Cardiotrophin-1 alters airway smooth muscle structure and mechanical properties in airway explants

Xueyan Zheng,* Danyi Zhou,* Chun Y. Seow, and Tony R Bai

The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia, St. Paul's Hospital, Vancouver, British Columbia, Canada V6Z 1Y6

Submitted 12 May 2004 ; accepted in final form 20 July 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Induction of hypertrophy and inhibition of apoptosis may be important mechanisms contributing to increased airway smooth muscle (ASM) mass in asthma. Data from our laboratory indicate that cardiotrophin-1 (CT-1) induces hypertrophy and inhibits apoptosis in isolated human ASM cells. To determine whether these novel effects of CT-1 also occur in the airway tissue milieu and to determine whether structural changes are accompanied by functional changes, matched pairs of guinea pig airway explants were treated with or without CT-1 for 7 days, and structural features as well as isometric and isotonic contractile and relaxant mechanical properties were measured. CT-1 (0.2–5 ng/ml) increased both myocyte mass and extracellular matrix in a concentration-dependent fashion. CT-1 (10 ng/ml)-treated tissues exhibited a significant increase in passive tension at all lengths on day 7; at optimal length, passive tension generated by CT-1-treated tissues was 1.72 ± 0.12 vs. 1.0 ± 0.1 g for control. Maximal isometric stress was decreased in the CT-1-treated group on day 7 (0.39 ± 0.10 kg/cm2) vs. control (0.77 ± 0.15 kg/cm2, P < 0.05). Isoproterenol-induced relaxant potency was reduced in CT-1-treated tissues, log EC50 being –7.28 ± 0.34 vs. –8.12 ± 0.25 M in control, P < 0.05. These data indicate that CT-1 alters ASM structural and mechanical properties in the tissue environment and suggest that structural changes found in the airway wall in asthma are not necessarily associated with increased responsiveness.

tracheal explants; mechanics; cytokines; asthma; matrix; smooth muscle hypertrophy


INCREASED SMOOTH MUSCLE MASS has been repeatedly documented in asthmatic subjects (3, 11, 29, 33, 34), but the mechanisms responsible for the increased mass are unclear. The amount of both airway smooth muscle (ASM) and smooth muscle-associated matrix is greater with increased duration of asthma and may be an important determinant of the severity of airway responsiveness (3, 15). Yet the functional alterations in ASM in asthma are uncertain (29, 34). When bronchial segments from asthmatics have been studied in vitro, the majority of studies have shown no change in mechanical properties, although isolated cells from asthmatics show a higher velocity of shortening (16). Tracheal tissue offers some advantages in studies of ASM behavior in that the muscle is more uniform in orientation, and less matrix elements are present. The only study of asthmatic trachea, in severe asthma, showed a nonspecific increase in force generation, suggesting increased contractility. However, force was normalized to tissue weight, not muscle mass (1); thus, the changes could have been a reflection of increased muscle.

Cardiotrophin-1 (CT-1), a member of the IL-6 family, was initially defined as a factor that has the capacity to induce cardiac myocyte hypertrophy (26). Further study showed that CT-1 caused both enhanced survival and hypertrophy of differentiated cardiac muscle cells and inhibited cardiac myocyte apoptosis after serum deprivation or cytokine stimulation (25, 30, 36, 37, 40). CT-1, acting through the gp130 receptor common to the IL-6 family, has been reported to be critical in the induction of hypertrophy in response to biomechanical overload and is overexpressed in cardiomyopathy, as part of a panel of "fetal" genes induced in response to damage (14, 28).

We have demonstrated that CT-1 is detected in abundance in freshly isolated and minced normal adult human lung and is expressed in both fetal and adult human primary cultured bronchial smooth muscle cells (HBSMC), predominantly in synthetic phenotypes. CT-1 induces a significant increase in HBSMC size, as judged by protein-to-DNA ratio and flow cytometry, and reduces the apoptosis induced both by serum deprivation and by Fas antibody/TNF-{alpha} treatment in HBSMC (38).

We postulated that, in the tissue milieu, CT-1 would also increase ASM mass and thus alter mechanical properties. To test these hypotheses, two protocols were used: initially, matched pairs of guinea pig airway explants were treated with or without CT-1 to determine structural changes. Second, in vitro smooth muscle function was evaluated.


