Augmented epithelial endothelin-1 expression in refractory asthma

Article Outline

• Abstract
• Methods
  • Subjects
  • Fiberoptic bronchoscopy and laser-capture microdissection
  • Immunohistochemistry and morphometry
  • Statistical analysis
• Results
  • Transcript levels in microdissected bronchial epithelium
  • Assessment of protein expression by means of immunohistochemistry
  • Epithelial integrity and features of airway remodeling
  • Correlation analyses
• Discussion
• Acknowledgment
• Appendix. Supplementary data
• References
• Copyright

Background

Airway remodeling in patients with severe steroid-refractory asthma might result from a reduced ability of steroid therapy to limit the transcription of remodeling factors by the bronchial epithelium.

Objective

We sought to compare the levels of transcripts encoding remodeling factors in bronchial epithelium of healthy volunteers and of asthmatic patients with either steroid-sensitive or steroid-refractory disease and to correlate these levels with hallmarks of airway remodeling.

Methods

By means of real-time quantitative PCR, we assessed the levels of 14 transcripts encoding remodeling factors, matrix metalolproteinases, and extracellular matrix proteins in laser-capture microdissected bronchial epithelium of healthy volunteers, patients with mild steroid-untreated asthma, and patients with steroid-sensitive and steroid-refractory asthma (n = 8-10 in each group). Histologic features of airway remodeling and endothelin-1 (EDN1) immunolocalization were determined by using frozen specimens.

Results

Patients with steroid-refractory asthma had greater levels of EDN1 transcripts (4.1-fold increase, P = .026) and protein (P = .0009) in their bronchial epithelium compared with patients with steroid-sensitive asthma. EDN1 mRNA levels and protein expression in asthmatic patients were negatively correlated with prebronchodilator and postbronchodilator FEV₁ value (r² ≥ 0.193, P ≤ .03), and they were positively related to airway smooth muscle areas (r² = 0.253, P = .01 and r² = 0.281, P = .005 for EDN1 mRNA and protein expression, respectively).

Conclusion

Increased EDN1 synthesis by the bronchial epithelium characterizes severe refractory asthma and correlates with airway remodeling and airflow obstruction.

Clinical implications

Targeting EDN1 might represent a novel therapeutic strategy for severe steroid-refractory asthma.

Key words: Endothelin-1, severe asthma, laser microdissection, fibrosis, airway smooth muscle

Abbreviations used: ASM, Airway smooth muscle, CCL2, Chemokine (C-C motif) ligand 2, CXCL8, Chemokine (C-X-C motif) ligand 8, ECM, Extracellular matrix, EDN1, Endothelin-1, MCP, Monocyte chemotactic protein, MMP, Matrix metalloproteinase, SBM, Subepithelial basement membrane

Refractory asthma is a heterogeneous condition with different patterns of severity and diverse reasons for loss of control. Clinically, patients with refractory asthma can present with a variety of separate or overlapping conditions, including frequent exacerbations, irreversible airflow obstruction, and reduced sensitivity or resistance to corticosteroids.¹ The mechanisms behind the inability of corticosteroids to reverse airflow obstruction in refractory asthma are unclear. Persistent architectural alterations, defined as airway remodeling, might increase the thickness of the bronchial wall, thus contributing to irreversible airway narrowing in these patients.², ³

Airway remodeling is characterized by subepithelial fibrosis, with thickening of the subepithelial basement membrane (SBM), fibroblast and myofibroblast accumulation, greater expression of fibrogenic growth factors, and augmented extracellular matrix (ECM) protein deposition in the subepithelial areas of the proximal airways.² Other features of airway remodeling include an increase in airway smooth muscle (ASM) mass caused by hypertrophy and hyperplasia, goblet cell hyperplasia, and new blood vessel formation.² Notably, steroid-based therapy poorly affects the fibrotic response and the increase in ASM mass in patients with severe asthma,⁴, ⁵, ⁶, ⁷ suggesting that certain molecular pathways involved in the onset of these processes are less suppressible with corticosteroids.

