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An inflammation-independent contraction mechanophenotype of airway smooth muscle in asthma

2016; Elsevier BV; Volume: 138; Issue: 1 Linguagem: Inglês

10.1016/j.jaci.2015.12.1315

1097-6825

Steven S. An, Wayne Mitzner, Wan‐Yee Tang, Kwangmi Ahn, A‐Rum Yoon, Jessie Huang, Onur Kilic, Hwan Mee Yong, Jed W. Fahey, Sarvesh Kumar, Shyam Biswal, Stephen T. Holgate, Reynold A. Panettieri, Julian Solway, Stephen B. Liggett,

Myofascial pain diagnosis and treatment

Asthma is characterized by airway inflammation and bronchial obstruction due to airway smooth muscle (ASM) contraction. However, the underlying basis of the ASM hypercontractile state in asthma is not known. It remains equally unclear whether ASM from those with asthma has an intrinsic (genetic or epigenetic) property of increased basal tone and enhanced contractile responses.1de Jongste J.C. Mons H. Bonta I.L. Kerrebijin K.F. In vitro responses of airways from an asthmatic patient.Eur J Respir Dis. 1987; 71: 23-29PubMed Google Scholar, 2Bai T.R. Abnormalities in airway smooth muscle in fatal asthma.Am Rev Respir Dis. 1990; 141: 552-557Crossref PubMed Scopus (151) Google Scholar, 3Bjorck T. Gustafsson L.E. Dahlen S.E. Isolated bronchi from asthmatics are hyperresponsive to adenosine, which apparently acts indirectly by liberating of leukotrienes and histamine.Am Rev Respir Dis. 1992; 145: 1087-1091Crossref PubMed Scopus (205) Google Scholar, 4Chin L.Y. Bossé Y. Pascoe C. Hackett T.L. Seow C.Y. Paré P.D. Mechanical properties of asthmatic airway smooth muscle.Eur Respir J. 2012; 40: 45-54Crossref PubMed Scopus (76) Google Scholar, 5Ijpma G. Kachmar L. Matusovsky O.S. Bates J.H. Benedetti A. Martin J.G. et al.Human trachealis and main bronchi smooth muscle are normoresponsive in asthma.Am J Respir Crit Care Med. 2015; 191: 884-893Crossref PubMed Scopus (46) Google Scholar Furthermore, current dogma suggests that any altered mechanical property of the smooth muscle, if it exists in the disease, is from airway inflammation. Here, we sought to establish, using highly quantitative methods, whether the contractile state of ASM from those with asthma has an inflammation-independent component. We applied recently developed single-cell technologies to probe the mechanical properties of isolated, passaged, primary human ASM cells.6An S.S. Fabry B. Trepat X. Wang N. Fredberg J.J. Do biophysical properties of the airway smooth muscle in culture predict airway hyperresponsiveness?.Am J Respir Cell Mol Biol. 2006; 35: 55-64Crossref PubMed Scopus (90) Google Scholar, 7Deshpande D.A. Wang W.C. Mcllmoyle E.L. Robinett K.S. Schillinger R.M. An S.S. et al.Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction.Nat Med. 2010; 16: 1299-1304Crossref PubMed Scopus (354) Google Scholar This approach potentially avoids the interactions between airway epithelium and smooth muscle that are encountered in bronchial sections, the limited availability of fresh tissues from donors with and without asthma, and the nonspecific effects of acute dissociation of ASM cells from other tissues in biopsies. This approach may also minimize the acute effects of drugs such as β-agonists that would be expected to be administered during attempts to treat a severe asthma exacerbation. The methods used, Fourier transform traction microscopy and magnetic twisting cytometry, can be performed on the living cells adhered to matrices of varying rigidities across a pathophysiologic spectrum. As shown in Fig 1, A, we used Fourier transform traction microscopy to measure ASM mechanics, with direct measurement of traction stress, and the derived net contractile moments, so as to characterize the physical properties of human ASM cells derived from donor lungs from patients with and without asthma (see Table E1 in this article's Online Repository at www.jacionline.org for subjects' demographic characteristics). For these studies, we used cells that had been passaged in standard culture media, in the absence of inflammatory mediators, and studied them under identical experimental conditions using tuned elastic matrices (ie, mimicking the physiological extremes of airway wall rigidity). Human ASM cells isolated from both sets of donor lungs showed the expected between-cell and between-donor variation in cell spreading (Fig 1, B) and traction (root mean square) averaged over the entire cell-projected area (Fig 1, C). Nested design analysis8Krzywinski M. Altman N. Blainey P. Points of significance: nested designs.Nat Methods. 