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1.  Pleiotropic effects of interleukin-6 in a “two-hit” murine model of acute respiratory distress syndrome 
Pulmonary Circulation  2014;4(2):280-288.
Patients with acute respiratory distress syndrome (ARDS) exhibit elevated levels of interleukin-6 (IL-6), which correlate with increased morbidity and mortality. The exact role of IL-6 in ARDS has proven difficult to study because it exhibits either pro- or anti-inflammatory actions in mouse models of lung injury, depending on the model utilized. In order to improve understanding of the role of this complex cytokine in ARDS, we evaluated IL-6 using the clinically relevant combination of lipopolysaccharide (LPS) and ventilator-induced lung injury (VILI) in IL-6−/− mice. Bronchoalveolar lavage fluid (BAL), whole-lung tissue, and histology were evaluated for inflammatory markers of injury. Transendothelial electrical resistance was used to evaluate the action of IL-6 on endothelial cells in vitro. In wild-type mice, the combination model showed a significant increase in lung injury compared to either LPS or VILI alone. IL-6−/− mice exhibited a statistically significant decrease in BAL cellular inflammation as well as lower histologic scores for lung injury, changes observed only in the combination model. A paradoxical increase in BAL total protein was observed in IL-6−/− mice exposed to LPS, suggesting that IL-6 provides protection from vascular leakage. However, in vitro data showed that IL-6, when combined with its soluble receptor, actually caused a significant increase in endothelial cell permeability, suggesting that the protection seen in vivo was likely due to complex interactions of IL-6 and other inflammatory mediators rather than to direct effects of IL-6. These studies suggest that a dual-injury model exhibits utility in evaluating the pleiotropic effects of IL-6 in ARDS on inflammatory cells and lung endothelium.
doi:10.1086/675991
PMCID: PMC4070787  PMID: 25006447
acute respiratory distress syndrome (ARDS); interleukin-6 (IL-6); lipopolysaccharide (LPS); ventilator-induced lung injury (VILI)
2.  MicroRNAs Implicated in Dysregulation of Gene Expression Following Human Lung Transplantation 
Translational respiratory medicine  2013;1(1):10.1186/2213-0802-1-12.
Background
Lung transplantation remains the only viable treatment option for the majority of patients with advanced lung diseases. However, 5-year post-transplant survival rates remain low primarily secondary to chronic rejection. Novel insights from global gene expression profiles may provide molecular phenotypes and therapeutic targets to improve outcomes after lung transplantation.
Methods
Whole-genome gene expression profiling was performed in a cohort of patients that underwent lung transplantation as well as healthy controls using the Affymetrix Human Exon 1.0ST Array. To explore the potential roles of microRNAs (miRNAs) in regulating lung transplantation-associated gene dysregulation, miRNA expression levels were also profiled in the same samples using the Exiqon miRCURY™ LNA Array.
Results
In a cohort of 18 lung transplant patients, 364 dysregulated genes were identified in Caucasian lung transplant patients relative to normal individuals. Pathway enrichment analysis of the dysregulated genes pointed to Gene Ontology biological processes such as “defense response”, “immune response” and “response to wounding”. We then compared the expression profiles of potential regulating miRNAs, suggesting that dysregulation of a number of lung transplantation-associated genes (e.g., ATR, FUT8, LRRC8B, NFKBIA) may be attributed to the dysregulation of their respective regulating miRNAs.
Conclusions
Following human lung transplantation, a substantial proportion of genes, particularly those genes involved in certain biological processes like immune response, were dysregulated in patients relative to their healthy counterparts. This exploratory analysis of the relationships between miRNAs and their gene targets in the context of lung transplantation warrants further investigation and may serve as novel therapeutic targets in lung transplant complications.
doi:10.1186/2213-0802-1-12
PMCID: PMC3886917  PMID: 24416715
lung transplant; gene expression; microRNA; pathway; gene ontology
3.  Sphingosine-1–Phosphate Receptor–3 Is a Novel Biomarker in Acute Lung Injury 
The inflamed lung exhibits oxidative and nitrative modifications of multiple target proteins, potentially reflecting disease severity and progression. We identified sphingosine-1–phosphate receptor–3 (S1PR3), a critical signaling molecule mediating cell proliferation and vascular permeability, as a nitrated plasma protein in mice with acute lung injury (ALI). We explored S1PR3 as a potential biomarker in murine and human ALI. In vivo nitrated and total S1PR3 concentrations were determined by immunoprecipitation and microarray studies in mice, and by ELISA in human plasma. In vitro nitrated S1PR3 concentrations were evaluated in human lung vascular endothelial cells (ECs) or within microparticles shed from ECs after exposure to barrier-disrupting agonists (LPS, low-molecular-weight hyaluronan, and thrombin). The effects of S1PR3-containing microparticles on EC barrier function were assessed by transendothelial electrical resistance (TER). Nitrated S1PR3 was identified in the plasma of murine ALI and in humans with severe sepsis-induced ALI. Elevated total S1PR3 plasma concentrations (> 251 pg/ml) were linked to sepsis and ALI mortality. In vitro EC exposure to barrier-disrupting agents induced S1PR3 nitration and the shedding of S1PR3-containing microparticles, which significantly reduced TER, consistent with increased permeability. These changes were attenuated by reduced S1PR3 expression (small interfering RNAs). These results suggest that microparticles containing nitrated S1PR3 shed into the circulation during inflammatory lung states, and represent a novel ALI biomarker linked to disease severity and outcome.
