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1 Meakins Christie Laboratories, Royal Victoria Hospital, and 2 Genetics Unit, Shriner's Hospital for Crippled Children, McGill University, Montreal, Quebec, Canada H2X 2P2
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We tested the hypothesis that matrix glycosaminoglycans contribute to lung tissue viscoelasticity. We exposed lung parenchymal strips to specific degradative enzymes (chondroitinase ABC, heparitinase I, and hyaluronidase) and determined whether the mechanical properties of the tissue were affected. Subpleural parenchymal strips were obtained from Sprague-Dawley rats and suspended in a Krebs-filled organ bath. One end of the strip was attached to a force transducer and the other to a servo-controlled lever arm that effected sinusoidal oscillations. Recordings of tension and length at different amplitudes and frequencies of oscillation were recorded before and after enzyme exposure. Resistance, dynamic elastance, and hysteresivity were estimated by fitting the equation of motion to changes in tension and length. Quasi-static stress-strain curves were also obtained. Exposure to chondroitinase and heparitinase I caused significant increases in hysteresivity, no decrement in resistance, and similar decreases in dynamic elastance relative to control strips exposed to Krebs solution only. Conversely, measures of static elastance were different in treated versus control strips. Hyaluronidase treatment did not alter any of the mechanical measures. These data demonstrate that digestion of chondroitin sulfate and heparan sulfate alters the mechanical behavior of lung parenchymal tissues.
chondroitin sulfate; heparan sulfate; hyaluronidase; hysteresivity
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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LUNG PARENCHYMAL TISSUES display prominent viscoelastic behavior. The anatomic elements potentially responsible for this mechanical behavior are several and include the collagen-elastin-proteoglycan matrix, the surface film, and contractile elements in the lung periphery (10, 18). The tissue matrix represents a composite of collagen fibers, elastic fibers, proteoglycans, and glycosaminoglycans (GAGs). Collagen and elastin fibers are essentially elastic in nature (11). However, when fibers are arranged in a network, the network may display hysteretic properties (3).
In addition, the lung tissue matrix contains proteoglycans and GAGs. Proteoglycans are macromolecules that consist of a protein core to which GAG side chains are covalently attached. These side chains are hydrophilic in nature and can attract water molecules into the matrix, potentially altering tissue turgor and viscoelasticity (29). Recently, different theories have been advanced to explain tissue viscoelastic behavior in terms of the movement of fibers within the matrix. Mijailovich et al. (22) proposed that tissue viscoelasticity reflects the mechanical friction generated as adjacent fibers slide across one another. In addition, they proposed that the nature of the mechanical interaction between adjacent fibers could be modified by the action of the lubricating film between fibers. Insofar as proteoglycans are known to coat individual collagen and elastic fibers (7, 30), it is possible that alterations in these molecules could affect the energy dissipated at the fiber interface. Suki et al. (35) have proposed that the matrix can be modeled as a polymer-like material; as molecules deform within a polymer-like gel, the strain generated causes conformational changes that result in energy dissipation or viscoelastic behavior.
To test the hypothesis that matrix GAGs are important in contributing to lung tissue viscoelasticity, we performed the following experiment. We exposed excised lung parenchymal strips to specific enzymes that degrade individual GAGs and determined whether the mechanical behavior of the strips was affected. We chose to investigate the effect of degradation of three different GAGs: chondroitin/dermatan sulfate (CS/DS), heparan sulfate (HS), and hyaluronic acid (HA). We chose these specific GAGs for the following reasons. CS/DS is attached to the decorin core protein, a proteoglycan that has been shown to bind to collagen and affect collagen fibril formation in lung tissue (30, 31). HS is attached to membrane-bound proteoglycans such as syndecan and glypican (4, 5) and the basement membrane proteoglycan perlecan (14, 26). These proteoglycans serve to anchor the cell to the surrounding matrix. Finally, HA is a large, highly charged GAG that forms aggregates with the proteoglycans versican and aggrecan and is likely to be important in determining tissue hydration (8). We reasoned that by degrading each of these molecules and measuring the effect on the oscillatory dynamics of isolated lung parenchymal strips, we could obtain important information on the contribution of each of these GAGs to viscoelastic behavior. Moreover, the differential effects of these enzymes might give insight into the relative importance of the different mechanistic pathways these molecules represent.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Tissue Preparation
