© 2003 American Thoracic Society DOI: 10.1165/rcmb.2002-0024OC Pulmonary Vascular Permeability and Ischemic Injury in Gelsolin-Deficient MiceDivision of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and Hematology Division, Brigham and Womens Hospital, Boston, Massachusetts Address correspondence to: Patrice M. Becker, Division of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: pbecker1{at}jhmi.edu
Gelsolin is a potent actin filament regulatory protein that controls cytoskeletal assembly and disassembly. Because cellular gelsolin deficiency leads to pronounced actin stress fiber formation and defective chemotaxis, and similar cytoskeletal remodeling results in endothelial barrier dysfunction, we hypothesized that gelsolin deficient mice would exhibit increased vascular permeability. To test this hypothesis, we compared baseline lung lavage (BAL) protein concentration, wet/dry weight ratio, and osmotic reflection coefficient for albumin (  alb) in gelsolin-deficient (gsn-/-) and C57BL/6 (wild-type) mice. In addition, we assessed lung permeability in response to ischemia by evaluating BAL protein concentration after 4, 8, or 24 h of left pulmonary arterial (LPA) occlusion, and lung wet/dry weight ratio and histology after 24 h of LPA occlusion, in gsn-/- and wild-type animals, as compared with control and sham-operated mice. Baseline measurements revealed that BAL protein concentration was 18-fold higher in gsn-/- than in wild-type mice, whereas alb averaged 0.62 + 0.15 in wild-type, as compared with 0.31 + 0.05 in gsn-/- animals, indicating that gelsolin deficiency caused increased pulmonary vascular permeability. Ischemia increased lung permeability (BAL protein and lung wet/dry weight) in both wild-type and gsn-/- mice. However, whereas the fold-increase in BAL protein concentration was less in gsn-/- mice (2- to 4-fold) as compared with wild-type (22- to 34-fold), the duration of ischemia-induced permeability changes was prolonged. Lung wet/dry weight and gross histology following ischemia were comparable in wild-type and gsn-/- animals. These data suggest that gelsolin significantly contributes to maintenance of vascular barrier function in the lung.
Abbreviations: baseline lung lavage, BAL • left pulmonary arterial, LPA • pulmonary artery, PA
Gelsolin is an 80-kD actin-binding protein which modulates cytoskeletal rearrangement via its ability to alter actin filament assembly/disassembly. When activated, gelsolin reduces actin cytoskeletal integrity by severing actin filaments, then binding to the barbed ends of severed filaments to prevent their extension. Analysis of cells derived from gelsolin-deficient mice suggests that gelsolin plays a critical role in fibroblast (1, 2) and neuronal (3) contraction and motility. Gelsolin deficiency also results in altered cellular calcium metabolism (4) and intracellular signaling (5–7) in these cell types. Gelsolin-deficient mice also exhibit delayed neutrophil migration in response to inflammatory stimuli, and have prolonged bleeding times, which have been attributed to platelet morphologic abnormalities (2). We have previously shown that the actin cytoskeleton is intimately involved in regulation of endothelial barrier function in vitro (8), and that agents which lead to cytoskeletal remodeling in vitro may also alter pulmonary vascular permeability in situ (9). Although gelsolin-null mice are more susceptible to ischemic brain injury than wild-type controls (4), the role of gelsolin in endothelial actin cytoskeletal remodeling and subsequent barrier function under basal conditions or in response to injurious stimuli has not yet been determined. Based upon the recognized effects of gelsolin on actin remodeling in other cell types, we hypothesized that gelsolin deficiency might decrease the integrity of the vascular endothelial barrier. Our prior studies have demonstrated that ischemic injury in the lung is manifest by markedly increased pulmonary vascular permeability to both water and protein (10, 11). Therefore, to determine the role of gelsolin in maintaining lung fluid balance, we evaluated pulmonary vascular permeability in gelsolin-deficient mice at baseline, and following up to 24 h of unilateral pulmonary arterial ischemia.
