RESUMEN
Skeletal muscle relies on coordination between myogenic and non-myogenic interstitial cells for homeostasis and for regeneration and response to injury. Fibroadipogenic progenitors (FAPs) have recently been recognized as key modulators of signaling to promote myogenesis following injury. FAPs are also responsible for the fibrosis and fatty replacement of muscle tissue seen in many diseased states. While extensive use of surface markers to purify FAPs has been undertaken in the mouse system, in particular PDGFRA, markers for human FAPs are less well understood. Here, we show that CD73 can be used as a single positive marker to purify FAPs from the lineage-negative (CD45-neg, CD31-neg) fraction of skeletal muscle mononuclear cells. Although CD73 was previously found to be expressed in cultured myogenic cells, we find that this marker is only acquired upon culture and that the CD73+ fraction of human skeletal muscle has no myogenic activity. We show that Lin-neg CD73+ cells from human muscle undergo fat differentiation as well as fibrogenesis when exposed to appropriate activating signals in vitro. This simple single positive marker approach effectively enables isolation of human FAPs from fresh human skeletal muscle biopsies.
RESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is caused by inappropriate expression of the double homeodomain protein DUX4. DUX4 has bimodal effects, inhibiting myogenic differentiation and blocking MyoD at low levels of expression, and killing myoblasts at high levels. Pax3 and Pax7, which contain related homeodomains, antagonize the cell death phenotype of DUX4 in C2C12 cells, suggesting some type of competitive interaction. Here, we show that the effects of DUX4 on differentiation and MyoD expression require the homeodomains but do not require the C-terminal activation domain of DUX4. We tested the set of equally related homeodomain proteins (Pax6, Pitx2c, OTX1, Rax, Hesx1, MIXL1 and Tbx1) and found that only Pax3 and Pax7 display phenotypic competition. Domain analysis on Pax3 revealed that the Pax3 homeodomain is necessary for phenotypic competition, but is not sufficient, as competition also requires the paired and transcriptional activation domains of Pax3. Remarkably, substitution mutants in which DUX4 homeodomains are replaced by Pax7 homeodomains retain the ability to inhibit differentiation and to induce cytotoxicity.
Asunto(s)
Proteínas de Homeodominio/genética , Células Musculares/metabolismo , Desarrollo de Músculos/genética , Proteína MioD/genética , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX7/genética , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Línea Celular , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Células Musculares/patología , Proteína MioD/metabolismo , Mioblastos/metabolismo , Mioblastos/patología , Factor de Transcripción PAX3/metabolismo , Factor de Transcripción PAX7/metabolismo , Dominios Proteicos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
BACKGROUND: Approximately 18-20% of all human breast cancers have overexpressed human epidermal growth factor receptor 2 (HER2). Standard clinical practice is to treat only overexpressed HER2 (HER2+) cancers with targeted anti-HER2 therapies. However, recent analyses of clinical trial data have found evidence that HER2-targeted therapies may benefit a sub-group of breast cancer patients with non-overexpressed HER2. This suggests that measurement of other biological factors associated with HER2 cancer, such as HER2 signaling pathway activity, should be considered as an alternative means of identifying patients eligible for HER2 therapies. METHODS: A new biosensor-based test (CELxTM HSF) that measures HER2 signaling activity in live cells is demonstrated using a set of 19 human HER2+ and HER2- breast cancer reference cell lines and primary cell samples derived from two fresh patient tumor specimens. Pathway signaling is elucidated by use of highly specific agonists and antagonists. The test method relies upon well-established phenotypic, adhesion-related, impedance changes detected by the biosensor. RESULTS: The analytical sensitivity and analyte specificity of this method was demonstrated using ligands with high affinity and specificity for HER1 and HER3. The HER2-driven signaling quantified ranged 50-fold between the lowest and highest cell lines. The HER2+ cell lines were almost equally divided into high and low signaling test result groups, suggesting that little correlation exists between HER2 protein expression and HER2 signaling level. Unexpectedly, the highest HER2-driven signaling level recorded was with a HER2- cell line. CONCLUSIONS: Measurement of HER2 signaling activity in the tumor cells of breast cancer patients is a feasible approach to explore as a biomarker to identify HER2-driven cancers not currently diagnosable with genomic techniques. The wide range of HER2-driven signaling levels measured suggests it may be possible to make a distinction between normal and abnormal levels of activity. Analytical validation studies and clinical trials treating HER2- patients with abnormal HER2-driven signaling would be required to evaluate the analytical and clinical validity of using this functional biomarker as a diagnostic test to select patients for treatment with HER2 targeted therapy. In clinical practice, this method would require patient specimens be delivered to and tested in a central lab.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Receptor ErbB-2/aislamiento & purificación , Técnicas Biosensibles , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Pronóstico , Receptor ErbB-2/genéticaRESUMEN
The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2-, n = 34 of which 33 were estrogen receptor positive) and healthy subjects (n = 16) were evaluated along with reference breast cancer cell lines (n = 19). Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2- breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI = 10%-37%) had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity (hyperactive or normal) (Regression analysis P = 0.144, R2 = 0.068). One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.
