RESUMO
Genome-wide association studies (GWAS) have uncovered numerous genetic variants (SNPs) that are associated with blood pressure (BP). Genetic variants may lead to BP changes by acting on intermediate molecular phenotypes such as coded protein sequence or gene expression, which in turn affect BP variability. Therefore, characterizing genes whose expression is associated with BP may reveal cellular processes involved in BP regulation and uncover how transcripts mediate genetic and environmental effects on BP variability. A meta-analysis of results from six studies of global gene expression profiles of BP and hypertension in whole blood was performed in 7017 individuals who were not receiving antihypertensive drug treatment. We identified 34 genes that were differentially expressed in relation to BP (Bonferroni-corrected p<0.05). Among these genes, FOS and PTGS2 have been previously reported to be involved in BP-related processes; the others are novel. The top BP signature genes in aggregate explain 5%-9% of inter-individual variance in BP. Of note, rs3184504 in SH2B3, which was also reported in GWAS to be associated with BP, was found to be a trans regulator of the expression of 6 of the transcripts we found to be associated with BP (FOS, MYADM, PP1R15A, TAGAP, S100A10, and FGBP2). Gene set enrichment analysis suggested that the BP-related global gene expression changes include genes involved in inflammatory response and apoptosis pathways. Our study provides new insights into molecular mechanisms underlying BP regulation, and suggests novel transcriptomic markers for the treatment and prevention of hypertension.
Assuntos
Pressão Sanguínea/genética , Estudo de Associação Genômica Ampla , Hipertensão/genética , Transcriptoma/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genótipo , Humanos , Hipertensão/patologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
RATIONALE: Patients with sickle cell disease (SCD) have markers of chronic inflammation, but the mechanism of inflammation and its relevance to patient survival are unknown. OBJECTIVE: To assess the relationship between iron, inflammation, and early death in SCD. METHODS AND RESULTS: Using peripheral blood mononuclear cell transcriptome profile hierarchical clustering, we classified 24 patients and 10 controls in clusters with significantly different expression of genes known to be regulated by iron. Subsequent gene set enrichment analysis showed that many genes associated with the high iron cluster were involved in the toll-like receptor system (TLR4, TLR7, and TLR8) and inflammasome complex pathway (NLRP3, NLRC4, and CASP1). Quantitative PCR confirmed this classification and showed that ferritin light chain, TLR4, and interleukin-6 expression were >100-fold higher in patients than in controls (P<0.001). Further linking intracellular iron and inflammation, 14 SCD patients with a ferroportin Q248H variant that causes intracellular iron accumulation had significantly higher levels of interleukin-6 and C-reactive protein compared with 14 matched SCD patients with the wild-type allele (P<0.05). Finally, in a cohort of 412 patients followed for a median period of 47 months (interquartile range, 24-82), C-reactive protein was strongly and independently associated with early death (hazard ratio, 3.0; 95% confidence interval, 1.7-5.2; P<0.001). CONCLUSIONS: Gene expression markers of high intracellular iron in patients with SCD are associated with markers of inflammation and mortality. The results support a model in which intracellular iron promotes inflammatory pathways, such as the TLR system and the inflammasome, identifying important new pathways for additional investigation.
Assuntos
Anemia Falciforme/sangue , Anemia Falciforme/genética , Marcadores Genéticos/genética , Ferro/sangue , Adulto , Anemia Falciforme/mortalidade , Estudos de Coortes , Feminino , Humanos , Inflamação/sangue , Inflamação/genética , Inflamação/mortalidade , Leucócitos Mononucleares/metabolismo , Masculino , Mortalidade/tendências , Estudos Prospectivos , Sistema de RegistrosRESUMO
Genome-wide association studies (GWAS) have identified numerous loci associated with blood pressure (BP). The molecular mechanisms underlying BP regulation, however, remain unclear. We investigated BP-associated molecular mechanisms by integrating BP GWAS with whole blood mRNA expression profiles in 3,679 individuals, using network approaches. BP transcriptomic signatures at the single-gene and the coexpression network module levels were identified. Four coexpression modules were identified as potentially causal based on genetic inference because expression-related SNPs for their corresponding genes demonstrated enrichment for BP GWAS signals. Genes from the four modules were further projected onto predefined molecular interaction networks, revealing key drivers. Gene subnetworks entailing molecular interactions between key drivers and BP-related genes were uncovered. As proof-of-concept, we validated SH2B3, one of the top key drivers, using Sh2b3(-/-) mice. We found that a significant number of genes predicted to be regulated by SH2B3 in gene networks are perturbed in Sh2b3(-/-) mice, which demonstrate an exaggerated pressor response to angiotensin II infusion. Our findings may help to identify novel targets for the prevention or treatment of hypertension.