    MATERIALS AND METHODS
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Animals

Female Duncan-Hartley adult guinea pigs, 500–600 g in weight, were used in this study, which was approved by the Biosafety and Animal Research Committee of the University of British Columbia.

Protocol 1: Structural Effects of CT-1 on Explanted Trachea

Explanted trachea. Tracheal ring explants were prepared as previously described from our laboratory (12), and matched pairs of guinea pig airway explants were used for the study. Briefly, the trachea was isolated, and all connective tissue and visible blood vessels were removed. Six to eight tracheal rings, 2.5–3 mm in width, were cut from each animal, and randomly chosen rings were placed individually in 12-well culture plates containing 1 ml CRML-1066 medium supplemented with 10% FBS, 2 mM glutamine, 5 µg/ml insulin, 0.25 µg/ml fungizone reagent, 100 U/ml penicillin, and 100 µg/ml streptomycin. Within each group, one ring served as the control, whereas the remaining rings were exposed to CT-1 (0.2, 1, or 5 ng/ml). Tissue culture plates were placed on a tray in a controlled atmosphere chamber that was flushed with a mixture of 45% O2, 50% nitrogen, and 5% CO2 at a flow rate of 4 l/min for 15 min. The chamber was then placed in a 37°C incubator on a rocking platform set at 10 cycles/min so that the tracheal lumen was intermittently exposed to media and gas mixture. The chamber was flushed with fresh gas mixture every 24 h. The media and CT-1 were changed every 48 h, and explants were cultured for 7 days.

ASM morphometry. The method used was based on a protocol established in our laboratory (31), with measurements on both circumferential and axial muscle profiles using different fixation techniques and section thickness. Different animals were used for axial (n = 6) and circumferential (n = 3) sections. Axial muscle profiles were obtained by longitudinal sectioning of tracheal rings fixed at reference length (the length at which isometric force was measured). Each of the tracheal rings was opened by cutting through the cartilage and then individually suspended in glass jars containing a solution of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, using suture thread and a weight conforming to preload at optimal length (Lmax; 0.8–1.2 g). After fixation, the strips were dehydrated in a graded series of concentrations of isopropyl alcohol and embedded in glycol methacrylate (JB-4) with the strips oriented such that the muscle was cut in axial cross section. Serial sections (2 µm) were cut on a JB-4 microtome (JB-4 Type Sorvall Microtome; Ivan Sorvall, Newtown, CT), using glass knives. Every twentieth section was mounted on glass slides and numbered until ~1 mm of smooth muscle had been sectioned. Sections were stained with Toluidine blue-O. To obtain circumferential muscle profiles, tracheal rings were fixed at reference length as above using 10% buffered formalin and paraffin embedded so that the strips were cut in cross section. Sections (5 µm) were cut on glass slides and stained with hematoxylin and eosin.

Basement membrane length and the subtended smooth muscle bundle area were traced on axial sections using the Bioquant (BQ) system software (R&M Biometrics, Nashville, TN), as previously described, at a final magnification of x250 (3, 31). The proportion of smooth muscle in the wall sections was also quantified by projecting a computer-generated grid of points on the microscope field. The area fraction of smooth muscle (Asm) in circumferential and axial sections was calculated as the number of points falling on muscle/total number of points on the wall at x250 magnification. The proportions of muscle cells, cell nuclei, and extracellular matrix within muscle bundles were determined by point counting on both axial and circumferential sections at a final magnification of x1,250, as previously described (3). Asm was calculated as: points falling on smooth muscle cells/total points within bundles multiplied by smooth muscle bundle points/total wall points at x 250 (Asm bundle/wall). Smooth muscle cell size was measured as the cytoplasm-to-nuclei ratio, that is, the number of points falling on cytoplasm divided by the total number of points falling on cytoplasm plus nuclei. This calculation assumes that cell nucleus size is not changing with treatments. The area fraction of extracellular matrix was calculated as the number of points falling on extracellular matrix divided by total number of points falling on the muscle bundle.

The mean value for the point counting was determined as the mean of eight observations on four random sections from each treatment. The distance between two sections was ~170 µm.

