RESUMEN
AIMS: The prolyl-4-hydroxylase domain (PHD) enzymes are representing novel therapeutic targets for ischemic tissue protection. Whereas the consequences of a knock out of the PHDs have been analyzed in the context of cardioprotection, the implications of PHD overexpression is unknown so far. METHODS AND RESULTS: We generated cardiomyocyte-specific PHD3transgenic mice (cPhd3tg). Resting cPhd3tg mice did not show constitutive accumulation of HIF-1α or HIF-2α or changes in HIF target gene expression in the heart. Cardiac function was followed up for 14 months in these mice and found to be unchanged. After challenging the cPhd3tg mice with ligation of the left anterior descending artery, HIF-1α/-2α accumulation in the left ventricles was blunted. This was associated with a significantly increased infarct size of the cPhd3tg compared to wild type mice. CONCLUSION: Whereas overexpression of PHD3 in the resting state does not significantly influence cardiac function, it is crucial for the cardiac response to ischemia by affecting HIFα accumulation in the ischemic tissue.
Asunto(s)
Ventrículos Cardíacos/metabolismo , Infarto del Miocardio/genética , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Animales , Aorta Torácica/cirugía , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica , Ventrículos Cardíacos/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ligadura , Ratones , Ratones Transgénicos , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/patología , Miocitos Cardíacos/patología , Procolágeno-Prolina Dioxigenasa/metabolismo , Transducción de Señal , TransgenesRESUMEN
Several genetically modified mouse models implicated that prolyl-4-hydroxylase domain (PHD) enzymes are critical mediators for protecting tissues from an ischemic insult including myocardial infarction by affecting the stability and activation of hypoxia-inducible factor (HIF)-1 and HIF-2. Thus, the current efforts to develop small-molecule PHD inhibitors open a new therapeutic option for myocardial tissue protection during ischemia. Therefore, we aimed to investigate the applicability and efficacy of pharmacological HIFα stabilization by a small-molecule PHD inhibitor in the heart. We tested for protective effects in the acute phase of myocardial infarction after pre- or post-conditional application of the inhibitor. Application of the specific PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) resulted in HIF-1α and HIF-2α accumulation in heart muscle cells in vitro and in vivo. The rapid and robust responsiveness of cardiac tissue towards ICA was further confirmed by induction of the known HIF target genes heme oxygenase-1 and PHD3. Pre- and post-conditional treatment of mice undergoing myocardial infarction resulted in a significantly smaller infarct size. Tissue protection from ischemia after pre- or post-conditional ICA treatment demonstrates that there is a therapeutic time window for the application of the PHD inhibitor (PHI) post-myocardial infarction, which might be exploited for acute medical interventions.
Asunto(s)
Cardiotónicos/uso terapéutico , Glicina/análogos & derivados , Isoquinolinas/farmacología , Infarto del Miocardio/tratamiento farmacológico , Inhibidores de Prolil-Hidroxilasa/uso terapéutico , Animales , Glicina/farmacología , Glicina/uso terapéutico , Factor 1 Inducible por Hipoxia/metabolismo , Poscondicionamiento Isquémico , Precondicionamiento Isquémico Miocárdico , Isoquinolinas/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND/AIMS: The hypoxia inducible factor-1 (HIF-1) is a suitable marker for tissue oxygenation. We intended to develop cardiomyocytes (CMs) expressing the oxygen-dependent degradation domain of HIF-1α fused to the firefly luciferase (ODD-Luc) followed by proof-of-concept for its applicability in the assessment of heart muscle oxygenation. METHODS AND RESULTS: We first generated embryonic stem cell (ESC) lines (ODD-Luc ESCs) from a Tg ROSA26 ODD-Luc/+ mouse. Subsequent CMs selection was facilitated by stable integration of an antibiotic resistance expressed under the control of the αMHC promoter. ODD-Luc ESCs showed a strong Luc-signal within 1 h of hypoxia (1% oxygen), which coincided with endogenous HIF-1α. Engineered heart muscle (EHM) constructed with ODD-Luc CMs confirmed the utility of the model to sense hypoxia, and monitor reoxygenation also in a multicellular heart muscle model. Pharmacologically induced inotropy/chronotropy under isoprenaline resulted in enhanced Luc-signal suggesting enhanced oxygen consumption, leading to notable myocardial hypoxia. CONCLUSIONS: ODD-Luc-CMs can be used to monitor dynamic changes of cardiomyocyte oxygenation in living heart muscle samples. We provide proof-of-concept for pharmacologically induced myocardial interventions and envision applications of the developed model in drug screens and fundamental studies of ischemia/reperfusion injury.
