L. Moreno

Suppression of endotoxin-induced inflammation by taxol

The pathogenesis of acute lung injury includes transendothelial diapedesis of leukocytes into lung tissues and disruption of endothelial/epithelial barriers leading to protein-rich oedema. In vitro studies show that the microtubule network plays a role in the regulation of endothelial permeability as well as in neutrophil locomotion. It was hypothesised that the microtubule-stabilising agent, taxol, might attenuate inflammation and vascular leak associated with acute lung injury in vivo. The effect of intravenously delivered taxol was assessed using a model of murine lung injury induced by intratracheal lipopolysaccharide (LPS) administration. Parameters of lung injury and inflammation were assessed 18 h after treatment. Intravenously delivered taxol significantly reduced inflammatory histological changes in lung parenchyma and parameters of LPS-induced inflammation: infiltration of proteins and inflammatory cells into bronchoalveolar lavage fluid, lung myeloperoxidase activity, and extravasation of Evans blue-labelled albumin into lung tissue. Taxol alone (in the absence of LPS) had no appreciable effect on these parameters. In addition to lung proteins, intravenous taxol reduced accumulation of leukocytes in ascitic fluid in a model of LPS-induced peritonitis. Taken together, the present data demonstrate that microtubule stabilisation with taxol systemically attenuates lipopolysaccharide-induced inflammation and vascular leak.

Sphingolipids and Lung Vascular Barrier Regulation

Long thought to function primarily as the structural components of lipid membranes, sphingolipids are now also recognized as vitally important signaling mediators regulating a diverse range of functions. We recently described the potent vascular barrier-regulating properties of one of these sphingolipids, the lipid and angiogenic factor sphingosine 1-phosphate (S1P) (Garcia et al, 2001). Since disruption of vascular barrier integrity commonly occurs in highly morbid inflammatory lung conditions, a better understanding of the mechanism of barrier regulation would have important clinical implications. In this chapter, we provide a brief overview of vascular barrier regulation before detailing the mechanisms underlying potent barrier-enhancing effects of S1P in vitro and in vivo in models of acute lung injury (ALI) syndromes. The potential ramifications of these findings for the development of specific therapeutic interventions for patients with ALI syndromes are then discussed.

76 ATTENUATION OF A RODENT MODEL OF PULMONARY HYPERTENSION: A NOVEL ROLE FOR SORAFENIB, A MULTIKINASE INHIBITOR.

Drawing from new drug discovery studies is the observation that severe pulmonary hypertension (PH) and cancer pathophysiology share common signal transduction pathways leading to abnormal smooth muscle and endothelial cell (EC) interactions and angioproliferative vasculopathy. Sorafenib (Sor), a chemotherapeutic agent in clinical trials for the treatment of renal cell cancer, is an inhibitor of multiple kinases, including Raf-1 kinase, MAPK, VEGFR-2, and VEGFR-3, genes implicated in angiogenesis, proliferation, and the inhibition of apoptosis. We therefore tested the hypothesis that Sor will attenuate the development of PH using an established rodent model of the disease. We performed two 3-week hypoxia (FiO2 10%) and SU5416 (a selective VEGFR-2 inhibitor known to dramatically augment hypoxia-induced PH) studies to induce PH in Dahl salt-sensitive rats (SS). Rat groups were normoxia/vehicle (Norm), hypoxia/vehicle (H), H-Su, hypoxia/sorafenib (H-Sor), and hypoxia/sorafenib/ SU5416 (H-Su-Sor). Except for Norm, all rats were kept in hypoxia, whereas the H-Su group received SU5416 at day 1 (20 mg/kg, sc) and Sor was gavaged daily (2.5 mg/kg). Echocardiography, pulmonary artery pressures (PAPs), right ventricular pressures (RVPs), and lung gene microarray analyses were assessed at 3 weeks. Our results showed that H-Su rats developed severe PH compared with Norm, rats in the H alone group had mildly elevated pressures compared with Norm, and no changes were seen in pressures, weights, or remodeling in the H-Sor or H-SU-Sor groups compared with Norm. The H-Su-Sor rats showed significant reductions in PAP (56%), RVP (55%), and RV hypertrophy (52%). Gene expression profiling data were compared with Norm using GCRMA normalization in R and SAM (> .639, MFC > 1.7). With false discovery rates (FDRs) of 5.1% and 0.7%, respectively, 356 and 293 genes were up- or down-regulated. Forty-seven of the 356 H genes were recapitulated from previous H studies in the rodent model. In addition, 45 genes were differentially expressed between H-Su and H-Su-Sor (FDR 12%), with ECM, cytoskeleton, and angiogenesis gene ontologies, and 81 genes were changed in the H and H-Su groups but not in the H-Su-Sor group. These studies suggest Sor as a powerful novel treatment in PH.

