RESUMO
Familial hypercholesterolemia is an inherited disorder that remains underdiagnosed. Conventional genetic testing methods such as next-generation sequencing (NGS) or target PCR are based on the amplification process. Due to the efficiency limits of polymerase and ligase enzymes, these methods usually target short regions and do not detect large mutations straightforwardly. This study combined the long-read nanopore sequencing and CRISPR-Cas9 system to sequence the target DNA molecules without amplification. We originally designed and optimized the CRISPR-RNA panel to target the low-density lipoprotein receptor gene (LDLR) and proprotein convertase subtilisin/kexin type 9 gene (PCSK9) from human genomic DNA followed by nanopore sequencing. The average coverages for LDLR and PCSK9 were 106× and 420×, versus 1.2× for the background genome. Among them, continuous reads were 52x and 307x, respectively, and spanned the entire length of LDLR and PCSK9. We identified pathogenic mutations in both coding and splicing donor regions in LDLR. We also detected an 11,029 bp large deletion in another case. Furthermore, using continuous long reads generated from the benchmark experiment, we demonstrated how a false-positive 670 bp deletion caused by PCR amplification errors was easily eliminated.
Assuntos
Hiperlipoproteinemia Tipo II , Sequenciamento por Nanoporos , Humanos , Pró-Proteína Convertase 9/genética , Sistemas CRISPR-Cas/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Mutação , Genômica , DNARESUMO
AIMS: As heart failure (HF) progresses, ATP levels in myocardial cells decrease, and myocardial contractility also decreases. Inotropic drugs improve myocardial contractility but increase ATP consumption, leading to poor prognosis. Kyoto University Substance 121 (KUS121) is known to selectively inhibit the ATPase activity of valosin-containing protein, maintain cellular ATP levels, and manifest cytoprotective effects in several pathological conditions. The aim of this study is to determine the therapeutic effect of KUS121 on HF models. METHODS AND RESULTS: Cultured cell, mouse, and canine models of HF were used to examine the therapeutic effects of KUS121. The mechanism of action of KUS121 was also examined. Administration of KUS121 to a transverse aortic constriction (TAC)-induced mouse model of HF rapidly improved the left ventricular ejection fraction and improved the creatine phosphate/ATP ratio. In a canine model of high frequency-paced HF, administration of KUS121 also improved left ventricular contractility and decreased left ventricular end-diastolic pressure without increasing the heart rate. Long-term administration of KUS121 to a TAC-induced mouse model of HF suppressed cardiac hypertrophy and fibrosis. In H9C2 cells, KUS121 reduced ER stress. Finally, in experiments using primary cultured cardiomyocytes, KUS121 improved contractility and diastolic capacity without changing peak Ca2+ levels or contraction time. These effects were not accompanied by an increase in cyclic adenosine monophosphate or phosphorylation of phospholamban and ryanodine receptors. CONCLUSIONS: KUS121 ameliorated HF by a mechanism totally different from that of conventional catecholamines. We propose that KUS121 is a promising new option for the treatment of HF.
Assuntos
Cálcio , Insuficiência Cardíaca , Humanos , Camundongos , Animais , Cães , Cálcio/metabolismo , Proteína com Valosina/metabolismo , Volume Sistólico , Universidades , Função Ventricular Esquerda , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , Doença Crônica , Trifosfato de Adenosina/metabolismo , Modelos Animais de DoençasRESUMO
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
Abdominal aortic aneurysm (AAA) is a lethal disease, but no beneficial therapeutic agents have been established to date. Previously, we found that AAA formation is suppressed in microRNA (miR)-33-deficient mice compared with wild-type mice. Mice have only one miR-33, but humans have two miR-33 s, miR-33a and miR-33b. The data so far strongly support that inhibiting miR-33a or miR-33b will be a new strategy to treat AAA. We produced two specific anti-microRNA oligonucleotides (AMOs) that may inhibit miR-33a and miR-33b, respectively. In vitro studies showed that the AMO against miR-33b was more effective; therefore, we examined the in vivo effects of this AMO in a calcium chloride (CaCl2)-induced AAA model in humanized miR-33b knock-in mice. In this model, AAA was clearly improved by application of anti-miR-33b. To further elucidate the mechanism, we evaluated AAA 1 week after CaCl2 administration to examine the effect of anti-miR-33b. Histological examination revealed that the number of MMP-9-positive macrophages and the level of MCP-1 in the aorta of mice treated with anti-miR-33b was significantly reduced, and the serum lipid profile was improved compared with mice treated with control oligonucleotides. These results support that inhibition of miR-33b is effective in the treatment for AAA.
