RESUMEN
RATIONALE: Myocardial infarction (MI) is a leading cause of death worldwide. Because endogenous cardiac repair mechanisms are not sufficient for meaningful tissue regeneration, MI results in loss of cardiac tissue and detrimental remodeling events. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in a sequence dependent manner. Our previous data indicate that miRNAs are dysregulated in response to ischemic injury of the heart and actively contribute to cardiac remodeling after MI. OBJECTIVE: This study was designed to determine whether miRNAs are dysregulated on ischemic damage in porcine cardiac tissues and whether locked nucleic acid (LNA)-modified anti-miR chemistries can target cardiac expressed miRNAs to therapeutically inhibit miR-15 on ischemic injury. METHODS AND RESULTS: Our data indicate that the miR-15 family, which includes 6 closely related miRNAs, is regulated in the infarcted region of the heart in response to ischemia-reperfusion injury in mice and pigs. LNA-modified chemistries can effectively silence miR-15 family members in vitro and render cardiomyocytes resistant to hypoxia-induced cardiomyocyte cell death. Correspondingly, systemic delivery of miR-15 anti-miRs dose-dependently represses miR-15 in cardiac tissue of both mice and pigs, whereas therapeutic targeting of miR-15 in mice reduces infarct size and cardiac remodeling and enhances cardiac function in response to MI. CONCLUSIONS: Oligonucleotide-based therapies using LNA-modified chemistries for modulating cardiac miRNAs in the setting of heart disease are efficacious and validate miR-15 as a potential therapeutic target for the manipulation of cardiac remodeling and function in the setting of ischemic injury.
Asunto(s)
MicroARNs/antagonistas & inhibidores , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Animales , Supervivencia Celular/efectos de los fármacos , Femenino , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/efectos de los fármacos , Modelos Animales , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Oligonucleótidos/farmacología , Oligonucleótidos/uso terapéutico , PorcinosRESUMEN
BACKGROUND: Diastolic dysfunction in response to hypertrophy is a major clinical syndrome with few therapeutic options. MicroRNAs act as negative regulators of gene expression by inhibiting translation or promoting degradation of target mRNAs. Previously, we reported that genetic deletion of the cardiac-specific miR-208a prevents pathological cardiac remodeling and upregulation of Myh7 in response to pressure overload. Whether this miRNA might contribute to diastolic dysfunction or other forms of heart disease is currently unknown. METHODS AND RESULTS: Here, we show that systemic delivery of an antisense oligonucleotide induces potent and sustained silencing of miR-208a in the heart. Therapeutic inhibition of miR-208a by subcutaneous delivery of antimiR-208a during hypertension-induced heart failure in Dahl hypertensive rats dose-dependently prevents pathological myosin switching and cardiac remodeling while improving cardiac function, overall health, and survival. Transcriptional profiling indicates that antimiR-208a evokes prominent effects on cardiac gene expression; plasma analysis indicates significant changes in circulating levels of miRNAs on antimiR-208a treatment. CONCLUSIONS: These studies indicate the potential of oligonucleotide-based therapies for modulating cardiac miRNAs and validate miR-208 as a potent therapeutic target for the modulation of cardiac function and remodeling during heart disease progression.
Asunto(s)
Terapia Genética , Insuficiencia Cardíaca Diastólica/tratamiento farmacológico , Corazón/fisiopatología , Terapia Molecular Dirigida , Oligonucleótidos Antisentido/uso terapéutico , Animales , Vías de Administración de Medicamentos , Evaluación Preclínica de Medicamentos , Electrocardiografía , Perfilación de la Expresión Génica , Insuficiencia Cardíaca Diastólica/diagnóstico por imagen , Insuficiencia Cardíaca Diastólica/etiología , Insuficiencia Cardíaca Diastólica/genética , Hipertensión/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/sangre , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Pesadas de Miosina/genética , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacología , Interferencia de ARN , Ratas , Ratas Endogámicas Dahl , Transcripción Genética/efectos de los fármacos , Ultrasonografía , Remodelación Ventricular/efectos de los fármacosRESUMEN
Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin. Together with results obtained from cultured cells and mouse models, these clinical studies have yielded novel insights into the unexpected roles of Ig domain proteins in mechanotransduction and signaling to the nucleus. An emerging theme in this field is that cytoskeleton-associated Ig domain proteins are more than structural elements of the cell, and may have evolved to fill different needs in different cellular compartments. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
Asunto(s)
Citoplasma/metabolismo , Citoesqueleto/metabolismo , Proteínas Portadoras/metabolismo , Conectina , Proteínas del Citoesqueleto/metabolismo , Humanos , Proteínas de Microfilamentos , Modelos Biológicos , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismoRESUMEN
Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase-anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase-anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus.
Asunto(s)
Anafase , Metafase , Miosina Tipo II/química , Quinasa de Cadena Ligera de Miosina/metabolismo , Óvulo/citología , Óvulo/fisiología , Erizos de Mar/citología , Animales , Fenómenos Biomecánicos , Birrefringencia , Señalización del Calcio/fisiología , Citocinesis/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microtúbulos/metabolismo , Fosforilación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/química , Quinasas Asociadas a rhoRESUMEN
Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA have been lagging behind. The miR-29 family has gained a lot of attention for its clear function in tissue fibrosis. This fibroblast-enriched miRNA family is downregulated in fibrotic diseases which induces a coordinate increase of many extracellular matrix genes. Here, we show that intravenous injection of synthetic RNA duplexes can increase miR-29 levels in vivo for several days. Moreover, therapeutic delivery of these miR-29 mimics during bleomycin-induced pulmonary fibrosis restores endogenous miR-29 function whereby decreasing collagen expression and blocking and reversing pulmonary fibrosis. Our data support the feasibility of using miRNA mimics to therapeutically increase miRNAs and indicate miR-29 to be a potent therapeutic miRNA for treating pulmonary fibrosis.
