News Physiol Sci 16: 66-71, 2001;
1548-9213/01 $5.00
News in Physiological Sciences, Vol. 16, No. 2, 66-71,
April 2001
© 2001 Int. Union Physiol. Sci./Am. Physiol. Soc.
Development of Lung Edema: Interstitial Fluid Dynamics and Molecular Structure
Giuseppe Miserocchi,
Daniela Negrini,
Alberto Passi and
Giancarlo De Luca
G. Miserocchi is in the Department of Experimental and Environmental Medicine and Biotechnology, University of Milano-Bicocca, Milano; D. Negrini is in the Department of Medicine, Surgery and Dentistry, Ospedale S. Paolo, University of Milano; and A. Passi and G. De Luca are in the Department of Experimental and Clinical Biomedical Sciences, University of Insubria, Varese, Italy.
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Abstract
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Pulmonary interstitium is maintained dehydrated at subatmospheric pressure (–10 cmH2O) through low capillary permeability, low tissue compliance, and an efficient lymphatic drainage. Enzymatic degradation of proteoglycans disrupts the endothelial basal membrane and the matrix structure, triggering the development of pulmonary edema.
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Introduction
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Pulmonary gas diffusion occurs at the alveolocapillary membrane, which is well designed to fulfill this task because its specific anatomic feature is to be only 0.1- to 0.5-µm thick. The alveolar and capillary walls delimit the pulmonary interstitium, a thin compartment made of a fiber system serving as a scaffold, other macromolecules forming the capillary, and the alveolar basement membranes, which fill the extravascular space. The thinness of the alveolocapillary membrane reflects a condition of minimum hydration volume of the interstitial compartment, and indeed one may think of the lung interstitium as a functionally "dry" tissue space. Lung water content depends on several factors, such as the transcapillary balance of pressures (Starling balance), the tissue forces transmitted through the interstitial matrix related to the degree of lung expansion, the forces arising from surface tension phenomena at the alveolar-air interface, and the lymph fluid drainage.
The hydraulic pressure of the liquid phase of the pulmonary interstitium (Pip) is a key variable in understanding the water balance at the interstitial level. Indeed, Pip reflects the dynamic situation resulting from the complex interaction between the various factors mentioned above. Any change in one set of forces would influence the other ones, so any of them can be regarded as either an independent or a dependent variable. The result of such a complex interaction might involve a perturbation of the steady state extravascular water balance. This is of relevance in particular if one considers that a major failure of respiratory function occurs when extravascular water increases, leading to lung edema. It is therefore not surprising that physiologists have been interested in accurately measuring Pip.
In this review, we present Pip values obtained with the micropuncture technique in in situ control lungs and in the transition phase toward the development of lung edema. Pip values were correlated to structural and functional modifications of extracellular matrix components, focusing in particular on proteoglycans, which are largely responsible for lung tissue compliance.
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Micropuncture of in situ lung
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Pulmonary micropuncture has been performed in the past on isolated perfused lung kept inflated at positive alveolar pressure. This experimental condition obviously did not preserve the physiological extravascular water balance for two main reasons: 1) the experimental manipulation caused an increase in extravascular lung water, so that lungs were either in a subedematous or edematous state, and 2) lung expansion was maintained with positive alveolar pressure, whereas in physiological conditions, lungs are kept expanded in the chest with zero alveolar pressure and subatmospheric pleural pressure. For any given transpulmonary pressure, one may expect interstitial pressure to differ in the two conditions; in fact, lung tissue is subjected to a compressive stress with positive alveolar pressure and to a tensile stress with zero alveolar pressure and subatmospheric pleural pressure. These considerations prompted us to develop an experimental approach to micropuncture in situ lungs by preserving the integrity of lung-chest wall coupling (5). We prepared a "window" in an intercostal space by resecting the intercostal muscles down to the endothoracic fascia; next, under stereomicroscopic view, we carefully stripped the fascia down to the parietal pleura that is transparent and allows a neat view of the lung surface. This preparation allowed us to insert 2- to 3-µm tip glass micropipettes through the intact parietal pleura to reach the pulmonary interstitial space or the microvessels in the subpleural region. We mostly micropunctured the perivascular interstitial space that is wide enough (20-30 µm) to host the micropipette tip. This experimental approach proved useful to relate interstitial fluid dynamics to the mechanical properties of the lung tissue. Indeed, since the micropuncture technique is minimally invasive, it allowed us to monitor early deviations in steady state interstitial fluid dynamics following small experimentally induced perturbations of pulmonary fluid balance.
