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1.
Nat Biomed Eng ; 5(8): 880-896, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34426676

RESUMO

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.


Assuntos
Células Endoteliais/transplante , Coração/fisiologia , Infarto do Miocárdio/terapia , Miócitos de Músculo Liso/transplante , Regeneração , Animais , Ácido Ascórbico/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Reprogramação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Junções Comunicantes/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Neovascularização Fisiológica , Transcriptoma
2.
Behav Brain Res ; 401: 113065, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33321164

RESUMO

Despite a widespread expression pattern in the central nervous system, the role of the sodium bicarbonate cotransporter NBCn1/Slc4a7 has not been investigated for locomotor activity, emotion and cognition. Here, we addressed the behavioral consequences of NBCn1 knockout and evaluated hearing and vision that are reportedly impaired in an earlier line of NBCn1 knockout mice and may contribute to behavioral changes. In a circular open field, the knockout mice traveled a shorter distance, especially in the periphery of the chamber, than wildtype littermates. The knockout mice also traveled a shorter total distance in a home cage-like open field. Rearing and grooming behaviors were reduced. The knockout and control mice displayed similar time spent and number of open and closed arms in the elevated plus maze test, indicating negligible change in anxiety. In the Morris water maze test, both groups of mice learned the location of an escape platform within comparable time on the training trials and showed similar platform identification on the probe trial. The knockout mice maintained normal visual responses in the optokinetic drum and produced evoked potentials in response to light stimuli. However, these mice failed to produce auditory evoked potentials. qPCR revealed a robust expression of an alternatively transcribed NBCn1 variant in the knockout mouse retina. These results indicate that NBCn1 deletion leads to reduced locomotor activity in mice by affecting their exploratory behaviors or emotionality. The deletion also causes hearing loss, but its effect on vision varies between different lines of knockout mice.

3.
Biochem Biophys Res Commun ; 522(1): 1-7, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31735334

RESUMO

Hepatocellular adenoma/carcinoma (HCA/HCC) is a long-term complication of the metabolic disorder glycogen storage disease type Ia (GSD-Ia) deficient in glucose-6-phosphatase-α (G6PC or G6Pase-α). We have shown previously that hepatic G6Pase-α deficiency leads to autophagy impairment, mitochondrial dysfunction, enhanced glycolysis, and augmented hexose monophosphate shunt, all of which can contribute to hepatocarcinogenesis. However, the mechanism underlying HCA/HCC development in GSD-Ia remains unclear. We now show that G6Pase-α deficiency-mediated hepatic autophagy impairment leads to sustained accumulation of an autophagy-specific substrate p62 which can activate tumor-promoting pathways including nuclear factor erythroid 2-related factor 2 (Nrf2) and mammalian target of rapamycin complex 1 (mTORC1). Consistently, the HCA/HCC lesions developed in the G6Pase-α-deficient livers display marked accumulation of p62 aggregates and phosphorylated p62 along with activation of Nrf2 and mTORC1 signaling. Furthermore, the HCA/HCC lesions exhibit activation of additional oncogenic pathways, ß-catenin and Yes-associated protein (YAP) which is implicated in autophagy impairment. Intriguingly, hepatic levels of glucose-6-phosphate and glycogen which are accumulated in the G6Pase-α-deficient livers were significantly lower in HCC than those in HCA. Conversely, compared to HCA, the HCC lesion display increased expression of many oncogenes and the M2 isoform of pyruvate kinase (PKM2), a glycolytic enzyme critical for aerobic glycolysis and tumorigenesis. Collectively, our data show that hepatic G6Pase-α-deficiency leads to persistent autophagy impairment and activation of multiple tumor-promoting pathways that contribute to HCA/HCC development in GSD-Ia.


Assuntos
Carcinoma Hepatocelular/etiologia , Doença de Depósito de Glicogênio Tipo I/complicações , Neoplasias Hepáticas/etiologia , Animais , Autofagia , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Glucose-6-Fosfatase/metabolismo , Doença de Depósito de Glicogênio Tipo I/metabolismo , Doença de Depósito de Glicogênio Tipo I/patologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais
4.
Cytotherapy ; 21(4): 433-443, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30879964

RESUMO

Critical limb ischemia, a severe manifestation of peripheral artery disease, is emerging as a major concern in aging societies worldwide. Notably, cell-based gene therapy to induce angiogenesis in ischemic tissue has been investigated as treatment. Despite many studies demonstrating the efficacy of this approach, better therapies are required to prevent serious sequelae such as claudication, amputation and other cardiovascular events. We have now established a simplified method to enhance the effects of therapeutic transgenes by selecting for and transplanting only transduced cells. Herein, mesenchymal stromal cells were transfected to co-express vascular endothelial growth factor as angiogenic factor and enhanced green fluorescent protein as marker. Transfected cells were then collected using flow cytometry based on green fluorescence and transplanted into ischemic hind limbs in mice. Compared with unsorted or untransfected cells, purified cells significantly improved blood perfusion within 21days, suggesting that transplanting only cells that overexpress vascular endothelial growth factor enhances therapeutic angiogenesis. Importantly, this approach may prove to be useful in cell-based gene therapy against a wide spectrum of diseases, simply by replacing the gene to be delivered or the cell to be transplanted.


