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1Department of Experimental and Clinical Biomedical Sciences, University of Insubria, Varese, Italy; and 2Department of Biomedicine, University of Bergen, Bergen, Norway
Submitted 15 July 2005 ; accepted in final form 3 October 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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collagenase; heparanase; interstitial matrix; interstitial edema; mechanical stiffness
The early development of interstitial pulmonary edema has been shown to be associated to a degradation of the PG molecular family and a weakening of the intermolecular bonds between fibers resulting in a disorganization of the three-dimensional matrix fiber mesh. The loss of intermolecular constraints makes the pulmonary interstitial space mechanically more compliant (6, 12), increasing the amount of water that can be accommodated in the tissue and progressively leading to interstitial and, eventually, alveolar lung edema. This behavior has been demonstrated to come into play in the development of hydraulic edema caused by slow infusion of saline solution (6) as well as in lesional edema induced by increasing the plasma concentration of the proteolytic enzyme pancreatic elastase (11). Notwithstanding the completely different etiology of lung edema in those studies, the mechanical response of the solid interstitial matrix was similar in the two types of edema and consisted on one hand in counteracting the initial onset of edema formation and on the other in allowing fluid accumulation once the matrix integrity was disrupted.
Although these sets of experiments revealed the important role of the solid matrix in controlling lung tissue hydration, saline as well as elastase infusion resulted in a nonspecific cleavage of several PG families, failing to attribute a specific role to any of the matrix macromolecules. Hence, in the present study we aimed at describing the mechanical properties and the biochemical structural modification of the pulmonary parenchyma after challenging the lung tissue with more specific lesional agents, i.e., collagenase or heparanase. The two enzymes display different properties; in fact, collagenase is a wide-spectrum proteolytic enzyme acting mainly on collagen type I fibers that build the three-dimensional environment of the interstitial space, whereas heparanase has a more specific activity, cleaving the heparan sulfate chains belonging to the PG molecules (15). On considering that the heparan sulfate proteoglycans (HS-PG) primarily lay in the basement membrane of the endothelial and alveolar epithelial monolayer, the present data will be discussed in light of the contribution of the matrix macromolecules to the mechanical strength of the tissue but also of the role played by the endothelial barrier in edema onset.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The trachea was cannulated with a T-tracheal tube that was connected to a heated pneumotacograph (Hans Rudolph, model 8420) equipped with a dedicated pneumotach amplifier (Hans Rudolph, model 1110A). Saline-filled catheters were inserted in the carotid artery and into a jugular vein and were connected via three-way stopcocks to physiological pressure transducers (model P23XL, Gould Electronics) conveying the pressure signals to the corresponding amplifiers and to a signal conditioner (model 6600, Gould Electronics). The pressures and the flow signals were then digitized with an analog-to-digital board and displayed on the monitor screen using a dedicated LabView software (National Instruments). In addition, the flow signal was electronically integrated using with the same LabView software, and the respiratory tidal volume was also displayed on the monitor screen.
Measurement of pulmonary interstitial pressure in control situation and after lesional edema.
A first group of animals [n = 10, body wt = 2.3 ± 0.3 (SD) kg] was used to measure pulmonary interstitial fluid pressure (Pip) by micropuncture in control situations and after induction of lesional edema. With the spontaneously breathing animal lying supine, a "pleural window" was prepared on the right side of the chest at the level of the 6th or 7th intercostal space by resecting the external and internal intercostal muscles down to the endothoracic fascia. Under stereomicroscopic view, the endothoracic fascia was then carefully stripped over a surface area of 
0.2 cm2 leaving intact the underlying parietal pleura, whose thickness (30 µm) and transparency allowed observation of the intact lung surface. The prepared area was wet with saline solution and kept covered with Parafilm to avoid tissue dehydration before proceeding to micropuncture.
