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Department of Experimental, Environmental Medicine, and Biotechnology, University of Milano-Bicocca, Monza 20052, Italy
Submitted 28 February 2003 ; accepted in final form 4 June 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema. caveolae; lipid rafts; mechanotransduction; interstitial pressure
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Pulmonary interstitial edema formation. The right superior jugular vein was cannulated, and pulmonary interstitial edema was induced by infusing saline at a rate of 0.5 ml·kg-1·min-1 for 3 h. The experimental protocol was shown to cause a slow development of interstitial edema (14) due to a decrease in plasma colloid osmotic pressure that leads to a greater microvascular filtration rate; in addition, the increase in plasma volume (12-15%) also causes a greater lung perfusion and, therefore, a greater filtration area. These animals will be simply referred to in the text as edematous or treated. Previous investigation showed that sham animals (exposed to anesthesia for 3 h) did not present any morphological difference relative to control animals (19).
Chemical. The reagents used (analytic grade) and high-performance thin-layer chromatography (HPTLC) plates (Kieselgel 60) were purchased from Merck (Darmstadt, Germany). 3-[Cyclohexylamino]1-propanesulfonic acid, MES, Percoll, PMSF, and horseradish peroxidase-labeled cholera toxin B subunit were from Sigma Chemical (Milano, Italy). The antibodies against caveolin-1 (C2297) and flotillin (F65020 [GenBank] ) were from Transduction Laboratories (Lexington, KY). The antibodies against aquaporin (AQP)-1 and -5 (sc-9878 and sc-9890) were from Santa Cruz Biotechnology (Santa Cruz, CA). All of the material for the electrophoresis was from Bio-Rad (Milano, Italy). Autoradiographic films were from Amersham Pharmacia Biotech (Uppsala, Sweden).
Lung tissue preparation. Eight rabbits were used for the biochemical analysis of PM: one group of animals was killed immediately after anesthesia and tracheotomy (control; n = 4); another group received intravenous saline infusion for 3 h (edematous; n = 4). The chest was opened, the pulmonary artery was cannulated, and, after the left atrium was sectioned, the lungs were perfused with mammalian Ringer solution (without calcium) containing nitroprusside (20 mg/ml) for 3-5 min at room temperature (18, 19). Then the lungs were flushed with 50 ml of 0.25 M sucrose and 20 mM tricine, pH 7.4, with protease inhibitors (solution A) and excised from the chest. Tissue samples were used to determine the wet weight-to-dry weight ratio (W/D), whereas the rest were immersed in ice-cold solution A. The lung tissue was then finely minced with a scalpel on a cold glass plate, and the minute lung pieces were finally placed in a tube with 30 ml of solution A to be homogenized. The W/D was calculated from the weight of the fresh tissue samples and after drying in the oven at 70°C for at least 24 h.
Purification of PM and preparation of DRFs. Lung homogenates obtained from control and edematous lungs were subjected to Percoll gradient centrifugation, with minor modifications (19), to isolate a plasmalemmal fraction from control and edematous lungs (PMC and PME, respectively) (19). Briefly, the lung tissues were homogenized in buffer (0.25 sucrose, 1 mM EDTA, 20 mM tricine, pH 7.8), and the lung homogenate was filtered sequentially through 53- and 30-µm filters. The homogenate was subjected to centrifugation (1,000 g for 10 min) at 4°C, and the supernatant was saved. The resulting pellet was resuspended in 3 ml of buffer and subjected again to homogenization and centrifugation as above. The two supernatants were combined and overlaid over 25 ml of 30% Percoll in buffer. After centrifugation by using an SW28 rotor at 84,000 g for 45 min at 4°C, we collected a single membranous band readily visible about two-thirds from the bottom of the tube. To reduce the volumes and concentrate the membranes, they were pelleted by diluting the suspension threefold with PBS before centrifugation at 100,000 g for 45 min at 4°C. The pellets obtained from tissue homogenates in control and edema were called PMC and PME, respectively.
To maintain a constant protein-to-detergent ratio in all of the experiments, the pellet containing PM was submitted to protein assay, and a proper volume, corresponding to 4.5-mg PM proteins, was submitted to centrifugation. PMC and PME pellets were incubated in 2 ml of 1% Triton X-100 in 25 mM MES buffer, pH 6.5, containing 150 mM NaCl, 1 mM PMSF, and 75 U/ml aprotinin (MBS buffer), for 30 min on ice. The lysate was submitted to discontinuous sucrose density gradient centrifugation, as described (20, 26). Briefly, the lysate was diluted with an equal volume of 80% (wt/vol) sucrose in MBS lacking Triton X-100 and placed at the bottom of a discontinuous sucrose concentration gradient (30-5%) in MBS lacking Triton X-100. After centrifugation at 250,000 g for 18 h at 4°C with SW-41 rotor (Beckman Instruments), 1-ml fractions were collected from the top of the gradient and submitted to further analysis. Fraction 5 (from the top) is referred to as DRF; fractions 6-8 as intermediate density fraction; and fractions 9-12 as high-density fraction (HDF).