Assuntos
Membro Posterior/irrigação sanguínea , Membro Posterior/patologia , Isquemia/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Modelos Animais de Doenças , Fibrose , Expressão Gênica , Terapia Genética , Humanos , Isquemia/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Necrose , Perfusão , Plasmídeos/metabolismo , Ratos Sprague-Dawley , Transfecção
5.
Theranostics ; 7(7): 2067-2077, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28638487

RESUMO

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) are considered a most promising option for cell-based cardiac repair. Hence, various protocols have been developed for differentiating hPSCs into CMs. Despite remarkable improvement in the generation of hPSC-CMs, without purification, these protocols can only generate mixed cell populations including undifferentiated hPSCs or non-CMs, which may elicit adverse outcomes. Therefore, one of the major challenges for clinical use of hPSC-CMs is the development of efficient isolation techniques that allow enrichment of hPSC-CMs. In this review, we will discuss diverse strategies that have been developed to enrich hPSC-CMs. We will describe major characteristics of individual hPSC-CM purification methods including their scientific principles, advantages, limitations, and needed improvements. Development of a comprehensive system which can enrich hPSC-CMs will be ultimately useful for cell therapy for diseased hearts, human cardiac disease modeling, cardiac toxicity screening, and cardiac tissue engineering.


Assuntos
Separação Celular/métodos , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular , Humanos
6.
PLoS One ; 10(12): e0144491, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26649571

RESUMO

Developments of non-viral carriers have headed toward reducing cytotoxicity, which results from the use of conventional gene carriers, and enhancing gene delivery efficiency. Cys-(d-R9)-Cys repeated reducible poly(oligo-D-arginine) (rPOA) is one of the most efficient non-viral carriers for gene therapy; however, while its efficiency has been verified in the lung and brain, it is necessary to confirm its activity in each organ or tissue since there are differences of gene carrier susceptibility to among tissue types. We therefore tested the compatibility of rPOA in cardiac tissue by in vitro or in vivo experiments and confirmed its high transfection efficiency and low cytotoxicity. Moreover, substantial regenerative effects were observed following transfection with rPOA/pVEGF expression vector complexes (79% decreased infarct size) compared to polyethyleneimine (PEI) (34% decreased infarct size) in a rat myocardial infarction (MI) model. These findings suggest that rPOA efficiently enables DNA transfection in cardiac tissue and can be used as a useful non-viral therapeutic gene carrier for gene therapy in ischemic heart disease.


Assuntos
Técnicas de Transferência de Genes , Peptídeos , Transfecção , Animais , DNA , Terapia Genética/métodos , Humanos , Infarto do Miocárdio/genética , Isquemia Miocárdica/genética , Isquemia Miocárdica/terapia , Ratos , Fator A de Crescimento do Endotélio Vascular
7.
Biochem Biophys Res Commun ; 443(3): 924-31, 2014 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-24361894

RESUMO

mNOSTRIN is the mouse ortholog of hNOSTRIN. Unlike hNOSTRIN, which is alternatively spliced to produce two isoforms (α and ß), only a single isoform of mNOSTRIN has been detected in either the nucleus or cytoplasm/membrane. Because mNOSTRIN represses its own transcription through direct binding onto its own promoter, this protein is constantly expressed in a temporally regulated pattern during differentiation of F9 embryonic carcinoma cells. In this study, we identified the specific cis-element in the mNOSTRIN regulatory region that is responsible for negative autogenous control. This element exhibits inverted dyad symmetry. Furthermore, we identified a putative bZIP motif in the middle region of mNOSTRIN, which is responsible for DNA binding, and showed that disruption of the leucine zippers abolished the DNA-binding activity of mNOSTRIN. Here, we report that a single form of mNOSTRIN functions in both the nucleus and cytoplasm/membrane. In the nucleus, mNOSTRIN acts as a transcriptional repressor by binding to the cis-element through its bZIP motif.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , DNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/química , Sítios de Ligação , Células COS , Diferenciação Celular/genética , Chlorocebus aethiops , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Sequências Repetidas Invertidas/genética , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína
8.
Biochem Biophys Res Commun ; 337(1): 75-81, 2005 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-16171775

RESUMO

The mDaIP2 protein is a mouse orthologue of human Nostrin (a regulator of eNos). The absence of eNos activity in RA-treated F9 cell implies that the protein plays somehow different role from Nostrin. In this experiment, this protein has been shown to repress the expression of its own gene, via a feedback mechanism which involves binding to the promoter region. Data from cotransfection, DNAP, mDaIP2-silenced F9 cell, and EMSA analyses clearly support the repression activity and direct binding of the protein to the promoter region. The protein contains N-terminal FCH domain and C-terminal SH3 domain. The SH3 domain is known to interact with the proline-rich domain of mDab2, while even no function has been reported about the FCH domain. Here, we report a novel function of mDaIP2 as a transcriptional repressor and suggest the possible association of the FCH domain with transcriptional regulation.


Assuntos
Regulação da Expressão Gênica , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Região 5'-Flanqueadora , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Ensaio de Desvio de Mobilidade Eletroforética , Camundongos , Óxido Nítrico Sintase/antagonistas & inibidores , Interferência de RNA , Transcrição Genética
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