RESUMO
Individuals are exposed to a wide arrays of hazardous chemicals on a daily basis through various routes, many of which have not undergone comprehensive toxicity assessments. While traditional developmental toxicity tests involving pregnant animals are known for their reliability, they are also associated with high costs and time requirements. Consequently, there is an urgent demand for alternative, cost-efficient, and rapid in vitro testing methods. This study aims to address the challenges related to automating and streamlining the screening of early developmental toxicity of chemicals by introducing a mouse embryoid body test (EBT) model in a 384-ultra low attachment well format. Embryoid bodies (EBs) generated in this format were characterized by a spontaneous differentiation trajectory into cardiac mesoderm by as analyzed by RNA-seq. Assessing prediction accuracy using reference compounds suggested in the ICH S5(R3) guideline and prior studies resulted in the establishment of the acceptance criteria and applicability domain of the EBT model. The results indicated an 84.38% accuracy in predicting the developmental toxicity of 23 positive and 9 negative reference compounds, with an optimized cutoff threshold of 750 µM. Overall, the developed EBT model presents a promising approach for more rapid, high-throughput chemical screening, thereby facilitating well-informed decision-making in environmental management and safety assessments.
RESUMO
Current cerebral organoid technology provides excellent in vitro models mimicking the structure and function of the developing human brain, which enables studies on normal and pathological brain; however, further improvements are necessary to overcome the problems of immaturity and dearth of non-parenchymal cells. Vascularization is one of the major challenges for recapitulating processes in the developing human brain. Here, we examined the formation of blood vessel-like structures in cerebral organoids induced by vascular endothelial growth factor (VEGF) in vitro. The results indicated that VEGF enhanced differentiation of vascular endothelial cells (ECs) without reducing neuronal markers in the embryonic bodies (EBs), which then successfully developed into cerebral organoids with open-circle vascular structures expressing an EC marker, CD31, and a tight junction marker, claudin-5, characteristic of the blood-brain barrier (BBB). Further treatment with VEGF and Wnt7a promoted the formation of the outer lining consisting of pericyte-like cells, which surrounded the vascular tubes. RNA sequencing revealed that VEGF upregulated genes associated with tube formation, vasculogenesis, and the BBB; it also changed the expression of genes involved in brain embryogenesis, suggesting a role of VEGF in neuronal development. These results indicate that VEGF treatment can be used to generate vessel-like structures with mature BBB characteristics in cerebral organoids in vitro.
Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Organoides/crescimento & desenvolvimento , Células Cultivadas , Córtex Cerebral/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Organoides/metabolismoRESUMO
BACKGROUND: Necroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied. METHODS: We performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown. RESULTS: Ilk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF-ß signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals' injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice. CONCLUSIONS: The study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.