Lipid analysis. All of the fractions withdrawn from the two gradients obtained from PMC and PME were dialyzed against distilled water at 4°C and then lyophilized. Lipids were extracted according to Bligh and Dyer (3) with small modifications, as described (38). Phospholipids and sphingomyelin in the organic phases were separated by HPTLC (solvent system chloroform-methanol-acetic acid-water 60: 45:4:2 vol/vol/vol/vol) and then sprayed with anisaldehyde reagent. For cholesterol visualization, the extracted lipid samples were separated by HPTLC (solvent system hexanediethylether-acetic acid 20:35:1 vol/vol/vol) and then sprayed with anisaldehyde reagent. For the analysis of the plasmalogens, the phospholipids were chromatographed in chloroform-methanol-acetic acid-water (60:45:4:2 vol/vol/vol/vol). Then the plates were exposed to HCl vapors for 10 min and subsequently chromatographed for second dimension in chloroform-methanol-acetone-acetic acid-water (50:15:15:10:5 vol/vol/vol/vol/vol).
After the plate was heated at 180°C for 15 min, the HPTLC plates were submitted to densitometric scanning. Quantification was made on the basis of known amounts of standard lipids loaded on the same plate. In the case of the glicosphyngolipid GM1, HPTLC separation, blotting with horse-radish peroxidase-labeled cholera toxin B subunit, enhanced chemiluminescence (ECL) detection, and quantification were performed as described (19, 38).
Protein analysis. All fractions and also the homogenates and PM, in some cases, were subjected to trichloroacetic acid precipitation. Fractions 1-4, having very low protein content, were pooled before treatment with trichloroacetic acid to obtain a sufficient protein amount for the electrophoresis. The pellets, washed with acetone, were suspended in water for protein assay by bicinchoninic acid method (Sigma Chemical) and then submitted to SDS-PAGE (10% acrylamide, 7 µg protein/lane), followed by Western blotting. Proteins were transferred to nitrocellulose membranes. After blocking, blots were incubated for 2 h with the primary antibody diluted in PBS-Tween-20 (PBS-T)/milk (anti-cav1 1:1,000, anti-flotillin 1:250, anti-AQP-1 1:100, anti-AQP-5 1:100) and then for 2 h with horseradish peroxidase-conjugated anti-rabbit/mouse/goat IgG (5,000- to 10,000-fold diluted in PBST/milk). Control experiments by omitting the primary antibody were also performed. Proteins were detected by ECL using the SuperSignal detection kit (Pierce, Rockford, IL).
To evaluate the enrichment of a given protein in DRF in control and edematous lungs, quantification of the corresponding bands in the ECL film was performed with a model GS-710 imaging densitometer on Molecular Analyst software (Bio-Rad Laboratory).
Immunofluorescence. Lungs from control and edematous animals were prepared for immunofluorescence. The lungs were first washed with saline for 3 min and then perfused with 4% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M phosphate buffer for 25-30 min. After fixation, the lungs were diced, and tissue samples were infiltrated with 18% sucrose solution overnight. The samples were then frozen in isopentane cooled by liquid nitrogen to about -140°C. Frozen sections (10 µm) were treated shortly with 0.1 M glycin in PBS, pH 7.4, and then exposed to a dilution buffer solution (DB) (3% Triton X-100, 15% filtered goat serum, 0.45 M NaCl, and 10 mM phosphate buffer, pH 7.4) for 15 min. The sections were then incubated (overnight at 4°C) with the primary antibody against caveolin-1 (mouse monoclonal, C2297) diluted in DB at 1:20. They were then exposed to a rodamin-conjugated goat anti-mouse IgG (1:100 in DB) for 1 h and mounted in glycerol. As controls, the primary antibody was omitted or replaced with nonimmune mouse IgG. Confocal microscopy was carried out on a Radiance 2100 microscope (Bio-Rad Laboratories, Hercules, CA) equipped with a krypton-argon laser. Noise reduction was achieved by Kalman filtering during acquisition.
Statistical analysis. Biochemical determinations were repeated three times for each animal. Biochemical results were expressed as means ± SD (n = 12 determinations), averaging data from the different animals. The significance of the differences among groups was determined by using one-way ANOVA and t-test.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Protein analysis. The DRF obtained from control lung contained a lower proportion of proteins (2.2 ± 0.6%) relative to the edematous lung (4.6 ± 0.9%) (Fig. 1, left). The majority of proteins recovered in the sucrose gradient (>80%) was present in HDF, and the total amount recovered was similar in control and edematous lungs (Fig. 1, right).
