INTRODUCTION

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Increased airway narrowing in response to nonspecific stimuli is a characteristic feature of human obstructive airways diseases (1). This abnormality is an important aspect of the disease, although the pathophysiological changes leading to this hyperresponsiveness are still unknown. Several mechanisms have been postulated to explain hyperresponsiveness, including alterations in the neurohumoral control of airway smooth muscle (ASM) (2, 3), increased sensitivity of ASM (4), increased mucosal permeability (7), increased mucosal secretions (8, 9), and mechanical factors related to remodeling of the airways (10, 11). In spite of the potential contributions of some of these mechanisms, the reversibility and rapid onset of airway narrowing indicate that airway smooth muscle contraction is central to the pathophysiology underlying exaggerated airway narrowing.

To determine whether abnormalities in ASM play a role in airway hyperresponsiveness, a number of studies have been carried out to examine in vitro contractility of isolated human airway smooth muscle from patients who have varying degrees of airflow obstruction and airway responsiveness. While in most studies a relationship between in vitro and in vivo responsiveness has not been shown (12), in a few a relation between force generation and airway responsiveness has been found in chronic obstructive pulmonary disease (COPD) (17), and asthma (18). These inconsistent findings may be attributable to several problems in study design, such as the use of a small sample size, sampling from different parts of the tracheobronchial tree, lack of normalization of force, and absence of measurements of isotonic shortening.

In the present study we used a modified, previously described technique (22, 23), to measure both isometric force generation and isotonic shortening in peripheral airway rings (2.5 ± 0.8 mm; mean ± SD) from patients who had varying degrees of airflow obstruction. The hypothesis is that altered ASM contractility and/or airway smooth muscle mass contribute to the pathogenesis of obstructive airways disease. The aims of this study were to characterize the passive and active mechanical properties of peripheral airways from the lungs of normal subjects and patients who had COPD and asthma, and to relate in vitro contractility to pulmonary function measured before surgery. The main distinguishing feature of this study is the measurement of force and smooth muscle mass and the use of stress to make more valid comparisons than those previously reported.

	METHODS

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Study Population

Lung tissue was obtained from 30 patients who had a surgical resection of a lung or lobe for a solitary peripheral lung lesion at St. Paul's Hospital (Vancouver, BC, Canada). A patient was not included in this study if a lung lesion obstructed a segmental or larger bronchus or if pneumonitis was diagnosed. Subjects were studied with the approval of both the University of British Columbia and St. Paul's Hospital Ethics Committees and after obtaining informed consent from the subjects. Patients were characterized on the basis of a detailed questionnaire concerning respiratory symptoms, smoking history, and allergies before surgery.

Pulmonary function tests were done before surgery and included maximal expiratory flow-volume curves. Twenty-eight of the patients had a diagnosis of bronchogenic carcinoma, while the remaining 2 had a diagnosis of carcinoid tumor. Twenty-one of the patients had been life-long smokers, 4 were ex-smokers (had not smoked for 7, 15, 40, and 50 years), and 5 were nonsmokers. Only four patients had a past history of allergies. Preoperative medications included sedatives (lorazepam; Ativan), laxatives (docusate sodium), heparin, antibiotics (cefazolin sodium), analgesics (acetaminophen), and in some cases -agonists (albuterol [Ventolin], terbutaline [Bricanyl]). Preliminary studies showed no differences in in vitro contractility as a result of the various medications received while in hospital (our unpublished observations, 1998). Asthma and COPD were defined on the basis of clinical criteria, using the definition of the American Thoracic Society (24). Table 1 summarizes the data for the patients in this study.

