RESUMO
Recent reports, including ours, have indicated that microRNA (miR)-33 located within the intron of sterol regulatory element binding protein (SREBP) 2 controls cholesterol homeostasis and can be a potential therapeutic target for the treatment of atherosclerosis. Here, we show that SPAST, which encodes a microtubule-severing protein called SPASTIN, was a novel target gene of miR-33 in human. Actually, the miR-33 binding site in the SPAST 3'-UTR is conserved not in mice but in mid to large mammals, and it is impossible to clarify the role of miR-33 on SPAST in mice. We demonstrated that inhibition of miR-33a, a major form of miR-33 in human neurons, via locked nucleic acid (LNA)-anti-miR ameliorated the pathological phenotype in hereditary spastic paraplegia (HSP)-SPG4 patient induced pluripotent stem cell (iPSC)-derived cortical neurons. Thus, miR-33a can be a potential therapeutic target for the treatment of HSP-SPG4.
Assuntos
Terapia Genética/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/genética , Células-Tronco Neurais/metabolismo , Neuritos/metabolismo , Oligonucleotídeos/genética , Paraplegia Espástica Hereditária/terapia , Espastina/genética , Regiões 3' não Traduzidas , Sítios de Ligação , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Células-Tronco Neurais/patologia , Neuritos/patologia , Neurogênese , Oligonucleotídeos/metabolismo , Fenótipo , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Paraplegia Espástica Hereditária/patologia , Espastina/metabolismoRESUMO
RATIONALE: Heart failure and atherosclerosis share the underlying mechanisms of chronic inflammation followed by fibrosis. A highly conserved microRNA (miR), miR-33, is considered as a potential therapeutic target for atherosclerosis because it regulates lipid metabolism and inflammation. However, the role of miR-33 in heart failure remains to be elucidated. OBJECTIVE: To clarify the role of miR-33 involved in heart failure. METHODS AND RESULTS: We first investigated the expression levels of miR-33a/b in human cardiac tissue samples with dilated cardiomyopathy. Increased expression of miR-33a was associated with improving hemodynamic parameters. To clarify the role of miR-33 in remodeling hearts, we investigated the responses to pressure overload by transverse aortic constriction in miR-33-deficient (knockout [KO]) mice. When mice were subjected to transverse aortic constriction, miR-33 expression levels were significantly upregulated in wild-type left ventricles. There was no difference in hypertrophic responses between wild-type and miR-33KO hearts, whereas cardiac fibrosis was ameliorated in miR-33KO hearts compared with wild-type hearts. Despite the ameliorated cardiac fibrosis, miR-33KO mice showed impaired systolic function after transverse aortic constriction. We also found that cardiac fibroblasts were mainly responsible for miR-33 expression in the heart. Deficiency of miR-33 impaired cardiac fibroblast proliferation, which was considered to be caused by altered lipid raft cholesterol content. Moreover, cardiac fibroblast-specific miR-33-deficient mice also showed decreased cardiac fibrosis induced by transverse aortic constriction as systemic miR-33KO mice. CONCLUSION: Our results demonstrate that miR-33 is involved in cardiac remodeling, and it preserves lipid raft cholesterol content in fibroblasts and maintains adaptive fibrotic responses in the remodeling heart.
Assuntos
Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , MicroRNAs/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Remodelação Ventricular/fisiologia , Adulto , Idoso , Animais , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibrose/metabolismo , Fibrose/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Ratos , Ratos Sprague-DawleyRESUMO
Objective- Atherosclerosis is a common disease caused by a variety of metabolic and inflammatory disturbances. MicroRNA (miR)-33a within SREBF2 (sterol regulatory element-binding factor 2) is a potent target for treatment of atherosclerosis through regulating both aspects; however, the involvement of miR-33b within SREBF1 remains largely unknown. Although their host genes difference could lead to functional divergence of miR-33a/b, we cannot dissect the roles of miR-33a/b in vivo because of lack of miR-33b sequences in mice, unlike human. Approach and Results- Here, we analyzed the development of atherosclerosis using miR-33b knock-in humanized mice under apolipoprotein E-deficient background. MiR-33b is prominent both in human and mice on atheroprone condition. MiR-33b reduced serum high-density lipoprotein cholesterol levels and systemic reverse cholesterol transport. MiR-33b knock-in macrophages showed less cholesterol efflux capacity and higher inflammatory state via regulating lipid rafts. Thus, miR-33b promotes vulnerable atherosclerotic plaque formation. Furthermore, bone marrow transplantation experiments strengthen proatherogenic roles of macrophage miR-33b. Conclusions- Our data demonstrated critical roles of SREBF1-miR-33b axis on both lipid profiles and macrophage phenotype remodeling and indicate that miR-33b is a promising target for treating atherosclerosis.
