Expression of smooth muscle and extracellular matrix proteins in relation to airway function in asthma Annelies M. Slats, MD,a Kirsten Janssen, BHe,a Annemarie van Schadewijk, MSc,a Dirk T. van der Plas, BSc,a Robert Schot, BSc,a Joost G. van den Aardweg, MD, PhD,b Johan C. de Jongste, MD, PhD,c Pieter S. Hiemstra, PhD,a Thais Mauad, MD,d Klaus F. Rabe, MD, PhD,a and Peter J. Sterk, MD, PhDa,e Leiden, Alkmaar, Rotterdam, and Amsterdam, The Netherlands, and Sa˜o Paulo, Brazil Background: Smooth muscle content is increased within the selective expression of airway smooth muscle proteins and airway wall in patients with asthma and is likely to play a role in components of the extracellular matrix. (J Allergy Clin airway hyperresponsiveness. However, smooth muscle cells Immunol 2008;121:1196-202.) express several contractile and structural proteins, and each of these proteins may influence airway function distinctly. Key words: Actin, desmin, elastin, airway smooth muscle, extracel- Objective: We examined the expression of contractile and lular matrix, lung function, hyperresponsiveness, deep inspiration- structural proteins of smooth muscle cells, as well as induced bronchodilation, bronchial biopsies extracellular matrix proteins, in bronchial biopsies of patients with asthma, and related these to lung function, airway hyperresponsiveness, and responses to deep inspiration. Asthma is characterized by chronic airway inflammation, Methods: Thirteen patients with asthma (mild persistent, which is presumed to contribute to variable airways obstruction 1 atopic, nonsmoking) participated in this cross-sectional study. and bronchial hyperresponsiveness. However, recent studies FEV1% predicted, PC20 methacholine, and resistance of the have led to a reappraisal of the role of airway smooth muscle in 2 respiratory system by the forced oscillation technique during asthma pathophysiology. Because smooth muscle contraction tidal breathing and deep breath were measured. Within 1 week, leads to airway narrowing, abnormalities in airway smooth mus- a bronchoscopy was performed to obtain 6 bronchial biopsies cle size, mass, or function could easily lead to exaggerated airway that were immunohistochemically stained for a-SM-actin, narrowing. In addition, mast cells within the airway smooth desmin, myosin light chain kinase (MLCK), myosin, calponin, muscle bundles have been associated with airway hyperrespon- 3 vimentin, elastin, type III collagen, and fibronectin. The level of siveness, and more recently, we observed a similar association expression was determined by automated densitometry. with impaired deep inspiration-induced bronchodilation in 4 Results: PC20 methacholine was inversely related to the patients with asthma. expression of a-smooth muscle actin (r 520.62), desmin Increased smooth muscle mass has been demonstrated in 5-7 8,9 (r 520.56), and elastin (r 520.78). In addition, FEV1% bronchial biopsies as well as in resected lung tissue from pa- predicted was positively related and deep inspiration-induced tients with asthma compared with healthy subjects. Mathematical bronchodilation inversely related to desmin (r 520.60), MLCK models have shown that increased smooth muscle mass can (r 520.60), and calponin (r 520.54) expression. explain exaggerated airway narrowing to contractile stimuli in pa- 10 11 Conclusion: Airway hyperresponsiveness, FEV1% predicted, tients with asthma, especially at high lung volumes. Interest- and airway responses to deep inspiration are associated with ingly, although increased smooth muscle area in bronchial biopsies has been associated with impaired lung function,5,7 no relationship was found with airway hyperresponsiveness. Never- From athe Department of Pulmonology, Leiden University Medical Center; bthe Depart- theless, in vitro studies have shown that smooth muscle cells ob- ment of Pulmonology, Medical Center Alkmaar; cthe Department of Pediatrics, Eras- tained from bronchial biopsies of patients with asthma exhibit an mus University Medical Center, Sophia Children’s Hospital, Rotterdam; dthe increase in isotonic shortening12 and shortening velocity com- e Department of Pathology, Sa˜o Paulo University Medical School; and the Department pared with controls without asthma.13 of Respiratory Medicine, Academic Medical Center, University of Amsterdam. Supported by the Netherlands Asthma Foundation (3.2.02.34). Smooth muscle cells express several contractile and structural 14,15 Disclosure of potential conflict of interest: J. C. de Jongste has received research support proteins. Cultured airway smooth muscle cells with a con- from Aerocrine. K. F. Rabe has consulting arrangements with AstraZeneca, tractile phenotype are relatively rich in smooth muscle myosin Boehringer