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Pulmonary interstitial pressure and lung water balance under physiological conditions
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Under physiological conditions, Pip is subatmospheric, averaging –10 cmH2O (5), in line with the relative dryness of the pulmonary interstitium. Micropuncture of pulmonary microvessels also allowed us to describe the pressure profile along the microcirculation (7) and to estimate pulmonary capillary pressure. The Starling balance of forces at the capillary level sustains fluid filtration from capillaries into pulmonary interstitium, from which interstitial fluid is drained by lymphatics (5). A minimum interstitial volume results from the combined action of a low permeability of the capillary endothelium and a powerful lymphatic pump that can generate a rather subatmospheric pressure (6).
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Time course of pulmonary interstitial pressure in the transition from physiological conditions to interstitial edema
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To detect early events leading to the formation of lung edema, we have developed experimental protocols that cause a slow increase in extravascular lung water. We used two models to induce a moderate increase in microvascular filtration in anesthetized rabbits: 1) a "hydraulic" type of edema due to slow intravenous injection of saline solution (0.5 ml•min–1•kg–1) causing only ~15% increase in plasma volume in 60 min (4), and 2) a "lesional" type of edema obtained by injecting a single bolus of pancreatic elastase (200 µg, 7 IU) (8). Elastase is an omnivorous proteolytic enzyme with broad affinity for a variety of soluble and insoluble protein substrates, including the components of the extracellular matrix.
Figure 1
shows the time course of Pip as microvascular filtration is increased in the hydraulic or lesional type of edema (4, 8). At left, the time scale is expressed relative to the attainment of peak Pip value. One can appreciate that, although the time course of Pip is not exactly the same in the two edema models, it nevertheless displays two common features. At the onset of edema development (mild edema), Pip increases from the control value to ~5 cmH2O, decreasing thereafter toward zero as edema formation progresses with time. Figure 1 also shows (at right) that the initial marked increase in Pip is not accompanied, in either edema model, by a corresponding increase in the amount of extravascular lung water, indexed by the wet weight-to-dry weight ratio of the lung (W/D). In this phase of mild edema, the tissue compliance of the lung interstitium is very low, estimated at 0.5 ml•mmHg–1•100 g wet wt–1, a value ~20-fold lower compared with other tissues (4). A low compliance provided by the structure of the matrix represents an important "tissue safety factor" to counteract further progression of pulmonary edema; in fact, the attainment of a positive Pip value buffers and even nullifies the Starling pressure gradient that causes microvascular fluid filtration. Figure 1 also shows that, as the severity of edema progresses, Pip drops back to zero and subsequently remains unchanged despite a marked increase in W/D; mechanically, this reflects a marked increase in tissue compliance. Note that, as edema develops toward a more severe condition, fluid filtration occurs down a transendothelial Starling pressure gradient that is smaller compared with the control condition, due to the progressive increase of interstitial fluid pressure (from –10 cmH2O in control to slightly above or around 0 cmH2O) as W/D increases. Accordingly, our data suggest that an increase in permeability of the capillary endothelium has occurred to account for the elevated microvascular fluid filtration leading to edema development (4). Hence, one can deduce that at least two factors interact to determine the development of pulmonary edema: the loss of the tissue safety factor, which reflects an increase in tissue compliance (as from data of Fig. 1) coupled with an increase in microvascular permeability.
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FIGURE 1. Left: time course of pulmonary interstitial pressure (Pip) during development of hydraulic or lesional lung edema. Time is expressed relative to the attainment of the positive peak pressure value. Right: Pip data are plotted as a function of the corresponding wet weight-to-dry weight lung ratio (W/D). The marked increase in Pip with negligible change in W/D during the initial phase reflects a low tissue compliance; edema progression (increased W/D) in the face of a small decrease and no further change in Pip suggests an augmented tissue compliance.
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On the basis of this rationale, a biochemical analysis was carried out to investigate whether the native architecture of the extracellular matrix was altered in the transition phase from control to a condition of modest increase in extravascular lung water.
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The structure of the pulmonary extracellular matrix
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The tissue matrix provides a strong yet elastic framework for the delicate alveolar epithelial-capillary structure and consists mainly of collagen, elastic fibers, and proteoglycans. Proteoglycans are responsible for two important aspects of microvascular and interstitial fluid dynamics, namely the sieving properties of the capillary membrane and of the matrix and the compliance of the interstitial tissue. Proteoglycans include families of multidomain core proteins covalently linked to one or more glycosaminoglycan chains. Versican is a large chondroitin sulfate (CS) proteoglycan found in the interstitial matrix, where it forms aggregates with hyaluronic acid. The relatively high number of CS chains gives a high anion charge to the macromolecule, allowing it to display marked hydrophilic properties and to control the hydration of the interstitial tissues. Perlecan and syndecan are heparan sulfate (HS) proteoglycans found in the basal membrane and in the cell membrane, respectively. The copolymeric nature of HS chains accounts for specific interaction properties in basement membrane organization, receptor functions, and cell-cell and cell-matrix interactions (1).
