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1.
Am J Pathol ; 188(7): 1640-1652, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29684358

RESUMO

Acetaminophen overdose is the most common cause of acute liver injury (ALI) or acute liver failure in the United States. Its pathogenetic mechanisms are incompletely understood. Additional studies are warranted to identify new genetic risk factors for more mechanistic insights and new therapeutic target discoveries. The objective of this study was to explore the role and mechanisms of nicotinamide phosphoribosyltransferase (NAMPT) in acetaminophen-induced ALI. C57BL/6 Nampt gene wild-type (Nampt+/+), heterozygous knockout (Nampt+/-), and overexpression (NamptOE) mice were treated with overdose of acetaminophen, followed by histologic, biochemical, and transcriptomic evaluation of liver injury. The mechanism of Nampt in acetaminophen-induced hepatocytic toxicity was also explored in cultured primary hepatocytes. Three lines of evidence have convergently demonstrated that acetaminophen overdose triggers the most severe oxidative stress and necrosis, and the highest expression of key necrosis driving genes in Nampt+/- mice, whereas the effects in NamptOE mice were least severe relative to Nampt+/+ mice. Treatment of P7C3-A20, a small chemical molecule up-regulator of Nampt, ameliorated acetaminophen-induced mouse ALI over the reagent control. These findings support the fact that NAMPT protects against acetaminophen-induced ALI.


Assuntos
Acetaminofen/toxicidade , Analgésicos não Narcóticos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Citocinas/fisiologia , Nicotinamida Fosforribosiltransferase/fisiologia , Substâncias Protetoras , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo
2.
J Histochem Cytochem ; 66(8): 577-584, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29596030

RESUMO

Rhabdomyosarcoma (RMS) is a common malignancy of soft tissue, subclassified as alveolar (ARMS), pleomorphic (PRMS), spindle cell/sclerosing (SRMS), and embryonal (ERMS) types. The Yes-associated protein (YAP) is a member of the Hippo pathway and a transcriptional regulator that controls cell proliferation. We have studied the immunohistochemical expression of YAP in different RMSs, arranged in tissue microarray (TMA) and whole slide formats. Pertinent clinical data including patient age, gender, tumor location, and clinical stage were collected. Out of 96 TMA cases, 30 cases (31%) were pleomorphic, 27 (28%) were embryonal, 24 (25%) alveolar, and 15 (16%) spindle cell. Positive nuclear YAP staining was seen in the PRMS (17/30, 56.7%), SRMS (7/15, 46.7%), ERMS (19/27 or 70%), and less in ARMS (37.5%). YAP nuclear staining was significantly more prevalent in ERMS than ARMS ( p=0.02). Of the 41 whole slide cases, nuclear staining was detected in all ARMS but was restricted in distribution to <30% of the cells, in contrast to ERMS and SRMS, which had diffuse or >30% staining. These results highlight the role of YAP in RMS tumorigenesis, a fact that can be useful in engineering targeted therapy. Restricted nuclear YAP staining (<30% of cells) may be of value in the diagnosis of ARMS.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/análise , Imuno-Histoquímica/métodos , Fosfoproteínas/análise , Rabdomiossarcoma/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/patologia , Núcleo Celular/patologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Coloração e Rotulagem/métodos , Análise Serial de Tecidos/métodos , Fatores de Transcrição , Proteínas de Sinalização YAP , Adulto Jovem
3.
J Pharmacol Exp Ther ; 365(1): 96-106, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29420256

RESUMO

Lower plasma nicotinamide phosphoribosyltransferase (NAMPT) levels are associated with improved response to methotrexate (MTX) in patients with juvenile idiopathic arthritis. Cell-based studies confirmed that reduced cellular NAMPT activity potentiates the pharmacologic activity of MTX; however, the mechanism of this interaction has yet to be defined. Therefore, in this study, we investigate the mechanism of enhanced pharmacologic activity of MTX in NAMPT-deficient A549 cells. Small interfering RNA-based silencing of NAMPT expression resulted in a greater than 3-fold increase in sensitivity to MTX (P < 0.005) that was completely reversed by supplementation with folinic acid. Despite a 68% reduction in cellular NAD levels in NAMPT-deficient cells, no change in expression or activity of dihydrofolate reductase was observed and uptake of MTX was not significantly altered. MTX did not potentiate the depletion of cellular NAD levels, but NAMPT-deficient cells had significant elevations in levels of intermediates of de novo purine biosynthesis and were 4-fold more sensitive to depletion of ATP by MTX (P < 0.005). Supplementation with hypoxanthine and thymidine completely reversed the antiproliferative activity of MTX in NAMPT-deficient cells and corresponded to repletion of the cellular ATP pool without any effect on NAD levels. Together, these findings demonstrate that increased MTX activity with decreased NAMPT expression is dependent on the antifolate activity of MTX and is driven by enhanced sensitivity to the ATP-depleting effects of MTX. For the first time, these findings provide mechanistic details to explain the increase in pharmacological activity of MTX under conditions of reduced NAMPT activity.


