Journal of Investigative Medicine:
EB Symposium Manuscripts
Pharmacologic Therapies on the Horizon for Acute Lung Injury/Acute Respiratory Distress Syndrome
Jacobson, Jeffrey R. MD
From the Section of Pulmonary and Critical Care Medicine, Pritzker School of Medicine, University of Chicago, Chicago, IL.
Received July 30, 2009, and in revised form September 3, 2009.
Accepted for publication September 3, 2009.
Reprints: Jeffrey R. Jacobson, MD, Section of Pulmonary and Critical Care Medicine, Pritzker School of Medicine, University of Chicago, Chicago, IL. E-mail: email@example.com.
This work acknowledges support from the Parker B. Francis Foundation, and the symposium was supported in part by a grant from the National Center for Research Resources (R13 RR023236).
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a spectrum of diseases that are commonly encountered in the intensive care unit and are associated with high mortality. Although significant advances have been made with respect to the ventilatory management of patients with ALI/ARDS with proven beneficial effects on outcomes, pharmacologic therapies remain nonexistent. Because the cardinal feature of ALI/ARDS is an increase in lung vascular permeability, often precipitated by an exuberant inflammatory response with subsequent endothelial barrier disruption, strategies aimed at promoting endothelial barrier function could serve as novel therapies in this setting. We have identified several promising agonists in this regard including sphingosine 1-phosphate, activated protein C, and statins, a class of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. These agonists all have in common the ability to directly mediate endothelial cell signaling and induce characteristic actin cytoskeletal rearrangement leading to endothelial cell barrier protection. Our in vitro findings have been extended to animal models of ALI/ARDS and suggest that effective pharmacologic therapies for patients with ALI/ARDS may soon be available.
Derangements in lung vascular permeability, particularly in the context of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), represent a common yet difficult clinical problem associated with significant morbidity and mortality.1 Effective therapies for the vascular leak associated with ALI/ARDS are currently not available. One known precipitant for increased lung leak is exposure to mechanical ventilation with excessive tidal volumes now recognized as potentially directly harmful in susceptible patients. The clinical presentation of ventilator-associated lung injury (VALI) is identical to that of other causes of ALI and is similarly characterized by increased lung inflammation and edema. However, the etiology and specific mechanisms involved in VALI have only recently come under investigation. Previously proposed mechanisms include increased hydrostatic pressure, direct damage to endothelial cells (EC) or stress failure, and more subtle effects on cell-signaling pathways that alter vascular permeability. Although animal models of ALI/ARDS exhibit increased hydrostatic pressure, the changes in transmural pressures measured in these models are too small to account for the degree of pulmonary edema frequently associated with VALI. Studies by Parker et al.,2 Webb and Tierney,3 and others4,5 demonstrate changes in microvascular permeability in isolated lung and intact animal models exposed to increased airway pressures. Thus, evidence has emerged that these changes in permeability may in large part be attributed to the effects of mechanical stimuli on various cell signaling pathways.6,7 The results from these studies led to new strategies in clinical practice regarding mechanical ventilation and the benefits of lower tidal volumes to achieve lower airway pressures, thus reducing lung cell stretch in patients with ALI/ARDS was subsequently established by the landmark ARDSNet findings.8 Nonetheless, increased vascular leak remains a serious, life-threatening clinical challenge in the setting of ALI/ARDS and novel treatments are needed.
ENDOTHELIAL CELL CYTOSKELETAL REARRANGEMENT AND VASCULAR PERMEABILITY
The EC actin cytoskeleton serves as a scaffold promoting cell integrity and cell-cell interactions but is also a determinant of intracellular tensile forces generated by the formation of transcellular stress fibers that drive cell contraction resulting in paracellular gaps and increased vascular permeability. Dynamic regulation of the actin cytoskeleton in response to various stimuli may result in an imbalance between these competing forces. Among the various signaling molecules involved in cytoskeletal regulation are the small guanosine triphosphatases (GTPases) RhoA and Rac1 as well as myosin light chain kinase (MLCK; for full review, see Dudek and Garcia9). RhoA activates Rho kinase, thereby inhibiting myosin light chain (MLC) phosphatase while the Ca++/calmodulin-dependent MLCK phosphorylates MLC at threonine 18 and serinine 19. The combined activities of Rho kinase and MLCK result in increased levels of MLC phosphorylation that drives actomyosin contraction and stress fiber formation. Conversely, activation of the small GTPase Rac1 is associated with lamelopodial formation and cortical actin polymerization. Accordingly, inhibition of either Rho kinase or MLCK or the activation of Rac1 may be associated with decreased agonist-induced actin stress fiber formation, thereby promoting EC barrier integrity and attenuating vascular permeability. In this regard, we have identified 3 agents of particular interest including sphingosine 1-phosphate (S1P), activated protein C (APC), and statins, which affect cytoskeletal dynamics (Fig. 1) and may serve as potentially novel therapeutic strategies for ALI/ARDS.
