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1.
Stem Cell Res Ther ; 15(1): 19, 2024 01 17.
Article in English | MEDLINE | ID: mdl-38229180

ABSTRACT

BACKGROUND: After myocardial infarction, the lost myocardium is replaced by fibrotic tissue, eventually progressively leading to myocardial dysfunction. Direct reprogramming of fibroblasts into cardiomyocytes via the forced overexpression of cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) offers a promising strategy for cardiac repair. The limited reprogramming efficiency of this approach, however, remains a significant challenge. METHODS: We screened seven factors capable of improving direct cardiac reprogramming of both mice and human fibroblasts by evaluating small molecules known to be involved in cardiomyocyte differentiation or promoting human-induced pluripotent stem cell reprogramming. RESULTS: We found that vitamin C (VitC) significantly increased cardiac reprogramming efficiency when added to GMT-overexpressing fibroblasts from human and mice in 2D and 3D model. We observed a significant increase in reactive oxygen species (ROS) generation in human and mice fibroblasts upon Doxy induction, and ROS generation was subsequently reduced upon VitC treatment, associated with increased reprogramming efficiency. However, upon treatment with dehydroascorbic acid, a structural analog of VitC but lacking antioxidant properties, no difference in reprogramming efficiency was observed, suggesting that the effect of VitC in enhancing cardiac reprogramming is partly dependent of its antioxidant properties. CONCLUSIONS: Our findings demonstrate that VitC supplementation significantly enhances the efficiency of cardiac reprogramming, partially by suppressing ROS production in the presence of GMT.


Subject(s)
Antioxidants , Ascorbic Acid , Humans , Mice , Animals , Reactive Oxygen Species , Ascorbic Acid/pharmacology , Antioxidants/pharmacology , Cellular Reprogramming , T-Box Domain Proteins/genetics , MEF2 Transcription Factors/genetics , Myocytes, Cardiac , Vitamins , Fibroblasts
3.
Nat Metab ; 5(11): 1911-1930, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37973897

ABSTRACT

Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair.


Subject(s)
Cell Plasticity , Cellular Reprogramming , Animals , Mice , Vitamin B 12 , Wound Healing , Vitamins
4.
Stem Cell Res Ther ; 14(1): 296, 2023 10 15.
Article in English | MEDLINE | ID: mdl-37840130

ABSTRACT

BACKGROUND: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. METHODS: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. RESULTS: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA-expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. CONCLUSIONS: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.


Subject(s)
Myocardial Infarction , Myocytes, Cardiac , Mice , Animals , Myocytes, Cardiac/metabolism , Ventricular Remodeling , T-Box Domain Proteins/genetics , MEF2 Transcription Factors/genetics , Myocardial Infarction/therapy , Myocardial Infarction/drug therapy , Fibroblasts , Cellular Reprogramming/genetics
5.
Int J Mol Sci ; 24(15)2023 Jul 27.
Article in English | MEDLINE | ID: mdl-37569414

ABSTRACT

Acute myeloid leukemia (AML) is the most common and incurable leukemia subtype. Despite extensive research into the disease's intricate molecular mechanisms, effective treatments or expanded diagnostic or prognostic markers for AML have not yet been identified. The morphological, immunophenotypic, cytogenetic, biomolecular, and clinical characteristics of AML patients are extensive and complex. Leukemia stem cells (LSCs) consist of hematopoietic stem cells (HSCs) and cancer cells transformed by a complex, finely-tuned interaction that causes the complexity of AML. Microenvironmental regulation of LSCs dormancy and the diagnostic and therapeutic implications for identifying and targeting LSCs due to their significance in the pathogenesis of AML are discussed in this review. It is essential to perceive the relationship between the niche for LSCs and HSCs, which together cause the progression of AML. Notably, methylation is a well-known epigenetic change that is significant in AML, and our data also reveal that microRNAs are a unique factor for LSCs. Multiple-targeted approaches to reduce the risk of epigenetic factors, such as the administration of natural compounds for the elimination of local LSCs, may prevent potentially fatal relapses. Furthermore, the survival analysis of overlapping genes revealed that specific targets had significant effects on the survival and prognosis of patients. We predict that the multiple-targeted effects of herbal products on epigenetic modification are governed by different mechanisms in AML and could prevent potentially fatal relapses. Thus, these strategies can facilitate the incorporation of herbal medicine and natural compounds into the advanced drug discovery and development processes achievable with Network Pharmacology research.