Protocol 2: Functional Effects of CT-1 on ASM

In vitro organ bath studies were carried out 7 days after explant culture with or without CT-1 (10 ng/ml) to characterize mechanical properties as previously described (12, 24). To minimize bias, the operator did not know the treatment of the tissues.

Tissue preparation. Guinea pig explanted tracheal rings were cut through the cartilage adjacent to the smooth muscle and mounted as strips. Each tracheal strip was suspended using clips attached at each smooth muscle-cartilage interface. One clip was hooked on a fixed point, whereas the other clip was hooked on the force transducer arm of a servo-controlled myograph (Raytech Instruments, Vancouver, BC). The tracheal strips were then placed in Krebs-Henseleit (KH) solution and bubbled with 95% O2-5% CO2, pH 7.4, 37°C. Indomethacin (2 µM) was added to the KH reservoir and was present for the duration of the experiment to inhibit prostaglandin synthesis and thus to stabilize muscle intrinsic tone (12). The weight of the tissue was incorporated in a balancing current in the electronic circuit to achieve zero tension. The initial length (Li) of the muscle was measured with an optical micrometer to an accuracy of 0.01 mm. All subsequent changes in length were referenced to this value so that changes in length could be calculated. Each airway ring was slowly stretched three times by loads that determined Lmax during preliminary experiments and then allowed to equilibrate in the organ bath with no load for 1 h, with fresh KH solution rinses every 15 min. After equilibration, passive, isometric, and isotonic length-tension relationships were obtained (12, 24).

Passive length-tension relationship. Measurements of passive tension were made by stretching the muscle and recording the tension. The stretching preloads were performed at graded loads between 0 and 2.5 g. At each length, stable resting tension was recorded (4, 5, 12, 24).

Isometric and isotonic preloaded contraction. Muscle contractions were elicited by ACh (10–5 M). Measurement of isometric force was first made at lengths below the level at which passive tension was first detected. A small preload was then applied to the strip to stretch it, and the length was recorded once it stabilized. An isometric contraction with this preload was performed by maintaining the airway length constant during ACh addition. Once the force returned to baseline at each preload, an isotonic contraction was elicited and the length change measured. At each preload, before isometric or isotonic stimulation, the baths were rinsed three times over 10 min, and the strips were allowed to relax to their prestimulated baseline tone. Complete isometric and isotonic length-tension relationships were obtained by serially increasing the smooth muscle length to 10–20% more than the length at which maximal force occurred. From the isometric force-length data, Lmax was determined as the length at which maximal isometric force (Fmax) occurred. In the instances when the maximal force at two lengths was similar, the shorter of the two lengths was chosen (4, 5, 12, 24).

Phamacodynamic studies of ASM responsiveness. After the full length-tension relationship was determined, the muscle strips were allowed to equilibrate in the organ bath with no load for 1 h, and then the strips were reset at Lmax. All of the contractile and relaxation curves were performed at Lmax. A cumulative concentration-effect relationship (CCER) to histamine (10–9 to 10–4 M) was performed, and the effective concentration that produced 50% of the maximal response (EC50) was subsequently determined. Thereafter, a relaxation CCER to isoproterenol (10–9 to 10–4 M) was conducted in tissue contracted with histamine (10–4 M). A maximal relaxation was subsequently induced by the addition of 10–4 M papaverine and 10–3 M EDTA (12).

Morphological studies. At the completion of the organ bath experiments, the strips were fixed in glutaraldehyde at Lmax and processed as previously described. Axial 2-µm sections were used to determine muscle area by point counting.

Data analysis. Force and length measurements were standardized, respectively, and expressed as %Fmax and %Lmax or percentage Li (%Li). The muscle stress was calculated by dividing force values by the axial cross-sectional muscle area and expressed as kilograms per square centimeter. Isotonic shortening was analyzed by dividing the shortening by the length from which the preparation started shortening, expressed as %Li. (4, 12, 23, 24). The EC50 was calculated as previously described (27).

