Therapies to limit the life-threatening vascular leak observed in patients with acute lung injury (ALI) are currently lacking. We explored the effect of simvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor that mediates endothelial cell barrier protection in vitro, in a murine inflammatory model of ALI. C57BL/6J mice were treated with simvastatin (5 or 20 mg/kg body wt via intraperitoneal injection) 24 h before and again concomitantly with intratracheally administered LPS (2 μg/g body wt). Inflammatory indexes [bronchoalveolar lavage (BAL) myeloperoxidase activity and total neutrophil counts assessed at 24 h with histological confirmation] were markedly increased after LPS alone but significantly reduced in mice that also received simvastatin (20 mg/kg; ∼35–60% reduction). Simvastatin also decreased BAL albumin (∼50% reduction) and Evans blue albumin dye extravasation into lung tissue (100%) consistent with barrier protection. Finally, the sustained nature of simvastatin-mediated lung protection was assessed by analysis of simvastatin-induced gene expression (Affymetrix platform). LPS-mediated lung gene expression was significantly modulated by simvastatin within a number of gene ontologies (e.g., inflammation and immune response, NF-κB regulation) and with respect to individual genes implicated in the development or severity of ALI (e.g., IL-6, Toll-like receptor 4). Together, these findings confirm significant protection by simvastatin on LPS-induced lung vascular leak and inflammation and implicate a potential role for statins in the management of ALI.
- acute lung injury
derangements in lung vascular permeability are a cardinal feature of inflammation and the initial cause of the profound physiological abnormalities observed in patients with acute lung injury (ALI), a common yet difficult clinical problem associated with significant morbidity and mortality (26). Although accurate statistics are elusive, the incidence of ALI in the United States has been estimated at up to 100,000 cases per year with a mortality rate of ∼35–40% (33). Unfortunately, despite the enormity of the problem, effective therapies to ameliorate the vascular leak associated with ALI are currently not available.
The mechanisms relevant to the maintenance of lung fluid balance and vascular barrier regulation are complex. The pulmonary microvasculature regulates solute transport between vascular compartments and surrounding tissues with the vascular endothelium functioning as a semipermeable cellular barrier that is dynamically regulated by the actin cytoskeleton (10). Cytoskeletal rearrangement, a mandatory event for dynamic barrier regulation, is mediated by the coordinate activity of myosin light chain kinase and the small GTPase Rho, which regulate levels of myosin light chain phosphorylation, drive actin stress fiber formation, and ultimately lead to increased cell contraction, the development of paracellular gaps, and increased permeability (10). Conversely, another small GTPase, Rac, is an important determinant of endothelial cell barrier protection (5, 11, 35) via its critical involvement in cortical actin polymerization and enhanced cell-cell adhesion. However, efforts to precisely characterize these events and identify potential mediators are currently incomplete.
Our research efforts have focused on identifying potential effectors of vascular barrier restoration with several candidates proposed recently (13, 24, 31). We recently reported the potential for the class of drugs known as the statins (e.g., simvastatin) to serve as in vitro endothelial cell barrier-regulatory agents. Use of statins for their lipid-lowering properties and beneficial effects on the morbidity and mortality associated with coronary artery disease is now firmly established. However, it is also now abundantly clear that the beneficial effects of the statins in a variety of settings including cancer metastases, Alzheimer's severity, and stroke area, cannot be attributed to reductions in serum cholesterol levels alone (1, 12, 22, 34). In addition to these pleiotropic effects, we recently demonstrated simvastatin confers endothelial cell barrier protection in vitro with significant reductions in thrombin-mediated barrier disruption (19). These findings were tightly linked to dramatic cytoskeletal protein rearrangement driven by Rac GTPase with significant induced expression of genes specifically involved in cell signaling and cytoskeletal regulation (19). As these observations implicated the statins as a potentially novel therapeutic tool in ALI, we sought to extrapolate this work to a relevant clinical setting and have now examined the effect of simvastatin in an established animal model of inflammatory lung injury. We observed marked attenuation of vascular leak and inflammation by simvastatin in a murine model of endotoxin-induced ALI in association with identifiable histological protection. Furthermore, whole lung tissue microarray analysis revealed clear differential expression of genes involved in several gene ontologies relevant to ALI (inflammation and immune response, transcription, cell signaling). Together, these studies strongly implicate a potential role for statins in the management of patients with ALI.
