RESUMO
Previous experiments suggest a connection between the N-alpha-acetylation of proteins and sensitivity of cells to apoptotic signals. Here, we describe a biochemical assay to detect the acetylation status of proteins and demonstrate that protein N-alpha-acetylation is regulated by the availability of acetyl-CoA. Because the antiapoptotic protein Bcl-xL is known to influence mitochondrial metabolism, we reasoned that Bcl-xL may provide a link between protein N-alpha-acetylation and apoptosis. Indeed, Bcl-xL overexpression leads to a reduction in levels of acetyl-CoA and N-alpha-acetylated proteins in the cell. This effect is independent of Bax and Bak, the known binding partners of Bcl-xL. Increasing cellular levels of acetyl-CoA by addition of acetate or citrate restores protein N-alpha-acetylation in Bcl-xL-expressing cells and confers sensitivity to apoptotic stimuli. We propose that acetyl-CoA serves as a signaling molecule that couples apoptotic sensitivity to metabolism by regulating protein N-alpha-acetylation.
Assuntos
Sobrevivência Celular , Proteínas/metabolismo , Proteína bcl-X/metabolismo , Acetilação , Animais , Apoptose , Caspase 2/metabolismo , Linhagem Celular , Embrião de Mamíferos/citologia , Técnicas de Inativação de Genes , Células HeLa , Humanos , Células Jurkat , Camundongos , Processamento de Proteína Pós-TraducionalRESUMO
Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.
Assuntos
Lisofosfolipídeos , Osteogênese , Receptores de Lisofosfolipídeos , Perda de Dente , Animais , Camundongos , Diferenciação Celular/fisiologia , Lisofosfolipídeos/metabolismo , Osteoblastos/metabolismo , Ligamento Periodontal/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Receptores de Lisofosfolipídeos/metabolismoRESUMO
Supernumerary tooth (ST) may arise from uncertain developmental abnormalities or underlying genetic causes, and the extraction at the early age is recommended. Dental pulp stem cells (DPSCs) are the valuable resource for the regeneration of tooth and related craniofacial structures. DPSCs isolated from ST (sDPSCs) have not been fully characterized despite the potential in the applications. The objectives of this study are the efficient isolation of sDPSCs and the analysis of the properties as stem cells. sDPSCs were established by hammer-cracking and separation of the intact pulp from ST. sDPSCs in the culture were examined by light microscope and flow cytometer for the morphology and the surface marker expression. sDPSCs exhibited the cellular morphology of typical mesenchymal stem cells and expressed CD44, CD73, CD90, CD105 and CD166, but not CD14, CD34 or CD45. sDPSCs showed the differentiation potential toward osteogenic, chondrogenic and adipogenic lineages. During osteogenic differentiation, the stimulation by Oncostatin M enhanced the differentiation and significantly increased the expression of genes involved in the hard tissue repair, such as BMP2, BMP4, BMP6 and RUNX2. sDPSCs can be effectively derived from ST and displays the characteristics of mesenchymal stem cells in the maintenance and the differentiation. sDPSCs satisfies the quality as DPSCs thus provide the valuable resource to the regenerative therapy.
Assuntos
Polpa Dentária/citologia , Oncostatina M/metabolismo , Osteogênese , Células-Tronco/citologia , Dente Supranumerário/metabolismo , Diferenciação Celular , Células Cultivadas , Polpa Dentária/metabolismo , Humanos , Células-Tronco/metabolismoRESUMO
In the present study, we examined the bone healing capacity of Meox2, a homeobox gene that plays essential roles in the differentiation of a range of developing tissues, and identified its putative function in palatogenesis. We applied the knocking down of Meox2 in human periodontal ligament fibroblasts to examine the osteogenic potential of Meox2. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of Meox2 knockdown for 1 and 2 weeks in bone healing processes. We examined the detailed histomorphological changes using Masson's trichrome staining and micro-computed tomography evaluation. Moreover, we observed the localization patterns of various signaling molecules, including α-SMA, CK14, IL-1ß, and MPO to examine the altered bone healing processes. Furthermore, we investigated the process of bone formation using immunohistochemistry of Osteocalcin and Runx2. On the basis of the results, we suggest that the knocking down of Meox2 via the activation of osteoblast and modulation of inflammation would be a plausible answer for bone regeneration as a gene therapy. Additionally, we propose that the purpose-dependent selection and application of developmental regulation genes are important for the functional regeneration of specific tissues and organs, where the pathological condition of tooth loss lesion would be.