Subject(s)
Leukemia, Myeloid, Acute , MicroRNAs , Humans , Aged , Cellular Reprogramming , Neoplastic Stem Cells/metabolism , Leukemia, Myeloid, Acute/genetics , Hematopoietic Stem Cells/metabolism , MicroRNAs/metabolism
6.
Nucleic Acids Res ; 50(19): 10981-10994, 2022 10 28.
Article in English | MEDLINE | ID: mdl-36305821

ABSTRACT

Dendritic cells (DCs), the most potent antigen-presenting cells, are necessary for effective activation of naïve T cells. DCs' immunological properties are modulated in response to various stimuli. Active DNA demethylation is crucial for DC differentiation and function. Vitamin C, a known cofactor of ten-eleven translocation (TET) enzymes, drives active demethylation. Vitamin C has recently emerged as a promising adjuvant for several types of cancer; however, its effects on human immune cells are poorly understood. In this study, we investigate the epigenomic and transcriptomic reprogramming orchestrated by vitamin C in monocyte-derived DC differentiation and maturation. Vitamin C triggers extensive demethylation at NF-κB/p65 binding sites, together with concordant upregulation of antigen-presentation and immune response-related genes during DC maturation. p65 interacts with TET2 and mediates the aforementioned vitamin C-mediated changes, as demonstrated by pharmacological inhibition. Moreover, vitamin C increases TNFß production in DCs through NF-κB, in concordance with the upregulation of its coding gene and the demethylation of adjacent CpGs. Finally, vitamin C enhances DC's ability to stimulate the proliferation of autologous antigen-specific T cells. We propose that vitamin C could potentially improve monocyte-derived DC-based cell therapies.


Subject(s)
Ascorbic Acid , Dendritic Cells , Epigenesis, Genetic , NF-kappa B , Humans , Ascorbic Acid/pharmacology , Cell Differentiation/genetics , NF-kappa B/metabolism , T-Lymphocytes/metabolism , Cellular Reprogramming
7.
Int J Oncol ; 60(5)2022 May.
Article in English | MEDLINE | ID: mdl-35322860

ABSTRACT

Prostate cancer mortality is ranked second among all cancer mortalities in men worldwide. There is a great need for a method of efficient drug screening for precision therapy, especially for patients with existing drug­resistant prostate cancer. Based on the concept of bacterial cell culture and drug sensitivity testing, the traditional approach of cancer drug screening is inadequate. The current and more innovative use of cancer cell culture and in vivo tumor models in drug screening for potential individualization of anti­cancer therapy is reviewed and discussed in the present review. An ideal screening model would have the ability to identify drug activity for the targeted cells resembling what would have occurred in the in vivo environment. Based on this principle, three available cell culture/tumor screening models for prostate cancer are reviewed and considered. The culture conditions, advantages and disadvantages for each model together with ideas to best utilize these models are discussed. The first screening model uses conditional reprogramed cells derived from patient cancer cells. Although these cells are convenient to grow and use, they are likely to have different markers and characteristics from original tumor cells and thus not likely to be informative. The second model employs patient derived xenograft (PDX) which resembles an in vivo approach, but its main disadvantages are that it cannot be easily genetically modified and it is not suitable for high­throughput drug screening. Finally, high­throughput screening is more feasible with tumor organoids grown from patient cancer cells. The last system still needs a large number of tumor cells. It lacks in situ blood vessels, immune cells and the extracellular matrix. Based on these current models, future establishment of an organoid data bank would allow the selection of a specific organoid resembling that of an individual's prostate cancer and used for screening of suitable anticancer drugs. This can be further confirmed using the PDX model. Thus, this combined organoid­PDX approach is expected to be able to provide the drug sensitivity testing approach for individualization of prostate cancer therapy in the near future.


Subject(s)
Cellular Reprogramming , Drug Evaluation, Preclinical/methods , Heterografts , Organoids , Precision Medicine/methods , Prostatic Neoplasms/drug therapy , Animals , Disease Models, Animal , Humans , Male , Prostatic Neoplasms/pathology , Tumor Cells, Cultured
8.
Stem Cell Res ; 60: 102673, 2022 04.
Article in English | MEDLINE | ID: mdl-35074713

ABSTRACT

In this paper, we describe the generation and validation of human induced pluripotent stem cell (hiPSC) lines from peripheral blood mononuclear cells (PBMCs) from 6 epilepsy patients using a non-integrative Sendai virus vector. These human cellular models will enable patient-specific drug screening to improve outcomes for individuals with this disorder.