88 EFFECT OF THE S1P1 GENE KO ON LIPOPOLYSACCHARIDE-INDUCED MURINE ACUTE LUNG INJURY.

Acute lung injury ALI/ARDS, a significant cause of morbidity and mortality, is characterized by a diffuse inflammatory parenchymal process with pulmonary EC vascular leak and alveolar flooding. This syndrome remains a significant cause of intensive care unit mortality, and more effective therapeutic interventions are needed. Our in vitro studies indicate that sphingosine 1-phosphate (S1P), a phospholipid angiogenic factor and a major barrier-protective product of platelets, produces endothelial cell barrier enhancement through ligation of the S1P family of receptors, especially S1P1, a G protein-coupled receptor expressed on vascular endothelial cells. Our previous data show that S1P, via S1P1, has impressive protective effects in both murine and canine models of ALI (McVerry et al, 2004). To better understand S1P receptors in barrier regulation, we examined LPS-induced ALI in S1P1 receptor heterozygous (S1P1R+/−) mice. Our data demonstrate that the S1P1-R+/− mice exhibit increased barrier disruption compared with wild-type mice, reflected by an increase in protein (25%) and inflammatory cell count (20%) in bronchoalveolar lavage (BAL) fluid. To confirm the role of S1P1R on the S1P barrier-protective effect, we administered S1P (UM final blood concentration, iv) simultaneously with LPS (2.5 mg/kg) and evaluated lung inflammation 18 hours later. LPS-treated wild-type mice treated with S1P demonstrated profound reductions (> 50%) in BAL protein, whereas S1P1+/− mice similarly treated with S1P only exhibited only a ≈10% increase in BAL protein. In conclusion, our data using genetically engineered mice demonstrate a critical need for S1P1 receptors in vivo, particularly in conditions of endotoxemia.

52 A NOVEL MYOSIN LIGHT CHAIN KINASE INHIBITOR, PIK, PROTECTS FROM LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of acute respiratory failure that are associated with high morbidity and mortality. These disorders are characterized by a significant pulmonary inflammatory response resulting in injury to alveolar epithelial and endothelial barriers followed by pulmonary edema. Our prior studies demonstrated that myosin light chain kinase phosphorylation of myosin light chains (MLC) is associated with increases in both epithelial and endothelial barrier permeability. We previously have identified a novel oligopeptide, PIK, that inhibits MLC kinase in vitro, is membrane permeant, decreases intracellular MLC phosphorylation, and causes increased intestinal epithelial cell barrier function (Turner et al. Gastroenterology 2002). We hypothesized that PIK-mediated inhibition of MLC kinase activity would attenuate inflammation and vascular leak associated with acute lung injury. To test this hypothesis, we used a murine acute lung injury model with intratracheal administration of endotoxin/lipopolysaccharide (LPS, 2.5 mg/kg). Optimal PIK responses were observed at 125 μM PIK when tested over a range of PIK concentrations. PIK administered intravenously simultaneously with LPS resulted in significantly attenuated lung inflammation reflected by decreasing accumulation of bronchoalveolar lavage (BAL) proteins (25% reduction, p < .02) and BAL cells (25% reduction, p < .05). IV administration of PIK decreased LPS-induced tissue MPO activity (a reflection of leukocytes in lung tissue) (15% reduction) and LPS-mediated MLC phosphorylation in lung homogenates (25% reduction). Our data suggest that PIK stabilizes epithelial-endothelial permeability and has significant therapeutic potential in ALI.