Assuntos
Pressão Sanguínea/genética , Hipertensão/genética , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Angiotensina II/metabolismo , Animais , Índice de Massa Corporal , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Redes Reguladoras de Genes , Loci Gênicos , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Lineares , Masculino , Proteínas de Membrana , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Biologia de Sistemas , Transcriptoma , Adulto JovemRESUMO
In adults with sickle cell disease (SCD), markers of iron burden are associated with excessive production of the angiogenic protein placenta growth factor (PlGF) and high estimated pulmonary artery pressure. Enforced PlGF expression in mice stimulates production of the potent vasoconstrictor endothelin-1, producing pulmonary hypertension. We now demonstrate heme-bound iron (hemin) induces PlGF mRNA >200-fold in a dose- and time-dependent fashion. In murine and human erythroid cells, expression of erythroid Krüppel-like factor (EKLF) precedes PlGF, and its enforced expression in human erythroid progenitor cells induces PlGF mRNA. Hemin-induced expression of PlGF is abolished in EKLF-deficient murine erythroid cells but rescued by conditional expression of EKLF. Chromatin immunoprecipitation reveals that EKLF binds to the PlGF promoter region. SCD patients show higher level expression of both EKLF and PlGF mRNA in circulating blood cells, and markers of iron overload are associated with high PlGF and early mortality. Finally, PlGF association with iron burden generalizes to other human diseases of iron overload. Our results demonstrate a specific mechanistic pathway induced by excess iron that is linked in humans with SCD and in mice to markers of vasculopathy and pulmonary hypertension. These trials were registered at www.clinicaltrials.gov as #NCT00007150, #NCT00023296, #NCT00081523, and #NCT00352430.
Assuntos
Anemia Falciforme/sangue , Células Eritroides/metabolismo , Heme/metabolismo , Ferro/sangue , Fatores de Transcrição Kruppel-Like/sangue , Proteínas da Gravidez/sangue , Adulto , Anemia Falciforme/complicações , Anemia Falciforme/genética , Animais , Diferenciação Celular , Células Eritroides/patologia , Hemina/metabolismo , Humanos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/etiologia , Sobrecarga de Ferro/sangue , Sobrecarga de Ferro/genética , Células K562 , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Fator de Crescimento Placentário , Proteínas da Gravidez/genética , Regiões Promotoras Genéticas , RNA Mensageiro/sangue , RNA Mensageiro/genéticaRESUMO
The apolipoprotein E (apoE) is a classic example of a gene exhibiting pleiotropism. We examine potential pleiotropic associations of the apoE2 allele in three biodemographic cohorts of long-living individuals, offspring, and spouses from the Long Life Family Study, and intermediate mechanisms, which can link this allele with age-related phenotypes. We focused on age-related macular degeneration, bronchitis, asthma, pneumonia, stroke, creatinine, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, diseases of heart (HD), cancer, and survival. Our analysis detected favorable associations of the ε2 allele with lower LDL-C levels, lower risks of HD, and better survival. The ε2 allele was associated with LDL-C in each gender and biodemographic cohort, including long-living individuals, offspring, and spouses, resulting in highly significant association in the entire sample (ß = -7.1, p = 6.6 × 10-44). This allele was significantly associated with HD in long-living individuals and offspring (relative risk [RR] = 0.60, p = 3.1 × 10-6) but this association was not mediated by LDL-C. The protective effect on survival was specific for long-living women but it was not explained by LDL-C and HD in the adjusted model (RR = 0.70, p = 2.1 × 10-2). These results show that ε2 allele may favorably influence LDL-C, HD, and survival through three mechanisms. Two of them (HD- and survival-related) are pronounced in the long-living parents and their offspring; the survival-related mechanism is also sensitive to gender. The LDL-C-related mechanism appears to be independent of these factors. Insights into mechanisms linking ε2 allele with age-related phenotypes given biodemographic structure of the population studied may benefit translation of genetic discoveries to health care and personalized medicine.