Male Sprague-Dawley rats weighing ~280 g were obtained from Charles River (St. Constant, PQ) and were housed in a regular animal facility at McGill University (Montreal, PQ). Each animal was anesthetized with an intraperitoneal injection of pentobarbital sodium (30 mg/kg). After tracheotomy, a metal cannula (2-mm internal diameter) was inserted into the trachea and tightly bound. Through an abdominal incision, the diaphragm was cut, and a bilateral pneumothorax was induced. To degas the lung, the animal was mechanically ventilated with 100% O2. A cannula (PE-90) was inserted into the inferior vena cava. The thorax was opened, and after 10 min, the tracheal cannula was clamped and the O2 remaining in the lungs was absorbed into the bloodstream. The inferior vena cava was infused with sterile Hanks' balanced salt solution (pH 7.4, calcium and magnesium free) to rinse the vasculature. The animal was exsanguinated, and the heart, lungs, and trachea were carefully resected en bloc. The lungs were then filled and rinsed via the trachea with modified Krebs solution (in mM: 118 NaCl, 4.5 KCl, 25.5 NaHCO3, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 10 glucose, pH 7.35) at 6°C. Lung parenchymal strips (1.5 × 1.5 × 12 mm) were cut in an orientation parallel to the pleural surface, and after the pleura was dissected, the unloaded or resting length (Lr) and wet weight (W) of each strip were recorded.Experimental Apparatus
Metal clips were glued to either end of the tissue strip with cyanoacrylate glue. Steel music wires (0.5-mm diameter) were attached to the clips, and the strip was 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 while preventing the Krebs solution from leaking out. The bath was filled with 15 ml of Krebs solution maintained at 37°C and continuously bubbled with 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 compliance of 1 µm/g, and the other end was connected to a servo-controlled lever arm (model 300B, Cambridge Technologies). The lever arm was capable of peak-to-peak length excursions of 8 mm and a length resolution of 1 µm and was, in turn, connected to a function generator (model 3030, B & K Precision, Dynascan, Chicago, IL) that controlled the frequency (f), amplitude, and waveform of the oscillation. The resting tension (T) was set by movement of a thumbwheel screw system that effected slow vertical displacements of the force transducer. Length and force signals were converted with an analog-to-digital converter (DT2801-A, Data Translation, Marlborough, MA) and recorded on an A/T-compatible computer.The linearity and hysteresis of the system were tested by measuring the
moduli of a steel spring that was of a stiffness comparable to that of
the tissue strip. The spring was suspended in the bath by music wire in
the same manner as the strip. The f and amplitude dependence
of the system were assessed over a range of frequencies (0.1-10
Hz). The spring stiffness did not show any dependence on oscillatory
f < 5 Hz. The hysteresivity (
) of the system was independent of f and had a value of <0.003.
Experimental Protocol
Oscillatory measurements.
One strip per animal was obtained. Each lung parenchymal strip was
preconditioned by slowly cycling the applied tension from 0 to 2 g
three times. On the fourth cycle, the strip was unloaded to a stress
(
) of 45 g/cm2 and allowed to stabilize for 45 min. The
final resting was ~30 g/cm2. Strips were then
oscillated at different amplitudes (0.5, 1.0, 2.5, and 5.0%) of
Lr and different frequencies (0.3, 0.6, 1.0, and
3.0 Hz) in random order; the only exception was that the 5% amplitude
was sampled last. Twenty-second recordings were made at each amplitude
and f.
Quasi-static measurements.
Stress-strain curves were obtained from the same strips before and
after incubation with enzyme after oscillatory measurements were
completed. Length was unloaded in stepwise increments (~200 mg) from
a maximum of ~60 to 0 g/cm2. Recordings were made
after 20 s at each step, at which time values of had reached a
relative plateau. and extensibility at the inflection point (IP)
were calculated from the stress-strain curves as defined by Sata et al.
(28).
Calculation of parenchymal mechanics.
The W (in g) and unloaded Lr (in cm) were used
to obtain the average unstressed cross-sectional area
(Ao; in cm2) with the following
formula
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(1) |
is the mass density of the tissue (1.06 g/cm3).
The T (in g) required to achieve the required (in
g/cm2) was calculated with the following equation
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(2) |
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(3) |
, the
equation used was
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(4) |
is a dimensionless variable that represents the
coefficient of coupling between elastic and dissipative stresses.
For quasi-static measurements, strain (
) was defined as
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(5) |
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(6) |
Enzymes and Enzyme Assays
To establish the optimum conditions (i.e., concentration and incubation time) for each enzyme, preliminary experiments were conducted. Tissue was collected from male Sprague-Dawley rats as described in Tissue Preparation. Each strip was placed in 1 ml of modified Krebs solution containing the enzyme of interest (chondroitinase ABC from Proteus vulgaris, heparitinase I from Flavobacterium heparinum, and hyaluronidase from Streptomyces hyaluroticus). A range of concentrations and incubation times was used. Experiments with hyaluronidase and heparitinase were conducted at 37°C; those with chondroitinase ABC were conducted at 20°C. This latter modification was done to maintain tissue viability because the incubation period was relatively long. Karlinsky et al. (16) previously showed that incubation at this temperature does not cause alterations in the quasi-static mechanical properties of lung parenchyma. At the end of the different incubation periods, the strips were removed and rinsed thoroughly in modified Krebs solution to remove the enzyme, placed in 10 mM sodium acetate buffer, pH 6.0 (200 ml), and frozen at
80°C.