2014; 11: 977-978Crossref PubMed Scopus (19) Google Scholar revealed significant differences in both the projected area (7779 ± 303 vs 6345 ± 199 μm2; P = .0136, nested ANOVA) and the average traction stress (166 ± 10 vs 119 ± 8 Pa; P = .0316, nested ANOVA) between those with and without asthma; there were no within-group differences. In particular, compared with ASM from those without asthma, ASM from those with asthma showed an approximately 2-fold higher (47.4 ± 4.8 vs 26.4 ± 2.4 pNm; P = .0015, nested ANOVA) net contractile moment, which is a scalar measure of the cell's resting contractile amplitude. These differences in physical force generation between those with and without asthma were maintained in culture with increasing passage number (Fig 1, D) and across the range of matrix rigidity of approximately 1 to 8 kPa (Fig 1, E). These results establish an unequivocal difference in resting force of cultured ASM cells between those with and without asthma, which is persistent and is apparent across a wide range of matrix rigidities mimicking that of healthy and diseased airways in the absence of the inflammatory airway milieu. To ascertain whether ASM cells from those with asthma display increased responsiveness to locally generated spasmogens, we then measured dynamic changes in cytoskeleton stiffness in response to histamine and methacholine with magnetic twisting cytometry, as an index of single-cell contractility.6An S.S. Fabry B. Trepat X. Wang N. Fredberg J.J. Do biophysical properties of the airway smooth muscle in culture predict airway hyperresponsiveness?.Am J Respir Cell Mol Biol. 2006; 35: 55-64Crossref PubMed Scopus (90) Google Scholar, 7Deshpande D.A. Wang W.C. Mcllmoyle E.L. Robinett K.S. Schillinger R.M. An S.S. et al.Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction.Nat Med. 2010; 16: 1299-1304Crossref PubMed Scopus (354) Google Scholar Here, RGD-coated ferrimagnetic beads are attached to ASM cell surface integrin receptors and subjected to an external magnetic field, with measurement of lateral displacement of the beads during drug exposure (see this article's Methods section in the Online Repository at www.jacionline.org). For these experiments, we used primary cells derived from 12 additional donor lungs of those with and without asthma (see Table E2 in this article's Online Repository at www.jacionline.org). On the basis of dose-response relationships (Fig E1), and experience using isolated human ASM cells, herein we chose a single dose (10 μM) of histamine or methacholine to contract the cells (Fig 2). There was heterogeneity in responses from cells of both those with and without asthma, although the latter showed greater within-group variability in the responses to both histamine (Fig 2, A and C) and methacholine (Fig 2, B and D). Using the nested effect model8Krzywinski M. Altman N. Blainey P. Points of significance: nested designs.Nat Methods. 2014; 11: 977-978Crossref PubMed Scopus (19) Google Scholar to control for random effects from multiple cells from the same donor, and the repeated measurements, we found significant differences in the cell-stiffening response to histamine (Fig 2, E) and methacholine (Fig 2, F) between those with and without asthma. These series of studies, taken together, establish for the first time that, in the resting state, ASM from those with asthma has increased basal tone and enhanced contractility to known asthmatic spasmogens. Almost 150 years ago, Henry Hyde Salter9Salter H.H. Asthma: its pathology and treatment.2nd ed. Churchill, London1868Google Scholar posited that "the vice in asthma consists, not in the production of any special irritant, but in the irritability of the part irritated." Defining the asthmatic ASM mechanical phenotype, however, has been somewhat elusive.1de Jongste J.C. Mons H. Bonta I.L. Kerrebijin K.F. In vitro responses of airways from an asthmatic patient.Eur J Respir Dis. 1987; 71: 23-29PubMed Google Scholar, 2Bai T.R. Abnormalities in airway smooth muscle in fatal asthma.Am Rev Respir Dis. 1990; 141: 552-557Crossref PubMed Scopus (151) Google Scholar, 3Bjorck T. Gustafsson L.E. Dahlen S.E. Isolated bronchi from asthmatics are hyperresponsive to adenosine, which apparently acts indirectly by liberating of leukotrienes and histamine.Am Rev Respir Dis. 1992; 145: 1087-1091Crossref PubMed Scopus (205) Google Scholar, 4Chin L.Y. Bossé Y. Pascoe C. Hackett T.L. Seow C.Y. Paré P.D. Mechanical properties of asthmatic airway smooth muscle.Eur Respir J. 2012; 40: 45-54Crossref PubMed Scopus (76) Google Scholar, 5Ijpma G. Kachmar L. Matusovsky O.S. Bates J.H. Benedetti A. Martin J.G. et al.Human trachealis and main bronchi smooth muscle are normoresponsive in asthma.Am J Respir Crit Care Med. 2015; 191: 884-893Crossref PubMed Scopus (46) Google Scholar We hypothesized that an asthmatic mechanical phenotype of ASM, if it existed, would be intrinsic to asthmatic cells, and thus allow us to study cultured ASM in the absence of inflammatory mediators, drugs used in the treatment of asthma