1Meakins Christie Laboratories and 3Genetics Unit, Shriner's Hospital for Crippled Children, McGill University, Montreal, Quebec, Canada; and 2Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania
Submitted 15 March 2004 ; accepted in final form 14 September 2004
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ABSTRACT |
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complex impedance; pressure-volume curves; length-stress curves
Decorin has been shown to be present in tissues with fibrillar collagen, such as the lung (2, 15, 16). Decorin has been described in the submucosal region of the airway wall as well as in the lung vasculature (15, 16). Decorin may also play a role in the remodeling associated with such respiratory diseases as pulmonary fibrosis and asthma (2, 16). The process of remodeling involves the deposition of new matrix, including decorin-regulated collagen fibrillogenesis. Decorin has the ability to bind transforming growth factor- (22), a cytokine implicated in pathological processes characterized by remodeling (12, 21).
Danielson et al. (5) have recently published studies in mice in which the decorin gene has been disrupted such that decorin mRNA is not transcribed and the decorin protein is not produced. They described that the collagen fibers in the skin of these decorin-deficient (Dcn/) mice were more heterogeneous, both in terms of size and structure, than those in Dcn+/+ animals. In studies characterizing the mechanical behavior of the skin, they documented increased skin fragility, altered compliance, and reduced tensile strength in the Dcn/ mice compared with Dcn+/+ controls.
We questioned whether lung mechanical properties would also be altered in Dcn/ mice. We were particularly interested in the behavior at the top portion of the quasistatic pressure vs. volume (P-V) curve, as it is thought the mechanical behavior of collagen is more readily sampled at higher lung volumes (11). Therefore, we studied the dynamic and quasistatic mechanical lung tissue properties, both in vivo and in vitro, of Dcn/ and Dcn+/+ mice. We measured complex impedance of the respiratory system as well as quasistatic P-V curves in vivo. Additionally, we measured the complex impedance and the length-stress relationship of parenchymal tissue strips in vitro. We also performed Western blotting and immunohistochemistry to quantify and localize both decorin and biglycan, another SLRP. Biglycan, like decorin, belongs to the class 1 subfamily of SLRPs (8). Of the SLRPs, biglycan and decorin show the highest internal homology in amino acid sequence (57%) (8). They have the same types of glycosaminoglycan sidechains, i.e., dermatan and/or chondroitin sulfate. Finally, and perhaps most relevant to this study, biglycan and decorin deficiency seem to have similar effects on collagen fibril structure, at least in the skin of mice (4). There is published evidence for upregulation of one member of an SLRP family in response to a deficiency in another family member. In the case of the fibromodulin-null mouse, lumican protein increases fourfold in the tail tendon compared with the tail tendon of wild-type mice (20). We reasoned, therefore, that biglycan might be upregulated in the Dcn/ mice to compensate for the lack of decorin and subserve its usual physiological function.
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METHODS |
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Breeding was initiated with a male C57Bl/6 mouse heterozygous for decorin Dcn+/ and Dcn+/+ C57Bl/6 females. Mice were housed in a regular animal facility at McGill University (Montreal, PQ). The mutation of the decorin gene was created by targeted disruption of exon 2 of murine decorin by the insertion of the Pgk-neo cassette (5). The genotype of the offspring was determined by extracting DNA from the tail tissue using a Genomic DNA Purification kit (Promega, Madison, WI) according to the manufacturer's instructions. Real-time PCR (Roche, Basel, Switzerland) was used to analyze DNA. Sense and antisense primers corresponding to exon 2 of murine decorin, and an additional primer corresponding to the Pgk promoter of the Pgk-neo cassette (Pgk primer), were generated (5). The reaction mixture consisted of 1 µl DNA, 1 µl sense primer, 1 µl antisense primer or 1 µl PGK primer, 7 µl H2O, and 10 µl SBr-green mix (Qiagen, Hilden, Germany and Qiagen, Mississauga, ON). Reaction conditions were as follows: melting, 95°C, 900 s; PCR (45 cycles), segment 1, 94°C, 15 s; segment 2, 57°C, 20 s; segment 3, 72°C, 25 s; melting curve, 57°C, 45 s; cooling, 30°C, 30 s.
To verify that the products of the RT-PCR reaction were of the appropriate size, PCR products were run on 1.8% agarose gel for 1.5 h at 60 V.
