1 Dipartimento di Biochimica
"A. Castellani, We extracted and
isolated proteoglycans from lung tissue samples obtained
from three groups of anesthetized rabbits:
1) control animals (C; n = 8) killed by overdose after 180 min;
2) animals receiving an
intravenous saline infusion (S; n = 4, 1.5 ml · kg-1 · min-1)
for 180 min; 3) animals receiving an
intravenous bolus of 200 µg of pancreatic elastase (E;
n = 4), killed after 200 min. The lung
dry weight-to-wet weight ratio in the three groups was 5.2 ± 0.2, 6.0 ± 0.4, and 5.6 ± 0.5, respectively. Gel-filtration analysis
showed a massive fragmentation of the versican family of the
extracellular matrix (ECM) in the S groups and a marked degradation of
heparan sulfate-containing proteoglycans, including perlecan of the
basement membrane, in the E group. The binding properties of total
proteoglycans to other ECM components were lowered in both groups
relative to control. The decrease in proteoglycan binding was more
pronounced for collagen type IV in the E group relative to C
(
extracellular matrix; basement membrane; gelatinase; interstitial
fluid dynamics
RECENT STUDIES HAVE CLARIFIED some aspects of pulmonary
interstitial fluid dynamics in physiological conditions and in the transition toward the development of lung edema (8, 10, 11). In
physiological conditions, transendothelial pressure gradients sustain
net filtration from pulmonary microvessels into lung interstitium from
which fluid is drained by pulmonary lymphatics. Pulmonary interstitial
pressure (PIP) is rather subatmospheric in physiological conditions
(approximately In conditions of increased microvascular filtration induced in rabbits
by saline infusion (hydrostatic type of edema; Refs. 8, 10) or by
intravenous injection of pancreatic elastase (lesional edema; Ref. 11),
we observed a similar time course for PIP, reflecting two functional
phases. In the early phase of edema development, PIP increased with a
minimal increase in extravascular lung water, a mechanical behavior
consistent with a low compliance of the extracellular matrix (ECM);
this feature indeed provides a strong "tissue safety factor"
against the development of lung edema. However, as edema progressed,
PIP tended to decrease in the face of a substantial increase in
extravascular lung water, suggesting a marked increase in tissue
compliance. This change in the mechanical tissue properties was related
to the biochemical modifications of proteoglycans, which are largely
responsible for the compliance and fluid balance of the lung (1, 2, 16)
and are also involved in other dynamic biological processes, including
the regulation of cell adhesion, cell migration, and cell proliferation
(1, 2, 16).
Proteoglycans are a heterogeneous family of genetically unrelated core
proteins covalently linked to one or more glycosaminoglycan (GAG)
chains. In lung parenchyma, proteoglycans are
multifunctional components of ECM, basement membranes, and plasma
membranes (1, 2, 16) and include versican, a large chondroitin sulfate (CS)-containing proteoglycan, which may form aggregates with hyaluronic acid in the ECM and contribute to the hydration and to the
biomechanical properties of the tissue (1, 2, 16); decorin, a small dermatan sulfate-containing proteoglycan, which is associated with
fibrillar collagen and may play a role in fiber formation (20);
perlecan, a heparan sulfate (HS)-containing proteoglycan, which acts as
a selective permeability barrier in the basement membrane and also
binds growth factors, cytokines, proteinases, and proteinase inhibitors
(5); and the family of syndecans, HS-containing proteoglycans, which
are involved in cell adhesion, in cell-cell and cell-ECM interactions,
and in the cytokine network (1, 2, 12, 16).
In both the hydrostatic and the lesional type of lung edema (10, 11),
we found that the biochemical modifications of proteoglycans included
1) a progressive weakening of the
noncovalent bonds linking proteoglycans to other ECM components and
2) a fragmentation of proteoglycans,
leading to a loss of the native architecture of the ECM. Although the
time course of PIP is similar during development of hydraulic or
lesional lung edema, one may hypothesize a different involvement of
proteoglycan families because of the different noxious stimuli.
In the present study, we specifically aim at determining
1) possible differences in the
sequential degradation of proteoglycan families in the two edema
models, 2) interaction properties of proteoglycans to some major constituents of ECM, and
3) the possible involvement of
proteinase activities in the degradation of ECM protein components.