Asunto(s)
Técnicas Biosensibles , Neoplasias de la Mama/metabolismo , Adhesión Celular , Receptor ErbB-2/deficiencia , Transducción de Señal , Adulto , Anciano , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Inmunológicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/farmacología , Femenino , Humanos , Persona de Mediana Edad , Receptor ErbB-2/agonistas , Receptor ErbB-2/antagonistas & inhibidores , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is caused by mutations leading to ectopic expression of the transcription factor DUX4, and encompasses both muscle-related and non-muscle phenotypes. Mouse models bearing this gene represent valuable tools to investigate which pathologies are due to DUX4 expression, and how DUX4 leads to these pathologies. The iDUX4(2.7) mouse contains an X-linked doxycycline-inducible DUX4 gene that shows low level basal expression in the absence of doxycycline, leading to male lethality, generally in embryo, but always before 8 weeks of age. Here, we describe additional non-muscle phenotypes in this animal model. We find that iDUX4(2.7) female carriers are extremely hyperactive, spending large amounts of time ambulating and much less time resting. Rare 3-week old males, although hypophagic, runted and extremely fragile, are capable of high activity, but show periods of catatonic torpor in which animals appear dead and respiration is virtually absent. We also examine a non-muscle phenotype of interest to FSHD, high frequency hearing loss. We find that young iDUX4(2.7) females are significantly impaired in their ability to hear at frequencies above 8 kHz. These phenotypes make the iDUX4(2.7) mouse an attractive model in which to study non-muscle activities of DUX4.
Asunto(s)
Pérdida Auditiva de Alta Frecuencia/genética , Proteínas de Homeodominio/fisiología , Hipercinesia/genética , Animales , Composición Corporal/genética , Cromatina/genética , Modelos Animales de Enfermedad , Doxiciclina/farmacología , Insuficiencia de Crecimiento/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Genes Letales , Heterocigoto , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Proteínas Recombinantes de Fusión/biosíntesis , Trastornos Respiratorios/genética , Caracteres Sexuales , Letargo/genética , Transgenes/efectos de los fármacos , Cromosoma X/genética , Inactivación del Cromosoma XRESUMEN
WNT/ß-CATENIN signaling promotes the hematopoietic/endothelial differentiation of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs). The transient addition of a GSK3ß inhibitor (GSKi) has been found to facilitate in vitro endothelial cell differentiation from hESCs/hiPSCs. Because hematopoietic and endothelial cells are derived from common progenitors (hemogenic endothelial progenitors [HEPs]), we examined the effect of transient GSKi treatment on hematopoietic cell differentiation from hiPSCs. We found that transient GSKi treatment at the start of hiPSC differentiation induction altered the gene expression profile of the cells. Multiple CDX/HOX genes, which are expressed in the posterior mesoderm of developing embryos, were significantly upregulated by GSKi treatment. Further, inclusion of the GSKi in a serum- and stroma-free culture with chemically defined medium efficiently induced HEPs, and the HEPs gave rise to various lineages of hematopoietic and endothelial cells. Therefore, transient WNT/ß-CATENIN signaling triggers activation of the CDX/HOX pathway, which in turn confers hemogenic posterior mesoderm identity to differentiating hiPSCs. These data enhance our understanding of human embryonic hematopoietic/endothelial cell development and provide a novel in vitro system for inducing the differentiation of hematopoietic cells from hiPSCs.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genes Homeobox , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Células Madre Pluripotentes/citología , Glucógeno Sintasa Quinasa 3 beta , Humanos , Células Madre Pluripotentes/enzimologíaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is an enigmatic disease associated with epigenetic alterations in the subtelomeric heterochromatin of the D4Z4 macrosatellite repeat. Each repeat unit encodes DUX4, a gene that is normally silent in most tissues. Besides muscular loss, most patients suffer retinal vascular telangiectasias. To generate an animal model, we introduced a doxycycline-inducible transgene encoding DUX4 and 3' genomic DNA into a euchromatic region of the mouse X chromosome. Without induction, DUX4 RNA was expressed at low levels in many tissues and animals displayed a variety of unexpected dominant leaky phenotypes, including male-specific lethality. Remarkably, rare live-born males expressed DUX4 RNA in the retina and presented a retinal vascular telangiectasia. By using doxycycline to induce DUX4 expression in satellite cells, we observed impaired myogenesis in vitro and in vivo. This mouse model, which shows pathologies due to FSHD-related D4Z4 sequences, is likely to be useful for testing anti-DUX4 therapies in FSHD.
