Role of c-Met/phosphatidylinositol 3-kinase (PI3k)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells.

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

c-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury.

Paxillin is phosphorylated at multiple residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and acute lung injury (ALI) remains unclear. We used siRNA and site-specific nonphosphorylable mutants of paxillin to abrogate the function of paxillin to determine its role in lung endothelial permeability and ALI. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (HLMVECs) resulted in enhanced tyrosine phosphorylation of paxillin at Y31 and Y118 with no significant change in Y181 and significant barrier dysfunction. Knockdown of paxillin with siRNA attenuated LPS-induced endothelial barrier dysfunction and destabilization of VE-cadherin. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase, but not by Src and focal adhesion kinase. c-Abl siRNA significantly reduced LPS-induced endothelial barrier dysfunction. Transfection of HLMVECs with paxillin Y31F, Y118F, and Y31/118F double mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization. In vivo, the c-Abl inhibitor AG957 attenuated LPS-induced pulmonary permeability in mice. Together, these results suggest that c-Abl mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury.

The sphingosine kinase 1/sphingosine-1-phosphate pathway in pulmonary arterial hypertension.

RATIONALE: Sphingosine kinases (SphKs) 1 and 2 regulate the synthesis of the bioactive sphingolipid sphingosine-1-phosphate (S1P), an important lipid mediator that promotes cell proliferation, migration, and angiogenesis. OBJECTIVES: We aimed to examine whether SphKs and their product, S1P, play a role in the development of pulmonary arterial hypertension (PAH). METHODS: SphK1(-/-), SphK2(-/-), and S1P lyase heterozygous (Sgpl1(+/-)) mice, a pharmacologic SphK inhibitor (SKI2), and a S1P receptor 2 (S1PR2) antagonist (JTE013) were used in rodent models of hypoxia-mediated pulmonary hypertension (HPH). S1P levels in lung tissues from patients with PAH and pulmonary arteries (PAs) from rodent models of HPH were measured. MEASUREMENTS AND MAIN RESULTS: mRNA and protein levels of SphK1, but not SphK2, were significantly increased in the lungs and isolated PA smooth muscle cells (PASMCs) from patients with PAH, and in lungs of experimental rodent models of HPH. S1P levels were increased in lungs of patients with PAH and PAs from rodent models of HPH. Unlike SphK2(-/-) mice, SphK1(-/-) mice were protected against HPH, whereas Sgpl1(+/-) mice were more susceptible to HPH. Pharmacologic SphK1 and S1PR2 inhibition prevented the development of HPH in rodent models of HPH. Overexpression of SphK1 and stimulation with S1P potentially via ligation of S1PR2 promoted PASMC proliferation in vitro, whereas SphK1 deficiency inhibited PASMC proliferation. CONCLUSIONS: The SphK1/S1P axis is a novel pathway in PAH that promotes PASMC proliferation, a major contributor to pulmonary vascular remodeling. Our results suggest that this pathway is a potential therapeutic target in PAH.

The mitochondrial cardiolipin remodeling enzyme lysocardiolipin acyltransferase is a novel target in pulmonary fibrosis.

RATIONALE: Lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, is necessary for maintaining normal mitochondrial function and vascular development. We hypothesized that modulation of LYCAT expression in lung epithelium regulates development of pulmonary fibrosis. OBJECTIVES: To define a role for LYCAT in human and murine models of pulmonary fibrosis. METHODS: We analyzed the correlation of LYCAT expression in peripheral blood mononuclear cells (PBMCs) with the outcomes of pulmonary functions and overall survival, and used the murine models to establish the role of LYCAT in fibrogenesis. We studied the LYCAT action on cardiolipin remodeling, mitochondrial reactive oxygen species generation, and apoptosis of alveolar epithelial cells under bleomycin challenge. MEASUREMENTS AND MAIN RESULTS: LYCAT expression was significantly altered in PBMCs and lung tissues from patients with idiopathic pulmonary fibrosis (IPF), which was confirmed in two preclinical murine models of IPF, bleomycin- and radiation-induced pulmonary fibrosis. LYCAT mRNA expression in PBMCs directly and significantly correlated with carbon monoxide diffusion capacity, pulmonary function outcomes, and overall survival. In both bleomycin- and radiation-induced pulmonary fibrosis murine models, hLYCAT overexpression reduced several indices of lung fibrosis, whereas down-regulation of native LYCAT expression by siRNA accentuated fibrogenesis. In vitro studies demonstrated that LYCAT modulated bleomycin-induced cardiolipin remodeling, mitochondrial membrane potential, reactive oxygen species generation, and apoptosis of alveolar epithelial cells, potential mechanisms of LYCAT-mediated lung protection. CONCLUSIONS: This study is the first to identify modulation of LYCAT expression in fibrotic lungs and offers a novel therapeutic approach for ameliorating lung inflammation and pulmonary fibrosis.