Lung Function Studies

Lung function studies were done within 1 wk before surgery. Methods have been previously described (25). Briefly, the subjects were seated in a volume displacement, pressure-compensated body plethysmograph, with a nose clip in place. Volume was measured with a Krogh water-seated spirometer coupled to a linear displacement transducer (type 300 HR; Shaevitz, Pennsauken, NJ). Flow was measured with a Fleisch No. 3 pneumotachometer coupled to a Sanborn 270 differential pressure transducer (MP 45-28, ± 3.5 cm H₂O; Validyne, Northridge, CA). Functional residual capacity (FRC) was determined by the Boyle's law technique, and total lung capacity (TLC) was calculated by adding the inspiratory capacity to FRC. Maximal expiratory flow- volume curves were used to calculate the forced expiratory volume in 1 s of the forced vital capacity (FEV₁), the forced vital capacity (FVC), the forced expiratory flow at 50% of FVC (FEF₅₀), and the forced expiratory flow between 25-75% of FVC (FEF_25-75). The FEV₁ was expressed as a percentage of the predicted value, using the equations of Crapo and co-workers (26). The ratio of FEV₁ to FVC (FEV₁/FVC) was also calculated and was used as an indicator of airflow obstruction. A value of 70% is approximately the lower limit of the normal range as defined by 95% confidence intervals for FEV₁/FVC (26), and was therefore used to categorize the subjects in this study. At least two expiratory efforts were carried out and were accepted when the two values for each variable measured were within 5% of each other.

In Vitro Studies

Specimens were selected from a macroscopically tumor-free part of the specimen, and immediately transferred to ice-cold sterile Krebs-Henseleit buffer (in mM: 118 NaCl, 4.7 KCl, 2.5 CaCl₂H₂O, 1.2 MgSO₄ · 7 H₂O, 1.2 KH₂PO₄, 25 NaHCO₃, and 11.1 glucose). Segments (30 mm long) from third- and fourth-order bronchi were dissected free of blood vessels and surrounding parenchyma. Segments were obtained from each subject and one airway ring was randomly selected for studies in physiology. Tissues were stored overnight in a large volume of cooled (4° C) Krebs buffer that was previously aerated with carbogen (95% O₂-5% CO₂). This procedure has been shown to wash out drug residues and any substances liberated during the dissection (22). The pH of the solution ranged between 7.2 and 7.3. Viability of responses after storage was tested on a number of the airways by eliciting a near maximal contraction with acetylcholine (10⁵ M) the day of thoracotomy, and on the next day at the beginning and at the end of the experiment. The responses to cholinergic agonist remained unchanged up to 48 h, and were reproducible throughout the experiment (data not shown), which is consistent with previous findings (22).

In vitro studies were carried out the day after thoracotomy to characterize the length-tension curves for each airway ring as previously described (23). Passive tension, isotonic shortening, and isometric force generation were measured in vitro by a servo-controlled myograph. The motor arm of this apparatus could be fixed to achieve an isometric contraction or maintain constant force allowing an isotonic contraction (G325D pen motor [General Scanning, Watertown, MA]; Kulite BG-10 force transducer [Durham Instruments, Pickering, ON, Canada]). The frequency response was 100 Hz and the resolution was 0.02 g for force and 0.01 mm for length measurements.

Protocol. Each airway ring was placed in an organ bath that contained fresh Krebs-Henseleit buffer that was replaced continuously, and bubbled with 95% O₂-5% CO₂ at 37° C. The airway was mounted between a force transducer and a motor arm and allowed to equilibrate at zero resting tension for 1 h. The weight of the tissue was incorporated into a balancing current in the electronic circuit in order to achieve zero tension. The initial length 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 optimal length during preliminary experiments, and then allowed to equilibrate in the organ bath with no load for 1 h. After equilibration, passive, isometric, and isotonic length-force relationships were obtained. Contractions were elicited by acetylcholine (10⁵ M). Measurement of isometric force was first performed at lengths below the level at which passive tension was first detected. A small preload was then applied to the ring 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 while stimulating and measuring the change in force. Once the force returned to baseline (same preload), an isotonic contraction was elicited and the length change measured. Complete isometric and isotonic length- force curves were obtained by serially increasing the smooth muscle length to 10-20% more than the length at which maximal force occurred. For each length, an isometric contraction was followed by an isotonic contraction. From the isometric force-length curve, Lmax was determined as the length at which maximal isometric force (Fmax) occurred. In the rare instances when the maximal force at two lengths was similar, the shorter of the two lengths was chosen. Lmax was used to calculate the diameter at Lmax (Dmax) by assuming that in the stretched ring, the airway perimeter was twice the measured length at Fmax [diameter = (2 × length)/].