MATERIALS AND METHODS
Materials and reagents.
All reagents were purchased from Sigma (St. Louis, MO) unless otherwise specified. C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME).
Animal care and treatment.
All experiments were approved by the Johns Hopkins Animal Care and Use Committee. Male C57BL/6J mice (8–10 wk old) were administered intraperitoneal (IP) simvastatin (5 or 20 mg/kg) or vehicle 24 h before and again at the time of endotoxin administration to induce lung injury. Mice were anesthetized with IP ketamine (150 mg/kg) and acetylpromazine (15 mg/kg), and intratracheal LPS (2 μg/g) or vehicle (nuclease-free water) was administered via a 20-gauge catheter as we have previously described (31). Animals were allowed to recover and were observed for 24 h at which time an assessment of injury was obtained as detailed below. Anesthesia was again administered before the delivery of Evans blue albumin dye (EBA), and animals were then maintained under anesthesia for the duration of the experiment.
Measurement of bronchoalveolar lavage protein, EBA concentration, and cell counts.
EBA (20 mg/kg) was injected into the internal jugular vein 30 min before termination of the experiment to assess vascular leak as we previously described (30). After thoracotomy, mice were exsanguinated via abdominal aorta transection. The pulmonary artery was cannulated, the left atrial appendage was excised, and 0.5–0.75 ml of PBS containing 5 mM EDTA were perfused through the pulmonary circulation to remove blood-borne elements and plasma. Right lungs were then tied off, and the left lung was lavaged by intratracheal injection of three sequential 0.3-ml aliquots of Hanks' balanced salt solution, followed by aspiration. Recovered fluid was pooled and centrifuged. Supernatants were preserved for measurement of total albumin concentration using an enzyme-linked immunoassay (Bethyl Labs, Montgomery, TX). The leukocyte pellet was resuspended in extraction buffer [50 mM potassium phosphate buffer containing 0.5% hexadecyl trimethylammonium bromide (HTAB)] as we have recently described (31). Half of this volume was frozen for subsequent analysis of myeloperoxidase (MPO) activity. In the remaining volume, red blood cells were lysed with ACK lysing buffer (Biosource International, Camarillo, CA), and samples were then processed for cell count with differential. Results were adjusted for total lung weight. Separately, right lungs were excised en bloc, blotted dry, weighed, and snap-frozen in liquid nitrogen. Lungs were then homogenized in PBS (1 ml/100 μg of tissue), incubated with 2 vol formamide (18 h, 60°C), and centrifuged at 5,000 g for 30 min, and the optical density of the supernatant was determined spectrophotometrically at 620 nm. Extravasated EBA concentration in lung homogenate was calculated against a standard curve (μg EBA per lung) (15).
Bronchoalveolar lavage (BAL) MPO activity, an indicator of neutrophil extravasation, was measured by resuspension of BAL cell pellets as described above. Suspensions were then subjected to three cycles of freezing in liquid nitrogen and thawing in a cold water bath followed by centrifugation at 13,000 g for 15 min at 4°C. Supernatants were assayed for MPO activity by kinetic readings over 30 s (20-μl sample with 180-μl reaction buffer containing potassium phosphate buffer, 0.5% HTAB, 0.167 mg/ml O-dianisidine dihydrochloride, and 0.0006% H2O2). The rate of change in absorbance was measured at 405 nm on a Vmax kinetic microplate reader (Molecular Devices, Sunnyvale, CA) with the results adjusted for total lung weight and presented as MPO units/lung.
To characterize the histological alterations immediately after death, right lungs from two animals in each experimental group were inflated to 30 cmH2O with 0.2% of low-melting agarose for histological evaluation by hematoxylin and eosin staining as we have previously described (31).