Assuntos
Túbulos Renais Coletores/patologia , Rim/patologia , Necroptose , Nefrite/etiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Células Cultivadas , Fibrose , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Aquaporin-2 (AQP2) is a water channel protein expressed in principal cells (PCs) of the kidney collecting ducts (CDs) and plays a critical role in mediating water reabsorption and urine concentration. AQP2 undergoes both regulated trafficking mediated by vasopressin (VP) and constitutive recycling, which is independent of VP. For both pathways, actin cytoskeletal dynamics is a key determinant of AQP2 trafficking. We report here that manganese chloride (MnCl2) is a novel and potent regulator of AQP2 trafficking in cultured cells and in the kidney. MnCl2 treatment promoted internalization and intracellular accumulation of AQP2. The effect of MnCl2 on the intracellular accumulation of AQP2 was associated with activation of RhoA and actin polymerization without modification of AQP2 phosphorylation. Although the level of total and phosphorylated AQP2 did not change, MnCl2 treatment impeded VP-induced phosphorylation of AQP2 at its serine-256, -264, and -269 residues and dephosphorylation at serine 261. In addition, MnCl2 significantly promoted F-actin polymerization along with downregulation of RhoA activity and prevented VP-induced membrane accumulation of AQP2. Finally, MnCl2 treatment in mice resulted in significant polyuria and reduced urinary concentration, likely due to intracellular relocation of AQP2 in the PCs of kidney CDs. More importantly, the reduced urinary concentration caused by MnCl2 treatment in animals was not corrected by VP. In summary, our study identified a novel effect of MnCl2 on AQP2 trafficking through modifying RhoA activity and actin polymerization and uncovered its potent impact on water diuresis in vivo.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Actinas/metabolismo , Aquaporina 2/metabolismo , Cloretos/toxicidade , Capacidade de Concentração Renal/efeitos dos fármacos , Túbulos Renais Coletores/efeitos dos fármacos , Poliúria/induzido quimicamente , Citoesqueleto de Actina/metabolismo , Animais , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/fisiopatologia , Células LLC-PK1 , Masculino , Compostos de Manganês , Camundongos Endogâmicos C57BL , Fosforilação , Polimerização , Poliúria/metabolismo , Poliúria/fisiopatologia , Transporte Proteico , Transdução de Sinais/efeitos dos fármacos , Suínos , Vasopressinas/farmacologia , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
During the past decade, microRNAs have continuously been suggested as a promising therapeutic tool due to their beneficial effects, such as their multi-targets and multi-functions in pathologic conditions. As a pathologic phenotype is generally regulated by multiple signaling pathways, in this study we identified a microRNA regulating multiple target genes within cardiac hypertrophic signaling pathways. microRNA-133a is known to play a crucial role in cardiac hypertrophy. However, the role of microRNA-133a, which may regulate several signaling pathways in norepinephrine-induced cardiac hypertrophy via multi-targeting, has not been investigated. In the current study, we showed that microRNA-133a can protect cardiomyocyte hypertrophy against norepinephrine stimulation in neonatal rat ventricular cardiomyocytes via new targets, PKCδ and Gq, all of which are related to downstream signaling pathways of the α1-adrenergic receptor. Taken together, these results suggest the advantages of the therapeutic use of microRNAs as an effective potential drug regulating multiple signaling pathways under pathologic conditions.
Assuntos
Cardiomegalia/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Quinase C-delta/metabolismo , Transdução de Sinais , Animais , Cardiomegalia/patologia , Miócitos Cardíacos/patologia , Ratos , Receptor A1 de Adenosina/metabolismoRESUMO
Dynamin-related protein-1 (Drp1) plays a critical role in mitochondrial fission which allows cell proliferation and Mdivi-1, a specific small molecule Drp1 inhibitor, is revealed to attenuate proliferation. However, few molecular mechanisms-related to Drp1 under stimulus for restenosis or atherosclerosis have been investigated in vascular smooth muscle cells (vSMCs). Therefore, we hypothesized that Drp1 inhibition can prevent vascular restenosis and investigated its regulatory mechanism. Angiotensin II (Ang II) or hydrogen peroxide (H2 O2 )-induced proliferation and migration in SMCs were attenuated by down-regulation of Drp1 Ser 616 phosphorylation, which was demonstrated by in vitro assays for migration and proliferation. Excessive amounts of ROS production and changes in mitochondrial membrane potential were prevented by Drp1 inhibition under Ang II and H2 O2 . Under the Ang II stimulation, activated Drp1 interacted with PKCδ and then activated MEK1/2-ERK1/2 signaling cascade and MMP2, but not MMP9. Furthermore, in ex vivo aortic ring assay, inhibition of the Drp1 had significant anti-proliferative and -migration effects for vSMCs. A formation of vascular neointima in response to a rat carotid artery balloon injury was prevented by Drp1 inhibition, which shows a beneficial effect of Drp1 regulation in the pathologic vascular condition. Drp1-mediated SMC proliferation and migration can be prevented by mitochondrial division inhibitor (Mdivi-1) in in vitro, ex vivo and in vivo, and these results suggest the possibility that Drp1 can be a new therapeutic target for restenosis or atherosclerosis.