Ingelheim, Novartis, Pfizer, Altana, GlaxoSmithKline, and Roche; has heavy chain (sm-MHC), a-smooth muscle actin (a-SM-actin), received research support from AstraZeneca, Merck, Altana, and Boehringer calponin, desmin, and myosin light chain kinase (MLCK), Ingelheim; and is on the speakers’ bureau for AstraZeneca, Boehringer Ingelhim, Novartis, Pfizer, Altana, GlaxoSmithKline, and Roche. P. S. Hiemstra has received whereas when proliferating they express less sm-MHC, calponin, research support from AltanaPharma, Novartis, Bayer, AstraZeneca, Pfizer, Merck, a-SM-actin, and desmin, and significantly more vimentin. Be- Exhale Therapeutics, Boehringer Ingelheim, Roche, and GlaxoSmithKline. The rest of nayoun et al5 examined the expression of some of these contrac- the authors have declared that they have no conflict of interest. tile proteins in bronchial biopsies in relation to asthma severity. Received for publication August 9, 2007; revised January 3, 2008; accepted for publica- tion February 14, 2008. MLCK expression correlated inversely with lung function, but Available online April 14, 2008. this was not the case for the proteins a-SM-actin or myosin. Reprint requests: Annelies M. Slats, MD, Leiden University Medical Center, Department This suggests that the level of expression of these proteins may of Pulmonology (C2-P-62), PO Box 9600, 2300 RC Leiden, The Netherlands. E-mail: have different functional consequences. We selected several con- [email protected]. tractile and structural proteins that may influence airway respon- 0091-6749/$34.00 Ó 2008 American Academy of Allergy, Asthma & Immunology siveness and function, namely a-SM-actin, myosin, desmin, doi:10.1016/j.jaci.2008.02.017 vimentin, calponin, and MLCK. Furthermore, it has been shown 1196 J ALLERGY CLIN IMMUNOL SLATS ET AL 1197 VOLUME 121, NUMBER 5 4 single dose of methacholine that induced a 20% fall in FEV1. Within Abbreviations used 1 week of the last visit, a bronchoscopy was performed to obtain 6 bronchial a-SM-actin: a-Smooth muscle actin biopsies. MLCK: Myosin light chain kinase Rrs: Resistance of the respiratory system Airway hyperresponsiveness RrsExp: Resistance of the respiratory system during tidal expirations Methacholine bromide in normal saline was used for the bronchial challenges that were performed by standardized methodology.26 At 5-minute RrsInsp: Resistance of the respiratory system during tidal inspirations intervals, aerosolized serial doubling concentrations of methacholine (0.15- sm-MHC: Smooth muscle myosin heavy chain 40 mmol/L) were inhaled by tidal breathing (DeVilbiss, Somerset, Pa) for 2 minutes with the nose clipped. The challenge was stopped when FEV1 drop- ped by more than 20% from baseline, and the response was expressed as the provocative concentration causing a 20% fall in FEV1 PC20. that stretch of smooth muscle cells can increase the expression of contractile proteins.16,17 Because smooth muscle cells are most Airway responses to deep inspiration likely stretched during deep inspiration, we analyzed the relation- Deep inspiration–induced bronchodilation was measured using a single- ship between protein expression and airway responses to deep dose methacholine challenge to induce a fall in FEV1 of 20% in the absence of inspiration. deep inspirations before methacholine inhalation. Baseline measurements of In addition, the smooth muscle bundles are embedded in and FEV1 and Rrs were followed by a period of 20 minutes without deep inspira- also contain extracellular matrix. The amount and the composi- tions. A single dose of methacholine (approximately the cumulative dose of tion of the matrix may have functional consequences by altering the PC20 of the previous challenge) was inhaled, and 2 minutes later, Rrs 18-20 was measured during tidal breathing, a deep inspiration to total lung capacity, the physical properties of the airway wall and can also influ- a passive expiration, and again tidal breathing. This was directly followed by ence the proliferation of smooth muscle cells.21-23 Therefore, we spirometry to measure the fall in FEV1. The forced oscillation technique with analyzed the expression of different extracellular matrix proteins an applied oscillation frequency of 8 Hz and an amplitude of 61 cmH2O was (type III collagen, fibronectin, and elastin) within and surround- used to measure Rrs continuously during tidal breathing and a deep inspiration ing the smooth muscle bundles. (Woolcock Institute, Sydney, Australia).4 Deep inspiration–induced broncho- We hypothesized that a higher level of expression of the selected dilation was expressed as the reduction in Rrs during tidal breathing induced contractile and structural proteins of smooth muscle cells, as well by the deep inspiration. as components of the extracellular matrix, in bronchial