RESUMO
Embryonic Stem Cells not only hold a lot of potential for use in regenerative medicine, but also provide an elegant and efficient way to study specific developmental processes and pathways in mammals when whole animal gene knock out experiments fail. We have investigated a pathway through which HDAC1 affects cardiovascular and more specifically cardiomyocyte differentiation in ES cells by controlling expression of SOX17 and BMP2 during early differentiation. This data explains current discrepancies in the role of HDAC1 in cardiovascular differentiation and sheds light into a new pathway through which ES cells determine cardiovascular cell fate.
Assuntos
Proteína Morfogenética Óssea 2/genética , Diferenciação Celular/genética , Proteínas HMGB/genética , Histona Desacetilase 1/genética , Miócitos Cardíacos/metabolismo , Fatores de Transcrição SOXF/genética , Animais , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Imunofluorescência , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas HMGB/metabolismo , Histona Desacetilase 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Miócitos Cardíacos/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXF/metabolismo , Transdução de Sinais/genética , Fatores de TempoRESUMO
Epigenetic and chromatin modifications play particularly important roles in embryonic and induced pluripotent stem cells (ESCs and iPSCs) allowing for the cells to both differentiate and dedifferentiate back to a pluripotent state. We analyzed how the loss of a key chromatin-modifying enzyme, histone deacetylase 1 (HDAC1), affects early and cardiovascular differentiation of both ESCs and iPSCs. We also investigated potential differences between these two cell types when differentiation is induced. Our data indicate an essential role for HDAC1 in deacetylating regulatory regions of key pluripotency-associated genes during early differentiation. Although HDAC1 functions primarily as a HDAC, its loss also affects DNA methylation in ESCs and iPSCs both during pluripotency and differentiation. We show that HDAC1 plays a crucial, nonredundant role in cardiomyocyte differentiation and maturation. Our data also elucidate important differences between ESCs and iPSCs, when levels of this enzyme are reduced, that affect their ability to differentiate into functional cardiomyocytes. As varying levels of chromatin-modifying enzymes are likely to exist in patient-derived iPSCs, understanding the molecular circuitry of these enzymes in ESCs and iPSCs is critical for their potential use in cardiovascular therapeutic applications
Assuntos
Diferenciação Celular , Histona Desacetilase 1/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Sinalização do Cálcio , Conexina 43/metabolismo , Metilação de DNA , Corpos Embrioides/enzimologia , Corpos Embrioides/fisiologia , Epigênese Genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Histona Desacetilase 1/deficiência , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/enzimologia , Células NIH 3T3 , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Fatores de Transcrição SOXB1/genética , Análise de Sequência de DNA , Troponina T/genética , Troponina T/metabolismoRESUMO
Cardiovascular diseases are still the leading cause of death worldwide. Despite the improvement shown in the prognosis of patients with acute MI, there remains still a significant mortality risk. Since the main underlying problem after an MI is the loss of cardiomyocytes and microvasculature, treatment strategies aimed at preserving or regenerating myocardial tissue have been examined as potential therapeutic modalities. Toward this goal, many cell types are being investigated as potent sources of cardiomyocytes for cell transplantation. The progress made toward the generation of induced Pluripotent Stem (iPS) cells hold great potential for future use in myocardial repair. We review critical aspects of these cell's potential, such as their generation, their differentiating ability, the known epigenetic mechanisms that allow for their reprogramming, maintenance of pluripotency, their cardiovascular differentiation and therapeutic potential, and the possibility of an epigenetic memory. Understanding the molecular circuitry of these cells will provide a better understanding of their potential as well as limitations in future clinical use.
Assuntos
Sistema Cardiovascular/citologia , Sistema Cardiovascular/metabolismo , Epigênese Genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , HumanosRESUMO
BACKGROUND: Inflammation plays a critical role in adverse cardiac remodeling and heart failure. Therefore, approaches geared toward inhibiting inflammation may provide therapeutic benefits. We tested the hypotheses that genetic deletion of interleukin-10 (IL-10), a potent antiinflammatory cytokine, exacerbates pressure overload-induced adverse cardiac remodeling and hypertrophy and that IL-10 therapy inhibits this pathology. METHODS AND RESULTS: Cardiac hypertrophy was induced in wild-type and IL-10 knockout mice by isoproterenol (ISO) infusion. ISO-induced left ventricular dysfunction and hypertrophic remodeling, including fibrosis and fetal gene expression, were further exaggerated in knockout mice compared with wild-type mice. Systemic recombinant mouse IL-10 administration markedly improved left ventricular function and not only inhibited but also reversed ISO-induced cardiac remodeling. Intriguingly, a very similar cardioprotective response of IL-10 was found in transverse aortic constriction-induced hypertrophy and heart failure models. In neonatal rat ventricular myocytes and H9c2 myoblasts, ISO activated nuclear factor-κB and inhibited signal transducers and activators of transcription 3 (STAT3) phosphorylation. Interestingly, IL-10 suppressed ISO-induced nuclear factor-κB activation and attenuated STAT3 inhibition. Moreover, pharmacological and genetic inhibition of STAT3 reversed the protective effects of IL-10, whereas ectopic expression of constitutively active STAT3 mimicked the IL-10 responses on the ISO effects, confirming that the IL-10-mediated inhibition of nuclear factor-κB is STAT3 dependent. CONCLUSION: Taken together, our results suggest IL-10 treatment as a potential therapeutic approach to limit the progression of pressure overload-induced adverse cardiac remodeling.