Assuntos
Reestenose Coronária/metabolismo , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Proteína Quinase C-delta/metabolismo , Angiotensina II/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Peróxido de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neointima/metabolismo , Fosforilação , RatosRESUMO
The proliferation and migration of smooth muscle cells (SMCs) are considered to be key steps in the progression of atherosclerosis and restenosis. Certain stimuli, such as, interleukin-3 (IL-3) are known to stimulate proliferation and migration in vascular diseases. Meanwhile, microRNAs (miRs) have been revealed as critical modulators of various diseases in which miR-29b is known to regulate cell growth by targeting Mcl-1 and MMP2. However, roles of miR-29b in vascular smooth muscle cells remain almost unknown. We hypothesized that miR-29b may control the proliferation and migration processes induced by IL-3 stimulation by inhibiting its own specific targets in SMCs. MiR-29b significantly suppressed the proliferation and migration of SMCs through the inhibition of the signaling pathway related to Mcl-1 and MMP2. We also found that miR-29b expression levels significantly declined in balloon-injured rat carotid arteries and that the overexpression of miR-29b by local oligonucleotide delivery can inhibit neointimal formation. Consistent with the critical role of miR-29b in vitro, we observed down-regulated expression levels of Mcl-1 and MMP2 from the neointimal region. These results indicate that miR-29b suppressed the proliferation and migration of SMCs, possibly through the inhibition of Mcl-1 and MMP2, and suggest that miR-29b may serve as a useful therapeutic tool to treat cardiovascular diseases such as, atherosclerosis and restenosis.
Assuntos
Lesões das Artérias Carótidas/genética , Interleucina-3/farmacologia , MicroRNAs/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Neointima/genética , Animais , Lesões das Artérias Carótidas/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Metaloproteinase 2 da Matriz/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Miócitos de Músculo Liso/citologia , Ratos , Ratos Sprague-DawleyRESUMO
Under distinct pathological heart conditions, the expression of a single miRNA can display completely opposite patterns. However, the mechanism underlying the bidirectional regulation of a single miRNA and the clinical implications of this regulation remain largely unknown. To address this issue, we examined the regulation of miR-1, one of the most abundant miRNAs in the heart, during cardiac hypertrophy and ischemia/reperfusion (I/R). Our data indicated that different magnitudes and chronicities of ROS levels in cardiomyocytes resulted in differential expression of miR-1, subsequently altering the expression of myocardin. In animal models, the administration of a miR-1 mimic attenuated cardiac hypertrophy by suppressing the transverse aortic constriction-induced increase in myocardin expression, whereas the administration of anti-miR-1 ameliorated I/R-induced cardiac apoptosis and deterioration of heart function. Our findings indicated that a pathologic stimulus such as ROS can bidirectionally alter the expression of miRNA to contribute to the development of pathological conditions exhibiting distinct phenotypes and that the meticulous adjustment of the pathological miRNA levels is required to improve clinical outcomes.