Morphological Studies

Tissue preparation and airway sampling. After completion of the isometric and isotonic length-force measurements, each airway ring was removed from the organ bath and fixed in a solution of 2.5% (vol/vol) glutaraldehyde (Polysciences, Warrington, PA) in 0.1 M sodium cacodylate buffer (Polysciences) at the tension that stretched each ring to Lmax. The tissue specimens were dehydrated through a series of graded ethanols and embedded in glycol methacrylate plastic (JB-4; Polysciences). The maximal measured area shrinkage factor has been shown to be less than 4% by this method (27).

The airway rings were oriented within the JB-4 blocks such that cross-sectional profiles of the smooth muscle fibers could be measured within the axial sections of the airway wall. JB-4 blocks were sectioned at a thickness of 2 µm on a microtome (JB-4 type Sorvall microtome; Ivan Sorvall, Newtown, CT), using glass knives prepared on an LKB knife maker (LKB knife maker type 7801B; LKB-Produkter AB, Stockholm, Sweden). The entire specimen was sectioned but only every 50th section (approximately every 100 µm) was mounted on a glass slide and stained with an aqueous solution of 1% (wt/vol) toluidine blue O (C.I. 52040; Sigma, St. Louis, MO) and 1% (wt/vol) sodium borate at pH 8.7. Three slides were selected by a systematic random method as described by Gundersen and Jensen (28).

Stereologic Methods

Cross-sectional areas of the smooth muscle and the total amount of tissue in the airway wall were measured at a magnification of ×200 (×20 objective lens), using a Nikon microscope with a camera lucida (Nikon Labophot; Nikon Canada, Mississauga, ON, Canada), a digitizing board (Summasketch II model MM II 1201; Summagraphics, Seymour, CT), and the Bioquant BQ system software (R&M Biometrics, Nashville, TN) on an IBM-compatible personal computer. The intra- and interobserver coefficient of variation was less than 5% for each variable measured. Measurements were made on three slides and a mean value was obtained for each variable. The fraction of the airway wall made up of muscle was calculated by dividing the smooth muscle area by the total area of tissue in the airway wall (WA).

Data Analysis

Data have been expressed as means ± SD unless otherwise stated. Force and length measurements were standardized, respectively, and expressed as %Fmax and %Lmax. The muscle stress was calculated by dividing force values by the cross-sectional areas of muscle and expressed as mN/mm². This method of force normalization was recommended by Stephens and coworkers (29) for comparison of muscle strips from the same or different species. Although a more precise denominator would be to measure the total number of myosin filaments parallel to the force vector, by densitometry of myosin heavy chain bands or by quantitative immunohistochemistry of transverse sections, this method was not applied as it did not aid the specific aims of this study. The amount of smooth muscle was normalized by the airway diameter measured at Lmax (Dmax). The amount of isotonic shortening was analyzed in three ways: (1) by dividing the shortening by the length from which the preparation started shortening, and expressing it as a percentage of the initial length, %Li; (2) by dividing shortening by Lmax, and expressing it as %Lmax; and (3) by calculating shortening at the operating length of the muscle at FRC (see the next section). Force, stress, and shortening were related to the FEV₁, and to FEV₁/FVC values, by Pearson correlations. Differences between obstructed and nonobstructed groups in force, stress, shortening, smooth muscle (ASM, ASM/Dmax, ASM/WA), FEV₁, FEV₁/FVC, FEF₅₀, FEF_25-75, FRC, and RV were analyzed by unpaired t tests. Bonferroni corrections were made for multiple comparisons. p Values were considered to be significant when less than 0.05.