Experimental protocol. The experiments
were performed on adult New Zealand rabbits [body weight = 2.23 ± 0.17 kg (mean ± SD), range = 2-2.5 kg]
anesthetized with a cocktail of 2.5 ml/kg of 25% (wt/vol)
urethan and 1.5 ml of pentobarbital sodium (60 mg/kg) injected into an
ear vein. Additional anesthesia was given throughout the experiment as
judged by arousal of ocular reflexes. The animals were tracheotomized,
and the trachea was cannulated to allow spontaneous breathing. A
carotid artery and a jugular vein were also cannulated. We studied
three groups of animals: 1) control
(C; n = 8);
2) animals receiving slow saline
infusion through the jugular vein (S; 1.5 ml · kg-1 · min-1;
n = 4; hydraulic edema model); and
3) intravenous administration of a
single bolus of 200 µg (7 IU) of pancreatic elastase (E, lesional
edema model; n = 4). Animals were
killed by an overdose of anesthetic (10, 11) after 180 min for C and S
groups and after 200 min for the E group. After complete bleeding
through the carotid artery, the lungs were excised, washed with PBS
containing a cocktail of protease inhibitors (10, 11), pooled into the three groups (C, S, and E) and cut into small slices, separating the
pulmonary parenchyma from the terminal bronchioles. Gross and
microscopic examination excluded that the lungs were affected by
infective processes. Tissue water content was determined by measuring
the wet weight-to-dry weight ratio (W/D) on aliquots of the pooled lung
slices, representing about 10% of the total sample. Each specimen was
weighed immediately after sampling and after oven drying at 70°C
for at least 24 h.
Proteoglycan extraction and
isolation. Pooled lung slices from C,
S, and E groups were treated with 0.4 M guanidine hydrochloride (GuHCl)
at 4°C for 24 h in the presence of protease inhibitors to break
intermolecular noncovalent bonds and extract proteoglycans (4). The
extracts were gel filtered on Bio-Gel P2 columns, and proteoglycans
were then isolated from proteins and hyaluronic acid by duplicate
ion-exchange chromatography on DEAE-Sephacel columns (10, 11). The
recovery of proteoglycans was quantitated by hexuronate
determination after precipitation with cold ethanol (10, 11).
Proteoglycan characterization.
Aliquots of isolated proteoglycans were radiolabeled with
125I (11), freed of
nonincorporated radioactive material by gel filtration on PD 10 columns, and then analyzed on Sepharose CL-4B under dissociative
conditions (10, 11). Radiolabeled fractions were counted in a Cobra II
gamma counter (Canberra-Packard).
We chose the Sepharose CL-4B column because it is adequate for
separation of molecular weights ranging from
105 to
108. The resolution of gel
filtration is evaluated by the index
Kav = (Ve The different types of GAG chains linked to proteoglycan core proteins
were determined by digestion with specific eliminases: 1) chondroitinase (Chase) ABC, which
degrades galactosamine-containing GAGs such as CS (22); and
2) heparanase plus heparitinase,
which digest glucosamine-containing HS chains (21). The unsaturated disaccharides, released by Chase ABC digestion from
galactosamine-containing GAGs and by heparanase plus heparitinase
digestion from glucosamine-containing GAGs, were fractionated and
identified by capillary electrophoresis performed on a Biofocus 3000 apparatus, as recently reported (11).
Binding properties of proteoglycans.
The binding properties of
125I-proteoglycans for type I and
type IV collagen and hyaluronic acid were studied by solid-phase
binding assay (19).
125I-proteoglycans were tested as
specific ligands interacting with the macromolecular substrates
immobilized on the wells of the microtiter plates. In all assays, the
bound radioactivity was corrected for background values, which were the
radioactivity levels recovered in the wells coated with albumin
(nonspecific substrate), and are expressed as percent
increase in 125I-proteoglycan
binding with specific ligands compared with the nonspecific binding to
albumin. Binding assays were repeated six times for each ligand.