Assuntos
Cardiomegalia/metabolismo , Insuficiência Cardíaca/metabolismo , MicroRNAs/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transativadores/metabolismo , Animais , Apoptose , Cardiomegalia/genética , Células Cultivadas , Regulação da Expressão Gênica/genética , Insuficiência Cardíaca/genética , MicroRNAs/genética , Proteínas Nucleares/genética , Ratos , Ratos Sprague-Dawley , Transativadores/genéticaRESUMO
Myocardial Ca(2+) overload induced by ischemia/reperfusion (I/R) is a major element of myocardial dysfunction in heart failure. Phospholipase C (PLC) plays important roles in the regulation of the phosphoinositol pathway and Ca(2+) homeostasis in various types of cells. Here, we investigated the protective role of PLCδ1 against myocardial I/R injury through the regulation of Ca(2+) homeostasis. To investigate its role, PLCδ1 was fused to Hph1, a cell-permeable protein transduction domain (PTD), and treated into rat neonatal cardiomyocytes and rat hearts under respective hypoxia-reoxygenation (H/R) and ischemia-reperfusion conditions. Treatment with Hph1-PLCδ1 significantly inhibited intracellular Ca(2+) overload, reactive oxygen species generation, mitochondrial permeability transition pore opening, and mitochondrial membrane potential elevation in H/R neonatal cardiomyocytes, resulting in the inhibition of apoptosis. Intravenous injections of Hph1-PLCδ1 in rats with I/R-injured myocardium caused significant reductions in infarct size and apoptosis and also improved systolic and diastolic cardiac functioning. Furthermore, a small ions profile obtained using time-of-flight secondary ion mass spectrometry showed that treatment with Hph1-PLCδ1 leads to significant recovery of calcium-related ions toward normal levels in I/R-injured myocardium. These results suggest that Hph1-PLCδ1 may manifest as a promising cardioprotective drug due to its inhibition of the mitochondrial apoptotic pathway in cells suffering from I/R injury.
Assuntos
Cálcio/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Fosfolipase C gama/administração & dosagem , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Injeções , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagemRESUMO
BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.
Assuntos
Apoptose , Glioma/patologia , Hexoquinase/metabolismo , Peróxido de Hidrogênio/toxicidade , Células-Tronco Mesenquimais/patologia , MicroRNAs/metabolismo , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Glioma/metabolismo , Humanos , Peróxido de Hidrogênio/administração & dosagem , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , MicroRNAs/antagonistas & inibidores , Mitocôndrias/enzimologia , Invasividade Neoplásica , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase em Tempo Real , Semaforinas/genética , Semaforinas/metabolismoRESUMO
Osteoarthritis (OA) is a chronic, progressive, and irreversible degenerative joint disease. Conventional OA treatments often result in complications such as pain and limited activity. However, transplantation of mesenchymal stem cells (MSCs) has several beneficial effects such as paracrine effects, anti-inflammatory activity, and immunomodulatory capacity. In addition, MSCs can be differentiated into several cell types, including chondrocytes, osteocytes, endothelia, and adipocytes. Thus, transplantation of MSCs is a suggested therapeutic tool for treatment of OA. However, transplanted naïve MSCs can cause problems such as heterogeneous populations including differentiated MSCs and undifferentiated cells. To overcome this problem, new strategies for inducing differentiation of MSCs are needed. One possibility is the application of microRNA (miRNA) and small molecules, which regulate multiple molecular pathways and cellular processes such as differentiation. Here, we provide insight into possible strategies for cartilage regeneration by transplantation of differentiated MSCs to treat OA patients.
Assuntos
Condrogênese , Transplante de Células-Tronco Mesenquimais/métodos , Osteoartrite/terapia , Animais , Humanos , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genéticaRESUMO
Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of disease progression in atherosclerosis. Cell proliferation is regulated by cell cycle regulatory proteins. MicroRNAs (miR) have been reported to act as important gene regulators and play essential roles in the proliferation and migration of VSMCs in a cardiovascular disease. However, the roles and mechanisms of miRs in VSMCs and neointimal formation are far from being fully understood. In this study, cell cycle-specific cyclin D1 was found to be a potential target of miR-365 by direct binding. Through an in vitro experiment, we showed that exogenous miR-365 overexpression reduced VSMC proliferation and proliferating cell nuclear antigen (PCNA) expression, while miR-365 was observed to block G1/S transition in platelet-derived growth factor-bb (PDGF-bb)-induced VSMCs. In addition, the proliferation of VSMCs by various stimuli, including PDGF-bb, angiotensin II (Ang II), and serum, led to the downregulation of miR-365 expression levels. The expression of miR-365 was confirmed in balloon-injured carotid arteries. Taken together, our results suggest an anti-proliferative role for miR-365 in VSMC proliferation, at least partly via modulating the expression of cyclin D1. Therefore, miR-365 may influence neointimal formation in atherosclerosis patients.