Asunto(s)
Genes Dominantes , Genes Ligados a X , Proteínas de Homeodominio/genética , Distrofia Muscular Facioescapulohumeral/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Eucromatina/genética , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Ratones Transgénicos , Distrofia Muscular Facioescapulohumeral/patología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retina/metabolismo , Retina/patologíaRESUMEN
Mesp1 is regarded as the master regulator of cardiovascular development, initiating the cardiac transcription factor cascade to direct the generation of cardiac mesoderm. To define the early embryonic cell population that responds to Mesp1, we performed pulse inductions of gene expression over tight temporal windows following embryonic stem cell differentiation. Remarkably, instead of promoting cardiac differentiation in the initial wave of mesoderm, Mesp1 binds to the Tal1 (Scl) +40 kb enhancer and generates Flk-1+ precursors expressing Etv2 (ER71) and Tal1 that undergo hematopoietic differentiation. The second wave of mesoderm responds to Mesp1 by differentiating into PDGFRα+ precursors that undergo cardiac differentiation. Furthermore, in the absence of serum-derived factors, Mesp1 promotes skeletal myogenic differentiation. Lineage tracing revealed that the majority of yolk sac and many adult hematopoietic cells derive from Mesp1+ precursors. Thus, Mesp1 is a context-dependent determination factor, integrating the stage of differentiation and the signaling environment to specify different lineage outcomes.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Tipificación del Cuerpo , Corazón/embriología , Sistema Hematopoyético/embriología , Mesodermo/embriología , Músculo Esquelético/embriología , Células Madre/citología , Envejecimiento/metabolismo , Animales , Emparejamiento Base/genética , Células de la Médula Ósea/citología , Diferenciación Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Elementos de Facilitación Genéticos/genética , Hematopoyesis , Sistema Hematopoyético/citología , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Endogámicos mdx , Desarrollo de Músculos , Músculo Esquelético/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Células Satélite del Músculo Esquelético/patología , Células Madre/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de Tiempo , Factores de Transcripción/metabolismo , Saco Vitelino/metabolismoRESUMEN
Misexpression of the double homeodomain protein DUX4 in muscle is believed to cause facioscapulohumeral muscular dystrophy (FSHD). Although strategies are being devised to inhibit DUX4 activity in FSHD, there is little known about the normal function of this protein. Expression of DUX4 has been reported in pluripotent cells and testis. To test the idea that DUX4 may be involved in initiating a germ lineage program in pluripotent cells, we interrogated the effect of expressing the human DUX4 gene at different stages during in vitro differentiation of murine embryonic stem (ES) cells. We find that expression of even low levels of DUX4 is incompatible with pluripotency: DUX4-expressing ES cells downregulate pluripotency markers and rapidly differentiate even in the presence of leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). Transcriptional profiling revealed unexpectedly that DUX4 induced a neurectodermal program. Embryoid bodies exposed to a pulse of DUX4 expression displayed severely inhibited mesodermal differentiation, but acquired neurogenic potential. In a serum-containing medium in which neurogenic differentiation is minimal, DUX4 expression served as a neural-inducing factor, enabling the differentiation of Tuj1+ neurites. These data suggest that besides effects in muscle and germ cells, the involvement of DUX4 in neurogenesis should be considered as anti-DUX4 therapies are developed.