Assuntos
Envelhecimento/genética , Alelos , Apolipoproteína E2/genética , Estado Terminal/mortalidade , Predisposição Genética para Doença/epidemiologia , Predisposição Genética para Doença/genética , Longevidade/genética , Distribuição por Idade , Doença Crônica/mortalidade , Medicina Baseada em Evidências , Feminino , Marcadores Genéticos , Humanos , Internacionalidade , Masculino , Pessoa de Meia-Idade , Prevalência , Locos de Características Quantitativas/genética , Fatores de Risco , Distribuição por Sexo , Taxa de SobrevidaRESUMO
The very low density lipoprotein receptor (VLDLR) is a member of the low-density lipoprotein receptor family that binds multiple ligands and plays a key role in brain development. Although the VLDLR mediates pleiotropic biological processes, only a limited amount of information is available regarding its role in adaptive immunity. In this study, we identify an important role for the VLDLR in attenuating house dust mite (HDM)-induced airway inflammation in experimental murine asthma. We show that HDM-challenged Vldlr(-/-) mice have augmented eosinophilic and lymphocytic airway inflammation with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia. A genome-wide analysis of the lung transcriptome identified that mRNA levels of CD209e (DC-SIGNR4), a murine homolog of DC-SIGN, were increased in the lungs of HDM-challenged Vldlr(-/-) mice, which suggested that the VLDLR might modify dendritic cell (DC) function. Consistent with this, VLDLR expression by human monocyte-derived DCs was increased by HDM stimulation. In addition, 55% of peripheral blood CD11c(+) DCs from individuals with allergy expressed VLDLR under basal conditions. Lastly, the adoptive transfer of HDM-pulsed, CD11c(+) bone marrow-derived DCs (BMDCs) from Vldlr(-/-) mice to the airways of wild type recipient mice induced augmented eosinophilic and lymphocytic airway inflammation upon HDM challenge with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia, as compared with the adoptive transfer of HDM-pulsed, CD11c(+) BMDCs from wild type mice. Collectively, these results identify a novel role for the VLDLR as a negative regulator of DC-mediated adaptive immune responses in HDM-induced allergic airway inflammation.
Assuntos
Imunidade Adaptativa , Células Dendríticas/imunologia , Pyroglyphidae , Receptores de LDL/imunologia , Hipersensibilidade Respiratória/imunologia , Animais , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/patologia , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Masculino , Camundongos , Camundongos Knockout , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de LDL/genética , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/patologia , Células Th2/imunologia , Células Th2/patologiaRESUMO
RATIONALE: Mitochondria are semiautonomous cellular organelles with their own genome, which not only supply energy but also participate in cell death pathways. MicroRNAs (miRNAs) are usually 19 to 25 nt long, noncoding RNAs, involved in posttranscriptional gene regulation by binding to the 3'-untranslated regions of target mRNA, which impact on diverse cellular processes. OBJECTIVE: To determine if nuclear miRNAs translocate into the mitochondria and regulate mitochondrial function with possible pathophysiological implications in cardiac myocytes. METHODS AND RESULTS: We find that miR-181c is encoded in the nucleus, assembled in the cytoplasm, and finally translocated into the mitochondria of cardiac myocytes. Immunoprecipitation of Argonaute 2 from the mitochondrial fraction indicates binding of cytochrome c oxidase subunit 1 (mt-COX1) mRNA from the mitochondrial genome with miR-181c. Also, a luciferase reporter construct shows that mi-181c binds to the 3'UTR of mt-COX1. To study whether miR-181c regulates mt-COX1, we overexpressed precursor miR-181c (or a scrambled sequence) in primary cultures of neonatal rat ventricular myocytes. Overexpression of miR-181c did not change mt-COX1 mRNA but significantly decreased mt-COX1 protein, suggesting that miR-181c is primarily a translational regulator of mt-COX1. In addition to altering mt-COX1, overexpression of miR-181c results in increased mt-COX2 mRNA and protein content, with an increase in both mitochondrial respiration and reactive oxygen species generation in neonatal rat ventricular myocytes. Thus, our data show for the first time that miR-181c can enter and target the mitochondrial genome, ultimately causing electron transport chain complex IV remodeling and mitochondrial dysfunction. CONCLUSIONS: Nuclear miR-181c translocates into the mitochondria and regulates mitochondrial genome expression. This unique observation may open a new dimension to our understanding of mitochondrial dynamics and the role of miRNA in mitochondrial dysfunction.
Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Genoma Mitocondrial/genética , MicroRNAs/fisiologia , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/fisiologia , Animais , Células Cultivadas , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To identify transcriptomic biomarkers of coronary heart disease (CHD) in 188 cases with CHD and 188 age- and sex-matched controls who were participants in the Framingham Heart Study. APPROACH AND RESULTS: A total of 35 genes were differentially expressed in cases with CHD versus controls at false discovery rate<0.5, including GZMB, TMEM56, and GUK1. Cluster analysis revealed 3 gene clusters associated with CHD, 2 linked to increased erythrocyte production and a third to reduced natural killer and T cell activity in cases with CHD. Exon-level results corroborated and extended the gene-level results. Alternative splicing analysis suggested that GUK1 and 38 other genes were differentially spliced in cases with CHD versus controls. Gene Ontology analysis linked ubiquitination and T-cell-related pathways with CHD. CONCLUSIONS: Two bioinformatically defined groups of genes show consistent associations with CHD. Our findings are consistent with the hypotheses that hematopoesis is upregulated in CHD, possibly reflecting a compensatory mechanism, and that innate immune activity is disrupted in CHD or altered by its treatment. Transcriptomic signatures may be useful in identifying pathways associated with CHD and point toward novel therapeutic targets for its treatment and prevention.
Assuntos
Doença das Coronárias/epidemiologia , Doença das Coronárias/genética , DNA Recombinante/genética , Predisposição Genética para Doença/epidemiologia , Transcriptoma/genética , Distribuição por Idade , Idoso , Estudos de Casos e Controles , Análise por Conglomerados , Éxons/genética , Feminino , Granzimas/genética , Humanos , Incidência , Masculino , Proteínas de Membrana , Proteínas dos Microfilamentos , Pessoa de Meia-Idade , Neurofibromina 2/genética , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Reprodutibilidade dos Testes , Fatores de Risco , Distribuição por SexoRESUMO
OBJECTIVE: Mice genetically deficient in endothelial nitric oxide synthase (eNOS(-/-)) are hypertensive with lower circulating nitrite levels, indicating the importance of constitutively produced nitric oxide (NOâ¢) to blood pressure regulation and vascular homeostasis. Although the current paradigm holds that this bioactivity derives specifically from the expression of eNOS in endothelium, circulating blood cells also express eNOS protein. A functional red cell eNOS that modulates vascular NO⢠signaling has been proposed. APPROACH AND RESULTS: To test the hypothesis that blood cells contribute to mammalian blood pressure regulation via eNOS-dependent NO⢠generation, we cross-transplanted wild-type and eNOS(-/-) mice, producing chimeras competent or deficient for eNOS expression in circulating blood cells. Surprisingly, we observed a significant contribution of both endothelial and circulating blood cell eNOS to blood pressure and systemic nitrite levels, the latter being a major component of the circulating NO⢠reservoir. These effects were abolished by the NOS inhibitor L-NG-nitroarginine methyl ester and repristinated by the NOS substrate L-arginine and were independent of platelet or leukocyte depletion. Mouse erythrocytes were also found to carry an eNOS protein and convert (14)C-arginine into (14)C-citrulline in NOS-dependent fashion. CONCLUSIONS: These are the first studies to definitively establish a role for a blood-borne eNOS, using cross-transplant chimera models, that contributes to the regulation of blood pressure and nitrite homeostasis. This work provides evidence suggesting that erythrocyte eNOS may mediate this effect.
Assuntos
Pressão Sanguínea/fisiologia , Homeostase/fisiologia , Hipertensão/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Nitritos/sangue , Animais , Arginina/sangue , Arginina/farmacocinética , Radioisótopos de Carbono , Citrulina/sangue , Citrulina/farmacocinética , Endotélio Vascular/enzimologia , Inibidores Enzimáticos/farmacologia , Eritrócitos/enzimologia , Hipertensão/genética , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: Genetic approaches have identified numerous loci associated with coronary heart disease (CHD). The molecular mechanisms underlying CHD gene-disease associations, however, remain unclear. We hypothesized that genetic variants with both strong and subtle effects drive gene subnetworks that in turn affect CHD. APPROACH AND RESULTS: We surveyed CHD-associated molecular interactions by constructing coexpression networks using whole blood gene expression profiles from 188 CHD cases and 188 age- and sex-matched controls. Twenty-four coexpression modules were identified, including 1 case-specific and 1 control-specific differential module (DM). The DMs were enriched for genes involved in B-cell activation, immune response, and ion transport. By integrating the DMs with gene expression-associated single-nucleotide polymorphisms and with results of genome-wide association studies of CHD and its risk factors, the control-specific DM was implicated as CHD causal based on its significant enrichment for both CHD and lipid expression-associated single-nucleotide polymorphisms. This causal DM was further integrated with tissue-specific Bayesian networks and protein-protein interaction networks to identify regulatory key driver genes. Multitissue key drivers (SPIB and TNFRSF13C) and tissue-specific key drivers (eg, EBF1) were identified. CONCLUSIONS: Our network-driven integrative analysis not only identified CHD-related genes, but also defined network structure that sheds light on the molecular interactions of genes associated with CHD risk.