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Alteration in matrix integrity during development of lung edema
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To relate pulmonary interstitial pressure to the functional state of the matrix, we studied the structure of perlecan and versican in control conditions and during the development of hydraulic and lesional edema (8, 9, 12, 13).
At various stages during the development of hydraulic or lesional edema, the lungs were removed, washed with phosphate-buffered saline, and put in liquid nitrogen. Proteoglycans were extracted from lung samples using 0.4 and 4 M guanidine hydrochloride that breaks the noncovalent bonds of proteoglycans with other matrix components. Proteoglycans were then purified by gel filtration and ion-exchange chromatography. The first important finding was that in both hydraulic and lesional mild edema proteoglycan extractability was increased, suggesting a weakening of their noncovalent bonds with other components of the matrix.
To proceed into the biochemical analysis, dissociative gel-filtration chromatography was performed on purified radiolabeled 125I proteoglycans. Proteoglycans eluted in the different peaks of the chromatography were identified by digestion of the glycosaminoglycan chains with specific eliminases, namely 1) chondroitinase (Chase) ABC, which degrades galactosamine-containing CS chains, and 2) heparinase plus heparitinase, which digest glucosamine-containing HS chains (12, 13).
The gel-filtration chromatography pattern in control conditions (Fig. 2) shows three peaks. Peak 1 corresponds to the large molecular weight proteoglycans and, being very sensitive to Chase ABC digestion, was identified as versican. Peak 2 included proteoglycans of smaller molecular weight exhibiting a lower sensitivity to Chase ABC digestion; the combined treatment with heparinase and heparitinase released unsaturated disaccharides that are typical of perlecan. Peak 3 included small molecular weight proteoglycans carrying both CS and HS chains, probably representing degradation products of larger proteoglycans. In animals receiving saline infusion (Fig. 2; W/D = 6 ± 0.4), peak 1 almost disappeared, indicating a massive fragmentation of the versican family. With respect to control, the fragmentation products should be recovered in peaks 2 and 3 that markedly increased relative to peak 1; note that peak 3 might also contain fragmentation products coming from intermediate molecular weight perlecan originally lying under peak 2. In elastase-treated animals (Fig. 2; W/D = 5.6 ± 0.5), the relative contents of peak 1 and 2 were lower with respect to control, whereas peak 3 was definitely larger, indicating fragmentation of both versican and perlecan. In elastase-induced edema, versican degradation was less severe compared with hydraulic edema.
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FIGURE 2. Dissociative gel-filtration chromatography profile of 125I-radiolabeled proteoglycans isolated from 0.4 M guanidine hydrochloride extracts of lung tissue in control (C), after saline loading (S), and after elastase injection (E). The relative content of large molecular size proteoglycans (peak 1, including versican) decreased markedly, particularly after saline loading. The relative content of intermediate size proteoglycans (peak 2, including perlecan) decreased, particularly after elastase injection. Peak 3 likely contains degradation products of larger proteoglycan populations.
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Hence, some differences are observed in the sequence of proteoglycan involvement during development of hydraulic or lesional edema. In the first case, the increase in filtration rate is due to capillary recruitment and to a decrease in plasma colloid osmotic pressure; this model would minimally affect endothelial permeability to water and proteins, as indicated by the major degradation of versican. In the lesional model, diffusion of elastase (mol mass = 25.9 kDa) through the endothelial wall induces fragmentation of perlecan of the basal membrane, leading to increased microvascular permeability.
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Binding properties of proteoglycans
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Binding properties of proteoglycans from edematous lungs to collagen type I, type IV, and hyaluronic acid were markedly decreased relative to control. Binding to collagen type IV, a component of the basement membrane, was more markedly decreased in the lesional model, in which fragmentation of perlecan was observed. Conversely, the decrease in binding to hyaluronan was larger in hydraulic edema, in accordance with the major degradation of versican that under physiological conditions forms aggregates with hyaluronic acid in the matrix. Hyaluronan degradation also occurs in interstitial edema caused by experimentally induced alveolitis (10).
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Detection of gelatinase activities
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One possible hypothesis for proteoglycan fragmentation relates to the tissue stresses developing within the matrix as extravascular water; this mechanism may also progressively weaken the noncovalent bonds of proteoglycans with other matrix molecules. A further mechanism leading to fragmentation may reside in the activation of tissue proteinases. The turnover and remodeling of lung matrix involves a family of structurally related zinc-dependent endopeptidases known as matrix metalloproteinases (MMPs) that can degrade almost all components of the matrix (13). In particular, the MMP subfamily of gelatinases (gelatinase A, also called MMP-2, and gelatinase B, also called MMP-9) degrades collagen type IV, fibronectin, and elastin (14). Gelatinase A is ubiquitously distributed in lung parenchyma, whereas gelatinase B is found in free intra-alveolar macrophages and in alveolar epithelial cells. MMPs are also involved in some pathological processes, like pulmonary emphysema (2), interstitial lung disease (3), and hyperoxia-induced lung injury (11).
