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The paucity of therapeutic strategies to reduce the severity of radiation-induced lung fibrosis (RILF), a life-threatening complication of intended or accidental ionizing radiation exposure, is a serious unmet need. We evaluated the contribution of eNAMPT (extracellular nicotinamide phosphoribosyltransferase), a damage-associated molecular pattern (DAMP) protein and TLR4 (Toll-like receptor 4) ligand, to the severity of whole-thorax lung irradiation (WTLI)-induced RILF. Wild-type (WT) and

Rationale Vascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregulation remain unclear, thereby impairing the development of desperately needed effective therapeutics. We have shown that sphingosine-1-phosphate (S1P) and 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) analogues are useful tools for exploring vascular barrier regulation mechanisms. Objective To experimentally define the effects of FTY720 regioisomers on lung endothelial cell barrier regulation. Methods Specific barrier-regulatory receptor and kinase inhibitors were utilized to probe signaling mechanisms involved in FTY720 regioisomer-mediated human lung endothelial cell barrier responses (trans-endothelial electrical resistance, TER). Docking simulations with the S1P1 receptor were performed to further evaluate FTY720 regioisomer signaling. Results FTY720 regioisomers produced potent endothelial cell barrier disruption reflected by declines in TER alterations. Pharmacologic inhibition of Gi-coupled S1P receptors (S1P1, S1P2, S1P3) failed to alter FTY720 regioisomer-mediated barrier disruption; findings that were corroborated by docking simulations demonstrating FTY720 regiosomers were repelled from S1P1 docking, in contrast to strong S1P1 binding elicited by S1P. Inhibition of either the barrier-disrupting PAR-1 receptor, the VEGF receptor, Rho-kinase, MAPK, NFkB, or PI3K failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. While FTY720 regioisomers significantly increased protein phosphatase 2 (PP2A) activity, PP2A inhibitors failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. Conclusions Together, these results imply a vexing model of pulmonary vascular barrier dysregulation in response to FTY720-related compounds and highlight the need for further insights into mechanisms of vascular integrity required to promote the development of novel therapeutic tools to prevent or reverse the pulmonary vascular leak central to ARDS outcomes.

Therapeutic strategies to prevent or reduce the severity of radiation pneumonitis are a serious unmet need. We evaluated extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a damage-associated molecular pattern protein (DAMP) and Toll-Like Receptor 4 (TLR4) ligand, as a therapeutic target in murine radiation pneumonitis. Radiation-induced murine and human NAMPT expression was assessed in vitro, in tissues (IHC, biochemistry, imaging), and in plasma. Wild type C57Bl6 mice (WT) and Nampt(+/−) heterozygous mice were exposed to 20Gy whole thoracic lung irradiation (WTLI) with or without weekly IP injection of IgG(1) (control) or an eN-AMPT-neutralizing polyclonal (pAb) or monoclonal antibody (mAb). BAL protein/cells and H&E staining were used to generate a WTLI severity score. Differentially-expressed genes (DEGs)/pathways were identified by RNA sequencing and bioinformatic analyses. Radiation exposure increases in vitro NAMPT expression in lung epithelium (NAMPT promoter activity) and NAMPT lung tissue expression in WTLI-exposed mice. Nampt(+/−) mice and eNAMPT pAb/mAb-treated mice exhibited significant histologic attenuation of WTLI-mediated lung injury with reduced levels of BAL protein and cells, and plasma levels of eNAMPT, IL-6, and IL-1 β. Genomic and biochemical studies from WTLI-exposed lung tissues highlighted dysregulation of NFkB/cytokine and MAP kinase signaling pathways which were rectified by eNAMPT mAb treatment. The eNAMPT/TLR4 pathway is essentially involved in radiation pathobiology with eNAMPT neutralization an effective therapeutic strategy to reduce the severity of radiation pneumonitis.

