1 Istituto di Fisiologia Umana, We evaluated the
effect of pancreatic elastase (7 IU iv) on pulmonary interstitial
pressure (Pip) in in situ rabbit
lungs by a micropuncture technique through the intact parietal pleura. Pip was
heparan sulfate proteoglycans; chondroitin sulfate proteoglycans; pulmonary interstitial pressure; elastase
PULMONARY FUNCTION RELIES on the maintenance of a
"dry" interstitial tissue as indicated by the fact that the
interstitial pressure (Pip) is
much lower in the lung parenchyma (approximately The ECM of lung alveoli consists of a meshwork of fibrous proteins
(mainly collagen and elastin) that provides a mechanical scaffolding
for proteoglycans, which are the major component of the nonfibrillar
compartment of the interstitium. Proteoglycans include various families
of multidomain core proteins, which are genetically unrelated and
contain one or more covalently linked glycosaminoglycan (GAG) chains
(8, 27). Proteoglycans are not only involved in the determination and
maintenance of tissue mechanical properties but also participate in a
number of dynamic biological processes that include regulation of cell
adhesion, cell migration, and cell proliferation as well as modulation
of the biological activities of matrix-bound growth factors and
cytokines (8, 27). Moreover, proteoglycans may also act as protease inhibitors (12, 25, 37).
Different proteoglycan populations are present in lung alveoli. The
large chondroitin sulfate (CS)-containing proteoglycan (versican) may
form aggregates with hyaluronic acid in the interstitial matrix,
whereas small dermatan sulfate (DS)-containing proteoglycans (mainly
decorin) are associated with collagen fibrils (27). Heparan sulfate
(HS)-containing proteoglycans include perlecan of the epithelial
basement membrane and syndecans of the cell surface (4, 27, 41). These
molecules are organized in the ECM through noncovalent interactions,
which are affected in various forms of lung injury such as
saline-induced edema, where a partial breakdown of proteoglycans occurs
(21).
The ECM is a dynamic structure, and proteoglycans turn over rapidly
(3). Therefore, a balance must exist between the synthetic and
degradative pathways to maintain a normal content of these molecules
that are responsible for the structural integrity of the alveolar wall.
It is well known that elastase treatment induces lung injury in many
animal models (5, 12, 22, 25, 33), causing emphysema. Elastase is an
omnivorous proteolytic enzyme at neutral pH, with broad affinity for a
variety of soluble and insoluble protein substrates, including all
structural components of the lung ECM, as well as for proteoglycans
(34).
In the present study, we evaluated the effect of an intravenous
injection of elastase in rabbits on the mechanical properties of the
lung interstitium and on the molecular structure of the ECM by focusing
on proteoglycans because compliance of the lung ECM is related to
proteoglycan structure.
Adult New Zealand rabbits [n = 26; body weight = 2.23 ± 0.17 (SD) kg, range 2-2.5 kg]
were 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 with an infant tube to allow spontaneous
breathing. The carotid artery was cannulated via a saline-filled
catheter, and the line was connected to a pressure transducer (model
4-327-I, TransAmerica Delaval, Pasadena, CA) for continuous monitoring of systemic arterial pressure
(Psys). A superior jugular vein was also cannulated for intravenous elastase administration.
General and hemodynamic variables. In
four supine rabbits, esophageal pressure
(Pes), pulmonary arterial
pressure (Ppa), left atrial
pressure (Pla), and cardiac
output ( Ppa and
Pla were measured with a surgical
procedure previously developed in our laboratory (18, 20) that allows
for continuous monitoring of pulmonary vascular parameters in
spontaneously breathing rabbits. The surgical procedure consisted of
removing the skin and the external intercostal muscle insertions on the
medial portion of the sternum. The latter was then widely opened by a
midsternal incision running from the manubrium to the xiphoid process;
after the soft mediastinal connective tissue and the thymus were moved, the pericardium was exposed and cut along a medial longitudinal line. A
saline-filled catheter inserted into the right ventricular wall was
driven into the pulmonary artery, and another catheter was placed in
the left atrium. Both catheters were linked to pressure transducers
placed at the same level as their tips. Blood samples ( Measurements of Pip and intercostal
interstitial pressure.
