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Actin released from damaged cells after a variety of tissue injuries appears to be involved in multiple organ dysfunction syndrome. Under experimental conditions, when the quantity of actin present in plasma is made to exceed the protective capacity of the actin-scavenging mechanism, microembolism and pulmonary vascular angiopathy have been noted in rats. It remains to be determined whether this injury is a result of a direct toxic effect or occurs indirectly via platelet activation or fibrin interactions. We examined the effect of sera from patients with adult respiratory distress syndrome (ARDS), as well as G-actin added to normal serum, on the viability, morphology, and function of cultured sheep pulmonary artery endothelial cells (SPAEC). Both patient sera and normal sera to which actin was added were toxic in the cell culture model; this toxicity could be abrogated, at least partially, by preincubation with gelsolin, which is known to complex with actin. A significant portion of the toxicity of sera from patients with ARDS was sensitive to heat (56° C), suggesting an important role of complement. Sera from patients with ARDS were shown to contain filaments of F-actin by immunoblot and rhodamine phalloidin staining after ultracentrifugation. Thus, saturation of the actin-scavenging system by addition of exogenous G-actin to plasma produces direct pulmonary endothelial cell injury. Furthermore, plasma from patients with ARDS secondary to bacterial pneumonia is toxic to SPAEC, and a small but significant contributory role of actin is apparent in these studies.
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Actin is the major protein of the microfilament system and has
critical and ubiquitous roles in cell structure and motility. When actin is released by dying cells, however, it may have adverse pathophysiologic consequences. A complex actin-scavenger system has evolved in vascular and extracellular spaces
(1). In Figure 1, we summarize recent observations indicating
that angiopathic pathology may occur when the release of monomeric (globular or G-actin) and polymerized (filamentous
or F-actin) actin exceeds the clearance capacity of the actin-scavenging system (group-specific component [vitamin D binding protein or Gc protein] and gelsolin). Conditions associated
with increased release of cellular actin and intracellular actin-binding proteins (gelsolin, profilin, and thymosin 
4) include
fulminant hepatic necrosis (2), adult respiratory distress syndrome (ARDS) (3), septic shock (4), and complicated pregnancies (5). Gc-protein, by binding monomeric G-actin and
releasing gelsolin, allows gelsolin to cap F-actin (inhibiting addition of monomers) and bind to the sides of F-actin (severing
actin filaments) in a substoichiometric fashion. Gc-protein-G-actin complex is then cleared by the reticuloendothelial system (Figure 1, top panel ). Several clinical and experimental
observations suggest that multiple organ dysfunction may
arise when circulating actin exceeds the protective function of
actin scavenging systems (Figure 1, bottom panel ). In ARDS,
elevations in actin-gelsolin complexes are apparent in humans
(3) and experimental models of acute lung injury (6). Saturation of actin-scavenging system by administration of exogenous G-actin in intact rats results in selective pulmonary endothelial cell injury (7). Although this latter in vivo study
strongly supports a contributory role of intravascular actin in
causing acute lung injury, it was unclear if endothelial cell injury was direct or secondary to embolic damage of actin, platelet activation, or interaction with fibrin during clot formation.
In the current study, we used cultured sheep pulmonary artery
endothelial cells (SPAEC) to show that (1) exogenous G-actin
is directly toxic to pulmonary endothelium, and (2) there are
gelsolin-sensitive mediators of SPAEC toxicity in serum of patients with ARDS.
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Patient Samples
Blood samples were obtained from eight patients with ARDS caused by community-acquired bacterial pneumonia (six-pneumococcal; one-staphylococcal; one-legionella pneumonia), eight healthy volunteers, and eight patients with fulminant hepatic necrosis (FHF). Diagnosis of ARDS was established according to American European Consensus Conference criteria for ARDS. Three of eight patients with ARDS were proven to have bacteremia. Six of eight patients with FHF had viral hepatitis, and two had an overdose of acetaminophen. No patients with FHF were found to have ARDS. An informed consent was obtained in all cases according to University of Pittsburgh Medical Center Institutional Review Board.