Asunto(s)
Ingeniería Genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Miocitos Cardíacos/metabolismo , Animales , RatonesRESUMEN
The prolyl-4-hydroxylase domain 1-3 (PHD1-3) enzymes are regulating the protein stability of the α-subunit of the hypoxia-inducible factor-1 (HIF-1), which mediates oxygen-dependent gene expression. PHD2 is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. In human cancers, HIF-1α is overexpressed as a result of intratumoral hypoxia which in turn promotes tumor progression. The role of PHD2 for tumor progression is in contrast far from being thoroughly understood. Therefore, we established PHD2 knockdown clones of MDA-MB-231 breast cancer cells and analyzed their tumor-forming potential in a SCID mouse model. Tumor progression was significantly impaired in the PHD2 knockdown MDA-MB-231 cells, which could be partially rescued by re-establishing PHD2 expression. In a RNA profile screen, we identified the secreted phosphoprotein 1 (SPP1) as one target, which is differentially regulated as a consequence of the PHD2 knockdown. Knockdown of PHD2 drastically reduced the SPP1 expression in MDA-MB-231 cells. A correlation of SPP1 and PHD2 expression was additionally verified in 294 invasive breast cancer biopsies. In subsequent analyses, we identified that PHD2 alters the processing of transforming growth factor (TGF)-ß1, which is highly involved in SPP1 expression. The altered processing capacity was associated with a dislocation of the pro-protein convertase furin. Thus, our data demonstrate that in MDA-MB-231 cells PHD2 might affect tumor-relevant TGF-ß1 target gene expression by altering the TGF-ß1 processing capacity.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Ratones , Osteopontina/genética , Transducción de Señal , Carga Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIMS: The hypoxia-inducible factor-1 (HIF-1) is the master modulator of hypoxic gene expression. The effects of chronically stabilized cardiac HIF-1α and its role in the diseased heart are not precisely known. The aims of this study were as follows: (i) to elucidate consequences of HIF-1α stabilization in the heart; (ii) to analyse long-term effects of HIF-1α stabilization with ageing and the ability of the HIF-1α overexpressing hearts to respond to increased mechanical load; and (iii) to analyse HIF-1α protein levels in failing heart samples. METHODS AND RESULTS: In a cardiac-specific HIF-1α transgenic mouse model, constitutive expression of HIF-1α leads to changes in capillary area and shifts the cardiac metabolism towards glycolysis with a net increase in glucose uptake. Furthermore, Ca(2+) handling is altered, with increased Ca(2)(+) transients and faster intracellular [Ca(2+)] decline. These changes are associated with decreased expression of sarcoplasmic/endoplasmic reticulum calcium ATPase 2a but elevated phosphorylation of phospholamban. HIF-1α transgenic mice subjected to transverse aortic constriction exhibited profound cardiac decompensation. Moreover, cardiomyopathy was also seen in ageing transgenic mice. In parallel, we found an increased stabilization of HIF-1α in heart samples of patients with end-stage heart failure. CONCLUSION: Changes induced with transgenic cardiac HIF-1α possibly mediate beneficial effects in the short term; however, with increased mechanical load and ageing they become detrimental for cardiac function. Together with the finding of increased HIF-1α protein levels in samples from human patients with cardiomyopathy, these data indicate that chronic HIF-1α stabilization drives autonomous pathways that add to disease progression.
Asunto(s)
Cardiomiopatías/metabolismo , Insuficiencia Cardíaca/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Miocitos Cardíacos/metabolismo , Factores de Edad , Animales , Señalización del Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Capilares/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Glucosa/metabolismo , Glucólisis/genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Transgénicos , Fosforilación , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Factores de Tiempo , Regulación hacia Arriba , Función Ventricular IzquierdaRESUMEN
Oligodendroglial damage and loss are typical characteristics of demyelinating diseases such as multiple sclerosis (MS) and the leukodystrophies. Axonal loss is the underlying cause of permanent neurological deficits in MS and it is thought to arise from a combination of immune-mediated axonal damage and the loss of trophic support to axons from myelin sheaths after demyelination. Prevention of oligodendroglial damage or death and demyelination are therefore attractive neuroprotective treatment strategies. However, a better understanding of mechanisms leading to oligodendroglial damage and demyelination is a prerequisite for the development of such treatment options. Here, we demonstrate that X-linked inhibitor of apoptosis (XIAP), the most potent member of the inhibitor of apoptosis proteins (IAP) family is expressed in oligodendrocytes in vivo and in vitro. Increased expression of XIAP is associated with protection against selected cell death pathways, whereas decreased expression increases oligodendroglial cell death in vitro. However, lack of XIAP does not modulate oligodendroglial cell death in toxic demyelination in vivo.