Assuntos
Aneurisma da Aorta Abdominal , MicroRNAs , Animais , Antagomirs/metabolismo , Antagomirs/farmacologia , Antagomirs/uso terapêutico , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Cloreto de Cálcio/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismoRESUMO
BACKGROUND: The mechanisms leading to atrial fibrosis in patients with atrial fibrillation (AF), especially in relation to inflammation, remain unclear. METHODS AND RESULTS: Forty biomarkers were measured in peripheral blood samples collected prior to catheter ablation, and the association with left atrial (LVZ) was evaluated in 16 consecutive patients. The median %LVZ was 17%. In Pearson's correlation analysis, interleukin(IL)-17A and interferon(IFN)-γ showed the most significant positive and negative correlations with %LVZ (R = 0.35 and 0.43, P < 0.001). Furthermore, the IL-17A/IFN-γ ratio was significantly associated with %LVZ (R = 0.65, P = 0.007), as was the macrophage inflammatory protein (MIP)-1δ/IFN-γ ratio (R = 0.73, P = 0.001). The area under the receiver operator characteristics curves of the IL-17A/IFN-γ and MIP-1δ/IFN-γ ratios for detecting severe LVZ (%LVZ ≥ 10% as a reference standard) were 0.88 and 0.90, respectively. The IL-17A/IFN-γ ratio was significantly higher in patients with severe LVZ than those without (1.41 versus 0.97, P = 0.01). Furthermore, the sensitivity, specificity, and accuracy for detecting severe LVZ were 60%, 100%, and 75.0%, respectively, at a cut-off value of 1.3. CONCLUSIONS: Among inflammatory biomarkers, the serum IL-17A/IFN-γ ratio was associated with severe left atrial LVZ in patients with AF. However, further studies are needed to clarify the role of inflammatory biomarkers in the development and progression of atrial fibrosis in patients with AF.
RESUMO
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
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
Objective: This study aims to investigate the time-dependent prognostic utility of two fibrosis markers representing organ fibrogenesis (N-terminal propeptide of procollagen III (PIIINP) and type IV collagen 7S (P4NP 7S)) in patients with acute heart failure (HF). Methods: 390 patients with acute HF were dichotomised based on the median value of fibrosis markers at discharge. The primary outcome measure was a composite of cardiac death and HF hospitalisation. Results: P4NP 7S significantly declined during hospitalisation, whereas PIIINP did not. The cumulative 90-day and 365-day incidence of the primary outcome measure was 16.6% vs 16.0% (p=0.42) and 33.3% vs 28.4% (p=0.34) in the patients with high versus low PIIINP; 19.9% vs 13.0% (p=0.04) and 32.3% vs 29.0% (p=0.34) in the patients with high and low P4NP 7S, respectively. After adjusting for confounders, high P4NP 7S correlated with significant excess risk relative to low P4NP 7S for both 90-day and 365-day primary outcome measure (adjusted HR, 1.50; 95% CI, 1.02 to 2.21; p=0.04 and adjusted HR, 1.89; 95% CI, 1.11 to 3.26; p=0.02, respectively), which was driven by significant association of high P4NP 7S with higher incidence of HF hospitalisation. Furthermore, P4NP 7S exhibited an additive value to conventional prognostic factors for predicting 90-day outcome (p=0.038 for net reclassification improvement; p=0.0068 for integrated discrimination improvement). High PIIINP did not correlate with significant excess risk for both 90-day and 365-day outcome. Conclusions: This study suggests a possible role of P4NP 7S in the risk stratification of patients with acute HF.