GAGs were then extracted to measure the extent of degradation. On the
day of extraction, the tissue was placed in 20 µl of 20 mM sodium
acetate (pH 6.8) containing 5 mM EDTA and 10 mM cysteine. Papain was
added at 1 mg/20 mg tissue. The mixture was incubated at 37°C for
24 h. The reaction was terminated by the addition of
iodoacetamide. The volume was increased to 80 µl by the addition of
50 mM Tris · HCl (pH 8.0). CS/DS and HS degradation were
measured by the dimethylmethylene blue (DMMB) assay method, where the
optical density (OD) was measured at a wavelength of 525 nm
(
525). The extent of HA degradation was assessed
by radiometric assay, which uses specific HA binding protein isolated
from bovine cartilage.
Exposure for 16 h at 20°C to chondroitinase ABC (0.6 U/ml)
resulted in maximal degradation of 37 ± 8% of total sulfated
GAGs. This maximal digestion was confirmed by further exposing the
papain-digested tissue to chondroitinase ABC. There was no further
reduction in OD at 525 as measured by the DMMB assay,
indicating that CS/DS had been completely degraded in this tissue
during the initial exposure. Exposure to heparitinase I, 0.006 U/ml for
5 h at 37°C, resulted in maximal degradation of 73% of total
sulfated GAGs. Exposure to hyaluronidase at a concentration of 60 turbidity reducing units/ml for 3 h at 37°C resulted in 80%
degradation of total HA in the parenchymal strips.
Wet-to-Dry Weight Ratio
In separate experiments, the W of treated and control strips was measured after incubation of the strips in the presence and absence of chondroitinase ABC or heparitinase I. The strips were then lyophilized for 9 h, the weight was measured again, and the wet-to-dry weight ratio was calculated.Immunohistochemistry
In additional experiments, immunohistochemical staining for HS was performed on 5-µm-thick sections from control and enzyme-treated parenchymal strips. The slides were blocked by incubation with universal blocking solution for 10 min at room temperature. Sections were then rinsed with Tris-buffered saline (TBS; 0.5 M Tris, pH 7.6, and 1.5 M NaCl) and incubated with the primary antibody, a monoclonal anti-mouse HS (1:50 dilution). After being washed with TBS, the tissue was incubated with a 1:200 biotin-labeled goat anti-mouse antibody in TBS for 1 h, rinsed with TBS, and then further incubated with 1:100 alkaline phosphatase-conjugated avidin in TBS for 1 h. After further washing, sections were developed with Fast Red salt, 1 mg/ml in alkaline phosphatase substrate, for 15 min at room temperature. Sections were counterstained with Gill's hematoxylin for 45 s and then washed with water. The sections were covered with a thin layer of crystal mount and dried in an oven at 37°C overnight.Materials
The HA radiometric assay kit was purchased from Pharmacia (Quebec City, PQ). Heparitinase I (from F. heparinum) and monoclonal anti-HS antibody (clone F58-10E4) were purchased from Seikagaku (Tokyo, Japan). This enzyme has been shown to specifically cleave HS at the N-acetylglucosaminide-D-glucuronic acid linkage, yielding unsaturated oligosaccharides, and has no activity against heparin (24). Chondroitinase ABC (from P. vulgaris, protease free), hyaluronidase (EC 4.2.2.1 from S. hyaluroticus), NaCl, KCl, NaHCO3, CaCl2, MgSO4, KH2PO4, glucose, sodium acetate, papain, DMMB, EDTA, iodoacetamide, Hanks' balanced salt solution, chondroitin sulfate A (shark cartilage), Tris, Fast Red salt, alkaline phosphatase substrate, hematoxylin, and cysteine were purchased from Sigma-Aldrich (St. Louis, MO). Chondroitinase ABC has been shown to be highly specific against CS and DS (37). Similarly, hyaluronidase has been reported to use only HA as its substrate (33). The universal blocking solution, biotin-labeled goat anti-mouse antibody, and alkaline phosphatase-conjugated avidin were purchased from Dako Diagnostics (Mississauga, ON).Statistical Analysis
To compare R, Edyn, and before and after incubation in control and treated groups, three-way
ANOVA (f, amplitude, and incubation) with a
Bonferroni correction was applied. The same test was also used to
compare the change in R,
Edyn, and between the control and
treated groups. (Because the changes in R,
Edyn, and with incubation were not dependent
on the f or amplitude, the values for each group were
pooled.) Change in a given parameter was defined as the percentage of
the value after the incubation relative to the value obtained before
incubation. Two-way ANOVA was used to compare the
Estat change between the control and treated
groups at the three different stress levels. Student's
t-test was used to compare the wet-to-dry weight ratio
between control and enzyme-treated strips. All values are expressed as
means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Chondroitinase ABC
Values of R, Edyn, and versus f before and after incubation are shown in Figs.