Pip and intercostal interstitial
pressure were measured in eight animals in which the variables listed
in General and hemodynamic variables were not measured to avoid a
difficult surgical procedure. The animal breathed spontaneously through
the tracheotomy while in the supine position, and the superficial
thoracic tissues were removed on one side of the chest. A "pleural
window" (surface area 1 cm2)
was prepared at approximately the same level as the heart by removing
the internal intercostal muscles down to the endothoracic fascia. In
three animals, two adjacent pleural windows were prepared in the sixth
(midaxillary line) and seventh (dorsal) intercostal spaces at an
~2-cm distance from each other. The endothoracic fascia was carefully
stripped with iridectomy forceps under steromicroscopic view (SMZ-2T,
Nikon) to expose ~0.2 cm2 of the
parietal pleura. This preparation allowed us to obtain a clear view of
the pulmonary structures (microvasculature, alveoli, and septa) through
the intact parietal pleura when the lungs were expanded at a
physiologically negative intrapleural pressure and zero alveolar
pressure so that the integrity of the lung-chest wall coupling and of
the entire pulmonary circulation were preserved.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
10.8 ± 2.2 (SD)
cmH2O in the control condition,
increased to +5.1 ± 1.7 cmH2O
at ~60 min [condition referred to as mild edema (ME)],
and subsequently decreased to
0.15 ± 0.8 cmH2O, remaining steady from 80 up
to 200 min with a marked increase in lung wet-to-dry weight ratio
[condition referred to as severe edema (SE)], suggesting an
increase in tissue compliance. We functionally correlated the measured
Pip to structural modifications of
proteoglycans, the major interfibrillar component of the
extracellular matrix (ECM). The strength of the noncovalent bonds
linking proteoglycans to other ECM components decreased with increasing
severity of edema, as indicated by the increased extractability of
proteoglycans with guanidine hydrochloride. Total proteoglycan recovery
(expressed as µg hexuronate/g dry tissue) increased from 436.8 ± 14 in the control condition to 495.3 ± 23 and 547.0 ± 10 in ME
and SE, respectively. Gel-filtration chromatography showed in ME a
fragmentation of heparan sulfate proteoglycans, suggesting that
elastase treatment first affected basement membrane integrity, whereas
large chondroitin sulfate proteoglycans were degraded only in SE.
Elastase caused a fragmentation only of the core protein of
proteoglycans, the binding properties of which to collagens,
fibronectin, and hyaluronic acid were markedly decreased, as indicated
by a solid-phase binding assay. The sequential degradation of heparan
sulfate and chondroitin sulfate proteoglycans may account for the
initial increase in microvascular permeability, followed by a loss of
the native architecture of the ECM, which may be responsible for the
increase in tissue compliance.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
10
cmH2O; see Refs. 15, 16) compared
with other tissues in control conditions. To minimize interstitial
fluid volume, the lung requires 1) a
selective endothelial barrier limiting capillary to interstitium fluid
filtration by restricting plasma protein escape (31),
2) an efficient interstitial fluid
drainage provided by pulmonary lymphatics, and
3) a very low tissue matrix
compliance that depends mainly on the macromolecular organization of
extracellular matrix (ECM). These functions add to provide a large
"tissue safety factor" that limits edema formation.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
) were measured in the control condition
and up to ~200 min after intravenous infusion of 200 µg (7 IU) of
porcine pancreatic elastase (Sigma Chemical, St. Louis, MO).
Pes, an index of the average
transpulmonary pressure during the respiratory cycle, was recorded via
a small latex esophageal balloon fixed to the tip of an air-filled
catheter connected to a three-way stopcock. The balloon was driven down
to the second third of the esophagus, and the line was attached to a
pressure transducer for continuous recording.
was
measured via an ultrasonic flow probe (model T201, Transonic Systems,
Ithaca, NY) secured around either the ascending aorta or the pulmonary artery. This approach, which is possible in rabbits because the pleural
spaces are ventrally separated, allowed us to preserve the integrity of
the pleural sacs.
0.5 ml each) were withdrawn in the control condition
and after elastase administration; after separation by centrifugation,
the total plasma protein concentration was determined through an
optical refractometer (model SPR-N, Atago).
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RESULTS |
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Hemodynamic parameters.
