Alterations in lung mechanics in decorin-deficient mice

Anita Fust,1 Frederique LeBellego,1 Renato V. Iozzo,2 Peter J. Roughley,3 and Mara S. Ludwig1

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


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Decorin, a small leucine-rich proteoglycan with a widespread tissue distribution, is required for the normal fibrillogenesis of collagen in most tissues. Because collagen is important in determining the elastic behavior of the lung, we hypothesized that lung tissue mechanics would be altered in a mutant mouse in which the single decorin gene was abrogated by targeted deletion (Dcn–/–). Complex impedance of the respiratory system was measured in C57Bl/6 mice (Dcn–/– and Dcn+/+) using a small animal ventilator that delivers a volume signal with multiple frequencies to the trachea. A constant-phase model was fit to calculate airway resistance (Raw), tissue damping, and tissue elastance. Compliance of the respiratory system (Crs) was measured from a pressure volume curve during stepwise deflations. Lungs were excised, and parenchymal tissue strips were mounted in an organ bath for in vitro measurement of tissue impedance and quasistatic length-stress curves. In addition, pulmonary tissue was examined by immunohistochemistry and immunoblotting. In vivo, in the Dcn–/– mice, Raw was decreased and Crs was increased. Similarly, in vitro, length-stress curves showed increased compliance of the strips in the Dcn–/– mice. These alterations in lung tissue mechanical behavior in Dcn–/– mice support a critical role for decorin in the formation of the lung collagen network.

complex impedance; pressure-volume curves; length-stress curves


THE VISCOELASTIC BEHAVIOR of the lung parenchymal tissues is determined primarily by the extracellular matrix and its components. The tissue matrix comprises collagen and elastin fibers, glycoproteins, and proteoglycans. Collagen and elastin fibers are essentially elastic, but when arranged in a network, they display prominent hysteretic properties (3). Decorin, a small leucine-rich proteoglycan (SLRP), plays a key role in regulating collagen fibril formation and the spatial arrangement of collagen fibers in the matrix (8). Via this mechanism, decorin may play an important role in lung tissue mechanics.

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-{beta} (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.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Determination of Genotype

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 (8–12 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

(1)
where P is cylinder pressure (cmH2O) and V is piston displacement volume (ml). Both are functions of angular frequency ({omega}). dV({omega})/dt is the derivative of piston displacement volume with respect to time (t). The Zrs({omega}) was fit with the constant-phase model (17)

(2)
where R is airway (flow-dependent) resistance, I is airway inertance, G is tissue damping, H is tissue elastance, j is the imaginary unit, and {alpha} = (2/{pi})tan–1(H/G).

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)

(3)
where V is volume, P is pressure, and k is an exponential constant. A and B are constants, and e is the exponential constant = 2.718. (We applied curve-fitting criteria to test the exponential curve fit and the usual r2 values >0.99.) The value of k was determined for each animal. Compliance of the respiratory system (Crs) was calculated from the fitted curve between 1.5 and 6.5 cmH2O and between 15 and 20 cmH2O as

(4)
where {Delta}V is change in volume and {Delta}P is change in pressure over the given range.

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 6–7 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 ({epsilon}), 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 (0.1–10 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

(5)
where T is tension in mg, l = length in mm, and t = time (in seconds).

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

(6)
where W0 is the weight of the strip in milligrams, {rho} is the mass density of the tissue taken as 1.06 mg/mm3, and lo is the length of the strip in millimeters.

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 ({sigma}) calculated as T/Ao. The data points were fit with a five-parameter exponential curve

(7)
where a, b, c, and d are constants, and e is the exponential constant = 2.718. Compliance (Ctis) of the strip was calculated from the fitted curve between 300 and 500 mg/mm2 as

(8)

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.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sample results of the genotyping of the mice are shown in Fig. 1. Primers for the wild-type allele produced a product of 161 bp. Primers for the disrupted allele were chosen so that the 3' end began in the region of the inserted Pgk-neo cassette. The product for the disrupted allele was 250 bp. The melting points of the PCR products of the wild-type and disrupted alleles were ~79 and ~84°C, respectively. As shown in Fig. 1A, Dcn+/+ mice had only one product that melted at ~79°C. Heterozygous mice had two products, one melting at ~79°C and the other at ~84°C. Dcn–/– mice had one product that melted at ~84°C. In Fig. 1B, agarose gels of PCR products from the respective animals are shown.



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Fig. 1. A: melting point derivatives for the real-time PCR product of genomic DNA from wild-type (+/+), heterozygous (+/–), and decorin-deficient (–/–) mice. –d(F1)/dT is the negative derivative of fluorescence with respect to temperature. B: PCR product of genomic DNA from wild-type, heterozygous, and decorin-deficient mice on a 1.8% agarose gel. The band at 250 bp corresponds to the disrupted allele. The band at 161 bp corresponds to the wild-type allele.