Assuntos
Regeneração Óssea , Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Ligamento Periodontal/metabolismo , Perda de Dente/metabolismo , Animais , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Transdução de Sinais , Perda de Dente/genéticaRESUMO
Our previous study reported that cancer upregulated gene (CUG)2, a novel oncogene, induces both faster cell migration and anti-cancer drug resistance. We thus wonder whether CUG2 also induces stemness, a characteristic of cancer stem cells (CSCs) and further examine the molecular mechanism of this phenotype. To test that CUG2 induces stemness, we examined expression of stemness-related factors. Overexpression of CUG2 enhanced expression levels of stemness-related factors in human lung carcinoma A549 and immortalized bronchial BEAS-2B cells. Consequently, CUG2 increased cellular spherical cluster forming ability. Overexpression of CUG2 also induced tumor formation in xenotransplanted nude mice whereas transplantation of control cells failed to, implying that CUG2 possesses malignant tumorigenic potential. We paid attention to nucleophosmin (NPM1) for its known interaction with CUG2. Suppression of NPM1 hindered the CUG2-mediated stemness-like phenotypes and diminished TGF-ß transcriptional activity and signaling. TGF-ß increased stemness-like phenotypes in the control cells whereas TGF-ß inhibitor blocked induction of the phenotypes, indicating that NPM1 is required for CUG2-mediated stemness-like phenotypes through TGF-ß signaling. Furthermore, the suppression of Smad- and non-Smad-dependent TGF-ß signaling pathways also prevented CUG2 from inducing stemness-like phenotypes. Altogether, we suggest that the novel CUG2 oncogene promotes cellular transformation and stemness, mediated by nuclear NPM1 protein and TGF-ß signaling.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Células A549 , Animais , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nucleofosmina , FenótipoRESUMO
Increasing evidence suggests that circulating angiogenic cells (CACs) promote repair of ischemic tissues. Activation of formyl peptide receptor 2 (Fpr2) has been reported to stimulate repair of ischemic heart. This study was conducted to investigate the role of Fpr2 on CAC mobilization and cardiac protection in myocardial infarction (MI). WKYMVm, a strong agonist for Fpr2, was administered in a murine model of acute MI, and mobilization of CACs including endothelial progenitor cells (CD34+ Flk1+ or Sca1+ Flk1+ cells) in peripheral blood was monitored. CAC mobilization by daily injection of WKYMVm for the first 4 days after MI was as efficient as granulocyte colony-stimulating factor and provided myocardial protection from apoptosis with increased vascular density and preservation of cardiac function. Transplantation of bone marrow (BM) from green fluorescent protein mice showed that BM-derived cells homed to ischemic heart after WKYMVm treatment and contributed to tissue protection. Transplantation of BM from Fpr2 knockout mice showed that Fpr2 in BM cells is critical in mediation of WKYMVm-stimulated myocardial protection and neovascularization after MI. These results suggest that activation of Fpr2 in BM after WKYMVm treatment provides cardiac protection through mobilization of CACs after MI, which may lead to the development of a new clinical protocol for treating patients with ischemic heart conditions. Stem Cells 2017;35:654-665.
Assuntos
Células Progenitoras Endoteliais/citologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Neovascularização Fisiológica , Receptores de Formil Peptídeo/metabolismo , Regeneração , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Cardiotônicos/farmacologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Testes de Função Cardíaca , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/fisiopatologia , Neovascularização Fisiológica/efeitos dos fármacos , Oligopeptídeos/farmacologia , Regeneração/efeitos dos fármacosRESUMO
Hematopoietic and vascular development share many common features, including cell surface markers and sites of origin. Recent lineage-tracing studies have established that definitive hematopoietic stem and progenitor cells arise from vascular endothelial-cadherin(+) hemogenic endothelial cells of the aorta-gonad-mesonephros region, but the genetic programs underlying the specification of hemogenic endothelial cells remain poorly defined. Here, we discovered that Notch induction enhances hematopoietic potential and promotes the specification of hemogenic endothelium in differentiating cultures of mouse embryonic stem cells, and we identified Foxc2 as a highly upregulated transcript in the hemogenic endothelial population. Studies in zebrafish and mouse embryos revealed that Foxc2 and its orthologs are required for the proper development of definitive hematopoiesis and function downstream of Notch signaling in the hemogenic endothelium. These data establish a pathway linking Notch signaling to Foxc2 in hemogenic endothelial cells to promote definitive hematopoiesis.