Asunto(s)
Células Madre Embrionarias/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Neurogénesis/genética , Animales , Proteína Morfogenética Ósea 4/metabolismo , Línea Celular , Cuerpos Embrioides/metabolismo , Expresión Génica , Proteínas de Homeodominio/biosíntesis , Humanos , Factor Inhibidor de Leucemia/metabolismo , Ratones , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/metabolismoRESUMEN
OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disease with an unclear genetic mechanism. Most patients have a contraction of the D4Z4 macrosatellite repeat array at 4qter, which is thought to cause partial demethylation (FSHD1) of the contracted allele. Demethylation has been surveyed at 3 restriction enzyme sites in the first repeat and only a single site across the entire array, and current models postulate that a generalized D4Z4 chromatin alteration causes FSHD. The background of normal alleles has confounded the study of epigenetic alterations; however, rare patients (FSHD2) have a form of the disease in which demethylation is global, i.e., on all D4Z4 elements throughout the genome. Our objective was to take advantage of the global nature of FSHD2 to identify where disease-relevant methylation changes occur within D4Z4. METHODS: Using bisulfite sequencing of DNA from blood and myoblast cells, methylation levels at 74 CpG sites across 3 disparate regions within D4Z4 were measured in FSHD2 patients and controls. RESULTS: We found that rates of demethylation caused by FSHD2 are not consistent across D4Z4. We identified a focal region of extreme demethylation within a 5' domain, which we named DR1. Other D4Z4 regions, including the DUX4 ORF, were hypomethylated but to a much lesser extent. CONCLUSIONS: These data challenge the simple view that FSHD is caused by a broad "opening" of D4Z4 and lead us to postulate that the region of focal demethylation is the site of action of the key D4Z4 chromatin regulatory factors that go awry in FSHD.
Asunto(s)
Cromatina/genética , Islas de CpG/genética , Metilación de ADN/genética , Distrofia Muscular Facioescapulohumeral/genética , Mioblastos/fisiología , Cromosomas Humanos Par 10 , Cromosomas Humanos Par 4 , Proteínas de Homeodominio/genética , Humanos , Fenotipo , Mapeo RestrictivoRESUMEN
AIMS: Oxidative stress and inflammation are leading risk factors for age-associated functional declines. We assessed aspirin effects on endogenous oxidative-stress levels, lifespan, and age-related functional declines, in the nematode Caenorhabditis elegans. RESULTS: Both aspirin and its salicylate moiety, at nontoxic concentrations (0.5-1 mM), attenuated endogenous levels of reactive oxygen species (p<0.001), and upregulated antioxidant genes encoding superoxide dismutases (especially sod-3, p<0.001), catalases (especially ctl-2, p<0.0001), and two glutathione-S-transferases (gst-4 and gst-10; each p<0.005). Aspirin, and to a lesser degree salicylate, improved survival of hydrogen peroxide, and in the absence of exogenous stress aspirin extended lifespan by 21%-23% (each p<10(-9)), while salicylate added 14% (p<10(-6)). Aspirin and salicylate delayed age-dependent declines in motility and pharyngeal pumping (each p<0.005), and decreased intracellular protein aggregation (p<0.0001)-all established markers of physiological aging-consistent with slowing of the aging process. Aspirin fails to improve stress resistance or lifespan in nematodes lacking DAF-16, implying that it acts through this FOXO transcription factor. INNOVATION: Studies in mice and humans suggest that aspirin may protect against multiple age-associated diseases by reducing all-cause mortality. We now demonstrate that aspirin markedly slows many measures of aging in the nematode. CONCLUSIONS: Aspirin treatment is associated with diminished endogenous oxidant stress and enhanced resistance to exogenous peroxide, both likely mediated by activation of antioxidant defenses. Our evidence indicates that aspirin attenuates insulin-like signaling, thus protecting against oxidative stress, postponing age-associated functional declines and extending C. elegans lifespan under benign conditions.