Studies with bronchial biopsy samples and with isolated cells suggested that an aberrant repair of the airway epithelium to injury is a major event that precipitates airway remodeling in asthma.⁸ Thus abnormally repaired epithelium becomes a prominent source of ECM components, fibrogenic cytokines, chemokines, and growth factors that sustain activation and proliferation of fibroblasts and ASM cells and promote the differentiation of fibroblasts into myofibroblasts.⁸ Although this hypothesis has been widely discussed in the literature, only a limited number of studies addressed this issue in patients with refractory asthma.⁹, ¹⁰, ¹¹, ¹², ¹³ In addition, none of these reports has used a quantitative approach targeting specifically epithelial cell transcript expression.

In the present study we hypothesized that the epithelial expression of certain transcripts encoding remodeling factors would be relatively resistant to suppression by steroid therapy in patients with refractory asthma. We applied the technique of laser-capture microdissection to collect bronchial epithelial cells from endobronchial biopsy samples of healthy control individuals, patients with mild steroid-untreated asthma, and patients with severe steroid-sensitive and steroid-refractory asthma. We assessed quantitatively the levels of the transcripts encoding fibrogenic cytokines, chemokines, and growth factors, namely IL-8/chemokine (C-X-C motif) ligand 8 (CXCL8),¹⁴ monocyte chemotactic protein (MCP) 1/chemokine (C-C motif) ligand 2 (CCL2),¹⁵ TGF-α, TGF-β1 and TGF-β2,¹⁶ the fibrotic and ASM-proliferating peptide endothelin-1 (EDN1),², ¹⁷ and some ECM proteins and their degrading matrix metalloproteinases (MMPs).¹⁸ The immunolocalization of selected encoded proteins was determined in frozen tissue sections. The expression of the epithelial transcripts encoding factors involved in airway remodeling was correlated with the degree of airflow obstruction and with hallmarks of airway remodeling, including the thickness of the SBM, the accumulation of fibroblasts/myofibroblasts in the bronchial submucosa, and the increase in ASM area.

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Methods 

Subjects 

Ten healthy volunteers, 10 patients with mild steroid-untreated asthma, and 18 patients with severe steroid-treated asthma fulfilling National Institute of Health criteria¹⁹ were recruited (see Table E1 in the Online Repository at www.jacionline.org). All subjects had never smoked. A flow-volume curve was performed in all subjects, and FEV₁ was assessed before and after the inhalation of 400 μg of salbutamol. All subjects provided written informed consent, and the protocol was approved by the Hôtel Dieu hospital Ethics Committee (CP 02819). Patients with severe steroid-treated asthma were further classified as steroid sensitive (n = 10) or steroid refractory (n = 8) on the basis of the reversibility of their airflow obstruction (prebronchodilator and postbronchodilator FEV₁ of ≥80% and ≤60% of predicted value, respectively) on 12 to 18 months of short- and long-lasting β₂-agonist and high-dose inhaled steroid therapy (mean daily dose of inhaled beclomethasone equivalents, 3200 and 3600 μg in patients with steroid-sensitive and steroid-refractory asthma, respectively) and at least 1 course of oral prednisone (see Table E1 and the Methods section of the Online Repository at www.jacionline.org).¹

Fiberoptic bronchoscopy and laser-capture microdissection 

Bronchial biopsy specimens were obtained by means of fiberoptic bronchoscopy,⁷, ²⁰ and bronchial epithelial cells were collected by means of laser-capture microdissection (PALM Microlaser Technologies, Bernried, Germany) on toluidine blue–stained frozen tissue sections (see Fig E1 in this article's Online Repository at www.jacionline.org). Total RNA was extracted, and RNA polyA was amplified and reverse transcribed with Moloney Murine Leukemia Virus enzyme (Invitrogen, Cergy-Pontoise, France). The expression of 17 transcripts, including 3 housekeeping genes, was evaluated by using quantitative real-time PCR with the Mx 3000P apparatus (Stratagene Europe, Amsterdam, The Netherlands). Only genes with cycle thresholds of less than 35 were included in the analysis. The expression of each transcript was normalized to the geometric mean of the levels of transcripts encoding the 3 selected most stable housekeeping genes: ubiquitin C, succinate dehydrogenase, and ribosomal protein 13a. Selection of housekeeping genes, calculation, and normalization were performed with geNorm software.²¹ Primers were designed with Primer Express 2 Software (Applied Biosystems, Foster City, Calif) and were synthesized with Genosys (Sigma, Lyon, France) (see Table E2 in this article's Online Repository at www.jacionline.org).