Assuntos
Aterosclerose/metabolismo , MicroRNAs/metabolismo , Placa Aterosclerótica , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Aterosclerose/genética , Aterosclerose/patologia , Transplante de Medula Óssea , Estudos de Casos e Controles , HDL-Colesterol/sangue , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Absorção Intestinal , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Microdomínios da Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , MicroRNAs/genética , Pessoa de Meia-Idade , Fenótipo , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Triglicerídeos/sangueRESUMO
OBJECTIVE: Abdominal aortic aneurysm (AAA) is an increasingly prevalent and ultimately fatal disease with no effective pharmacological treatment. Because matrix degradation induced by vascular inflammation is the major pathophysiology of AAA, attenuation of this inflammation may improve its outcome. Previous studies suggested that miR-33 (microRNA-33) inhibition and genetic ablation of miR-33 increased serum high-density lipoprotein cholesterol and attenuated atherosclerosis. APPROACH AND RESULTS: MiR-33a-5p expression in central zone of human AAA was higher than marginal zone. MiR-33 deletion attenuated AAA formation in both mouse models of angiotensin II- and calcium chloride-induced AAA. Reduced macrophage accumulation and monocyte chemotactic protein-1 expression were observed in calcium chloride-induced AAA walls in miR-33-/- mice. In vitro experiments revealed that peritoneal macrophages from miR-33-/- mice showed reduced matrix metalloproteinase 9 expression levels via c-Jun N-terminal kinase inactivation. Primary aortic vascular smooth muscle cells from miR-33-/- mice showed reduced monocyte chemotactic protein-1 expression by p38 mitogen-activated protein kinase attenuation. Both of the inactivation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were possibly because of the increase of ATP-binding cassette transporter A1 that is a well-known target of miR-33. Moreover, high-density lipoprotein cholesterol derived from miR-33-/- mice reduced expression of matrix metalloproteinase 9 in macrophages and monocyte chemotactic protein-1 in vascular smooth muscle cells. Bone marrow transplantation experiments indicated that miR-33-deficient bone marrow cells ameliorated AAA formation in wild-type recipients. MiR-33 deficiency in recipient mice was also shown to contribute the inhibition of AAA formation. CONCLUSIONS: These data strongly suggest that inhibition of miR-33 will be effective as a novel strategy for treating AAA.
Assuntos
Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/prevenção & controle , Aortite/prevenção & controle , Mediadores da Inflamação/metabolismo , MicroRNAs/metabolismo , Angiotensina II , Animais , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Aortite/induzido quimicamente , Aortite/genética , Aortite/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Transplante de Medula Óssea , Cloreto de Cálcio , Linhagem Celular , Quimiocina CCL2/metabolismo , HDL-Colesterol/sangue , Dilatação Patológica , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fenótipo , Transdução de Sinais , Fatores de Tempo , Transfecção , Remodelação Vascular , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