doi:10.1165/rcmb.2012-0048OC
PMCID: PMC3547106  PMID: 22771388
acute lung injury; sphingosine-1–phosphate receptor–3; microparticles; nitration; biomarker
4.  Hydrogen Sulfide Attenuates Particulate Matter–Induced Human Lung Endothelial Barrier Disruption via Combined Reactive Oxygen Species Scavenging and Akt Activation 
Exposure to particulate air pollution is associated with increased cardiopulmonary morbidity and mortality, although the pathogenic mechanisms are poorly understood. We previously demonstrated that particulate matter (PM) exposure triggers massive oxidative stress in vascular endothelial cells (ECs), resulting in the loss of EC integrity and lung vascular hyperpermeability. We investigated the protective role of hydrogen sulfide (H2S), an endogenous gaseous molecule present in the circulation, on PM-induced human lung EC barrier disruption and pulmonary inflammation. Alterations in EC monolayer permeability, as reflected by transendothelial electrical resistance (TER), the generation of reactive oxygen species (ROS), and murine pulmonary inflammatory responses, were studied after exposures to PM and NaSH, an H2S donor. Similar to N-acetyl cysteine (5 mM), NaSH (10 μM) significantly scavenged PM-induced EC ROS and inhibited the oxidative activation of p38 mitogen-activated protein kinase. Concurrent with these events, NaSH (10 μM) activated Akt, which helps maintain endothelial integrity. Both of these pathways contribute to the protective effect of H2S against PM-induced endothelial barrier dysfunction. Furthermore, NaSH (20 mg/kg) reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in bronchoalveolar lavage fluids in a murine model of PM-induced lung inflammation. These data suggest a potentially protective role for H2S in PM-induced inflammatory lung injury and vascular hyperpermeability.
doi:10.1165/rcmb.2011-0248OC
PMCID: PMC3488621  PMID: 22592920
particulate matter; hydrogen sulfide; endothelial permeability; Akt
5.  A Common Cortactin Gene Variation Confers Differential Susceptibility to Severe Asthma 
Genetic epidemiology  2008;32(8):757-766.
Genomic regions with replicated linkage to asthma-related phenotypes likely harbor multiple susceptibility loci with relatively minor effects on disease susceptibility. The 11q13 chromosomal region has repeatedly been linked to asthma with five genes residing in this region with reported replicated associations. Cortactin, an actin-binding protein encoded by the CTTN gene in 11q13, constitutes a key regulator of cytoskeletal dynamics and contractile cell machinery, events facilitated by interaction with myosin light chain kinase; encoded by MYLK, a gene we recently reported as associated with severe asthma in African Americans. To evaluate potential association of CTTN gene variation with asthma susceptibility, CTTN exons and flanking regions were re-sequenced in 48 non-asthmatic multiethnic samples, leading to selection of nine tagging polymorphisms for case-control association studies in individuals of European and African descent. After ancestry adjustments, an intronic variant (rs3802780) was significantly associated with severe asthma (odds ratio [OR]: 1.71; 95% confidence interval [CI]: 1.20-2.43; p = 0.003) in a joint analysis. Further analyses evidenced independent and additive effects of CTTN and MYLK risk variants for severe asthma susceptibility in African Americans (accumulated OR: 2.93, 95% CI: 1.40-6.13, p = 0.004). These data suggest that CTTN gene variation may contribute to severe asthma and that the combined effects of CTTN and MYLK risk polymorphisms may further increase susceptibility to severe asthma in African Americans harboring both genetic variants.
doi:10.1002/gepi.20343
PMCID: PMC3774307  PMID: 18521921
CTTN; MLCK; cytoskeleton; SNP; asthma
6.  PHOSPHOTYROSINE PROTEIN DYNAMICS IN CELL MEMBRANE RAFTS OF SPHINGOSINE-1-PHOSPHATE-STIMULATED HUMAN ENDOTHELIUM: ROLE IN BARRIER ENHANCEMENT 
Cellular signalling  2009;21(12):1945-1960.
Sphingosine-1-phosphate (S1P), a lipid growth factor, is critical to the maintenance and enhancement of vascular barrier function via processes highly dependent upon cell membrane raft-mediated signaling events. Anti-phosphotyrosine 2 dimensional gel electrophoresis (2-DE) immunoblots confirmed that disruption of membrane rafts formation (via methyl-β-cyclodextrin) inhibits S1P-induced protein tyrosine phosphorylation. To explore S1P-induced dynamic changes in membrane rafts, we used 2-D techniques to define proteins within detergent-resistant cell membrane rafts which are differentially expressed in S1P-challenged (13M, 5 min) human pulmonary artery endothelial cells (EC), with 57 protein spots exhibiting >3-fold change. S1P-induced the recruitment of over 20 cell membrane raft proteins exhibiting increasing levels of tyrosine phosphorylation including known barrier-regulatory proteins such as focal adhesion kinase (FAK), cortactin, p85α phosphatidylinositol 3-kinase (p85αPI3K), myosin light chain kinase (nmMLCK), filamin A/C, and the non-receptor tyrosine kinase, c-Abl. Reduced expression of either FAK, MLCK, cortactin, filamin A or filamin C by siRNA transfection significantly attenuated S1P-induced EC barrier enhancement. Furthermore, S1P induced cell membrane raft components, p-caveolin-1 and glycosphingolipid (GM1), to the plasma membrane and enhanced co-localization of membrane rafts with p-caveolin-1 and p-nmMLCK. These results suggest that S1P induces both the tyrosine phosphorylation and recruitment of key actin cytoskeletal proteins to membrane rafts, resulting in enhanced human EC barrier function.