Glass micropipettes with tips beveled to 2–3 µm and a taper of 200 µm were used. The micropipettes were filled with 1 M NaCl solution previously filtered (0.1-µm Millipore filter). Each pipette was calibrated (pressure range ± 30 cmH2O) after being seated in a holder and connected to a pressure transducer (Gould P23XL) motor driven by a servo-null system (model 5; Vista Electronics, Ramona, CA). The pressure signal was amplified, digitized, and analyzed as described for the arterial and venous pressure signals.
Before each measurement the electrical zero was obtained by insertion of the pipette in a saline pool placed at the height of the pleural window and grounded to the animal. Before micropuncture the animals were paralyzed with pancuronium bromide (2 mg/kg body wt) and mechanically ventilated through a ventilator delivering room air at the same tidal volume and frequency spontaneously chosen by the animal during the basal phase of spontaneous breathing.
The lung surface was observed though a stereomicroscope (magnification x60–100, Zeiss SV 11) whose image was captured by a digital video camera (Axiocam, Zeiss), processed by a dedicated board, and displayed on a color monitor with a maximal magnification on the video screen of x200. Final magnification on the video screen was twofold higher. Digital images on the screen were stored through image analysis software (KS300, Zeiss).
At the time of micropuncture, the animal was placed in lateral decubitus, and the ventilator was disconnected for a period of 5–10 min; in the apneic phase the animal was oxygenated by a constant flow of 50% O2 in nitrogen delivered through a thin intratracheal tubing with an outlet pressure of 
1 cmH2O. Under stereomicroscopic observation and with the aid of a three-dimensional hydraulic micromanipulators (Joystick Micromanipulator MO-188 or MO-109; Narishighe, Tokyo, Japan) equipped with a fourth micromanipulator movement, the pipette was advanced through the pleural surfaces and the pleural space until its tip was positioned into the perivascular interstitial space surrounding a pulmonary vessel (diameter from 20 to 200 µm) running over the lung surface. To minimize indentation of the tissues during insertion (7) the pipette was inclined at an angle of 45° relative to the lung surface. Height of measurement was 60% of lung height relative to the lowest point of the chest in the lateral decubitus. Between successive micropunctures the animal was connected to the ventilator and passively ventilated with room air until the next measurement. The criteria for accepting micropipette recordings were: 1) an unchanged electrical zero on withdrawal of the pipette, 2) a stable recording for at least 1 min, and 3) similar values (within±2 cmH2O) obtained from the same site on successive recordings. The latter criterion was met on a regular basis by repeating the pressure registration at the same site within a period of 3–5 min.
To favor the development of lesional pulmonary edema, a subgroup (group A, n = 5) of rabbits received an intravenous bolus of 10,000 collagen digestion units of collagenase (type VII; Sigma, St. Louis, MO), whereas a different subgroup (group B, n = 5) received 2 IU of heparanase (type III, Sigma). Measurements of all parameters, including Pip, were performed during the initial baseline control phase, before the enzymes injection and every 10–20 min after collagenase or heparanase injection up to 180 min.
Blood samples (0.2 ml) were taken every 30 min throughout the experiment via heparanized tubing and syringes (heparin solution: 0.1 mg/ml). The blood was stored in presence of 1 mg/ml EDTA and subsequently partly used to determine hematocrit through a microcentrifuge; the remaining blood was centrifuged, and plasma protein concentration was directly measured through refractometry.
After 3 h from collagenase or heparanase intravenous injection, the animals were killed with an anesthesia overdose. The chest was widely opened, and four samples of lung tissues were taken from each lung. The wet weight-to-dry weight (W/D) ratio of tissue samples from the lung was determined by weighing the samples fresh and after drying in an oven at 70°C for 24 h. To take into account the variability of the local W/D ratio within the same lung tissue due to the patchy nature of edema development, the W/D ratio was calculated on multiple samples of lung tissue excised from the ventral and dorsal areas.