Intermittent hypoxia-induced endothelial barrier dysfunction requires ROS-dependent MAP kinase activation.

The objective of the present study was to determine the impact of simulated apnea with intermittent hypoxia (IH) on endothelial barrier function and assess the underlying mechanism(s). Experiments were performed on human lung microvascular endothelial cells exposed to IH-consisting alternating cycles of 1.5% O2 for 30s followed by 20% O2 for 5 min. IH decreased transendothelial electrical resistance (TEER) suggesting attenuated endothelial barrier function. The effect of IH on TEER was stimulus dependent and reversible after reoxygenation. IH-exposed cells exhibited stress fiber formation and redistribution of cortactin, vascular endothelial-cadherins, and zona occludens-1 junction proteins along with increased intercellular gaps at cell-cell boundaries. Extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) were phosphorylated in IH-exposed cells. Inhibiting either ERK or JNK prevented the IH-induced decrease in TEER and the reorganization of the cytoskeleton and junction proteins. IH increased reactive oxygen species (ROS) levels, and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride, a membrane-permeable antioxidant, prevented ERK and JNK phosphorylation as well as IH-induced changes in endothelial barrier function. These results demonstrate that IH via ROS-dependent activation of MAP kinases leads to reorganization of cytoskeleton and junction proteins resulting in endothelial barrier dysfunction.

Sphingosine kinase 1 deficiency confers protection against hyperoxia-induced bronchopulmonary dysplasia in a murine model: role of S1P signaling and Nox proteins.

Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1(-/-) (Sphk1(-/-)), sphingosine kinase 2(-/-) (Sphk2(-/-)), and S1P lyase(+/-) (Sgpl1(+/-)) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1(-/-), but not Sphk2(-/-) or Sgpl1(+/-), mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling-regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia.

Novel role for non-muscle myosin light chain kinase (MLCK) in hyperoxia-induced recruitment of cytoskeletal proteins, NADPH oxidase activation, and reactive oxygen species generation in lung endothelium.

We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.

Hydroxyalkenals and oxidized phospholipids modulation of endothelial cytoskeleton, focal adhesion and adherens junction proteins in regulating endothelial barrier function.

Lipid peroxidation of polyunsaturated fatty acids generates bioactive aldehydes, which exhibit pro- and anti-inflammatory effects in cells and tissues. Accumulating evidence indicates that 4-hydroxynonenal (4-HNE), a major aldehyde derived from lipid peroxidation of n-6 polyunsaturated fatty acids trigger signals that modulates focal adhesion and adherens junction proteins thereby inducing endothelial barrier dysfunction. Similarly, oxidized phospholipids (Ox-PLs) generated by lipid peroxidation of phospholipids with polyunsaturated fatty acids have been implicated in atherogenesis, inflammation and gene expression. Interestingly, physiological concentration of Ox-PLs is anti-inflammatory and protect against endotoxin- and ventilator-associated acute lung injury. Thus, excess generation of bioactive hydroxyalkenals and Ox-PLs during oxidative stress contributes to pathophysiology of various diseases by modulating signaling pathways that regulate pro- and anti-inflammatory responses and barrier regulation. This review summarizes the role of 4-HNE and Ox-PLs affecting cell signaling pathways and endothelial barrier dysfunction through modulation of the activities of proteins/enzymes by Michael adducts formation, enhancing the level of protein tyrosine phosphorylation of the target proteins, and by reorganization of cytoskeletal, focal adhesion, and adherens junction proteins. A better understanding of molecular mechanisms of hydroxyalkenals- and Ox-PLs-mediated pro-and anti-inflammatory responses and barrier function may lead to development of novel therapies to ameliorate oxidative stress related cardio-pulmonary disorders.

Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation.

BACKGROUND: Exposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. OBJECTIVES: We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC) barrier integrity and enhanced cardiopulmonary dysfunction. METHODS: Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER) in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 µm). Biochemical assessment of ROS generation and Ca2+ mobilization were also measured. RESULTS: PM exposure induced tight junction protein Zona occludens-1 (ZO-1) relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and ß-catenin). N-acetyl-cysteine (NAC, 5 mM) reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2), in a ROS-dependent manner with subsequent activation of the Ca2+-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. CONCLUSIONS: These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.

Protection of LPS-induced murine acute lung injury by sphingosine-1-phosphate lyase suppression.

A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL(+/-) mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.

Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and ß-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.

Regulation of COX-2 expression and IL-6 release by particulate matter in airway epithelial cells.

Particulate matter (PM) in ambient air is a risk factor for human respiratory and cardiovascular diseases. The delivery of PM to airway epithelial cells has been linked to release of proinflammatory cytokines; however, the mechanisms of PM-induced inflammatory responses are not well-characterized. This study demonstrates that PM induces cyclooxygenase (COX)-2 expression and IL-6 release through both a reactive oxygen species (ROS)-dependent NF-kappaB pathway and an ROS-independent C/EBPbeta pathway in human bronchial epithelial cells (HBEpCs) in culture. Treatment of HBEpCs with Baltimore PM induced ROS production, COX-2 expression, and IL-6 release. Pretreatment with N-acetylcysteine (NAC) or EUK-134, in a dose-dependent manner, attenuated PM-induced ROS production, COX-2 expression, and IL-6 release. The PM-induced ROS was significantly of mitochondrial origin, as evidenced by increased oxidation of the mitochondrially targeted hydroethidine to hydroxyethidium by reaction with superoxide. Exposure of HBEpCs to PM stimulated phosphorylation of NF-kappaB and C/EBPbeta, while the NF-kappaB inhibitor, Bay11-7082, or C/EBPbeta siRNA attenuated PM-induced COX-2 expression and IL-6 release. Furthermore, NAC or EUK-134 attenuated PM-induced activation of NF-kappaB; however, NAC or EUK-134 had no effect on phosphorylation of C/EBPbeta. In addition, inhibition of COX-2 partly attenuated PM-induced Prostaglandin E2 and IL-6 release.

Protective effects of high-molecular weight polyethylene glycol (PEG) in human lung endothelial cell barrier regulation: role of actin cytoskeletal rearrangement.

Acute lung injury represents the result of multiple pathways initiated by local or systemic insults and is characterized by profound vascular permeability, pulmonary edema, and life-threatening respiratory failure. Permeability-reducing therapies are of potential clinical utility but are currently unavailable. We hypothesized that polyethylene glycol (PEG) compounds, inert and non-toxic polymers that serve as a surrogate mucin lining in intestinal epithelium, may attenuate agonist-mediated lung endothelial cell (EC) barrier dysfunction. High molecular weight PEG (PEG15-20) produced rapid, dose-dependent increases in transendothelial electrical resistance (TER) in human lung endothelium cultured on gold microelectrodes, reflecting increased paracellular integrity. The maximal effective concentration of 8% PEG induced a sustained 125% increase in TER (40 h), results similar to barrier-enhancing agonists such as sphingosine 1-phosphate (40% increase in TER). Maximal PEG barrier enhancement was achieved at 45-60 min and PEG effectively reversed both thrombin- and LPS-induced EC barrier dysfunction. Consistent with the increase in TER, immunofluorescent studies demonstrated that PEG produced significant cytoskeletal rearrangement with formation of well-defined cortical actin rings and lamellipodia containing the actin-binding proteins, cortactin and MLCK, known participants in cell-matrix and cell-cell junctional adhesion. Finally, PEG challenge induced rapid alterations in levels of MAP kinase and MLC phosphorylation. In summary, PEG joins a number of EC barrier-regulatory agents which rapidly activate barrier-enhancing signal transduction pathways which target the cytoskeleton and provides a potential therapeutic strategy in inflammatory lung injury.