Cultured Sheep Pulmonary Artery Endothelial Cells
SPAEC were isolated and cultured from collagenase-digested pulmonary arteries as described previously (8). Cells were subcultured in Optimem (GIBCO, Grand Island, NY) with endothelial cell growth supplement (15 µg/ml; Collaborative Biomedical Products, Bedford, MA), heparin sulfate (10 U/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% sheep serum (Sigma Chemical Co., St. Louis, MO). Cells were cultured in the above medium at 37° C in 95% air- 5% CO2. They were routinely passaged 1:3 by detaching cells with a balanced salt solution containing trypsin (0.05%) and EDTA (0.02%) and were used between passages 6 and 12. Human umbilical vein endothelial cells (HUVEC) were isolated and cultured from collagenase-digested human umbilical veins and subcultured in modified Eagle medium and 20% fetal bovine serum. At passage 5, HUVEC were maintained in short-term culture as described above for SPAEC, and actin toxicity was determined. Mouse aortic smooth muscle cells (MASMC) were started from explants prepared from thoracic aorta and allowed to initially attach to gelatin-coated tissue culture flasks. Cells that migrated rapidly from the explant were removed from gelatin and expanded by passaging with trypsin and EDTA. MASMC were maintained in MEM with 20% fetal bovine serum and penicillin and streptomycin and used between passage 4 and 6.
Endothelial Cell Toxicity
Viability of SPAEC after exposure to human sera was determined by quantifying reduction of a fluorogenic indicator (Alamar blue; Alamar Biosciences, Sacramento, CA). SPAEC (1 × 103) were allowed to attach in 96-well tissue culture dishes and were exposed to 20% serum from normal volunteers and from patients with fulminant hepatic necrosis or ARDS (see below). At 24-h intervals, Alamar blue was added to the medium, and 3 h later fluorescence was determined in a cytofluorometer (Cytofluor II; PerSeptive Biosystems, Framingham, MA). It has previously been shown that oxidized Alamar blue is taken up by cells and is reduced by intracellular dehydrogenases, and the water-soluble changes in fluorescence emission (590 nm) are utilized as an index of viability (9, 10).
In a separate group of SPAEC, 4 h prior to daily (24- to 72-h) measurements of cell viability, 2 µCi/ml of [3H]thymidine ([3H]TdR: [6- 3H]thymidine, 6.7 Ci/mmol; New England Nuclear, Boston, MA) were added to the medium. DNA synthesis was quantified by measuring the uptake of [3H]TdR into trichloroacetic acid precipitable fraction.
Structural correlates of endothelial cell injury were determined at transmission electron microscopic level. SPAEC were cultured on 6-well dishes and exposed for 72 h to either normal serum or normal serum and actin, with or without gelsolin, and were then fixed in 2% paraformaldehyde/0.1% glutaraldehyde for 60 min, preserved in 30% sucrose, and then subjected to electron microscopic examination. Similar experiments were performed with serum from patients with ARDS.
Identification of Actin in Plasma Samples
Relative amounts of actin present in serum from either control subjects or patients with ARDS were determined by Western blot or rhodamine-phalloidin staining. Total protein was determined in serum sample by Bio-Rad reagent (Bio-Rad Laboratories, Hercules, CA). Twenty µg/ ml of protein was loaded with Laemmli buffer into each well of a 12% precast TRIS-glycine minigel (Bio-Rad). Electrophoresis was performed at 120 V. Protein was transferred to nitrocellulose membrane (Optitran BA-S 83; Schleicher and Schuell, Keene, NH) and blocked with 5% nonfat dried milk in phosphate-buffered saline. The blot was incubated with 1:1,000 mouse antiactin monoclonal antibody (Chemicon International Inc., Temecula, CA) in 1% NFDM in PBS for 2 h. After rinsing in NFDM/PBS/0.05% Tween 20, the blot was incubated in 1:5,000 goat antimouse horseradish peroxidase conjugated with secondary antibody for 1 h. The blot was rinsed in this latter buffer and exposed to enhanced chemiluminescence reagent (New England Nuclear Life Science Products, Boston, MA). A photographic film (Kodak MR) was exposed for 5 min to reveal signal.