Transpulmonary pressure at Lmax (Ptp). To relate the in vitro mechanics to the operating length-tension curves of peripheral airways in vivo, the transpulmonary pressures were calculated for each length of the in vitro length-tension curves. The transpulmonary pressure at Lmax, the length at FRC, passive tension at FRC, and shortening at FRC were derived. The basic assumption made in estimating these variables is that in the absence of smooth muscle contraction the interstitial pressure surrounding the airways in vivo is equal to the pleural pressure. The transpulmonary pressure at Lmax was calculated using the LaPlace equation, P = T/r, where T is the wall tension obtained from the passive tension at Lmax in millinewtons per millimeter of airway width, and r is the airway radius at Lmax, calculated from the measured airway perimeter.

	RESULTS

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Lung Function Studies

Table 2 shows the mean lung function values for the patients in this study. Fifteen patients had an FEV₁/FVC < 70% and were considered to have airflow obstruction. The functional residual capacity, residual volume, and total lung capacity were increased (p < 0.05), while the FEF₅₀ and FEF_25-75 were significantly lower (p < 0.001) in the obstructed patients.

In Vitro Studies

Figure 1 shows the cumulative frequency distribution for the airways studied. Thirty airways with a mean diameter of 2.5 mm at Lmax were used for the in vitro studies. Fifteen airway rings were obtained from obstructed patients, and 15 from nonobstructed patients. Both groups had similar-sized airways, with most airways having a diameter between 2 and 3 mm (at Lmax). There was no difference in the diameter at Lmax between the two patient groups.

View larger version (43K): [in this window] [in a new window]   Figure 1.   The cumulative frequency distribution for the peripheral airways used in this study (n = 30) are shown classified according to their airway diameter at Lmax, for obstructed and nonobstructed patient groups. There were 15 airways in each group. The distribution of airways on the basis of size was similar in both groups.

The passive, isometric, and isotonic length-force relationships from one airway ring from the nonobstructed group are shown in Figure 2. Total force, as a percentage of Fmax, is plotted as a function of length, expressed as a percentage of Lmax. The active isometric force was obtained by subtracting the passive tension from the total force. It increased until a peak occurred at Lmax, beyond which increments in length produced no further increases in active force. The passive tension increased as the ring was stretched and was substantial at Lmax. Peripheral airway smooth muscle was able to develop force at lengths as low as 35%Lmax (data not shown). Preloaded isotonic shortening has also been plotted and is indicated at Lmax by an arrow. On average, maximal shortening occurred at ~ 80%Lmax, and was negligible near 40%Lmax.

View larger version (13K): [in this window] [in a new window]   Figure 2.   The length-tension relationships for one peripheral airway ring are shown. The force, expressed as a percentage of the maximal isometric force (Fmax), has been plotted versus the length of the airway preparation, expressed as a percentage of Lmax, the length at which Fmax occurs. The plot shows the passive tension (empty circles), the active isometric force (filled circles), the total tension (filled squares), and the active isotonic shortening (crosses). The active isometric force (total force  passive tension) increases at each length as the airway is stretched until it reaches a maximal value. Beyond that point, further increases in length result in a decrease in isometric force. The active isotonic shortening was higher at lengths greater than 70%Lmax and less than 100%Lmax. Isotonic shortening at Lmax is shown by an arrow.

The mean values for maximal isometric force, passive tension at Lmax, maximal isotonic shortening, and length for maximal shortening are shown in Table 3 for obstructed (OB, n = 15) and nonobstructed (NOB, n = 15) patients. Maximal isometric force and stress were significantly higher in the obstructed group (p < 0.05), but there was no difference in the passive tension between obstructed and nonobstructed patients. In both patient groups there was large variability in the passive tension that ranged between values of 30 to near 400%Fmax.

In addition, there was no difference in the maximal isotonic shortening between the two patient groups, whether it was normalized by the initial shortening length (29.7 ± 15.1%Li, OB; 22.4 ± 12.7%Li, NOB), or by the length at maximal isometric force (23.5 ± 10.6%Lmax, OB; 18.7 ± 10.3%Lmax, NOB). The length at which maximal shortening occurred was below Lmax and was similar between the two patient groups. When normalized by Lmax, the values for shortening occurred at a length about 10% higher than when normalized by Li.