Lysophosphatidic acid modulates c-Met redistribution and hepatocyte growth factor/c-Met signaling in human bronchial epithelial cells through PKC delta and E-cadherin.

Previously we demonstrated that ligation of lysophosphatidic acid (LPA) to G protein-coupled LPA receptors induces transactivation of receptor tyrosine kinases (RTKs), such as platelet-derived growth factor receptor beta (PDGF-Rbeta) and epidermal growth factor receptor (EGF-R), in primary cultures of human bronchial epithelial cells (HBEpCs). Here we examined the role of LPA on c-Met redistribution and modulation of hepatocyte growth factor (HGF)/c-Met pathways in HBEpCs. Treatment of HBEpCs with LPA-induced c-Met serine phosphorylation and redistribution to plasma membrane, while treatment with HGF-induced c-Met internalization. Pretreatment with LPA reversed HGF-induced c-Met internalization. Overexpression of dominant negative (Dn)-PKC delta or pretreatment with Rottlerin or Pertussis toxin (PTx) attenuated LPA-induced c-Met serine phosphorylation and redistribution. Co-immnuoprecipitation and immunocytochemistry showed that E-cadherin interacted with c-Met in HBEpCs. LPA treatment induced E-cadherin and c-Met complex redistribution to plasma membranes. Overexpression of Dn-PKC delta attenuated LPA-induced E-cadherin redistribution and E-cadherin siRNA attenuated LPA-induced c-Met redistribution to plasma membrane. Furthermore, pretreatment of LPA attenuated HGF-induced c-Met tyrosine phosphorylation and downstream signaling, such as Akt kinase phosphorylation and cell motility. These results demonstrate that LPA regulates c-Met function through PKC delta and E-cadherin in HBEpCs, suggesting an alternate function of the cross-talk between G-protein-coupled receptors (GPCRs) and RTKs in HBEpCs.

Lipid phosphate phosphatase-1 regulates lysophosphatidic acid-induced calcium release, NF-kappaB activation and interleukin-8 secretion in human bronchial epithelial cells.

LPA (lysophosphatidic acid), a potent bioactive phospholipid, elicits diverse cellular responses through activation of the G-protein-coupled receptors LPA1-LPA4. LPA-mediated signalling is partially regulated by LPPs (lipid phosphate phosphatases; LPP-1, -2 and -3) that belong to the phosphatase superfamily. This study addresses the role of LPPs in regulating LPA-mediated cell signalling and IL-8 (interleukin-8) secretion in HBEpCs (human bronchial epithelial cells). Reverse transcription-PCR and Western blotting revealed the presence and expression of LPP-1-3 in HBEpCs. Exogenous [3H]oleoyl LPA was hydrolysed to [3H]-mono-oleoylglycerol. Infection of HBEpCs with an adenoviral construct of human LPP-1 for 48 h enhanced the dephosphorylation of exogenous LPA by 2-3-fold compared with vector controls. Furthermore, overexpression of LPP-1 partially attenuated LPA-induced increases in the intracellular Ca2+ concentration, phosphorylation of IkappaB (inhibitory kappaB) and translocation of NF-kappaB (nuclear factor-kappaB) to the nucleus, and almost completely prevented IL-8 secretion. Infection of cells with an adenoviral construct of the mouse LPP-1 (R217K) mutant partially attenuated LPA-induced IL-8 secretion without altering LPA-induced changes in intracellular Ca2+ concentration, phosphorylation of IkappaB, NF-kappaB activation or IL-8 gene expression. Our results identify LPP-1 as a key regulator of LPA signalling and IL-8 secretion in HBEpCs. Thus LPPs could represent potential targets in regulating leucocyte infiltration and airway inflammation.

"Pulmonary Endothelial Cell Barrier Enhancement by Novel FTY720 Analogs: Methoxy-FTY720, Fluoro-FTY720, and ß-Glucuronide-FTY720".