Biochemical analysis of matrix macromolecules in lesional edema. A separate group of animals (n = 15, body wt = 2.6 ± 0.2 kg) was used to assess the effect of collagenase and heparanase on the pulmonary interstitial PG structure and content. The animals were first anesthetized as described for the micropuncture experiments. After tracheotomy and cannulation of a jugular vein, they received an intravenous injection of either collagenase (n = 6) or heparanase (n = 6) and were then left to breathe spontaneously while supine. In each group, three rabbits were killed at 60 min and three at 180 min. A third group (n = 3) was used as control and received a single lethal dose of anesthesia.
Immediately after death the chest was opened, and the lung was excised and frozen in liquid nitrogen to proceed with the biochemical analysis of the pulmonary tissue matrix.
Total PG content of lung tissue samples was evaluated by hexuronate assay, after papain digestion of the tissue (11, 12). After centrifugation of the digested material (15,000 g for 1 h at 4°C), glycosaminoglycans (the polysaccharide component of PGs plus HA) were precipitated with 4 volumes of cold ethanol at –20°C and assayed for hexuronate content.
PGs were extracted from small tissue slices using the chaotropic agent guanidinium chloride (GuHCl, 4 M) in 50 mM sodium acetate buffer, pH 6.0 containing protease inhibitors (11, 12) at 4°C under mild stirring for 24 h. The extract was dialyzed against 8 M urea in 50 mM sodium acetate buffer, pH 5.8, 0.5% (vol/vol) Triton X-100, protease inhibitors (buffer A) containing 0.15 M NaCl, and PGs were then purified on DEAE Sephacel column (1.6 x 50 cm, flow 12 ml/h) and washed with buffer A containing 0.15 M and 0.3 M NaCl. PGs were eluted with buffer A containing 1.2 M NaCl, and PGs containing peaks were concentrated using a concentrator equipped with a PM 10 membrane. Concentrated samples were precipitated with 9 volumes of ethanol at 4°C overnight and analyzed for uronate content, as previously described (12). Aliquots (50 µm as protein) of purified PGs were radiolabeled using 125I as described elsewhere (11) and were analyzed by gel filtration chromatography using a Sepharose CL-4B column (1 x 50 cm, flow 6 ml/h) eluted with 4 M GuHCl in 50 mM sodium acetate buffer, pH 6.0, containing protease inhibitors (11, 12). The elution profiles were obtained measuring radioactive PGs in the fractions by gamma counter (11).
Data analysis. Data are reported as means ± SD. Absolute values were compared by one-way ANOVA. Differences between mean values were considered significant at P < 0.05. Whenever one-way ANOVA detected a significant difference between mean values, all pairwise multiple-comparison procedures were performed (Bonferroni t-test) through a standard statistical program (Sigmastat).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Pip was recorded at heart level from the perivascular interstitial space surrounding arterioles on the costal surface of the lungs physiologically expanded at zero alveolar pressure, while the animals were lying supine. In control condition [time (t) = 0], the average Pip value amounted to –9.3 ± 1.5 cmH2O (n = 5) and was significantly different from zero (P < 0.001). Subsequent recordings attained every 10–20 min after intrajugular addition of collagenase (Fig. 1A) showed that in all individual experiments (n = 5) Pip rose from the subatmospheric control value toward a positive peak to return thereafter toward zero or slightly negative values. Although the time course of individual Pip values differed slightly, the general Pip behavior was similar among individuals: hence, data presented in Fig. 1A were normalized by horizontally translating the Pip time course so as to consider the time at the attainment of the peak value as the zero reference time (Fig. 1B). After this alignment and as described by the bold solid line in Fig. 1B, an average peak Pip of 4.5 ± 0.3 cmH2O (n = 5) was attained at 45 ± 24 min from the addition of collagenase; subsequently Pip dropped to zero in 120 min. The average peak Pip was significantly different from both the initial control value (P < 0.001) and from zero (P < 0.001).