RATIONALE: In some patients with type 2 diabetes mellitus (DM) without hypertension, cardiac hypertrophy and attenuated cardiac function are observed, and this insult is termed diabetic cardiomyopathy. To date, microRNA (miRNAs or miR) functions in diabetic cardiomyopathy remain to be elucidated. OBJECTIVE: To clarify the functions of miRNAs involved in diabetic cardiomyopathy caused by type 2 DM. METHODS AND RESULTS: C57BL/6 mice were fed a high-fat diet (HFD) for 20 weeks, which induced obesity and type 2 DM. miRNA microarray analyses and real-time polymerase chain reaction revealed that miR-451 levels were significantly increased in the type 2 DM mouse hearts. Because excess supply of saturated fatty acids is a cause of diabetic cardiomyopathy, we stimulated neonatal rat cardiac myocytes with palmitic acid and confirmed that miR-451 expression was increased in a dose- and time-dependent manner. Loss of miR-451 function ameliorated palmitate-induced lipotoxicity in neonatal rat cardiac myocytes. Calcium-binding protein 39 (Cab39) is a scaffold protein of liver kinase B1 (LKB1), an upstream kinase of AMP-activated protein kinase (AMPK). Cab39 was a direct target of miR-451 in neonatal rat cardiac myocytes and Cab39 overexpression rescued the lipotoxicity. To clarify miR-451 functions in vivo, we generated cardiomyocyte-specific miR-451 knockout mice. HFD-induced cardiac hypertrophy and contractile reserves were ameliorated in cardiomyocyte-specific miR-451 knockout mice compared with control mice. Protein levels of Cab39 and phosphorylated AMPK were increased and phosphorylated mammalian target of rapamycin (mTOR) was reduced in cardiomyocyte-specific miR-451 knockout mouse hearts compared with control mouse hearts. CONCLUSIONS: Our results demonstrate that miR-451 is involved in diabetic cardiomyopathy through suppression of the LKB1/AMPK pathway.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Cardiomegalia/metabolismo , Dieta Hiperlipídica/efeitos adversos , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Recém-Nascidos , Cardiomegalia/patologia , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
MicroRNAs (miRNAs; miRs) are small non-protein-coding RNAs that negatively regulate gene expression. They bind to the 3' UTR of specific mRNAs and either inhibit translation or promote mRNA degradation. There is emerging evidence linking miR-33a/b to lipid homoeostasis, targeting ABCA1,SREBF1, etc and it would appear that they have acted as "thrifty genes" during evolution to maintain cholesterol levels both at the cellular and whole body level. As we are now living in a period of "satiation", miR-33a/b no longer seem to be useful and could be potential therapeutic targets for lipid disorders and/or atherosclerosis. In this review, we describe the current understanding of the function of miR-33a/b in lipid homeostasis, focusing on the "thrifty" aspect.
Assuntos
Regiões 3' não Traduzidas , Aterosclerose/metabolismo , Metabolismo dos Lipídeos , MicroRNAs/metabolismo , Estabilidade de RNA , Transportador 1 de Cassete de Ligação de ATP/biossíntese , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Aterosclerose/genética , Aterosclerose/patologia , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
MicroRNAs (miRNAs) are small non-protein-coding RNAs that negatively regulate gene expression. They bind to the 3'-untranslated region of specific mRNAs and inhibit translation or promote mRNA degradation. Dyslipidemia/hyperlipidemia is a well-accepted risk factor for the development of atherosclerosis. The pathogenesis factors involved in lipid abnormalities are being examined extensively, and there is emerging evidence linking miRNAs to lipid metabolism. Among them, recent studies, including ours, have demonstrated that miRNAs control the expression of genes associated with high-density lipoprotein (HDL) cholesterol (HDL-C) metabolism, including ABCA1, ABCG1, and scavenger receptor class B, type I. Moreover, HDL-C itself was proved to carry miRNAs and deliver them to several different types of cells. In this review, we describe the current understanding of the functions of miRNAs in HDL metabolism and their potential in therapy for treating cardiometabolic diseases.