There was a significant correlation between force and shortening (%Li) (r = 0.389, p < 0.05), force and shortening (%Lmax) (r = 0.442, p < 0.05), stress and shortening (%Li) (r = 0.488, p < 0.02), and stress and shortening (%Lmax) (r = 0.538, p < 0.01) (Figure 3A-3D).

View larger version (22K): [in this window] [in a new window]   Figure 3.   Correlations between shortening and force. Shortening as a percentage of the starting length (Li) has been plotted versus maximal isometric force in grams (A) (r = 0.389, p < 0.05) and versus maximal isometric stress (mN/mm2) (B) (r = 0.488, p < 0.02). (C ) Shortening (%Lmax) versus force (r = 0.442, p < 0.05). (D) Shortening (%Lmax) versus stress (mN/mm2) (r = 0.538, p < 0.01).

Correlations between In Vivo and In Vitro Function

Figures 4A-4D show the relationships between force and stress, and two measures of pulmonary function, the FEV₁ (%pred) and FEV₁/FVC (%). There was a negative correlation between force and the FEV₁ (%pred) (r = 0.579, p < 0.004), and between force and the FEV₁/FVC (%) (r = 0.720, p < 0.003). Stress was also negatively correlated with the FEV₁/FVC (%) (r = 0.611, p < 0.002), but was not correlated with the FEV₁ (%pred). Shortening was not correlated with either measure of pulmonary function.

View larger version (19K): [in this window] [in a new window]   Figure 4.   (A) Force (g) versus FEV1 (%pred), r = 0.579, p < 0.004; (B) stress versus FEV1 (%pred), r = < 0.328, p = NS; (C ) force versus FEV1/ FVC (%), r = 0.720, p < 0.003; (D) stress (mN/mm2) versus FEV1/FVC (%) r = 0.611, p < 0.002, for all airways (n = 30) from obstructed and nonobstructed individuals. When the subject with extreme values is removed the correlations change as follows: force (g) versus FEV1 (%pred), r = 0.423, p < 0.04; stress (mN/mm2) versus FEV1 (%pred), r = 0.158, p = NS; force versus FEV1/FVC (%), r = 0.554, p < 0.004; stress (mN/mm2) versus FEV1/FVC (%), r = 0.484, p < 0.02.

Stereological Analysis

The mean values obtained from morphometry are shown in Table 4. Results for the amount of smooth muscle, the normalized smooth muscle (ASM/Dmax), and the total airway wall area are shown for airways from obstructed and nonobstructed patients. Both the amount of ASM and ASM/Dmax were significantly increased in the obstructed group (p < 0.05 and p < 0.01, respectively). Although there was no difference in the total airway wall cross-sectional area between the two patient groups, the fraction of the total airway wall made up of muscle was significantly higher (p < 0.02) in airways from obstructed patients.

Transpulmonary pressure at Lmax (Ptp). Table 5 shows the values calculated for Ptp at Lmax, the length at FRC, the passive tension at FRC, and the shortening at FRC. Ptp at Lmax was significantly higher in the obstructed group (p < 0.05), while airway length and passive tension at FRC were significantly reduced in the obstructed group (p < 0.05). There was no difference in the amount of shortening at FRC between the patient groups. Figure 5 shows significant correlations between Ptp at Lmax and FEV₁/FVC (%) (r = 0.390, p < 0.05), and between passive tension at FRC and FEV₁/FVC (%) (r = 0.412, p < 0.02).