Assuntos
Doença da Artéria Coronariana/epidemiologia , Doença da Artéria Coronariana/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes/genética , Predisposição Genética para Doença/epidemiologia , Biologia de Sistemas/métodos , Distribuição por Idade , Idoso , Teorema de Bayes , Estudos de Casos e Controles , Doença da Artéria Coronariana/fisiopatologia , Feminino , Variação Genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Valores de Referência , Medição de Risco , Distribuição por SexoRESUMO
It is well recognized that there is sex-dimorphic expression of mRNA and protein in the heart; however, the underlying mechanism is poorly understood. Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiac function, and the expression levels of eNOS differ between male and female hearts. The aim of this study was to examine whether expression of specific microRNA (miRNA, miR) in males and females contributes to changes in the expression of eNOS. miRNA was extracted from the myocardium of male and female C57BL/6 mice and subjected to an Affymetrix miRNA array. Decreased expression of miR-222 was discovered in females and confirmed by qRT-PCR from whole heart or isolated cardiomyocytes. The transcription factor V-ets erythroblastosis virus E26 oncogene homolog-1 (ets-1) was identified as a potential target of miR-222 using TargetScan, and fivefold increased ets-1 protein expression in females was confirmed by Western blot. Targeting of ets-1 by miR-222 was determined in HEK293 cells overexpressing luciferase under regulation of either the ets-1 3'-UTR, a null 3'-UTR control, or a scrambled ets-1 3'-UTR and treated with a small molecule miR-222 mimic or inhibitor. Additionally qRT-PCR confirmed that mRNA levels of the ets-1 transcriptional target, eNOS, were 25% higher in females. Compared with untreated myocyte controls, 50% inhibition of eNOS expression was achieved by treatment with a miR-222 mimic, compared with a 25% increase due to miR-222 inhibitor. Our findings indicate that sex-dependent miR-222 regulation alters the expression of the cardiac regulatory protein eNOS.
Assuntos
MicroRNAs/metabolismo , Miocárdio/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Caracteres Sexuais , Animais , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/enzimologia , Óxido Nítrico Sintase Tipo III/genética , Proteína Proto-Oncogênica c-ets-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-ets-1/genéticaRESUMO
Nitrite (NO(2)(-)) is an intrinsic signaling molecule that is reduced to NO during ischemia and limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain. Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both acute and delayed (24 h before ischemia) exposure to physiological concentrations of nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion injury. This cytoprotection is associated with increases in mitochondrial oxidative phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed by reoxygenation were directly protected by nitrite administered both in vitro during anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-dependently modifies and inhibits complex I by posttranslational S-nitrosation; this dampens electron transfer and effectively reduces reperfusion reactive oxygen species generation and ameliorates oxidative inactivation of complexes II-IV and aconitase, thus preventing mitochondrial permeability transition pore opening and cytochrome c release. These data suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury and may represent an effector of the cell-survival program of ischemic preconditioning and the Mediterranean diet.