Assuntos
HDL-Colesterol , Dislipidemias/genética , MicroRNAs/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Aterosclerose/prevenção & controle , HDL-Colesterol/genética , HDL-Colesterol/metabolismo , Dislipidemias/metabolismo , Dislipidemias/prevenção & controle , Regulação da Expressão Gênica , Humanos , Biossíntese de Proteínas/genética , Receptores Depuradores Classe B/genéticaRESUMO
BACKGROUND: It is controversial whether angiotensin-converting enzyme inhibitors (ACEI)/angiotensin receptor blockers (ARB) provide significant survival benefits in patients with coronary artery disease (CAD) but without myocardial infarction (MI). This study investigated whether the association of ACEI/ARB therapy with clinical outcome in patients undergoing percutaneous coronary intervention (PCI) was affected by history of MI. METHODS AND RESULTS: A total of 11,590 patients undergoing first PCI were divided into 2 groups: those with MI and those without MI. All-cause and cardiovascular mortality were compared between the patients with and without ACEI/ARB at discharge in each group. In patients with MI, significantly lower 3-year all-cause/cardiovascular mortality for patients with ACEI/ARB relative to those without ACEI/ARB was noted in the total patients (all-cause: 6.6% vs. 11.7%, P<0.0001; cardiovascular: 3.8% vs. 6.9%, P<0.0001) and in the 1,007 propensity score-matched pairs (all-cause: 8.2% vs. 11.3%, P=0.018; cardiovascular: 3.7% vs. 5.7%, P=0.014). In patients without MI, however, all-cause (5.2% vs. 5.6%, P=0.56) and cardiovascular (3.2% vs. 3.0%, P=0.23) mortality were similar regardless of whether ACEI/ARB were used or not; and similarly in the 2,061 propensity score-matched pairs (all-cause: 4.1% vs. 5.4%, P=0.33; cardiovascular: 1.4% vs. 2.1%, P=0.30). CONCLUSIONS: Use of ACEI/ARB at hospital discharge was associated with lower all-cause/cardiovascular mortality in revascularized CAD patients with MI, but not in those without MI.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Doença da Artéria Coronariana/terapia , Infarto do Miocárdio/terapia , Idoso , Distribuição de Qui-Quadrado , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/mortalidade , Alta do Paciente , Intervenção Coronária Percutânea/efeitos adversos , Intervenção Coronária Percutânea/mortalidade , Pontuação de Propensão , Modelos de Riscos Proporcionais , Sistema de Registros , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
The human gaze is directed at various locations from moment to moment in acquiring information necessary to recognize the external environment at the fine resolution of foveal vision. Previous studies showed that the human gaze is attracted to particular locations in the visual field at a particular time, but it remains unclear what visual features produce such spatiotemporal bias. In this study, we used a deep convolutional neural network model to extract hierarchical visual features from natural scene images and evaluated how much the human gaze is attracted to the visual features in space and time. Eye movement measurement and visual feature analysis using the deep convolutional neural network model showed that the gaze was more strongly attracted to spatial locations containing higher-order visual features than to locations containing lower-order visual features or to locations predicted by conventional saliency. Analysis of the time course of gaze attraction revealed that the bias to higher-order visual features was prominent within a short period after the beginning of observation of the natural scene images. These results demonstrate that higher-order visual features are a strong gaze attractor in both space and time, suggesting that the human visual system uses foveal vision resources to extract information from higher-order visual features with higher spatiotemporal priority.
Assuntos
Atenção , Fixação Ocular , Humanos , Visão Ocular , Campos Visuais , Fóvea Central , Percepção VisualRESUMO
Congenital heart disease often arises from perturbations of transcription factors (TFs) that guide cardiac development. ISLET1 (ISL1) is a TF that influences early cardiac cell fate, as well as differentiation of other cell types including motor neuron progenitors (MNPs) and pancreatic islet cells. While lineage specificity of ISL1 function is likely achieved through combinatorial interactions, its essential cardiac interacting partners are unknown. By assaying ISL1 genomic occupancy in human induced pluripotent stem cell-derived cardiac progenitors (CPs) or MNPs and leveraging the deep learning approach BPNet, we identified motifs of other TFs that predicted ISL1 occupancy in each lineage, with NKX2.5 and GATA motifs being most closely associated to ISL1 in CPs. Experimentally, nearly two-thirds of ISL1-bound loci were co-occupied by NKX2.5 and/or GATA4. Removal of NKX2.5 from CPs led to widespread ISL1 redistribution, and overexpression of NKX2.5 in MNPs led to ISL1 occupancy of CP-specific loci. These results reveal how ISL1 guides lineage choices through a combinatorial code that dictates genomic occupancy and transcription.
Assuntos
Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Miócitos Cardíacos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/genética , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismoRESUMO
Nonalcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma in their advanced stages; however, there are currently no approved therapies. Here, we show that microRNA (miR)-33b in hepatocytes is critical for the development of NASH. miR-33b is located in the intron of sterol regulatory element-binding transcription factor 1 and is abundantly expressed in humans, but absent in rodents. miR-33b knock-in (KI) mice, which have a miR-33b sequence in the same intron of sterol regulatory element-binding transcription factor 1 as humans and express miR-33b similar to humans, exhibit NASH under high-fat diet feeding. This condition is ameliorated by hepatocyte-specific miR-33b deficiency but unaffected by macrophage-specific miR-33b deficiency. Anti-miR-33b oligonucleotide improves the phenotype of NASH in miR-33b KI mice fed a Gubra Amylin NASH diet, which induces miR-33b and worsens NASH more than a high-fat diet. Anti-miR-33b treatment reduces hepatic free cholesterol and triglyceride accumulation through up-regulation of the lipid metabolism-related target genes. Furthermore, it decreases the expression of fibrosis marker genes in cultured hepatic stellate cells. Thus, inhibition of miR-33b using nucleic acid medicine is a promising treatment for NASH.