Immunohistochemistry and morphometry 

Five-micrometer frozen biopsy sections were incubated with mouse mAbs directed against EDN1, α-actin, or propyl-4-hydroxylase (fibroblasts/myofibroblasts).⁷ An anti-mouse antibody conjugated to biotin was then added, followed by the avidin–alkaline phosphatase complex from the Vectastain kit (Vector, Burlingame, Calif), Fast Red staining, and light nuclear Mayer's hematoxylin counterstaining.⁸ Epithelial cells expressing EDN1 (in percentage of total epithelial cells) and propyl-4-hydroxylase (fibroblasts/myofibroblasts)–positive cells per millimeter of SBM were enumerated.⁷ Computer-assisted image analysis was used to assess morphologically intact epithelium (in percentages), SBM thickness (in micrometers) and ASM area (in percentage of total biopsy area) on Mayer's hematoxylin– and α-actin–stained tissue sections, respectively.⁷, ²²

Statistical analysis 

Data were analyzed statistically by using the StatView SE+Graphics program for Macintosh (Abacus Concepts, Berkeley, Calif). Results are expressed as medians (interquartile ranges). A nonparametric Kruskal-Wallis analysis of variance with the Benjamini and Hochberg correction for multiple testing of the 14 transcripts was performed. When the results of this test were significant, the Mann-Whitney U test, followed by the Benjamini and Hochberg correction, was used to determine significance across the groups. An overall P value of .05 or less was considered significant.

Additional information on the methods used is provided in the Online Repository at www.jacionline.org.

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Results 

Transcript levels in microdissected bronchial epithelium 

Using quantitative real-time PCR, we found detectable amounts of the transcripts encoding all the factors tested (Table I; Table E3 in the Online Repository at www.jacionline.org). The highest expression was found for tenascin C, TGF-α, MMP-2, and fibronectin 1 (values of cycle threshold ranging from 22.6 to 25.6, Table I). Exemplary original quantitative real-time PCR amplification plots are shown in Fig E2 of the Online Repository at www.jacionline.org.

Table I. Levels of transcripts in laser-capture microdissected bronchial epithelium from control subjects and patients with steroid-untreated and steroid-treated asthma

Ct, Cycle threshold; FN1, fibronectin 1; TNC, tenascin C; COL, collagen.

By using the correction for multiple testing, only the differences in levels of the transcripts encoding IL-8/CXCL8 and EDN1 across the 4 study groups reached statistical significance (Table I; Table E3). Changes in the epithelial expression of MCP-1/CCL2 and MMP-9 mRNA was not significant by using the correction for multiple comparisons, despite the overall significance with use of the Kruskal-Wallis test (P = .011 and P = .04, respectively; Table I; Table E3).

When compared with control subjects, patients with mild steroid-untreated asthma had significantly higher epithelial amounts of the transcripts encoding EDN1 (2.6-fold increase, P = .038) and a 4.7-fold increase in IL-8/CXCL8 mRNA expression, although this latter difference failed to reach significance (P = .227). The levels of IL-8/CXCL8 and EDN1 mRNA were similar in patients with steroid-sensitive asthma compared with those in their counterparts with mild steroid-untreated asthma (Table I). In contrast, patients with steroid-refractory asthma had greater epithelial expression of EDN1 mRNA compared with tat seen in control subjects, patients with mild steroid-untreated asthma, and patients with steroid-sensitive asthma (8.8-, 3.2-, and 4.1-fold increase and P = .001, P = .024, and P = .026, respectively; Table I). This was accompanied by higher levels of IL-8/CXCL8 transcripts in relation to those seen in patients with steroid-sensitive asthma (P = .021, Table I).

Assessment of protein expression by means of immunohistochemistry 

As the most relevant and novel finding related to changes in the epithelial levels of the transcripts encoding EDN1, the immunolocalization of the corresponding encoded protein was assessed in frozen biopsy sections collected from the same control and asthmatic subjects used for microdissection studies (Fig 1).