1-3.
Strips incubated for 16 h with chondroitinase ABC
(n = 10) showed no change in R, but
Edyn decreased significantly (P < 0.001), and as a result, increased
significantly (P < 0.001). Incubation of control
strips for 16 h (n = 8) resulted in a significant reduction in both R and Edyn
(P < 0.001); remained unchanged (data not
shown; P = 0.06). These changes are likely due to the effects of stress relaxation. Compared with the control group, the
change in R and after chondroitinase ABC treatment was
significantly different (P < 0.001; Fig.
4). The change in
Edyn was similar between the control
and the chondroitinase-treated groups. Quasi-static stress versus
strain curves are shown in Fig. 5. In
control strips (n = 5), Estat at
20, 30, and 40 g/cm2 decreased significantly
(P < 0.01) with time. Conversely, in treated strips,
there was no change in Estat over time
(n = 6). The difference between the two groups was
significant (P < 0.05; Fig.
6). Table
1 shows wet-to-dry weight
ratios for tissues exposed to chondroitinase ABC. Enzyme treatment
caused a significant increase in wet-to-dry weight ratio compared with
that in control tissues, i.e., treated tissues retained more water.
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Heparitinase I
Strips incubated with heparitinase I for 5 h (n = 5) showed no change in R, but Edyn decreased significantly (P < 0.001). As a result, increased significantly (P < 0.001). Incubation of control strips for 5 h (n = 5) resulted in a significant reduction of both R and
Edyn (P < 0.01 and
P < 0.02, respectively), whereas remained
unchanged. Compared with the control group, there was a significant
increase in in the strips exposed to the enzyme (P < 0.001; Fig. 7). The
decrement in R in control strips versus treated strips was
borderline significant (P = 0.06; Fig. 7). Estat in both the control and the
heparitinase-treated groups showed a similar decrease over time.
However, there was an obvious upward shift in the curve in the
heparitinase-treated strips (Fig. 8).
This was evidenced by a significant increase in stress at the
IP (P < 0.05; Table
2). The wet-to-dry weight ratio was not altered in heparitinase I-treated tissues compared with that in control
tissues (Table 1). Immunohistochemical staining revealed that in
control tissues HS was prominent in alveolar wall and blood vessel
endothelia. After exposure to heparitinase I, there was a substantial
decrease in staining for HS (Fig. 9).
Furthermore, there was no obvious change in tissue morphology in the
enzyme-treated parenchymal strips.
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Hyaluronidase
Degradation of HA did not cause any change in R, Edyn, or compared with baseline values
obtained from the same strips (n = 7) before digestion.
Similarly, control strips (n = 7) showed no change in
R, Edyn, and with time. Thus
there was no significant difference in the changes in these parameters
between the control and hyaluronidase-treated groups (Fig.
10). The lack of a decrement in
R and Edyn in this group of control
strips compared with the previous groups may reflect the shorter
incubation time (3 h) and, therefore, less stress-relaxation effect.
The quasi-static stress-strain curve, Estat at
20, 30, or 40 g/cm2, and the change in IP were not
significantly altered after hyaluronidase treatment when compared with
that in control strips.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Energy is required during breathing to overcome the resistance of the lung. Much of that energy cost is accounted for by the viscoelastic behavior of the parenchymal lung tissues. (19). The stress-bearing structural elements potentially responsible for tissue viscoelasticity in intact lung include the extracellular matrix (ECM), the air-liquid interface (surfactant), and peripheral contractile elements (10). Although the precise contribution of each of these elements is not known, the mechanical behavior of the collagen-elastin-proteoglycan matrix likely accounts for a large proportion of the energy dissipated during breathing (10, 21). Studies from this laboratory (27) have shown that excised parenchymal strips in which the air-liquid interface has been removed and/or the basal tone has been ablated demonstrate substantial viscoelastic behavior.