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30 min from beginning of the enzymatic treatment. The difference between treatments became more evident (t = 8.53, P < 0.001) with the attainment of a positive Pip peak (points b') after collagenase but not heparanase (point b) treatment. Thereafter, the Pip time courses became more similar, although Pip remained somehow always slightly lower in heparanase compared with collagenase-treated lungs.
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At the end of the micropuncture experiments (180 min) the W/D ratio was significantly increased, with respect to controls (W/D = 4.23 ± 0.14, n = 3), both in collagenase (W/D = 5.7 ± 0.1, n = 5, P < 0.001)- and in heparanase (W/D = 5.6 ± 0.3, n = 5, P < 0.001)-treated lungs. However, as indicated by data presented in Table 1 for lungs used for biochemical determination of pulmonary PGs, extravascular lung fluid volume significantly increased already at 60 min both after collagenase (W/D = 5.33 ± 0.14, n = 3, P < 0.05) and heparanase (W/D = 5.48 ± 0.3, n = 3, P < 0.05) treatment. Although significantly elevated with respect to control, the lung W/D ratios observed in the treated lungs at both 60 and 180 min are compatible with a condition of mild to severe edema limited to the interstitial compartment. In fact, intratracheal foam, an index of alveolar edema, was never observed in the present experiments. Hence, the phenomena described in the present study pertain to the initial phase of pulmonary edema development.
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To separate the different PG families, sequential gel chromatography was performed on the lung tissue extracts (Fig. 4, A and B). Gel exclusion liquid chromatography was performed twice on specimens of the same pooled tissues, and the areas under the elution curves were averaged. The elution patterns were highly reproducible with deviation between corresponding samples of <1%. The elution pattern of the same control samples is reported in Fig. 4, A and B, top. The chromatography profile showed three different peaks corresponding to three different PG families: large chondroitin-sulfate proteoglycans (CS-PG), mainly versican of >1 x 106 Da (peak a); HS-PG, mainly perlecan, of 0.1–0.5 x 106 Da (peak b); and small CS-PGs, mainly decorin of <0.1 x 106 Da (peak c). After 60 min of enzymatic treatment, the versican peak was reduced, although not significantly, by both enzymatic treatments. However, the most important modification of the elution pattern consisted in the increase of material eluted in peak c, representing PG fragments likely deriving from cleavage of HS-PGs fraction belonging to peak b. After 180 min of both collagenase (Fig. 4A) and heparanase (Fig. 4B) treatment, the versican component was further reduced with a relative increase of peak c.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Because at 1 h from the beginning of the enzymatic treatment the lung W/D ratio was essentially similar, a comparable increased in fluid filtration likely occurred in the two conditions. However, the novelty of the present data was to show that the mechanical response of the tissue, witnessed by the change in Pip, differed in that it was maintained after collagenase, but not after heparanase. This result, substantiated by both the Pip time course and the biochemical evidence, suggests that the integrity of the heparan-sulfate components of the pulmonary extracellular tissue matrix is required to maintain the three-dimensional architecture of the matrix itself and, in turn, to guarantee its mechanical response to increased fluid filtration. Hence, the HS-PG chains would play an important role not only in setting the sieving properties of the endothelial barrier, as constituents of the basement membrane, but also as stabilizing bonds between the other matrix macromolecules.
The results obtained in the present study following the treatment with collagenase extends and refines those obtained in a series of previous studies, showing that, in the intact rabbit lungs, the time course of Pip during development of interstitial lung edema appeared to be independent of the edema etiology. In fact, Pip invariably described a biphasic behavior, rising from a negative control value to a positive peak in the very initial phase to subsequently decline toward values approximately equal to atmospheric pressure, consistent with the complete release of local tissue stress.