Rationale: The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle isoform of myosin light chain kinase (nmMLCK), a cytoskeletal protein centrally involved in regulation of vascular integrity. As MYLK coding SNPs are associated with severe inflammatory disorders (asthma, acute respiratory distress syndrome (ARDS)), we explored clinically relevant inflammatory stimuli and promoter SNPs in nmMLCK promoter regulation. Methods: Full-length or serially deleted MYLK luciferase reporter promoter activities were measured in human lung endothelial cells (ECs). SNP-containing non-muscle MYLK (nmMYLK) DNA fragments were generated and nmMYLK promoter binding by transcription factors (TFs) detected by protein–DNA electrophoretic mobility shift assay (EMSA). Promoter demethylation was evaluated by 5-aza-2′-deoxycytidine (5-Aza). A preclinical mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) was utilized for nmMLCK validation. Results: Lung EC levels of nmMLCK were significantly increased in LPS-challenged mice and LPS, tumor necrosis factor-α (TNF-α), 18% cyclic stretch (CS) and 5-Aza each significantly up-regulated EC nmMYLK promoter activities. EC exposure to FG-4592, a prolyl hydroxylase inhibitor that increases hypoxia-inducible factor (HIF) expression, increased nmMYLK promoter activity, confirmed by HIF1α/HIF2α silencing. nmMYLK promoter deletion studies identified distal inhibitory and proximal enhancing promoter regions as well as mechanical stretch-, LPS- and TNFα-inducible regions. Insertion of ARDS-associated SNPs (rs2700408, rs11714297) significantly increased nmMYLK promoter activity via increased transcription binding (glial cells missing homolog 1 (GCM1) and intestine-specific homeobox (ISX), respectively). Finally, the MYLK rs78755744 SNP (−261G/A), residing within a nmMYLK CpG island, significantly attenuated 5-Aza-induced promoter activity. Conclusion: These findings indicate nmMYLK transcriptional regulation by clinically relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.

RationaleThe severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target.MethodsWild-type C57BL/6J or endothelial cell (EC)-cNAMPT−/−knockout mice (targeted ECNAMPTdeletion) were exposed to either a lipopolysaccharide (LPS)-induced (“one-hit”) or a combined LPS/ventilator (“two-hit”)-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were usedin vitroandin vivo.ResultsImmunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models.In vitrohuman lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption.In vivostudies in wild-type and EC-cNAMPT−/−mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models.ConclusionsThese findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker andNAMPTgenotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.

Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y 464 and Y 471 phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK , the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y 464 phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y 464 phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.

Inflammatory mediators released in acute lung injury (ALI) trigger the disruption of interendothelial junctions, leading to loss of vascular barrier function, protein-rich pulmonary edema, and severe hypoxemia. Genetic signatures that predict patient recovery or disease progression are poorly defined, but recent genetic screening of ALI patients has identified an association between lung inflammatory disease and a single nucleotide polymorphism (SNP) in the gene for the actin-binding and barrier-regulatory protein cortactin. This study investigated the impact of this disease-linked cortactin variant on wound healing processes that may contribute to endothelial barrier restoration. A microfabricated platform was used to quantify wound healing in terms of gap closure speed, lamellipodia dynamics, and cell velocity. Overexpression of wild-type cortactin in endothelial cells (ECs) improved directional cell motility and enhanced lamellipodial protrusion length, resulting in enhanced gap closure rates. By contrast, the cortactin SNP impaired wound closure and cell locomotion, consistent with the observed reduction in lamellipodial protrusion length and persistence. Overexpression of the cortactin SNP in lung ECs mitigated the barrier-enhancing activity of sphingosine 1-phosphate. These findings suggest that this common cortactin variant may functionally contribute to ALI predisposition by impeding endothelial wound healing.

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 β-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.

