      Small airways disease in mild and moderate chronic obstructive pulmonary disease: a cross-sectional study.
      ]. Both inflammatory and remodeling processes in the peripheral airways of COPD are believed to be related to the resultant obstructive changes [
      • Hogg J.C.
      • McDonough J.E.
      • Suzuki M.
      Small airway obstruction in COPD: new insights based on micro-CT imaging and MRI imaging.
      ,
      • Jones R.L.
      • Noble P.B.
      • Elliot J.G.
      • James A.L.
      Airway remodeling in COPD: it's not asthma.
      ]. In addition, COPD is also characterized by excessive mucus secretion. An uncontrolled and prolonged inflammatory response may cause excessive mucus secretion, as chronic inflammation continues even after smoking cessation. Therefore, increased airway microvascular permeability in former and current smokers with COPD can contribute to formation of luminal mucus plugs, thereby reducing the airway lumen, which in turn causes airway obstruction [
      • Allinson J.P.
      • Hardy R.
      • Donaldson G.C.
      • Shaheen S.O.
      • Kuh D.
      • Wedzicha J.A.
      The presence of chronic mucus hypersecretion across adult life in relation to chronic obstructive pulmonary disease development.
      ]. In contrast, since smokers without COPD lacks the high intensity of small airway inflammation and remodeling, there was no significant correlation between obstructive changes in lung function and AMPI. The smoking-related airway microvascular hyperpermeability in the peripheral airways may, partly, impose a major risk for the development of COPD.
      The bronchoscopic microsampling technique has methodological advantages compared with induced sputum or bronchoalveolar lavage fluid (BAL) sampling technique by enabling quantitative analysis. These advantages were enable us to investigate airway inflammation in the central or peripheral airways separately [
      • Kanazawa H.
      • Kodama T.
      • Asai K.
      • Matsumura S.
      • Hirata K.
      Increased levels of Nε-(Carboxymethyl)Lysine in epithelial lining fluid from peripheral airways in patients with chronic obstructive pulmonary disease: a pilot study.
      ]. Thus, using our microsampling technique, we for the first time determined that increased airway microvascular permeability may a pivotal role on airway obstruction in patients with COPD.
      However, there are several limitations in this study to reinforce our results. First, the number of subjects enrolled in our study was relatively small. However, it is difficult that a large number of non-smoking subjects were enrolled in this study for an ethical reason. Second, all COPD patients in this study were mild and moderate in GOLD classification. In the future study, we will be required to investigate for the severe to very severe COPD patients. Third, there are various methods for establishing the pathophysiological characteristics of COPD, each with their own merits and limitations. There is also considerable diversity in the microsampling techniques adopted to perform and no agreed gold standard. However, the weight of evidence supports the wide use of our microsampling method for the investigating the pathogenesis of COPD.

      5. Conclusions

      In conclusion, increased microvascular permeability in the peripheral airways may be a characteristic feature in COPD. Therefore, our findings may shed new light on the role of airway microvascular permeability in the pathogenesis of COPD. It is also clear that airway microvascular permeability in the peripheral airways is a potentially interesting target for new pharmacological treatments in COPD patients. These findings are of great clinical significance, and highlight the need to consider the peripheral airways as a target in any therapeutic strategy for the treatment of COPD.

      Funding

      This work was supported by JSPS KAKENHI Grant Number 26461166 & 17K09622 .

      Authors contributions

      Dr. Kyomoto: contributed to study design; data collection and analysis; manuscript writing and preparation; and reviewing, editing, and approving the manuscript.
      Dr. Kanazawa: contributed to study design; data collection and analysis; manuscript writing and preparation; and reviewing, editing, and approving the manuscript.
      Dr. Tochino: contributed to data collection and reviewing, editing, and approving the manuscript.
      Dr. Watanabe: contributed to data collection and reviewing, editing, and approving the manuscript.
      Dr. Asai: contributed to data collection and reviewing, editing, and approving the manuscript.
      Dr. Kawaguchi: contributed to study design; participant recruitment; data analysis; manuscript writing and preparation; and reviewing, editing, and approving the manuscript.

      Competing interests

      All authors have no conflicts of interest to disclose.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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