Assuntos
Neoplasias Hepáticas , MicroRNAs , Hepatopatia Gordurosa não Alcoólica , Camundongos , Humanos , Animais , Hepatopatia Gordurosa não Alcoólica/genética , Antagomirs , MicroRNAs/genética , MicroRNAs/metabolismo , Colesterol , Neoplasias Hepáticas/patologia , Fatores de TranscriçãoRESUMO
Chronic inflammation and tissue fibrosis are common stress responses that worsen organ function, yet the molecular mechanisms governing their crosstalk are poorly understood. In diseased organs, stress-induced changes in gene expression fuel maladaptive cell state transitions and pathological interaction between diverse cellular compartments. Although chronic fibroblast activation worsens dysfunction of lung, liver, kidney, and heart, and exacerbates many cancers, the stress-sensing mechanisms initiating the transcriptional activation of fibroblasts are not well understood. Here, we show that conditional deletion of the transcription co-activator Brd4 in Cx3cr1-positive myeloid cells ameliorates heart failure and is associated with a dramatic reduction in fibroblast activation. Analysis of single-cell chromatin accessibility and BRD4 occupancy in vivo in Cx3cr1-positive cells identified a large enhancer proximal to Interleukin-1 beta (Il1b), and a series of CRISPR deletions revealed the precise stress-dependent regulatory element that controlled expression of Il1b in disease. Secreted IL1B functioned non-cell autonomously to activate a p65/RELA-dependent enhancer near the transcription factor MEOX1, resulting in a profibrotic response in human cardiac fibroblasts. In vivo, antibody-mediated IL1B neutralization prevented stress-induced expression of MEOX1, inhibited fibroblast activation, and improved cardiac function in heart failure. The elucidation of BRD4-dependent crosstalk between a specific immune cell subset and fibroblasts through IL1B provides new therapeutic strategies for heart disease and other disorders of chronic inflammation and maladaptive tissue remodeling.
RESUMO
The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current therapies and known causes of familial hypercholesterolemia. We found that cold shock domain-containing protein E1 (CSDE1) enhanced hepatic LDLR messenger RNA (mRNA) decay via its 3' untranslated region and regulated atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we identified 40 specific regulators of the LDLR that were not previously identified by observational human genetic studies. Among these, we demonstrated that, in HepG2 cells, CSDE1 regulated the LDLR at least as strongly as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In addition, we showed that hepatic gene silencing of Csde1 treated diet-induced dyslipidemia in mice to a similar degree as Pcsk9 silencing. These results suggest the therapeutic potential of targeting CSDE1 to manipulate the posttranscriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. Our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, may serve as a generalizable template for the identification of therapeutic targets in other human disease states.
Assuntos
Resposta ao Choque Frio , Proteínas de Ligação a DNA/metabolismo , Pró-Proteína Convertase 9 , Proteínas de Ligação a RNA/metabolismo , Animais , Humanos , Camundongos , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , RNA Mensageiro/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transcrição GênicaRESUMO
We have developed a West Nile virus (WNV) subgenomic replicon harboring the secreted alkaline phosphatase (SEAP) reporter gene instead of viral structural genes (designated repWNV/SEAP). The repWNV/SEAP allowed easy evaluation of viral replication efficiency by direct measurement of SEAP secretion in the cell culture medium in physical containment level 2 facilities. Furthermore, we validated the availability of this system using a known anti-flavivirus gene, mouse oligoadenylate synthetase 1b (Oas1b). The Oas1b-transfected cells were more resistant to repWNV/SEAP replication than the original cells. Thus, this system not only affords a useful tool for identification/evaluation of anti-flavivirus genes/drugs in terms of safety, ease of use and reliability, but should be able to reduce or replace the bioassay using laboratory animals.