Assuntos
Trifosfato de Adenosina/metabolismo , Citocinas/deficiência , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Metotrexato/farmacologia , Nicotinamida Fosforribosiltransferase/deficiência , Células A549 , Transporte Biológico , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Ácido Fólico/metabolismo , Inativação Gênica , Homeostase/efeitos dos fármacos , Humanos , Nicotinamida Fosforribosiltransferase/genética
4.
Circulation ; 135(16): 1532-1546, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28202489

RESUMO

BACKGROUND: Pulmonary arterial hypertension is a severe and progressive disease, a hallmark of which is pulmonary vascular remodeling. Nicotinamide phosphoribosyltransferase (NAMPT) is a cytozyme that regulates intracellular nicotinamide adenine dinucleotide levels and cellular redox state, regulates histone deacetylases, promotes cell proliferation, and inhibits apoptosis. We hypothesized that NAMPT promotes pulmonary vascular remodeling and that inhibition of NAMPT could attenuate pulmonary hypertension. METHODS: Plasma, mRNA, and protein levels of NAMPT were measured in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension and in the lungs of rodent models of pulmonary hypertension. Nampt+/- mice were exposed to 10% hypoxia and room air for 4 weeks, and the preventive and therapeutic effects of NAMPT inhibition were tested in the monocrotaline and Sugen hypoxia models of pulmonary hypertension. The effects of NAMPT activity on proliferation, migration, apoptosis, and calcium signaling were tested in human pulmonary artery smooth muscle cells. RESULTS: Plasma and mRNA and protein levels of NAMPT were increased in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension, as well as in lungs of rodent models of pulmonary hypertension. Nampt+/- mice were protected from hypoxia-mediated pulmonary hypertension. NAMPT activity promoted human pulmonary artery smooth muscle cell proliferation via a paracrine effect. In addition, recombinant NAMPT stimulated human pulmonary artery smooth muscle cell proliferation via enhancement of store-operated calcium entry by enhancing expression of Orai2 and STIM2. Last, inhibition of NAMPT activity attenuated monocrotaline and Sugen hypoxia-induced pulmonary hypertension in rats. CONCLUSIONS: Our data provide evidence that NAMPT plays a role in pulmonary vascular remodeling and that its inhibition could be a potential therapeutic target for pulmonary arterial hypertension.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Nicotinamida Fosforribosiltransferase/uso terapêutico , Artéria Pulmonar/fisiopatologia , Remodelação Vascular/efeitos dos fármacos , Animais , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nicotinamida Fosforribosiltransferase/administração & dosagem , Nicotinamida Fosforribosiltransferase/farmacologia , Ratos , Ratos Sprague-Dawley , Transfecção
5.
BMC Pulm Med ; 15: 95, 2015 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-26285698