Data comply with the Minimum Information About Microarray Experiments (MIAME) standard (6) and may be accessed via the National Center for Biotechnology Information's GEO database (http://www.ncbi.nlm.nih.gov/geo, accession nos. GSM29706, GSM29707, GSM29708, GSM29709, GSM29710, GSM29711, and GSM29712).
Synthesis of cDNA and hybridization probes.
Murine lung tissue was homogenized in 2 ml of TRIzol (Life Technologies, Frederick, MD). The samples were separated into two tubes (1 ml/tube) and centrifuged for 1 min at 15,000 g. The supernatant was collected, 0.2 ml of chloroform were added, and samples were briefly centrifuged at 4°C. Supernatant containing RNA was preserved, and 0.5 ml of isopropanol were added, followed by repeat centrifugation. The supernatant was again preserved, and 100 μl of diethyl pyrocarbonate-treated water and 200 μl of cold ethanol were added. Before cDNA synthesis, RNA was purified using RNeasy Mini Kit (Qiagen, Valencia, CA) and quantified by spectrophotometric analysis. Double-stranded cDNA was synthesized from total RNA, and biotin-labeled cRNA was prepared as previously described (25).
Expression profiling was performed (Affymetrix GeneChip system) at the Gene Expression Profiling Core of the Johns Hopkins University Center for Translational Respiratory Medicine as previously described (8). Samples hybridized (45°C, 16 h) to an Affymetrix MG-430 version 2.0 Array (∼45,000 transcripts representing ∼34,000 genes) and were stained with streptavidin phycoerythrin conjugate. Fluorescence signal intensity subsequent to hybridization was read using the Agilent Gene Array Scanner and converted into GeneChip Cell files (CEL) using GCOS 1.1.1 software (Affymetrix, Santa Clara, CA). The analysis of the probe level data (available in the .CEL files) was performed using the Bioconductor affy package (18). This package was used for extraction of probe level data and conversion to expression levels of individual probe pairs using background correction, across array normalization, and summarization. Poorly performing probe pairs identified by this analysis were masked, and arrays were reanalyzed by GCOS 1.1.1. Absent calls for each GeneChip were then replaced with the chip background value as we have previously described (16). The modified data set was analyzed using a two-tailed unequal variance independent t-test (3 controls vs. 3 corresponding experimental conditions), and change in the gene expression with P < 0.05 was determined. The fold-change ratio was computed from the mean values produced by t-test for corresponding comparisons.
For gene ontology (GO) analysis, MAPPFinder 2.0 beta software was employed. Probe sets (45038 excluding internal controls) were linked to GO terms using expression change >20% (1.2-fold change) and P < 0.05 value as filtering cutoffs. Resulting GO biological processes were filtered by z-score (>2) and the number of hits in the first GO node (>5). Analysis followed a progressive filtering algorithm as we have recently described for expression profiling data and GO analysis (16).
Two-way ANOVA was used to compare the means of data from two or more different experimental groups. If significant difference was present by ANOVA (P < 0.05), a least significant differences test was performed post hoc. Subsequently, differences between groups were considered statistically significant when P < 0.05. Results are expressed as means ± SE.
Effect of simvastatin on LPS-induced murine lung inflammation.
Anesthetized mice were exposed to simvastatin (5 or 20 mg/kg IP) or to vehicle 24 h before and again concomitant with intratracheal LPS (2 μg/g) or vehicle. After a 24-h exposure to LPS, animals were immediately killed, and left lungs were lavaged. BAL fluid was used for assay of MPO activity and total neutrophil count as independent assessments of alveolar inflammation. In LPS-treated mice, a significant reduction in MPO (34%, P < 0.05) was achieved by pretreatment with 20 mg/kg of simvastatin relative to vehicle (Fig. 1). Similarly, whereas a significant effect was not observed with 5 mg/kg of simvastatin (no appreciable reduction), total lavage neutrophil counts were reduced by ∼60% in LPS-treated mice that received 20 mg/kg of simvastatin relative to vehicle (P < 0.05). In addition, we observed a trend toward decreased lavage MPO activity and total neutrophils with simvastatin treatment in mice that did not receive LPS relative to vehicle controls (independently of LPS).
