RESUMO
The release of the neurotransmitter norepinephrine (NE) is modulated by presynaptic adenosine receptors. In the present study we investigated the effect of a partial activation of this feedback mechanism. We hypothesized that partial agonism would have differential effects on NE release in isolated hearts as well as on heart rate in vivo depending on the genetic background and baseline sympathetic activity. In isolated perfused hearts of Wistar and Spontaneously Hypertensive Rats (SHR), NE release was induced by electrical stimulation under control conditions (S1), and with capadenoson 6 · 10(-8) M (30 µg/l), 6 · 10(-7) M (300 µg/l) or 2-chloro-N(6)-cyclopentyladenosine (CCPA) 10(-6) M (S2). Under control conditions (S1), NE release was significantly higher in SHR hearts compared to Wistar (766+/-87 pmol/g vs. 173+/-18 pmol/g, p<0.01). Capadenoson led to a concentration-dependent decrease of the stimulation-induced NE release in SHR (S2/S1â = â0.90 ± 0.08 with capadenoson 6 · 10(-8) M, 0.54 ± 0.02 with 6 · 10(-7) M), but not in Wistar hearts (S2/S1â =â 1.05 ± 0.12 with 6 · 10(-8) M, 1.03 ± 0.09 with 6 · 10(-7) M). CCPA reduced NE release to a similar degree in hearts from both strains. In vivo capadenoson did not alter resting heart rate in Wistar rats or SHR. Restraint stress induced a significantly greater increase of heart rate in SHR than in Wistar rats. Capadenoson blunted this stress-induced tachycardia by 45% in SHR, but not in Wistar rats. Using a [(35)S]GTPγS assay we demonstrated that capadenoson is a partial agonist compared to the full agonist CCPA (74+/-2% A(1)-receptor stimulation). These results suggest that partial adenosine A(1)-agonism dampens stress-induced tachycardia selectively in rats susceptible to strong increases in sympathetic activity, most likely due to a presynaptic attenuation of NE release.
Assuntos
Agonistas do Receptor A1 de Adenosina/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Norepinefrina/metabolismo , Receptor A1 de Adenosina/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Feminino , Técnicas In Vitro , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Restrição FísicaRESUMO
Ectopic expression of defined sets of genetic factors can reprogram somatic cells to create induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle cells, and endothelial cells arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2.5. Here we show, using genetic fate-mapping, that Isl1(+) multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all 3 cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1(+) progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.-Moretti, A., Bellin, M., Jung, C. B., Thies, T.-M., Takashima, Y., Bernshausen, A., Schiemann, M., Fischer, S., Moosmang, S., Smith, A. G., Lam, J. T., Laugwitz, K.-L. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1(+) cardiovascular progenitors.
Assuntos
Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Multipotentes/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Citometria de Fluxo , Heterozigoto , Humanos , Imuno-Histoquímica , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Multipotentes/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Reação em Cadeia da Polimerase , Fatores de TranscriçãoRESUMO
Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1(+) precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1(+) cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1(+)/Nkx2.5(+)/flk1(+) defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1(+) cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.
Assuntos
Células-Tronco Embrionárias/fisiologia , Células Endoteliais/citologia , Proteínas de Homeodomínio/genética , Células-Tronco Multipotentes/fisiologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos de Músculo Liso/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem da Célula , Células Clonais , Coração/embriologia , Heterozigoto , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Endogâmicos , Fatores de TranscriçãoRESUMO
Bearing in mind the limited success of available treatment modalities for the therapy of multidrug-resistant tumor cells, alternative and complementary strategies need to be developed. It is known that the transcriptional activation of genes, such as MDR1 and MRP1, which play a major role in the development of a multidrug-resistant phenotype in tumor cells, involves the Y-box protein YB-1. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance. In addition, it has been reported previously that YB-1 is an important factor in adenoviral replication because it activates transcription from the adenoviral E2-late promoter. Here, we report that an oncolytic adenovirus, named Xvir03, expressing the viral proteins E1B55k and E4orf6, leads to nuclear translocation of YB-1 and in consequence to viral replication and cell lysis in vitro and in vivo. Moreover, we show that Xvir03 down-regulates the expression of MDR1 and MRP1, indicating that recruiting YB-1 to the adenoviral E2-late promoter for viral replication is responsible for this effect. Thus, nuclear translocation of YB-1 by Xvir03 leads to resensitization of tumor cells to cytotoxic drugs. These data reveal a link between chemotherapy and virotherapy based on the cellular transcription factor YB-1 and provide the basis for formulating a model for a novel combined therapy regimen named Mutually Synergistic Therapy.