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• Fig 1. 

  Representative immunostaining of EDN1 in bronchial biopsy specimens from control subjects (A), a patient with mild steroid-untreated asthma (B), and patients with severe steroid-sensitive (C) and steroid-refractory (D) asthma is shown. Scale bars = 250 μm. E, proportion of EDN1-positive epithelial cells per millimeter of SBM in bronchial biopsy specimens from control subjects and the 3 groups of asthmatic patients. Bars represent median values, and error bars denote interquartile ranges.

Normal bronchial epithelium failed to show significant immunoreactivity for EDN1 (Fig 1, A), and scarce positive cells were found in patients with mild steroid-untreated asthma (Fig 1, B). Patients with steroid-sensitive asthma exhibit an irregular pattern of expression, with the majority of EDN1 expression being confined on the luminal surface of the epithelium (Fig 1, C). Some weak and discontinuous EDN1 immunostaining was also seen in basal cells, irrespective of the group of asthmatic patients analyzed (ie, patients with mild steroid-untreated or steroid-sensitive asthma; Fig 1, B and C). In patients with steroid-refractory asthma EDN1 immunostaining was visibly increased and occurred uniformly throughout the epithelial layer (Fig 1, D). Quantification analysis showed a significantly greater proportion of epithelial cells staining for EDN1 in the severe asthma groups compared with that in the control group (1.9- and 2.6-fold increase and P = .0002 and P = .0004, respectively; Fig 1, E). EDN1 epithelial immunostaining was significantly increased in patients with steroid-refractory asthma in relation to that seen in patients with mild steroid-untreated asthma (1.7-fold increase and P = .002; Fig 1, E) and patients with steroid-sensitive asthma (1.4-fold increase and P = .0009; Fig 1, E).

Epithelial integrity and features of airway remodeling 

A significant decrease in the proportion of the epithelial layer with an intact structure was seen in patients with mild steroid-untreated asthma compared with that seen in control subjects (Fig 2, A). Patients with severe steroid-sensitive and steroid-refractory asthma showed an apparent epithelial restitution, and the extent of morphologically intact epithelium in these latter groups was comparable with that measured in control subjects (Fig 2, A). SBM was thicker in the 3 groups of asthmatic patients than in the control subjects. Both patients with steroid-sensitive and steroid-refractory asthma had greater SBM thickness in relation to patients with mild steroid-untreated asthma (Fig 2, B). Scant mucosal fibroblasts/myofibroblasts (propyl-4-hydroxylase–positive cells) were enumerated in control individuals (Figs 1, E, and 3, C). These numbers were similar in patients with mild steroid-untreated asthma (Fig 3, B and E), and they were significantly augmented in patients with severe asthma with either steroid-sensitive or steroid-refractory disease (Fig 3, C, D, and E). ASM area was larger in patients with steroid-sensitive and steroid-refractory asthma in relation to that seen in control subjects (Fig 2, C). However, patients with steroid-refractory asthma had the highest ASM areas, with values statistically significant compared with those of patients with mild steroid-untreated asthma (P = .049) but not those of patients with severe steroid-sensitive asthma (P = 0.112; Fig 2, C).

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• Fig 2. 

  Epithelial integrity (A), SBM thickness (B), and ASM areas (C) in bronchial biopsy specimens from control subjects and asthmatic patients are shown. Bars represent median values, and error bars denote interquartile ranges.

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• Fig 3. 

  Representative immunostaining of fibroblasts and myofibroblasts (propyl-4-hydroxylase–positive cells) in bronchial biopsy specimens from a control subject (A), a patient with mild steroid-untreated asthma (B), and patients with severe steroid-sensitive (C) and refractory (D) asthma is shown. Scale bars = 250 μm. E, Numbers of fibroblasts/myofibroblasts per millimeter of SBM in bronchial biopsy specimens from control subjects and the 3 groups of asthmatic patients. Bars represent median values, and error bars denote interquartile ranges.

Correlation analyses 

By means of univariate regression analysis (the Spearman rank-order method), we found that the individual epithelial levels of the transcripts encoding IL-8/CXCL8 and EDN1 correlated negatively with prebronchodilator and postbronchodilator FEV₁, whereas a positive correlation was found for MCP-1/CCL2 mRNA (Table II). In addition, EDN1 and IL-8/CXCL8 transcripts correlated positively between each other (r² = 0.323, P = .005), and EDN1 mRNA was positively related to ASM areas (Table II).