RESUMO

BACKGROUND: Accumulated to-date gene microarray data on Acute Respiratory Distress Syndrome (ARDS) in the Gene Expression Omnibus (GEO) represent a rich source for identifying new unsuspected targets and mechanisms of ARDS. The recently developed expression-based genome-wide association study (eGWAS) for analysis of GEO data was successfully used for analysis of gene expression of comparatively noncomplex adipose tissue, 75 % of which is represented by adipocytes. Although lung tissue is more heterogenic and does not possess a prevalent cell type for driving gene expression patterns, we hypothesized that eGWAS of ARDS samples will generate biologically meaningful results. METHODS: The eGWAS was conducted according to (Proc Natl Acad Sci U S A 109:7049-7054, 2012) and genes were ranked according to p values of chi-square test. RESULTS: The search of GEO retrieved 487 ARDS related entries. These entries were filtered for multiple qualitative and quantitative conditions and 219 samples were selected: mouse n sham/ARDS = 67/92, rat n = 13/13, human cells n = 11/11, canine n = 6/6 with the following ARDS model distributions: mechanical ventilation (MV)/cyclic stretch n = 11; endotoxin (LPS) treatment n = 8; MV + LPS n = 3; distant organ injury induced ARDS n = 3; chemically induced ARDS n = 2; Staphylococcus aureus induced ARDS n = 2; and one experiment each for radiation and shock induced ARDS. The eGWAS of this dataset identified 42 significant (Bonferroni threshold P < 1.55 × 10(-6)) genes. 66.6 % of these genes, were associated previously with lung injury and include the well known ARDS genes such as IL1R2 (P = 4.42 × 10(-19)), IL1ß (P = 3.38 × 10(-17)), PAI1 (P = 9.59 × 10(-14)), IL6 (P = 3.57 × 10(-12)), SOCS3 (P = 1.05 × 10(-10)), and THBS1 (P = 2.01 × 10(-9)). The remaining genes were new ARDS candidates. Expression of the most prominently upregulated genes, CLEC4E (P = 4.46 × 10(-14)) and CD300LF (P = 2.31 × 10(-16)), was confirmed by real time PCR. The former was also validated by in silico pathway analysis and the latter by Western blot analysis. CONCLUSIONS: Our first in the field application of eGWAS in ARDS and utilization of more than 120 publicly available microarray samples of ARDS not only justified applicability of eGWAS to complex lung tissue, but also discovered 14 new candidate genes which associated with ARDS. Detailed studies of these new candidates might lead to identification of unsuspected evolutionarily conserved mechanisms triggered by ARDS.


Assuntos
Biomarcadores/metabolismo , DNA/genética , Expressão Gênica , Estudo de Associação Genômica Ampla/métodos , Síndrome do Desconforto Respiratório/metabolismo , Animais , Western Blotting , Cães , Humanos , Camundongos , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Síndrome do Desconforto Respiratório/genética
6.
PLoS One ; 9(11): e111953, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25372662

RESUMO

Acute respiratory distress syndrome (ARDS) is a lung condition characterized by impaired gas exchange with systemic release of inflammatory mediators, causing pulmonary inflammation, vascular leak and hypoxemia. Existing biomarkers have limited effectiveness as diagnostic and therapeutic targets. To identify disease-associating variants in ARDS patients, whole-exome sequencing was performed on 96 ARDS patients, detecting 1,382,399 SNPs. By comparing these exome data to those of the 1000 Genomes Project, we identified a number of single nucleotide polymorphisms (SNP) which are potentially associated with ARDS. 50,190SNPs were found in all case subgroups and controls, of which89 SNPs were associated with susceptibility. We validated three SNPs (rs78142040, rs9605146 and rs3848719) in additional ARDS patients to substantiate their associations with susceptibility, severity and outcome of ARDS. rs78142040 (C>T) occurs within a histone mark (intron 6) of the Arylsulfatase D gene. rs9605146 (G>A) causes a deleterious coding change (proline to leucine) in the XK, Kell blood group complex subunit-related family, member 3 gene. rs3848719 (G>A) is a synonymous SNP in the Zinc-Finger/Leucine-Zipper Co-Transducer NIF1 gene. rs78142040, rs9605146, and rs3848719 are associated significantly with susceptibility to ARDS. rs3848719 is associated with APACHE II score quartile. rs78142040 is associated with 60-day mortality in the overall ARDS patient population. Exome-seq is a powerful tool to identify potential new biomarkers for ARDS. We selectively validated three SNPs which have not been previously associated with ARDS and represent potential new genetic biomarkers for ARDS. Additional validation in larger patient populations and further exploration of underlying molecular mechanisms are warranted.


Assuntos
Exoma , Estudos de Associação Genética , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Polimorfismo de Nucleotídeo Único , Síndrome do Desconforto Respiratório/genética , Adolescente , Alelos , Estudos de Casos e Controles , Criança , Pré-Escolar , Comorbidade , Feminino , Genômica , Humanos , Masculino , Síndrome do Desconforto Respiratório/diagnóstico , Síndrome do Desconforto Respiratório/mortalidade , Transdução de Sinais , Adulto Jovem
7.
BMC Hematol ; 14(1): 7, 2014 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-24656173