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Caveolin-1 content, a specific constituent of caveolae used as marker of PM purification (18), increased 20 times on comparison of homogenate to PM in both experimental conditions (Fig. 2A). Figure 2B shows the distribution of caveolin-1 in the fractions of the sucrose gradient, and one can appreciate that the DRF was highly enriched in caveolin-1, with the total amount being 15% higher in edematous relative to control lungs. Control experiments gave no labeling. Lung parenchyma from control and edematous animals (Fig. 2, C and D, respectively) was also examined by immunofluorescence for the presence of caveolin-1. In control lungs, the immunoreaction appeared to be fairly uniformly distributed in the cells of the alveolar septa, showing a fine punctate-staining pattern. In interstitial edema, caveolin-1 immunofluorescence shows a less homogeneous distribution with respect to control, with areas of intense staining. Both omission of primary antibodies or replacement with nonimmune mouse IgG gave no labeling (not shown).
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Flotillin, AQP-1, and AQP-5 content in PM was assessed by Western blotting analysis. Flotillin (Fig. 3A) was enriched in DRF from control lungs and increased by 50% in DRF from edematous lungs. AQP-1 (Fig. 3B) greatly increased in DRF of edematous lungs; it was also present in control lung but could only be demonstrated by increasing the exposure time of ECL film (data not shown). AQP-5 (Fig. 3C), instead, was localized in HDF, with similar concentration in control and edematous lungs.
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Lipid analysis. The GM1 concentrations in PM obtained from control and edematous lung, normalized to total protein content, were similar (44.1 ± 2.5 and 47.2 ± 2.9 pmol/mg protein, respectively). The DRF was enriched with GM1 ganglioside and cholesterol (Fig. 4, A and B, respectively), with the values being higher in edematous lungs relative to control.
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Figure 5, A-E, presents the distribution of different lipids in the fractions obtained from the sucrose gradient and clearly shows an increase in the percentage of all lipid components in DRF. Figure 5F shows the phospholipid content in DRF expressed as micromoles of phosphorous per milligram protein recovered from DRF. The amount of all different lipids in fraction 5 increased from two- to threefold in edematous relative to control lungs, when normalized to DRF protein content. Because the DRF protein content was doubled in edema, the observed increase in phospholipids was larger than that reported in Fig. 5E.
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The concentration of ethanolamine plasmalogen (included in phosphatidylethanolamine) increased from 0.07 to 0.16 µmol/mg protein (128% increase), but remained unchanged as a fraction of total phosphatidylethanolamine (40%).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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5 cmH2O for a minor increase in water content, and this represents a strong "tissue safety factor" against the development of severe edema (14). It was also shown that this resistance relies on the role of matrix proteoglycans to oppose interstitial water accumulation (16, 17, 22, 23). In fact, even in mild interstitial edema, some proteoglycan fragmentation was shown to occur due to increased parenchymal tissue stresses and to the action of metalloproteases (16, 17, 22, 23). It was also found that severe edema develops when the fragmentation of matrix proteoglycans has progressed to a point at which tissue compliance is increased and, therefore, the tissue safety factor is completely lost (16, 17, 22, 23). In this paper, we focus again on the early events accompanying extravascular fluid accumulation, because recent experimental data from our group show a functional correlation among initial extravascular water accumulation (6), gene expression (25), and membrane cellular activation (19). This latter aspect is being analyzed further in the present work, considering in particular "lipid rafts microdomains," where proteins regulating cellular transduction are concentrated. Sphingomyelin and glicosphyngolipids are components of microdomains. These lipids tend to possess longer, more saturated fatty acyl groups, and, therefore, bilayers containing these compounds form an immobile gel (i.e., solid-like phase) at biological temperature. Membrane cholesterol tends to dissolve into this region, creating a liquid-ordered phase with fluidity that is intermediate between gel-ordered and mobile-fluid phase. Only membranes displaying these features (i.e., caveolae and lipid raft) are insoluble in 1% Triton X-100 at 0-4°C. No other lipids from either interstitial matrix, alveolar surfactant, or other compartments are structurally organized so as to possess the physicochemical feature to resist Triton detergent action at low temperature. Lipid rafts are dynamic flat domains enriched in glicosphyngolipid GM1, cholesterol, and glycosylphosphatidylinositol-anchored proteins (5, 15, 32, 37), whereas caveolae, a special form of lipid raft (1), contain specific markers like caveolin-1 and flotillin. The doubling in protein concentration in the DRF (see Fig. 1) reflects the increase in caveolar proteins (caveolin, AQP-1, and flotillin). Because the lipid-to-protein ratio, on average, doubles in DRF (considering GM1 and phospholipids), this indicates that lipid concentration increased about four times relative to control, suggesting a parallel increase in caveolae and lipid rafts in edema. The increase in protein and lipids may reflect compartmental translocation and/or synthesis.