Statistical Analysis

All values are reported as means ± SE. ANOVA or paired t-tests were used to determine significance. P values <0.05 were considered significant.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Protocol 1: Effects of CT-1 on Explanted Trachea Structure

CT-1-exposed guinea pig tracheal explants exhibited increased smooth muscle mass, and the increase was apparent in both circumferential (mean data from 3 animals) and axial sections (mean data from 6 animals; see Figs. 1 and 2). Exposure to CT-1 (0.2–5 ng/ml) resulted in an increase in smooth muscle mass in a concentration-dependent manner compared with controls, as demonstrated by both tracing muscle bundle area at all concentrations of CT-1 tested (Fig. 2A) and by point counting area fractions after control treatment or 5 ng/ml CT-1 (Fig. 2B). The axial muscle bundle area in CT-1 (5 ng/ml)-treated airways was increased 26.2 ± 8.2% (P = 0.007) above control values, as measured by tracing bundle size (control = 0.06 ± 0.005, 5 ng CT-1 = 0.075 ± 0.007 mm2/mm perimeter of basement membrane). As measured by point counting, absolute muscle area, when within-bundle matrix counts were excluded from the calculations, was increased by 14.4 ± 5.7% (Asm control = 12.9 ± 0.8, 5 ng CT-1 = 14.7 ± 0.8, P <0.05). Including bundle matrix in the counts, smooth muscle area was increased by 18.6 ± 5.7%.



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Fig. 1. Photomicrographs of sections from tracheal explants. Circumferential (A and B, hematoxylin and eosin) and axial (C and D, Toluidine blue-O) muscle profiles are shown. A and C: control explants. B and D: adjacent tissues from the same animal treated with cardiotrophin-1 (CT-1), 5 ng/ml for 7 days. Scale is 5 µm.

 


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Fig. 2. A: CT-1 increases smooth muscle mass. Tracheal rings were treated with vehicle control or CT-1 at different concentrations (0.2, 1, or 5 ng/ml) for 7 days. Measurements were performed on a 2-µm-thick cross section of JB-4-embedded, Toluidine blue-O-stained tissue by tracing the muscle bundle area at x250 magnification. The muscle area (mm2) was normalized per basement membrane length (mm). Pbm, perimeter of basement membrane. B: CT-1 increases smooth muscle cell size and extracellular matrix within bundles. Experimental protocol is the same as in A. Muscle cell size and number and matrix area within muscle bundles were assessed by point counting at a magnification of x1,250. Asm, smooth muscle area; Acytoplasm/nuclei, cytoplasm/nuclei area; AECM, extracellular matrix area. See MATERIALS AND METHODS for more details. Data are reported as means ± SE, comparing 5 ng/ml CT-1 with control; n = 6 (axial cross section) and 3 (circumferential section), *P < 0.01 and **P < 0.05.

 
To further evaluate whether the changes in smooth muscle mass in CT-1 (5 ng/ml)-treated airways were because of an increase in cell size or number, or to an increase in the amount of extracellular matrix, we assessed the muscle bundles at a final magnification of x1,250 (Fig. 2B). The muscle cytoplasm-to-nuclei area fraction in CT-1-treated airways was 130.6 ± 7.7% of the control in axial cross sections (P < 0.05) and 119.3 ± 5.7% in circumferential sections (P < 0.05). This change, coupled with an increase in the Asm cell points within bundles (an increase of 14.4 ± 5.7% over the control in axial section and 28.3 ± 10.9% in circumferential sections), indicated that hypertrophy rather than hyperplasia was the predominant change in ASM after CT-1 exposure. The extracellular matrix area fraction was also significantly increased (P < 0.01) in CT-1-treated airways (with an increase of 31.7 ± 20% of control values); however, this change was only detected in the axial sections (Fig. 2B).

Protocol 2: Functional Effects of CT-1 on ASM

Effects of CT-1 on ASM passive length-tension characteristics. Passive tension measurements of the muscle are shown in Fig. 3. At all lengths, the CT-1-treated tissues generated higher passive tensions when compared with controls (Fig. 3A). After treating the airway trachea with CT-1 (10 ng/ml) for 7 days, we found a decrease in the length relative to Lmax at all passive loads compared with the controls (Fig. 3A). At Lmax, passive tension generated by CT-1-treated groups was 1.72 ± 0.12 vs. 1.0 ± 0.1 g for controls, n = 5, P < 0.05 (Fig. 3B).