Asunto(s)
Envejecimiento/fisiología , Aspirina/farmacología , Caenorhabditis elegans/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Esperanza de Vida , Locomoción , Microscopía FluorescenteRESUMEN
Angiotensin II via type 1 receptor activation upregulates the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and LOX-1 activation, in turn, upregulates angiotensin II type 1 receptor expression. We postulated that interruption of this positive feedback loop might attenuate the genesis of angiotensin II-induced hypertension and subsequent cardiac remodeling. To examine this postulate, LOX-1 knockout and wild-type mice were infused with angiotensin II or norepinephrine (control for angiotensin II) for 4 weeks. Angiotensin II-, but not norepinephrine-, induced hypertension was attenuated in LOX-1 knockout mice. Angiotensin II-induced cardiac remodeling was also attenuated in LOX-1 knockout mice. Importantly, angiotensin II type 1 receptor expression was reduced, and the expression and activity of endothelial NO synthase were preserved in the tissues of LOX-1 knockout mice given angiotensin II. Reactive oxygen species generation, nicotinamide-adenine dinucleotide phosphate oxidase expression, and phosphorylation of p38 and p44/42 mitogen-activated protein kinases were also much less pronounced in the LOX-1 knockout mice given angiotensin II. These alterations in biochemical and structural abnormalities were associated with preservation of cardiac hemodynamics in the LOX-1 knockout mice. To confirm that fibroblast function is modulated in the absence of LOX-1, cardiac fibroblasts from wild-type and LOX-1 knockout mice were treated with angiotensin II. Indeed, LOX-1 knockout mice cardiac fibroblasts revealed an attenuated profibrotic response on treatment with angiotensin II. These observations provide strong evidence that LOX-1 is a key modulator of the development of angiotensin II-induced hypertension and subsequent cardiac remodeling.
Asunto(s)
Angiotensina II/farmacología , Hipertensión/metabolismo , Hipertensión/fisiopatología , Receptores Depuradores de Clase E/genética , Vasoconstrictores/farmacología , Remodelación Ventricular/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Células Cultivadas , Fibroblastos/citología , Fibroblastos/fisiología , Eliminación de Gen , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Ratones , Ratones Noqueados , Miocardio/citología , Miocardio/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo III , Norepinefrina/metabolismo , Norepinefrina/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Receptor de Angiotensina Tipo 1/genética , Receptores Depuradores de Clase E/metabolismoRESUMEN
AIMS: Collagen, as a component of the extracellular matrix, has been linked to atherosclerotic plaque formation and stability. Activation of LOX-1, a lectin-like oxidized low-density lipoprotein (LDL) receptor-1, exerts a significant role in collagen formation. We examine the hypothesis that LOX-1 deletion may inhibit collagen accumulation in atherosclerotic arteries in LDL receptor (LDLR) knockout (KO) mice. METHODS AND RESULTS: We generated LOX-1 KO and LOX-1/LDLR double KO mice on a C57BL/6 (wild-type mice) background and fed a 4% cholesterol/10% cocoa butter diet for 18 weeks. Vessel wall collagen accumulation was increased in association with atherogenesis in the LDLR KO mice (P < 0.01 vs. wild-type mice), but much less so in the double KO mice (P < 0.01 vs. LDLR KO mice). Collagen accumulation data were corroborated with pro-collagen I measurements. Expression/activity of osteopontin, fibronectin, and matrix metalloproteinases (MMP-2 and MMP-9) was also increased in the LDLR KO mice (P < 0.01 vs. wild-type mice), but not in the mice with LOX-1 deletion (P < 0.01 vs. LDLR KO mice). The expression of NADPH oxidase (p47(phox), p22(phox), gp91(phox), and Nox-4 subunits) and nitrotyrosine was increased in the LDLR KO mice (P < 0.01 vs. wild-type mice) and not in mice with LOX-1 deletion (P < 0.01 vs. LDLR KO mice). Phosphorylation of Akt-1 and endothelial nitric oxide synthase and expression of haem-oxygenase-1 were found to be reduced in the LDLR KO mice (P < 0.01 vs. wild-type mice), but not in the mice with LOX-1 deletion (P < 0.01 vs. LDLR KO mice). CONCLUSION: LOX-1 deletion reduces enhanced collagen deposition and MMP expression in atherosclerotic regions via inhibition of pro-oxidant signals.