doi:10.1016/j.cellsig.2009.09.002
PMCID: PMC3758232  PMID: 19755153
endothelial cells; cell membrane rafts; 2-DE; Mass spectrometry; sphingosine 1-phosphate (S1P)
7.  Protective Effects of High-Molecular Weight Polyethylene Glycol (PEG) in Human Lung Endothelial Cell Barrier Regulation: Role of Actin Cytoskeletal Rearrangement 
Microvascular research  2008;77(2):174-186.
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.
doi:10.1016/j.mvr.2008.11.007
PMCID: PMC3736723  PMID: 19121327
PEG; LPS; thrombin; endothelium; barrier function
8.  HMGB1 induces human lung endothelial cell cytoskeletal rearrangement and barrier disruption 
Microvascular research  2010;81(2):189-197.
Acute lung injury (ALI) results from loss of alveolar-capillary barrier integrity and the evolution of high-permeability pulmonary edema resulting in alveolar flooding and significant morbidity and mortality. HMGB1 is a late mediator of sepsis which uniquely participates in the evolution of sepsis and sepsis-induced ALI. The molecular events by which HMGB1 contributes to ALI remain poorly characterized. We characterized the role of HMGB1 in endothelial cell (EC) cytoskeletal rearrangement and vascular permeability, events essential to paracellular gap formation and barrier dysfunction characteristic of ALI. Initial experiments demonstrated HMGB1-mediated dose-dependent (5–20 μg/ml) decreases in transendothelial cell electrical resistance (TER) in human pulmonary artery EC, a reflection of loss of barrier integrity. Furthermore, HMGB1 produced dose-dependent increases in paracellular gap formation in concert with loss of peripheral organized actin fibers, dissociation of cell-cell junctional cadherins, and development of central stress fibers, a phenotypic change associated with increased contractile activity and increased EC permeability. Using siRNA strategies directed against known HMGB1 receptors (RAGE, TLR2, TLR4), we systematically determined that the receptor for advanced glycation end products (RAGE) is the primary receptor signaling HMGB1-induced TER decreases and paracellular gap formation via p38 MAP kinase activation and phosphorylation of the actin-binding protein, Hsp27. These studies add to understanding of HMGB1-induced inflammatory events and vascular barrier disruption and offer the potential for clinical intervention in sepsis-induced ALI.
doi:10.1016/j.mvr.2010.11.010
PMCID: PMC3678727  PMID: 21146549
HMGB1; RAGE; acute lung injury; endothelium; MAP kinase; Hsp27
9.  Integrating genomic and clinical medicine: Searching for susceptibility genes in complex lung diseases 
The integration of molecular, genomic, and clinical medicine in the post-genome era provides the promise of novel information on genetic variation and pathophysiologic cascades. The current challenge is to translate these discoveries rapidly into viable biomarkers that identify susceptible populations and into the development of precisely targeted therapies. In this article, we describe the application of comparative genomics, microarray platforms, genetic epidemiology, statistical genetics, and bioinformatic approaches within examples of complex pulmonary pathobiology. Our search for candidate genes, which are gene variations that drive susceptibility to and severity of enigmatic acute and chronic lung disorders, provides a logical framework to understand better the evolution of genomic medicine. The dissection of the genetic basis of complex diseases and the development of highly individualized therapies remain lofty but achievable goals.
doi:10.1016/j.trsl.2007.10.005
PMCID: PMC3616408  PMID: 18355765
10.  Conflicting Physiological and Genomic Cardiopulmonary Effects of Recruitment Maneuvers in Murine Acute Lung Injury 
Low tidal volume ventilation, although promoting atelectasis, is a protective strategy against ventilator-induced lung injury. Deep inflation (DI) recruitment maneuvers restore lung volumes, but potentially compromise lung parenchymal and vascular function via repetitive overdistention. Our objective was to examine cardiopulmonary physiological and transcriptional consequences of recruitment maneuvers. C57/BL6 mice challenged with either PBS or LPS via aspiration were placed on mechanical ventilation (5 h) using low tidal volume inflation (TI; 8 μl/g) alone or in combination with intermittent DIs (0.75 ml twice/min). Lung mechanics during TI ventilation significantly deteriorated, as assessed by forced oscillation technique and pressure–volume curves. DI mitigated the TI-induced alterations in lung mechanics, but induced a significant rise in right ventricle systolic pressures and pulmonary vascular resistances, especially in LPS-challenged animals. In addition, DI exacerbated the LPS-induced genome-wide lung inflammatory transcriptome, with prominent dysregulation of a gene cluster involving vascular processes, as well as increases in cytokine concentrations in bronchoalveolar lavage fluid and plasma. Gene ontology analyses of right ventricular tissue expression profiles also identified inflammatory signatures, as well as apoptosis and membrane organization ontologies, as potential elements in the response to acute pressure overload. Our results, although confirming the improvement in lung mechanics offered by DI, highlight a detrimental impact in sustaining inflammatory response and exacerbating lung vascular dysfunction, events contributing to increases in right ventricle afterload. These novel insights should be integrated into the clinical assessment of the risk/benefit of recruitment maneuver strategies.
doi:10.1165/rcmb.2011-0306OC
PMCID: PMC3359949  PMID: 22135358
mechanical ventilation; microarray; pulmonary hypertension; right ventricle; acute lung injury
11.  GADD45a Promoter Regulation by a Functional Genetic Variant Associated with Acute Lung Injury 
PLoS ONE  2014;9(6):e100169.