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Fig. 3. Effects of CT-1 on airway smooth muscle (ASM) passive force. A: passive length-tension curve from tracheal smooth muscle preparations treated with or without CT-1 (10 ng/ml) for 7 days. Passive tension expressed as percentage maximal isometric force (%Fmax) and length as percentage length at which maximal isometric force was obtained (%Lmax). B: passive tension at Lmax (g) from tracheal smooth muscle preparations treated with or without CT-1 (10 ng/ml) for 7 days. Values are means ± SE; n = 5, *P < 0.05.

 
Effects of CT-1 on ASM isometric stress. Treatment of airway explants with CT-1 for 7 days induced a decrease in maximal ACh-induced isometric stress, 0.39 ± 0.1 in CT-1-treated group vs. 0.77 ± 0.15, P < 0.05, (Fig. 4). The Fmax to histamine was also significantly reduced in the CT-1-treated groups vs. control groups (1.09 ± 0.79 in CT-1-treated tissues vs. 1.51 ± 0.85 in control, n = 5, P = 0.02; Fig. 4).



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Fig. 4. Effect of CT-1 on ASM active isometric force. Maximal (Max) stress generation (Fmax) to ACh and histamine from tracheal smooth muscle preparations treated with or without CT-1 (10 ng/ml) for 7 days. Fmax was standardized by the cross-sectional muscle area and expressed as maximal stress (kg/cm2). Values are means ± SE; n = 5, *P < 0.05.

 
Effects of CT-1 on ASM isotonic shortening. We expressed isotonic shortening as %Li, dividing the change in length by the length from which the preparation started shortening [{Delta}L/Li (mm)]. Because CT-1 altered the passive mechanical properties of the explants, we compared shortening only at similar preloads (0.25, 0.5, and 0.8 g). At these three preloads, the isotonic shortening generated by CT-1-treated tissue was less (6.89 ± 2.24, 5.81 ± 2.33, and 2.92 ± 1.25 %Li), compared with the controls shortening under the same preloads (7.08 ± 2.72, 8.31 ± 1.65, and 3.22 ± 0.32 %Li). Although shortening was less in CT-1-treated explants, this difference was not significant (Fig. 5). Data were missing for one pair because of equipment malfunction.



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Fig. 5. Effect of CT-1 on ASM isotonic shortening. The amounts of isotonic shortening generated at three preloads (0.25, 0.5, and 0.80 g) in CT-1-treated (7 days, 10 ng/ml) group and controls are shown. Isotonic contractions were elicited with 10–5 M ACh. Values are expressed as percentage of the initial (relaxed) muscle length (%Li). Values are means ± SE; n = 4.

 
Effect of CT-1 on ASM relaxation responses to isoproterenol. Mean CCER to isoproterenol are shown Fig. 6A. Isoproterenol produces concentration-dependent and cumulative isometric relaxation in guinea pig tracheal strips pretreated with histamine. After treatment of the explant with CT-1 (10 ng/ml) for 7 days, isoproterenol-induced relaxation was significantly reduced. The relaxation curve shifts to the right, which indicates that CT-1-treated tissues are less sensitive to isoproterenol compared with controls (Fig. 6A). Maximal relaxation, induced by addition of 10–4 M papaverine and 10–3 M EDTA, was not different from controls. Figure 6B shows the sensitivity to isoproterenol expressed as EC50 (the concentration required to give 50% of maximal response).



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Fig. 6. Effect of CT-1 on ASM relaxation to isoproterenol. A: mean cumulative concentration-response relationships (CCEC) to isoproterenol from tracheal smooth muscle preparations treated with or without CT-1 (10 ng/ml) for 7 days. All preparations were precontracted with histamine. Responses were calculated as a percentage of the maximal relaxation produced by 10–4 M papaverine and 10–3 M EDTA. Values are means ± SE; n = 5. B: concentration required to give 50% of maximal relaxation (EC50) to isoproterenol is less in CT-1-treated group compared with controls; n = 5, *P < 0.05.