Assuntos
Colágeno Tipo IV/sangue , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/diagnóstico , Miocárdio/metabolismo , Fragmentos de Peptídeos/sangue , Pró-Colágeno/sangue , Doença Aguda , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Causas de Morte , Feminino , Fibrose , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/terapia , Hospitalização , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Miocárdio/patologia , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Fatores de TempoRESUMO
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.
Assuntos
Aterosclerose/genética , Hepatócitos/metabolismo , Fígado/metabolismo , MicroRNAs/genética , Placa Aterosclerótica/genética , Animais , Apolipoproteínas B/metabolismo , Transplante de Medula Óssea , Colesterol/metabolismo , Colesterol na Dieta , Dieta Hiperlipídica , Progressão da Doença , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Camundongos Knockout para ApoE , Camundongos Transgênicos , MicroRNAs/metabolismo , Triglicerídeos/metabolismoRESUMO
BACKGROUND: Copeptin, a surrogate marker of pro-arginine vasopressin, is expected to be a marker in cardiovascular diseases. Its utility for predicting long-term clinical outcomes in heart failure (HF), however, has not been adequately evaluated in daily clinical practice in Japan. METHODS: To assess the relationship of serum copeptin at admission with long-term clinical outcomes, we evaluated serum copeptin at admission in consecutive 107 patients hospitalized for HF between April 2011 and July 2012. The primary outcome measure was defined as a composite of all-cause death and re-admission for HF (all-cause death/HF). RESULTS: In this study population, median serum copeptin at admission was 15.5 (6.7-32.0)pmol/L. As compared with the low-copeptin group (<18pmol/L, N=60), the high-copeptin group (≥18pmol/L, N=47) included more male patients and those with prior myocardial infarction, prior HF, low left ventricular ejection fraction, and chronic kidney disease. During median 4.5 (1.0-5.5) years of clinical follow-up, the cumulative incidence of all-cause death/HF was significantly higher in the high-copeptin than in the low-copeptin group (63.4% versus 33.0% at 1 year, and 85.2% versus 77.2% at 5 years, log-rank p=0.03). After adjusting for confounders, high-copeptin was still an independent predictor for all-cause death/HF [hazard ratio (95% confidence interval): 1.77 (1.04-3.01), p=0.03]. CONCLUSION: Copeptin was suggested as a useful marker for predicting long-term clinical outcomes in patients with HF.
Assuntos
Glicopeptídeos/sangue , Insuficiência Cardíaca/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Causas de Morte , Feminino , Insuficiência Cardíaca/fisiopatologia , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/complicações , Prognóstico , Modelos de Riscos Proporcionais , Volume Sistólico , Função Ventricular EsquerdaRESUMO
AIMS: Collagen-derived peptides such as collagen I C-terminal telopeptide (CITP) and procollagen III N-terminal propeptide (PIIINP) have been conventionally used as markers of cardiac fibrosis. Collagen IV 7S domain (P4NP 7S) has been recently reported to be correlated with haemodynamics in patients with acute heart failure. We investigated whether these markers reflect cardiac remodelling and myocardial collagen expression. METHODS AND RESULTS: In 80 patients with dilated cardiomyopathy, relationships of CITP, PIIINP, and P4NP 7S to clinical and echocardiographic variables were analysed. CITP and PIIINP were inversely correlated with estimated glomerular filtration rate (r = -0.41, P < 0.001 and r = -0.32, P = 0.004, respectively); P4NP 7S was positively correlated with B-type natriuretic peptide (r = 0.32, P = 0.003) and γ-glutamyltransferase (r = 0.38, P < 0.001). These correlations were significant even after adjustment by potential confounders, whereas all three collagen markers were not independently correlated with ejection fraction nor with left ventricular (LV) diastolic diameter. In 33 patients undergoing endomyocardial biopsy, myocardial collagen I and III mRNA expressions were correlated with LV end-diastolic volume index (r = 0.42, P = 0.02 and r = 0.54, P = 0.002, respectively), whereas myocardial collagen IV mRNA expression was not correlated with LV end-diastolic volume index nor with ejection fraction. Each collagen-derived peptide was not significantly correlated with the myocardial expression of their corresponding collagen mRNA. CONCLUSIONS: Our study shows that CITP, PIIINP, and P4NP 7S do not reflect myocardial collagen mRNA expression but presumably reflect extra-cardiac organ injury in heart failure.
