Dataset Information

Hypoxia-Activated PI3K/Akt Inhibits Oxidative Stress via the Regulation of Reactive Oxygen Species in Human Dental Pulp Cells.

ABSTRACT: In order to use stem cells as a resource for tissue regeneration, it is necessary to induce expansion in vitro. However, during culture, stem cells often lose functional properties and become senescent. Increasing evidence indicates that hypoxic preconditioning with physiological oxygen concentration can maintain the functional properties of stem cells in vitro. The purpose of the current study was to test the hypothesis that hypoxic preconditioning with physiological oxygen concentration can maintain the functional properties of stem cells in culture by reducing oxidative stress. In vitro studies were performed in primary human dental pulp cells (hDPCs). Reduced levels of oxidative stress and increased cellular "stemness" in response to physiological hypoxia were dependent upon the expression of reactive oxygen species (ROS). Subsequently, RNA-sequencing analysis revealed the increased expression of phosphoinositide 3-kinase (PI3K)/Akt signaling in culture, a pathway which regulates oxidative stress. Furthermore, we found evidence that PI3K/Akt signaling might affect intracellular ROS production by negatively regulating expression of the downstream protein Forkhead Box Protein O1 (FOXO1) and Caspase 3. Collectively, our data show that the PI3K/Akt pathway is activated in response to hypoxia and inhibits oxidative stress in a ROS-dependent manner. This study identified redox-mediated hypoxic preconditioning regulatory mechanisms that may be significant for tissue regeneration.

SUBMITTER: Liu F

PROVIDER: S-EPMC6343138 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Hypoxia-Activated PI3K/Akt Inhibits Oxidative Stress via the Regulation of Reactive Oxygen Species in Human Dental Pulp Cells.

Liu Fei F Huang Xin X Luo Zhenhua Z He Jingjun J Haider Farhan F Haider Farhan F Song Ci C Peng Ling L Chen Ting T Wu Buling B

Oxidative medicine and cellular longevity 20190109

In order to use stem cells as a resource for tissue regeneration, it is necessary to induce expansion <i>in vitro</i>. However, during culture, stem cells often lose functional properties and become senescent. Increasing evidence indicates that hypoxic preconditioning with physiological oxygen concentration can maintain the functional properties of stem cells <i>in vitro</i>. The purpose of the current study was to test the hypothesis that hypoxic preconditioning with physiological oxygen concen ...[more]

PMID: 30728888

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Project description:BackgroundDental pulp stem cells (DPSCs) have been developed as a potential source of mesenchymal stem cells (MSCs) for regeneration of dental pulp and other tissues. However, further strategies to isolate highly functional DPSCs beyond the colony-forming methods are required. We have demonstrated the safety and efficacy of DPSCs isolated by G-CSF-induced mobilization and cultured under normoxia (mobilized DPSCs, MDPSCs) for pulp regeneration. The device for isolation of MDPSCs, however, is not cost-effective and requires a prolonged cell culture period. It is well known that MSCs cultured under hypoxic-preconditions improved MSC proliferation activity and stemness. Therefore, in this investigation, we attempted to improve the clinical utility of DPSCs by hypoxia-preconditioned DPSCs (hpDPSCs) compared with MDPSCs to improve the potential clinical utility for pulp regeneration in endodontic dentistry.MethodsColony-forming DPSCs were isolated and preconditioned with hypoxia in a stable closed cultured system and compared with MDPSCs isolated from the individual dog teeth. We examined the proliferation rate, migration potential, anti-apoptotic activity, and gene expression of the stem cell markers and angiogenic/neurotrophic factors. Trophic effects of the conditioned medium (CM) were also evaluated. In addition, the expression of immunomodulatory molecules upon stimulation with IFN-γ was investigated. The pulp regenerative potential and transplantation safety of hpDPSCs were further assessed in pulpectomized teeth in dogs by histological and immunohistochemical analyses and by chemistry of the blood and urine tests.ResultshpDPSCs demonstrated higher proliferation rate and expression of a major regulator of oxygen homeostasis, HIF-1α, and a stem cell marker, CXCR-4. The direct migratory activity of hpDPSCs in response to G-CSF was significantly higher than MDPSCs. The CM of hpDPSCs stimulated neurite extension. However, there were no changes in angiogenic, migration, and anti-apoptotic activities compared with the CM of MDPSCs. The expression of immunomodulatory gene, PTGE was significantly upregulated by IFN gamma in hpDPSCs compared with MDPSCs. However, no difference in nitric oxide was observed. The regenerated pulp tissue was quantitatively and qualitatively similar in hpDPSC transplants compared with MDPSC transplants in dog teeth. There was no evidence of toxicity or adverse events of the hpDPSC transplantation.ConclusionsThese results demonstrated that the efficacy of hpDPSCs for pulp regeneration was identical, although hpDPSCs improved stem cell properties compared to MDPSCs, suggesting their potential clinical utility for pulp regeneration.