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Aterosclerosis/metabolismo , Colesterol en la Dieta/farmacología , Colágeno/metabolismo , Receptores de LDL/metabolismo , Receptores Depuradores de Clase E/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/metabolismo , Osteopontina/metabolismo , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de LDL/genética , Receptores Depuradores de Clase E/genéticaRESUMEN
Transforming growth factor beta(1) (TGFbeta(1)) activation leads to tissue fibrosis. Here, we report on the role of LOX-1, a lectin-like 52-kDa receptor for oxidized low density lipoprotein, in TGFbeta(1)-mediated collagen expression and underlying signaling in mouse cardiac fibroblasts. TGFbeta(1) was overexpressed in wild-type (WT) and LOX-1 knock-out mouse cardiac fibroblasts by transfection with adeno-associated virus type 2 vector carrying the active TGFbeta(1) moiety (AAV/TGFbeta (ACT)(1)). Transfection of WT mouse cardiac fibroblasts with AAV/TGFbeta (ACT)(1) markedly enhanced the expression of NADPH oxidases (p22(phox), p47(phox), and gp91(phox) subunits) and LOX-1, formation of reactive oxygen species, and collagen synthesis, concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). The TGFbeta(1)-mediated increase in collagen synthesis was markedly attenuated in the LOX-1 knock-out mouse cardiac fibroblasts as well as in WT mouse cardiac fibroblasts treated with a specific anti-LOX-1 antibody. Treatment with anti-LOX-1 antibody also reduced NADPH oxidase expression and MAPK activation. The NADPH oxidase inhibitors and gp91phox small interfering RNA reduced LOX-1 expression, MAPK activation, and collagen formation. The p38 MAPK inhibitors as well as the p44/42 MAPK inhibitors reduced collagen formation without affecting LOX-1 expression in cardiac fibroblasts. These observations suggest that collagen synthesis in cardiac fibroblasts involves a facilitative interaction between TGFbeta(1)-NADPH oxidase and LOX-1. Further, the activation of MAPK pathway appears to be downstream of TGFbeta(1)-reactive oxygen species-LOX-1 cascade.
Asunto(s)
Colágeno/metabolismo , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica , Miocardio/metabolismo , Receptores Depuradores de Clase E/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Grupo Citocromo b/metabolismo , Sistema de Señalización de MAP Quinasas , Glicoproteínas de Membrana/metabolismo , Ratones , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno , Receptores Depuradores de Clase E/genética , Transducción de SeñalRESUMEN
Atherogenesis is associated with inflammation and oxidative stress. Activation of renin-angiotensin system with generation of angiotensin II and type 1 receptor (AT1R) stimulation has been amply reported in atherosclerosis. Since angiotensin II type 2 receptor (AT2R) activity is purported to oppose the effects of AT1R, we hypothesized that AT2R (agtr2) over-expression would inhibit atherogenesis. We prepared recombinant adeno-associated virus type-2 (AAV) carrying AT2R cDNA (AAV/AT2R), and homozygous LDLR-deficient (KO) mice were given AAV/AT2R, AAV/Neo or saline. All mice were placed on a high cholesterol diet. After 18 weeks, AT2R was found to be over-expressed systemically in AAV/AT2R-treated mice. Atherogenesis in aorta was reduced in the AAV/AT2R group by approximately 50% compared to other LDLR KO mice groups. Expression of NADPH oxidase, nitrotyrosine and NF-kappaB was increased in aortic tissues of the LDLR KO mice given saline or AAV/Neo, but not in mice with AT2R upregulation. Expression of endothelial nitric oxide synthase (eNOS) and heme-oxygenase-1 (HO-1) was decreased and that of the lectin-like oxidized-LDL receptor (LOX-1) increased in the LDLR KO mice, but not in the mice with AT2R over-expression. Further, Akt-1 phosphorylation was reduced in the LDLR KO mice, but not in the mice with AT2R over-expression. Thus, AT2R upregulation can reduce atherogenesis, possibly by modulating oxidative stress and the pro-inflammatory cascade, mediated via Akt-1. Over-expression of AT2R may be an important therapeutic approach in atherosclerosis.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Regulación de la Expresión Génica , Hemo-Oxigenasa 1/metabolismo , Receptor de Angiotensina Tipo 2/biosíntesis , Receptor de Angiotensina Tipo 2/genética , Receptores Depuradores de Clase E/metabolismo , Angiotensinas/metabolismo , Animales , Aterosclerosis/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo , Receptores de LDL/metabolismoRESUMEN
Angiotensin (Ang) II, via type 1 receptor activation, exerts a significant role in atherogenesis and collagen synthesis. To test the hypothesis that Ang II type 2 receptor (AT2R) upregulation delivered with adeno-associated virus type 2 (AAV/AT2R) would inhibit collagen synthesis in atherosclerotic arteries, LDLR knockout mice were injected with AAV/AT2R and fed 4% cholesterol diet for 18 weeks. LDLR knockout mice treated with saline or AAV/Neo exhibited extensive vessel wall collagen accumulation, which was reduced by about 50% with AT2R over-expression. AT2R upregulation completely blocked the alterations in the expression of procollagen-I, osteopontin, fibronectin, CD68, and matrix metalloproteinases (MMP-2 and MMP-9), as well as phosphorylation of p38 and p44/42 MAPKs. Activity of superoxide dismutase was reduced in the LDLR KO mice and it increased with AT2R upregulation. This study demonstrates that AT2R over-expression reduces enhanced collagen accumulation, MMP expression and activity in atherosclerotic regions via inhibition of pro-oxidant signals.
