RESUMEN
Soluble guanylate cyclase (sGC), a key enzyme of the nitric oxide signaling pathway, is formed as a heterodimer by various isoforms of its α and ß subunit. GUCY1A3, encoding the α1 subunit, was identified as a risk gene for coronary artery disease and myocardial infarction, but its specific contribution to atherosclerosis remains unclear. This study sought to decipher the role of Gucy1a3 in atherosclerosis in mice. At age 32 weeks and after 20 weeks of standard or high-fat diet, Gucy1a3(-/-)/Ldlr(-/-) mice exhibited a significant reduction of the atherosclerotic plaque size at the aortic root and the aorta for high-fat diet animals as compared with Ldlr(-/-) control mice. Collagen content in plaques in the aortic root was reduced, suggesting an alteration of smooth muscle cell function. Proliferation and migration were reduced in Gucy1a3(-/-) primary aortic smooth muscle cells (AoSMCs), and proliferation was also reduced in human AoSMCs after inhibition of sGC by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one. Gucy1a3 deficiency in AoSMCs prevents their phenotypic switching, as indicated by the differential expression of marker proteins. The inherited Gucy1a3(-/-) loss exerts an atheroprotective effect. We suggest that sGC activity promotes the phenotypic switching of smooth muscle cells from a contractile to a synthetic state, fostering the formation of atherosclerosis. Preventing this switch by sGC inhibition may provide a novel target in atherosclerotic disease.
Asunto(s)
Aterosclerosis/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Animales , Aterosclerosis/genética , Western Blotting , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Femenino , Genotipo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Guanilil Ciclasa Soluble/genéticaRESUMEN
AIMS: Recent genome-wide association (GWA) studies identified 10 chromosomal loci for coronary artery disease (CAD) or myocardial infarction (MI). However, these loci explain only a small proportion of the genetic variability of these pertinent diseases. We sought to identify additional CAD/MI loci by applying a three-stage approach. METHODS AND RESULTS: We genotyped n = 1157 MI cases and n = 1748 controls from a population-based study population [German MI Family Study (GerMIFS) III (KORA)] with genome-wide SNP arrays. At this first stage, n = 462 SNPs showed association with MI at P<1 × 10(-3) in two-sided logistic regression. In a second stage, 415 of these SNPs were evaluated in silico in two independent GWA samples, the GerMIFS I (875 cases/1644 controls) and GerMIFS II (1222 cases/1298 controls). Nine SNPs, representing three regions, displayed consistent replication in this in silico analysis (P<0.05 for each GWA sample): five SNPs at 9p21.3, a well-known CAD/MI locus, two SNPs at 10p11.21, and two SNPs at 2p24.3. Wet-lab replication, i.e. the third stage, of SNP rs3739998 (representing the novel locus at 10p11.21, p.S1002T in the KIAA1462 gene) in additional 5790 cases and 5302 controls confirmed the association (P=9.54 × 10(-4)), but not for the 2p24.3 locus. The combined P-value across all stages for SNP rs3739998 is P=1.27 × 10(-11) [odds ratio (OR) = 1.15 (1.11-1.20)]. CONCLUSION: Analysis of a GWA study followed by in silico and wet-lab replication steps identified the KIAA1462 gene, encoding a yet uncharacterized protein, on chromosome 10p11.23 with genome-wide significant association for CAD/MI. Further studies are needed to characterize the functional role of this locus in the aetiology of these diseases.
Asunto(s)
Cromosomas Humanos Par 10/genética , Enfermedad de la Arteria Coronaria/genética , Infarto del Miocardio/genética , Polimorfismo de Nucleótido Simple/genética , Adulto , Moléculas de Adhesión Celular/genética , Cromosomas Humanos Par 9/genética , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Técnicas de Genotipaje/métodos , Humanos , Masculino , Persona de Mediana Edad , Linaje , Factores de Riesgo , Adulto JovenRESUMEN
Compelling evidence suggests that Foxp3-expressing CD25(+)CD4(+) regulatory T cells (Treg) are generated within the thymus as a separate lineage. However, Foxp3(+)CD4(+) Treg can also be generated de novo in a TGF-beta-dependent process from naive T cells by TCR triggering. Recently, we have shown that naturally occurring, but not in vitro TGF-beta-induced Foxp3(+) Treg display stable Foxp3 expression that was associated with selective demethylation of an evolutionarily conserved element within the Foxp3 locus named TSDR (Treg-specific demethylated region). Here, we report that inhibition of DNA methylation by azacytidine, even in absence of exogenous TGF-beta, not only promoted de novo induction of Foxp3 expression during priming, but also conferred stability of Foxp3 expression upon restimulation. Most notably, such stable Foxp3 expression was found only for cells displaying enhanced TSDR demethylation. In contrast, in vitro TSDR methylation diminished its transcriptional activity. Foxp3(+) Treg generated in vivo by DEC-205-mediated targeting of agonist ligands to dendritic cells showed long-term survival in the absence of the inducing antigen and exhibited efficient TSDR demethylation. Together, our data suggest that TSDR is an important methylation-sensitive element regulating Foxp3 expression and demonstrate that epigenetic imprinting in this region is critical for establishment of a stable Treg lineage.