Asunto(s)
Citoprotección/genética , Enfermedades Desmielinizantes/patología , Regulación hacia Abajo/fisiología , Oligodendroglía/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Animales , Animales Modificados Genéticamente , Línea Celular , Quelantes/toxicidad , Cuprizona/toxicidad , Citoprotección/efectos de los fármacos , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/patología , Cultivo Primario de Células , Ratas , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/deficienciaRESUMEN
Prolylhydroxylase domain proteins (PHD) are cellular oxygen-sensing molecules that regulate the stability of the α-subunit of the transcription factor hypoxia inducible factor (HIF)-1. HIF-1 affects cardiac development as well as adaptation of the heart toward increased pressure overload or myocardial infarction. We have disrupted PHD2 in cardiomyocytes (cPhd (-/-)) using Phd2(flox/flox) mice in combination with MLCvCre mice, which resulted in HIF-1α stabilization and activation of HIF target genes in the heart. Although cPhd2(-/-) mice showed no gross abnormalities in cardiac filament structure or function, we observed a significant increased cardiac capillary area in those mice. cPhd2 (-/-) mice did not respond differently to increased mechanical load by transverse aortic constriction compared with their wild-type (wt) littermates. After ligation of the left anterior descending artery, however, the area at risk and area of necrosis were significantly smaller in the cPhd2(-/-) mice compared with Phd2 wt mice in line with the described pivotal role of HIF-1α for tissue protection in case of myocardial infarction. This correlated with a decreased number of apoptotic cells in the infarcted myocardium in the cPhd2(-/-) mice and significantly improved cardiac function 3 weeks after myocardial infarction.
Asunto(s)
Infarto del Miocardio/enzimología , Miocardio/enzimología , Procolágeno-Prolina Dioxigenasa , Enfermedad Aguda , Animales , Apoptosis/genética , Técnicas de Silenciamiento del Gen , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Ratones , Ratones Transgénicos , Infarto del Miocardio/patología , Miocardio/patología , Necrosis/enzimología , Necrosis/patología , Estabilidad ProteicaRESUMEN
The αLß2-integrin LFA-1 (CD11a/CD18) is known as an important molecule for leukocyte migration. However, the precise role of LFA-1 in the pathogenesis of EAE has so far remained unclear. We describe here the disease development in LFA-1(-/-) mice compared with WT controls. Ablation of LFA-1 resulted in more severe EAE with increased demyelination and increased numbers of myelin oligodendrocyte glycoprotein-reactive CD4(+) T cells in the CNS. However, the production of the pro-inflammatory cytokines IL-17 and IFN-γ was unchanged on the level of antigen-specific T cells. Interestingly, LFA-1-deficient mice showed a clearly reduced frequency of Treg in the inflamed CNS. Moreover, Treg counts in spleens and thymi of unimmunized LFA-1(-/-) mice were lower in comparison to the WT controls, indicating an impairment of Treg generation. In combination, these results suggest a substantial role of LFA-1 in Treg generation and subsequent expansion of effector T cells and highlight the importance of Treg in limiting EAE.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Antígeno-1 Asociado a Función de Linfocito/genética , Linfocitos T Reguladores/metabolismo , Animales , Encéfalo/inmunología , Encéfalo/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Recuento de Células , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Mediadores de Inflamación/metabolismo , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Antígeno-1 Asociado a Función de Linfocito/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , Médula Espinal/inmunología , Médula Espinal/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Oligodendroglial cell death is a frequent phenomenon of many neurological diseases, e.g. in demyelinating diseases such as multiple sclerosis (MS). The underlying mechanisms are largely unknown. Here, we demonstrate that in the toxic demyelination cuprizone model, oligodendroglial cell death and downregulation of myelin genes start days after initiation of the cuprizone diet and weeks before demyelination is obvious. In early - but not in later - stages, dying oligodendrocytes express activated caspase 3, suggesting a switch from classical apoptotic pathways to caspase 3-independent mechanisms during the course of the cuprizone diet. The expression level of FAS in the corpus callosum, a cell death receptor crucial for oligodendroglial cell death in experimental autoimmune encephalomyelitis (EAE), correlates with the expression of activated caspase 3 in oligodendrocytes. However, mice lacking FAS in oligodendrocytes are not protected against cuprizone-induced oligodendroglial cell death, showing that FAS is dispensable for oligodendroglial cell death in the cuprizone model.