Rationale
Growth arrest DNA damage inducible alpha (GADD45a) is a stress-induced gene we have shown to participate in the pathophysiology of ventilator-induced lung injury (VILI) via regulation of mechanical stress-induced Akt ubiquitination and phosphorylation. The regulation of GADD45a expression by mechanical stress and its relationship with acute lung injury (ALI) susceptibility and severity, however, remains unknown.
Objectives
We examined mechanical stress-dependent regulatory elements (MSRE) in the GADD45a promoter and the contribution of promoter polymorphisms in GADD45a expression and ALI susceptibility.
Methods and Results
Initial studies in GADD45a knockout and heterozygous mice confirmed the relationship of GADD45a gene dose to VILI severity. Human lung endothelial cells (EC) transfected with a luciferase vector containing the full length GADD45a promoter sequence (−771 to +223) demonstrated a >4 fold increase in GADD45a expression in response to 18% cyclic stretch (CS, 4 h) compared to static controls while specific promoter regions harboring CS-dependent MSRE were identified using vectors containing serial deletion constructs of the GADD45a promoter. In silico analyses of GADD45a promoter region (−371 to −133) revealed a potential binding site for specificity protein 1 (SP1), a finding supported by confirmed SP1 binding with the GADD45a promoter and by the significant attenuation of CS-dependent GADD45a promoter activity in response to SP1 silencing. Separately, case-control association studies revealed a significant association of a GADD45a promoter SNP at −589 (rs581000, G>C) with reduced ALI susceptibility. Subsequently, we found allelic variation of this SNP is associated with both differential GADD45a expression in mechanically stressed EC (18% CS, 4 h) and differential binding site of interferon regulatory factor 7 (IRF7) at this site.
Conclusion
These results strongly support a functional role for GADD45a in ALI/VILI and identify a specific gene variant that confers risk for ALI.
doi:10.1371/journal.pone.0100169
PMCID: PMC4062486  PMID: 24940746
12.  Genetic variants associated with idiopathic pulmonary fibrosis susceptibility and mortality: a genome-wide association study 
The lancet. Respiratory medicine  2013;1(4):309-317.
Summary
Background
Idiopathic pulmonary fibrosis (IPF) is a devastating disease that probably involves several genetic loci. Several rare genetic variants and one common single nucleotide polymorphism (SNP) of MUC5B have been associated with the disease. Our aim was to identify additional common variants associated with susceptibility and ultimately mortality in IPF.
Methods
First, we did a three-stage genome-wide association study (GWAS): stage one was a discovery GWAS; and stages two and three were independent case-control studies. DNA samples from European-American patients with IPF meeting standard criteria were obtained from several US centres for each stage. Data for European-American control individuals for stage one were gathered from the database of genotypes and phenotypes; additional control individuals were recruited at the University of Pittsburgh to increase the number. For controls in stages two and three, we gathered data for additional sex-matched European-American control individuals who had been recruited in another study. DNA samples from patients and from control individuals were genotyped to identify SNPs associated with IPF. SNPs identified in stage one were carried forward to stage two, and those that achieved genome-wide significance (p<5 × 10−8) in a meta-analysis were carried forward to stage three. Three case series with follow-up data were selected from stages one and two of the GWAS using samples with follow-up data. Mortality analyses were done in these case series to assess the SNPs associated with IPF that had achieved genome-wide significance in the meta-analysis of stages one and two. Finally, we obtained gene-expression profiling data for lungs of patients with IPF from the Lung Genomics Research Consortium and analysed correlation with SNP genotypes.
Findings
In stage one of the GWAS (542 patients with IPF, 542 control individuals matched one-by-one to cases by genetic ancestry estimates), we identified 20 loci. Six SNPs reached genome-wide significance in stage two (544 patients, 687 control individuals): three TOLLIP SNPs (rs111521887, rs5743894, rs5743890) and one MUC5B SNP (rs35705950) at 11p15.5; one MDGA2 SNP (rs7144383) at 14q21.3; and one SPPL2C SNP (rs17690703) at 17q21.31. Stage three (324 patients, 702 control individuals) confirmed the associations for all these SNPs, except for rs7144383. Linkage disequilibrium between the MUC5B SNP (rs35705950) and TOLLIP SNPs (rs111521887 [r2=0.07], rs5743894 [r2=0.16], and rs5743890 [r2=0.01]) was low. 683 patients from the GWAS were included in the mortality analysis. Individuals who developed IPF despite having the protective TOLLIP minor allele of rs5743890 carried an increased mortality risk (meta-analysis with fixed-effect model: hazard ratio 1.72 [95% CI 1.24–2.38]; p=0.0012). TOLLIP expression was decreased by 20% in individuals carrying the minor allele of rs5743890 (p=0.097), 40% in those with the minor allele of rs111521887 (p=3.0 × 10−4), and 50% in those with the minor allele of rs5743894 (p=2.93 × 10−5) compared with homozygous carriers of common alleles for these SNPs.