Reagents The generation of mice lacking gelsolin has been previously described (2). Gelsolin-deficient mice exhibit no overt phenotype, and reproduce normally (2). The absence of gelsolin in lung tissue from gelsolin-/- mice (n = 3) was confirmed by evaluation of gelsolin protein expression, measured by Western blot analysis, in lung homogenates obtained by freeze-clamp biopsy, and compared with gelsolin expression in the lungs of control (C57/B6; n = 3) animals. Monoclonal anti-gelsolin (GS2C4) antibodies and anti–  -actin antibodies were purchased from Sigma (St. Louis, MO) and Chemicon International (Temecula, CA), respectively. Ten percent paraformaldehyde was purchased from J. T. Baker (Phillipsburg, NJ), and diluted in 0.1 M phosphate buffer (pH 7.2) for histology experiments. All other chemicals and reagents were purchased from Sigma (St. Louis, MO).
Preparation To determine whether gelsolin deficiency would potentiate increased vascular permeability of the lung following ischemia, BAL protein concentration and lung wet/dry weight ratios were also compared during 24 h of left pulmonary arterial ischemia. After endotracheal intubation, ventilation was maintained with compressed oxygen at a rate of 120 breaths/min and a tidal volume of 0.2 ml. A left thoracotomy was performed in the second intercostal space, and the left hilum was exposed. The left pulmonary artery (PA) was isolated and ligated using 6.0 suture, then the lungs were hyperinflated to reverse atelectasis, and positive end-expiratory pressure of 3 cm H2O was added. The thoracotomy incision was then closed, and the mice were allowed to recover from anesthesia. After durations of left PA ligation specified below under PROTOCOLS, mice were anesthetized and then killed by rapid exsanguination via the left ventricle, and lungs were excised for measurement of wet/dry lung weight ratios, or lung lavage was performed.
Protocols Protocol II. In the next series of experiments, lung lavage protein concentration was measured after 4 (n = 4–5), 8 (n = 4–5), or 24 (n = 4–5) h of left PA ligation in gelsolin-/- and C57BL/6 mice, and results were compared with sham-operated (n = 4–6) animals. In separate groups of wild-type and gelsolin-deficient mice (n = 4–5) exposed to 24 h of left PA ligation, both left and right lungs were excised for measurement of wet/dry lung weight ratio, and results were compared with sham (n = 6). Protocol III. In a third series of experiments, lungs from wild type (n = 4) and gelsolin-/- (n = 4) mice were fixed for histologic evaluation by the intratracheal instillation of 4% paraformaldehyde (20 cm H20), under either control conditions (n = 2), or after 24 h of left PA occlusion (n = 2). Lungs were imbedded in paraffin, then sections were stained with hematoxylin–eosin for histologic evaluation using light microscopy.
Measurements Lung lavage protein concentration. Evaluation of pulmonary vascular permeability was first evaluated by measurement of the protein concentration in lung lavage fluid, indicative of protein leak from the vascular to the alveolar space. Lavage was performed by intratracheal instillation of 0.5 ml saline immediately after the animal was anesthetized, with aspiration of fluid via the endotracheal tube and careful recording of the amount of fluid recovered (11). Protein concentration was measured in undiluted lavage fluid (13) (BioRad, Hercules, CA). Wet/dry lung weight. As a separate measure of pulmonary edema formation, wet/dry lung weight ratios were determined for both left and right lungs. The lungs were excised separately, then each lung was rapidly weighed on pre-tared dishes for determination of wet lung weight. The samples were then dried in an oven (Fisher Isotemp, 65°C) for 3–4 d, and weighed daily to establish dry lung weight. Wet/dry lung weight ratio would be expected to increase with an increase in extravascular lung water, increased blood volume, or both.
Western blot analysis.