Table II. Univariate correlation analyses in asthmatic patients (n = 28, Spearman rank-order method)

NS, Not significant.

None of these transcripts correlated with the extent of epithelial integrity. In addition, the proportion of epithelial cells expressing EDN1 negatively correlated with prebronchodilator and postbronchodilator FEV₁ values (P = .0004 and P = .022, respectively; Table II) and was positively related to ASM areas (P = .002, Table II) and to EDN1 mRNA (r² = 0.195, P = .02).

The main significant correlations are shown in Figs E3 and E4 in the Online Repository at www.jacionline.org.

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Discussion 

Using laser-capture microdissection coupled to quantitative real-time PCR, we demonstrated that the levels of IL-8/CXCL8 and EDN1 mRNA were significantly augmented in the bronchial epithelium from patients with refractory asthma. These subjects' FEV₁ values failed to improve to greater than 70% of predicted value and less than 15% from baseline value after 14 days of treatment with 0.75 mg/kg/d oral prednisone, and they maintain severe airflow obstruction despite long-term treatment with high doses of inhaled steroids. A group of patients with sensitive asthma (ie, asthmatic patients normalizing their lung function on a similar treatment regimen) was examined in parallel to establish whether the observed transcriptional modulations were due to asthma severity or to steroid use. Finally, a group of patients with mild steroid-untreated asthma was included to determine the levels of the expression of the different transcripts in the absence of a potential confounding effect linked to steroid therapy. The expression of EDN1 mRNA was correlated with epithelial integrity and with hallmarks of airway remodeling that have been previously associated with asthma severity, such as SBM thickness, mucosal fibroblast numbers, and ASM area.⁷, ¹⁰, ²³

When compared with healthy control individuals, patients with mild asthma had significantly higher epithelial levels of the transcripts encoding EDN1 and a trend toward a greater expression IL-8/CXCL8 mRNA. This was accompanied by a significant SBM thickness, thus corroborating previous observations showing that the production of these molecules, together with features of airway remodeling, are present in patients with mild asthma with normal respiratory function.²⁴, ²⁵, ²⁶ In our hands the levels of TGF-α, TGF-β1, TGF-β2, and MMP-9 mRNA were not significantly modulated in patients with mild asthma, suggesting that the bronchial epithelium might not represent the main source of these factors in the airways. Supporting this hypothesis, previous studies have shown that TGF-β1, TGF-β2, and MMP-9 originate from mucosal eosinophils²³, ²⁷ and that neutrophils contribute substantially to MMP-9 production in the airways.⁴

An increase in the epithelial levels of IL-8/CXCL8 was observed in patients with steroid-refractory asthma compared with that seen in patients with steroid-sensitive asthma, a finding that extends previous observations showing a greater immunoreactivity for this chemokine in the bronchial epithelium and higher levels in induced sputum of patients with severe asthma.¹², ²⁸ IL-8/CXCL8 overexpression is believed to contribute to neutrophilic inflammation but also to airway remodeling by promoting ASM cell contraction and migration.¹⁴

The most salient finding of the current report relates to epithelial EDN1 transcript, the increased levels of which distinguished patients with steroid-refractory asthma from patients with steroid-sensitive asthma. Immunohistochemical analysis corroborated the data obtained with quantitative real-time PCR because they illustrate a striking disease-related increase in the proportion of epithelial cells expressing EDN1, with the highest immunostaining observed in patients with refractory asthma.