Assuntos
Mitocôndrias/metabolismo , Nitritos/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Aconitato Hidratase/metabolismo , Administração Oral , Animais , Citocromos c/metabolismo , Citoproteção/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Precondicionamento Isquêmico , Fígado/irrigação sanguínea , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Mimetismo Molecular/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Nitritos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
Chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B lymphocytes, involves blood, bone marrow, and secondary lymphoid organs such as the lymph nodes (LN). A role of the tissue microenvironment in the pathogenesis of CLL is hypothesized based on in vitro observations, but its contribution in vivo remains ill-defined. To elucidate the effects of tumor-host interactions in vivo, we purified tumor cells from 24 treatment-naive patients. Samples were obtained concurrently from blood, bone marrow, and/or LN and analyzed by gene expression profiling. We identified the LN as a key site in CLL pathogenesis. CLL cells in the LN showed up-regulation of gene signatures, indicating B-cell receptor (BCR) and nuclear factor-κB activation. Consistent with antigen-dependent BCR signaling and canonical nuclear factor-κB activation, we detected phosphorylation of SYK and IκBα, respectively. Expression of BCR target genes was stronger in clinically more aggressive CLL, indicating more effective BCR signaling in this subtype in vivo. Tumor proliferation, quantified by the expression of the E2F and c-MYC target genes and verified with Ki67 staining by flow cytometry, was highest in the LN and was correlated with clinical disease progression. These data identify the disruption of tumor microenvironment interactions and the inhibition of BCR signaling as promising therapeutic strategies in CLL. This study is registered at http://clinicaltrials.gov as NCT00019370.
Assuntos
Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Adulto , Proliferação de Células , Separação Celular , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfonodos/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
Bortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines. Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We conclude that plasmacytic differentiation in the absence of an increased secretory load can enable cells to withstand the stress of proteasome inhibition. Expression of CD38 and IRF4 could serve as markers of bortezomib resistance in MCL. This study has been registered at http://clinicaltrials.gov as NCT00131976.
Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Linfoma de Célula do Manto/tratamento farmacológico , Plasmócitos/patologia , Pirazinas/farmacologia , ADP-Ribosil Ciclase 1/biossíntese , Idoso , Western Blotting , Bortezomib , Diferenciação Celular , Linhagem Celular Tumoral , Separação Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Fatores Reguladores de Interferon/biossíntese , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
RATIONALE: An increased tricuspid regurgitation jet velocity (TRV > 2.5 m/s) and pulmonary hypertension defined by right heart catheterization both independently confer increased mortality in sickle cell disease (SCD). OBJECTIVES: We explored the usefulness of peripheral blood mononuclear cell-derived gene signatures as biomarkers for an elevated TRV in SCD. METHODS: Twenty-seven patients with SCD underwent echocardiography and peripheral blood mononuclear cell isolation for expression profiling and 112 patients with SCD were genotyped for single-nucleotide polymorphisms. MEASUREMENTS AND MAIN RESULTS: Genome-wide gene and miRNA expression profiles were correlated against TRV, yielding 631 transcripts and 12 miRNAs. Support vector machine analysis identified a 10-gene signature including GALNT13 (encoding polypeptide N-acetylgalactosaminyltransferase 13) that discriminates patients with and without increased TRV with 100% accuracy. This finding was then validated in a cohort of patients with SCD without (n = 10) and with pulmonary hypertension (n = 10, 90% accuracy). Increased TRV-related miRNAs revealed strong in silico binding predictions of miR-301a to GALNT13 corroborated by microarray analyses demonstrating an inverse correlation between their expression. A genetic association study comparing patients with an elevated (n = 49) versus normal (n = 63) TRV revealed five significant single-nucleotide polymorphisms within GALNT13 (P < 0.005), four trans-acting (P < 2.1 × 10(-7)) and one cis-acting (P = 0.6 × 10(-4)) expression quantitative trait locus upstream of the adenosine-A2B receptor gene (ADORA2B). CONCLUSIONS: These studies validate the clinical usefulness of genomic signatures as potential biomarkers and highlight ADORA2B and GALNT13 as potential candidate genes in SCD-associated elevated TRV.