Detection of gelatinase
activities. Lung specimens from C, E,
and S rabbit groups were homogenized at 4°C in 50 mM
Tris · HCl buffer, pH 7.6, containing 150 mM NaCl, 5 mM CaCl2, 10 mM EDTA, and 0.02%
Tween 20. The samples were cleaned by centrifugation and incubated for
1 h with gelatin-Sepharose resin (100 µg protein/50 µl resin) (6)
to isolate gelatin-degrading enzymes. The resin was then recovered,
washed carefully, resuspended in 4× Laemmli sample buffer and
used for electrozymograms. SDS-PAGE was carried out using a 5-10%
gradient gel copolymerized with gelatin (1 mg/ml). After overnight
incubation of the gel in an activating buffer (37°C in 50 mM
Tris · HCl buffer, pH 7.5, containing 200 mM NaCl and
5 mM CaCl2) and staining with
0.5% (wt/vol) Coomassie brilliant blue R-250 (6), gelatinolytic
activity was detected as clear bands of gel against the blue
background, indicating areas where the gelatin was degraded by the
enzymes. Molecular weights of the gelatinolytic bands were estimated by
using molecular-weight markers (Pharmacia). The gels were scanned by a
laser Ultroscan densitometer (LKB). To analyze metalloproteinase
inhibition, gels were incubated in the presence of 20 mM EDTA.
Immunoblot analysis was performed after SDS-PAGE, and the Western
transfer to nitrocellulose with a semidry transfer cell (Bio-Rad) was
carried out at 15 V for 20 min. The identification of
gelatinases was then performed with specific sheep antisera (1:100; The
Binding Site, Birmingham, UK). Data are expressed as the
arithmetic means ± SD.
The W/D of lung used for biochemical investigations was 5.2 ± 0.2 for C (n = 8), 5.6 ± 0.5 for E
(n = 4), and 6.0 ± 0.4 for S
(n = 4) groups, respectively.
The molecular size distribution of
125I-proteoglycans isolated from
0.4 M GuHCl extracts was analyzed by gel filtration, performed under
dissociative conditions to prevent molecular interactions (11).
The elution pattern of the radiolabeled material isolated
from control lungs showed that three primary peaks were different (Fig.
1, left,
C). Peak
1 was eluted in the void volume of the column and should mainly consist of versican; in fact, this material was markedly sensitive to Chase ABC digestion, and capillary
electrophoresis analysis demonstrated that this enzymatic treatment
released unsaturated disaccharides typical of galactosamine-containing
GAGs such as CS. Peak
2 was included in the gel and
exhibited a lower sensitivity to Chase ABC digestion. The combined
treatment of this material with heparinase and heparitinase released
unsaturated disaccharides, which were typical constituents of HS
chains, as assessed by capillary electrophoresis analysis. Therefore,
peak
2 consisted of proteoglycans carrying
mainly HS chains. The treatment of the low-molecular-weight material
eluted in peak
3 with Chase ABC and with heparanase
plus heparitinase released unsaturated disaccharides containing
galactosamine and glucosamine, respectively. Therefore,
peak
3 included both CS and HS chains,
which probably represented degradation products of larger
proteoglycans.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
93.5%, P < 0.05) and for
hyaluronic acid in the S groups (
85.8%,
P < 0.05). These findings suggest
that elastase treatment produces a major degree of damage to the
organization of basement membrane, whereas saline loading affects more
markedly the architecture of interstitial ECM. Qualitative zymography
performed on lung extracts showed increased gelatinase activities in
both S and E groups, providing direct evidence that the activation of
tissue proteinases may play a role in acute lung injury.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
10 cmH2O)
(9), reflecting the absorptive pressure of the lymphatic pump.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Vo)/(Vt
Vo),
where Ve is volume eluted for each
peak, Vo is void volume, and
Vt is total volume of the column.
A good resolution is obtained when
Kav is close to zero
for the largest molecules and
Kav is close to one for
the smallest ones.
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
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Fig. 1.
Gel-filtration chromatography of
125I-radiolabeled proteoglycans
isolated from 0.4 M guanidine hydrochloride (GuHCl) extracts of lungs
in control conditions (C), after saline loading (S), and after
intravenous elastase administration (E). Determination of counts/min
(cpm) is expressed in thousands. Black, stippled, and open areas depict
relative contribution of material eluted in void, with gel included,
and total volume of column, respectively.