RESUMO

BACKGROUND: Chronic graft-versus-host disease (cGVHD) is a major factor of morbidity and mortality for allogeneic stem cell transplantation (aSCT). The skin and internal organ involvement is the most common systemic complication of cGVHD and closely resembles systemic sclerosis (SSc). Circulating lymphocytes characterize the autoimmune nature of both conditions. Therefore we hypothesized that the common clinical manifestation (systemic organ and skin injury) and the common underlying players (lymphocytes) justify the combined meta-analysis of these diseases. RESULTS: The aSCT and SSc datasets were uploaded from Gene Expression Omnibus (GEO), a public functional genomics data repository. The available microarray studies of peripheral blood mononuclear cells (PBMCs) and isolated lymphocytes were limited to well established microarray platforms (Affymetrix, Agilent, Canvac, and Illumina) and experimental settings with ≥10 patients per group. The resulting pools of data were merged by unique gene identifier and analyzed by the expression genome-wide association studies (eGWAS) coupled with the subtraction of the cGVHD+ and cGVHD- molecular signatures. The eGWAS was applied to 47 and 50 lymphocyte profiles from aSCT and SSc patients, respectively. The identified 35 candidates were represented by 8 known cGVHD genes (including CXCR4, LTBR and PML) and 28 new candidate genes (including SEPX1 and DNJGB1). The further mutual subtraction of cGVHD+ and cGVHD- candidates and pathway analysis identified a list of 25 genes. Seven of these genes belong to the fibroblast development and function pathway, consisting of the well known cGVHD genes CCND1, JUN, and FOS, and the new molecular targets MMP2, FOSB, TNFAIP8, and DUSP1. These genes become primary candidates for a potential link of systemic effects of cGVHD and SSc. CONCLUSIONS: We designed a new approach for meta-analysis by combining data from different diseases using common clinical manifestation as a linker. This allowed us to power up the insufficient standalone meta-analysis of aSCT microarray studies, by adding SSc samples to the data pool. This new method has successfully identified novel molecular targets for systemic effects of both aSCT and SSc. We believe that this approach is generalizable and can be applied to an array of diseases with common clinical manifestations.

8.
BMC Nephrol ; 14: 231, 2013 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-24152794

RESUMO

BACKGROUND: Accumulated to-date microarray data on ischemia reperfusion injury (IRI) of kidney represent a powerful source for identifying new targets and mechanisms of kidney IRI. In this study, we conducted a meta-analysis of gene expression profiles of kidney IRI in human, pig, rat, and mouse models, using a new scoring method to correct for the bias of overrepresented species. The gene expression profiles were obtained from the public repositories for 24 different models. After filtering against inclusion criteria 21 experimental settings were selected for meta-analysis and were represented by 11 rat models, 6 mouse models, and 2 models each for pig and human, with a total of 150 samples. Meta-analysis was conducted using expression-based genome-wide association study (eGWAS). The eGWAS results were corrected for a rodent species bias using a new weighted scoring algorithm, which favors genes with unidirectional change in expression in all tested species. RESULTS: Our meta-analysis corrected for a species bias, identified 46 upregulated and 1 downregulated genes, of which 26 (55%) were known to be associated with kidney IRI or kidney transplantation, including LCN2, CCL2, CXCL1, HMOX1, ICAM1, ANXA1, and TIMP1, which justified our approach. Pathway analysis of our candidates identified "Acute renal failure panel" as the most implicated pathway, which further validates our new method. Among new IRI candidates were 10 novel (<5 published reports related to kidney IRI) and 11 new candidates (0 reports related to kidney IRI) including the most prominent candidates ANXA2, CLDN4, and TYROBP. The cross-species expression pattern of these genes allowed us to generate three workable hypotheses of kidney IRI, one of which was confirmed by an additional study. CONCLUSIONS: Our first in the field kidney IRI meta-analysis of 150 microarray samples, corrected for a species bias, identified 10 novel and 11 new candidate genes. Moreover, our new meta-analysis correction method improved gene candidate selection by identifying genes that are model and species independent, as a result, function of these genes can be directly extrapolated to the disease state in human and facilitate translation of potential diagnostic or therapeutic properties of these candidates to the bedside.


Assuntos
Rim/irrigação sanguínea , Rim/metabolismo , Proteínas/metabolismo , Traumatismo por Reperfusão/epidemiologia , Traumatismo por Reperfusão/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos , Prevalência , Ratos , Especificidade da Espécie , Suínos
10.
J Med Chem ; 55(16): 7290-4, 2012 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-22889195

RESUMO

Herein we report the use of carboranes to significantly increase the potency of small molecule inhibitors of nicotinamide phosphoribosyltranferase (Nampt), an enzyme that is central to metabolism and cell survival. We compare the inclusion of carborane with other similarly sized substituents and demonstrate that, compared with their purely organic counterparts, these molecules exhibit up to 10-fold greater antiproliferative activity against cancer cells in vitro and a 100-fold increase in Nampt inhibition.