In Vivo Measurements
Animal preparation. Dcn/ and Dcn+/+ C57Bl/6 mice (812 wk old) were anesthetized with an injection of xylazine (12 mg/kg ip) followed 5 min later by an injection of pentobarbital sodium (40 mg/kg ip). After tracheostomy, an 18-gauge metal cannula was inserted into the trachea and tightly bound. The mouse was connected via the tracheal cannula to a computer-controlled small animal ventilator (FlexiVent, Montreal, PQ) (18). Mice were mechanically ventilated at 150 breaths/min with a tidal volume of 6 ml/kg at a positive end-expiratory pressure (PEEP) of 1.5 cmH2O. The animals were paralyzed with an injection of pancuronium bromide (1.2 mg/kg ip). Heart rate was monitored by a 3-lead electrocardiogram. All animals received humane care in compliance with the Guide to the Care and Use of Experimental Animals formulated by the Canadian Council of Animal Care, and an institutional animal ethics committee approved the protocol.
Measurement of complex impedance.
A computer-generated volume signal composed of 19 mutually primed sinusoids ranging from 0.25 to 19.625 Hz was applied to the airway opening. The amplitudes of the sinusoids decreased hyperbolically with frequency such that each frequency component had equal power. The phase of each component was chosen to minimize the peak-to-peak amplitude excursions of the complex signal. The signal had a peak-to-peak volume of 0.17 ml and lasted 16 s. Piston displacement (ml) and cylinder pressure (cmH2O) were measured in a cylinder of known radius during the application of the signal. A total lung capacity (TLC; in the mouse 25 cmH2O) was delivered to standardize volume history. One minute later, complex impedance of the respiratory system (Zrs) was measured at a PEEP of 1.5 cmH2O. Measurements were repeated three or four times for each mouse, and the parameter estimates from each of the signal applications were averaged.
Calculation of parameters.
Zrs was determined using the equation
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Measurement of P-V curve. After the complex impedance measurements, quasistatic measurements of the P-V relationship of the respiratory system were performed. Lungs were slowly inflated to TLC and then stepwise deflated over 18 s. The signal was applied three times, and the data were pooled to make a single P-V curve.
The expiratory limb of the P-V curve was characterized by the equation (17)
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In Vitro Measurements
Tissue preparation. Mice were disconnected from the small animal ventilator after the in vivo experiments were completed. Through an abdominal incision, the diaphragm was cut and a bilateral pneumothorax was induced. The thorax was opened, the animal was exsanguinated, and the heart, lungs, and trachea were carefully resected en bloc ensuring that the lungs were not punctured. The lungs were rinsed with a modified Krebs solution (mM: 118 NaCl, 4.5 KCl, 25.5 NaHCO3, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 10 glucose, pH 7.40, 6°C; Sigma, St. Louis, MO) by filling the lungs three times to TLC. The right lung was reserved for pathological study, and a strip (1 x 1 x 67 mm) was cut from the periphery of the left lung. The remainder of the left lung was set aside for protein extraction. The pleura was removed, and the unloaded length (lo) and wet weight (W0) were recorded. The strips were kept in a bath of iced Krebs solution that was continuously bubbled with 95% O2-5% CO2.
Experimental setup.
Metal clips were glued to either end of the tissue strip with cyanoacrylate. Steel music wires (0.5-mm diameter) were attached to the clips, and the strip suspended vertically in an organ bath. A mercury bead was placed in the bottom of the organ bath, allowing the wire to pass through the bath but preventing the Krebs solution from leaking out. The bath was filled with 20 ml of Krebs solution, maintained at 37°C and continuously bubbled with the 95% O2-5% CO2. One end of the strip was attached to a force transducer (model 400A; Cambridge Technologies, Watertown, MA) that had an operating range of ±10 g, a resolution of ±200 µg, and a compliance of 1 µm/g, whereas the other end was connected to a servo-controlled lever arm (model 300B; Cambridge Technologies) that delivered length perturbations to the mounted strip. The lever arm was capable of peak-to-peak length excursions of 8 mm and length resolution of 1 µm and was in turn connected to a computer that controlled the frequency, amplitude (), and wave form of the oscillation. Movement of a screw thumbwheel system, which effected slow vertical displacements of the force transducer, set the resting tension (T). Length and tension signals, as obtained by the lever arm and force or tension transducer, respectively, were converted from analog to digital with an analog-to-digital converter (DT2801-A; Data Translation, Marlborough, MA) low-pass filtered and recorded on a computer at a sampling frequency of 256 Hz.