Lung histology analysis 24 h after the administration of LPS (2 μg/g) or vehicle revealed evidence of a dramatic increase in lung neutrophils in response to LPS (Fig. 2), results consistent with the BAL findings noted above. Simvastatin (20 mg/kg) significantly attenuated LPS-induced inflammation with a decreased prevalence of lung neutrophils in tissue, achieving a level of inflammation comparable to that observed in uninjured lungs. Of note, simvastatin alone had no appreciable effect on lung histology.
Effect of simvastatin on LPS-induced lung vascular leak.
We recently reported that simvastatin confers endothelial cell barrier protection in vitro (19). Accordingly, we hypothesized that LPS-induced changes in murine lung vascular leak would be attenuated by simvastatin treatment as measured by both lavage albumin and lung EBA extravasation from the vascular space into surrounding lung tissue. In all groups, mice receiving LPS demonstrated significantly higher concentrations of lavage albumin than vehicle-treated mice (Fig. 3A), findings that were commensurate with lung injury. LPS-treated mice receiving pretreatment with 5 mg/kg of simvastatin did not exhibit any appreciable effect on BAL albumin, whereas a 50% decrease in lavage albumin was observed with 20 mg/kg of simvastatin relative to vehicle. Interestingly, in companion experiments, we observed lung EBA leakage to be significantly attenuated in LPS-challenged mice receiving either 5 or 20 mg/kg of simvastatin treatment after the results in each LPS group were normalized to their respective controls performed in the same experiment to adjust for assay variability (Fig. 3B). This response appeared to be concentration dependent as lung EBA extravasation was reduced by 80% in injured mice receiving 5 mg/kg of simvastatin relative to controls, whereas EBA extravasation was completely abolished in mice treated with 20 mg/kg of simvastatin.
Differential lung gene expression by simvastatin in LPS-induced ALI.
We have recently noted that the in vitro effects of simvastatin are prolonged and associated with dramatic alterations in endothelial cell gene expression (19). To evaluate the sustained nature of the simvastatin-mediated lung protection, the effect of simvastatin on LPS-mediated lung gene expression was assessed utilizing the Affymetrix microarray platform and RNA extracted from whole lung. Subsequently, we employed MAPPFinder (9), a tool that incorporates annotations of the GO project with GenMAPP (2), a free software package that allows microarray data to be analyzed by microarray pathway profiles (MAPPs).
Relative to vehicle controls, the combination of simvastatin pretreatment (20 mg/kg) followed by LPS challenge resulted in the identification of >400 genes with significantly altered expression (>2-fold change) that were not significantly affected by LPS alone at 24 h. Furthermore, comparing gene expression data from the simvastatin and LPS group to that from the LPS alone, we identified specific ontologies in which genes were differentially regulated (inflammation and immune response, NF-κB regulation, chemotaxis, cell adhesion) (Fig. 4). Notably, within these ontologies, simvastatin significantly altered LPS-induced expression of several individual genes previously implicated in the development of ALI (e.g., IL-6, Toll-like receptor 4) (Table 1).
ALI is characterized clinically by excessive vascular permeability, and inflammation and is associated with significant morbidity and mortality. Unfortunately, effective therapies for ALI are currently not available, but the identification of potential molecular targets involved in vascular barrier regulation provides an attractive approach. We have recently reported that simvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor, confers endothelial cell barrier protection in vitro via prominent cytoskeletal activation and rearrangement. In addition, these studies revealed a potent effect of simvastatin on human endothelial gene expression. These in vitro findings led us to investigate the potential role of this drug in attenuating vascular leak in an established animal model of ALI. We now report that simvastatin significantly decreased LPS-induced murine lung vascular leak and inflammation in association with differential lung gene expression.