 
Morphometric analysis. The area of smooth muscle, %total axial area, was significantly increased in CT-1-treated tissues vs. controls (control 14.5 ± 1.30, CT 17.6 ± 1.40, similar results to protocol 1). This result demonstrates that CT-1 reproducibly increases ASM mass at a concentration twofold higher than the maximal used in protocol 1.


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The findings in this study indicate that, in the tissue milieu, CT-1, a cytokine associated with synthetic ASM phenotype, causes airway structural changes by enhancing smooth muscle mass, resulting from both hypertrophy and increased matrix expression, changes found in asthma (3). The results validate and extend our findings in isolated human ASM cells, with the finding of a relatively greater magnitude of change in matrix area than change in smooth muscle mass. These novel effects of CT-1 were accompanied by functional changes, including decreased ASM stress generation, increased tissue passive tension (decreased compliance), and reduced smooth muscle relaxation to isoproterenol. The results indicate that the structural changes in asthma may not necessarily be accompanied by increased responsiveness; rather, the converse may apply.

We employed an explant model developed in our laboratory (12). Our prior experience with 72 h or more of culture with platelet-derived growth factor (10 ng/ml), histamine (0.1 mM), leukotriene D4 (1 µm), and TNF-{alpha} (0.1 µg/ml) indicated that cytokines and mediators of asthma known to induce smooth muscle proliferative or functional changes in isolated cells do not do so in airway explants, illustrating the selectivity and novelty of our results (17). We compared two morphometric methods that provide different orientations of the ASM, including axial cross-sectional profiles and circumferential profiles of smooth muscle, since muscle orientation plus methodological differences in preparation have been reported to result in a wide variation in the assessment of muscle mass (31, 32). Although we reproducibly detected the increased muscle mass and hypertrophy in both sections, the matrix changes after CT-1 treatment were only detected in the axial cross sections. This variation between the cross-sectional and the circumferential sections may be the result of section thickness and fixative or may represent a type 2 error because only three animals were used for circumferential profiles. With the thicker sections used for circumferential measurements, overlapping muscle cells make it more difficult to distinguish matrix from the muscle cells. The axial cross section also has advantages for matrix measurements in that it allows clearer boundaries between individual smooth muscle cells. We noted in protocol 1 that smooth muscle area by point counting increased 14.4 ± 5.7% in the axial cross section compared with 28.3 ± 10.9% in the circumferential section. In addition to fixation and thickness differences, some of this variation may, in part, be the result of a greater increase in cell length rather than in cell width induced by CT-1, as demonstrated in cardiac myocytes (37).

Although increased smooth muscle in asthmatic airways has been found repeatedly, the mechanism for the increase in cell size is uncertain and has not been well defined. A recent report suggests HBSMC hypertrophy is secondary to cell cycle arrest (39); however, in prior experiments, we have not observed an effect of CT-1 on cell cycle (Ref. 38 and unpublished data). Moreover, activation of the gp130 receptor has been reported to induce cardiac myocyte hypertrophy both in vivo and in vitro (10, 37). Further reports have shown that the JAK-STAT pathway, especially the STAT3-mediated pathway, appears to be essential in the induction of cardiac myocyte hypertrophy through gp130 (13, 14). This study is the first to demonstrate that CT-1, which acts through a gp130/leukemia inhibitory factor receptor heterodimer, has the ability to induce ASM hypertrophy in the tissue milieu and that the hypertrophy is associated with increased deposition of extracellular matrix, findings consistent with our studies in severe asthma (3).

Given that there is more ASM in asthma of long duration, greater active force generation has been proposed to exist simply because of the increased muscle mass (1, 7, 8, 29). However, muscle contractility and tissue elastance are also influenced by cytokines, matrix-degrading enzymes, and other inflammatory mediators present in the airways of asthmatic subjects (19–21, 29, 35). Our study demonstrates that the extracellular matrix area fraction is significantly increased in CT-1-treated airways (increased by 31.7 ± 20% of control values). The passive mechanic properties reflect the contribution from both the smooth muscle element and the parallel elastic elements represented in connective tissue (23). Several studies have shown that extracellular matrix may represent a parallel elastic element, reducing maximal muscle shortening, but the effect on isometric stress generation is less certain (5, 1921, 24, 29). In the simplest models, no direct effect on isometric stress is expected unless more matrix alters the alignment of muscle cells. Indirectly, signaling between matrix and cells via integrins and other receptors may lead to alteration in the contractility of the smooth muscle. The reduction in active stress generation by CT-1 in the current study therefore presumably represents a change in the contractile properties of the smooth muscle in explant culture, or one of the effects listed above. Furthermore, the phenomenon of length adaptation (plasticity) needs further study using this explant system, with fixation of length of tracheal rings during explantation and tight control of muscle loading history, length, and activation, now that we have established a reproducible model of structural airway change.