Assuntos
Cardiomegalia/tratamento farmacológico , Interleucina-10/farmacologia , Interleucina-10/uso terapêutico , NF-kappa B/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Disfunção Ventricular Esquerda/tratamento farmacológico , Remodelação Ventricular/efeitos dos fármacos , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Fibrose , Interleucina-10/genética , Isoproterenol/efeitos adversos , Isoproterenol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mioblastos Cardíacos/citologia , Mioblastos Cardíacos/efeitos dos fármacos , Mioblastos Cardíacos/metabolismo , Miocárdio/patologia , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia , Remodelação Ventricular/fisiologiaRESUMO
Interstitial cystitis/painful bladder syndrome is a chronic bladder inflammatory disease of unknown etiology that is often regarded as a neurogenic cystitis. Interstitial cystitis is associated with urothelial lesions, voiding dysfunction, and pain in the pelvic/perineal area. In this study, we used a murine neurogenic cystitis model to identify genes participating in the development of pelvic pain. Neurogenic cystitis was induced by the injection of Bartha's strain of pseudorabies virus (PRV) into the abductor caudalis dorsalis (tail base) muscle of female C57BL/6J mice. Mice infected with PRV developed progressive pelvic pain. The sacral spinal cord was harvested on postinfection days (PID) 2 and 4, and gene expression was analyzed by microarrays and confirmed by quantitative RT-PCR. On PID 2, the overall expression profile was similar to that of uninfected sacral spinal cord; by PID 4, there were substantial differences in expression of multiple functional classes of genes, especially inflammation. Analysis of pain-signaling pathways at the dorsal horn suggested that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) contributes to neurogenic cystitis pelvic pain. Consistent with this, CaMKIIδ expression exhibited a mast cell-dependent increase in the sacral spinal cord at the mRNA level, and phospho-CaMKII immunoreactivity in the dorsal horn was increased on postinfection day (PID) 4 during PRV infection. Finally, intrathecal injection of the CaMKII inhibitor KN-93 attenuated the PRV pain response. These data suggest that CaMKII plays a functional role in pelvic pain due to neurogenic cystitis.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cistite/complicações , Cistite/enzimologia , Dor Pélvica/enzimologia , Dor Pélvica/etiologia , Animais , Comportamento Animal/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Cistite/virologia , Relação Dose-Resposta a Droga , Feminino , Herpesvirus Suídeo 1 , Hiperalgesia/etiologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Injeções Espinhais , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Dor Pélvica/psicologia , Fosforilação , Células do Corno Posterior/enzimologia , RNA/biossíntese , RNA/isolamento & purificação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Medula Espinal/metabolismo , Transcrição GênicaRESUMO
RATIONALE: Although bone marrow endothelial progenitor cell (EPC)-based therapies improve the symptoms in patients with ischemic heart disease, their limited plasticity and decreased function in patients with existing heart disease limit the full benefit of EPC therapy for cardiac regenerative medicine. OBJECTIVE: We hypothesized that reprogramming mouse or human EPCs, or both, using small molecules targeting key epigenetic repressive marks would lead to a global increase in active gene transcription, induce their cardiomyogenic potential, and enhance their inherent angiogenic potential. METHOD AND RESULTS: Mouse Lin-Sca1(+)CD31(+) EPCs and human CD34(+) cells were treated with inhibitors of DNA methyltransferases (5-Azacytidine), histone deacetylases (valproic acid), and G9a histone dimethyltransferase. A 48-hour treatment led to global increase in active transcriptome, including the reactivation of pluripotency-associated and cardiomyocyte-specific mRNA expression, whereas endothelial cell-specific genes were significantly upregulated. When cultured under appropriate differentiation conditions, reprogrammed EPCs showed efficient differentiation into cardiomyocytes. Treatment with epigenetic-modifying agents show marked increase in histone acetylation on cardiomyocyte and pluripotent cell-specific gene promoters. Intramyocardial transplantation of reprogrammed mouse and human EPCs in an acute myocardial infarction mouse model showed significant improvement in ventricular functions, which was histologically supported by their de novo cardiomyocyte differentiation and increased capillary density and reduced fibrosis. Importantly, cell transplantation was safe and did not form teratomas. CONCLUSIONS: Taken together, our results suggest that epigenetically reprogrammed EPCs display a safe, more plastic phenotype and improve postinfarct cardiac repair by both neocardiomyogenesis and neovascularization.