Measurement of complex impedance. For measurements of complex impedance, an 8-s broad-band pseudorandom displacement input signal composed of 17 mutually primed frequencies ranging from 0.5 to 19.75 Hz with a maximal amplitude of 0.18 mm was generated by a computer and delivered to the lever arm. The linearity and hysteresis of the system were tested by measuring the moduli of a steel spring of stiffness comparable with that of the tissue strip. The spring was suspended in the bath by music wire in the same manner as the strip. The frequency and amplitude dependence of the system was assessed over a range of frequencies (0.110 Hz). The spring stiffness did not show any dependence on oscillatory frequency or amplitude in the tested range. The hysteresivity of the system was independent of frequency and amplitude and had a value <0.003.
Lung parenchymal strips were preconditioned by slowly cycling tension from 0 to 2 g three times; on the third cycle, the strip was unloaded to a stress of 500 mg/mm2 and was allowed to stabilize for 45 min, at the end of which time the stress was
450 mg/mm2. Complex impedance was then measured. Measurements consisted of eight consecutive 8-s recordings; the average of these eight recordings was calculated.
Calculation of parameters.
Tissue impedance (Ztis) was calculated as
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Tension and length signals were obtained and recorded as a function of time, Fourier transformed to functions of frequency, and then complex impedance was calculated. The mechanical parameters were estimated by fitting the constant-phase model (7) to the impedance data according to Eq. 2. In this instance, the parameter R reflects the Newtonian resistance of the tissues. Results were standardized for strip size by multiplying the values of G and H by lo/Ao. Ao is the unstressed cross-sectional area of the lung parenchymal strip obtained from the formula
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Measurement of length-stress curves.
When the complex impedance measurements were completed, the strip was stretched to 3-g tension by adjusting the screw thumbwheel system and then relaxed in a stepwise fashion, allowing 5 s between steps. This generated a length-tension curve, which was then converted to a length-stress curve, with stress (
) calculated as T/Ao. The data points were fit with a five-parameter exponential curve
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Tissue Fixation and Immunocytochemistry
The right lung was filled with histocon, submerged in optimum cutting temperature compound (OCT), and immediately frozen in isopentane cooled in liquid nitrogen. Sagital sections (7-mm thickness) were cut from medial and lateral aspects of the lobe. Sections were incubated overnight at 4°C with the primary antibody [either a 1:200 dilution of rabbit anti-mouse decorin antiserum (LF-113) or a 1:100 dilution of rabbit anti-mouse biglycan antiserum (LF-159) (6)]. The slides were then washed and subsequently incubated for 30 min in a 1:100 dilution of biotinylated swine antibody against rabbit immunoglobulin (E 0353; Dako, Glostrup, Denmark). Finally, the slides were incubated for 30 min in a 1:100 dilution of alkaline phosphatase-conjugated avidin (D 0365; Dako). The slides were developed with Fast Red TR (F-2768; Sigma, Oakville, ON), counterstained with Gill II hematoxylin, and fixed in lithium carbonate. After being allowed to dry, the slides were covered with a thin layer of crystal mount.
Protein Extraction and Western Blotting
Proteoglycans were extracted from the left lung in 4 M guanidine HCl. The extract was dialyzed extensively and the pellet resuspended. The amount of protein was determined with the Bio-Rad Protein assay (Bio-Rad, Mississauga, ON) An aliquot of 10 µg of protein from each sample was incubated with protease-free chondroitinase ABC (0.1 U/ml; Sigma). Proteins were separated on 10% polyacrylamide gels under reducing conditions and transferred to nitrocellulose membranes (Hybond ECL; Amersham Biosciences, Baie d'Urfé, PQ). Membranes were incubated in a 1:1,000 dilution of a rabbit anti-mouse decorin antiserum (LF-113) or a rabbit anti-mouse biglycan antiserum (LF-159) that were generated against synthetic decorin and biglycan peptides of the mouse protein (6). Membranes were then incubated with biotin-labeled swine anti-rabbit secondary antibody (Dako) and finally with streptavidin-biotinylated horseradish peroxidase complex (Amersham Biosciences). The antigen/antibody complexes were detected with ECL detection reagents (Amersham Biosciences) according to the manufacturer's instructions. Cartilage extracts were used as a positive control. Membranes were then stripped, reblocked, and probed with polyclonal antibody against actin. Densitometric analysis of biglycan and actin was accomplished with image analyzer software (Fluorchem; Alpha Innotech, San Leandro, CA), which measures the sum of all the pixel values after background correction.