Sphingosine 1-phosphate is a platelet-derived phospholipid that directly activates EC via ligation of specific S1P receptors on the EC surface. We have characterized S1P as a potent enhancer of barrier function in vitro able to induce a marked increase in transmonolayer electrical resistance (TER) across EC monolayers.10 Sphingosine 1-phosphate-induced EC barrier enhancement occurs primarily as a result of phosphorylation of the S1P1 receptor by phosphatidylinositol 3-kinase (PI3-kinase)/Akt activation with subsequent signaling through G protein-dependent pathways. In addition, S1P significantly attenuates thrombin-induced barrier disruption and is able to rapidly restore barrier integrity when added subsequent to thrombin stimulation. Underlying these effects is S1P activation of Rac1 and translocation of the actin-binding protein cortactin.11 In addition, S1P drives MLCK translocation to the cell periphery and leads to increased MLC phosphorylation in a cortical distribution, events associated with dynamic actin cytoskeletal rearrangement characterized by enhanced cortical actin and decreased transcellular actin stress fibers. Moreover, these changes are associated with a reduction in paracellular gaps consistent with augmentation of EC barrier integrity.
We have also confirmed the protective effects of S1P in both murine and canine models of ALI. In our murine model, animals were administered intratracheal lipopolysacharide (LPS) to induce lung injury followed 1 hour later by intravenous S1P (1 μmol/L) or vehicle, and indices of lung vascular permeability and inflammation were then measured 24 hours later.12 Compared with animals that received LPS alone, S1P conferred significant protection in this model as lung neutrophils and myeloperoxidase activity were decreased as was brochoalveolar lavage (BAL) albumin content and Evans blue dye albumin extravasation into lung tissue. Moreover, lung histologic examination results demonstrated an attenuation of LPS-induced neutrophil infiltration and fewer areas of alveolar hemorrhage in mice treated with S1P. In our canine model, animals were administered intrabronchial LPS and then subjected to high tidal volume mechanical ventilation (17 mL/kg, 6 hours) to induce lung injury.13 These studies also confirmed the protective effects of S1P (85 μg/kg, intravenous) administered concomitantly with LPS as evidenced by an attenuation of BAL protein and the degree of shunt formation by venous admixture. In addition, computed tomographic scans of animals from each group revealed dramatically reduced lung edema associated with S1P treatment.
Activated Protein C
Protein C is present in the circulation as a proenzyme that is activated by the thrombomodulin-thrombin complex, an event that is augmented by ligation of the endothelial protein C receptor (EPCR) by protein C. Activated protein C then affects decreased thrombin generation via binding to protein S and the cleavage of the coagulation cofactors VIIIa and Va. Although the landmark PROWESS trial reported a survival benefit in patients with severe sepsis treated with APC that was thought to be due to its anticoagulant and anti-inflammatory properties,14 the mechanisms underlying these effects were not clearly established. This led us to investigate the possibility that APC is able to regulate EC barrier function directly.