The presence of polymerized actin in normal serum samples treated with actin and gelsolin, as well as serum from patients with ARDS (with and without exogenous gelsolin), was determined by rhodamine-phalloidin staining. Briefly, 100 µl of rhodamine-phalloidin complex in phosphate-buffered saline (PBS) were added to dried serum sediment on a glass slide and washed after 30 min with PBS, and F-actin filaments were visualized by fluorescence microscopy.
Experimental Protocols
Effect of serum and exogenous actin on endothelial cell viability. SPAEC were seeded at initial concentrations of 103 cells per well in 96-well microplasty reader plates (Corning Glass Works, Corning, NY) in media consisting of low glucose DME, L-glutamine, penicillin G, and streptomycin with 20% serum from: (1) healthy volunteers, (2) patients with fulminant hepatic necrosis, or (3) 20% fetal bovine serum. Rabbit skeletal muscle G-actin (Sigma Chemical) was prepared in G-actin buffer (0.01 M TRIS/0.2 mM CaCl2/0.5 mM sodium ATP at pH 7.5) and added to samples from normal volunteers at a final concentration of 500 µg/ml with or without gelsolin (120 µg/ml). In a subset of normal volunteer sera, the effect of G-actin buffer alone was assessed. Triplicate aliquots of the above samples were analyzed at 24, 48, and 72 h for cell viability (Alamar blue or [3H]TdR uptake). These studies were repeated in sera from eight normal volunteers, and each data point represents the mean of a triplicate determination. In addition, SPAEC, HUVEC, or MASMC were exposed to actin (0 to 500 µg/ml) for 4 h and viability assessed by Alamar blue, including sensitivity of toxicity to gelsolin (280 µg/ml).
Effect of serum from patients with ARDS on endothelial cell viability. Viability of SPAEC was assessed after exposure to 20% serum obtained from patients with ARDS (n = 8). Cell viability was quantified from reduction of Alamar blue, DNA synthesis from [3H]TdR incorporation, and electron microscopy. Two maneuvers were used to affect ARDS serum-induced changes in SPAEC viability: (1) heat-inactivation of serum components by exposing serum to 56° C for 30 min, and (2) adding gelsolin (120 µg/ml) at the time of exposure.
Statistical Analysis
All values are expressed as mean ± SE. The effect of time of exposure to various serum samples was determined by one-way analysis of variance. Comparison of the effect of serum from patients with fulminant hepatic necrosis of ARDS with normal subjects was performed by one-way ANOVA and multiple means compared by Newman-Keuls post-hoc analysis. The effect of actin, actin and gelsolin, gelsolin, or heat inactivation was studied at each time point within respective protocols by one-way ANOVA. Significance was established at p < 0.05 (11).
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Effect of G-actin on Endothelial Cell Viability
There was a time-dependent increase in the reduction of a redox-sensitive substrate for mitochondrial respiration (Figure 2) and incorporation of [3H]TdR into DNA (Figure 3) after exposure of SPAEC to medium containing 20% serum from normal volunteers over a 3-d period. There was no significant difference in either mitochondrial respiration (Figure 2) or DNA synthesis (Figure 3) over 72 h when medium contained 20% serum from patients with fulminant hepatic necrosis. Similar time-dependent increases in reduction of Alamar blue or DNA synthesis (data not shown) were noted in SPAEC exposed to 20% fetal bovine serum alone. Thus, SPAEC continued to proliferate upon exposure to either normal serum or serum from patients with fulminant hepatic necrosis. Addition of G-actin (500 µg/ml) to serum from healthy volunteers, however, significantly decreased reduction of Alamar blue (Figure 2) or incorporation of [3H]TdR (Figure 3). Addition of gelsolin (120 µg/ ml) along with G-actin largely reversed the effect (Figures 2 and 3), suggesting that the toxicity of G-actin was probably secondary to its polymerization to F-actin in the medium. Specifically, addition of gelsolin to normal serum in the presence of G-actin resulted in viability that was significantly (p < 0.01) greater than serum with actin alone but was still significantly less viable than control (Figures 2 and 3). There was no significant effect of the buffer in which G-actin was prepared.