Pes,
Psys,
Ppa,
Pla, and in the
control condition and after elastase administration are reported in
Table 1. Over time,
Psys remained essentially
unchanged, whereas Ppa and
Pla increased slightly.
tended to decrease, although not significantly,
during edema development. As a result, the total flow resistance
offered by the pulmonary vascular bed
(Rtot), calculated as
Rtot = (Ppa
Pla)/
,
increased over time, although not significantly. No significant change
in end-expiratory Pes was
observed.
|
Total plasma protein concentration
(Cp) averaged 5.73 ± 0.38 g/dl in the control condition and significantly decreased to 5.25 ± 0.23 g/dl at 67 min, 4.55 ± 0.33 g/dl at 131 min, and 4.75 ± 0.73 g/dl at 207 min after elastase administration
(P > 0.05); Cp data can be fitted by the
linear regression, Cp = 5.79 0.0058t (r2 = 0.67),
where t is time in minutes. Plasma
colloid osmotic pressure (
; calculated as
= 4.64Cp + 0.0027C2p; see Ref. 19) was 26.6 cmH2O in the control condition and
significantly decreased by 2.3 and 4.6 cmH2O at 67 and 207 min,
respectively (P > 0.05 by paired
t-test).
Micropuncture measurements. In three rabbits (Fig. 1), we measured Pip after elastase administration (injection at t = 0) in two recording sites (sixth intercostal space on the midaxillary line and seventh intercostal space dorsal). Although the time course of Pip differs among animals, it appears similar for the two recording sites within each animal.
|
Figure
2A
shows Pip in the control condition
and at different times after elastase administration for all the
animals studied; for the three animals shown in Fig. 1, average values
from the two recording sites are shown. Despite an individual
variability in Pip measurements,
in all experiments, a similar trend occurred over time. After elastase
administration, Pip increased from
the control value (10.8 ± 2.2 cmH2O;
n = 5 animals) to positive values, subsequently decreased to zero, and remained essentially unchanged. In
Fig. 2B, the data of Fig.
2A were normalized by plotting the Pip values as a function of time,
with zero time corresponding to the peak positive
Pip value. On average, a positive
peak pressure of 5.1 ± 1.7 cmH2O was attained at 56 ± 16 min after elastase administration, followed by a pressure decline that
was essentially zero at ~60 min after the peak positive
Pip value. This trend is similar
to that observed in a previous study during development of hydrostatic
edema (Fig. 2B, dashed line; Ref. 21).
Pepl was
1.9 ± 0.8 cmH2O in the control condition and
increased slightly after elastase administration according to the
equation Pepl =
1.19 + 0.008t
(r2 = 0.37).
|
The lung W/D averaged 4.9 ± 0.2 in the control condition (n = 5 animals) and 5.23 ± 0.07 (n = 4 animals) and 5.6 ± 0.5 (n = 6 animals) in experiments lasting 90 ± 7 and 138 ± 4 min, respectively, after elastase administration. The intercostal muscles W/D was 3.28 ± 0.1 in the control condition (n = 4 animals) and increased to 3.87 ± 0.17 (n = 2 animals) and 3.64 ± 0.2 (n = 3 animals) at 88 and 132 min, respectively, after elastase administration.
Biochemical analysis. The total recovery of proteoglycans after sequential extraction of lung specimens with 0.4 and 4 M GuHCl, evaluated by hexuronate determination on isolated proteoglycans, increased with the development of elastase-induced edema, resulting in 436.8 ± 14, 495.3 ± 23, and 547.0 ± 10 µg hexuronate/g dry tissue (n = 3 lungs) in the control condition, ME, and SE, respectively. The increase in efficiency of proteoglycan extraction with edema development was particularly evident with the lowest GuHCl concentration. Hexuronate recovery in the 0.4 M extracts was 222.2 ± 23, 260.5 ± 28, and 332.7 ± 15 µg hexuronate/g dry tissue in the control condition, ME, and SE, respectively.