Assuntos
Adenoviridae/fisiologia , Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/metabolismo , Genes MDR/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Terapia Viral Oncolítica/métodos , Neoplasias da Próstata/terapia , Adenoviridae/genética , Proteínas E2 de Adenovirus/genética , Animais , Núcleo Celular/metabolismo , Terapia Combinada , Daunorrubicina/farmacologia , Docetaxel , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Nucleares , Regiões Promotoras Genéticas , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/virologia , Taxoides/farmacologia , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína 1 de Ligação a Y-BoxRESUMO
Conditionally replicating adenoviruses are a promising new modality for the treatment of cancer. However, early clinical trials demonstrate that the efficacy of current vectors is limited. Interestingly, DNA replication and production of viral particles do not always correlate with virus-mediated cell lysis and virus release depending on the vector utilized for infection. However, we have previously reported that nuclear accumulation of the human transcription factor YB-1 by regulating the adenoviral E2 late promoter facilitates viral DNA replication of E1-deleted adenovirus vectors which are widely used for cancer gene therapy. Here we report the promotion of virus-mediated cell killing as a new function of the human transcription factor YB-1. In contrast to the E1A-deleted vector dl312 the first-generation adenovirus vector AdYB-1, which overexpresses YB-1 under cytomegalovirus promoter control, led to necrosis-like cell death, virus production, and viral release after infection of A549 and U2OS tumor cell lines. Our data suggest that the integration of YB-1 in oncolytic adenoviruses is a promising strategy for developing oncolytic vectors with enhanced potency against different malignancies.
Assuntos
Adenoviridae/fisiologia , Proteínas de Ligação a DNA/genética , Vetores Genéticos/fisiologia , Terapia Viral Oncolítica , Adenoviridae/genética , Proteínas E1B de Adenovirus/fisiologia , Proteínas E3 de Adenovirus/análise , Apoptose , Núcleo Celular/virologia , Efeito Citopatogênico Viral , Proteínas de Ligação a DNA/fisiologia , Vetores Genéticos/genética , Humanos , Proteínas Nucleares , Recombinação Genética , Vírion/fisiologia , Replicação Viral , Proteína 1 de Ligação a Y-BoxRESUMO
Resistance to radiation and chemotherapy remains an obstacle to the treatment of brain tumors. We have demonstrated that the replication-deficient adenovirus d1520, which lacks the E1A 13S protein, replicates efficiently and exhibits oncolytic potential in multidrug-resistant cells with nuclear localization of the human transcription factor YB-1. However, besides others, key factors regarding oncolytic virotherapy are limited tumor transduction rate and low replication efficiency. The objective of this study was to determine whether the chemotherapeutic agent irinotecan, by enhancing nuclear localization of YB-1, and the histone deacetylase inhibitor trichostatin A, by upregulating coxsackievirus-adenovirus receptor (CAR) expression, could augment replication of and cell lysis by adenovirus dl520 in glioblastomas in vitro. We found that trichostatin A upregulated CAR expression and that irinotecan caused increased nuclear localization of YB-1 in both glioblastoma cell lines. Irinotecan alone, and trichostatin A alone, enhanced replication of and cell lysis by dl520. Importantly, when combining both agents, the replication efficiency (maximum, 27-fold) and induction of cytopathic effect (maximum, 3.8-fold) of dl520 were further augmented significantly. These results support the hypothesis that the enhanced oncolytic effect of dl520, after incubation with chemotherapeutic agents, is mediated by an increased accumulation of YB-1 in the nucleus (due to irinotecan) and by upregulation of CAR (due to trichostatin A). Thus, therapy combining virotherapy, chemotherapy, and histone deacetylase inhibitor treatment is a novel approach to enhance the oncolytic efficacy of dl520.
Assuntos
Neoplasias Encefálicas/terapia , Inibidores Enzimáticos/uso terapêutico , Glioblastoma/terapia , Inibidores de Histona Desacetilases , Vírus Oncolíticos/fisiologia , Adenoviridae/efeitos dos fármacos , Adenoviridae/fisiologia , Antineoplásicos Fitogênicos/farmacologia , Southern Blotting/métodos , Neoplasias Encefálicas/patologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Camptotecina/uso terapêutico , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Deleção de Genes , Expressão Gênica , Violeta Genciana , Glioblastoma/patologia , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Imuno-Histoquímica/métodos , Irinotecano , Vírus Oncolíticos/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Receptores Virais/análise , Receptores Virais/metabolismo , Células Tumorais CultivadasRESUMO
Resistance to chemotherapy is responsible for a failure of current treatment regimens in cancer patients. We have reported previously that the Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug resistant-tumor phenotype. YB-1 predicts drug resistance and patient outcome in breast cancer. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance. In drug-resistant cancer cells and in adenovirus-infected cells YB-1 is found in the nucleus. Nuclear accumulation of YB-1 in adenovirus-infected cells is a function of the E1 region, and we have shown that YB-1 facilitates adenovirus replication. Here we report that E1A-deleted or mutant adenovirus vectors, such as Ad312 and Ad520, replicate efficiently in multidrug-resistant (MDR) cancer cells and induce an adenovirus cytopathic effect resulting in host cell lysis. Thus, replication-defective adenoviruses are a previously unrecognized vector system for a selective elimination of MDR cancer cells. Our work forms the basis for the development of novel oncolytic adenovirus vectors for the treatment of MDR malignant diseases in the clinical setting.