Assuntos
Células-Tronco Embrionárias/citologia , Endotélio Vascular/citologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Receptor Notch1/metabolismo , Animais , Apoptose , Western Blotting , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Endotélio Vascular/metabolismo , Fatores de Transcrição Forkhead/genética , Células-Tronco Hematopoéticas/metabolismo , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Notch1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Ovarian cancer shows high mortality due to development of resistance to chemotherapy and relapse. Cancer stem cells (CSCs) have been suggested to be a major contributor in developing drug resistance and relapse in ovarian cancer. In this study, we isolated CSCs through sphere culture of A2780, SKOV3, OVCAR3 epithelial ovarian cancer cells and primary ovarian cancer cells from patients. We identified heat-stable factors secreted from ovarian CSCs stimulated migration and proliferation of CSCs. Mass spectrometry and ELISA analysis revealed that lysophosphatidic acid (LPA) was significantly elevated in CSC culture media compared with non-CSC culture media. Treatment of CSCs with LPA resulted in augmented CSC characteristics such as sphere-forming ability, resistance to anticancer drugs, tumorigenic potential in xenograft transplantation, and high expression of CSC-associated genes, including OCT4, SOX2, and aldehyde dehydrogenase 1. Treatment of CSCs with LPA receptor 1-specific inhibitors or silencing of LPA receptor 1 expression abrogated the LPA-stimulated CSC properties. Autotaxin, an LPA-producing enzyme, is highly secreted from ovarian CSCs, and pharmacological inhibition or knockdown of autotaxin markedly attenuated the LPA-producing, tumorigenic, and drug resistance potentials of CSCs. Clinicopathological analysis showed a significant survival disadvantage of patients with positive staining of autotaxin. In addition, we further identified that AKT1 activity was upregulated in ovarian CSCs through an LPA-dependent mechanism and silencing of AKT1 expression led to suppression of CSC characteristics. These results suggest that autotaxin-LPA-LPA receptor 1-AKT1 signaling axis is critical for maintaining CSC characteristics through an autocrine loop and provide a novel therapeutic target for ovarian CSCs.
Assuntos
Lisofosfolipídeos/administração & dosagem , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Diester Fosfórico Hidrolases/genética , Receptores de Ácidos Lisofosfatídicos/genética , Ataxina-1/genética , Comunicação Autócrina/efeitos dos fármacos , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
During development, the hematopoietic lineage transits through hemogenic endothelium, but the signaling pathways effecting this transition are incompletely characterized. Although the Hedgehog (Hh) pathway is hypothesized to play a role in patterning blood formation, early embryonic lethality of mice lacking Hh signaling precludes such analysis. To determine a role for Hh signaling in patterning of hemogenic endothelium, we assessed the effect of altered Hh signaling in differentiating mouse ES cells, cultured mouse embryos, and developing zebrafish embryos. In differentiating mouse ES cells and mouse yolk sac cultures, addition of Indian Hh ligand increased hematopoietic progenitors, whereas chemical inhibition of Hh signaling reduced hematopoietic progenitors without affecting primitive streak mesoderm formation. In the setting of Hh inhibition, induction of either Notch signaling or overexpression of Stem cell leukemia (Scl)/T-cell acute lymphocytic leukemia protein 1 rescued hemogenic vascular-endothelial cadherin(+) cells and hematopoietic progenitor formation. Together, our results reveal that Scl overexpression is sufficient to rescue the developmental defects caused by blocking the Hh and Notch pathways, and inform our understanding of the embryonic endothelial-to-hematopoietic transition.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Endotélio/fisiologia , Proteínas Hedgehog/metabolismo , Células-Tronco Hematopoéticas/citologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Ensaio de Unidades Formadoras de Colônias , Embrião de Mamíferos , Feminino , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/fisiologia , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Peixe-ZebraRESUMO