Asunto(s)
MicroARNs/genética , Imitación Molecular/genética , Fibrosis Pulmonar/genética , Animales , Bleomicina , Northern Blotting , Línea Celular , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Imitación Molecular/fisiología , Células 3T3 NIH , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIMS: Recent studies have shown that microRNAs (miRNAs), besides being potent regulators of gene expression, can additionally serve as circulating biomarkers of disease. The aim of this study is to determine if plasma miRNAs can be used as indicators of disease progression or therapeutic efficacy in hypertension-induced heart disease. METHODS AND RESULTS: In order to define circulating miRNAs that change during hypertension-induced heart failure and that respond to therapeutic treatment, we performed miRNA arrays on plasma RNA from hypertensive rats that show signs of heart failure. Array analysis indicated that approximately one-third of the miRNAs on the array are detectable in plasma. Quantitative real-time polymerase chain reaction (PCR) analysis for a selected panel of miRNAs indicated that circulating levels of miR-16, miR-20b, miR-93, miR-106b, miR-223, and miR-423-5p were significantly increased in response to hypertension-induced heart failure, while this effect was blunted in response to treatment with antimiR-208a as well as an ACE inhibitor. Moreover, treatment with antimiR-208a resulted in a dramatic increase in one miRNA, miR-19b. A time course study indicated that several of these miRNA changes track with disease progression. CONCLUSIONS: Circulating levels of miRNAs are responsive to therapeutic interventions and change during the progression of hypertension-induced heart disease.
Asunto(s)
Biomarcadores/sangre , Progresión de la Enfermedad , Insuficiencia Cardíaca/tratamiento farmacológico , Hipertensión/tratamiento farmacológico , MicroARNs/sangre , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Captopril/uso terapéutico , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/diagnóstico , Hipertensión/inducido químicamente , Hipertensión/diagnóstico , Masculino , MicroARNs/genética , Péptido Natriurético Encefálico/sangre , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Endogámicas Dahl , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Cloruro de Sodio/toxicidad , Resultado del TratamientoRESUMEN
Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a characteristic pattern of early metastasis, which is driving a search for biomarkers that can be used to detect the cancer at an early stage. Recently, the actin-associated protein palladin was identified as a candidate biomarker when it was shown that palladin is mutated in a rare inherited form of PDA, and overexpressed in many sporadic pancreas tumors and premalignant precursors. In this study, we analyzed the expression of palladin isoforms in murine and human PDA and explored palladin's potential use in diagnosing PDA. We performed immunohistochemistry and immunoblot analyses on patient samples and tumor-derived cells using an isoform-selective monoclonal antibody and a pan-palladin polyclonal antibody. Immunoblot and real-time quantitative reverse transcription-PCR were used to quantify palladin mRNA levels in human samples. We show that there are two major palladin isoforms expressed in pancreas: 65 and 85-90 kDa. The 65 kDa isoform is expressed in both normal and neoplastic ductal epithelial cells. The 85-90 kDa palladin isoform is highly overexpressed in tumor-associated fibroblasts (TAFs) in both primary and metastatic tumors compared to normal pancreas, in samples obtained from either human patients or genetically engineered mice. In tumor-derived cultured cells, expression of palladin isoforms follows cell-type specific patterns, with the 85-90 kDa isoform in TAFs, and the 65 kDa isoform predominating in normal and neoplastic epithelial cells. These results suggest that upregulation of 85-90 kDa palladin isoform may play a role in the establishment of the TAF phenotype, and thus in the formation of a desmoplastic tumor microenvironment. Thus, palladin may have a potential use in the early diagnosis of PDA and may have much broader significance in understanding metastatic behavior.
Asunto(s)
Carcinoma Ductal Pancreático/diagnóstico , Proteínas del Citoesqueleto/genética , Fosfoproteínas/genética , Animales , Biomarcadores de Tumor , Proteínas del Citoesqueleto/análisis , Fibroblastos/patología , Humanos , Inmunoensayo , Ratones , Mutación , Proteínas de Neoplasias , Fosfoproteínas/análisis , Isoformas de Proteínas , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Fertilization triggers a reorganization of oocyte cytoskeleton, and in sea urchins, there is a dramatic increase in cortical F-actin. However, the role that myosin II plays during fertilization remains largely unexplored. Myosin II is localized to the cortical cytoskeleton both before and after fertilization and to examine myosin II contractility in living cells, Lytechinus pictus eggs were observed by time-lapse microscopy. Upon sperm binding, a cell surface deflection traversed the egg that was followed by and dependent on the calcium wave. The calcium-dependence of surface contractility could be reproduced in unfertilized eggs, where mobilization of intracellular calcium in unfertilized eggs under compression resulted in a marked contractile response. Lastly, inhibition of myosin II delayed absorption of the fertilization cone, suggesting that myosin II not only responds to the same signals that activate eggs but also participates in the remodeling of the cortical actomyosin cytoskeleton during the first zygotic cell cycle.