Tissue levels of gelsolin were determined by Western blot analysis (14). Lung tissue from gelsolin-/- and wild-type mice was snap frozen in liquid nitrogen, then homogenized in ice-cold buffer containing 1x phosphate-buffered saline, 1% Nonidet P-40 (Amaresco, Solon, OH), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and Complete protease inhibitor cocktail (Boehringer Mannheim, acquired by Roche Applied Science, Indianapolis, IN). After centrifugation, protein concentration of the supernatant was determined, and 25 µg of protein was loaded onto 4–12% gradient sodium dodecyl sulfate–polyacrylamide gels for electrophoresis. Proteins were electrophoretically transferred to nitrocellulose membranes, then immunoreacted with gelsolin antibodies (1:5,000). After washing away the primary antibody, peroxidase-conjugated avidin secondary antibody was used for visualization. Blots were stripped and probed with
Statistical Analysis
Effects of Gelsolin Deficiency on Pulmonary Vascular Permeability in Normal Murine Lungs As shown in the upper panel of Figure 1, BAL protein concentration under control conditions was 18-fold higher in gelsolin-deficient mice (0.426 ± 0.179 µg/µl) as compared with wild-type mice (0.024 ± 0.052 µg/µl). Interestingly, mean lung wet/dry weight ratio was not significantly altered as a result of gelsolin deficiency, although values for lung wet/dry weight ratios were highly variable in the knockout animals (Figure 1, middle panel). To confirm that pulmonary vascular permeability was increased in mice lacking gelsolin, we estimated the osmotic reflection coefficient for albumin ( alb) in lungs isolated from wild-type and gelsolin-deficient animals. As shown in the lower panel of Figure 1, the mean alb in lungs from gelsolin-deficient mice (0.31 ± 0.05) was significantly lower than in lungs isolated from wild-type mice (0.62 ± 0.15), indicating increased lung vascular permeability to protein in these animals. Differences in pulmonary vascular permeability to protein between gelsolin-deficient and C57BL/6 mice were not due to background strain–related alterations in alveolar capillary permeability, as values for basal BAL protein concentration (0.15 ± 0.07) and mean alb (0.56 ± 0.10) in 129/Sv mice did not differ significantly from those measured in C57BL/6 mice.
Effects of Gelsolin Deficiency on Pulmonary Vascular Permeability Changes during Ischemia As we have described in prior studies (11), lung lavage protein concentration in C57BL/6 mice increased significantly at all time points following left pulmonary arterial ligation. As shown in Figure 2, BAL protein concentration increased after 4 h, peaked after 8 h, and began to decrease after 24 h of left PA ischemia in wild-type animals. Despite an overall response to ischemia that was similar to wild-type mice, gelsolin-/- mice demonstrated differences in the magnitude and time course of the BAL protein response after PA occlusion. Whereas lavage protein concentration peaked following 8 h of unilateral PA ischemia in C57BL/6 animals, the time course of increased permeability was delayed in gelsolin-deficient mice, with maximal protein concentration developing only after 24 h of PA occlusion. Additionally, the fold increase in BAL protein in response to ischemia was significantly less in gelsolin-/- mice when compared with wild-type (2- to 4-fold versus 22- to 34-fold increase in gelsolin-/- versus wild-type mice, respectively), potentially reflecting the higher basal permeability in the knockout animals.
To independently evaluate ischemia-induced changes in pulmonary vascular permeability to water, we also compared lung wet/dry weight ratios in wild-type and gelsolin-/- mice. As shown in Figure 3, wet/dry weight ratio for both right and left lungs from gelsolin-/- mice exhibited substantial variability when compared with wild-type mice. Left lung wet/dry weight ratio increased following 24 h of left PA occlusion in both wild-type and gelsolin-deficient animals, whereas right lung wet/dry weight ratios did not differ. No significant differences in the magnitude of the ischemia-induced increase were detected between the two groups of mice. Similarly, histologic evaluation of lungs by light microscopy demonstrated comparable vascular congestion and mild intra-alveolar hemorrhage after 24 h of left PA ligation in both wild-type and gelsolin-/- mice (Figure 4), an observation similar to previous reports (2).