Periostin, an extracellular matrix protein, is expressed in injured tissues, such as the heart with myocardial infarction, and promotes angiogenesis and tissue repair. However, the molecular mechanism associated with periostin-stimulated angiogenesis and tissue repair is still unclear. In order to clarify the role of periostin in neovascularization, we examined the effect of periostin in angiogenic potentials of human endothelial colony forming cells (ECFCs) in vitro and in an ischemic limb animal model. Recombinant periostin protein stimulated the migration and tube formation of ECFCs. To identify the functional domains of periostin implicated in angiogenesis, five fragments of periostin, including four repeating FAS-1 domains and a carboxyl terminal domain, were expressed in Escherichia coli and purified to homogeneity. Of the five different domains, the first FAS-1 domain stimulated the migration and tube formation of human ECFCs as potent as the whole periostin. Chemotactic migration of ECFCs induced by the full length and the first FAS-1 domain of periostin was abrogated by blocking antibodies against ß3 and ß5 integrins. Intramuscular injection of the full length and the first FAS-1 domain of periostin into the ischemic hindlimb of mice attenuated severe limb loss and promoted blood perfusion and homing of intravenously administered ECFCs to the ischemic limb. These results suggest that the first FAS-1 domain is responsible for periostin-induced migration and angiogenesis and it can be used as a therapeutic tool for treatment of peripheral artery occlusive disease by stimulating homing of ECFCs.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Isquemia/prevenção & controle , Neovascularização Patológica/prevenção & controle , Proteínas Recombinantes/metabolismo , Indutores da Angiogênese/farmacologia , Animais , Western Blotting , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Endotélio Vascular/citologia , Imunofluorescência , Membro Posterior/irrigação sanguínea , Humanos , Injeções Intramusculares , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/genéticaRESUMO
Oct4 has been implicated in regulation of pluripotency in embryonic stem cells (ESCs) and reprogramming of somatic cells into induced pluripotent stem cells. However, the molecular mechanisms involved in Oct4-dependent regulation of pluripotency and reprogramming have not been clear. To gain insight into the mechanism of regulation of Oct4-mediated self-renewal of ESCs and reprogramming of somatic cells, we attempted to identify Oct4-binding proteins using affinity purification and mass spectrometry. We identified Reptin, a key component of ATP-dependent chromatin remodeling complexes, as an Oct4-binding protein. Depletion of endogenous Reptin using lentiviral short hairpin RNA (shRNA) led to a decrease in the number and size of alkaline phosphatase-positive colonies of mouse ESCs. In addition, shRNA-mediated silencing of Reptin resulted in decreased expression of pluripotency-specific marker genes, including Oct4, Sox2, Nanog, and SSEA-1. Results of the Oct4 reporter assay showed synergism between Oct4 and Reptin, and depletion of endogenous Reptin abolished Oct4 transcriptional activity. Results of a chromatin immunoprecipitation assay showed the overlapping interaction of Reptin and Oct4 to CR4 in the Oct4 enhancer in ESCs. Knockdown of Reptin using shRNA suppressed the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells, whereas overexpression of Reptin resulted in enhanced efficiency of induced pluripotent stem cell generation. These results strongly suggest that Reptin plays a key role in maintaining the pluripotency of ESCs and in establishing the pluripotency during reprogramming of somatic cells by regulation of Oct4-mediated gene regulation.
Assuntos
Diferenciação Celular/fisiologia , Reprogramação Celular , DNA Helicases/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Células Cultivadas , Reprogramação Celular/fisiologia , CamundongosRESUMO
Endothelial colony-forming cells (ECFCs) are recruited to the sites of ischemic injury in order to contribute to neovascularization and repair of injured tissues. However, therapeutic potential of ECFCs is limited due to low homing and engraftment efficiency of transplanted ECFCs. The G-protein-coupled formyl peptide receptor (FPR) 2 has been implicated in regulation of inflammation and angiogenesis, while the role of FPR2 in homing and engraftment of ECFCs and neovascularization in ischemic tissues has not been fully defined. This study was undertaken to investigate the effects of WKYMVm, a selective FPR2 agonist isolated by screening synthetic peptide libraries, on homing ability of ECFCs and vascular regeneration of ischemic tissues. WKYMVm stimulated chemotactic migration, angiogenesis, and proliferation ability of human ECFCs in vitro. Small interfering RNA-mediated silencing of FPR2, but not FPR3, abrogated WKYMVm-induced migration and angiogenesis of ECFCs. Intramuscular