This behavior has been repeatedly observed, although with some time course variability, during development of edema induced by 1) slow saline infusion (6), 2) unspecific protease treatment [pancreatic elastase (11)], and 3) acute exposure to hypoxia [6 h at 12% O2 in nitrogen (9)]. The common behavior of Pip has been interpreted in terms of the mechanical response of the solid tissue fibers framework to water accumulation: in the normal lung and until the solid interstitial scaffold remains integer, tissue stiffness provides a mechanical safeguard in that increased fluid entrance in the interstitial compartment determines a Pip increase that opposes further filtration into the tissue. The progressive rise toward positive Pip values observed during fluid accumulation is consistent with the development of a compressive tissue stress secondary to the protective role of the solid matrix. The latter, however, fails whenever the mechanical compliance of the tissue increases, a condition that has been observed to be associated with fragmentation either of large structural CS-PGs in case of hydraulic edema (6, 12) or of the HS-PGs in case of the nonspecific elastase-induced edema (11). If it is true that the degradation of all PG families exposes the extracellular matrix to fluid accumulation, the present data reveal that even the mechanical response attributable to the CS-PGs component requires the integrity of the HS-PGs to be exerted.
In the normal tissue, resident blood monocytes and alveolar macrophages express various types of native collagenases belonging to a group of matrix metalloproteinases (MMP). The endogenous collagenases MMP-1 and MMP-9 specifically hydrolyze both intact and damaged collagen acting both intracellularly and extracellularly (1, 16). Endogenous collagenases potentially degrade various types of native collagen, including type I and IV (16). In addition, collagenases may cleave PGs by directly hydrolyzing their protein core; in fact, it has been shown that large chondroitin-sulfate-containing PGs (versican) isolated from the lung parenchyma of rabbits may be cleaved by purified gelatinase A (MMP-2) or B (MMP-9) (13). The increase in MMP expression and activity, depending either on direct addition of the enzyme in the circulating plasma, as in the collagenase or elastase treatments, or on indirect activation triggered by the mechanical stress imposed to the fiber scaffold, like during the development of hydraulic edema, seems to be the unifying factor motivating the similarity of the Pip time course during edema development. The fact that Pip attains a positive peak during the early phase of edema development suggests that, although MMPs activate a process of interstitial remodeling, interstitial stiffness is still close to normal and high enough to provide an effective mechanical response. From data in Figs. 4 and 5, it is clear that at 1 h after beginning of collagenase treatment, the high molecular versican fraction still accounts for the greatest proportion of the PG families. However, the integrity of versican (Fig. 5B), likely linked to the collagen fibers, does not seem to guarantee per se the efficiency of the tissue safety factor. In fact, for a similar versican fraction and content, the mechanical response of the matrix fibers to fluid accumulation was still present after 60 min of collagenase, whereas it was lost after 60 min of heparanase. Hence heparanase, by specifically cleaving the HS-PG of the basement membranes, more significantly affected the mechanical response of the tissue to fluid influx. The architecture of the basement membrane acts not only as a barrier for fluid and solute efflux from the microvasculature, but also as a fiber network connecting the outer vascular wall to the surrounding interstitial space. Hence, fragmentation of the HS-PGs and, likely, of the other basement membrane macromolecules might functionally disconnect the rigid macromolecular interstitial scaffold from the microvascular wall, causing the loss of mechanical response of the tissue to fluid accumulation.
At 3 h of treatment, in the same manner and similarly to what was observed in the previous models of lesional (11) and hydraulic (12) edema, a significant degradation of all families of PGs is invariably associated to a nullified mechanical response of the matrix. In fact, high versican degradation observed after 3 h of collagenase treatment in the present study (Figs. 4 and 5) is associated with Pip values approaching zero (Figs. 1–3), suggesting that mechanical tissue stress is completely released in this condition.