GuHCl extraction allowed the recovery of different proteoglycan populations from each lung specimen. A-PAGE analysis of proteoglycans isolated from the 0.4 M extracts showed two main bands of different electrophoretic mobility, in agreement with recent findings (21). The determination of their GAG composition by Chase ABC digestion and nitrous acid treatment indicated that the slower moving band, consisting of large-size proteoglycans, contained mainly CS and DS chains because 73% of this band disappeared after Chase ABC digestion. Therefore, this large material is likely versican. The broad, fast-moving band, consisting of heterogeneous, lower molecular size proteoglycans, contained mainly HS chains because 78% of this material resisted Chase ABC digestion, disappearing completely after nitrous acid treatment. HS-containing proteoglycans extracted from the basement membrane are probably included in this material. Similar results were obtained in the analysis of the 4 M extracts.
125I-radiolabeled proteoglycans,
isolated from the 0.4 and 4 M extracts, were gel filtered under
dissociative conditions (in the presence of 4 M GuHCl) to prevent
molecular interactions, which may affect molecular size analysis (9).
Under these experimental conditions, radiolabeled proteoglycans
isolated from the 0.4 M extracts were resolved into three main
different populations (Fig. 3,
top). Part of the radioactivity was
eluted in the void volume of the column ( peak
L). It likely corresponds to the population of
large-size proteoglycans, which shows slower electrophoretic mobility
in A-PAGE and carries CS chains such as versican. This interpretation
was confirmed by the marked sensitivity of this material to Chase ABC
treatment; moreover, the unsaturated disaccharides released by this
digestion were identified by capillary electrophoresis analysis as the
typical constituents of galactosamine containing GAG chains:
-
4,5-unsaturated hexuronic
acid-[1
3]N-acetylgalactosamine 4-sulfate,
-
4,5-unsaturated hexuronic
acid-[1
3]N-acetylgalactosamine 6-sulfate, and
-
4,5-unsaturated hexuronic
acid-2-sulfate-[1
3]N-acetylgalactosamine 4-sulfate (40). Part of the radioactivity was included in the gel,
consisting of a heterogeneous mixture of smaller size proteoglycans ( peak I) that contained mainly
HS chains as indicated by the lower sensitivity to Chase ABC digestion.
Moreover, capillary electrophoresis analysis showed that treatment of
this material with heparinase and heparitinase released unsaturated
monosulfated disaccharides that contained glucosamine and represented
typical constituents of HS chains (
-
4,5-unsaturated hexuronic
acid-[1
4]N-sulfate-glucosamine and
-
4,5-unsaturated hexuronic
acid-[1
4]N-acetylglucosamine 6-sulfate) (32). Finally, part of the radioactivity was eluted in the
total volume of the column and consisted of low-molecular-size material
( peak S).
|
The relative content of the different proteoglycan populations changed markedly with the development of elastase-induced edema (Fig. 3, ME and SE). The percentage of radioactivity recovered in peak S increased progressively from the control condition to ME and SE (10.7, 25.7, and 37.2%, respectively). Treatment of this material with Chase ABC and heparanase plus heparitinase released unsaturated disaccharides containing galactosamine and glucosamine, respectively, as indicated by capillary electrophoresis analysis. Therefore, peak S contained GAG chains, including both CS and HS, which are likely to represent degradation products of larger size proteoglycans.
In ME, the relative content of large-size proteoglycans ( peak L) was unaffected, suggesting that the increase in fragmentation products in this phase might depend mainly on partial degradation of smaller size proteoglycans containing mostly HS chains. However, in SE, the relative content of large proteoglycans was greatly reduced, indicating that the further development of elastase-induced lung injury was coupled with a fragmentation of large CS-containing proteoglycans.
Proteoglycans eluted in both peak L and peak I from control lungs were degraded by further incubation with pancreatic elastase (90 mIU/ml in 0.1 M Tris-acetate buffer, pH 7.0, 24 h, 37°C). Gel-filtration chromatography of the elastase-treated material showed a complete disappearance of both peaks. This finding confirms in vitro that lung interstitial proteoglycans can become a substrate for pancreatic elastase.
Similar results were obtained in the study of radiolabeled proteoglycans isolated from the 4 M extracts.
Size-distribution analysis of native GAG chains freed of proteins was performed by gel-filtration HPLC. In all the samples, the chain size was dispersed within the same range of values, but material of very low molecular size was never detected, indicating that GAG chains were not subjected to extensive degradation during the development of elastase-induced edema. This finding is in agreement with the observation that pancreatic elastase is devoid of digesting activity on free HS chains (33). On this basis, the elastase-induced degradation of lung proteoglycans is likely to depend only on the digestion of the core protein.