injection of WKYMVm resulted in attenuation of severe hind limb ischemia and promoted neovascularization in ischemic limb. ECFCs transplanted via tail vein into nude mice were incorporated into capillary vessels in the ischemic hind limb, resulting in augmented neovascularization and improved ischemic limb salvage. Intramuscular injection of WKYMVm promoted homing of exogenously administered ECFCs to the ischemic limb and ECFC-mediated vascular regeneration. Silencing of FPR2 expression in ECFCs resulted in abrogation of WKYMVm-induced in vivo homing of exogenously transplanted ECFCs to the ischemic limb, neovascularization, and ischemic limb salvage. These results suggest that WKYMVm promotes repair of ischemic tissues by stimulating homing of ECFCs and neovascularization via a FPR2-dependent mechanism.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Oligopeptídeos/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/transplante , Membro Posterior/patologia , Humanos , Injeções Intramusculares , Isquemia/fisiopatologia , Salvamento de Membro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oligopeptídeos/administração & dosagem , Perfusão , Receptores de Formil Peptídeo/agonistas , Receptores de Formil Peptídeo/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacosRESUMO
Diabetes is one of the most common human diseases and 15% of the 200 million diabetics worldwide suffer from diabetic wounds. Development of new therapeutic agents is needed for treatment of diabetic wounds. Wound healing is mediated by multiple steps, including inflammation, epithelialization, neoangiogenesis, and granulation. Formyl peptide receptor 2 has been known to stimulate angiogenesis, which is essential for tissue repair and cutaneous wound healing. In this study, we explored the therapeutic effects of WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH2), a synthetic peptide agonist of formyl peptide receptor 2, on cutaneous wounds in streptozotocin-induced diabetic rats. Topical application of WKYMVm onto cutaneous wounds stimulated formation of von Willebrand factor-positive capillary and α-smooth muscle actin-positive arteriole with a maximal stimulation on day 6, suggesting WKYMVm-stimulated angiogenesis. Infiltration of immune cells could be detected on early phase during wound healing and WKYMVm treatment acutely augmented infiltration of CD68-positive macrophages. In addition, reepithelialization and granulation tissue formation were accelerated by treatment with WKYMVm. These results suggest that WKYMVm has therapeutic effects on diabetic wounds by stimulating angiogenesis and infiltration of immune cells.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Oligopeptídeos/administração & dosagem , Receptores de Formil Peptídeo/agonistas , Úlcera Cutânea/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Administração Tópica , Animais , Fatores Quimiotáticos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Pele/irrigação sanguínea , Pele/efeitos dos fármacos , Pele/patologia , Úlcera Cutânea/etiologia , Úlcera Cutânea/metabolismo , Resultado do TratamentoRESUMO
Mesenchymal stem cells (MSCs) accelerate regeneration of ischemic or injured tissues by stimulation of angiogenesis through a paracrine mechanism. Tumor necrosis factor-α (TNF-α)-activated MSCs secrete pro-angiogenic cytokines, including IL-6 and IL-8. In the present study, using an ischemic hindlimb animal model, we explored the role of IL-6 and IL-8 in the paracrine stimulation of angiogenesis and tissue regeneration by TNF-α-activated MSCs. Intramuscular injection of conditioned medium derived from TNF-α-treated MSCs (TNF-α CM) into the ischemic hindlimb resulted in attenuated severe limb loss and stimulated blood perfusion and angiogenesis in the ischemic limb. Immunodepletion of IL-6 and IL-8 resulted in attenuated TNF-α CM-stimulated tissue repair, blood perfusion, and angiogenesis. In addition, TNF-α CM induced migration of human cord blood-derived endothelial progenitor cells (EPCs) through IL-6- and IL-8-dependent mechanisms in vitro. Intramuscular injection of TNF-α CM into the ischemic limb led to augmented homing of tail vein-injected EPCs into the ischemic limb in vivo and immunodepletion of IL-6 or IL-8 from TNF-α CM attenuated TNF-α CM-stimulated homing of EPCs. In addition, intramuscular injection of recombinant IL-6 and IL-8 proteins resulted in increased homing of intravenously transplanted EPCs into the ischemic limb and improved blood perfusion in vivo. These results suggest that TNF-α CM stimulates angiogenesis and tissue repair through an increase in homing of EPCs through paracrine mechanisms involving IL-6 and IL-8.
