Effect of glycosaminoglycan degradation on lung tissue
viscoelasticity
Rehab
Al Jamal1,
Peter
J.
Roughley2, and
Mara S.
Ludwig1
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 |
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 |
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 |
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.
The strip was removed from the bath and exposed to the enzyme of
interest (chondroitinase ABC, heparitinase I, or hyaluronidase) at a
predetermined concentration and for a predetermined incubation period
(see Enzymes and Enzyme Assays). Control strips were
incubated in the same manner with no enzyme added. After incubation,
the Krebs solution was changed to remove the enzyme, and the weight and
length of the tissue were recorded again. The tissue was then resuspended in the organ bath, and recordings were repeated as described above. Tissue viability was confirmed at the end of each
experiment by adding methacholine (1 µM) to the organ bath to elicit
a contractile response.
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
|
(1)
|
where
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
|
(2)
|
For oscillatory experiments, resistance (R) and
dynamic elastance (Edyn) were estimated by
fitting the equation of motion to changes in tension (T) and length
(L)
|
(3)
|
where dL/dt is the rate of
L change per unit of time, and K is a constant.
All the data were standardized for strip size. To calculate
, the
equation used was
|
(4)
|
where
is a dimensionless variable that represents the
coefficient of coupling between elastic and dissipative stresses.
For quasi-static measurements, strain (
) was defined as
|
(5)
|
Quasi-static elastance (Estat) was
calculated at three different stresses with the following formula
|
(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.
 |
RESULTS |
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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Fig. 1.
Resistance (R) vs. frequency curves at
indicated amplitudes of oscillation at a stress of 30 g/cm2. Solid and dashed lines, before and after incubation
with chondroitinase, respectively. No change in R was
detected.
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Fig. 2.
Dynamic elastance (Edyn) vs.
frequency curves at indicated amplitudes of oscillation at a stress of
30 g/cm2. There was a significant decrease in
Edyn after 16 h of incubation with
chondroitinase (P < 0.001).
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Fig. 3.
Hysteresivity ( ) vs. frequency curves at indicated
amplitudes of oscillation at a stress of 30 g/cm2. A
significant increase was observed in after 16 h of incubation
with chondroitinase (P < 0.001).
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Fig. 4.
Changes in R, Edyn, and
in control and chondroitinase-treated tissues. n, No. of
strips. The decrement in R and the increase in were
significantly different between the control and treated groups
(*P < 0.001).
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Fig. 6.
Percent change in quasi-static elastance
(Estat) at indicated stresses for both control
and chondroitinase-treated groups before and after the 16-h incubation.
n, No. of strips. At all stresses, the change in
Estat was significantly different between the 2 groups after 16 h of incubation (*P
<0.05).
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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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Fig. 7.
Changes in R, Edyn, and
in control and heparitinase-treated tissues. n, No. of
strips. The change in was significantly greater in the strips
exposed to the enzyme (* P < 0.001). The change in
R after heparitinase I treatment did not achieve
significance.
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Fig. 8.
Mean quasi-static stress-strain curves of lung
parenchymal strips before and after heparan sulfate degradation. IP,
inflection point.
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Fig. 9.
Immunohistochemical staining for heparan sulfate in control
(A) and heparitinase I-treated (B) parenchymal
strips. Original magnification, ×400.
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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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Fig. 10.
Changes in R, Edyn,
and in control and hyaluronidase-treated tissues. n, No.
of strips. Changes in R,
Edyn, and after hyaluronidase
treatment were not different from those in control strips.
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DISCUSSION |
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.
 |
ACKNOWLEDGEMENTS |
This study was supported by the J. T. Costello Memorial
Research Fund and the Medical Research Council of Canada.
 |
FOOTNOTES |
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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