There are several reasons to postulate that proteoglycans and GAGs in the matrix play an important role in contributing to the viscosity of the tissues. The hydrophilic nature of these molecules attracts ions and fluid into the matrix and thereby potentially influences the viscoelastic behavior of the tissue (29). Mijailovich et al. (22) have proposed that energy dissipation occurs not at the molecular level within collagen or elastin but rather at the level of fiber-fiber contact and by the shearing of GAGs, which provide the lubricating film between adjacent fibers. Suki et al. (35) have developed the idea that the mechanical properties of the tissue matrix can be understood if one models matrix behavior as a polymer. At higher frequencies, viscoelastic behavior occurs because of conformational changes in the molecules that obey the Rousse theory. This theory proposes that external stresses cause a velocity gradient in the solution that continuously alters the configuration of the various polymer molecules. At lower frequencies, the process of "reptation" becomes more important, wherein step changes in molecular conformation create distortions that, as the molecules reassume their equilibrium shape over time, dissipate energy. One possible anatomic correlate for these polymer-like molecules would be the proteoglycan and GAG molecules that comprise the "ground substance" of the matrix.
Some information is available regarding the role of GAGs in determining viscoelastic behavior in other systems that may be pertinent to the lung. Schmidt et al. (29) have shown in articular cartilage preparations exposed to chondroitinase that the kinetics of the creep response (creep is a characteristic of viscoelastic materials) is influenced by the GAG content of the tissue. They hypothesized that GAGs may act to "brake" the load applied when a sudden tensile stress is applied to the cartilage. In a further study published by this group (41), changes in shear modulus and energy dissipation were noted when articular cartilage was exposed to either chondroitinase ABC or hyaluronidase. More recently, Gandley et al. (12) examined the stiffness of rat mesenteric arteries and determined that exposure to chondroitinase ABC resulted in increased arterial wall stiffness, much as was observed in the present study.
To date, a number of proteoglycans and GAGs have been described in the lung. Versican is the hyalectin, or large aggregating proteoglycan, that has been demonstrated in the lung (1). HA has been shown to be present in the lung in a number of studies (2, 8). In the peripheral lung, lumican has been shown to be the predominant small proteoglycan (6), although biglycan and decorin have also been described in airway and blood vessel walls and in alveolar tissue (39). Finally, the basement membrane HS proteoglycan perlecan has been identified in lung samples (24, 40).
Our data demonstrate that the degradation of CS/DS in the lung
parenchymal matrix resulted in a significant increase in the index of
mechanical friction, . The decrement in R over time observed in control strips did not occur in tissues exposed to chondroitinase. Hence, more energy was dissipated as the tissues were
oscillated. Similar results were observed with heparitinase treatment.
Although the percent change in R between control and treated
strips did not reach significance (P = 0.06), a
definite trend was observed (Fig. 7). Conversely, exposure to
hyaluronidase did not affect any of the dynamic mechanical parameters
measured despite an 80% digestion of HA in the tissues. The results of the quasi-static experiment also showed an effect of the degradation of
CS/DS and HS on the parameters reflecting tissue stiffness. Again, HA
degradation did not result in any appreciable change compared with that
in control tissue.
There are few data available in the pulmonary literature to which our results can be directly compared. Most of the previous studies (15, 32) employing degradative enzymes, including one from our own laboratory (23), have measured only quasi-static behavior or have utilized papain, collagenase, or elastase, relatively nonspecific enzymes capable of degrading many of the matrix components. Nonetheless, some studies warrant mention insofar as they pertain to the results of the current experiment. Sata et al. (28) measured the effects of hyaluronidase treatment on stress-strain curves in parenchymal strips previously exposed to collagenase or elastase. They found no change in several measures of tissue mechanics. Similarly, Martin and Sugihara (20) found no effect of hyaluronidase on peak force, slope of the length-tension curve, or hysteresis ratio in alveolar wall preparations. Gleisner and Martin (13) and Shimura et al. (34) published data on lung parenchymal strips in which GAGs and/or matrix proteins were nonspecifically leached out of the tissues by prolonged immersion in phosphate-buffered saline. These authors demonstrated that the decay in peak tissue tension (TTD) after extension of the strip was affected by removal of GAGs from the tissue. Moreover, the rate of TTD was correlated with the rate of GAG loss. Furthermore, they observed that agents that specifically enhanced HS loss resulted in the greatest change in TTD, suggesting a relative importance of this particular GAG in determining mechanical behavior.