Assuntos
Movimento Celular , Meios de Cultivo Condicionados/farmacologia , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana/citologia , Isquemia/tratamento farmacológico , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Células-Tronco/citologia , Fator de Necrose Tumoral alfa/farmacologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Imunofluorescência , Membro Posterior/metabolismo , Membro Posterior/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-6/deficiência , Interleucina-6/imunologia , Interleucina-8/deficiência , Interleucina-8/imunologia , Isquemia/metabolismo , Isquemia/patologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Necrose , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , CicatrizaçãoRESUMO
Mesenchymal stem cells in the dental tissue indicate a disposition for differentiation into diverse dental lineages and contain enormous potential as the important means for regenerative medicine in dentistry. Among various dental tissues, the dental pulp contains stem cells, progenitor cells and odontoblasts for maintaining dentin homeostasis. The conventional culture of stem cells holds a limit as the living tissue constitutes the three-dimensional (3D) structure. Recent development in the organoid cultures have successfully recapitulated 3D structure and advanced to the assembling of different types. In the current study, the protocol for 3D explant culture of the human dental pulp tissue has been established by adopting the organoid culture. After isolating dental pulp from human tooth, the intact tissue was placed between two layers for Matrigel with addition of the culture medium. The reticular outgrowth of pre-odontoblast layer continued for a month and the random accumulation of dentin was observed near the end. Electron microscopy showed the cellular organization and in situ development of dentin, and immunohistochemistry exhibited the expression of odontoblast and stem cell markers in the outgrowth area. Three-dimensional explant culture of human dental pulp will provide a novel platform for understanding stem cell biology inside the tooth and developing the regenerative medicine.
RESUMO
Haematopoietic stem cell (HSC) homeostasis is tightly controlled by growth factors, signalling molecules and transcription factors. Definitive HSCs derived during embryogenesis in the aorta-gonad-mesonephros region subsequently colonize fetal and adult haematopoietic organs. To identify new modulators of HSC formation and homeostasis, a panel of biologically active compounds was screened for effects on stem cell induction in the zebrafish aorta-gonad-mesonephros region. Here, we show that chemicals that enhance prostaglandin (PG) E2 synthesis increased HSC numbers, and those that block prostaglandin synthesis decreased stem cell numbers. The cyclooxygenases responsible for PGE2 synthesis were required for HSC formation. A stable derivative of PGE2 improved kidney marrow recovery following irradiation injury in the adult zebrafish. In murine embryonic stem cell differentiation assays, PGE2 caused amplification of multipotent progenitors. Furthermore, ex vivo exposure to stabilized PGE2 enhanced spleen colony forming units at day 12 post transplant and increased the frequency of long-term repopulating HSCs present in murine bone marrow after limiting dilution competitive transplantation. The conserved role for PGE2 in the regulation of vertebrate HSC homeostasis indicates that modulation of the prostaglandin pathway may facilitate expansion of HSC number for therapeutic purposes.
Assuntos
Dinoprostona/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Vertebrados , Animais , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Dinoprostona/agonistas , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Proteínas Proto-Oncogênicas c-myb/genética , Vertebrados/embriologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
INTRODUCTION: The aim of this study was to compare the physicochemical properties and biocompatibility of various calcium silicate-based bioceramic sealers (CSBSs). METHODS: Four recently developed CSBSs, including AH Plus Bioceramic Sealer (AHB), EndoSequence BC Sealer (ESB), TotalFill BC Sealer (TTB), and Bio-C Sealer (BIC), were compared with the epoxy resin-based sealer AH Plus (AHP). Their physical properties, including flow, setting time, radiopacity, dimensional stability, and pH, were evaluated according to the International Organization for Standardization (ISO) 6876. Their cytotoxicity in human periodontal ligament fibroblast (hPDLF) was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and compared. Additionally, cell attachment to the sealer surface was analyzed using green fluorescent protein and confocal laser scanning microscopy to evaluate cell viability. Data were analyzed using a one-way analysis of variance to determine the difference between groups for categorical variables, followed by Tukey's post hoc test at the significance level of 95%. RESULTS: The flow, setting time, and radiopacity of all tested CSBSs satisfied the ISO 6876/2012 standards. Further, these CSBSs showed shrinkage after immersion in distilled water for 30 days and complied with the ISO 6876/2001 requirements. The pH values of AHB, ESB, TTB, and BIC were greater than 11, whereas AHP had a pH of 6.69 after 4 weeks. CSBS showed excellent biocompatibility compared with that of AHP (P < .05). Confocal laser scanning microscopy showed that alive hPDLFs were attached well to all the tested CSBSs but not to AHP. CONCLUSIONS: CSBSs have similar physical characteristics within the ISO standards and higher biocompatibility than epoxy resin-based sealers.