In animals receiving saline infusion (Fig. 1, middle, S), peak 1 nearly disappeared, indicating an almost complete fragmentation of the versican family. The fragmentation products should be recovered in peaks 2 and 3, which indeed show a larger increase relative to the C group.
In elastase-treated animals (Fig. 1, right, E), the relative content of peaks 1 and 2 was lower with respect to the C group; meanwhile, the recovery of radioactivity under peak 3 increased, indicating a marked fragmentation of proteoglycans.
The profiles reflect the relative content of the different proteoglycan families in the pooled samples. Pooling was necessary to collect enough material for the biochemical analysis; moreover, this procedure overcomes the variability of the animals within the groups and within the same animal for the different lung regions. The high reproducibility of the gel-filtration technique of our experimental conditions on a total of eight runs is indicated by 1) total recovery of the radioactivity eluted from the column, which was always >90% of the loaded amount and 2) Kav of the peaks eluted in the Vo (largest molecules) and in the Vt (fragments of smaller molecular weight), which were 0.03 ± 0.01 and 0.93 ± 0.06, respectively. Accordingly, one may consider the differences in the gel-filtration profiles among groups as indicative of corresponding differences in the relative proportions of proteoglycan families.
The binding properties of total 125I-proteoglycans isolated from 0.4 M GuHCl extracts, examined by solid-phase binding assay for type I and type IV collagen and hyaluronic acid, decreased markedly in E and S groups (Fig. 2) relative to the C group. In particular, the proteoglycan ability to bind collagen type IV was significantly reduced (unpaired t-test, df = 10) relative to C group, to E group (t = 12.7, P < 0.001), and to a smaller extent to S group (t = 4.4, P < 0.01). Binding to hyaluronic acid was more markedly reduced in S group (t = 29.2, P < 0.001) than in E group (t = 8.6, P < 0.001). The decrease in proteoglycan binding to type I collagen was similar and significant in the E (t = 14.2, P < 0.001) and S (t = 8.5, P < 0.001) groups.
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The pattern of qualitative zymography for the detection of gelatinase activities (Fig. 3) showed two main gelatinolytic bands in control (top, C) at about 92 kDa (band b) and 72 kDa (band a). These bands were identified by specific antisera as the inactive proenzymes gelatinase B and A, respectively. Two minor bands (b' and a') migrated faster than the corresponding proenzymes and likely represented the proteolytically activated forms of the zymogens. We also observed a slight gelatinolytic band of very high molecular mass (>200 kDa; band c), which may represent a multiple form of gelatinase B (3). All the gelatinolytic activities were inhibited by 20 mM EDTA, thus confirming the identification as metalloproteinases (7). The proteolytically activated forms of gelatinases increased in the S and E groups, being particularly pronounced in the latter case (Fig. 3, middle, S, and bottom, E).
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DISCUSSION |
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In this study we purposefully adopted two models to cause an increase in microvascular filtration by different mechanisms. Saline infusion caused plasma expansion (~15% at 60 min) (8) and increased the filtration rate due to capillary recruitment and lower plasma colloid osmotic pressure; therefore, this model would minimally affect hydraulic and protein endothelial permeability. By contrast, elastase injection caused a lesion of the endothelial and basal membrane barriers, thus involving a specific increase in water and protein permeability coefficients. On the basis of these premises, we thought it useful to describe possible differences in the sequence of events affecting the native architecture of ECM during the development of hydraulic or lesional lung edema, involving in particular the proteoglycan families.
The lung W/D of the animals used in this study in the S and E groups is higher than the control value, although not significantly, and may be considered as corresponding to the transition phase toward the development of severe alveolar edema, leading to a much higher W/D. Therefore, analysis of lung tissue samples may prove useful to detect possible differences in the sequence of degradation of the various proteoglycan families in the two experimental edema models.