While we can only postulate on the precise mechanisms whereby degradation of CS/DS and HS results in alterations in mechanical friction in these tissues, the functions ascribed to these different molecules offer some possibilities. CS/DS is covalently attached to decorin, which is present in the d band of collagen bundles (30). CS/DS attached to decorin interacts electrostatically across the bundle thereby aiding in collagen organization (31). Removal of CS/DS may have resulted in the disruption of the collagen fibril, which may have led to increased friction and energy dissipation during fiber-fiber interactions. A further possibility is that CS attached to the large aggregating proteoglycan versican may have contributed to this effect. Versican, together with HA, forms aggregates that are highly hydrophilic. Disruption of the aggregate by CS degradation could have altered the hydration of the tissue. Altered hydration could potentially change the nature of the contact and friction between the tissue components, leading to increased energy dissipation and stiffness. Our results of wet-to-dry weight ratios speak to this possibility. Chondroitinase ABC treatment resulted in an increase in wet-to-dry weight ratio or tissue hydration, whereas heparitinase I treatment had no effect. Conversely, the effects of digestion with these two enzymes on the mechanical behavior was very similar. This argues that hydration per se is not the key determinant of viscoelastic behavior. That the wet-to-dry weight ratio should increase with loss of charged CS side chains is, perhaps, surprising. However, a similar result was observed in articular cartilage in vitro (41) and in intervertebral disks injected with chondroitinase ABC into the disk (36). Degradation of CS may alter the assembly of the collagen fiber in such a way as to "loosen" the fibrils and thereby increase the exposure of ionic charges to the extracellular environment (30, 31).
HS is a major component of the basement membrane (14, 24, 40) where it plays an important role in regulating diffusion and permeability. HS has been located at the surface of elastic fibers (7). It is also associated with cell surface molecules such as syndecan (4) and glypican (5). These molecules mediate cell-matrix and cell-cell adhesion and connect the ECM to the cytoskeleton (14, 17). Syndecan interacts via HS with fibronectin and laminin through its extracellular domain. It is also connected to the cytoskeleton via its cytoplasmic domain (4). One possible explanation for the change in mechanical behavior of the tissues with HS degradation is the effect on basement membrane integrity and cell-matrix interaction. Disruption of the connection between the cytoskeleton and the ECM may alter the stress transfer between the ECM and the cellular cytoskeleton (38). Gandley et al. (12) proposed such a mechanism to explain the effects of chondroitinase on mesenteric artery. Results from immunohistochemical analysis revealed a decrease in HS within the alveolar wall, although the precise site of HS loss is difficult to ascertain. Nonetheless, these data are consistent with the idea that HS digestion may interfere with the normal transfer of stress between the matrix and the cellular component.
Whereas CS/DS and HS degradation altered tissue viscoelasticity, HA degradation did not. HA is a large GAG that has no covalent interaction with a proteoglycan core protein. It forms large aggregates with the hyalectins aggrecan and versican. This highly negatively charged molecule plays an important role in tissue water balance (8). The failure of HA degradation to alter tissue mechanics reinforces the idea that the viscoelastic behavior of the tissues is not simply determined by the absolute amount of fluid present. It is interesting to note that Sata et al. (28) and Martin and Sugihara (20) were also unable to discern any effect of digestion with hyaluronidase on quasi-static tissue mechanics.
A final question relates to the differential elastance results obtained from dynamic oscillations and quasi-static stress-strain curves. Whereas chondroitinase and heparitinase had no effect on Edyn compared with that in control strips, chondroitinase exposure resulted in a greater value of Estat compared with that in control strips at all levels of stress sampled (Fig. 6). Although heparitinase did not affect Estat, it did cause a significant increase in stress at the IP (Table 2). The reasons for this disparity in elastance results are not clear but may relate to the technique of measurement. Edyn was sampled over a small length excursion (0.5-5.0% of Lr), whereas Estat was measured over a stress range of 0-60 g/cm2. The former may simply represent too small an amplitude perturbation to effectively sample elastic phenomena. Alternately, nonlinearities in the tissue may contribute.
In conclusion, we have demonstrated that degradation of CS/DS and HS with specific enzymes altered the viscoelastic behavior of excised parenchymal lung strips, resulting in a less efficient mechanical system in which energy dissipation or loss was greater. This change in viscoelastic behavior did not seem to depend on the amount of fluid in the tissues. Rather, the ability of GAGs to influence the biomechanical behavior of the system may lie in their interactions with other matrix molecules and/or the cell membrane. Alterations in these molecules in different diseases or in response to lung injury may account for the abnormal lung tissue mechanics observed in these processes.
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ACKNOWLEDGEMENTS |
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This study was supported by the J. T. Costello Memorial Research Fund and the Medical Research Council of Canada.
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FOOTNOTES |
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M. S. Ludwig is a Chercheur Boursier of the Fonds de la Recherche en Santé du Québec.