such as corticosteroids, β-agonists, and antagonists to histamine and acetylcholine receptors, or influences from the epithelium. Our results from the present study indicate that ASM cells from those with asthma have increased cell traction forces at baseline, and enhanced stiffening (contraction) in response to activation of physiologically relevant G protein–coupled receptors, the M3-muscarinic receptor, and the H1-histamine receptors, than do ASM cells from those without asthma. We thus conclude that ASM from those with asthma has intrinsic mechanical properties that are hard-wired to the development of airway hyperresponsiveness. These phenotypes are presumably from genetic or epigenetic mechanisms. To date, we have not been successful in ascertaining the distinct polymorphisms or methylation-specific variants that are common across our group of ASM cells derived from subjects with asthma that might account for this mechanical phenotype (data not shown). As might be expected, the number of variants found dictates that a much larger number of ASM samples are needed to infer statistical significance after correcting for multiple comparisons. Last, we have not addressed any additional contribution to ASM phenotypes that arise from airway inflammation, and indeed we contend that asthma susceptibility and exacerbations are dependent on both inflammation-dependent and independent mechanisms. Tissues for some of our studies were derived from the Gift of Hope Organ & Tissue Donor Network donor families; we thank them for their selfless gift. Unless otherwise noted, all reagents were obtained from Sigma-Aldrich with the exception of Dulbecco modified Eagle medium-Ham's F-12 (1:1), which was purchased from GIBCO (Grand Island, NY). The synthetic arginine-glycine-aspartic acid (RGD)-containing peptide was purchased from American Peptide Company (Sunnyvale, Calif). Human ASM cells were prepared from donor lungs unsuitable for transplantation in accordance with the respective institutional review boards at the University of Chicago and the University of Pennsylvania. Because the availability of large numbers of early passage primary ASM cells from donor lungs is a challenge, and because the propagation of cells from distal airways often necessitate greater numbers of passages in culture, here we harvested cells from the proximal airways (first- through third-order bronchi) as described.E1Deshpande D.A. Wang W.C. Mcllmoyle E.L. Robinett K.S. Schillinger R.M. An S.S. et al.Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction.Nat Med. 2010; 16: 1299-1304Crossref PubMed Scopus (478) Google Scholar Cells were maintained in serum-free media for 24 hours at 37°C in humidified air containing 5% CO2 before study. These conditions have been optimized for seeding cultured cells on collagen matrix and for assessing their mechanical properties.E1Deshpande D.A. Wang W.C. Mcllmoyle E.L. Robinett K.S. Schillinger R.M. An S.S. et al.Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction.Nat Med. 2010; 16: 1299-1304Crossref PubMed Scopus (478) Google Scholar, E2An S.S. Fabry B. Trepat X. Wang N. Fredberg J.J. Do biophysical properties of the airway smooth muscle in culture predict airway hyperresponsiveness?.Am J Respir Cell Mol Biol. 2006; 35: 55-64Crossref PubMed Scopus (104) Google Scholar, E3Fabry B. Maksym G.N. Butler J.P. Glogauer M. Navajas D. Fredberg J.J. Scaling the microrheology of living cells.Phys Rev Lett. 2001; 87: 148102Crossref PubMed Scopus (688) Google Scholar, E4Butler J.P. Tolic-Norrelykke I.M. Fabry B. Fredberg J.J. Traction fields, moments, and strain energy that cells exert on their surroundings.Am J Physiol. 2002; 282: C595-C605Crossref PubMed Scopus (771) Google Scholar, E5An S.S. Kim J. Ahn K. Trepat X. Drake K.J. Kumar S. et al.Cell stiffness, contractile stress and the role of extracellular matrix.Biochem Biophys Res Commun. 2009; 382: 697-703Crossref PubMed Scopus (50) Google Scholar Briefly, cells were plated sparsely on collagen-coated elastic gel blocks precisely tuned to mimic a (patho)physiological range of airway wall rigidity (Young's modulus from 1 to 8 kPa),E5An S.S. Kim J. Ahn K. Trepat X. Drake K.J. Kumar S. et al.Cell stiffness, contractile stress and the role of extracellular matrix.Biochem Biophys Res Commun. 2009; 382: 697-703Crossref PubMed Scopus (50) Google Scholar and allowed to adhere and stabilize for 24 hours. The contractile stress arising at the interface between each adherent cell and its substrate was measured with traction microscopy,E4Butler J.P. Tolic-Norrelykke I.M. Fabry B. Fredberg J.J. Traction fields, moments, and strain energy that cells exert on their surroundings.Am J Physiol. 