Assuntos
RNA Viral/genética , RNA Viral/metabolismo , Vírus do Nilo Ocidental/enzimologia , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Fosfatase Alcalina , Animais , Linhagem Celular , Cricetinae , Regulação Viral da Expressão Gênica/fisiologia , Genes Reporter , Replicon , Proteínas Virais , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/metabolismoRESUMO
Mapping the gene-regulatory networks dysregulated in human disease would allow the design of network-correcting therapies that treat the core disease mechanism. However, small molecules are traditionally screened for their effects on one to several outputs at most, biasing discovery and limiting the likelihood of true disease-modifying drug candidates. Here, we developed a machine-learning approach to identify small molecules that broadly correct gene networks dysregulated in a human induced pluripotent stem cell (iPSC) disease model of a common form of heart disease involving the aortic valve (AV). Gene network correction by the most efficacious therapeutic candidate, XCT790, generalized to patient-derived primary AV cells and was sufficient to prevent and treat AV disease in vivo in a mouse model. This strategy, made feasible by human iPSC technology, network analysis, and machine learning, may represent an effective path for drug discovery.
Assuntos
Valvopatia Aórtica/tratamento farmacológico , Estenose da Valva Aórtica/tratamento farmacológico , Valva Aórtica/patologia , Calcinose/tratamento farmacológico , Redes Reguladoras de Genes/efeitos dos fármacos , Aprendizado de Máquina , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Algoritmos , Animais , Valva Aórtica/efeitos dos fármacos , Valva Aórtica/metabolismo , Valva Aórtica/fisiopatologia , Valvopatia Aórtica/genética , Valvopatia Aórtica/fisiopatologia , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/fisiopatologia , Calcinose/genética , Calcinose/fisiopatologia , Modelos Animais de Doenças , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Haploinsuficiência , Humanos , Células-Tronco Pluripotentes Induzidas , Camundongos Endogâmicos C57BL , RNA-Seq , Receptor Notch1/genética , Bibliotecas de Moléculas PequenasRESUMO
Adaptive thermogenesis is essential for survival, and therefore is tightly regulated by a central neural circuit. Here, we show that microRNA (miR)-33 in the brain is indispensable for adaptive thermogenesis. Cold stress increases miR-33 levels in the hypothalamus and miR-33-/- mice are unable to maintain body temperature in cold environments due to reduced sympathetic nerve activity and impaired brown adipose tissue (BAT) thermogenesis. Analysis of miR-33f/f dopamine-ß-hydroxylase (DBH)-Cre mice indicates the importance of miR-33 in Dbh-positive cells. Mechanistically, miR-33 deficiency upregulates gamma-aminobutyric acid (GABA)A receptor subunit genes such as Gabrb2 and Gabra4. Knock-down of these genes in Dbh-positive neurons rescues the impaired cold-induced thermogenesis in miR-33f/f DBH-Cre mice. Conversely, increased gene dosage of miR-33 in mice enhances thermogenesis. Thus, miR-33 in the brain contributes to maintenance of BAT thermogenesis and whole-body metabolism via enhanced sympathetic nerve tone through suppressing GABAergic inhibitory neurotransmission. This miR-33-mediated neural mechanism may serve as a physiological adaptive defense mechanism for several stresses including cold stress.
Assuntos
MicroRNAs/metabolismo , Sistema Nervoso Simpático/fisiologia , Termogênese/genética , Tecido Adiposo Marrom/fisiologia , Animais , Temperatura Corporal/fisiologia , Peso Corporal , Encéfalo/metabolismo , Linhagem Celular , Temperatura Baixa , Dieta Hiperlipídica , Estresse do Retículo Endoplasmático , Humanos , Integrases/metabolismo , Masculino , Camundongos , Camundongos Obesos , MicroRNAs/genética , Consumo de Oxigênio/fisiologia , Fenótipo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMO
The ICGN mouse strain is a glomerulosclerosis (GS) model that shows characteristic proteinuria, podocyte morphological abnormalities and increased extracellular matrix accumulation in the glomeruli, which are the final common pathology associated with a variety of kidney diseases leading to end-stage renal failure. Previously, we performed a quantitative trait locus (QTL) analysis to identify the causative genes for GS in ICGN mice and found the deletion mutation of the tensin2 (Tns2) gene that creates both a premature stop codon and dramatically decreases mRNA expression levels within the region of the major QTL (this mutation was designated Tns2(nep)). The severity of GS varies considerably in humans and other animals, indicating the influence of several genes controlling the disease phenotype. In this study, to identify the modifier/resistant gene(s) for GS, we produced congenic strains carrying the Tns2(nep) mutation on the C57BL/6J (B6) genetic background and analyzed GS severity. Interestingly, the B6 congenic mice exhibited milder phenotypes than the ICGN strain mice. The results suggest that B6 mice have a modifier(s) of GS resistance. Therefore, identification of the modifier loci in B6 mice will provide important new information regarding gene interactions controlling GS.