In elastase-induced edema, gel-filtration analysis demonstrates that the morbid process affects only partially the interstitial versicans (reduction in peak 1, Fig. 1, E compared with C). Meanwhile, elastase induces marked fragmentation of HS-containing proteoglycan families, including perlecan of the basement membranes (near-total disappearance of peak 2, Fig. 1, E compared with C). Perlecan is likely to be the first proteoglycan population to be degraded by elastase, which destroys proteoglycan core proteins (18). In addition, solid-phase binding assay indicates a marked decrease in the binding properties of proteoglycans to collagen type IV, a major component of the basement membrane. A decrease in HS proteoglycan content in lung alveoli after intratracheal instillation of pancreatic elastase in rats (17) confirms the preferential fragmentation process of this enzyme on basement membranes. Therefore, the transition phase toward the development of lesional lung edema implies major damage to the architectural macromolecular organization of the basement membrane, including both a fragmentation process and a loss of binding properties of proteoglycans to the other ECM components. These modifications ought to play an important role in increasing transendothelial fluid filtration. Despite this, extravascular lung water was not significantly increased relative to control (from the W/D), a fact that can be interpreted by recalling that the integrity of a significant amount of large structural proteoglycans (versican) still provides a low compliance of the extracellular matrix. Indeed, a low compliance allows a marked increase in interstitial fluid pressure that, in turn, opposes further filtration (8, 10, 11).
In hydraulic edema, we found massive fragmentation of the versican proteoglycan family (reduction in peak 1, Fig. 1, S compared with C), whereas the involvement of HS-containing proteoglycans of the basement membrane seems to be less severe than in lesional edema, as suggested by the less marked decrease of proteoglycan-binding properties to collagen type IV. However, the indications of gel-filtration analysis are not conclusive because the coelution of versican fragments with HS-containing proteoglycans does not allow one to evaluate exactly the relative content of the latter family. The almost complete disappearance of interstitial versicans is in agreement with the marked decrease in proteoglycan-binding properties to hyaluronic acid, which may form aggregates with versican monomers in ECM (1, 2, 16).
In the hydraulic edema model, an increase in microvascular filtration occurs due to two related mechanisms, an increase in perfusion rate and a decrease in plasma colloid osmotic pressure (8). The data from the present study suggest that an increase in the hydration state of the matrix primarily leads to damage of the large extracellular proteoglycans, thus progressively abolishing their role in providing a tissue safety factor.
Some differences can be further discussed between the two models concerning the possible mechanisms leading to proteoglycan fragmentation. Qualitative zymography showed that the activities of the proteolytically activated forms of gelatinases A and B (Fig. 3, bands a' and b', respectively), which recognize collagen type IV as a substrate (7), were more markedly increased in E than in S, further suggesting a less severe involvement of the basement membrane in the latter model. Gelatinase A is ubiquitously distributed in lung parenchyma, whereas gelatinase B is found in free intra-alveolar macrophages and in alveolar epithelial cells (13). Elastase may proteolytically activate other proteases, such as gelatinase A and B, present in the tissue as zymogen forms, and it may cleave specific inhibitors of the endogenous proteases (18), reducing the antiproteolytic defense of the lung. Therefore, elastase may enhance the degradation of the protein components of ECM and in particular of basement membrane.
Also in the hydraulic edema model, proteoglycan degradation might result from the activation of latent proforms of metalloproteinases triggered by an inflammatory response involving macrophages and macrophage-derived cells releasing proteases and reactive oxygen species (14, 15).
Finally, one may note that the normal proteoglycan interaction with the fibrillar component of ECM is lost in both edema models, as suggested by the similar decrease in proteoglycan binding to collagen type I.
In conclusion, we observed an imbalance toward matrix degradation in both edema models, although the sequence of fragmentation of proteoglycan families was different; this may be relevant in the remodeling process leading to tissue repair.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Ruggero Tenni (Dept. of Biochemistry "A. Castellani," University of Pavia), who kindly provided human type I collagen.
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FOOTNOTES |
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This research was supported by grants from the Italian Ministry of the University and of Scientific and Technological Research (MURST 60% and "Cofinanziamento di Progetti di Interesse Nazionale") and Grant CT 96.03519 from the National Research Council.
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. §1734 solely to indicate this fact.
Address for reprint requests: G. Miserocchi, Istituto di Fisiologia Umana, Via Mangiagalli 32, 20133 Milan, Italy.
Received 17 April 1998; accepted in final form 3 June 1998.
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