We previously identified the intracellular nicotinamide phosphoribosyltransferase (iNAMPT, aka pre–B-cell colony enhancing factor) as a candidate gene promoting acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI) with circulating nicotinamide phosphoribosyltransferase potently inducing NF-κB signaling in lung endothelium. iNAMPT also synthesizes intracellular nicotinamide adenine dinucleotide (iNAD) in response to extracellular oxidative stress, contributing to the inhibition of apoptosis via ill-defined mechanisms. We now further define the role of iNAMPT activity in the pathogenesis of ARDS/VILI using the selective iNAMPT inhibitor FK-866. C57/B6 mice were exposed to VILI (40 ml/kg, 4 h) or LPS (1.5 mg/kg, 18 h) after osmotic pump delivery of FK-866 (100 mg/kg/d, intraperitoneally). Assessment of total bronchoalveolar lavage (BAL) protein, polymorphonuclear neutrophil (PMN) levels, cytokine levels (TNF-α, IL-6, IL-1α), lung iNAD levels, and injury scores revealed that FK-866–mediated iNAMPT inhibition successfully reduced lung tissue iNAD levels, BAL injury indices, inflammatory cell infiltration, and lung injury scores in LPS- and VILI-exposed mice. FK-866 further increased lung PMN apoptosis, as reflected by caspase-3 activation in BAL PMNs. These findings support iNAMPT inhibition via FK-866 as a novel therapeutic agent for ARDS via enhanced apoptosis in inflammatory PMNs.

Effective therapies are needed to reverse the increased vascular permeability that characterizes acute inflammatory diseases such as acute lung injury. FTY720 is a pharmaceutical analog of the potent barrier-enhancing phospholipid, sphingosine 1-phosphate. Because both FTY720 and sphingosine 1-phosphate have properties that may limit their usefulness in patients with acute lung injury, alternative compounds are needed for therapeutic use. The objective of this study is to characterize the effects of FTY720 (S)-phosphonate, a novel analog of FTY720-phosphate, on variables of pulmonary vascular permeability in vitro and alveolar-capillary permeability in vivo.University-affiliated research institute.Cultured human pulmonary endothelial cells; C57BL/6 mice.Endothelial cells were stimulated with sphingosine 1-phosphate receptor 1 agonists to determine effects on sphingosine 1-phosphate receptor 1 expression. Acute lung injury was induced in C57BL/6 mice with bleomycin to assess effects of sphingosine 1-phosphate receptor 1 agonists.FTY720 (S)-phosphonate potently increases human pulmonary endothelial cell barrier function in vitro as measured by transendothelial electrical resistance. Reduction of sphingosine 1-phosphate receptor 1 with small interference RNA significantly attenuates this transendothelial electrical resistance elevation. FTY720 (S)-phosphonate maintains endothelial sphingosine 1-phosphate receptor 1 protein expression in contrast to greater than 50% reduction after incubation with sphingosine 1-phosphate, FTY720, or other sphingosine 1-phosphate receptor 1 agonists. FTY720 (S)-phosphonate does not induce β-arrestin recruitment, sphingosine 1-phosphate receptor 1 ubiquitination, and proteosomal degradation that occur after other agonists. Intraperitoneal administration of FTY720 (S)-phosphonate every other day for 1 week in normal or bleomycin-injured mice maintains significantly higher lung sphingosine 1-phosphate receptor 1 expression compared with FTY720. FTY720 fails to protect against bleomycin-induced acute lung injury in mice, while FTY720 (S)-phosphonate significantly decreases lung leak and inflammation.FTY720 (S)-phosphonate is a promising barrier-promoting agent that effectively maintains sphingosine 1-phosphate receptor 1 levels and improves outcomes in the bleomycin model of acute lung injury.

Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

Novel therapies are needed to address the vascular endothelial cell (EC) barrier disruption that occurs in inflammatory diseases such as acute lung injury (ALI). We previously demonstrated the potent barrier-enhancing effects of both sphingosine 1-phosphate (S1P) and the structurally similar compound FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] in inflammatory lung injury. In this study, we examined the therapeutic potential of several novel FTY720 analogs to reduce vascular leak. Similar to S1P and FTY720, the (R)- and (S)-enantiomers of FTY720 phosphonate and enephosphonate analogs produce sustained EC barrier enhancement in vitro, as seen by increases in transendothelial electrical resistance (TER). In contrast, the (R)- and (S)-enantiomers of FTY720-regioisomeric analogs disrupt EC barrier integrity in a dose-dependent manner. Barrier-enhancing FTY720 analogs demonstrate a wider protective concentration range in vitro (1-50 microM) and greater potency than either S1P or FTY720. In contrast to FTY720-induced EC barrier enhancement, S1P and the FTY720 analogs dramatically increase TER within minutes in association with cortical actin ring formation. Unlike S1P, these FTY720 analogs exhibit differential phosphorylation effects without altering the intracellular calcium level. Inhibitor studies indicate that barrier enhancement by these analogs involves signaling via G(i)-coupled receptors, tyrosine kinases, and lipid rafts. Consistent with these in vitro responses, the (S)-phosphonate analog of FTY720 significantly reduces multiple indices of alveolar and vascular permeability in a lipopolysaccharide-mediated murine model of ALI (without significant alterations in leukocyte counts). These results demonstrate the capacity for FTY720 analogs to significantly decrease pulmonary vascular leakage and inflammation in vitro and in vivo.

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 hrs), 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.

Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 minutes) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.

Ventilator-induced inflammatory lung injury (VILI) is mechanistically linked to increased NAMPT transcription and circulating levels of nicotinamide phosphoribosyl-transferase (NAMPT/PBEF). Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown. We now report that NAMPT/PBEF induces lung NFκB transcriptional activities and inflammatory injury via direct ligation of Toll–like receptor 4 (TLR4). Computational analysis demonstrated that NAMPT/PBEF and MD-2, a TLR4-binding protein essential for LPS-induced TLR4 activation, share ~30% sequence identity and exhibit striking structural similarity in loop regions critical for MD-2-TLR4 binding. Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS. The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P1, the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P1 on threonine residues consistent with S1P1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P1 upon APC treatment. Targeted silencing of S1P1 expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis.

Increased nicotinamide phosphoribosyltransferase (NAMPT) transcription is mechanistically linked to ventilator-induced inflammatory lung injury (VILI), with VILI severity attenuated by reduced NAMPT bioavailability. The molecular mechanisms of NAMPT promoter regulation in response to excessive mechanical stress remain poorly understood. The objective of this study was to define the contribution of specific transcription factors, acute respiratory distress syndrome (ARDS)-associated single nucleotide polymorphisms (SNPs), and promoter demethylation to NAMPT transcriptional regulation in response to mechanical stress. In vivo NAMPT protein expression levels were examined in mice exposed to high tidal volume mechanical ventilation. In vitro NAMPT expression levels were examined in human pulmonary artery endothelial cells exposed to 5 or 18% cyclic stretch (CS), with NAMPT promoter activity assessed using NAMPT promoter luciferase reporter constructs with a series of nested deletions. In vitro NAMPT transcriptional regulation was further characterized by measuring luciferase activity, DNA demethylation, and chromatin immunoprecipitation. VILI-challenged mice exhibited significantly increased NAMPT expression in bronchoalveolar lavage leukocytes and in lung endothelium. A mechanical stress–inducible region (MSIR) was identified in the NAMPT promoter from −2,428 to −2,128 bp. This MSIR regulates NAMPT promoter activity, mRNA expression, and signal transducer and activator of transcription 5 (STAT5) binding, which is significantly increased by 18% CS. In addition, NAMPT promoter activity was increased by pharmacologic promoter demethylation and inhibited by STAT5 silencing. ARDS-associated NAMPT promoter SNPs rs59744560 (−948G/T) and rs7789066 (−2,422A/G) each significantly elevated NAMPT promoter activity in response to 18% CS in a STAT5-dependent manner. Our results show that NAMPT is a key novel ARDS therapeutic target and candidate gene with genetic/epigenetic transcriptional regulation in response to excessive mechanical stress.

Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.

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.

Introduction Acute lung injury (ALI) syndromes are highly morbid consequences of systemic inflammatory conditions such as sepsis. Inflammation-induced disruption during ALI of the endothelial cell (EC) barrier that lines the pulmonary vasculature results in leakage of fluid, protein, and cells into the airspaces of the lung, resulting in respiratory failure. We have previously described (Dudek et al. J Biol Chem 2004;279:24692-700) a critical role for the actin-binding protein cortactin in mediating EC cytoskeletal rearrangements that regulate in vitro barrier function. Methods/Results We now report immunofluorescence and coimmunoprecipitation data demonstrating increased association of cortactin with the junctional proteins β-catenin, focal adhesion kinase, and vinculin during the recovery phase following thrombin-induced permeability. Thus, cortactin interacts at critical cell-cell and cell-matrix junctional sites as the EC monolayer recovers after inflammation-induced disruption. As a translational approach building upon these in vitro observations, single-nucleotide polymorphism (SNP) discovery of the cortactin gene was performed using direct fluorescence-based resequencing. Cortactin SNP discovery in 36 patients, subdivided into those with sepsis-induced ALI, sepsis alone and healthy controls, identified 26 SNPs within the human cortactin gene, including a single novel coding variant. This SNP at amino acid position 484 results in a serine to asparagine change (Ser484Asn), which was enriched in patients with ALI and sepsis compared with controls. Since this S484N site is in close proximity to a critical p60src-targeted tyrosine residue (Y486), a major regulatory site of cortactin function, we generated wild-type and S484N mutant constructs for overexpression in human pulmonary ECs. These studies revealed greatly increased phosphorylation of cortactin at Y486 in the S484N mutant under baseline and EC barrier-altering conditions. In addition, overexpression of S484N in ECs inhibited the potent barrier-promoting effects of sphingosine 1-phosphate as well as delayed barrier recovery after the barrier disruption by thrombin. Summary Cortactin plays a critical role in mediating cytoskeletal and junctional protein rearrangements that regulate EC barrier function. Given that phosphorylation of Y486 modulates cortactin function, the S484N cortactin SNP may increase ALI susceptibility via this mechanism.

ARDS is characterized by sudden, life-threatening lung failure with diffuse alveolar infiltrate, reduced arterial oxygenation, and pulmonary edema. Acute lung injury (ALI) is the end result of common pathways initiated by a variety of local or systemic insults. Polyethylene glycol (PEG) compounds are inert and nontoxic polymers that act as a surrogate mucin lining providing protection against bacterial infections on intestinal epithelial cells. We hypothesized that PEG may provide similar protective effects on lung endothelium by attenuating endothelial cell (EC) activation that results in barrier dysfunction. This study examines the effects of a high-molecular-weight PEG (PEG15-20) on cultured human pulmonary microvessel EC from barrier disruptive agents. EC were cultured on microelectrodes and changes in transendothelial electrical resistances (TER) were measured to assess alterations in paracellular permeability. PEG induced a rapid, dose-dependent increase in TER similar to barrier-enhancing lipid such as sphingosine 1-phosphate (S1P). Optimal concentration of 7.5-9% PEG induced a robust increase in TER from 2,500 to 5,000 ohm, which was sustained for 40 hours. In comparison to S1P, which induced a 40% increase in resistance in 10-15 minutes, the maximum barrier enhancing effect of PEG was achieved in about 45 minutes, but with 100% increase in resistance. With a 2-fold increase in resistance, PEG is one of the most potent barrier-enhancing agents tested among all the barrier-enhancing agents, such as S1P, FTY720, phospho-FTY720, and HGF. Immunofluorescence data revealed that PEG altered the EC actin cytoskeleton to form a defined cortical actin ring that may help strengthen cell-cell junctional adhesion. PEG rapidly induced dephosphorylation of ERK and MLC as early as 1 minute and completely inhibited thrombin-induced ERK and MLC phosphorylation. More importantly, pretreatment with PEG for 1 hour attenuated thrombin-induced endothelial barrier dysfunction. In summary, PEG activates a rapid, actin-associated, barrier-enhancing signal transduction pathway in EC, which may have therapeutic potential to prevent and reverse pulmonary edema. Funded by HL058064 and GM062344.