To investigate whether increased activities of gelatinases account for proteoglycan breakdown, we performed SDS-PAGE gelatin zymography to identify proteins with gelatinolytic activities in enzyme extracts from rabbit lung (12, 13). We found that the activated forms of gelatinase A and B increased in hydraulic and, to a greater extent, lesional edema, this last finding being in line with major damage to collagen type IV of the basal membrane.
In addition, we demonstrated that versican is sensitive to MMP-2 and MMP-9 (13). Figure 3 shows the results of dissociative gel filtration chromatography of 125I-versican extracted and purified from rabbit lung incubated with buffer only (Fig. 3A). When 125I-versican was incubated with enzyme extracts from lungs with hydraulic edema (Fig. 3B), the main peak was decreased and a peak of smaller molecular weight appeared, including fragmentation products. HPLC gel filtration analysis showed that the breakdown of versican depended only on the degradation of core protein, because the size of glycosaminoglycan chains was unaffected by proteinase treatment.
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FIGURE 3. Dissociative gel-filtration chromatography of 125I-versican extracted and purified from normal rabbit lung. A: versican incubated with buffer only. B: versican incubation with enzyme extracts from lungs with mild hydraulic edema. Determination of counts per minute (cpm) is expressed in thousands. The peak corresponding to large size versican has disappeared, and one observes a peak of smaller molecular weight fragments resulting from proteolytic activity.
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Gelatin zymography of lung enzyme extracts included the high molecular mass forms of MMP-9 (135 and >200 kDa), which are characteristics of the zymography pattern of neutrophils (2). Therefore, even in mild hydraulic edema, a lesional component is present, because proteoglycan fragmentation results from proteolytic activity, possibly triggered by neutrophil activation. Our study shows that the upregulation of neutrophil gelatinases is triggered by a moderate perturbation of microvascular and interstitial fluid dynamics.
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Conclusion
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By studying the early alterations in pulmonary interstitial fluid balance induced by small perturbations of the steady state condition, we identified a sequence of events leading to an increased extravascular lung water that is summarized in Fig. 4. The early phase of interstitial edema implies an increase in interstitial fluid pressure with no significant change in interstitial fluid volume due to the low tissue compliance. Biochemical analysis of tissue structure reveals an initial fragmentation of CS-proteoglycan in hydraulic edema due to mechanical stress and/or proteolysis. In lesional edema, the partial fragmentation of HS-proteoglycan is mainly due to enzymatic activity. Progression toward severe edema is similar for both kinds of models because the activation of tissue metalloproteinases leads to extended fragmentation of CS-proteoglycan, causing a marked increase in tissue compliance and therefore a loss in tissue safety factor, and of HS-proteoglycan, leading to an increase in microvascular permeability. Using slowly developing edema models, we demonstrated that, once conditions of increased microvascular filtration are established, matrix remodeling proceeds fairly rapidly due to the activation of proteases. We have so far no data on the time course of tissue repair that implies switching from matrix destruction to matrix deposition. It may be of interest to recall that edema fluid is mostly redistributed from the interstitium to the alveoli in experimentally induced fibrosis (15); in this case, a massive matrix deposition would be expected to decrease interstitial compliance and therefore make the tissue safety factor stronger. Yet this possible defense against alveolar flooding does not seem to operate; accordingly, the preferential alveolar distribution of edema fluid can be interpreted if one considers that the extensive matrix remodeling led to a decreased hydraulic resistance of both the endothelial and the epithelial barrier and possibly of the interstitial matrix.
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FIGURE 4. Schematic diagram showing the pathophysiological mechanism leading to hydraulic and lesional lung edema. CS-PG, chondroitin sulfate proteoglycans (versican), one of the structures responsible for interstitial tissue compliance; HS-PG, heparan sulfate proteoglycans (perlecan) mainly controlling microvascular permeability. Physiological conditions are characterized by subatmospheric fluid interstitial pressure and a highly dehydrated interstitial space (A). Increased microvascular filtration leads to interstitial edema (B); interstitial pressure is increased and structures including CS-PG are put under tension, providing a low tissue compliance (tissue safety factor). Hydraulic edema causes a partial fragmentation of CS-PG; lesional edema causes partial fragmentation of HS-PG. In both edema models, the activation of tissue metalloproteases causes combined fragmentation of CS-PG (loss of tissue safety factor) and HS-PG (increase in microvascular permeability), leading to development of severe edema (C).
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Introduction
Micropuncture of in situ...