3 SUPPRESSION OF LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY BY TAXOL.

Our prior study demonstrated that similar to actin cytoskeleton, mictotubule (MT) network is a key participant in the regulation of endothelial (EC) permeability. Disassembly of MT by pharmacological inhibitors nocodazole and vinblastine results in rearrangement of actin cytoskeleton, stress fiber formation, EC contraction, and increased permeability. MT-stabilizing agent, Taxol, prevented EC barrier dysfunction induced by MT inhibitors and significantly attenuated permeability induced by proinflammatory agonists such as thrombin in cell culture model. We hypothesized that Taxol may prevent inflammation and vascular leak associated with lung injury. The effect of Taxol was assessed employing a model of murine lung injury induced by intratracheal LPS administration (2.5 mg/kg). Our data demonstrate that intravenous Taxol (8.5 mg/kg) injected simultaneously with LPS administration dramatically reduced inflammatory histological changes in lung parenchyma, decreased infiltration of proteins (40%, p < .001) and inflammatory cells in bronchoalveolar lavage (BAL) (51%, p < .01) and extravasation of Evans blue albumin dye into lung tissue. Taxol also significantly reduced LPS-induced release of inflammatory cytokines (tumor necrosis factor α and interleukin-6) into BAL (30% for both, p < .05). At the same time Taxol alone had no appreciable effect on the parameters described above. These data suggested that MT destabilization may be involved in LPS-induced lung injury in vivo. HL 58064; HL 60637.

27 PROTECTIVE EFFECT OF PURINERGIC AGONIST ATPgS AGAINST ACUTE LUNG INJURY.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of acute respiratory failure that are associated with high morbidity and mortality. These disorders are characterized by a significant pulmonary inflammatory response resulting in injury to alveolar epithelial and endothelial barriers of the lung and protein-rich pulmonary edema. The pharmacological treatment of ALI/ARDS is nonspecific and relies on good supportive care and control of initiating cause. ALI/ARDS pathogenesis is still only partly understood; however, pulmonary endothelium plays a major role by changing its barrier permeability, thus promoting pulmonary edema formation. Pulmonary endothelial functional and structural alterations are key components of increased vascular leak. Consequently, endothelium-related therapies may have beneficial effects in ALI/ARDS. Recently, much attention has been given to the therapeutic potential of purinergic agonists and antagonists for the treatment of cardiovascular and pulmonary diseases. Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface P2Y receptors. We have shown that ATP promotes endothelial cell (EC) barrier enhancement via a complex cell signaling. We hypothesize that activation of endothelial purinoreceptors would exert anti-inflammatory barrier-protective effect. To test this hypothesis we used two model systems: cultured pulmonary EC and murine model of ALI induced by intratracheal administration of endotoxin/lipopolysaccharide (LPS). In cell culture model, ATP inhibited intracellular gap formation and junctional permeability induced by inflammatory mediator thrombin. In murine model of ALI, nonhydrolyzed ATP analogue ATPgS (50 μM final blood concentration) administered intravenously attenuated inflammatory response by decreasing accumulation of cells (48%, p < .01) and proteins (57%, p < .01) in bronchioalveolar lavage (BAL) and by reducing neutrophil infiltration into alveoli. ATPgS slightly reduced LPS-induced release of inflammatory cytokines (tumor necrosis factor alpha and interleukin-6) into BAL. These findings suggest that purinergic receptor stimulation exerts a protective role against ALI, probably via decreasing endothelial junctional permeability.