Assuntos
Aterosclerose/patologia , Ciclina D1/biossíntese , MicroRNAs/genética , Músculo Liso Vascular/crescimento & desenvolvimento , Neointima/genética , Angiotensina II/farmacologia , Animais , Becaplermina , Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/metabolismo , Divisão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Regulação para Baixo , MicroRNAs/biossíntese , Músculo Liso Vascular/citologia , Antígeno Nuclear de Célula em Proliferação/biossíntese , Ligação Proteica , Proteínas Proto-Oncogênicas c-sis/farmacologia , Proteínas de Ligação a RNA , Ratos , Pontos de Checagem da Fase S do Ciclo Celular/genéticaRESUMO
Despite the safety and feasibility of mesenchymal stem cell (MSC) therapy, an optimal cell type has not yet emerged in terms of electromechanical integration in infarcted myocardium. We found that poor to moderate survival benefits of MSC-implanted rats were caused by incomplete electromechanical integration induced by tissue heterogeneity between myocytes and engrafted MSCs in the infarcted myocardium. Here, we report the development of cardiogenic cells from rat MSCs activated by phorbol myristate acetate, a PKC activator, that exhibited high expressions of cardiac-specific markers and Ca(2+) homeostasis-related proteins and showed adrenergic receptor signaling by norepinephrine. Histological analysis showed high connexin 43 coupling, few inflammatory cells, and low fibrotic markers in myocardium implanted with these phorbol myristate acetate-activated MSCs. Infarct hearts implanted with these cells exhibited restoration of conduction velocity through decreased tissue heterogeneity and improved myocardial contractility. These findings have major implications for the development of better cell types for electromechanical integration of cell-based treatment for infarcted myocardium.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Mesenquimais/metabolismo , Contração Miocárdica/fisiologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/fisiologia , Análise de Variância , Animais , Western Blotting , Conexina 43/metabolismo , Citocinas/imunologia , Eletrocardiografia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Isoproterenol/farmacologia , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Norepinefrina/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/metabolismoRESUMO
Background: Korean red ginseng (KRG) is a product from ginseng roots, which is enriched with ginsenosides and has been utilized for a long time as an adaptogen to alleviate various physiological or disease conditions. While KRG is generally considered safe, conducting a thorough toxicological assessment of the spray-dried powder G1899 during the juvenile period is essential to establish its safety profile. This study aimed to assess the safety of G1899 during the juvenile period using Sprague-Dawley rats. Methods: Two studies were conducted separately: a juvenile toxicity study and a uterotrophic bioassay. To assess the potential toxicity at systemic, postnatal developmental, and reproductive levels, G1899 was orally gavaged once a day in post-weaning juvenile Sprague-Dawley (SD) rats at 0, 1250, 2500, or 5000 mg/kg/day. Estrogenicity was assessed by orally gavaging G1899 in immature female SD rats at 0, 2500, or 5000 mg/kg/day on postnatal days (PND) 19-21, followed by a uterotrophic bioassay. These studies were conducted in accordance with the Good Laboratory Practice (GLP) regulations and regulatory test guidelines. Results: Regarding juvenile toxicity, no abnormalities related to the G1899 treatment were observed in any group during the experiment. Moreover, no uterotrophic responses were observed in the dosed female group. Based on these results, the no observed adverse effect level (NOAEL) of G1899 was determined to be at least 5000 mg/kg/day for general systemic function, developmental/reproductive function, and estrogenic activity. Conclusion: Our results suggest that G1899 is not toxic to juveniles at doses of up to 5000 mg/kg/day.