Project description:BackgroundThe repair of cranio-maxillofacial bone defects remains a formidable clinical challenge. The Ets variant 2 (ETV2) transcription factor, which belongs to the E26 transformation-specific (ETS) family, has been reported to play a key role in neovascularization. However, the role of ETV2 in the osteogenesis of human dental pulp stem cells (hDPSCs) remains unexplored.MethodsTransgenic overexpression of ETV2 was achieved using a lentiviral vector, based on a Dox-inducible system. The effects of Dox-induced overexpression of ETV2 on the osteogenesis of hDPSCs were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, immunofluorescence staining, alkaline phosphatase (ALP) staining, and Alizarin Red S (ARS) staining. Additionally, RNA-sequencing (RNA-Seq) analysis was performed to analyze the underlying mechanisms of ETV2-induced osteogenesis. Additionally, the role of ETV2 overexpression in bone formation in vivo was validated by animal studies with a rat calvarial defect model and a nude mice model.ResultsOur results demonstrated that ETV2 overexpression significantly upregulated the mRNA and protein expression levels of osteogenic markers, markedly enhanced ALP activity, and promoted matrix mineralization of hDPSCs. Moreover, the results of RNA-Seq analysis and western blot showed that the ERK/MAPK and PI3K-Akt signaling pathways were activated upon transgenic overexpression of ETV2. The enhanced osteogenic differentiation of hDPSCs due to ETV2 overexpression was partially reversed by treatment with inhibitors of ERK/MAPK or PI3K-AKT signaling. Furthermore, the results of in vivo studies demonstrated that ETV2 overexpression improved bone healing in a rat calvarial defect model and increased ectopic bone formation in nude mice.ConclusionsCollectively, our results indicated that ETV2 overexpression exerted positive effects on the osteogenesis of hDPSCs, at least partially via the ERK/MAPK and PI3K/AKT signaling pathways.

Project description:Gestational hypoxia inhibits steroid hormone-induced upregulation of Ca(2+)-activated K(+) (KCa) channel activities in uterine arteries. We tested the hypothesis that increased reactive oxygen species play an important role in hypoxia-mediated inhibition of KCa channel activities. Uterine arteries were isolated from nonpregnant (nonpregnant uterine artery) and near-term (?142-145 day) pregnant (pregnant uterine artery) sheep maintained at either sea level or high altitude (3820 m, Pao2: 60 mm Hg) for 110 days. In pregnant uterine arteries, hypoxia significantly decreased large conductance channel opener NS1619- and small conductance channel opener NS309-induced relaxations, which were partially restored by reactive oxygen species inhibitor N-acetylcysteine (NAC). NAC significantly increased large conductance KCa but not small conductance KCa current densities in uterine arterial smooth muscle cells in pregnant animals acclimatized to high altitude. The NAC-sensitive component of small conductance KCa-induced relaxations was diminished in endothelium-denuded arteries. In nonpregnant uterine arteries, NS1619- and NS309-induced relaxations were diminished compared with those in pregnant uterine arteries. Treatment of nonpregnant uterine arteries with 17?-estradiol and progesterone for 48 hours increased small conductance KCa type 3 protein abundance and NS1619- and NS309-induced relaxations, which were inhibited by hypoxia. This hypoxia-mediated inhibition was reversed by NAC. Consistently, steroid hormone treatment had no significant effects on large conductance KCa current density in nonpregnant uterine arteries of hypoxic animals in the absence of NAC but significantly increased it in the presence of NAC. These results suggest an important role of hypoxia-mediated reactive oxygen species in negatively regulating steroid hormone-mediated upregulation of KCa channel activity and adaptation of uterine vascular reactivity in pregnancy, which may contribute to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia.