Assuntos
Diferenciação Celular/genética , Células Endoteliais/fisiologia , Epigênese Genética/genética , Isquemia Miocárdica/genética , Miócitos Cardíacos/fisiologia , Transplante de Células-Tronco/métodos , Regulação para Cima/genética , Animais , Células Cultivadas , Células Endoteliais/patologia , Células Endoteliais/transplante , Endotélio Vascular/patologia , Endotélio Vascular/fisiologia , Endotélio Vascular/transplante , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Isquemia Miocárdica/patologia , Isquemia Miocárdica/cirurgia , Miócitos Cardíacos/patologia , Neovascularização Fisiológica/genética , Células-Tronco/patologia , Células-Tronco/fisiologia , Resultado do TratamentoRESUMO
RATIONALE: Endothelial progenitor cell (EPC) survival and function in the injured myocardium is adversely influenced by hostile microenvironment such as ischemia, hypoxia, and inflammatory response, thereby compromising full benefits of EPC-mediated myocardial repair. OBJECTIVE: We hypothesized that interleukin-10 (IL-10) modulates EPC biology leading to enhanced survival and function after transplantation in the ischemic myocardium. METHODS AND RESULTS: Myocardial infarction (MI)-induced mobilization of bone marrow EPC (Sca-1+Flk1+cells) into the circulation was significantly impaired in IL-10 knockout (KO) mice. Bone marrow transplantation to replace IL-10 KO marrow with wild-type (WT) marrow attenuated these effects. Impaired mobilization was associated with lower stromal cell-derived factor (SDF)-1 expression levels in the myocardium of KO mice. Interestingly, SDF-1 administration reversed mobilization defect in KO mice. In vitro, hypoxia-mediated increases in CXCR4 expression and cell survival were lower in IL-10-deficient EPCs. Furthermore, SDF-1-induced migration of WT EPCs was inhibited by AMD3100, an inhibitor of CXCR4. To further study the effect of IL-10 on in vivo EPC survival and engraftment into vascular structures, GFP-labeled EPC were injected intramyocardially after induction of MI, and the mice were treated with either saline or recombinant IL-10. The IL-10-treated group showed increased retention of transplanted EPCs in the myocardium and was associated with significantly reduced EPC apoptosis after MI. Interestingly, increased EPC retention and their association with the vascular structures was observed in IL-10-treated mice. Increased EPC survival and angiogenesis in the myocardium of IL-10-treated mice corroborated with improved left ventricular function, reduced infarct size, and fibrosis in the myocardium. In vitro, IL-10-induced increase in VEGF expression in WT EPC was abrogated by STAT3 inhibitor, suggesting IL-10 signals through STAT3 activation. CONCLUSIONS: Taken together, our studies demonstrate that MI-induced EPC mobilization was impaired in IL-10 KO mice and that IL-10 increases EPC survival and function possibly through activation of STAT3/VEGF signaling cascades, leading to attenuation of MI-induced left ventricular dysfunction and remodeling.
Assuntos
Células Endoteliais/patologia , Transplante de Células-Tronco Hematopoéticas , Interleucina-10/deficiência , Infarto do Miocárdio/patologia , Remodelação Ventricular/fisiologia , Animais , Células da Medula Óssea/citologia , Hipóxia Celular , Sobrevivência Celular , Quimiocina CXCL12/farmacologia , Sobrevivência de Enxerto , Coração/fisiologia , Interleucina-10/genética , Interleucina-10/farmacologia , Interleucina-10/fisiologia , Camundongos , Camundongos Knockout , Neovascularização Fisiológica , Receptores CXCR4/biossíntese , Receptores CXCR4/genética , Proteínas Recombinantes/farmacologia , Regeneração , Fator de Transcrição STAT3/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Through a multi-university and interdisciplinary project we have involved undergraduate biology and computer science research students in the functional annotation of maize genes and the analysis of their microarray expression patterns. We have created a database to house the results of our functional annotation of >4400 genes identified as being differentially regulated in the maize shoot apical meristem (SAM). This database is located at http://sam.truman.edu and is now available for public use. The undergraduate students involved in constructing this unique SAM database received hands-on training in an intellectually challenging environment, which has prepared them for graduate and professional careers in biological sciences. We describe our experiences with this project as a model for effective research-based teaching of undergraduate biology and computer science students, as well as for a rich professional development experience for faculty at predominantly undergraduate institutions.