2002; 282: C595-C605Crossref PubMed Scopus (771) Google Scholar and the computed traction field was used to obtain net contractile moment, which is a scalar measure of the cell's contractile amplitude. Net contractile moment is expressed in units of pico-Newton meters. Dynamic increases in cell stiffness to bronchoconstrictive agonists were measured as an indicator of the single-cell contraction of isolated human ASM cells as we have previously described.E1Deshpande D.A. Wang W.C. Mcllmoyle E.L. Robinett K.S. Schillinger R.M. An S.S. et al.Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction.Nat Med. 2010; 16: 1299-1304Crossref PubMed Scopus (478) Google Scholar, E2An S.S. Fabry B. Trepat X. Wang N. Fredberg J.J. Do biophysical properties of the airway smooth muscle in culture predict airway hyperresponsiveness?.Am J Respir Cell Mol Biol. 2006; 35: 55-64Crossref PubMed Scopus (104) Google Scholar, E3Fabry B. Maksym G.N. Butler J.P. Glogauer M. Navajas D. Fredberg J.J. Scaling the microrheology of living cells.Phys Rev Lett. 2001; 87: 148102Crossref PubMed Scopus (688) Google Scholar In brief, RGD-coated ferrimagnetic microbeads (4.5 μm in diameter) bound to the cytoskeleton through cell surface integrin receptors were magnetized horizontally and then twisted in a vertically aligned homogeneous magnetic field that was varying sinusoidally in time. This sinusoidal twisting magnetic field caused both a rotation and a pivoting displacement of the bead: as the bead moves, the cell develops internal stresses, which, in turn, resist bead motions.E3Fabry B. Maksym G.N. Butler J.P. Glogauer M. Navajas D. Fredberg J.J. Scaling the microrheology of living cells.Phys Rev Lett. 2001; 87: 148102Crossref PubMed Scopus (688) Google Scholar Lateral bead displacements in response to the resulting oscillatory torque were detected with a spatial resolution of approximately 5 nm, and the ratio of specific torque to bead displacements was computed and expressed here as the cell stiffness in units of Pascal per nanometer. For cell traction force measurements, we used nested design analysis to control for random effects from repeated measurements of multiple cells in the same subject, and to increase the power.E6Krzywinski M. Altman N. Blainey P. Points of significance: nested designs.Nat Methods. 2014; 11: 977-978Crossref PubMed Scopus (35) Google Scholar To satisfy the normal distribution assumptions associated with ANOVA, cell traction data were converted to log scale before analyses. Unless otherwise stated, we used Student t test and ANOVA with adjustment for multiple comparisons by applying Bonferroni's methods. All analyses were performed using SAS V.9.2 (SAS Institute Inc, Cary, NC), and 2-sided P values of less than .05 were considered significant.Table E1Characteristics of donor lungs used for cell traction force measurementsDonorAge (y)SexRace and ethnicityCause of deathNonasthma 125MaleUnknownMultiple trauma, anoxia 269MaleUnknownIntracranial bleed 353FemaleWhite, not HispanicIntracranial bleed 440MaleWhite, not HispanicDrug overdose 561MaleBlack, not HispanicIntracranial bleed 649MaleWhite, not HispanicIntracranial bleedAsthma 742MaleUnknownUnknown 844FemaleUnknownAsthma attack 945MaleWhite, not HispanicIntracranial bleed 1048FemaleBlack, not HispanicIntracranial bleed 1151FemaleBlack, unknownIntracranial bleed 1211MaleWhite, not HispanicAnoxiaHuman ASM cells used for cell traction force measurements were obtained from the University of Chicago, through the Gift of Hope Organ and Tissue Donor Network. Open table in a new tab Table E2Characteristics of donor lungs used for cell stiffness measurementsDonorAge (y)SexRace and ethnicityCause of deathNonasthma 1316FemaleWhite, not HispanicHead trauma 1437MaleBlack, not HispanicIntracranial bleed 1519MaleBlack, not HispanicClosed head injury 1619FemaleBlack, not HispanicHead trauma 1755FemaleHispanicHypertensive bleed 1855FemaleWhite, not HispanicCentral nervous system tumorAsthma 1913MaleWhite, not HispanicAsthma attack, anoxia 2044MaleHispanicAsthma attack, anoxia 2115FemaleHispanicAsthma attack, anoxia 2225FemaleWhite, not HispanicAnoxia 2338MaleWhite, not HispanicAsthma attack, anoxia 249MaleWhite, not HispanicAsthma attackHuman ASM cells used for cell stiffness measurements were obtained from the University of Pennsylvania. Lungs were procured through the National Disease Research Interchange and the International Institute for the Advancement of Medicine. Open table in a new tab Human ASM cells used for cell traction force measurements were obtained from the University of Chicago, through the Gift of Hope Organ and Tissue Donor Network. Human ASM cells used for cell stiffness measurements were obtained from the University of Pennsylvania. Lungs were procured through the National Disease Research Interchange and the International Institute for the Advancement of Medicine.