Assuntos
Cardiomiopatia Dilatada/sangue , Colágeno Tipo III/sangue , Colágeno Tipo IV/sangue , Colágeno Tipo I/sangue , Regulação da Expressão Gênica , Miocárdio/metabolismo , Volume Sistólico/fisiologia , Idoso , Biomarcadores/sangue , Biópsia , Cateterismo Cardíaco , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/fisiopatologia , Colágeno Tipo I/biossíntese , Colágeno Tipo III/biossíntese , Colágeno Tipo IV/biossíntese , Ecocardiografia , Feminino , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/metabolismo , Humanos , Masculino , Miocárdio/patologia , RNA/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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
Recent evidence suggests that the accumulation of macrophages as a result of obesity-induced adipose tissue hypoxia is crucial for the regulation of tissue fibrosis, but the molecular mechanisms underlying adipose tissue fibrosis are still unknown. In this study, we revealed that periostin (Postn) is produced at extraordinary levels by adipose tissue after feeding with a high-fat diet (HFD). Postn was secreted at least from macrophages in visceral adipose tissue during the development of obesity, possibly due to hypoxia. Postn-/- mice had lower levels of crown-like structure formation and fibrosis in adipose tissue and were protected from liver steatosis. These mice also showed amelioration in systemic insulin resistance compared with HFD-fed WT littermates. Mice deficient in Postn in their hematopoietic compartment also had lower levels of inflammation in adipose tissue, in parallel with a reduction in ectopic lipid accumulation compared with the controls. Our data indicated that the regulation of Postn in visceral fat could be beneficial for the maintenance of healthy adipose tissue in obesity.
Assuntos
Moléculas de Adesão Celular/deficiência , Celulite (Flegmão)/metabolismo , Gorduras na Dieta/efeitos adversos , Resistência à Insulina , Gordura Intra-Abdominal/metabolismo , Obesidade/metabolismo , Animais , Celulite (Flegmão)/induzido quimicamente , Celulite (Flegmão)/genética , Celulite (Flegmão)/patologia , Gorduras na Dieta/farmacologia , Fibrose , Gordura Intra-Abdominal/patologia , Camundongos , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/patologiaRESUMO
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
BACKGROUND: The mechanism of cardiac energy production against sustained pressure overload remains to be elucidated. METHODS AND RESULTS: We generated cardiac-specific kinase-dead (kd) calcium/calmodulin-dependent protein kinase kinase-ß (CaMKKß) transgenic (α-MHC CaMKKßkd TG) mice using α-myosin heavy chain (α-MHC) promoter. Although CaMKKß activity was significantly reduced, these mice had normal cardiac function and morphology at baseline. Here, we show that transverse aortic binding (TAC) in α-MHC CaMKKßkd TG mice led to accelerated death and left ventricular (LV) dilatation and dysfunction, which was accompanied by significant clinical signs of heart failure. CaMKKß downstream signaling molecules, including adenosine monophosphate-activated protein kinase (AMPK), were also suppressed in α-MHC CaMKKßkd TG mice compared with wild-type (WT) mice. The expression levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, which is a downstream target of both of CaMKKß and calcium/calmodulin kinases, were also significantly reduced in α-MHC CaMKKßkd TG mice compared with WT mice after TAC. In accordance with these findings, mitochondrial morphogenesis was damaged and creatine phosphate/ß-ATP ratios assessed by magnetic resonance spectroscopy were suppressed in α-MHC CaMKKßkd TG mice compared with WT mice after TAC. CONCLUSIONS: These data indicate that CaMKKß exerts protective effects on cardiac adaptive energy pooling against pressure-overload possibly through phosphorylation of AMPK and by upregulation of PGC-1α. Thus, CaMKKß may be a therapeutic target for the treatment of heart failure.