View larger version (13K): [in this window] [in a new window]   Figure 5.   (A) Transpulmonary pressure at Lmax versus FEV1/FVC (%) (r = 0.390, p < 0.05); (B) passive tension at FRC (%Fmax) versus FEV1/FVC (%) (r = 0.412, p < 0.02) (n = 30 airways).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Lung Function

The purpose of this study was to characterize the mechanical properties of peripheral airways, and to relate these properties to pulmonary function in patients who had variable airflow obstruction, predominantly secondary to chronic cigarette smoking. We categorized our patients as obstructed or nonobstructed on the basis of the ratio of FEV₁ to FVC. We used an FEV₁/FVC of less than 70% to define airflow obstruction. We also used measurements of FEV₁/FVC (%) and FEV₁ (%pred) as continuous variables to examine their relationships to airway smooth muscle force, stress, and shortening. As expected, patients categorized as being obstructed on the basis of FEV₁/FVC also showed hyperinflation and gas trapping with significant increases in FRC and residual volume. Only one of the patients had any features suggestive of asthma.

In Vitro Function and Correlations with In Vivo Function

Maximal isometric force was significantly higher in obstructed patients. In addition, when the force was normalized by the amount of smooth muscle, the values of stress developed by the smooth muscle were still significantly higher in the obstructed individuals, indicating that both contractility and muscle mass are increased in disease. Because we used a supramaximal concentration of acetylcholine as the agonist, a change in sensitivity cannot explain these results. An increase in muscle stress could be due to a fundamental change in the contractile apparatus of the muscle or to diminished activity of a mechanism(s) that normally attenuates muscle stress during maximal stimulation. A fundamental change in the contractile properties could be caused by intracellular reorganization of the contractile apparatus resulting in an increase in the number of myosin filaments or in the number of contractile units in parallel (30). Changes in the molecular mechanism underlying altered contractility could occur at the level of the enzymes involved in smooth muscle contraction. An increase in the amount and activity of myosin light chain kinase, and in the activity of myosin ATPase, have been found in sensitized canine airway smooth muscle (5, 6) but only in association with an increase in the velocity and capacity of shortening, and not force generation. Alternatively, there could be more myosin and actin per unit cross-sectional area of muscle or an alteration in the expression pattern of myosin isoforms. Increased stress could also be due to loss, in the tissue of obstructed patients, of an inhibitory factor or factors such as relaxant prostanoids or acetylcholinesterase normally produced by airway epithelial cells (31). The design of our study does not allow us to conclude which of these mechanisms led to increased ASM stress in the obstructed group.

In addition to studying airflow obstruction as a categorical variable we examined the relationships between ASM force, stress, and shortening and the severity of pulmonary dysfunction. The results of these analyses support the categorical analysis in that maximal isometric force was significantly related to both FEV₁/FVC (%) and FEV₁ (%pred) and FEV₁/ FVC (%) was significantly related to maximal isometric stress.

The observation that both FEV₁/FVC (%) and FEV₁ (%pred) are significantly related to maximal force but that the significance of the relationship with FEV₁ (%pred) is lost when maximal stress is used as the dependent variable suggests that, in part, the relationship between muscle force and airflow obstruction is due to an increase in the amount of muscle in the airways that were studied. This finding is consistent with previous reports of increased smooth muscle mass in both asthma (32, 33) and COPD (33, 34). However, the fact that a significant relationship still remains between stress and FEV₁/ FVC (%) supports the contention that there is also a change in the ASM contractile function in patients who have airflow obstruction.

The relationships between force and FEV₁/FVC (%) and force and FEV₁ (%pred) appear to be heavily influenced by a single individual who had the most severe airflow obstruction and an airway in which the muscle made considerable isometric force. The large amount of force generated by the airway from this individual is consistent with the fact that such airway had approximately twice the amount of smooth muscle as the mean of the rest of the group. The fact that there was a correlation between force and stress shows that the data from this individual are in keeping with the rest of the population. Even if this subject is removed from the study, all significant correlations remain. Nonetheless, it should be noted that this was the individual with the most severe obstructive lung disease in our group and was the only patient with an FEV₁/FVC (%) less than 50% included in this study. This censoring in the data to patients with relatively mild chronic airflow obstruction is related to the fact that patients with severe airflow obstruction are not selected for surgery.