Similar to S1P, APC induces dynamic rearrangement of the EC actin cytoskeleton characterized by augmentation of the cortical actin ring and decreased transcellular stress fibers.15 These changes are also associated with decreased paracellular gaps and improved monolayer integrity that corresponds to EC barrier protection in response to barrier-disruptive agonists such as thrombin as measured by TER. Activated protein C also induces activation of Rac1, an event that we have linked to its barrier-protective properties. We also confirmed a role for EPCR in EC barrier regulation by APC as both APC-mediated EC barrier protection as measured by TER and APC-induced MLC phosphorylation are attenuated by pretreatment with an EPCR blocking antibody. Moreover, we identified activation of the PI3-kinase/Akt with transactivation of S1P1 upon ligation of EPCR by APC. These findings support the notion that S1P and APC share significant mechanistic features with respect to their EC barrier protective properties, features that may account to a significant degree for the benefits of APC observed in patients with severe sepsis. Accordingly, studies examining the potential therapeutic role for APC in ALI/ARDS using relevant animal models are now underway.
The statins are a class of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors commonly used to lower serum cholesterol levels but are also known to have pleiotropic properties including direct effects on vascular function. These drugs inhibit cholesterol synthesis via inhibition of the prenylation pathway, a posttranslational modification of proteins that culminates in either farnesylation, the addition of a 15-carbon side chain, or geranylgeranylation, the addition of two 20-carbon side chains. Although a downstream product of farnesylation is cholesterol, members of the Rho family of small GTPases including Rho and Rac are dependent on geranylgeranylation for localization to the cell membrane and subsequent GTP loading that is required for their activation. Although statin inhibition of Rho and Rac activities at the cell membrane is well recognized, our laboratory and others have identified a paradoxical increase in GTP loading of cytosolic Rho GTPases of unclear functional significance.16,17 We found that simvastatin induces significant EC barrier protection as measured by both TER16 and fluorescein isothiocyanate-dextran monolayer flux,18 although the mechanisms underlying statin-mediated EC signaling are complex as evidenced by early (within 15 minutes) increased PI3-kinase/Akt signaling19 but late (12 hours) inhibition of this pathway.20 Nonetheless, statins inhibit EC Rho activation at the cell membrane that is associated with the characteristic actin cytoskeletal changes seen in response to other barrier protective agonists including S1P and APC as described previously. Statins also inhibit Rac1 at the cell membrane with consequent inhibition of the reduced form of the nicotinamide adenine dinucleotide phosphate oxidase complex assembly and an attenuation of agonist-induced reactive oxygen species generation, a separate mechanism of EC barrier protection.
Although inhibition of Rho GTPase geranylgeranylation is an early effect of statin treatment, we have also identified differential gene expression in EC after prolonged (24-hour) treatment with simvastatin.16 These include genes involved in cytoskeletal regulation such as caldesmon, an actin-binding protein, which is down-regulated (>2-fold decrease), and the up-regulation of thrombomodulin (>2-fold increase) that may contribute to barrier protection through activation of protein C as described previously. In addition, we found integrin β4 to be markedly up-regulated by simvastatin (>7-fold increase). Although little is known about the role of this integrin β subunit in EC function in general, the fact that the β integrins have been implicated in Rho GTPase regulation, as well as both reactive oxygen species and mitogen-activated protein kinase signaling,21 makes this finding particularly intriguing.
Evidence of EC barrier protection by simvastatin associated with actin cytoskeletal rearrangement, regulation of relevant EC signaling events, and differential gene expression led us to investigate the effects of this drug in our murine model of ALI.22 Simvastatin (20 mg/kg via intraperitoneal injection) administered 24 hours before and then concomitant with intratracheal LPS was associated with an attenuation of multiple indices of lung inflammation and vascular permeability measured 24 hours later, including decreased BAL neutrophils, myeloperoxidase activity, and albumin content, and decreased extravasation of Evans blue albumin into the surrounding lung tissue. These findings coincided with a significant decrease in histologic evidence of lung inflammation and edema in LPS-treated animals that received simvastatin compared with the LPS-treated controls.
ALI/ARDS is a challenging clinical problem in which derangements in vascular permeability are recognized as a key feature. We have identified specific agonists that are able to attenuate EC barrier dysfunction in this context as evidenced by relevant in vitro and in vivo studies. Although the specific extent to which their EC barrier-protective properties may contribute to their clinical effects remains to be defined, our data nonetheless firmly support the idea that pharmacologic approaches including the use of S1P, APC, or statins may represent novel therapeutic strategies for ALI/ARDS.
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sphingosine 1-phosphate; activated protein C; statins; permeability; ALI/ARDS
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