Asunto(s)
Aterosclerosis/metabolismo , Aterosclerosis/patología , Colágeno/metabolismo , Expresión Génica , Oxidantes/metabolismo , Receptor de Angiotensina Tipo 2/genética , Animales , Aorta/enzimología , Aorta/patología , Aterosclerosis/enzimología , Fibronectinas/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteopontina/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
LOX-1 is a newly described lectin-like receptor for oxidized-LDL (ox-LDL), which is over-expressed in the ischemic myocardium. To examine the pathogenic role of LOX-1 in the determination of ischemia-reperfusion (I-R) injury to the heart, we developed LOX-1 knockout (KO) mice, and subjected these mice to 60 min of left coronary artery occlusion followed by 60 min of reperfusion. I-R in the LOX-1 KO mice resulted in a significant reduction in myocardial injury as well as in accumulation of inflammatory cells in the I-R myocardium and lipid peroxidation (P<0.01 vs. wild-type mice). Concomitantly, there was significant preservation of cardiac function in the LOX-1 KO mice despite I-R (P<0.01 vs. the wild-type mice). The phosphorylation of oxidative stress-sensitive mitogen-activated protein kinase (p38MAPK) and protein kinase B/Akt-1, expression of nitrotyrosine and inducible nitric oxide synthase (iNOS), and superoxide dismutase activity were enhanced during I-R in the wild-type mice. These alterations in p38MAPK, Akt-1 and iNOS were much less pronounced in the LOX-1 KO mice. The superoxide dismutase activity increased further in the LOX-1 KO mice. These observations provide compelling evidence that LOX-1 may be a key modulator of myocardial I-R injury, and its effect is mediated by pro-oxidant signals. LOX-1 may be a potential target for therapy of myocardial ischemic injury.
Asunto(s)
Daño por Reperfusión Miocárdica/metabolismo , Receptores Depuradores de Clase E/deficiencia , Animales , Forma MB de la Creatina-Quinasa/sangre , Eliminación de Gen , Hemodinámica , Malondialdehído/sangre , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Daño por Reperfusión Miocárdica/enzimología , Miocardio/enzimología , Miocardio/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Superóxido Dismutasa/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Angiotensin II (Ang II) induces angiogenesis by stimulating reactive oxygen species-dependent vascular endothelial growth factor (VEGF) expression. Ang II via type 1 receptor upregulates the expression of LOX-1, a lectin-like receptor for oxidized low-density lipoprotein. LOX-1 activation, in turn, upregulates Ang II type 1 receptor expression. We postulated that interruption of the feedback loop between Ang II and LOX-1 might attenuate Ang II-induced VEGF expression and capillary formation. In vitro experiments showed that Ang II (1 nmol/L) induced the expression of LOX-1 and VEGF and enhanced capillary formation from human coronary endothelial cells in Matrigel assay. Ang II-mediated expression of LOX-1 and VEGF, capillary formation, intracellular reactive oxygen species generation, and phosphorylation of p38 as well as p44/42 mitogen-activated protein kinases, were suppressed by anti-LOX-1 antibody, nicotinamide-adenine dinucleotide phosphate oxidase inhibitor apocynin and the Ang II type 1 receptor blocker losartan, but not by the Ang II type 2 receptor blocker PD123319. Expression of VEGF and capillary formation induced by Ang II were also inhibited by the p44/42 mitogen-activated protein kinase inhibitor U0126 and the p38 mitogen-activated protein kinase inhibitor SB203580. In ex vivo experiments, Ang II stimulated capillary sprouting from aortic rings from wild-type mice, and this phenomenon was significantly attenuated by pretreatment of aortic rings with anti-LOX-1 antibody, apocynin, and losartan, but not by PD123319. Importantly, Ang II-induced capillary sprouting was minimal from aortic rings from LOX-1 null mice compared with wild-type mice. These findings suggest that small concentrations of Ang II promote capillary formation by inducing the expression of VEGF via Ang II type 1 receptor/LOX-1-mediated stimulation of the reactive oxygen species-mitogen-activated protein kinase pathway.