RESUMO
A change in intracellular free calcium (Ca(2+)) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca(2+) signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca(2+)-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H2O2-mediated Ca(2+) overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca(2+) overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca(2+)-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca(2+) overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Peróxido de Hidrogênio/metabolismo , MicroRNAs/farmacologia , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Telomeric repeat binding factor 1 (TRF1) plays an essential role in maintaining telomere length. Here, we established TRF1-knockout human pluripotent stem cells (hPSCs; hTRF1-KO) using the CRISPR/Cas9 technology. The hTRF1-KO cell lines expressed pluripotency markers and demonstrated a normal karyotype (46, XX) and DNA profile. In addition, hTRF1-KOcells spontaneously differentiated into all three germ layers in vitro. Thus, these cell lines could be useful models in various research fields.
Assuntos
Células-Tronco Embrionárias Humanas , Telômero , Humanos , Telômero/genética , Telômero/metabolismo , Sistemas CRISPR-Cas/genética , Células-Tronco Embrionárias Humanas/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Linhagem CelularRESUMO
To achieve effective regeneration of injured myocardium, it is important to find physiological way of improving the cardiogenic differentiation of stem cells. Previous studies demonstrated that cardiomyocytes from bone marrow-derived mesenchymal stem cells (BMSCs) activated with phorbolmyristate acetate (PMA), a protein kinase C (PKC) activator, restore electromechanical function in infarcted rat hearts. In this study, we investigated the effect of PMA on cardiogenic differentiation of adipose-derived MSCs (ASCs) for clinical applications. To confirm the effect of PMA, ASCs treated with 1µM PMA were grown for nine days. The expression of cardiac-specific markers (cardiac troponin T, myosin light chain, myosin heavy chain) in PMA-treated MSCs was demonstrated by immunocytochemistry. Alhough few α(1A) receptors exist in ASCs, α(1)-adrenergic receptor subtypes were preferentially expressed in PMA-treated ASCs. Moreover, expression of the ß-adrenergic and muscarinic receptors increased in PMA-treated ASCs compared to normal cells. The mRNA levels of Ca(2+)-related factors (SERCA 2a; sarcoplasmic reticulum Ca(2+)-ATPase, LTCC; L-type Ca(2+) channel) in treated ASCs were similar to the levels in cardiomyocytes. Following the transplantation of chemically activated cardiogenic ASCs into infarcted myocardium, histological analysis showed that infarct size, interstitial fibrosis, and apoptotic index were markedly decreased and cardiac function was restored. In conclusion, PMA might induce the cardiogenic differentiation of human ASCs as well as BMSCs. This result suggests successful use of human ASCs in cardiac regeneration therapy.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Miócitos Cardíacos/citologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Isquemia Miocárdica/terapia , Miócitos Cardíacos/transplante , Ratos , Ratos Sprague-Dawley , Regeneração/genéticaRESUMO
Myocardial ischemia is the major cause of morbidity and mortality due to cardiovascular diseases. This disease is a severe stress condition that causes extensive biochemical changes which trigger cardiac cell death. Stress conditions such as deprivation of glucose and oxygen activate the endoplasmic reticulum in the cytoplasm of cells, including cardiomyocytes, to generate and propagate apoptotic signals in response to these conditions. microRNAs (miRNAs) are a class of small non-coding RNAs that mediate posttranscriptional gene silencing. The miRNAs play important roles in regulating cardiac physiological and pathological events such as hypertrophy, apoptosis, and heart failure. However, the roles of miRNAs in reactive oxygen species (ROS)-mediated injury on cardiomyocytes are uncertain. In this study, we identified at the apoptotic concentration of H(2)O(2), miR-26a expression was increased. To determine the potential roles of miR-26a in H(2)O(2)-mediated cardiac apoptosis, miR-26a expression was regulated by a miR-26a or an anti-miR-26a. Overexpression of miR-26a increased apoptosis as determined by upregulation of Annexin V/PI positive cell population, caspase-3 activity and expression of pro-apoptotic signal molecules, whereas inhibition of miR-26a reduced apoptosis. We identified GSK3B as a direct downstream target of miR-26a. Furthermore, miR-26a attenuated viability and increased caspase-3 activity in normal cardiomyocytes. This study demonstrates that miR-26a promotes ROS-induced apoptosis in cardiomyocytes. Thus, miR-26a affects ROS-mediated gene regulation and cellular injury response.