 
Qualitatively, we observed that the skin, cartilage, and lung tissue of the Dcn–/– mice were more fragile and tore more readily than those of the Dcn+/+ mice.

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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Table 1. Dynamic mechanics in wild-type (Dcn+/+) and decorin-deficient (Dcn–/–) mice

 
The average P-V curves obtained in vivo from all animals are shown in Fig. 2. The k values were not different in the two groups of mice. However, Crs between 15 and 20 cmH2O was higher in the Dcn–/– mice compared with the Dcn+/+ mice (Crs = 2.03 ± 0.09 x 10–2 vs. 1.75 ± 0.04 x 10–2 ml/cm H2O, respectively, P < 0.02). The volume of the lungs at 18 cmH2O was also greater in the Dcn–/– mice than in the Dcn+/+ mice (0.865 ± 0.026 vs. 0.781 ± 0.026 ml, respectively, P < 0.05). We also calculated Crs on the lower portion of the P-V curve, between 1.5 and 6.5 cmH2O. These values were not significantly different (7.93 ± 0.51 x 10–2 vs. 7.27 ± 0.29 x 10–2 ml/cmH2O in Dcn–/– mice vs. Dcn+/+ mice, respectively, P = 0.15).



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Fig. 2. Pressure (P)-volume (V) curves during quasistatic deflation in vivo. The model V = A – BekP, where A and B are constants, was fit to the data to generate the curves. Representative data from 2 animals are shown (inset). The exponential constant (k) was derived from the model fit. Compliance of the respiratory system (Crs) was measured from the slope of the tangent to the curve between 15 and 20 cmH2O. Dcn+/+, wild-type animals; Dcn–/–, decorin-deficient animals; V18, volume of the lungs at 18 cmH2O. *P < 0.05 vs. Dcn+/+; +P < 0.02 vs. Dcn+/+.

 
Length-stress curves obtained in vitro are shown in Fig. 3. The average values for the slope of the tangent between 300 and 500 mg/mm2 (Ctis) for the Dcn–/– strips compared with the Dcn+/+ strips were 3.2 ± 0.2 x 10–3 vs. 2.2 ± 0.3 x 10–3 mm3/mg, respectively (P < 0.01). The length of the strip at 500 mg/mm2 for the Dcn–/– strips compared with the Dcn+/+ strips was 0.73 ± 0.06 vs. 0.53 ± 0.07 mm, respectively (P < 0.05).



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Fig. 3. Stress-length curves during stepwise relaxation of parenchymal strips in vitro. A 5-parameter exponential curve (l = lo + a[1 – e–b{sigma}] + c[1 – e–d{sigma}]), where a, b, c, and d are constants, and e is the exponential constant = 2.718, was fit to the data to generate the curves. Representative data from 2 strips are shown (inset). Compliance of the tissues (Ctis) was measured from the slope of the tangent to the curve between 300 and 500 mg/mm2. L500 is the length of the strip above lo at 500 mg/mm2. *P < 0.05 vs. Dcn+/+; **P < 0.01 vs. Dcn+/+.

 
Results of the Western immunoblotting done for decorin and biglycan are shown in Fig. 4. As expected, no decorin was detected in the Dcn–/– mice. Biglycan, when controlled for protein loading, was not different in the Dcn–/– vs. Dcn+/+ mice (5.25 ± 0.80 vs. 4.91 ± 1.22).



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Fig. 4. Expression of decorin and biglycan in Dcn–/– and Dcn+/+ mice. Cartilage (Ct) was used as a positive control and actin as a control for protein loading.

 
We identified the presence of decorin by immunohistochemical staining of the lung tissue. In the Dcn+/+ mice, decorin epitopes were detected in the airways and around the blood vessels (Fig. 5A), whereas the Dcn–/– lungs failed to reveal any decorin, as expected (Fig. 5B).



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Fig. 5. Photomicrographs of immunohistochemical staining for decorin in wild-type (A) and decorin-deficient (B) animals. In wild-type mice, decorin is prominent around airways (large arrow) and blood vessels (small arrow). Magnification, x200.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The main finding of this study is that, at high lung volumes or stresses, the lung tissue compliance of Dcn–/– mice is significantly different compared with Dcn+/+ mice. This is likely due to the interaction of decorin with collagen, a structural protein that is recruited at higher stresses.

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 30–50 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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This work was supported by the J. T. Costello Memorial Research Fund and Canadian Institutes of Health Research.


    ACKNOWLEDGMENTS
 
A. Fust is a recipient of a studentship award from the Montreal Chest Institute Research Centre.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. S. Ludwig, Meakins Christie Laboratories, McGill Univ., 3626 St. Urbain St., Montreal, Quebec, Canada H2X 2P2 (E-mail: mara.ludwig{at}mcgill.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.


    REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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