RESUMO
Nicotinamide phosphoribosyltransferase (NAMPT) upregulation in human pulmonary artery endothelial cells (hPAECs) is associated with pulmonary arterial hypertension (PAH) progression and pulmonary vascular remodeling. The underlying mechanisms regulating NAMPT expression are still not clear. In this study, we aimed to study the regulation of NAMPT expression by microRNA410 (miR410) in hPAECs and explore the role of miR410 in the pathogenesis of experimental pulmonary hypertension. We show that miR410 targets the 3' UTR of NAMPT and that, concomitant with NAMPT upregulation, miR410 is downregulated in lungs of mice exposed to hypoxia-induced pulmonary hypertension (HPH). Our results also demonstrate that miR410 directly inhibits NAMPT expression. Overexpression of miR410 in hPAECs inhibits basal and VEGF-induced proliferation, migration and promotes apoptosis of hPAECs, while miR410 inhibition via antagomirs has the opposite effect. Finally, administration of miR410 mimics in vivo attenuated induction of NAMPT in PAECs and prevented the development of HPH in mice. Our results highlight the role of miR410 in the regulation of NAMPT expression in hPAECs and show that miR410 plays a potential role in PAH pathobiology by targeting a modulator of pulmonary vascular remodeling.
Assuntos
Citocinas/metabolismo , Regulação da Expressão Gênica , Hipertensão Pulmonar/prevenção & controle , Hipóxia/complicações , MicroRNAs/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Artéria Pulmonar/citologia , Remodelação Vascular/fisiologia , Animais , Apoptose , Movimento Celular , Proliferação de Células , Células Cultivadas , Citocinas/genética , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Camundongos , Camundongos Endogâmicos C57BL , Nicotinamida Fosforribosiltransferase/genética , Artéria Pulmonar/metabolismoRESUMO
BACKGROUND: Sphingosine- 1-Phosphate (S1P) is a bioactive lipid and an intracellular as well as an extracellular signaling molecule. S1P ligand specifically binds to five related cell surface G-protein-coupled receptors (S1P1-5). S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. We previously reported that knock down of SphK1 (Sphk1 -/- ) in a neonatal mouse BPD model conferred significant protection against hyperoxia induced lung injury. To better understand the underlying molecular mechanisms, genome-wide gene expression profiling was performed on mouse lung tissue using Affymetrix MoGene 2.0 array. RESULTS: Two-way ANOVA analysis was performed and differentially expressed genes under hyperoxia were identified using Sphk1 -/- mice and their wild type (WT) equivalents. Pathway (PW) enrichment analyses identified several signaling pathways that are likely to play a key role in hyperoxia induced lung injury in the neonates. These included signaling pathways that were anticipated such as those involved in lipid signaling, cell cycle regulation, DNA damage/apoptosis, inflammation/immune response, and cell adhesion/extracellular matrix (ECM) remodeling. We noted hyperoxia induced downregulation of the expression of genes related to mitotic spindle formation in the WT which was not observed in Sphk1 -/- neonates. Our data clearly suggests a role for SphK1 in neonatal hyperoxic lung injury through elevated inflammation and apoptosis in lung tissue. Further, validation by RT-PCR on 24 differentially expressed genes showed 83% concordance both in terms of fold change and vectorial changes. Our findings are in agreement with previously reported human BPD microarray data and completely support our published in vivo findings. In addition, the data also revealed a significant role for additional unanticipitated signaling pathways involving Wnt and GADD45. CONCLUSION: Using SphK1 knockout mice and differential gene expression analysis, we have shown here that S1P/SphK1 signaling plays a key role in promoting hyperoxia induced DNA damage, inflammation, apoptosis and ECM remodeling in neonatal lungs. It also appears to suppress pro-survival cellular responses involved in normal lung development. We therefore propose SphK1 as a therapeutic target for the development drugs to combat BPD.
Assuntos
Displasia Broncopulmonar/complicações , Perfilação da Expressão Gênica , Hiperóxia/etiologia , Hiperóxia/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Animais Recém-Nascidos , Apoptose/genética , Displasia Broncopulmonar/tratamento farmacológico , Ciclo Celular/genética , Modelos Animais de Doenças , Deleção de Genes , Humanos , Hiperóxia/patologia , Lisofosfolipídeos/metabolismo , Camundongos , Terapia de Alvo Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Transcrição GênicaRESUMO
Genotyping is commonly used to define specific gene alterations or the presence of transgenes in mice. This procedure is typically done using DNA isolated from mouse tail tissue. Although there are commercially available kits for tail DNA isolation, they can be time consuming and costly for routine genotyping. In this study, we describe a rapid, "crude" DNA isolation method using mouse tail tissue and compare it to a frequently used, commercially available kit in the genotyping of over 1,000 total mice from 8 genetic lines. Our genotyping results were obtained faster and less expensively but with the same success rate (Crude method: 97.7 %, Kit method: 98.4 %). To our knowledge, this is the first systematic study to compare the reliability of this crude DNA isolation method for mouse genotyping compared to a commercially available kit.