Effective therapeutic agents are lacking for the prevention and reversal of vascular leak, a frequent pathophysiologic result of inflammatory processes such as acute respiratory distress syndrome (ARDS) and sepsis. We previously demonstrated the potent barrier-enhancing effects of related compounds sphingosine 1-phosphate (S1P), the pharmaceutical agent FTY720, and its analog (S)-FTY720 phosphonate (Tys) in models of inflammatory lung injury. In this study, we characterize additional novel FTY720 analogs for their potential to reduce vascular leak as well as utilize them as tools to better understand the mechanisms by which this class of agents modulates permeability. Transendothelial resistance (TER) and labeled dextran studies demonstrate that (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F), and ß-glucuronide-FTY720 (FTY-G) compounds display in vitro barrier-enhancing properties comparable or superior to FTY720 and S1P. In contrast, the (S)-methoxy-FTY720 ((S)-OMe-FTY) analog disrupts lung endothelial cell (EC) barrier integrity in TER studies in association with actin stress fiber formation and robust intracellular calcium release, but independent of myosin light chain or ERK phosphorylation. Additional mechanistic studies with (R)-OMe-FTY, FTY-F, and FTY-G suggest that lung EC barrier enhancement is mediated through lipid raft signaling, Gi-linked receptor coupling to downstream tyrosine phosphorylation events, and S1PR1-dependent receptor ligation. These results provide important mechanistic insights into modulation of pulmonary vascular barrier function by FTY720-related compounds and highlight common signaling events that may assist the development of novel therapeutic tools in the prevention or reversal of the pulmonary vascular leak that characterizes ARDS.

Role played by paxillin and paxillin tyrosine phosphorylation in hepatocyte growth factor/sphingosine-1-phosphate-mediated reactive oxygen species generation, lamellipodia formation, and endothelial barrier function.

Paxillin is a multifunctional and multidomain focal adhesion adaptor protein. It serves as an important scaffolding protein at focal adhesions by recruiting and binding to structural and signaling molecules. Paxillin tyrosine phosphorylation at Y31 and Y118 is important for paxillin redistribution to focal adhesions and angiogenesis. Hepatocyte growth factor (HGF) and sphingosine-1-phosphate (S1P) are potent stimulators of lamellipodia formation, a prerequisite for endothelial cell migration. The role played by paxillin and its tyrosine phosphorylated forms in HGF- or S1P-induced lamellipodia formation and barrier function is unclear. HGF or S1P stimulated lamellipodia formation, tyrosine phosphorylation of paxillin at Y31 and Y118, and c-Abl in human lung microvascular endothelial cells (HLMVECs). Knockdown of paxillin with small interfering RNA (siRNA) or transfection with paxillin mutants (Y31F or Y118F) mitigated HGF- or S1P-induced lamellipodia formation, translocation of p47 (phox) to lamellipodia, and reactive oxygen species (ROS) generation in HLMVECs. Furthermore, exposure of HLMVECs to HGF or S1P stimulated c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 in a time-dependent fashion, and down-regulation of c-Abl with siRNA attenuated HGF- or S1P-mediated lamellipodia formation, translocation of p47 (phox) to lamellipodia, and endothelial barrier enhancement. In vivo, knockdown of paxillin with siRNA in mouse lungs attenuated ventilator-induced lung injury. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates HGF- or S1P-mediated lamellipodia formation, ROS generation in lamellipodia, and endothelial permeability.

Pulmonary endothelial cell barrier enhancement by novel FTY720 analogs: methoxy-FTY720, fluoro-FTY720, and ß-glucuronide-FTY720.