Asunto(s)
Angiotensina II/fisiología , Capilares/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Receptores Depuradores de Clase E/fisiología , Angiotensina II/farmacología , Bloqueadores del Receptor Tipo 2 de Angiotensina II , Animales , Capilares/crecimiento & desarrollo , Células Cultivadas , Vasos Coronarios/citología , Células Endoteliales/citología , Retroalimentación Fisiológica/efectos de los fármacos , Retroalimentación Fisiológica/genética , Humanos , Imidazoles/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Oxidación-Reducción/efectos de los fármacos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genética , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: Chronic ischemia is associated with alterations in genes that result in myocardial remodeling. An important biochemical basis of cardiac remodeling is generation of reactive oxygen species (ROS). A few studies have suggested that acute ischemia triggers signals for remodeling. We examined the hypothesis that targeted deletion of lectin-like oxidized-LDL receptor (LOX-1) may inhibit signals related to cardiac remodeling. METHODS AND RESULTS: We generated LOX-1 knockout (KO) mice on C57BL/6 (wild-type mice) background, and subjected wild-type and KO mice to ischemia-reperfusion (I-R). The wild-type mice developed a marked reduction in left ventricular systolic pressure and +/-dp/dt(max) and an increase in left ventricular end-diastolic pressure following I-R, and this change was much less in the LOX-1 KO mice, indicating preservation of left ventricular function with LOX-1 deletion. There was evidence for marked oxidative stress (NADPH oxidase expression, malondialdehyde and 8-isoprostane) following I-R in the wild-type mice, much less so in the LOX-1 KO mice (P<0.01). In concert, collagen deposition (Masson's trichrome and Picro-sirius red staining) increased dramatically in the wild-type mice, but only half as much in the LOX-1 KO mice (P<0.01). Collagen staining data was corroborated with procollagen-I expression. Further, fibronectin and osteopontin expression increased in the wild-type mice, but to a much smaller extent in the LOX-1 KO mice (P<0.01). CONCLUSIONS: These findings provide compelling evidence that LOX-1 is a key modulator of cardiac remodeling which starts immediately following I-R.
Asunto(s)
Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/etiología , Receptores Depuradores de Clase E/fisiología , Transducción de Señal , Remodelación Ventricular , Animales , Colágeno Tipo I/metabolismo , Fibronectinas/genética , Metaloproteasas/genética , Ratones , Ratones Endogámicos C57BL , Osteopontina/genética , Estrés Oxidativo , ARN Mensajero/análisisRESUMEN
OBJECTIVE: Vascular endothelial growth factor (VEGF), a key angiogenic growth factor, stimulates angiogenesis. Low levels of reactive oxygen species (ROS) function as signaling molecules for angiogenesis. We postulated that low concentrations of oxLDL might induce low levels of ROS and initiate angiogenesis. METHODS AND RESULTS: An in vitro model of tube formation from human coronary artery endothelial cells (HCAECs) was used. oxLDL (0.1, 1, 2, 5 microg/mL) induced VEGF expression and enhanced tube formation. oxLDL-mediated VEGF expression and tube formation were suppressed by a specific blocking anti-LOX-1 antibody. Anti-LOX-1 antibody also reduced oxLDL-induced increase in the expression of NADPH oxidase (gp91(phox) and p47(phox) subunits) and subsequent intracellular ROS generation, phosphorylation of p38 as well as p44/42MAPK, and NF-kappaB p65 expression. gp91(phox) siRNA had a similar effect. The expression of VEGF and NF-kappaB p65 induced by oxLDL was also inhibited by the specific extracellular signal-regulated kinase (ERK) 1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580. Importantly, the NADPH oxidase inhibitor apocynin, gp91(phox) siRNA, U0126, and SB203580 all reduced tube formation in response to oxLDL. CONCLUSIONS: These findings suggest that small concentrations of oxLDL promote capillary tube formation by inducing the expression of VEGF via LOX-1-mediated activation of NADPH oxidase- MAPKs-NF-kappaB pathway.