Rationale Disruption of pulmonary vascular integrity occurs during inflammatory disease processes such as acute lung injury and sepsis, and as a result, regulation of this barrier is an important clinical objective. We have described rapid endothelial cell (EC) barrier enhancement by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P), mediated via S1P1 receptor (S1P1R) ligation. FTY720, an immunosuppressive drug currently in phase III clinical trials, is phosphorylated in vivo to form a structural analogue of S1P. We and others have demonstrated that FTY720 also induces EC barrier enhancement, and we now explore the role of S1P1R ligation in this process. Methods/Results FTY (1 μM) significantly increased cultured human pulmonary artery EC barrier function in a sustained manner as measured by transendothelial electrical resistance (TER), with a delayed onset and slower rate of rise relative to S1P (1 μM). Lipid extraction and 2-phase thin layer chromatography studies revealed that phosphorylation of FTY is not required for barrier enhancement. Both S1P- and FTY-induced barrier enhancement were abolished by pretreatment with pertussis toxin; however, reduction of S1P1R expression via siRNA significantly inhibited S1P-, but not FTY-induced TER elevation. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated threonine phosphorylation of S1P1R. Inhibition of this phosphorylation by preincubation with the PI3 kinase inhibitor LY294002 (10 μM) blocked S1P-, but not FTY-induced TER elevation. S1P, but not FTY, induced significant phosphorylation of ERK and myosin light chains within 5 min as detected by Western blotting and dramatic actin cytoskeletal rearrangement. Reduction of downstream effectors Rac1 or cortactin via siRNA strategies attenuated S1P-, but not FTY-induced TER elevation. Conclusion These results indicate that FTY720 augments EC barrier function through a novel, S1P1R-independent signaling pathway. HL58064, HL70013-01, FTY720 supplied by Novartis.

Rationale Acute lung injury (ALI) is a frequent complication of sepsis in which disruption of the pulmonary endothelial cell (EC) monolayer leads to increased vascular permeability and life-threatening pulmonary edema. Activated protein C (APC) reduces mortality in sepsis; however, the mechanism through which it exerts its protective effect remains undefined. We hypothesized that APC, via interaction with the endothelial protein C receptor (EPCR), and the EC cytoskeletal protein phosphorylated myosin light chain (phosphoMLC), enhances EC barrier function. Methods Transendothelial electrical resistance (TER) was measured in confluent human pulmonary artery EC monolayers cells treated with APC alone and with the addition of thrombin(0.2 U/mL). Activation of MLC via phosphorylation was assessed using Western blot analysis. Results APC dose-dependently attenuated the thrombin-induced reductions in TER at 1 hr and Western blot analysis of APC (1 μg/mL)-challenged EC demonstrated a 3 fold increase in phosphoMLC (5 min) compared to control. Addition of an EPCR blocking antibody abolished APC-induced barrier-protection and increases in phosphoMLC, while incubation with a control antibody did not alter APC-mediated changes in TER or phosphoMLC levels. Incubation with an antibody against the thrombin receptor, PAR1, had no effect on APC-mediated increases in phosphoMLC. Conclusion APC significantly reduces thrombin-induced barrier dysfunction in ECs via an EPCR-dependent mechanism. APC-mediated alterations in the cytoskeletal protein phosphoMLC was EPCR-dependent but independent of an interaction of APC with PAR1. Modulation of EC barrier function may play a crucial role in the improved survival in patients with sepsis and ALI treated with APC.