Microvascular permeability in normal blood vessels lined with nonfenestrated endothelium is determined largely by the flux of fluid and hydrophilic solutes between endothelial cells (16). Numerous studies have suggested that the convective movement of solute across a vascular barrier may be regulated by the balance between the contractile state of the endothelial cells lining the vessel, and the forces which tether endothelial cells to each other and their interstitial matrix. This balance of mechanical forces is intimately mediated by the endothelial cytoskeleton, a complex network of actin microfilaments, microtubules, and intermediate filaments (8). Actin filaments form an integral link between plasma membrane domains and proteins of the cortical cytoskeleton, and are intimately involved in maintenance of cellular shape and integrity. Dynamic changes in actin conformation studied in vitro lead to changes in cell configuration associated with cell motility, as well as attachment of the cell to substrates or adjacent cells. In addition, extracellular signals mediating multiple intracellular signaling cascades are transduced via cytoskeletal elements. Rearrangement of the actin cytoskeleton is a dynamic process, controlled by various actin binding, capping, nucleating and severing proteins (7, 17–19). Regulation of cellular contractile forces, via modulation of actin rearrangement, therefore plays a critical role in the maintenance of endothelial cell barrier function in vitro, a phenomenon that may be particularly important in organs such as the lung, where the surface area of the vasculature is extensive. Gelsolin is the prototype of a family of actin-binding proteins (7, 18, 19), and modulates the motility of fibroblasts (1) and osteoclasts (20), as well as contraction of both fibroblasts (1) and neurons (3). Because endothelial contraction has been demonstrated to decrease pulmonary endothelial cell barrier function in vitro in numerous models (21, 22), we hypothesized that gelsolin deficiency might contribute to impaired cytoskeletal rearrangement, resulting in the development of increased pulmonary vascular permeability in vivo. We believe our study to be the first to demonstrate an important role for gelsolin in regulation of vascular barrier properties.
To accurately evaluate pulmonary vascular protein permeability in gelsolin-deficient mice, we modified our previously published methods (12) to estimate the osmotic reflection coefficient for albumin in isolated mouse lungs. The baseline values for Our data demonstrating that gelsolin deficiency alters the barrier properties of the pulmonary vasculature support the hypothesis that the endothelial cytoskeleton is critically involved in maintenance of vascular barrier function. Prior studies from our laboratory revealed that destabilization of the actin cytoskeleton, using agents that disrupt microtubules also increased permeability in vitro and in isolated lungs (9). In addition, cytoskeletal changes seen with gelsolin deficiency of fibroblasts in vitro, e.g., increased F-actin stress fiber formation and loss of the cortical actin ring (1), have been linked with in vitro endothelial barrier dysfunction in response to multiple stimuli in prior studies from our laboratories (9, 21, 32) and others (29). Gelsolin deficiency has been shown to potentiate infarct volume following cerebral ischemia (4). We therefore evaluated whether increased pulmonary vascular permeability in response to ischemia was enhanced in gelsolin-deficient mice. As we have previously reported (11), both BAL protein concentration and left lung wet/dry weight ratio increased during 24 h of left pulmonary arterial ischemia in wild-type mice. These changes were accompanied by histologic evidence of congestion and mild alveolar hemorrhage. The response of gelsolin-deficient mice to pulmonary arterial ischemia was similar, with levels of BAL protein increasing significantly throughout 24 h of left pulmonary arterial occlusion. Interestingly, the magnitude of the ischemia-induced increases in BAL protein was 10-fold lower in gelsolin-deficient animals than in wild-type controls. This may reflect the enhanced baseline permeability observed in the knockout animals. However, prior studies have suggested that gelsolin may be involved in the recruitment or activation of intracellular second messengers, such as phosphoinositide 3-kinase (20, 30) and phospholipase C (31), which can mediate increased permeability (32–37). The role of these specific signaling pathways in mediating ischemic lung injury has not been fully explored, but these studies raise the possibility that gelsolin deficiency could alter cellular responses to ischemia. Alternatively, intracellular calcium and pH play an important role in activation of gelsolin, thereby increasing its actin severing activity (31, 38). Because intracellular calcium may increase during glucose or oxygen deprivation, the absence of gelsolin might attenuate calcium-regulated pathways, leading to actin remodeling during ischemia. Another notable difference in the response to ischemia was the prolonged duration of ischemia-induced pulmonary vascular permeability observed in gelsolin-deficient mice when compared with wild-type animals. BAL protein concentration peaked after 8 h of unilateral lung ischemia in control