Assuntos
Anemia Falciforme/genética , Anemia Falciforme/fisiopatologia , N-Acetilgalactosaminiltransferases/genética , Receptor A2B de Adenosina/genética , Insuficiência da Valva Tricúspide/genética , Insuficiência da Valva Tricúspide/fisiopatologia , Adulto , Anemia Falciforme/diagnóstico por imagem , Cateterismo Cardíaco , Estudos de Coortes , Ecocardiografia Doppler/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Marcadores Genéticos/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Hipertensão Pulmonar/diagnóstico por imagem , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Masculino , Análise em Microsséries/métodos , Polimorfismo de Nucleotídeo Único/genética , Artéria Pulmonar/diagnóstico por imagem , Artéria Pulmonar/fisiopatologia , Reprodutibilidade dos Testes , Máquina de Vetores de Suporte , Insuficiência da Valva Tricúspide/diagnóstico por imagem , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Despite a growing number of reports of gene expression analysis from blood-derived RNA sources, there have been few systematic comparisons of various RNA sources in transcriptomic analysis or for biomarker discovery in the context of cardiovascular disease (CVD). As a pilot study of the Systems Approach to Biomarker Research (SABRe) in CVD Initiative, this investigation used Affymetrix Exon arrays to characterize gene expression of three blood-derived RNA sources: lymphoblastoid cell lines (LCL), whole blood using PAXgene tubes (PAX), and peripheral blood mononuclear cells (PBMC). Their performance was compared in relation to identifying transcript associations with sex and CVD risk factors, such as age, high-density lipoprotein, and smoking status, and the differential blood cell count. We also identified a set of exons that vary substantially between participants, but consistently in each RNA source. Such exons are thus stable phenotypes of the participant and may potentially become useful fingerprinting biomarkers. In agreement with previous studies, we found that each of the RNA sources is distinct. Unlike PAX and PBMC, LCL gene expression showed little association with the differential blood count. LCL, however, was able to detect two genes related to smoking status. PAX and PBMC identified Y-chromosome probe sets similarly and slightly better than LCL.
Assuntos
Sangue/metabolismo , Doenças Cardiovasculares/sangue , Perfilação da Expressão Gênica , Leucócitos Mononucleares/metabolismo , Linfócitos/metabolismo , Adulto , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/metabolismo , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Linhagem Celular , Estudos de Coortes , Feminino , Humanos , Linfócitos/patologia , Masculino , Massachusetts , Análise em Microsséries , Pessoa de Meia-Idade , Projetos PilotoRESUMO
BACKGROUND: Exceptional longevity as manifested by the lower incidence and delayed onset of age-related disabilities/diseases that include cardiovascular disease, Alzheimer's disease, cancer is believed to be influenced by inherent protective molecular factors in exceptionally long-lived individuals. Unraveling these protective factors could lead to the discovery of therapeutic target(s) and interventions to promote healthy aging. METHODS: In this context, the National Institute on Aging has established a collection of translational longevity research projects (ie, the Long-Life Family Study, the Longevity Consortium, Longevity Genomics, and the Integrative Longevity Omics) which are generating large omics data sets spanning the human genome to phenome and have embarked on cross-species multiomic data analyses integrating human and nonhuman species that display wide variation in their life spans. RESULTS: It is expected that these studies will discover key signaling pathways that influence exceptional health span and identify therapeutic targets for translation to enhance health and life span. Other efforts related to translational longevity research include the "Comprehensive Evaluation of Aging-Related Clinical Outcomes and Geroproteins study," which focuses on potential effects in humans of polypeptides/proteins whose circulating levels change with age, and for which experimental evidence indicates reversal or acceleration of aging changes. The "Predictive Human Mechanistic Markers Network" is devoted to the development of predictive markers of aging, for target engagement when testing novel interventions for healthy aging. CONCLUSION: We describe here the significance, the unique study design, categories of data sets, analytical strategies, and a data portal to facilitate open science and sharing of resources from these longevity studies to identify and validate potential therapeutic targets for healthy aging.
Assuntos
Doença de Alzheimer , Longevidade , Estados Unidos , Humanos , Longevidade/genética , Pesquisa Translacional Biomédica , Envelhecimento/genética , National Institute on Aging (U.S.) , Doença de Alzheimer/epidemiologia , BiomarcadoresRESUMO
Aging is characterized by increased mortality, functional decline, and exponential increases in the incidence of diseases such as cancer, stroke, cardiovascular disease, neurological disease, respiratory disease, etc. Though the role of aging in these diseases is widely accepted and considered to be a common denominator, the underlying mechanisms are largely unknown. A significant age-related feature observed in many population cohorts is somatic mosaicism, the detectable accumulation of somatic mutations in multiple cell types and tissues, particularly those with high rates of cell turnover (e.g., skin, liver, and hematopoietic cells). Somatic mosaicism can lead to the development of cellular clones that expand with age in otherwise normal tissues. In the hematopoietic system, this phenomenon has generally been referred to as "clonal hematopoiesis of indeterminate potential" (CHIP) when it applies to a subset of clones in which mutations in driver genes of hematologic malignancies are found. Other mechanisms of clonal hematopoiesis, including large chromosomal alterations, can also give rise to clonal expansion in the absence of conventional CHIP driver gene mutations. Both types of clonal hematopoiesis (CH) have been observed in studies of animal models and humans in association with altered immune responses, increased mortality, and disease risk. Studies in murine models have found that some of these clonal events are involved in abnormal inflammatory and metabolic changes, altered DNA damage repair and epigenetic changes. Studies in long-lived individuals also show the accumulation of somatic mutations, yet at this advanced age, carriership of somatic mutations is no longer associated with an increased risk of mortality. While it remains to be elucidated what factors modify this genotype-phenotype association, i.e., compensatory germline genetics, cellular context of the mutations, protective effects to diseases at exceptional age, it points out that the exceptionally long-lived are key to understand the phenotypic consequences of CHIP mutations. Assessment of the clinical significance of somatic mutations occurring in blood cell types for age-related outcomes in human populations of varied life and health span, environmental exposures, and germline genetic risk factors will be valuable in the development of personalized strategies tailored to specific somatic mutations for healthy aging.