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The specificity of the acute (within 4 h) effect of exogenous G-actin was assessed by comparing G-actin-induced changes in Alamar blue reduction of SPAEC to changes noted in cultured human umbilical vein endothelial cells (HUVEC) or cultured murine aortic smooth muscle cells (MASMC). G-actin (10 to 500 µg/ml) was added to serum-free medium in the above three cell types, and their ability to reduce Alamar blue was quantified as described. G-actin caused a concentration-dependent decrease in the reduction of Alamar blue in all three cell types (Figure 4). There was a similar gelsolin (280 µg/ml) sensitive component of G-actin (500 µg/ml) toxicity in each of the cell types studied (data not shown).
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Effect of Sera from Patients with ARDS on Endothelial Cell Viability
In contrast to lack of effect of serum for the above two patient groups, addition of 20% serum from patients with ARDS profoundly decreased the ability of SPAEC to reduce Alamar blue (Figure 5) or incorporate [3H]TdR into their DNA (Figure 6) from 24 to 72 h. There was no reversibility of this effect of 72 h. Heating serum from patients with ARDS to 56° C, however, significantly reduced toxicity at each time point studied. The same concentration of gelsolin (120 µg/ml) that restored G-actin-exposed SPAEC viability to approximately 65 to 75% of control values at 72 h, only partly reversed the toxicity of patients with ARDS (Figures 5 and 6). Addition of gelsolin resulted in an approximately doubling of Alamar blue reduction and [3H]TdR incorporation towards normal serum values. Thus a small but significant portion of direct endothelial cell injury from serum of patients with ARDS is sensitive to gelsolin.
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We performed immunoblot and fluorescence assays to determine whether serum from patients with ARDS contained elevated levels of actin. There was no detectable immunoreactive actin in serum of two normal subjects (Figure 7, lanes 3 and 4). In contrast, immunoreactive actin was readily apparent in serum from three patients with ARDS (Figure 7, lanes 5, 6, and 7) and comigrated with chemiluminescence signal apparent after addition of G-actin to serum of normal subjects (Figure 7, lanes 1 and 2). Confirmation of the presence of F-actin in serum of patients with ARDS is shown in the example of histofluorescence in Figure 8. Intense fluorescence was apparent in serum from the patient with ARDS after labeling with rhodamine-phalloidin (Figure 8E); this fluorescence was sensitive to addition of gelsolin (Figure 8F) and was not detectable in serum from a healthy volunteer (Figure 8A). As a positive control, we examined gelsolin-sensitive fluorescence (Figure 8D) of serum from a healthy volunteer in which exogenous actin was added (Figure 8C). Serum from patients with FHF (Figure 8B) had considerably less fluorescence after labeling with rhodamine-phalloidin than serum from patients with ARDS.
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Structural Changes in SPAEC Exposed to Sera from Healthy Volunteers or Patients with ARDS
Electron microscopy revealed normal ultrastructure of SPAEC exposed to serum from either healthy volunteers (Figure 9A) or patients with FHF (Figure 9B). Addition of exogenous actin to this sample resulted in necrotic death of SPAEC (Figure 9C) that was inhibited by the addition of gelsolin along with actin (Figure 9D). Application of serum from patients with ARDS was highly toxic to SPAEC (Figure 9E). Addition of gelsolin to serum from patients with ARDS protected a significant portion of SPAEC from injury (see Figures 5 and 6), and this was manifested by the relatively normal ultrastructure of SPAEC that survived gelsolin-sensitive injury of ARDS serum (Figure 9F).