Data Analysis
All in vivo data manipulations were performed with FlexiVent software, whereas all in vitro data manipulations were performed with the ANADAT software package (RHT-InfoDat; Montreal, PQ). T-tests were used to assess whether mechanical parameters were different in Dcn/ vs. Dcn+/+ mice. Results were considered statistically significant at a probability level of 5%. Values are reported as means ± SE.
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RESULTS |
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The results for the dynamic measurements of complex impedance for both the in vivo and in vitro preparations are shown in Table 1. In vivo, airway resistance (Raw) was smaller in the Dcn/ mice than in the Dcn+/+ mice. However, tissue damping and elastance were not significantly different. In vitro, no differences were observed in the complex impedance of the tissue strips. Again, tissue damping and elastance were equivalent in the two groups.
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DISCUSSION |
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Certain technical issues warrant discussion. When comparing pressure vs. volume and length vs. stress curves from different animals, it is necessary to decide at which point to match the curves. We chose to match the curves at a constant pressure, rather than a constant volume, as it was not feasible to determine absolute lung volume in the closed-chested preparation in vivo. We chose to match for stress in vitro in part to be consistent with our in vivo approach and in part because determining lo, once the parenchymal strip was mounted in the organ bath, was problematic, for the reasons outlined below. Second, we chose to match at the lower, rather than the upper, end of the curves. A transpulmonary pressure of 1.5 cmH2O likely represents functional residual capacity in the mouse. In vitro, we matched the curves at the lower stress, again to be consistent with the in vivo approach, and also because of technical difficulties related to making measurements at the higher stress levels in the strips from the Dcn/ mice. As described in RESULTS, the lung tissue of the Dcn/ mice was quite fragile. Stress failure (tearing of the tissue) often occurred at higher stresses during measurement of the length-stress curve in the Dcn/ parenchymal strips. This reduced the range over which the length-stress curve could be obtained. Therefore, we had more consistent data in the lower portion of the stress-length curve and therefore chose to match the curves at this level. It is possible that our results may have varied slightly had we matched the curves for length or strain rather than stress, but this is difficult to predict. The two in vitro curves were sufficiently distinct to make this result unlikely.
Traditionally, a stress-strain curve [where strain = (l lo)/lo] is obtained to gather information about in vitro tissue mechanics. However, because of the different compliance of the Dcn/ vs. Dcn+/+ tissue strips, the resting length (lo) of the tissue strips, once mounted in the organ bath and the operating stress applied, was not consistent. Therefore, we measured the stress-length relationship, where length was the absolute change in length from the original length in the organ bath.
As shown in Fig. 2, the difference between the two groups of mice occurred in the values of the slope at higher pressures. The compliance of the Dcn/ and Dcn+/+ mice was equivalent in the lower portion of the curve and different in the higher pressure range. Tissue elastance, calculated from the complex impedance measured at PEEP = 1.5 cmH2O, was also similar in the two populations of mice. It has been suggested that elastic fibers account for lung compliance in the lower pressure range, i.e., around functional residual capacity, whereas collagen fibrils become more important as lung volume becomes limited. Mercer and Crapo (11) have conducted elegant morphometric studies examining the configuration of collagen and elastic fibers in the alveolar duct and wall. They concluded that at low levels of strain, collagen fibers have a "wave-like" configuration and are readily extensible; stress is borne by adjacent elastic fibers. At higher levels of strain, collagen fibers act to limit further distention. Recently, Sly et al. (19) reported a decrease in the hysteresivity of mouse lungs as lung volume increased. These authors suggest that their findings support the idea that at high lung volumes, the tissue matrix contributes less to the mechanics of the lung, whereas the individual collagen fibers become more important. Decorin binds collagen and is involved in both collagen fibrillogenesis and the spacial alignment of the fibers in the matrix. It seems reasonable, therefore, to expect that the absence of decorin would affect lung mechanics at a lung volume where the mechanical behavior of collagen becomes limiting.
In these experiments, we did not measure the actual TLC of the lungs from the two groups of animals. After in vivo measurements, it was necessary to quickly open the chest, excise the lungs, and prepare them for excision of parenchymal strips, fixation in OCT, and protein extraction. This precluded measurement of lung volume by water displacement, for example. We matched the P-V curves at PEEP = 1.5 cmH2O and measured lung volume at 18 cmH2O to obtain information about actual differences in lung volume at equivalent pressures. (We chose 18 cmH2O because this was the highest pressure for which we had data in all the animals.)