Effective therapeutic agents are lacking for the prevention and reversal of vascular leak, a frequent pathophysiologic result of inflammatory processes such as acute respiratory distress syndrome (ARDS) and sepsis. We previously demonstrated the potent barrier-enhancing effects of related compounds sphingosine 1-phosphate (S1P), the pharmaceutical agent FTY720, and its analog (S)-FTY720 phosphonate (Tys) in models of inflammatory lung injury. In this study, we characterize additional novel FTY720 analogs for their potential to reduce vascular leak as well as utilize them as tools to better understand the mechanisms by which this class of agents modulates permeability. Transendothelial resistance (TER) and labeled dextran studies demonstrate that (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F), and ß-glucuronide-FTY720 (FTY-G) compounds display in vitro barrier-enhancing properties comparable or superior to FTY720 and S1P. In contrast, the (S)-methoxy-FTY720 ((S)-OMe-FTY) analog disrupts lung endothelial cell (EC) barrier integrity in TER studies in association with actin stress fiber formation and robust intracellular calcium release, but independent of myosin light chain or ERK phosphorylation. Additional mechanistic studies with (R)-OMe-FTY, FTY-F, and FTY-G suggest that lung EC barrier enhancement is mediated through lipid raft signaling, Gi-linked receptor coupling to downstream tyrosine phosphorylation events, and S1PR1-dependent receptor ligation. These results provide important mechanistic insights into modulation of pulmonary vascular barrier function by FTY720-related compounds and highlight common signaling events that may assist the development of novel therapeutic tools in the prevention or reversal of the pulmonary vascular leak that characterizes ARDS.

Redox regulation of reactive oxygen species-induced p38 MAP kinase activation and barrier dysfunction in lung microvascular endothelial cells.

Reactive oxygen species (ROS)-mediated compromise of endothelial barrier integrity has been implicated in a number of pulmonary disorders, including adult respiratory distress syndrome, pulmonary edema, and vasculitis. The mechanisms by which ROS increase endothelial permeability are unclear. We hypothesized that ROS-induced changes in cellular redox status (thiols) may contribute to endothelial barrier dysfunction. To test this hypothesis, we used N-acetylcysteine (NAC) and diamide to modulate intracellular levels of cellular glutathione (GSH) and investigated hydrogen peroxide (H(2)O(2))-mediated mitogen-activated protein kinase (MAPK) activation and transendothelial electrical resistance (TER). Exposure of bovine lung microvascular endothelial cells (BLMVECs) to H(2)O(2), in a dose- and time-dependent fashion, increased endothelial permeability. Pretreatment of BLMVECs with NAC (5 mM) for 1 h resulted in partial attenuation of H(2)O(2)-induced TER (a measure of increase in permeability) and GSH. Furthermore, treatment of BLMVECs with diamide, which is known to reduce the intracellular GSH, resulted in significant reduction in TER, which was prevented by NAC. To understand further the role of MAPKs in ROS-induced barrier dysfunction, we examined the role of extracellular signal-regulated kinase (ERK) and p38 MAPK on H(2)O(2)- and diamide-mediated permeability changes. Both H(2)O(2) and diamide, in a dose-dependent manner, activated ERK and p38 MAPK in BLMVECs. However, SB203580, an inhibitor of p38 MAPK, but not PD98059, blocked H(2)O(2)- and diamide-induced TER. Also, NAC prevented H(2)O(2)- and diamide-induced p38 MAPK, but not ERK activation. These results suggest a role for redox regulation of p38 MAPK in ROS-dependent endothelial barrier dysfunction.

Influence of beta-amyloid fibrils on the interactions between red blood cells and endothelial cells.

Alzheimer's disease is associated with vascular amyloidosis. As blood flows through the microcirculation, red blood cells (RBCs) come in contact with the vasculature. RBCs as well as endothelial cells (ECs) are known to bind beta amyloid fibrils. This suggests that a potential effect of amyloidosis may involve the interactions of RBCs with ECs lining the wall of the blood vessels mediated by amyloid fibrils. We have studied the effect of beta-amyloid peptide[1-40] (Abeta1-40) fibrils on the interactions of murine RBCs with ECs derived from bovine lung microvascular endothelium (BLMVEC) as well as bovine pulmonary arterial endothelium (BPAEC) in culture. We show that the initial incorporation of Abeta fibrils onto either RBCs or ECs cause RBCs to adhere to the ECs with greater affinity for the microvascular cells than the arterial cells. In addition, there is a transfer of Abeta fibrils between the RBCs and the ECs. Both the transfer and adhesion occurs when the amyloid fibrils are on the ECs or on the RBCs. However, with the amyloid fibrils on the RBCs, the adhesion and the transfer are greater than with the fibrils on the ECs. These results suggest that amyloidosis may affect the flow of RBCs through the microcirculation and that RBCs may play a role in propagating amyloidosis through the vasculature.