Interpretation
Novel variants in TOLLIP and SPPL2C are associated with IPF susceptibility. One novel variant of TOLLIP, rs5743890, is also associated with mortality. These associations and the reduced expression of TOLLIP in patients with IPF who carry TOLLIP SNPs emphasise the importance of this gene in the disease.
Funding
National Institutes of Health; National Heart, Lung, and Blood Institute; Pulmonary Fibrosis Foundation; Coalition for Pulmonary Fibrosis; and Instituto de Salud Carlos III.
doi:10.1016/S2213-2600(13)70045-6
PMCID: PMC3894577  PMID: 24429156
13.  Type 2 Deiodinase and Host Responses of Sepsis and Acute Lung Injury 
The role of thyroid hormone metabolism in clinical outcomes of the critically ill remains unclear. Using preclinical models of acute lung injury (ALI), we assessed the gene and protein expression of type 2 deiodinase (DIO2), a key driver for synthesis of biologically active triiodothyronine, and addressed potential association of DIO2 genetic variants with ALI in a multiethnic cohort. DIO2 gene and protein expression levels in murine lung were validated by microarrays and immunoblotting. Lung injury was assessed by levels of bronchoalveolar lavage protein and leukocytes. Single-nucleotide polymorphisms were genotyped and ALI susceptibility association assessed. Significant increases in both DIO2 gene and D2 protein expression were observed in lung tissues from murine ALI models (LPS- and ventilator-induced lung injury), with expression directly increasing with the extent of lung injury. Mice with reduced levels of DIO2 expression (by silencing RNA) demonstrated reduced thyroxine levels in plasma and increased lung injury (increased bronchoalveolar lavage protein and leukocytes), suggesting a protective role for DIO2 in ALI. The G (Ala) allele of the Thr92Ala coding single-nucleotide polymorphism (rs225014) was protective in severe sepsis and severe sepsis–associated ALI after adjustments for age, sex, and genetic ancestry in a logistic regression model in European Americans. Our studies indicate that DIO2 is a novel ALI candidate gene, the nonsynonymous Thr92Ala coding variant of which confers ALI protection. Increased DIO2 expression may dampen the ALI inflammatory response, thereby strengthening the premise that thyroid hormone metabolism is intimately linked to the integrated response to inflammatory injury in critically ill patients.
doi:10.1165/rcmb.2011-0179OC
PMCID: PMC3262665  PMID: 21685153
acute respiratory distress syndrome; hypothyroidism; mechanical ventilation; sepsis
14.  Functional variants of sphingosine-1-phosphate receptor 1 gene associate with asthma susceptibility 
Background
The genetic mechanisms underlying asthma remain unclear. Increased permeability of the microvasculature is a feature of asthma and the sphingosine-1-phosphate receptor, S1PR1, is an essential participant regulating lung vascular integrity and responses to lung inflammation.
Objective
We explored the contribution of polymorphisms in the S1PR1 gene (S1PR1) to asthma susceptibility.
Methods
A combination of gene re-sequencing for SNP discovery, case-control association, functional evaluation of associated SNPs, and protein immunochemistry studies was utilized.
Results
Immunohistochemistry studies demonstrated significantly decreased S1PR1 protein expression in pulmonary vessels in asthmatic lungs compared to non-asthmatic individuals (p<0.05). Direct DNA sequencing of 27 multiethnic samples identified 39 S1PR1 variants (18 novel SNPs). Association studies were performed based on genotyping results from cosmopolitan tagging SNPs in three case-control cohorts from Chicago and New York totaling 1061 subjects (502 cases and 559 controls). Promoter SNP rs2038366 (−1557G/T) was found to be associated with asthma (p=0.03) in European Americans. In African Americans, an association was found for both asthma and severe asthma for intronic SNP rs3753194 (c.−164+170A/G) (p=0.006 and p=0.040, respectively) and for promoter SNP rs59317557 (−532C/G) with severe asthma (p=0.028). Consistent with predicted in silico functionality, alleles of promoter SNPs rs2038366 (−1557G/T) and rs59317557 (−532C/G) influenced the activity of a luciferase S1PR1 reporter vector in transfected endothelial cells exposed to growth factors (EGF, PDGF, VEGF) known to be increased in asthmatic airways.
Conclusion
These data provide strong support for a role for S1PR1 gene variants in asthma susceptibility and severity.
Clinical Implications
Our results indicate S1PR1 is a novel asthma candidate gene and an attractive target for future therapeutic strategies.