Assuntos
Antineoplásicos/síntese química , Boranos/síntese química , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Boranos/química , Boranos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Relação Estrutura-Atividade
11.
Genes Cells ; 17(5): 420-30, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22487217

RESUMO

This study profiled transcriptomes of human pulmonary microvascular endothelial cells (HMVEC-L) treated with pre-B-cell colony-enhancing factor (PBEF) siRNA or scrambled RNA to gain insight into transcriptional regulations of PBEF on the endothelial function using the Affymetrix GeneChips HG-U133 plus 2. Several important themes are emerged from this study. First, PBEF affected expressions of multiple genes in the endothelium. Expression of 373 genes was increased and 64 genes decreased by at least 1.3-fold in the PBEFsiRNA-treated HMVEC-L versus the scramble RNA control. Second, the microarray results confirmed previous reports of PBEF-mediated gene expressions in some pathways but provided a more complete repertoire of molecules in those pathways. Third, most of the affected canonical pathways have not previously been reported to be PBEF responsive. Fourth, network analysis supports that PBEF has pleiotropic functions. Our first transcriptome analysis of human pulmonary microvascular endothelial cells treated with PBEFsiRNA has provided important insights into the transcriptional regulation of gene expression in HMVEC-L cells by PBEF. Further in-depth analysis of these transcriptional regulations may shed light on molecular mechanisms underlying PBEF-mediated endothelial functions and dysfunctions in various diseases and provide new leads of therapeutic targets to those diseases.


Assuntos
Células Endoteliais/metabolismo , Nicotinamida Fosforribosiltransferase/genética , RNA Interferente Pequeno/genética , Transcriptoma , Células Cultivadas , Regulação da Expressão Gênica , Pleiotropia Genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Transfecção
12.
Mol Cell Biol ; 29(19): 5348-56, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19651900

RESUMO

E3 ubiquitin ligases, which target specific molecules for proteolytic destruction, have emerged as key regulators of immune functions. Several E3 ubiquitin ligases, including c-Cbl, Cbl-b, GRAIL, Itch, and Nedd4, have been shown to negatively regulate T-cell activation. Here, we report that the HECT-type E3 ligase AIP2 positively regulates T-cell activation. Ectopic expression of AIP2 in mouse primary T cells enhances their proliferation and interleukin-2 production by suppressing the apoptosis of T cells. AIP2 interacts with and promotes ubiquitin-mediated degradation of EGR2, a zinc finger transcription factor that has been found to regulate Fas ligand (FasL) expression during activation-induced T-cell death. Suppression of AIP2 expression by small RNA interference upregulates EGR2, inhibits EGR2 ubiquitination and FasL expression, and enhances the apoptosis of T cells. Therefore, AIP2 regulates activation-induced T-cell death by suppressing EGR2-mediated FasL expression via the ubiquitin pathway.


Assuntos
Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Apoptose , Biocatálise , Células Cultivadas , Proteína Ligante Fas/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Linfócitos T/citologia , Ubiquitina-Proteína Ligases/genética
13.
Am J Respir Crit Care Med ; 178(6): 605-17, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18658108

RESUMO

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-alpha, KC) compared with either challenge alone. Gene expression profiling identified induction of ALI- and VILI-associated gene modules (nuclear factor-kappaB, 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.


Assuntos
Citocinas/fisiologia , Nicotinamida Fosforribosiltransferase/fisiologia , Síndrome do Desconforto Respiratório/fisiopatologia , Ventiladores Mecânicos , Animais , Líquido da Lavagem Broncoalveolar , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Quimiotaxia de Leucócito/fisiologia , Perfilação da Expressão Gênica , Interleucina-6/análise , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Síndrome do Desconforto Respiratório/etiologia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Ventiladores Mecânicos/efeitos adversos
14.
Transl Res ; 150(1): 18-29, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17585860