Address for reprint requests and other correspondence: M. S. Ludwig, Meakins Christie Laboratories, 3626 St. Urbain St., Montreal, PQ, Canada H2X 2P2 (E-mail: mara{at}meakins.lan.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.
Received 23 September 1999; accepted in final form 9 August 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Bensadoun, ES,
Burke AK,
Hogg JC,
and
Roberts CR.
Proteoglycan deposition in pulmonary fibrosis.
Am J Respir Crit Care Med
154:
1819-1828,
1996[Abstract].
2.
Bray, BA,
Hsu W,
and
Turino GM.
Lung hyaluronan as assayed with a biotinylated hyaluronan-binding protein.
Exp Lung Res
20:
317-330,
1994[ISI][Medline].
3.
Bull, HB.
Protein structure and elasticity.
In: Tissue Elasticity, edited by Remington JW.. Washington, DC: Waverly, 1957, p. 33-42.
4.
Carey, DJ.
Syndecans: multifunctional cell-surface co-receptors.
Biochem J
327:
1-16,
1997.
5.
David, G.
Integral membrane heparan sulfate proteoglycans.
FASEB J
7:
1023-1030,
1993[Abstract].
6.
Dolhnikoff, M,
Morin J,
Roughley PJ,
and
Ludwig MS.
Expression of lumican in human lungs.
Am J Respir Crit Care Med
19:
582-587,
1998.
7.
Erlinger, R.
Glycosaminoglycans in porcine lung: an ultrastructural study using cupromeronic blue.
Cell Tissue Res
281:
473-483,
1995[ISI][Medline].
8.
Fraser, JR,
Laurent TC,
and
Laurent UB.
Hyaluronan: its nature, distribution, functions and turnover.
J Intern Med
242:
27-33,
1997[ISI][Medline].
9.
Fredberg, JJ,
Bunk D,
Ingenito E,
and
Shore SA.
Tissue resistance and the contractile state of lung parenchyma.
J Appl Physiol
74:
1387-1397,
1993
10.
Fredberg, JJ,
and
Stamenovic D.
On the imperfect elasticity of lung tissue.
J Appl Physiol
67:
2408-2419,
1989
11.
Fung, YC.
Biomechanics. New York: Springer-Verlag, 1993.
12.
Gandley, RE,
McLaughlin MK,
Koob TJ,
Little SA,
and
McGuffee LJ.
Contribution of chondroitin-dermatan sulfate-containing proteoglycans to the function of rat mesenteric arteries.
Am J Physiol Heart Circ Physiol
273:
H952-H960,
1997
13.
Gleisner, JM,
and
Martin CJ.
Lung tissue tension and glycosaminoglycans.
Respir Physiol
66:
247-258,
1986[ISI][Medline].
14.
Iozzo, RV.
Matrix proteoglycans: from molecular design to cellular function.
Annu Rev Biochem
67:
609-652,
1998[ISI][Medline].
15.
Johanson, WG, Jr,
and
Pierce AK.
Effects of elastase, collagenase, and papain on structure and function of rat lungs in vitro.
J Clin Invest
51:
288-293,
1972.
16.
Karlinsky, JB,
Bowers JT,
Fredette JV,
and
Evans J.
Thermoelastic properties of uniaxially deformed lung strips.
J Appl Physiol
58:
459-467,
1985
17.
Liu, W,
Litwack ED,
Stanley MJ,
Langford JK,
Lander AD,
and
Sanderson RD.
Heparan sulfate proteoglycans as adhesive and anti-invasive molecules. Syndecans and glypican have distinct functions.
J Biol Chem
273:
22825-22832,
1998
18.
Ludwig, MS.
Role of lung parenchyma.
In: Asthma, edited by Barnes PJ,
Grunstein MM,
Leff AR,
and Woolcock AJ.. Philadelphia, PA: Lippincott-Raven, 1997, p. 1319-1334.
19.
Ludwig, MS,
Dreshaj I,
Solway J,
Munoz A,
and
Ingram RH, Jr.
Partitioning of pulmonary resistance during constriction in the dog: effects of volume history.
J Appl Physiol
62:
807-815,
1987
20.
Martin, CJ,
and
Sugihara T.
Simulation of tissue properties in irreversible diffuse obstructive pulmonary syndromes. Enzyme digestion.
J Clin Invest
52:
1918-1924,
1973.
21.
Mijailovich, SM,
Stamenovic D,
Brown R,
Leith DE,
and
Fredberg JJ.
Dynamic moduli of rabbit lung tissue and pigeon ligamentum propatagiale undergoing uniaxial cyclic loading.