The binding properties of total proteoglycans isolated from the 0.4 and 4 M extracts (Fig. 4), assayed for different ECM components by solid-phase binding assay, changed with increasing severity of lung edema. On average, the binding properties of proteoglycans extracted with 0.4 M GuHCl were always lower than those extracted with 4 M GuHCl. This result is in agreement with the ability of the lowest GuHCl concentration to extract from the tissue only the molecules that interact more weakly with the other ECM components. In the case of both the 0.4 and 4 M extracts, proteoglycan ability to bind the different ligands was always markedly reduced in SE in comparison to the control condition, indicating that the complete development of elastase-induced edema was coupled with both structural and functional modifications of proteoglycans. On the contrary, the pattern of proteoglycan interaction properties was less homogeneous in ME, and significant differences were not always found with respect to the control condition, in particular in the case of proteoglycans isolated from the 4 M extract.
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DISCUSSION |
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Pulmonary edema was studied with an intravenous administration of elastase, a proteolytic enzyme that can be discharged into the intestinal lumen with other pancreatic enzymes. These enzymes may also be secreted into the blood after an injury affecting the permeability of the pancreatic cell membrane, as in acute pancreatitis. In this pathological condition, loss of the protective role of the ECM with respect to interstitial fluid load can be an important factor leading to the sudden development of severe pulmonary edema, adult respiratory distress syndrome, and multiorgan failure (13, 17, 39).
A single dose of 200 µg (7 IU) of pancreatic elastase, which provides an average plasma concentration of ~90 mIU/ml, was used. At this plasma concentration, elastase is unlikely to affect the elastic properties of the lung as suggested by the finding that end-expiratory Pes remains essentially unchanged during the development of edema (Table 1). Over a longer time frame, elastase has been used at similar concentrations to damage the elastic lung scaffold and cause pulmonary emphysema (1, 24, 33).
In the present study, we followed the progression of lung edema in the
intact lung on the basis of changes observed in
Pip. The similar pattern of
Pip changes at two different sites
within the same animal (Fig. 1) suggests a homogeneous time course for the cascade of events leading to edema. The different time courses of
Pip among the animals (Fig.
2A) can reflect either individual differences in the diffusion rate of elastase and/or different individual availability of proteinase inhibitors such as
1-proteinase inhibitor (34).
The initial phase of the increase in
Pip stems from an increased
microvascular filtration rate, whereas the subsequent decrease in
Pip with a marked increase in lung
W/D suggests an increase in tissue compliance.
From the macromolecular standpoint, the development of the edema associated with elastase administration reveals a sequential susceptibility of different proteoglycans to elastase-induced degradation, which affected only their core protein. In fact, in ME, large-size CS proteoglycans were unaffected, whereas intermediate-size HS proteoglycans were markedly reduced (Fig. 3). Because elastase, due to its low molecular mass (25.9 kDa; see Ref. 29), diffuses from the luminal toward the abluminal endothelial side, it is likely that HS proteoglycans of the basement membrane are the first to be degraded by the enzyme in the subendothelial compartment; this process appears to worsen in SE (Fig. 3).
The nonsignificant decrease in proteoglycan binding properties to collagen type IV in ME (Fig. 4) can be explained on the basis of the following considerations. Basement membrane HS proteoglycan contains three HS chains, which are attached to the NH2 terminus of the core protein and are peripherally oriented, that bind to collagen type IV (41). Therefore, any elastase-mediated cut of the core protein that does not affect the NH2-terminal domain will preserve the ability of HS chains to bind to collagen type IV; however, the fragmentation of the core protein implies a consistent alteration of the native architecture of the basement membrane. The further proteolytic degradation occurring in SE might then lead to a complete separation of HS chains, thus ultimately affecting their optimal spatial arrangement, which is likely to be critical for binding to collagen type IV.
The early damage of HS proteoglycans is likely to cause an increase in endothelial permeability to solutes and water, leading to increased fluid filtration from capillary to interstitium. This interpretation is also supported by the data of Guretzky et al. (6), who showed that loss of HS proteoglycan integrity caused a threefold increase in in vitro endothelial monolayer permeability. The microvascular wall permeability does not depend only on the integrity of the basement membrane but also on the glycocalix covering the endothelial luminal side and on the endothelial pores (26). We showed degradation of HS proteoglycans of the basement membrane in ME, although we cannot exclude that the initial permeability increase is due to endothelial junction opening.