As an initial course of investigation, we chose to administer simvastatin before LPS to establish a proof of principle. The combination of simvastatin pretreatment (24 h) and concomitant treatment resulted in significant lung vascular barrier protection. Although a strict extrapolation of our model to the clinical setting may not be realistic given the difficulty of accurately predicting the development of ALI to allow for early simvastatin treatment, our findings do have significant clinical relevance given the natural course of this disease. Specifically, early intervention to attenuate lung injury could have a marked impact on outcome, even if initiated after the onset of injury, since a prolonged clinical course is not uncommon in ALI, and patients who fare poorly often progress to acute respiratory distress syndrome over the course of several days (7).
The concentrations of simvastatin utilized in our studies warrant comment as these doses are obviously excessive compared with human dosing regimens. Truncated analysis of dose-dependent anti-inflammatory effects of simvastatin in our studies revealed minimal effects with 5 mg/kg of simvastatin but substantial protection with 20 mg/kg. While 20-mg/kg concentrations in the murine model do exceed clinically relevant levels of statins in humans, in mice the rapid upregulation of HMG-CoA reductase in response to statins necessitates the use of significantly higher concentrations (21). Notably, simvastatin doses as high as 40 mg/kg have been delivered to mice via IP injection without evidence of adverse consequences (23). However, a careful consideration of appropriate simvastatin doses is certainly indicated before extending our findings to humans or other animal models.
The attenuating effects of simvastatin on LPS-induced murine lung inflammation and vascular leak were striking. Indeed, in our murine model of ALI, the cumulative effects of simvastatin on the vascular barrier could account for both a decrease in vascular leak as well as a reduction in inflammation characterized by reduced lung neutrophil influx as a result of this tightened vascular barrier. Although the pleiotropic properties of the statins may suggest a multifactorial mechanism, our previous work strongly implicates a significant role for the effects of simvastatin on endothelial cell barrier protection via decreased geranylgeranylation and subsequent cytoskeletal rearrangement. As is well recognized, the statins inhibit prenylation, a covalent modification involving the addition of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) side chains. One product of the prenylation pathway is cholesterol; however, activation of the Rho family GTPases is also dependent on this modification. These small GTPases act as molecular switches controlling various cellular events by cycling between GTP-bound (active) and GDP-bound (inactive) states. GTPase activation is strongly reliant on geranylgeranylation and subsequent translocation to the cell membrane. Whereas several small GTPases have been identified, three in particular, Rho, Rac, and Cdc42, have been implicated in cytoskeletal regulation (27, 32). We previously demonstrated that inhibition of Rho by statins attenuates Rho kinase activation and subsequent actomyosin contraction. This precludes a cascade of subsequent events characterized by stress fiber formation, increased intracellular tension, intercellular gap formation, and, finally, vascular barrier dysfunction (10). Moreover, we also reported activation of Rac by simvastatin, a small GTPase implicated in peripheral actin polymerization. This finding, although contradictory to the predicted downstream effects of HMG-CoA reductase inhibition, is nonetheless consistent with the observed EC barrier protection that we have previously noted with other barrier protective strategies that are also dependent on Rac GTPase signaling (5, 11, 19, 24).
Although our measures of the effects of simvastatin on LPS-induced vascular leak and lung inflammation produced largely congruous data, we did appreciate a discrepancy with respect to the effects of low-dose simvastatin (5 mg/kg) on BAL protein (no effect) and EBA tissue content (80% reduction). In contrast, we observed significant attenuation of both indexes with high-dose simvastatin (20 mg/kg). One possible explanation for the disparity in our results with these assays is related to their very nature, since EBA serves an index of increased vascular leak during the final 30 min of LPS exposure, whereas BAL protein reflects leakage of protein into the alveolar space, across both the vascular and epithelial barriers, during the entire 24 h after LPS administration. In addition, the time courses of these two events are inherently different, as clearly protein must first translocate across the vasculature before it can cross the epithelium.