Capsule summary
This study identified novel polymorphisms in S1PR1, revealed the functional implications of S1PR1 genetic variants in different populations, and their association with asthma susceptibility and severity.
doi:10.1016/j.jaci.2010.04.036
PMCID: PMC3495167  PMID: 20624651
asthma; sphingosine-1-phosphate receptor 1; single nucleotide polymorphism; promoter activity
15.  A Sphingosine 1–Phosphate 1 Receptor Agonist Modulates Brain Death–Induced Neurogenic Pulmonary Injury 
Lung transplantation remains the only viable therapy for patients with end-stage lung disease. However, the full utilization of this strategy is severely compromised by a lack of donor lung availability. The vast majority of donor lungs available for transplantation are from individuals after brain death (BD). Unfortunately, the early autonomic storm that accompanies BD often results in neurogenic pulmonary edema (NPE), producing varying degrees of lung injury or leading to primary graft dysfunction after transplantation. We demonstrated that sphingosine 1–phosphate (S1P)/analogues, which are major barrier-enhancing agents, reduce vascular permeability via the S1P1 receptor, S1PR1. Because primary lung graft dysfunction is induced by lung vascular endothelial cell barrier dysfunction, we hypothesized that the S1PR1 agonist, SEW-2871, may attenuate NPE when administered to the donor shortly after BD. Significant lung injury was observed after BD, with increases of approximately 60% in bronchoalveolar lavage (BAL) total protein, cell counts, and lung tissue wet/dry (W/D) weight ratios. In contrast, rats receiving SEW-2871 (0.1 mg/kg) 15 minutes after BD and assessed after 4 hours exhibited significant lung protection (∼ 50% reduction, P = 0.01), as reflected by reduced BAL protein/albumin, cytokines, cellularity, and lung tissue wet/dry weight ratio. Microarray analysis at 4 hours revealed a global impact of both BD and SEW on lung gene expression, with a differential gene expression of enriched immune-response/inflammation pathways across all groups. Overall, SEW served to attenuate the BD-mediated up-regulation of gene expression. Two potential biomarkers, TNF and chemokine CC motif receptor-like 2, exhibited gene array dysregulation. We conclude that SEW-2871 significantly attenuates BD-induced lung injury, and may serve as a potential candidate to improve human donor availability.
doi:10.1165/rcmb.2010-0267OC
PMCID: PMC3262681  PMID: 21617203
neurogenic pulmonary edema; lung injury; sphingosine 1–phosphate; sphingolipids; lung transplant donors
16.  Genomic Investigations into Acute Inflammatory Lung Injury 
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are complex illnesses involving the interplay of both environmental (such as mechanical ventilation) and genetic factors. To understand better the underlying mechanisms of pathogenesis associated with ALI, we recently identified several candidate genes by global expression profiling in preclinical models of ALI and relevant single-nucleotide polymorphisms. We summarize here several strategies successfully used to identify novel ALI candidate genes and detail the validation of variants in these genes as contributing factors to ALI pathobiology, conclusions based on functional analyses, and specific genetic association studies conducted in ALI cohorts. Continued insights into ALI pathogenesis and identification of genetic variants, which confer ALI risk and severity, promise to reveal novel molecular therapeutic targets that can be translated into personalized treatments to reduce the very high, unacceptable mortality of this disorder.
doi:10.1513/pats.201101-002MS
PMCID: PMC3131835  PMID: 21543796
acute lung injury; SNP; microarray; PBEF; inflammation
17.  Simvastatin Attenuates Radiation-Induced Murine Lung Injury and Dysregulated Lung Gene Expression 
Novel therapies are desperately needed for radiation-induced lung injury (RILI), which, despite aggressive corticosteroid therapy, remains a potentially fatal and dose-limiting complication of thoracic radiotherapy. We assessed the utility of simvastatin, an anti-inflammatory and lung barrier–protective agent, in a dose- and time-dependent murine model of RILI (18–(25 Gy). Simvastatin reduced multiple RILI indices, including vascular leak, leukocyte infiltration, and histological evidence of oxidative stress, while reversing RILI-associated dysregulated gene expression, including p53, nuclear factor–erythroid-2–related factor, and sphingolipid metabolic pathway genes. To identify key regulators of simvastatin-mediated RILI protection, we integrated whole-lung gene expression data obtained from radiated and simvastatin-treated mice with protein–protein interaction network analysis (single-network analysis of proteins). Topological analysis of the gene product interaction network identified eight top-prioritized genes (Ccna2a, Cdc2, fcer1 g, Syk, Vav3, Mmp9, Itgam, Cd44) as regulatory nodes within an activated RILI network. These studies identify the involvement of specific genes and gene networks in RILI pathobiology, and confirm that statins represent a novel strategy to limit RILI.
doi:10.1165/rcmb.2010-0122OC
PMCID: PMC3095940  PMID: 20508068
radiation pneumonitis; lung vascular permeability; simvastatin; gene dysregulation; protein–protein interaction
18.  Genes Influenced by the Non-Muscle Isoform of Myosin Light Chain Kinase Impact Human Cancer Prognosis 
PLoS ONE  2014;9(4):e94325.