The structural modifications of proteoglycans associated with edema development markedly affected the strength of their interactions with other ECM components, as indicated by the progressive increase in proteoglycan extractability with GuHCl and by the decrease in proteoglycan binding properties in a solid-phase binding assay, which was particularly evident in SE (Fig. 4). In ME, this assay did not show significant modifications for most of the ligands, in particular in the case of the 4 M extract. This result might be explained by considering that proteoglycan degradation is only partial in ME and that the 4 M extract includes proteoglycans that interact strongly in the tissue and might have been poorly affected by the morbid process, thus maintaining their ability to interact with the specific ligands.
The damage of different proteoglycan populations found in ME and SE
actually depended on elastase activity. Under our experimental conditions, all proteoglycan populations extracted from lung specimens were degraded by in vitro treatment with pancreatic elastase, which
digested the core protein without affecting the size of the GAG chains.
Pancreatic elastase is known to degrade the main components of the
basal membrane and ECM (34), and its capability to digest proteoglycans
has also been demonstrated in rat lung using immunohistochemical
methods after intratracheal instillation of the enzyme (33). Moreover,
elastase can also degrade specific inhibitors of the endogenous
proteinases, such as
1-proteinase inhibitor, thus
reducing the antiproteolytic and anti-elastase defenses of the lung
(34). However, we cannot exclude that, under our experimental
conditions, proteoglycan degradation might also be performed by other
proteinases proteolytically activated by elastase.
The large subatmospheric Pip measured in the intact lung suggests that a normal respiratory function can be achieved through a dry, low-compliant pulmonary interstitium. This condition is attained through the matching of transcapillary fluid filtration, mechanical properties of the ECM, and fluid and solute removal through the lymphatic system. ME seems to occur as a consequence of an increased capillary permeability, whereas the structural integrity of large CS proteoglycans in the interstitial ECM is still preserved. Increased fluid filtration into a compact, low-compliant ECM determines a consistent increase in Pip (as shown in Figs. 1 and 2), with a minor change in extravascular water content going from the control condition to ME. The attainment of positive Pip values counteracts further fluid filtration into the perimicrovascular interstitial space; therefore, the maintenance of large CS proteoglycan integrity seems to be critical for ECM architecture and might be regarded as a powerful tissue safety factor, preventing a higher degree of tissue hydration.
From the mechanical standpoint, the loss of macromolecular assembly alters tissue compliance, as suggested by the fact that Pip drops to atmospheric values and remains unaltered as edema progresses (Fig. 2). An increase in tissue compliance after exposure to elastase has also been reported for the vascular intima (11). The present data are similar to previous results from our laboratory (14, 21) on the development of hydraulic edema, showing that the low tissue compliance is critical to minimize interstitial fluid volume and is coupled with the maintenance of normal proteoglycan structure. This behavior was not observed in the interstitium of the intercostal muscles, a tissue that can stand large hydrations without functional impairment. The concept of tissue compliance as a safety factor against the development of edema was originally proposed for subcutaneous tissue by Guyton (7) and subsequently developed by Wiig and Reed (38).
It is a common clinical observation that the transition from mild interstitial edema, normally undetectable with common diagnostic procedure, to accelerated alveolar edema occurs very rapidly. In light of the present results, lesional elastase-induced edema develops as a consequence of a cascade of the following events: 1) an increase in capillary permeability (loss of the structural integrity of HS proteoglycans) and 2) a decrease in interstitial tissue compliance (fragmentation of large-size CS proteoglycans) that abolishes the tissue safety factor.
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ACKNOWLEDGEMENTS |
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Human type I collagen and human fibronectin were kindly provided by Profs. Ruggero Tenni and Pietro Speziale, respectively (Department of Biochemistry "A. Castellani," Università degli Studi, Pavia, Italy).
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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 40 and 60%) and Grant CT 96.03519 from the National Research Council.
Address for reprint requests: D. Negrini, Istituto di Fisiologia Umana, Via Mangiagalli 32, 20133 Milan, Italy.
Received 4 August 1997; accepted in final form 29 October 1997.
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