J Appl Physiol
76:
773-782,
1994
22.
Mijailovich, SM,
Stamenovic D,
and
Fredberg JJ.
Toward a kinetic theory of connective tissue micromechanics.
J Appl Physiol
74:
665-681,
1993
23.
Moretto, A,
Dallaire M,
Romero P,
and
Ludwig M.
Effect of elastase on oscillation mechanics of lung parenchymal strips.
J Appl Physiol
77:
1623-1629,
1994
24.
Murdoch, AD,
Liu B,
Schwarting R,
Tuan RS,
and
Iozzo RV.
Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization.
J Histochem Cytochem
42:
239-249,
1994[Abstract].
25.
Nader, HB,
Porcionatto MA,
Tersariol IL,
Pinhal MA,
Oliveira FW,
Moraes CT,
and
Dietrich CP.
Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy.
J Biol Chem
265:
16807-16813,
1990
26.
Roberts, CR,
Wight TN,
and
Hascall VC.
Proteoglycans.
In: The Lung: Scientific Foundations (2nd ed.), edited by Barnes PJ,
Grunstein MM,
Leff AR,
and Woolcock AJ.. Philadelphia, PA: Lippincott-Raven, 1997, p. 757-767.
27.
Salerno, FG,
Kurosawa H,
Eidelman DH,
and
Ludwig MS.
Characterization of the anatomical structures involved in the contractile response of the rat lung periphery.
Br J Pharmacol
118:
734-740,
1996[ISI][Medline].
28.
Sata, M,
Takahashi K,
Sato S,
and
Tomoike H.
Structural and functional characteristics of peripheral pulmonary parenchyma in golden hamsters.
J Appl Physiol
78:
239-246,
1995
29.
Schmidt, MB,
Mow VC,
Chun LE,
and
Eyre DR.
Effects of proteoglycan extraction on the tensile behavior of articular cartilage.
J Orthop Res
8:
353-363,
1990[ISI][Medline].
30.
Scott, JE.
Proteoglycan-fibrillar collagen interaction.
Biochem J
252:
313-323,
1988[ISI][Medline].
31.
Scott, JE,
and
Thomlinson AM.
The structure of interfibrillar proteoglycan bridges (shape modules) in extracellular matrix of fibrous connective tissues and their stability in various chemical environments.
J Anat
192:
391-405,
1998.
32.
Senior, RM,
Bielefeld DR,
and
Abensohn MK.
The effects of proteolytic enzymes on the tensile strength of human lung.
Am Rev Respir Dis
111:
184-188,
1975[ISI][Medline].
33.
Shimada, E,
and
Matsumura G.
Degradation process of hyaluronic acid by Streptomyces hyaluronidase.
J Biochem (Tokyo)
88:
1015-1023,
1980
34.
Shimura, S,
Martin CJ,
Boatman ES,
and
Dhand
R. A role for interstitial matrix in tissue tension of alveolar wall.
Respir Physiol
62:
293-303,
1985[ISI][Medline].
35.
Suki, B,
Barabasi AL,
and
Lutchen KR.
Lung tissue viscoelasticity: a mathematical framework and its molecular basis.
J Appl Physiol
76:
2749-2759,
1994
36.
Takahashi, T,
Kurihara H,
Nakajima S,
Kato T,
Matsuzaka S,
Sekiguchi T,
Onaya M,
Miyauchi S,
Mizuno S,
Horie K,
Fujita Y,
and
Hirose T.
Chemonucleolytic effects of chondroitinase ABC on normal rabbit intervertebral discs. Course of action up to 10 days postinjection and minimum effective dose.
Spine
21:
2405-2411,
1996[ISI][Medline].
37.
Thurston, CF.
The kinetic of degradation of chondroitin of sulphates and hyaluronic acid by chondroitinase form Proteus vulgaris.
Biochem J
145:
397-400,
1975[ISI][Medline].
38.
Wang, N,
and
Ingber DE.
Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension.
Biophys J
66:
2181-2189,
1994
39.
Westergren-Thorsson, G,
Hernnas J,
Sarnstrand B,
Oldberg A,
Heinegard D,
and
Malmstrom A.
Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in developing bleomycin-induced pulmonary fibrosis in rats.
J Clin Invest
92:
632-637,
1993.
40.
Yurchenco, PD,
and
Schittny JC.
Molecular architecture of basement membranes.
FASEB J
4:
1577-1590,
1990[Abstract].
41.
Zhu, W,
Mow VC,
Koob TJ,
and
Eyre DR.
Viscoelastic shear properties of articular cartilage and the effects of glycosidase treatments.
J Orthop Res
11:
771-781,
1993[ISI][Medline].
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