Assuntos
Nefropatias Diabéticas/genética , Glomerulosclerose Segmentar e Focal/genética , Proteínas dos Microfilamentos/genética , Albuminúria/genética , Anemia/etiologia , Anemia/genética , Animais , Nitrogênio da Ureia Sanguínea , Cruzamentos Genéticos , Primers do DNA , Deleção de Genes , Hematócrito , Hemoglobinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Locos de Características Quantitativas , Índice de Gravidade de Doença , TensinasRESUMO
Recent high-throughput approaches have revealed a vast number of transcripts with unknown functions. Many of these transcripts are long noncoding RNAs (lncRNAs), and intergenic region-derived lncRNAs are classified as long intergenic noncoding RNAs (lincRNAs). Although Myosin heavy chain 6 (Myh6) encoding primary contractile protein is down-regulated in stressed hearts, the underlying mechanisms are not fully clarified especially in terms of lincRNAs. Here, we screen upregulated lincRNAs in pressure overloaded hearts and identify a muscle-abundant lincRNA termed Lionheart. Compared with controls, deletion of the Lionheart in mice leads to decreased systolic function and a reduction in MYH6 protein levels following pressure overload. We reveal decreased MYH6 results from an interaction between Lionheart and Purine-rich element-binding protein A after pressure overload. Furthermore, human LIONHEART levels in left ventricular biopsy specimens positively correlate with cardiac systolic function. Our results demonstrate Lionheart plays a pivotal role in cardiac remodeling via regulation of MYH6.
Assuntos
Coração/fisiopatologia , Pressão , RNA Longo não Codificante/genética , Sístole/genética , Animais , Biópsia , Dependovirus/metabolismo , Ventrículos do Coração/ultraestrutura , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/metabolismo , Ratos , Regulação para Cima/genéticaRESUMO
Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.
Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular , Polaridade Celular , Adesões Focais/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteína Substrato Associada a Crk/metabolismo , Forminas , Glioma/patologia , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Transporte Proteico , Interferência de RNA , RatosRESUMO
Background Micro RNA (miR)-33 targets cholesterol transporter ATP -binding cassette protein A1 and other antiatherogenic targets and contributes to atherogenic progression. Its inhibition or deletion is known to result in the amelioration of atherosclerosis in mice. However, mice lack the other member of the miR-33 family, miR-33b, which exists in humans and other large mammals. Thus, precise evaluation and comparison of the responsibilities of these 2 miRs during the progression of atherosclerosis has not been reported, although they are essential. Methods and Results In this study, we performed a comprehensive analysis of the difference between the function of miR-33a and miR-33b using genetically modified mice. We generated 4 strains with or without miR-33a and miR-33b. Comparison between mice with only miR-33a (wild-type mice) and mice with only miR-33b (miR-33a-/-/miR-33b+/+) revealed the dominant expression of miR-33b in the liver. To evaluate the whole body atherogenic potency of miR-33a and miR-33b, we developed apolipoprotein E-deficient/miR-33a+/+/miR-33b-/- mice and apolipoprotein E-deficient/miR-33a-/-/miR-33b+/+ mice. With a high-fat and high-cholesterol diet, the apolipoprotein E-deficient/miR-33a-/-/miR-33b+/+ mice developed increased atherosclerotic plaque versus apolipoprotein E-deficient/miR-33a+/+/miR-33b-/- mice, in line with the predominant expression of miR-33b in the liver and worsened serum cholesterol profile. By contrast, a bone marrow transplantation study showed no significant difference, which was consistent with the relevant expression levels of miR-33a and miR-33b in bone marrow cells. Conclusions The miR-33 family exhibits differences in distribution and regulation and particularly in the progression of atherosclerosis; miR-33b would be more potent than miR-33a.