EDN1 is a potent bronchoconstrictor 21-amino-acid peptide that plays an important role in the pathogenesis of airway remodeling through the activation of several functions in structural cells.¹⁷ These functions include the synthesis of ECM proteins from lung fibroblasts and bronchial epithelial cells,²⁹, ³⁰, ³¹ the differentiation of fibroblasts into myofibroblasts,³² and the proliferation of ASM cells, fibroblasts, and myofibroblasts.³³, ³⁴, ³⁵ Studies with bronchial biopsy samples demonstrated that EDN1 is weakly expressed by the normal airway epithelium and that this expression is upregulated in mild and moderate asthma.³⁶, ³⁷ Conflicting observations, however, have been reported concerning the effect of corticosteroids on EDN1 expression. Hence although immunoreactive EDN1 was downregulated in vivo by steroid therapy,³⁷ in vitro studies with human bronchial epithelial cells demonstrated failure by dexamethasone to prevent EDN1 synthesis in response to TNF-α, most likely because of the absence of any identifiable glucocorticoid-responsive element in the EDN1 promoter region.³⁸ It was speculated that in vivo inhibition of epithelial EDN1 expression observed in patients with steroid-treated asthma resulted from an indirect suppressive effect on the synthesis of TNF-α,³⁸ a cytokine the levels of which continue to be increased in the airways of patients with severe asthma despite steroid therapy.³⁹ Although the expression of TNF-α has not been examined in this study, these observations suggest that sustained local generation of this cytokine might contribute to the epithelial EDN1 overexpression presently reported.

Finally, we ascertained whether the epithelial levels of transcripts encoding IL-8/CXCL8 and EDN1 varied in relation with asthma severity and with the extent of epithelial integrity and airway remodeling. Although the assessment of airway epithelium integrity by means of bronchial biopsy is confounded by an artifactual cell desquamation caused by tissue sampling,⁴⁰ we found a loss of epithelial integrity in patients with mild steroid-untreated asthma, whereas in patients with severe asthma with either sensitive or refractory disease, the airway epithelium appeared fully restored, probably as a result of high-dose long-term steroid therapy.⁷, ⁴¹

The assessment of airway remodeling demonstrated the existence of larger ASM areas, greater SBM thickness, and higher mucosal fibroblast/myofibroblast numbers in patients with severe steroid-refractory and steroid-sensitive asthma compared with that seen in patients with mild asthma and control subjects. These results substantiate in part our previous observations performed in distinct subjects and showing that the main bronchial wall changes seen in patients with severe asthma consist of fibroblast/myofibroblast accumulation beneath the SBM and in increased ASM mass.⁷

Univariate regression analyses demonstrated that epithelial IL-8/CXCL8 and EDN1 transcripts, as well as the proportion of EDN1-positive epithelial cells, negatively correlated with prebronchodilator and postbronchodilator FEV₁ values. However, only EDN1 transcript and protein showed positive correlation with ASM areas. These correlations argue for an important role played by EDN1 in the pathogenesis of the remodeling response in the airways, particularly of ASM hypertrophy and hyperplasia, and in the respiratory function abnormalities seen in patients with refractory asthma. Finally, neither the levels of the EDN1 and IL-8/CXCL8 transcripts nor the extent of EDN1 immunostaining correlated with the proportion of morphologically intact epithelium, suggesting that the observed differences in the expression of these molecules between patients with severe steroid-sensitive and patients with steroid-refractory asthma reflect functional abnormalities of the bronchial epithelium rather than a disparity of its integrity.

Phase III trials are ongoing on the effects of selective EDN1 receptor antagonists in idiopathic pulmonary fibrosis,⁴² a severe and irreversible lung disease characterized by an abnormal repair of the alveolar epithelium and by the accumulation of fibroblasts and ECM components in the peripheral airways.⁴³ The current report proposes the new concept that limiting EDN1 production might also represent a novel and valuable therapeutic tool for attenuating airway remodeling and respiratory function deterioration in patients with severe steroid-refractory asthma.

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We thank the control and asthmatic subjects who participated in the study. We also thank the staff of the Service d'Explorations Fonctionnelles of the Hôpital Bichat who performed lung function measurements, Professor A. Janin and Dr L. Legrès (Inserm U728, Centre Hospitalier Universitaire St Louis), and Professor M. Peuchmaur (Platform of Microssection of the Inserm, Institut Fédératif de Recherche 02, Centre Hospitalier Universitaire Robert Debré, Université Paris 7) for technical help during initial laser-capture microdissection experiments, and Professor France Mentré (Inserm U738, Centre Hospitalier Universitaire Bichat-Claude Bernard) for expert statistical advice.

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Appendix. Supplementary data 

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