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Insuficiência Cardíaca/etiologia , Remodelação Ventricular/genética , Trifosfato de Adenosina , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Cadeias Pesadas de Miosina/genética , Fosforilação , Regiões Promotoras Genéticas , Transdução de Sinais , Regulação para Cima , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33a located within the intron of sterol regulatory element-binding protein (SREBP) 2 controls cholesterol homeostasis and can be a possible therapeutic target for treating atherosclerosis. Primates, but not rodents, express miR-33b from an intron of SREBF1. Therefore, humanized mice, in which a miR-33b transgene is inserted within a Srebf1 intron, are required to address its function in vivo. We successfully established miR-33b knock-in (KI) mice and found that protein levels of known miR-33a target genes, such as ABCA1, ABCG1, and SREBP-1, were reduced compared with those in wild-type mice. As a consequence, macrophages from the miR-33b KI mice had a reduced cholesterol efflux capacity via apoA-I and HDL-C. Moreover, HDL-C levels were reduced by almost 35% even in miR-33b KI hetero mice compared with the control mice. These results indicate that miR-33b may account for lower HDL-C levels in humans than those in mice and that miR-33b is possibly utilized for a feedback mechanism to regulate its host gene SREBF1. Our mice will also aid in elucidating the roles of miR-33a/b in different genetic disease models.
Assuntos
HDL-Colesterol/metabolismo , Íntrons/genética , MicroRNAs/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Animais , Sequência de Bases , Western Blotting , Células Cultivadas , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
BACKGROUND: Recently, we screened for cardiac genes induced by metabolic stress and identified neural cell adhesion molecule (NCAM) as a candidate. This study aimed to clarify the expression pattern of NCAM in human cardiomyopathy. METHODS AND RESULTS: A total of 64 cardiac tissue samples of patients with dilated cardiomyopathy were dichotomized according to the immunohistochemically determined signal intensity of NCAM staining (NCAM-high and NCAM-low groups). Clinical and hemodynamic data of the patients were compared between the 2 groups. Fibrosis area, left ventricular end-diastolic volume index, and left ventricular diastolic pressure were greater in the NCAM-high group (22.8% versus 11.6%, P<0.05; 130.3±57.6 versus 104.8±31.7 mL/m(2), P<0.05; 14.3±8.0 versus 8.8±4.7 mm Hg, P<0.005; respectively). Incidence of cardiac death and admission for worsening heart failure was higher in the NCAM-high group during a follow-up of 6.3 years (log-rank P<0.05). Another 18 tissue samples were analyzed to determine the relationships between expression level of NCAM and major metabolic genes as well as hemodynamic parameters. The mRNA level of NCAM correlated with the serum (r=0.58; P=0.01) and mRNA levels (r=0.61; P=0.008) of brain-derived natriuretic peptides. It was also correlated with the mRNA levels of proliferator-activated receptor-γ coactivator-1 α (r=0.69; P=0.002) and the nuclear respiratory factor 1 (r=0.74; P<0.001). CONCLUSIONS: Expression of NCAM was associated with worsening hemodynamic parameters and major metabolic genes. Together with our previous findings, these data support the involvement of NCAM in left ventricular remodeling, revealing new insights into the pathophysiology of heart failure.