The heparanase used in the present study was chosen among the available commercialized molecules because of its specific ability to recognize the heparan sulfate chains as a primary substrate. HS-PGs consist of a core protein to which heparanate sulfate chains are covalently linked. They are ubiquitously distributed in the extracellular tissue matrix, particularly in the basal membranes as well as on the cell surface. In the lung, HS-PGs include perlecan in the epithelial and endothelial basement membranes and syndecan at the cell surface (2, 14, 17). For their typical distribution and for the close interaction with collagen type IV, laminin, and fibronectin in the basement membranes (3, 5), HS-PGs may be involved in providing the sieving properties of the endothelial wall. Cleavage of HS-PGs may therefore modify the permeability of the microvasculature endothelium to plasma components, including fluid, solutes of different sizes, and cells. Increased expression of endogenous mammalian heparanase has in fact been reported in sera and urine of metastatic tumor-bearing animals and patients (15) and correlates with the metastatic potential of various tumor cell types (4). The results of the present study indicate that, unlike what is observed with collagenase, the protective role played by the solid component of the tissue matrix in counteracting fluid accumulation into the interstitial tissue is essentially lost as early as 60 min from the beginning of heparanase treatment. This suggests that the endoglycosidic cleavage operated by the heparanase on the HS-PG chains promotes a faster and/or more disruptive action on the extracellular fiber matrix compared with the cleavage of the protein component of the large PG molecules operated by collagenase. As evident from data of Fig. 5, A and B, although heparanase is specific for HS-PG, it also triggers the fragmentation of larger CS-containing PG, likely through activation of other endogenous metalloproteases, as suggested by finding that conditions as different as slow saline infusion (13) or hypoxia (9) induce the activation of systemic and /or tissue metalloproteases. Hence, one might conclude that the mechanical stiffness of the extracellular matrix is associated with the integrity of the glycosaminoglycan links of the PG macromolecules rather than with the core protein integrity.
Although the time course and the specific action of the two enzymes differed in their initial phase, the W/D ratio attained at the end of the experiments was similar (Table 1). On the average the extravascular water content was increased by >30% compared with control, identifying a condition of interstitial lung edema, with no invasion of the alveolar air space. The cause of the rise of Pip values observed in the first 60 min of both enzymatic treatments is an augmented fluid influx into the interstitial space, likely due to an increased permeability of the endothelial barrier to plasma water and solutes. The fact that interstitial but not alveolar edema develops may be attributed to different factors: 1) the increased Pip values reduces the net hydraulic pressure gradient across the endothelial barrier, limiting fluid filtration. This effect would be more pronounced in collagenase experiments because of the attainment of a positive Pip peak (Figs. 1 and 3); 2) since hemodynamic parameters, such as blood pressure and plasma protein concentration, remain constant throughout the experiments, the pressure gradients supporting fluid filtration are not likely to increase over time, but if at all they tend to decrease; and 3) the efficiency of the injected enzymes in plasma and pulmonary tissue might be progressively reduced with time due to the action of resident antiproteolytic enzymes. Hence, the cleaving action of the enzymes might attenuate over time, arresting the fragmentation of the extracellular matrix.
The increase in total hexuronate extraction (Table 1) indicates that, although the relative changes in PG fraction are significant in the present experimental protocol, matrix degradation is still limited. This may not be the case if the enzymes are endogenously produced. For example, it has been recently observed that mechanical ventilation activates both resident and tissue metalloproteases (MMP-2 and MMP-9) leading to matrix degradation and edema development (10). In this case the protracted lesional activity of the enzymes, if not inactivated, may actually determine a major lesion of the endothelial membrane and intra-alveolar extravasation of edema fluid.
In conclusion, the results of the present study indicate that the mechanical response of the pulmonary interstitial matrix in counteracting tissue fluid accumulation in situations of increased fluid filtration, a crucial factor in opposing interstitial edema development, not only is associated with the general macromolecular arrangement of the solid interstitial fiber matrix but specifically requires the integrity of the HS-PG components.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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, continuous line). A time interval ranging between 15 and 80 min was required for Pip to rise from the average control value (–9.2 ± 0.8 cmH2O, n = 5) to a significantly higher (P < 0.001) positive Pip peak at 4.5 ± 0.3 cmH2O. Bars represent ± 1 SD.
significantly different (P < 0.05) from the same enzymatic treatment at 60 min.