RESUMO

The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine central to the response to endotoxemia, is a putative biomarker in acute lung injury (ALI). To explore MIF as a molecular target and candidate gene in ALI, the MIF gene and protein expression were examined in murine and canine models of ALI (high tidal volume mechanical ventilation, endotoxin exposure) and in patients with either sepsis or sepsis-induced ALI. MIF gene expression and protein levels were significantly increased in each ALI model, with serum MIF levels significantly higher in patients with either sepsis or ALI compared with healthy controls (African- and European-descent). The association of 8 MIF gene polymorphisms (single-nucleotide polymorphisms (SNPs)) (within a 9.7-kb interval on chromosome 22q11.23) with the development of sepsis and ALI in European-descent and African-descent populations was studied next. Genotyping in 506 DNA samples (sepsis patients, sepsis-associated ALI patients, and healthy controls) revealed haplotypes located in the 3' end of the MIF gene, but not individual SNPs, associated with sepsis and ALI in both populations. These data, generated via functional genomic and genetic approaches, suggest that MIF is a relevant molecular target in ALI.


Assuntos
Biomarcadores/sangue , Oxirredutases Intramoleculares/sangue , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/sangue , Fatores Inibidores da Migração de Macrófagos/genética , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/metabolismo , Adulto , Negro ou Afro-Americano/genética , Idoso , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Estudos de Casos e Controles , Feminino , Haplótipos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Síndrome do Desconforto Respiratório/etnologia , Fatores de Risco , População Branca/genética
15.
Am J Physiol Lung Cell Mol Physiol ; 293(2): L292-302, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17468131

RESUMO

Increasing evidence supports the contribution of genetic influences on susceptibility/severity in acute lung injury (ALI), a devastating syndrome requiring mechanical ventilation with subsequent risk for ventilator-associated lung injury (VALI). To identify VALI candidate genes, we determined that Brown Norway (BN) and Dahl salt-sensitive (SS) rat strains were differentially sensitive to VALI (tidal volume of 20 ml/kg, 85 breaths/min, 2 h) defined by bronchoalveolar lavage (BAL) protein and leukocytes. We next exploited differential sensitivities and phenotyped both the VALI-sensitive BN and the VALI-resistant SS rat strains by expression profiling coupled to a bioinformatic-intense candidate gene approach (Significance Analysis of Microarrays, i.e., SAM). We identified 106 differentially expressed VALI genes representing gene ontologies such as "transcription" and "chemotaxis/cell motility." We mapped the chromosomal location of the differentially expressed probe sets and selected consomic SS rats with single BN introgressions of chromosomes 2, 13, and 16 (based on the highest density of probe sets) while also choosing chromosome 20 (low probe sets density). VALI exposure of consomic rats with introgressions of BN chromosomes 13 and 16 resulted in significant increases in both BAL cells and protein (compared to parental SS strain), whereas introgression of BN chromosome 2 displayed a large increase only in BAL protein. Introgression of BN chromosome 20 had a minimal effect. These results suggest that genes residing on BN chromosomes 2, 13, and 16 confer increased sensitivity to high tidal volume ventilation. We speculate that the consomic-microarray-SAM approach is a time- and resource-efficient tool for the genetic dissection of complex diseases including VALI.


Assuntos
Modelos Animais de Doenças , Genômica/métodos , Respiração Artificial/efeitos adversos , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/fisiopatologia , Animais , Biologia Computacional , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Dahl , Ratos Sprague-Dawley , Síndrome do Desconforto Respiratório/etiologia , Especificidade da Espécie
16.
Am J Physiol Lung Cell Mol Physiol ; 291(5): L851-61, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16782753

RESUMO

Human acute lung injury is characterized by heterogeneous tissue involvement, leading to the potential for extremes of mechanical stress and tissue injury when mechanical ventilation, required to support critically ill patients, is employed. Our goal was to establish whether regional cellular responses to these disparate local mechanical conditions could be determined as a novel approach toward understanding the mechanism of development of ventilator-associated lung injury. We utilized cross-species genomic microarrays in a unilateral model of ventilator-associated lung injury in anesthetized dogs to assess regional cellular responses to local mechanical conditions that potentially contribute pathogenic mechanisms of injury. Highly significant regional differences in gene expression were observed between lung apex/base regions as well as between gravitationally dependent/nondependent regions of the base, with 367 and 1,544 genes differentially regulated between these regions, respectively. Major functional groupings of differentially regulated genes included inflammation and immune responses, cell proliferation, adhesion, signaling, and apoptosis. Expression of genes encoding both acute lung injury-associated inflammatory cytokines and protective acute response genes were markedly different in the nondependent compared with the dependent regions of the lung base. We conclude that there are significant differences in the local responses to stress within the lung, and consequently, insights into the cellular responses that contribute to ventilator-associated lung injury development must be sought in the context of the mechanical heterogeneity that characterizes this syndrome.