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It has been formally proposed that an imbalance in the synthesis and scavenging of intravascular actin (see Figure 1) may contribute to multiple-organ dysfunction syndrome (1). Gelsolin-actin complexes were detected in serum of patients with ARDS (3) as well as rats after oleic-acid-induced lung injury (6), supporting the possibility that actin may be released from tissues during acute lung injury. Haddad and colleagues (7) showed that infusion of G-actin to intact rats led to increased levels of intravascular filamentous actin, microthombi, and a somewhat lung-specific vascular angiopathy. A critical question in this latter study was whether actin caused direct pulmonary vascular endothelial cell injury or whether such injury was secondary to microemboli, platelet activation, or interaction with fibrin. In the current study, we provide evidence that addition of G-actin to plasma and its conversion to gelsolin-sensitive F-actin is directly toxic to cultured pulmonary endothelial cells (Figures 2-4 and 9). Furthermore, serum from patients with ARDS contains detectable levels of immunoreactive (Figure 7) and histochemically detectable (Figure 8) actin. Addition of such serum to SPAEC results in severe toxicity to these cultured cells (Figures 5 and 6). Although a relatively larger component of such toxicity can be ascribed to a heat-sensitive molecule (e.g., complement), a small but significant fraction of such toxicity is gelsolin-sensitive (Figures 5, 6, and 9).
Effect of G-actin on Endothelial Cell Viability
Addition of G-actin to plasma from normal volunteers resulted in endothelial cell toxicity as manifested by a decreased capacity to reduce a flurogenic substrate for mitochondrial respiration (Alamar blue; Figure 2) and decreased DNA synthesis (Figure 3). At this dose of G-actin (500 µg/ml) the pattern of cell injury appeared to be necrotic, and there was no indication morphologically of an apoptotic cell death (Figure 9). G-actin is presumed to be rapidly polymerized to F-actin in medium of physiologic ionic strength and pH and this assumption was supported by the sensitivity of G-actin toxicity to exogenous gelsolin (Figures 2, 3, and 9). Presumably endogenous levels of either gelsolin and/or Gc protein, were inadequate in themselves to scavenge this large dose of G-actin in normal serum. The mechanism by which filamentous actin is directly toxic to SPAEC remains unclear, but it occurred in the absence of platelets and fibrin. It does, however, provide support for previous work in which exogenous G-actin infused into rats in vivo resulted in pulmonary endothelial cell injury (7). There was no difference in the effect of 20% human serum versus 20% fetal calf serum (data not shown), alone, on SPAEC viability nor was there a significant effect of the buffer in which G-actin was dissolved. The gelsolin-sensitive effect of exogenous G-actin (10 to 500 µg/ml) in serum-free medium was observed in cells cultured from sheep pulmonary artery or human umbilical vein endothelium or murine aortic smooth muscle, suggesting that the effect was neither species-specific nor limited to a particular cell of the vascular wall (Figure 4).