Assuntos
Resinas Epóxi , Materiais Restauradores do Canal Radicular , Humanos , Resinas Epóxi/farmacologia , Resinas Epóxi/química , Materiais Restauradores do Canal Radicular/farmacologia , Materiais Restauradores do Canal Radicular/química , Cavidade Pulpar , Teste de Materiais , Compostos de Cálcio/farmacologia , Compostos de Cálcio/química , Silicatos/farmacologia , Silicatos/químicaRESUMO
Dental pulp-derived stromal cells (DPSCs) are a crucial cell population for maintaining the tissue integrity of the pulp-dentin complex. The oxytocin receptor (OXTR), a member of the G protein-coupled receptor (GPCR) superfamily, plays versatile roles in diverse biological contexts. However, the role of OXTR in dental pulp has not yet been fully understood. Here, we demonstrate the biological functions and significance of OXTR in DPSCs through a multidisciplinary approach. Microarray data of 494 GPCR genes revealed high OXTR expression in human DPSCs (hDPSCs). Blocking OXTR activity increased the expression of osteogenic and odontogenic marker genes, promoting hDPSC differentiation. Additionally, we found that OXTR is involved in extracellular matrix (ECM) remodeling through the regulation of the gene expression related to ECM homeostasis. We further demonstrated that these genetic changes are mediated by trascriptional activity of Yes-associated protein (YAP). Based on the results, a preclinical experiment was performed using an animal model, demonstrating that the application of an OXTR inhibitor to damaged pulp induced significant hard tissue formation. These results provide new insight into the oxytocin-OXTR system in the regenerative process of pulp-dentin complex.
Assuntos
Receptores de Ocitocina , Células-Tronco , Animais , Humanos , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Proteínas/metabolismo , Matriz Extracelular , Diferenciação Celular/fisiologia , Dentina/fisiologia , Polpa Dentária , Células Cultivadas , Proliferação de CélulasRESUMO
Background/purpose: Human dental pulp stem cells (hDPSCs) possess excellent proliferative and osteogenic differentiation potentials. This study aimed to elucidate the role of lysophosphatidic acid (LPA) signaling in the proliferation and osteogenic differentiation of hDPSCs. Materials and methods: hDPSCs were treated with LPA and proliferation was measured using the cell counting kit-8 assay. Following the osteogenic differentiation of hDPSCs using osteogenic medium in the presence or absence of LPA, alkaline phosphatase (ALP) staining, ALP activity measurements, and RT-qPCR were performed to analyze the osteoblast differentiation. Small interfering RNA (siRNA)-mediated LPAR3 silencing and extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase inhibitors were used to elucidate the molecular mechanisms underlying LPA-induced proliferation and differentiation of hDPSCs. Results: LPA treatment significantly induced proliferation and osteogenic differentiation of hDPSCs. The depletion of LPAR3 expression by LPAR3-speicifc siRNA in hDPSCs diminished LPA-induced proliferation and osteogenic differentiation. The LPAR3-mediated proliferation and osteogenic differentiation of hDPSCs in response to LPA were significantly suppressed by U0126, a selective inhibitor of ERK. Conclusion: These findings suggest that LPA induces the proliferation and osteogenic differentiation of hDPSCs via LPAR3-ERK-dependent pathways.
RESUMO
Stem cells possess unique biological characteristics such as the ability to self-renew and to undergo multilineage differentiation into specialized cells. Whereas embryonic stem cells (ESC) can differentiate into all cell types of the body, somatic stem cells (SSC) are a population of stem cells located in distinct niches throughout the body that differentiate into the specific cell types of the tissue in which they reside in. SSC function mainly to restore cells as part of normal tissue homeostasis or to replenish cells that are damaged due to injury. Cancer stem-like cells (CSC) are said to be analogous to SSC in this manner where tumor growth and progression as well as metastasis are fueled by a small population of CSC that reside within the corresponding tumor. Moreover, emerging evidence indicates that CSC are inherently resistant to chemo- and radiotherapy that are often the cause of cancer relapse. Hence, major research efforts have been directed at identifying CSC populations in different cancer types and understanding their biology. Many factors are thought to regulate and maintain cell stemness, including bioactive lysophospholipids such as lysophosphatidic acid (LPA). In this review, we discuss some of the newly discovered functions of LPA not only in the regulation of CSC but also normal SSC, the similarities in these regulatory functions, and how these discoveries can pave way to the development of novel therapies in cancer and regenerative medicine.