Asunto(s)
Metilación de ADN , Factores de Transcripción Forkhead/genética , Linfocitos T Reguladores/metabolismo , Traslado Adoptivo , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/trasplante , Proteínas de Unión al ADN/genética , Decitabina , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Secuencias Reguladoras de Ácidos Nucleicos/genética , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
BACKGROUND: In order to further distinguish unique from general functions of connexin43, we have generated mice in which the coding region of connexin43 was replaced by that of connexin26. RESULTS: Heterozygous mothers showed impaired mammary gland development responsible for decreased lactation and early postnatal death of the pups which could be partially rescued by wild type foster mothers. Only about 17% of the homozygous connexin43 knock-in connexin26 mice instead of 25% expected according to Mendelian inheritance, were born and only 6% survived to day 21 post partum and longer. Neonatal and adult connexin43 knock-in connexin26 mice exhibited slowed ventricular conduction in their hearts, i.e. similar but delayed electrophysiological abnormalities as connexin43 deficient mice. Furthermore, connexin43 knock-in connexin26 male and female mice were infertile and exhibited hypotrophic gonads. In testes, tubuli seminiferi were developed and spermatogonia as well as some primary spermatocytes were present, but further differentiated stages of spermatogenesis were absent. Ovaries of female connexin43 knock-in connexin26 mice revealed only few follicles and the maturation of follicles was completely impaired. CONCLUSION: The impaired gametogenesis of homozygous males and females can explain their infertility.
Asunto(s)
Conexina 43/genética , Conexinas/genética , Sistema de Conducción Cardíaco/fisiopatología , Ovario/fisiopatología , Testículo/fisiopatología , Animales , Southern Blotting , Western Blotting , Conexina 26 , Electrocardiografía , Células Madre Embrionarias , Femenino , Ventrículos Cardíacos/fisiopatología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development and function of CD4(+) regulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity. Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as histone modifications within the conserved region in ex vivo isolated Foxp3(+)CD25(+)CD4(+) Tregs, but not in naïve CD25(-)CD4(+) T cells. Partial DNA demethylation is already found within developing Foxp3(+) thymocytes; however, Tregs induced by TGF-beta in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural Tregs, these TGF-beta-induced Foxp3(+) Tregs lose both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-beta. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions.
Asunto(s)
Epigénesis Genética , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Linfocitos T Reguladores/fisiología , Animales , Secuencia de Bases , Separación Celular , Islas de CpG , Metilación de ADN , Citometría de Flujo , Subunidad alfa del Receptor de Interleucina-2 , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Timo/citología , Timo/metabolismoRESUMEN
The scurfy mutant mouse strain suffers from a fatal lymphoproliferative disease leading to early death within 3-4 wk of age. A frame-shift mutation of the forkhead box transcription factor Foxp3 has been identified as the molecular cause of this multiorgan autoimmune disease. Foxp3 is a central control element in the development and function of regulatory T cells (T reg cells), which are necessary for the maintenance of self-tolerance. However, it is unclear whether dysfunction or a lack of T reg cells is etiologically involved in scurfy pathogenesis and its human correlate, the IPEX syndrome. We describe the generation of bacterial artificial chromosome-transgenic mice termed "depletion of regulatory T cell" (DEREG) mice expressing a diphtheria toxin (DT) receptor-enhanced green fluorescent protein fusion protein under the control of the foxp3 gene locus, allowing selective and efficient depletion of Foxp3+ T reg cells by DT injection. Ablation of Foxp3+ T reg cells in newborn DEREG mice led to the development of scurfy-like symptoms with splenomegaly, lymphadenopathy, insulitis, and severe skin inflammation. Thus, these data provide experimental evidence that the absence of Foxp3+ T reg cells is indeed sufficient to induce a scurfy-like phenotype. Furthermore, DEREG mice will allow a more precise definition of the function of Foxp3+ T reg cells in immune reactions in vivo.