Assuntos
Cardiomiopatias/genética , Regulação da Expressão Gênica , Miocárdio/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , RNA/genética , Disfunção Ventricular Esquerda/genética , Função Ventricular Esquerda/fisiologia , Biópsia , Cateterismo Cardíaco , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Miocárdio/patologia , Moléculas de Adesão de Célula Nervosa/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports have indicated that miR-33, which is located within the intron of sterol regulatory element-binding protein (SREBP) 2, controls cholesterol homoeostasis and may be a potential therapeutic target for the treatment of atherosclerosis. Here we show that deletion of miR-33 results in marked worsening of high-fat diet-induced obesity and liver steatosis. Using miR-33(-/-)Srebf1(+/-) mice, we demonstrate that SREBP-1 is a target of miR-33 and that the mechanisms leading to obesity and liver steatosis in miR-33(-/-) mice involve enhanced expression of SREBP-1. These results elucidate a novel interaction between SREBP-1 and SREBP-2 mediated by miR-33 in vivo.
Assuntos
Fígado Gorduroso/genética , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Obesidade/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Animais , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/metabolismo , Humanos , Íntrons , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , Obesidade/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
Tissue-specific patterns of gene expression play an important role in the distinctive features of each organ. Small CTD phosphatases (SCPs) 1-3 are recruited by repressor element 1 (RE-1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) to neuronal genes that contain RE-1 elements, leading to neuronal gene silencing in non-neuronal cells. SCPs are highly expressed in the heart and contain microRNAs (miR)-26b, 26a-2, and 26a-1 with the same seed sequence in their introns. Therefore, we tried to investigate the roles of miR-26b and its host gene in neonatal rat cardiomyocytes. Overexpression of miR-26b suppressed the mRNA expression levels of ANF, ßMHC, and ACTA1 and reduced the cell surface area in cardiomyocytes. We confirmed that miR-26b targets the 3' untranslated region (3'UTR) of GATA4 and canonical transient receptor potential channel (TRPC) 3. Conversely, silencing of the endogenous miR-26b family enhanced the expression levels of TRPC3 and GATA4. On the other hand, overexpression of SCP1 induced the mRNA expression of ANF and ßMHC and increased the cell surface area in cardiomyocytes. Next, we compared the effect of overexpression of SCP1 with its introns and SCP1 cDNA to observe the net function of SCP1 expression on cardiac hypertrophy. When the expression levels of SCP1 were the same, the overexpression of SCP1 cDNA had a greater effect at inducing cardiac hypertrophy than SCP1 cDNA with its intron. In conclusion, SCP1 itself has the potential to induce cardiac hypertrophy; however, the effect is suppressed by intronic miR-26b in cardiomyocytes. miR-26b has an antagonistic effect on its host gene SCP1.
Assuntos
Cardiomegalia/genética , Regulação da Expressão Gênica , Íntrons , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Animais , Animais Recém-Nascidos , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Genes Reporter , Luciferases , Masculino , Camundongos , MicroRNAs/metabolismo , Miócitos Cardíacos/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , RNA Interferente Pequeno/genética , Ratos , Sequências Reguladoras de Ácido Nucleico , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , TransfecçãoRESUMO
Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is a receptor for oxidized LDL, and is strongly expressed in endothelial cells at an early stage of atherosclerosis. LOX-1 expression in adipocytes is induced by PPARgamma (ligands and appears to be involved in adipocyte cholesterol metabolism. However, the role of adipose tissue LOX-1 in high-fat diet-induced obesity is unknown. We found that mRNA levels of adipose tissue LOX-1 were markedly increased in obese mice fed a high-fat diet (HFD) compared with those fed normal chow. The levels were closely correlated with those of a proinflammatory cytokine, monocyte chemoattractant protein-1 (MCP-1). Then, LOX-1 knockout (LOX-1-KO) and wild-type (WT) mice were fed HFD for 16weeks. HFD feeding increased the body and mesenteric fat weights similarly in WT and LOX-1-KO mice. HFD-induced expressions of proinflammatory cytokines such as MCP-1, MIP-1alpha, and IL-6 were significantly less in LOX-1-KO than WT mice. Thus, LOX-1 is required for the HFD-induced expression of proinflammatory cytokines in the adipose tissue of obese mice.