The decrease of the phosphatidylcholine-to-phosphatidylethanolamine ratio from 1.48 to 0.98 (as derived from data in Fig. 5) and of the cholesterol-tophospholipid ratio from 41.6 to 22.1 (as derived from data in Figs. 4 and 5) in DRF is in line with an increased membrane fluidity previously reported (19). The present finding of a relatively larger enrichment in the lipid compared with the protein component in DRF may suggest a possible relationship with the observed increase in plasma membrane fluidity.
Cellular deformation is known to induce vesicular formation and lipid redistribution in the PM, considering that sphyngolipids and cholesterol are floating platforms on the cell surface and, furthermore, that lipid trafficking may be stimulated from intracellular lipid stores. In cultured alveolar epithelial cells, lipid trafficking was shown to occur from lipid stores to the PM after inducing deformation of the basement membrane (36). Furthermore, an increase in traction causing deformation of cell surface was shown to elicit lipid translocation to PM (35, 36).
In interstitial edema, the compositional changes could be ascribed to mechanical stimulation on endothelial and epithelial cells, because they provide most of the PM surface extension at the level of the air-blood barrier. Endothelial cells are more likely to be involved, as a previous study revealed a marked increase in their luminal surface area in interstitial edema (19).
In the present experimental model, mechanical forces may arise as an increase in shear stress on the vascular wall and/or as an increase in parenchymal forces due to extravascular water accumulation. In a previous investigation, our laboratory estimated that the increase in shear stress would not exceed 30% (19). Conversely, parenchymal forces are expected to rise due to the extremely low pulmonary tissue compliance, as a consequence of a marked increase in interstitial hydraulic pressure in the face of a minimal increase of extravascular water (5%, in line with previous findings). In particular, parenchymal forces should develop at the level of the basement membrane and in the interfibrillar substance, where initial water accumulation has been described (6). These observations led us to hypothesize mechanotransduction as an early mechanism for endothelial cell activation in developing pulmonary interstitial edema.
The change observed in edematous lung of the immunostaining pattern of caveolin-1, together with the increase of this protein in the DRF, suggests its redistribution in the PM, leading to an increase in caveolar domains at cell surface. Caveolar traffic is critically dependent on actin cytoskeleton, because it is prevented by treatment with cytochalasin D (21, 31). The rapidity of the response in lipid translocation to the PM after mechanical stimulation led us to hypothesize that the cell might store vescicles in the region underneath the PM itself (35, 36).
Caveolae are thought to be involved in transcytosis for macromolecules and water. Recent data indicate that caveolin-1-deficient mice do not seem to show alterations in extravascular oncotic pressure (7); however, these animals show severe pathomorphological defects in the lung, in particular a thickening of the basement membrane in the air-blood barrier, that appear consistent with a condition of interstitial edema; this suggests that caveolae may be involved in the physiological mechanism that guarantees a minimum thickness of the air-blood barrier. The increased amount of extravascular water in caveolin-1-deficient mice was related to the increased microvascular permeability due to nitric oxide, whose action was not inhibited by caveolin (29). Conversely, in interstitial edema, no increase in microvascular permeability was observed (14), due to the inhibitory effect of caveolin on endothelial nitric oxide synthase activity (29).
AQP-1 and AQP-5 are water-transporting proteins present in microvascular endothelial and in type I alveolar epithelial cells, respectively (34). AQP-5 was not present in DRF, and no change was observed in interstitial edema. However, AQP-1 was enriched in DRF, in accordance with immunohistochemical data showing colocalization with caveolin-1 in endothelial cells (28). Because AQP-1 was greatly increased in the caveolar fraction after interstitial edema formation, this suggests a possible role for AQP-1 in water transport, even when the increase in extravascular water is still relatively small. As a next study, it appears of interest to relate caveolar formation and possible redistribution (31) to the marked change in morphology undergone by endothelial cells in interstitial edema (19), characterized by an increase in their luminal and interstitial surface profile.
The present data integrate well with our laboratory's recent results on gene expression analysis obtained in a similar model of interstitial edema (25). In fact, the pooled data stem from an early activation of endothelial cells as a consequence of mechanotransduction, due to a change in the cellular microenvironment. It appears, therefore, tempting to consider this activation as the first event leading to cytokine production that could, in turn, induce secondary effects, including interstitial matrix turnover.
In summary, the experimental evidence from the present work might help to trace the insight concerning the phase where the cellular involvement is in the critical balance between tissue damage or, conversely, tissue repair.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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