Assuntos
Apoptose/genética , Regulação Enzimológica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , MicroRNAs/biossíntese , Miócitos Cardíacos/fisiologia , Animais , Hipóxia Celular , Células Cultivadas , Glicogênio Sintase Quinase 3 beta , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
VSMC (vascular smooth muscle cell) proliferation contributes significantly to intimal thickening in atherosclerosis, restenosis and venous bypass graft diseases. Ang II (angiotensin II) has been implicated in VSMC proliferation though the activation of multiple growth-promoting signals. Although TZDs (thiazolidinediones) can inhibit VSMC proliferation and reduce Ang II-induced fibrosis, the mechanism underlying the inhibition of VSMC proliferation and fibrosis needs elucidation. We have used primary cultured rat aortic VSMCs and specific antibodies to investigate the inhibitory mechanism of rosiglitazone on Ang II-induced VSMC proliferation. Rosiglitazone treatment significantly inhibited Ang II-induced rat aortic VSMC proliferation in a dose-dependent manner. Western blot analysis showed that rosiglitazone significantly lowered phosphorylated ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt (also known as protein kinase B), mTOR (mammalian target of rapamycin), p70S6K (70 kDa S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E-binding protein) levels in Ang II-treated VSMCs. In addition, PPAR-γ (peroxisome-proliferator-activated receptor γ) mRNA increased significantly and CTGF (connective tissue growth factor), Fn (fibronectin) and Col III (collagen III) levels decreased significantly. The results demonstrate that the rosiglitazone directly inhibits the pro-atherosclerotic effect of Ang II on rat aortic VSMCs. It also attenuates Ang II-induced ECM (extracellular matrix) molecules and CTGF production in rat aortic VSMCs, reducing fibrosis. Importantly, PPAR-γ activation mediates these effects, in part, through the mTOR-p70S6K and -4EBP1 system.
Assuntos
Angiotensina II/metabolismo , Proliferação de Células , Músculo Liso Vascular/citologia , Serina-Treonina Quinases TOR/metabolismo , Tiazolidinedionas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Ratos , Rosiglitazona , Transdução de Sinais/efeitos dos fármacosRESUMO
Pathological stress including myocardial infarction and hypertension causes a negative effect on calcium regulation and homeostasis. Nevertheless, few studies reveal that Ca(2+) regulatory genes are related to pathological status in cardiomyocytes under early hypoxia. To determine the alteration of Ca(2+)-related gene in hypoxic myocytes, primary neonatal rat ventricular cardiomyocytes (NRVCMs) was isolated. Survival of hypoxic NRVCMs was significantly decreased in 6 h. We confirmed an increase of reactive oxygen species (ROS) generation and Ca(2+) overload in hypoxic NRVCMs by using 2',7'-dichlorodihydro-fluorescein diacetate (H2DCFDA) and FACS analysis. Furthermore, survival/apoptotic signals were also regulated in same condition. The expression profiles of more than 30,000 genes from NRVCMs that were subjected to early hypoxia revealed 630 genes that were differentially regulated. The intracellular Na(+) overload and Ca(2+) handling genes with at least two-fold changes were confirmed. The levels of Ca(2+)-handling proteins (calsequestrin, calmodulin, and calreticulin), ion channels (NCX, Na(+)-K(+)-ATPase, SERCA2a, and PLB), and stress markers (RyR2, ANP, and BNP) were significantly altered in early hypoxia. These results demonstrate that early hypoxia alters Ca(2+)-related gene expression in NRVCMs, leading to pathological status.