The multifunctional non-muscle isoform of myosin light chain kinase (nmMLCK) is critical to the rapid dynamic coordination of the cytoskeleton involved in cancer cell proliferation and migration. We identified 45 nmMLCK-influenced genes by bioinformatic filtering of genome–wide expression in wild type and nmMLCK knockout (KO) mice exposed to preclinical models of murine acute inflammatory lung injury, pathologies that are well established to include nmMLCK as an essential participant. To determine whether these nmMLCK-influenced genes were relevant to human cancers, the 45 mouse genes were matched to 38 distinct human orthologs (M38 signature) (GeneCards definition) and underwent Kaplan-Meier survival analysis in training and validation cohorts. These studies revealed that in training cohorts, the M38 signature successfully identified cancer patients with poor overall survival in breast cancer (P<0.001), colon cancer (P<0.001), glioma (P<0.001), and lung cancer (P<0.001). In validation cohorts, the M38 signature demonstrated significantly reduced overall survival for high-score patients of breast cancer (P = 0.002), colon cancer (P = 0.035), glioma (P = 0.023), and lung cancer (P = 0.023). The association between M38 risk score and overall survival was confirmed by univariate Cox proportional hazard analysis of overall survival in the both training and validation cohorts. This study, providing a novel prognostic cancer gene signature derived from a murine model of nmMLCK-associated lung inflammation, strongly supports nmMLCK-involved pathways in tumor growth and progression in human cancers and nmMLCK as an attractive candidate molecular target in both inflammatory and neoplastic processes.
doi:10.1371/journal.pone.0094325
PMCID: PMC3979809  PMID: 24714365
19.  Non–Muscle Myosin Light Chain Kinase Isoform Is a Viable Molecular Target in Acute Inflammatory Lung Injury 
Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non–muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (∼50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody–conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6–signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation.
doi:10.1165/rcmb.2009-0197OC
PMCID: PMC3028257  PMID: 20139351
endotoxin/lipopolysaccharide; nmMLCK; mice; lung injury; endothelial barrier
20.  Differential Effects of Sphingosine 1–Phosphate Receptors on Airway and Vascular Barrier Function in the Murine Lung 
The therapeutic options for ameliorating the profound vascular permeability, alveolar flooding, and organ dysfunction that accompanies acute inflammatory lung injury (ALI) remain limited. Extending our previous finding that the intravenous administration of the sphingolipid angiogenic factor, sphingosine 1–phosphate (S1P), attenuates inflammatory lung injury and vascular permeability via ligation of S1PR1, we determine that a direct intratracheal or intravenous administration of S1P, or a selective S1P receptor (S1PR1) agonist (SEW-2871), produces highly concentration-dependent barrier-regulatory responses in the murine lung. The intratracheal or intravenous administration of S1P or SEW-2871 at < 0.3 mg/kg was protective against LPS-induced murine lung inflammation and permeability. However, intratracheal delivery of S1P at 0.5 mg/kg (for 2 h) resulted in significant alveolar–capillary barrier disruption (with a 42% increase in bronchoalveolar lavage protein), and produced rapid lethality when delivered at 2 mg/kg. Despite the greater selectivity for S1PR1, intratracheally delivered SEW-2871 at 0.5 mg/kg also resulted in significant alveolar–capillary barrier disruption, but was not lethal at 2 mg/kg. Consistent with the S1PR1 regulation of alveolar/vascular barrier function, wild-type mice pretreated with the S1PR1 inverse agonist, SB-649146, or S1PR1+/− mice exhibited reduced S1P/SEW-2871–mediated barrier protection after challenge with LPS. In contrast, S1PR2−/− knockout mice as well as mice with reduced S1PR3 expression (via silencing S1PR3-containing nanocarriers) were protected against LPS-induced barrier disruption compared with control mice. These studies underscore the potential therapeutic effects of highly selective S1PR1 receptor agonists in reducing inflammatory lung injury, and highlight the critical role of the S1P delivery route, S1PR1 agonist concentration, and S1PR1 expression in target tissues.
doi:10.1165/rcmb.2009-0223OC
PMCID: PMC2951871  PMID: 19749179
SEW-2871; LPS; SB-649146; S1P; lung edema
21.  Nontuberculous Mycobacterial Disease Mortality in the United States, 1999–2010: A Population-Based Comparative Study 
PLoS ONE  2014;9(3):e91879.
Background
Environmental nontuberculous mycobacteria (NTM) are ubiquitous organisms with which humans commonly interact. The epidemiologic characteristics of NTM diseases including mortality rate and its associated factors remain largely unknown. In this study, we explored the geographical area of exposure and mortality and comorbid conditions of affected persons to determine environment, host, and host-pathogen interactive factors.
Methods
We analyzed mortality related to nontuberculous mycobacterial infections from 1999 through 2010 by examining multiple-cause-of-death data from the National Center for Health Statistics. Among those who died with these diseases, we analyzed age-adjusted mortality rates, trends, associations with demographic variables, and comorbid conditions and correlated this information with similar data for tuberculosis-related mortality during the same time.
Measurements and Mean Results
From 1999 through 2010, nontuberculous mycobacterial disease was reported as an immediate cause of death in 2,990 people in the United States with a combined overall mean age-adjusted mortality rate of 0.1 per 100,000 person-years. A significant increase in the number of NTM related deaths was seen from 1999 through 2010 (R2 = 0.72, p<0.0001), but it was not significant after adjustment for age. Persons aged 55 years and older, women, those living in Hawaii and Louisiana, and those of non-Hispanic, white ethnicity had higher mortality rates. Compared to tuberculosis-related mortality, chronic obstructive pulmonary disease, bronchiectasis, HIV, interstitial lung diseases, and tobacco use were significantly more common in persons with nontuberculous mycobacteria-related deaths.