Assuntos
Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/fisiopatologia , Animais , Modelos Animais de Doenças , Cães , Pulmão/diagnóstico por imagem , Pulmão/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos/normas , Reprodutibilidade dos Testes , Respiração Artificial/efeitos adversos , Síndrome do Desconforto Respiratório/diagnóstico por imagem , Especificidade da Espécie , Estresse Mecânico , Tomografia Computadorizada por Raios X
17.
Am J Respir Cell Mol Biol ; 34(4): 487-95, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16399953

RESUMO

The genetic basis of acute lung injury (ALI) is poorly understood. The myosin light chain kinase (MYLK) gene encodes the nonmuscle myosin light chain kinase isoform, a multifunctional protein involved in the inflammatory response (apoptosis, vascular permeability, leukocyte diapedesis). To examine MYLK as a novel candidate gene in sepsis-associated ALI, we sequenced exons, exon-intron boundaries, and 2 kb of 5' UTR of the MYLK, which revealed 51 single-nucleotide polymorphisms (SNPs). Potential association of 28 MYLK SNPs with sepsis-associated ALI were evaluated in a case-control sample of 288 European American subjects (EAs) with sepsis alone, subjects with sepsis-associated ALI, or healthy control subjects, and a sample population of 158 African American subjects (AAs) with sepsis and ALI. Significant single locus associations in EAs were observed between four MYLK SNPs and the sepsis phenotype (P<0.001), with an additional SNP associated with the ALI phenotype (P=0.03). A significant association of a single SNP (identical to the SNP identified in EAs) was observed in AAs with sepsis (P=0.002) and with ALI (P=0.01). Three sepsis risk-conferring haplotypes in EAs were defined downstream of start codon of smooth muscle MYLK isoform, a region containing putative regulatory elements (P<0.001). In contrast, multiple haplotypic analyses revealed an ALI-specific, risk-conferring haplotype at 5' of the MYLK gene in both European and African Americans and an additional 3' region haplotype only in African Americans. These data strongly implicate MYLK genetic variants to confer increased risk of sepsis and sepsis-associated ALI.


Assuntos
Desequilíbrio de Ligação , Quinase de Cadeia Leve de Miosina/genética , Polimorfismo de Nucleotídeo Único , Síndrome do Desconforto Respiratório/genética , Adolescente , Adulto , Negro ou Afro-Americano , Idoso , Estudos de Casos e Controles , Éxons , Feminino , Haplótipos , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Síndrome do Desconforto Respiratório/etiologia , Risco , Sepse/complicações , Sepse/genética , População Branca
18.
Am J Respir Cell Mol Biol ; 34(1): 108-18, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16195540

RESUMO

Long-term success in lung transplantation is limited by obliterative bronchiolitis, whereas T cell effector mechanisms in this process remain incompletely understood. Using the mouse heterotopic allogeneic airway transplant model, we studied T cell effector responses during obliterative airways disease (OAD). Allospecific CD8+ IFN-gamma+ T cells were detected in airway allografts, with significant coexpression of TNF-alpha and granzyme B. Therefore, using IFN-gamma as a surrogate marker, we assessed the distribution and kinetics of extragraft allo-specific T cells during OAD. Robust allospecific IFN-gamma was produced by draining the lymph nodes, spleen, and lung mononuclear cells from allograft, but not isograft recipients by Day 14, and significantly decreased by Day 28. Although the majority of allospecific T cells were CD8+, allospecific CD4+ T cells were also detected in these compartments, with each employing distinct allorecognition pathways. An influx of pluripotent CD8+ effector cells with a memory phenotype were detected in the lung during OAD similar to those seen in the allografts and secondary lymphoid tissue. Antibody depletion of CD8+ T cells markedly reduced airway lumen obliteration and fibrosis at Day 28. Together, these data demonstrate that allospecific CD8+ effector T cells play an important role in OAD and traffic to the lung after heterotopic airway transplant, suggesting that the lung is an important immunologic site, and perhaps a reservoir, for effector cells during the rejection process.