Project description:Dental pulp infection and necrosis are widespread diseases. Conventional endodontic treatments result in a devitalized and weakened tooth. In this work, we synthesized novel star-shaped polymer to self-assemble into unique nanofibrous spongy microspheres (NF-SMS), which were used to carry human dental pulp stem cells (hDPSCs) into the pulp cavity to regenerate living dental pulp tissues. It was found that NF-SMS significantly enhanced hDPSCs attachment, proliferation, odontogenic differentiation and angiogenesis, as compared to control cell carriers. Additionally, NF-SMS promoted vascular endothelial growth factor (VEGF) expression of hDPSCs in a 3D hypoxic culture. Hypoxia-primed hDPSCs/NF-SMS complexes were injected into the cleaned pulp cavities of rabbit molars for subcutaneous implantation in mice. After 4 weeks, the hypoxia group significantly enhanced angiogenesis inside the pulp chamber and promoted the formation of ondontoblast-like cells lining along the dentin-pulp interface, as compared to the control groups (hDPSCs alone group, NF-SMS alone group, and hDPSCs/NF-SMS group pre-cultured under normoxic conditions). Furthermore, in an in situ dental pulp repair model in rats, hypoxia-primed hDPSCs/NF-SMS were injected to fully fill the pulp cavity and regenerate pulp-like tissues with a rich vasculature and a histological structure similar to the native pulp.Statement of significanceVascularization is key to the regeneration of many vital tissues. However, it is challenging to create a suitable microenvironment for stem cells to regenerate vascularized tissue structure. This manuscript reports a novel star-shaped block copolymer that self-assembles into unique nanofibrous spongy microspheres, which as an injectable scaffold recapitulate the cell-cell and cell-matrix interactions in development. Using a clinically-relevant surgical procedure and a hypoxic treatment, the nanofibrous spongy microspheres were used to deliver stem cells and successfully regenerate dental pulp with a rich vasculature and a complex histologic structure similar to that of the native dental pulp. The novel microspheres can likely be used to regenerate many other vascularized tissues.

Project description:Background/purposeHigh glucose (HG)-induced aberrant proliferation, apoptosis and odontoblastic differentiation of dental pulp cells (DPCs) have been implicated in the pathogenesis of impaired diabetic pulp healing; however, the underlying mechanism remains unclear. This study aimed to investigate the role of mitochondrial reactive oxygen species (mtROS) and mitochondria in HG-induced dysfunction and apoptosis of DPCs.Materials and methodsHuman DPCs (hDPCs) were cultured in a low-glucose, high-glucose, mannitol, and MitoTEMPO medium in vitro. Methylthiazol tetrazolium assay, Annexin V-FITC/PI staining and scratch-wound assay were used to analyze cell proliferation, apoptosis and migration, respectively. Alkaline phosphatase staining and alizarin red S staining were used to evaluate cell differentiation. DCF-DA staining, MitoSOX staining, MitoTracker Red staining, JC-1 staining, and adenosine triphosphate (ATP) kit assay were performed to investigate total ROS and mtROS generation, mitochondrial density, mitochondrial membrane potential (MMP), and ATP synthesis, respectively. Quantitative PCR assay was performed to detect the mRNA expression of mitochondrial biogenesis- and dynamics-related markers. Transmission electron microscopy was used to observe the mitochondrial ultrastructure.ResultsHG augmented the production of total ROS and mtROS, and triggered mitochondrial damage in hDPCs, as reflected by decreased mitochondrial density, depolarized MMP, reduced ATP synthesis, altered mRNA expression of mitochondrial biogenesis- and dynamics-related markers, and abnormal mitochondrial ultrastructure. Supplementation of MitoTEMPO alleviated the mitochondrial damage and reversed the aberrant proliferation, apoptosis, migration and odontoblastic differentiation of HG-stimulated hDPCs.ConclusionHG triggers mitochondrial damage via augmenting mtROS generation, resulting in the inhibited proliferation, migration, and odontoblastic differentiation of hDPCs and enhanced their apoptosis.

Dataset Information

Hypoxia-Activated PI3K/Akt Inhibits Oxidative Stress via the Regulation of Reactive Oxygen Species in Human Dental Pulp Cells.

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Hypoxia-Activated PI3K/Akt Inhibits Oxidative Stress via the Regulation of Reactive Oxygen Species in Human Dental Pulp Cells.

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