RESUMO
Hematopoietic differentiation is strictly regulated by complex network of transcription factors that are controlled by ligands binding to cell surface receptors. Disruptions of the intricate sequences of transcriptional activation and suppression of multiple genes cause hematological diseases, such as leukemias, myelodysplastic syndromes, or myeloproliferative syndromes. From a clinical standpoint, deciphering the pattern of gene expression during hematopoiesis may help unravel disease-specific mechanisms in hematopoietic malignancies. Herein, we describe a human in vitro hematopoietic model system where lineage-specific differentiation of CD34(+) cells was accomplished using specific cytokines. Microarray and RNAseq-based whole transcriptome and exome analysis was performed on the differentiated erythropoietic, granulopoietic, and megakaryopoietic cells to delineate changes in expression of whole transcripts and exons. Analysis on the Human 1.0 ST exon arrays indicated differential expression of 172 genes (P < 0.0000001) and significant alternate splicing of 86 genes during differentiation. Pathway analysis identified these genes to be involved in Rac/RhoA signaling, Wnt/B-catenin signaling and alanine/aspartate metabolism. Comparison of the microarray data to next generation RNAseq analysis during erythroid differentiation demonstrated a high degree of correlation in gene (R = 0.72) and exon (R = 0.62) expression. Our data provide a molecular portrait of events that regulate differentiation of hematopoietic cells. Knowledge of molecular processes by which the cells acquire their cell-specific fate would be beneficial in developing cell-based therapies for human diseases.
Assuntos
Processamento Alternativo/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Análise de Sequência de DNA , Transcriptoma/genética , Antígenos CD34/metabolismo , Análise por Conglomerados , Células Eritroides/citologia , Células Eritroides/metabolismo , Éxons/genética , Citometria de Fluxo , Regulação da Expressão Gênica , Redes Reguladoras de Genes/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/genéticaRESUMO
BACKGROUND: High fat diets are known to be a risk factor for prostate cancer. In this study, we investigated the effect of high fat diet on mouse prostate gene expression. METHODS: C57BL/6J mice were fed either a control or high fat diet for 12 weeks. Microarray analyses were performed on mouse ventral prostate (VP) and dorsolateral prostate (DLP), followed by canonical pathway analysis and regulatory network identification. mRNA changes were confirmed by real time PCR. RESULTS: Approximately 2,125, and 1,194 genes responded significantly to the high fat diet in VP, DLP, respectively. Pathways and networks related to oxidative stress, glutathione metabolism, NRF-mediated oxidative stress response and NF-kappaB were all differentially regulated by high fat diet. Glutathione peroxidase 3 (GPx3) mRNA levels were decreased by approximately twofold by high fat diet in all three prostate lobes. In human non-transformed prostate cells (PrSC, PrEC, and BPH-1), cholesterol loading decreased GPx3 expression, and increased H2 O2 levels of culture medium. Troglitazone increased GPx3 expression in three normal prostate cells, and decreased H2 O2 levels. In addition, troglitazone attenuated cholesterol-induced H2 O2 increase. Tissue from prostate cancer biopsies had decreased GPx3 mRNA and its level was inversely related to the Gleason score. CONCLUSIONS: High fat diet alters pathways related to many genes concerned with oxidative stress. GPx3, a gene identified by this analysis, was found to be down-regulated by high fat diet and appears be decreased in human prostate cancers, suggesting that GPx3 may have a possible role in modulating carcinogenesis. Prostate 71:1499-1509, 2011. © 2011 Wiley-Liss, Inc.