mice, and began to return toward control values by 24 h. In contrast, ischemia-induced increased BAL protein concentration was most pronounced after 24 h of pulmonary arterial occlusion in the gelsolin-deficient mice. These data are consistent with the possibility that cytoskeletal rearrangement is necessary to restore barrier function after edemagenic agents, as we have previously demonstrated in vitro (39). Furthermore, in vitro, cytoskeletal alterations may be linked to regulation of programmed cell death (40). Preliminary data from our laboratory demonstrates evidence of apoptotic cell death after 24 h of ischemia in this model of lung injury in wild-type mice. Furthermore, broad spectrum caspase inhibition attenuated increased permeability following 24 h of unilateral lung ischemia, but had no effect on lung vascular leak after shorter durations of ischemia (41). Gelsolin is a recognized target for caspase cleavage (42), and may inhibit apoptotic cell death (43, 44). This raises the possibility that gelsolin deficiency may potentiate apoptosis after prolonged durations of pulmonary ischemia, thereby worsening injury following 24 h of pulmonary arterial occlusion, but not at earlier time points. We are currently assessing this intriguing possibility. Comparison of lung wet/dry weight ratios between gelsolin-deficient and control mice were discrepant with indices of protein permeability. Unlike control mice, lungs from gelsolin-deficient animals showed marked variability in wet/dry weight ratio under both control conditions and with sham thoracotomy. Although we might predict that lung water would increase with increased protein permeability, pore models of transvascular flux suggest that agonists mediating increased permeability may have nonuniform effects on hydraulic conductivity and protein permeability (16, 26, 45). For example, increased pulmonary vascular permeability to protein was demonstrated in awake sheep following intravenous Pseudomonas infusion, without demonstrable changes in lung water in the majority of animals studied (46). The converse has been demonstrated following intravascular pressure elevation in isolated lung preparations (47), where hydraulic conductivity increased without changes in protein permeability. It is also possible that water flux across the vascular barrier is increased with gelsolin deficiency, but that edema clearance mechanisms are unaltered, thus attenuating any differences in lung wet/dry weight between knockout and control animals. Prior studies in intact sheep demonstrated that 26–34% of edema fluid is cleared by lymphatics, whereas 23–29% of edema is cleared into the pleural space (48); thus significant compensatory mechanisms exist to protect the lung from fluid accumulation (49). Fukada and colleagues recently reported that more than 50% of extravascular lung water is cleared within 60 min in intact mice (50), suggesting that edema clearance mechanisms may be common across species. The absence of obvious histologic evidence of pulmonary edema by light microscopy in our studies is consistent with the lack of differences in lung water accumulation in gelsolin-deficient and wild-type animals. It has been speculated that plasma gelsolin functions as an important scavenger of actin released into the circulation with cellular damage, and that saturation of this scavenging system may potentiate lung injury. This speculation is based on prior studies suggesting a correlation between depression of plasma gelsolin levels and the severity of acute lung injury (51–53), and the recent demonstration that intravenous administration of recombinant human gelsolin modestly attenuated the inflammation associated with hyperoxic lung injury in mice (54). Cytoskeletal remodeling as a consequence of cellular gelsolin deficiency seems a more likely explanation for the pulmonary vascular barrier dysfunction seen under basal conditions than plasma gelsolin depletion. However, because both cellular and plasma gelsolin are absent in the gelsolin-/- mice, future experiments replacing plasma gelsolin will be necessary to eliminate plasma gelsolin deficiency as a contributing factor to the increased basal and ischemia-induced pulmonary vascular permeability seen in these animals. In summary, our data suggest that gelsolin is important for maintenance of pulmonary vascular permeability in vivo. Gelsolin deficiency may therefore have a significant impact on the development of acute lung injury under pathophysiologic conditions. Differences in the responses of gelsolin-deficient and wild-type animals to ischemic lung injury suggest that gelsolin may modulate increased pulmonary vascular permeability in response to ischemia, and will be the subject of further study.
The authors wish to thank Laura Welsh for outstanding technical support, and acknowledge Leslie Gregg and Kimberly Sullivan for excellent secretarial support. This work was supported by NIH HL60628 (P.M.B.) and HL58064 (J.G.N.G.), and an Established Investigator Award from the American Heart Association (D.B.P.). Received in original form February 15, 2002 Received in final form November 6, 2002
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Abstract
Introduction
0.05. 
P < 0.05 versus wild-type; 
P < 0.05 versus other wild-type PA occlusion; 
P < 0.05 versus other gsn-/- PA occlusion.