Assuntos
Bronquiolite Obliterante/imunologia , Linfócitos T CD8-Positivos/imunologia , Pulmão , Células-Tronco Pluripotentes/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Feminino , Rejeição de Enxerto/imunologia , Granzimas , Humanos , Interferon gama/imunologia , Isoantígenos/imunologia , Pulmão/citologia , Pulmão/imunologia , Pulmão/patologia , Transplante de Pulmão/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células-Tronco Pluripotentes/fisiologia , Serina Endopeptidases/imunologia , Transplante Homólogo/imunologia , Fator de Necrose Tumoral alfa/imunologia
19.
Microvasc Res ; 70(3): 142-51, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16188281

RESUMO

Prior genomic and genetic studies identified pre-B-cell colony-enhancing factor (PBEF) as a novel candidate gene and biomarker in acute lung injury (ALI). As increased vascular permeability is a cardinal feature of ALI, we assessed the role of PBEF in in vitro vascular barrier regulation using confluent human pulmonary artery endothelial cell (HPAEC) monolayers. Reductions in PBEF protein expression (>70%) by siRNA significantly attenuated EC barrier dysfunction induced by the potent edemagenic agent, thrombin, reflected by reductions in transendothelial electric resistance (TER, approximately 60% reduction). Furthermore, PBEF siRNA blunted thrombin-mediated increases in Ca(2+) entry, polymerized actin formation, and myosin light chain phosphorylation, events critical to the thrombin-mediated permeability response. Finally, PBEF siRNA also significantly inhibited thrombin-stimulated increase of IL-8 secretion in HPAEC, a chemokine known to induce actin fiber formation and intercellular gap formation of endothelial cells. Taken together, these studies demonstrate that PBEF may be required for complete expression of the thrombin-induced inflammatory response and reveal potentially novel role for PBEF in the regulation of EC Ca(2+)-dependent cytoskeletal rearrangement and endothelial barrier dysfunction. Ongoing studies will continue to address the molecular mechanisms by which PBEF contributes to ALI susceptibility.


Assuntos
Linfócitos B/metabolismo , Citocinas/fisiologia , Células Endoteliais/citologia , Endotélio Vascular/citologia , Pulmão/patologia , Trombina/metabolismo , Actinas/química , Sequência de Bases , Biomarcadores , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Citoesqueleto/metabolismo , Impedância Elétrica , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Inativação Gênica , Humanos , Inflamação , Interleucina-8/metabolismo , Microcirculação , Microscopia de Fluorescência , Dados de Sequência Molecular , Nicotinamida Fosforribosiltransferase , Fosforilação , Artéria Pulmonar/citologia , RNA Interferente Pequeno/metabolismo , Trombina/química , Fatores de Tempo , Transfecção
20.
Int J Cardiol ; 102(2): 333-40, 2005 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-15982506

RESUMO

BACKGROUND: Giant cell myocarditis is a rapidly progressive and often fatal condition without a clear etiology or treatment. A better understanding of giant cell myocarditis pathogenesis is critical to developing treatments to prevent progression and reverse damage. We compared the gene expression of giant cell myocarditis with that of nonfailing hearts. METHODS: Left ventricular samples from two giant cell myocarditis patients harvested during ventricular assist device placement and six unused donor hearts were examined using Affymetrix U133A microarrays. Differential gene expression was defined with a Bonferroni-adjusted p value < or = 0.05 from a Student's t-test and an absolute fold change > or = 2.0. Select gene expression was confirmed with quantitative PCR. RESULTS: Of 115 differentially expressed genes, most were upregulated in giant cell myocarditis and involved in immune response, transcriptional regulation, and metabolism. T-cell activation genes included chemokine receptor 4; chemokine ligands 5, 9, 13, and 18; interleukin-10 receptor alpha; and beta-2 integrin. CONCLUSIONS: Gene expression analysis of giant cell myocarditis offers novel insights into its pathogenesis, namely the role of T-cell activators of the Th1 subset and immune response genes previously implicated in heart failure. This forms the basis for future work aimed at defining novel therapeutic targets for giant cell myocarditis.


Assuntos
Expressão Gênica , Genes MHC da Classe II , Células Gigantes , Miocardite , RNA/genética , Linfócitos T/imunologia , Adulto , Biópsia , Análise por Conglomerados , Feminino , Genes MHC da Classe II/genética , Genes MHC da Classe II/imunologia , Células Gigantes/imunologia , Células Gigantes/patologia , Ventrículos do Coração/imunologia , Ventrículos do Coração/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Miocardite/genética , Miocardite/imunologia , Miocardite/patologia , Reação em Cadeia da Polimerase , RNA Mitocondrial , Reprodutibilidade dos Testes
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