Effect of Serum from Patients with ARDS on Endothelial Cell Function
In contrast to serum from normal patients, addition of serum from patients with ARDS led to profound decreases in endothelial cell viability (Figures 5, 6, and 9). The effect was relatively specific for this group of eight patients since serum from patients with fulminant hepatic necrosis was without significant effect on SPAEC (Figures 2 and 3). In this latter regard, none of the patients with fulminant hepatic necrosis experienced ARDS and none died. Although these patients with FHF had actin in serum complexed with Gc and probably gelsolin, they apparently did not exceed the capacity of the scavenger system. Patients had clinically documented ARDS according to current criteria (12) and all had bacterial pneumonia as either the underlying cause or the major contributing etiology. It remains to be determined if other causes of ARDS lead to stable mediators in serum that are toxic to cultured pulmonary endothelial cells. Furthermore, the precise nature of these toxic substances remains to be elucidated. Nonetheless the observation that a large and significant fraction of this material was sensitive to a 30-min exposure to 56° C is highly suggestive of a role for complement (13). As summarized by Pittet and colleagues (14), complement was one of the first mechanisms proposed to mediate acute lung injury (15), and elevations in plasma C5a correlate with severity of acidosis and shock (16). Although it is now quite clear that simple plasma determinations of C5a or other more stable complement products is of little prognostic use in ARDS (14), it is likely that one or more members of the complement family accounted for the heat-sensitive component of toxicity of ARDS serum in our experiments. Relevant to the current study is that a small but significant gelsolin-sensitive component of endothelial cell toxicity from ARDS serum existed (Figures 5, 6, and 9). Addition of gelsolin at the time of exposure of SPAEC to ARDS serum led to a nearly doubling of the surviving population. Indeed, those cells that were rescued by the addition of gelsolin had a remarkably normal appearance at electron microscopy (Figure 9).
All eight members of the group with ARDS had detectable levels of immunoreactive actin (see two examples presented in Figure 7) as well as rhodamine-phalloidin-detectable actin in their sera (Figure 8). We did not measure either gelsolin or Gc-protein in their serum, but it appears that the apparent imbalance depicted in Figure 1 may have been present in their intravascular space. The net effect on circulating actin was sufficient to increase actin levels to a toxic level for SPAEC that was only modestly inhibited by complementation of gelsolin to normal serum levels. It is possible that higher levels of gelsolin with or without addition of Gc-protein are required to further inhibit ARDS serum-induced toxicity to SPAEC. Lack of toxicity in patients with fulminant hepatic necrosis (Figure 9B) underscores the specificity of the effect of ARDS, especially since these former patients may have been expected to have decreased activity of actin-scavenging system (2). Thus it is apparent that a significant imbalance between production and scavenging of actin is required to result in serum that contains toxic gelsolin-sensitive actin polymers.
In summary, this study has shown that serum sampled from patients with ARDS within 24 h secondary to bacterial pneumonia is highly toxic to cultured sheep pulmonary artery endothelial cells. A significant portion of the toxicity is heat-sensitive consistent with a major contributory role of complement products. Alternatively, a small but significant component of this toxicity was gelsolin-sensitive consistent with elevated levels of immunoreactive actin and rhodamine-phalloidin-positive material in the serum of these patients. Although the mechanism by which actin is toxic to endothelial cells remains undetermined, addition of exogenous actin causes direct and profound toxicity that is gelsolin-sensitive.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. Bruce Pitt, Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. E-mail: Brucep{at}pitt.edu
(Received in original form June 15, 1998 and in revised form November 24, 1999).
Acknowledgments: The writers are grateful to Dr. William Calhoun (University of Pittsburgh) for his suggestions during preparation of the manuscript.
Supported by Grants HL-32154, PO1 GM-53789, and KO8 HL-03018, and by Postdoctoral Training Grants HL-07563 and HL-09627 from the National Institutes of Health.
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References |
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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
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1. Lee, W. M., and R. M. Galbraith. 1992. The extracellular actin-scavenger system and actin toxicity. N. Engl. J. Med. 326: 1335-1341 [Medline].
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M. J. TOBIN Critical Care Medicine in AJRCCM 2000 Am. J. Respir. Crit. Care Med., October 15, 2001; 164(8): 1347 - 1361. [Full Text] [PDF]
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 Z.-L. Tang, K. Wasserloos, C. M. St. Croix, and B. R. Pitt Role of zinc in pulmonary endothelial cell response to oxidative stress Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L243 - L249. [Abstract] [Full Text] [PDF]
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