This is the first study in which a significant relationship has been demonstrated between ASM force and stress and lung function. In previous studies correlations between in vitro ASM function and in vivo lung function or measures of airway responsiveness have been found inconsistently and the varied results may have been related to deficiencies in the design of the studies. In several of the positive studies the responses from only one patient with asthma have been compared with a group of control patients (18). With the exception of two studies (19, 35), the data reported to date has included measurements only of isometric force, and not isotonic shortening, which may be a more appropriate in vitro indicator of the airway narrowing that is occurring in vivo. In addition, there are few studies in which the measurements of force have been converted to stress by correction for muscle cross-sectional area. Only Ishida and coworkers (36) and Bramley and colleagues (19) measured both the force and smooth muscle in cross-section and calculated the stress generated by the muscle. In the study by Ishida and coworkers only main stem airways from 10 subjects were studied and comparisons between obstructed and nonobstructed individuals were not done. In our study both the isometric force and isotonic shortening were measured, and the force was normalized by the measured amount of smooth muscle. We did not, however, make measurements of velocity of shortening, which may be also be altered in chronically inflamed airways (37).

Maximal isotonic shortening was not significantly different between obstructed and nonobstructed subjects. This was surprising in light of the observation that there were positive correlations between ASM force and stress and the amount of shortening. An increase in force without a significant increase in shortening must be related to differences in the loads acting on the smooth muscle. Although not significant, the passive tension at Lmax tended to be lower in the obstructed individuals, and this ~ 20% reduction in passive preload could have influenced the subsequent isotonic shortening. We did not measure maximal unloaded shortening; the preload required to stretch the muscle to Lmax remained during shortening and constituted an elastic, auxotonic load.

An additional load on smooth muscle during shortening has been described by Meiss (38), who showed that if canine smooth muscle preparations, which normally shorten ~ 75%, are constrained by nonextensible silastic rings shortening is reduced. The connective tissue content of human peripheral airways is much higher than the relatively pure smooth muscle of canine trachealis and it is possible that this connective tissue radially constrains the ASM and reduces its ability to shorten. The high passive tension required to stretch the muscle to Lmax is a reflection of this high connective tissue content and the resultant radial constraint could explain the relatively lesser shortening in the human tissue as well as the failure to find a significant difference in maximal shortening despite differences in force and stress between obstructed and nonobstructed individuals.

Stereology of Smooth Muscle

To calculate smooth muscle stress the cross-sectional area of the smooth muscle was obtained by morphometry. Thomson and coworkers (39) showed that with morphometry some of what is usually considered smooth muscle area is really interstitial connective tissue. They found that this overestimation of muscle area was greatest when thick (> 5 µm) sections of longitudinally arranged smooth muscle bundles were examined. Although we used thin sections (2 µm) and performed the morphometric analysis on cross-sections of muscle to minimize this source of error, higher magnification would likely have allowed more precise quantification of muscle and thus larger values for stress. There is no reason to believe that this artifact would have been different in obstructed versus nonobstructed individuals.

Transpulmonary Pressure at Lmax

The estimated transpulmonary pressure at Lmax was higher in the airways from obstructed individuals, while the length of the muscle preparation at "FRC" was lower. The basic assumption made in estimating the transmural pressure at Lmax and the length at FRC is that in the absence of smooth muscle contraction in vivo the peribronchial interstitial pressure is equal to pleural pressure (i.e., the parenchymal attachments on the airway do not exert additional stretch in the absence of muscle shortening). This assumption has been validated by Sasaki and colleagues (40). The shorter length of the smooth muscle at FRC suggests that the airways of the obstructed group are stiffer and that more passive tension is required to stretch these airways to the length at which maximal isometric forces are developed.

In summary, we have demonstrated, for the first time, that the peripheral airway smooth muscle of patients who have COPD shows increased contractility in addition to increased volume. Airway remodeling occurs in the inflamed airways of patients who have COPD and asthma and is associated with airway hyperresponsiveness. In COPD the emphasis has been placed on loss of lung recoil and fibrosis of the small airways. These results suggest that altered peripheral airway smooth muscle function may be an important determinant of abnormal airway function in this group of patients.