Conclusions
Nontuberculous mycobacteria-related death numbers are rising and are unevenly distributed. The strong association of nontuberculous mycobacterial disease with age suggests that its prevalence will increase as the United States population ages.
doi:10.1371/journal.pone.0091879
PMCID: PMC3954860  PMID: 24632814
22.  Particulate Matter Disrupts Human Lung Endothelial Barrier Integrity via ROS- and p38 MAPK–Dependent Pathways 
Epidemiologic studies have linked exposure to airborne pollutant particulate matter (PM) with increased cardiopulmonary mortality and morbidity. The mechanisms of PM-mediated lung pathophysiology, however, remain unknown. We tested the hypothesis that PM, via enhanced oxidative stress, disrupts lung endothelial cell (EC) barrier integrity, thereby enhancing organ dysfunction. Using PM collected from Ft. McHenry Tunnel (Baltimore, MD), we assessed PM-mediated changes in transendothelial electrical resistance (TER) (a highly sensitive measure of barrier function), reactive oxygen species (ROS) generation, and p38 mitogen-activated protein kinase (MAPK) activation in human pulmonary artery EC. PM induced significant dose (10–100 μg/ml)- and time (0–10 h)-dependent EC barrier disruption reflected by reduced TER values. Exposure of human lung EC to PM resulted in significant ROS generation, which was directly involved in PM-mediated EC barrier dysfunction, as N-acetyl-cysteine (NAC, 5 mM) pretreatment abolished both ROS production and barrier disruption induced by PM. Furthermore, PM induced p38 MAPK activation and HSP27 phosphorylation, events that were both attenuated by NAC. In addition, PM-induced EC barrier disruption was partially prevented by the p38 MAP kinase inhibitor SB203580 (10 μM) as well as by reduced expression of either p38 MAPK β or HSP27 (siRNA). These results demonstrate that PM induces ROS generation in human lung endothelium, resulting in oxidative stress–mediated EC barrier disruption via p38 MAPK- and HSP27-dependent pathways. These findings support a novel mechanism for PM-induced lung dysfunction and adverse cardiopulmonary outcomes.
doi:10.1165/rcmb.2008-0402OC
PMCID: PMC2848737  PMID: 19520919
endothelial permeability; HSP27; particulate matter; p38 MAP kinase; ROS
23.  Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function 
Molecular Biology of the Cell  2010;21(22):4042-4056.
This study identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy and examined their influence on nmMLCK function and human lung endothelial barrier regulation. The data indicate an essential role for Abl kinase in vascular barrier regulation via phosphorylation of nmMLCK and the actin-binding protein cortactin.
Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.
doi:10.1091/mbc.E09-10-0876
PMCID: PMC2982111  PMID: 20861316
24.  A Novel Angiotensin I-Converting Enzyme Mutation (S333W) Impairs N-Domain Enzymatic Cleavage of the Anti-Fibrotic Peptide, AcSDKP 
PLoS ONE  2014;9(2):e88001.
Background
Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis.
Results
We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE.
Conclusions and Significance
A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis.
doi:10.1371/journal.pone.0088001
PMCID: PMC3913711  PMID: 24505347
25.  Essential Role of Pre–B-Cell Colony Enhancing Factor in Ventilator-induced Lung Injury 
Rationale: We previously demonstrated pre–B-cell colony enhancing factor (PBEF) as a biomarker in sepsis and sepsis-induced acute lung injury (ALI) with genetic variants conferring ALI susceptibility.
Objectives: To explore mechanistic participation of PBEF in ALI and ventilator-induced lung injury (VILI).
Methods: Two models of VILI were utilized to explore the role of PBEF using either recombinant PBEF or PBEF+/− mice.
Measurements and Main Results: Initial in vitro studies demonstrated recombinant human PBEF (rhPBEF) as a direct rat neutrophil chemotactic factor with in vivo studies demonstrating marked increases in bronchoalveolar lavage (BAL) leukocytes (PMNs) after intratracheal injection in C57BL/6J mice. These changes were accompanied by increased BAL levels of PMN chemoattractants (KC and MIP-2) and modest increases in lung vascular and alveolar permeability. We next explored the potential synergism between rhPBEF challenge (intratracheal) and a model of limited VILI (4 h, 30 ml/kg tidal volume) and observed dramatic increases in BAL PMNs, BAL protein, and cytokine levels (IL-6, TNF-α, KC) compared with either challenge alone. Gene expression profiling identified induction of ALI- and VILI-associated gene modules (nuclear factor-κB, leukocyte extravasation, apoptosis, Toll receptor pathways). Heterozygous PBEF+/− mice were significantly protected (reduced BAL protein, BAL IL-6 levels, peak inspiratory pressures) when exposed to a model of severe VILI (4 h, 40 ml/kg tidal volume) and exhibited significantly reduced expression of VILI-associated gene expression modules. Finally, strategies to reduce PBEF availability (neutralizing antibody) resulted in significant protection from VILI.
Conclusions: These studies implicate PBEF as a key inflammatory mediator intimately involved in both the development and severity of ventilator-induced ALI.
doi:10.1164/rccm.200712-1822OC
PMCID: PMC2542434  PMID: 18658108
visfatin; acute lung injury; chemotaxis; apoptosis; mechanical ventilation

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