Our recent work has demonstrated that CT-1 alters the HBSMC contractile protein phenotype (38), which could alter the smooth muscle component of passive tension. Treatment of cultured HBSMCs with CT-1 (10 ng/ml) for 96 h clearly induced formation of filamentous arrays of smooth muscle promoter (SM22), a contractile protein, oriented along the longitudinal axis of the cells, and increased expression of SM22, as measured by Western blot. In contrast, the cytostructural distribution of smooth muscle {alpha}-actin, vimentin, or nonmuscle myosin heavy chain appeared to be unaffected by treatment with CT-1 (38). Halayko and colleagues (9) have demonstrated that SM22 and other ASM contractile proteins, such as smooth muscle {alpha}-actin and smooth muscle myosin heavy chain, are all increased in "elongated" contractile smooth muscle cells. Thus we postulated that a similar effect in vitro in explant tissue would increase, not decrease, force generation. We emphasize that prolonged explant culture leads to less force generation and shortening under control conditions (unpublished data); thus, the effect of CT-1 observed in the present study may in some way represent a consequence of, and be limited to, explant conditions.

Parallel elastic elements or matrix deposition around smooth muscle may also contribute to the increased explant passive tension induced by CT-1, since our study demonstrates that the extracellular matrix area fraction was significantly increased by CT-1. Bramley et al. (5) demonstrated that collagenase treatment caused a significant decrease in passive tension, probably by degrading the collagenous matrix surrounding the smooth muscle in the airway wall, reducing the parallel elastic load. Thus, conversely, increased matrix in CT-1-treated airways may have resulted in an increase in passive tension. The radial constraint hypothesis postulates that matrix/connective tissue arranged radically can cause smooth muscle shortening to be limited (18, 21). Our study demonstrates, at three preloads (0.25, 0.5, and 0.80 g), that the isotonic shortening tended to be lower in the CT-1-treated group, although not significant, possibly because of the loss of a data pair through equipment malfunction. As discussed above, a reduction in the amount of shortening could be caused by matrix deposition or smooth muscle contractile element changes.

Structural changes in the airway wall do not necessarily imply airway hyperresponsiveness. Indeed, some data suggest that increased wall thickness may serve to limit airway narrowing, as shown by the report of Niimi and colleagues (22). In addition, ASM may contribute to airway hyperresponsiveness not only by its increased ability to shorten but also by an impairment of its ability to relax (2, 6). In our study, relaxation induced by a single pharmacological agent has been investigated. CT-1-treated tissues have less sensitivity to isoproterenol when compared with controls, but there was no effect on the maximal relaxation response. This result may be the result of the effect of several factors, such as {beta}2-adrenergic receptor dysfunction induced by CT-1, matrix deposition, or changes in smooth muscle contractile phenotype, and requires more study.

In summary, we have demonstrated that CT-1 increases ASM mass, via both increased myocyte hypertrophy and matrix deposition in airway explants, alters ASM mechanical proprieties, and reduces isoproterenol-induced ASM relaxation. Our findings provide data for modeling, and further study of, the mechanical consequences of the structural changes of airways found in asthma.


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This work was supported by Canadian Institutes of Health Research Grant 42537 and the British Columbia Lung Association.


    ACKNOWLEDGMENTS
 
We thank Drs. Peter Pare, Robert Schellenberg, Karen McKay, and Annabelle Opazo-Saez for advice.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. R. Bai, The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, Univ. of British Columbia, St. Paul's Hospital, 1081 Burrard St., Vancouver, B. C., Canada V6Z 1Y6 (E-mail: tbai{at}mrl.ubc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* X. Zheng and D. Zhou contributed equally to this research. Back


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