In vivo measurements include the mechanical behavior of many different elements in addition to the collagen-elastin proteoglycan matrix. Because the P-V curve was measured in closed-chested animals, we were also sampling mechanics of the chest wall. There is information in the literature on the contribution of the chest wall to measurement of complex impedance in mouse lungs. Sly et al. (19) reported that R and H are nearly identical in open- vs. closed-chested mice over a wide pressure range. G was somewhat higher in the close-chested preparation. Hence, attributing R in close-chested animals to Raw, and H to lung compliance, as we have done in the current study, is appropriate. The air-liquid interface, or surfactant, also contributes significantly to the compliance of the lungs (14). Studies by Bachofen and colleagues (1) measuring the relationships among surface tension, surface area, and alveolar geometry showed that, at low lung volumes, surface forces are important in determining mechanical behavior and hysteresis, whereas at higher lung volumes, tissue forces become predominant.
The differences in the stress vs. length curves measured in vitro address the issue of which elements are contributing to the difference in in vivo compliance observed in the Dcn/ vs. Dcn+/+ animals. The parenchymal strip is composed primarily of alveolar walls (9); hence, the behavior of the fibrous matrix is mainly sampled. Moreover, the preparation is fluid filled, thereby eliminating the contribution of surface forces. The consistent in vivo and in vitro results implicate the tissue matrix as the source of the different mechanical properties in the two populations of mice.
One issue that remains, however, is the question of why there were no observable differences in the values for G and H in the dynamic measurements of complex impedance. In vivo measurements were performed at 1.5 cmH2O PEEP, a relatively low transpulmonary pressure, where collagen was perhaps not yet recruited. In vitro measurements were performed at an operating stress of 500 mg/mm2. This represents a relatively high stress, roughly equivalent to an in vivo transpulmonary pressure of 3050 cmH2O (13). At this level of stress, collagen fibers should contribute to the mechanical behavior. One possibility to explain the lack of differences in dynamic measurements between the two sets of tissue strips is that in vitro measurements are made in a system where the tissue is strained in a uniaxial fashion, rather than in three dimensions, as is the case in vivo, and the relative effects of increasing strain and volume may therefore differ. Another possible explanation is that the amplitude of the length signal during impedance measurements was relatively modest (the largest amplitude component was 0.18 mm or 3% lo) compared with the change in length during the quasistatic measurement. A large length perturbation may be required for the compliance characteristics of collagen to be revealed, even at higher stresses.
One dynamic parameter that was different between the Dcn/ and Dcn +/+ mice was Raw (Table 1). Immunohistochemical staining demonstrated the presence of decorin in the wall of airways and blood vessels in the Dcn+/+ mice. This distribution of decorin has also been reported by Redington and coworkers (15) in the lungs of normal and asthmatic humans. Mice deficient in decorin may have decreased Raw because of the impact of decorin on the compliance of the airway wall. A more compliant airway wall could be relatively dilated because of the mechanical interdependence between airways and the surrounding parenchymal attachments (10). The parenchyma would more effectively tether open the more compliant airway wall, resulting in an airway with a larger luminal diameter and a lower Raw. It would be of interest to determine whether decorin-deficient mice respond differently to contractile challenge.
In addition to changes in the mechanical properties of the lung, we questioned whether, in the absence of decorin, other proteoglycans might be upregulated. Specifically, we hypothesized that biglycan, a SLRP structurally similar to decorin, would be increased. Decorin and biglycan have core proteins of approximately the same length and composition (8). Moreover, they both have chondroitin sulfate side chains; decorin has one and biglycan has two. A study recently reported by Svensson et al. (20) showed that lumican in the tail tendon of a fibromodulin-null mouse increased fourfold compared with the tail tendon of wild-type mice. However, we observed no measurable differences in the amount of biglycan in the lung tissues of Dcn/ and Dcn+/+ mice.
In conclusion, we have shown that lung tissue mechanics are altered in Dcn/ mice. Compliance was increased at higher pressures and stresses, and Raw was decreased. In Dcn+/+ mice, decorin was present in the vessel and airway walls. Biglycan was not upregulated to compensate for the absence of decorin. The alteration in tissue properties likely occurred as a result of the abnormal collagen fibril formation that occurs in Dcn/ mice (5). Further imaging studies to document the precise abnormalities in the formation of the fibrils comprising the lung matrix would be of interest.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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