We were not able to acquire wet-to-dry weight ratios, as those lungs that were not lavaged were instead used for analysis of Evans blue dye content. As a surrogate, normalized unlavaged lung-to-body weight ratios were analyzed and found to be consistent with our protein and Evans blue data (data not shown). However, we were unable to measure pulmonary vascular pressures and cannot exclude significant differences between groups in this respect. Nonetheless, the literature is now replete with evidence of the vasodilatory effects of statins (17, 28), and we cannot therefore exclude the possibility that the protective effects of simvastatin observed in our model are attributable to a combined effect on both vascular permeability and hemodynamics.
To extend our prior findings describing profound alterations in human lung endothelial cell gene expression by simvastatin, we evaluated murine lung tissue gene expression. Our results now suggest significant effects of simvastatin on LPS-induced murine lung gene expression. We analyzed our data by examining GO, a bioinformatic approach allowing for a global assessment of simvastatin effects on gene expression according to specific cellular pathways and processes. This analysis revealed simvastatin attenuated LPS-induced gene expression associated with both immune and inflammatory responses (e.g., IL-6, macrophage-derived chemoattractant), consistent with the observed protective effects of simvastatin, and affected both chemotaxis and cell adhesion genes, consistent with our prior in vitro analysis (19) as well as chemotaxis. We also noted differential regulation of NF-κB mediators, which is of considerable relevance given the importance of the NF-κB pathway in the pathogenesis of ALI via effects on immune and inflammatory responses and apoptosis (36). With respect to individual genes, we found LPS-induced IL-6 expression to be significantly downregulated by simvastatin. This finding is notable given the extensive literature implicating IL-6 as an important mediator of ALI (29, 37). Furthermore, these results are consistent with ours reported in in vitro studies where we found IL-6 to be markedly downregulated by both macro- and microvascular endothelial cells in response to simvastatin (unpublished data). In addition, macrophage-derived chemoattractant, a chemokine and potent inflammatory mediator via chemotactic effects on dendritic cells, natural killer cells, and monocytes, was significantly downregulated by simvastatin as well (14). Finally, Toll-like receptor 4 gene expression was also downregulated by simvastatin. As a recognized mediator of ALI and the LPS receptor responsible for subsequent signal transduction, the relevance of this finding with respect to the protective effects of simvastatin on LPS-induced injury is evident (3, 4). Although these findings are important by themselves, the mechanisms by which simvastatin is able to alter expression of these genes is unknown and warrants further investigation.
Although these results are clearly relevant to the sustained protective effect of simvastatin on inflammatory lung injury, it should be noted that we used a fold change threshold of 1.2 (P < 0.05) to identify genes that were differentially expressed. Although somewhat generous, this threshold is particularly suited for the identification of genes with elevated basal expression levels as upregulation of these genes may not produce a high fold change ratio and therefore would not otherwise be identified (20). Importantly, any potential increase in false positives using this approach should be consistent across ontologies and, therefore, relative effects on genes by ontology will not be affected.
In summary, we have now provided in vivo information that validates the barrier protective effects of simvastatin observed in vitro. Although additional research is warranted, particularly with respect to appropriate human dosages, routes of administration, and the effects of simvastatin treatment after injury has been sustained, recent recognition of the degree to which increased vascular permeability is central to the clinical manifestations of ALI has heightened the importance of relevant research in this area, specifically with respect to the identification of novel and highly precise therapeutic strategies. In this regard, our investigation of the potential role of simvastatin in the attenuation of vascular leak and inflammation in a murine model of ALI holds the promise of profound clinical significance, especially given the wide availability, affordability, and relatively favorable safety profile of this class of drugs.
This work acknowledges the support of the Center for Translational Respiratory Medicine, National Heart, Lung, and Blood Institute Grants HL-71411, HL-58064, HL-69340, and HL-66583, and the Dr. David Marine Endowment.
Special thanks to Shui Ye, Perry Iannaconi, Jr., and Denise Guise for invaluable efforts and assistance.
↵* J. R. Jacobson and J. W. Barnard contributed equally to this work.
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.
- Copyright © 2005 the American Physiological Society