Subject(s)
Epilepsy , Induced Pluripotent Stem Cells , Cell Differentiation , Cellular Reprogramming , Drug Evaluation, Preclinical , Humans , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear , Sendai virus
9.
Int J Mol Sci ; 22(20)2021 Oct 16.
Article in English | MEDLINE | ID: mdl-34681834

ABSTRACT

The selenoprotein family includes 25 members, many of which are antioxidant or redox regulating enzymes. A unique member of this family is Selenoprotein I (SELENOI), which does not catalyze redox reactions, but instead is an ethanolamine phosphotransferase (Ept). In fact, the characteristic selenocysteine residue that defines selenoproteins lies far outside of the catalytic domain of SELENOI. Furthermore, data using recombinant SELENOI lacking the selenocysteine residue have suggested that the selenocysteine amino acid is not directly involved in the Ept reaction. SELENOI is involved in two different pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, which are constituents of cellular membranes. Ethanolamine phospholipid synthesis has emerged as an important process for metabolic reprogramming that occurs in pluripotent stem cells and proliferating tumor cells, and this review discusses roles for upregulation of SELENOI during T cell activation, proliferation, and differentiation. SELENOI deficiency lowers but does not completely diminish de novo synthesis of PE and plasmenyl PE during T cell activation. Interestingly, metabolic reprogramming in activated SELENOI deficient T cells is impaired and this reduces proliferative capacity while favoring tolerogenic to pathogenic phenotypes that arise from differentiation. The implications of these findings are discussed related to vaccine responses, autoimmunity, and cell-based therapeutic approaches.


Subject(s)
Ethanolamine/metabolism , Ethanolaminephosphotransferase/physiology , Lymphocyte Activation , Phospholipids/metabolism , Selenoproteins/physiology , T-Lymphocytes/physiology , Cellular Reprogramming , Humans , Phosphatidylethanolamines/metabolism , Selenium/metabolism , Selenocysteine/metabolism , Selenoproteins/chemistry , Up-Regulation
10.
Stem Cell Reports ; 16(9): 2257-2273, 2021 09 14.
Article in English | MEDLINE | ID: mdl-34525385

ABSTRACT

Hair cell degeneration is a major cause of sensorineural hearing loss. Hair cells in mammalian cochlea do not spontaneously regenerate, posing a great challenge for restoration of hearing. Here, we establish a robust, high-throughput cochlear organoid platform that facilitates 3D expansion of cochlear progenitor cells and differentiation of hair cells in a temporally regulated manner. High-throughput screening of the FDA-approved drug library identified regorafenib, a VEGFR inhibitor, as a potent small molecule for hair cell differentiation. Regorafenib also promotes reprogramming and maturation of hair cells in both normal and neomycin-damaged cochlear explants. Mechanistically, inhibition of VEGFR suppresses TGFB1 expression via the MEK pathway and TGFB1 downregulation directly mediates the effect of regorafenib on hair cell reprogramming. Our study not only demonstrates the power of a cochlear organoid platform in high-throughput analyses of hair cell physiology but also highlights VEGFR-MEK-TGFB1 signaling crosstalk as a potential target for hair cell regeneration and hearing restoration.


Subject(s)
Cellular Reprogramming , Cochlea/metabolism , High-Throughput Screening Assays , Mitogen-Activated Protein Kinase Kinases/metabolism , Organoids/metabolism , Receptors, Vascular Endothelial Growth Factor/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Cell Culture Techniques, Three Dimensional/methods , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Cellular Reprogramming/genetics , Cochlea/cytology , Drug Discovery/methods , Drug Evaluation, Preclinical , Gene Expression Regulation/drug effects , Hair Cells, Auditory/cytology , Hair Cells, Auditory/drug effects , Hair Cells, Auditory/metabolism , Mice , Mice, Transgenic , Organoids/cytology , Phenylurea Compounds/pharmacology , Pyridines/pharmacology , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/drug effects
11.
Cells ; 10(7)2021 07 06.
Article in English | MEDLINE | ID: mdl-34359884

ABSTRACT

Hypoxia, low oxygen (O2) level, is a hallmark of solid cancers, especially hepatocellular carcinoma (HCC), one of the most common and fatal cancers worldwide. Hypoxia contributes to drug resistance in cancer through various molecular mechanisms. In this review, we particularly focus on the roles of hypoxia-inducible factor (HIF)-mediated metabolic reprogramming in drug resistance in HCC. Combination therapies targeting hypoxia-induced metabolic enzymes to overcome drug resistance will also be summarized. Acquisition of drug resistance is the major cause of unsatisfactory clinical outcomes of existing HCC treatments. Extra efforts to identify novel mechanisms to combat refractory hypoxic HCC are warranted for the development of more effective treatment regimens for HCC patients.


Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Cellular Reprogramming/drug effects , Drug Resistance, Neoplasm/genetics , Hypoxia/drug therapy , Liver Neoplasms/drug therapy , Antibodies, Monoclonal, Humanized/therapeutic use , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cellular Reprogramming/genetics , Gene Expression Regulation, Neoplastic , Humans , Hypoxia/genetics , Hypoxia/metabolism , Hypoxia/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immune Checkpoint Inhibitors/therapeutic use , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/genetics , Nivolumab/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyruvate Dehydrogenase Acetyl-Transferring Kinase/genetics , Pyruvate Dehydrogenase Acetyl-Transferring Kinase/metabolism , Sorafenib/therapeutic use , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics
12.
J Am Heart Assoc ; 10(14): e019473, 2021 07 20.
Article in English | MEDLINE | ID: mdl-34227403

ABSTRACT

Background It is known that dietary intake of polyunsaturated fatty acids may improve cardiac function. However, relatively high daily doses are required to achieve sufficient cardiac concentrations of beneficial omega-3 fatty acids. The liver X receptor (LXR) is a nuclear hormone receptor and a crucial regulator of lipid homeostasis in mammals. LXR activation has been shown to endogenously reprogram cellular lipid profiles toward increased polyunsaturated fatty acids levels. Here we studied whether LXR lipid reprogramming occurs in cardiac tissue and exerts cardioprotective actions. Methods and Results Male 129SV mice were treated with the LXR agonist AZ876 (20 µmol/kg per day) for 11 days. From day 6, the mice were injected with the nonselective ß-agonist isoproterenol for 4 consecutive days to induce diastolic dysfunction and subendocardial fibrosis while maintaining systolic function. Treatment with isoproterenol led to a marked impairment of global longitudinal strain and the E/e' ratio of transmitral flow to mitral annular velocity, which were both significantly improved by the LXR agonist. Histological examination showed a significant reduction in isoproterenol-induced subendocardial fibrosis by AZ876. Analysis of the cardiac lipid composition by liquid chromatography-high resolution mass spectrometry revealed a significant increase in cardiac polyunsaturated fatty acids levels and a significant reduction in saturated fatty acids by AZ876. Conclusions The present study provides evidence that the LXR agonist AZ876 prevents subendocardial damage, improves global longitudinal strain and E/e' in a mouse model of isoproterenol-induced cardiac damage, accompanied by an upregulation of cardiac polyunsaturated fatty acids levels. Cardiac LXR activation and beneficial endogenous cardiac lipid reprogramming may provide a new therapeutic strategy in cardiac disease with diastolic dysfunction.


Subject(s)
Aniline Compounds/pharmacology , Fatty Acids/metabolism , Heart Diseases/prevention & control , Isoproterenol , Myocardium/metabolism , Thiazoles/pharmacology , Animals , Cellular Reprogramming , Disease Models, Animal , Fibrosis , Heart Diseases/chemically induced , Heart Diseases/pathology , Liver X Receptors/agonists , Male , Mice , Mice, 129 Strain , Myocardium/pathology
13.
Cell ; 184(15): 3915-3935.e21, 2021 07 22.
Article in English | MEDLINE | ID: mdl-34174187

ABSTRACT

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.


Subject(s)
Epigenomics , Immunity/genetics , Influenza Vaccines/genetics , Influenza Vaccines/immunology , Single-Cell Analysis , Transcription, Genetic , Vaccination , Adolescent , Adult , Anti-Bacterial Agents/pharmacology , Antigens, CD34/metabolism , Antiviral Agents/pharmacology , Cellular Reprogramming , Chromatin/metabolism , Cytokines/biosynthesis , Drug Combinations , Female , Gene Expression Regulation , Histones/metabolism , Humans , Immunity, Innate/genetics , Influenza A Virus, H5N1 Subtype/drug effects , Influenza A Virus, H5N1 Subtype/immunology , Interferon Type I/metabolism , Male , Myeloid Cells/metabolism , Polysorbates/pharmacology , Squalene/pharmacology , Toll-Like Receptors/metabolism , Transcription Factor AP-1/metabolism , Transcriptome/genetics , Young Adult , alpha-Tocopherol/pharmacology
14.
Pharmacol Res ; 169: 105617, 2021 07.
Article in English | MEDLINE | ID: mdl-33872811

ABSTRACT

Traditional Chinese multi-herb-combined prescriptions usually show better performance than a single agent since a group of effective compounds interfere multiple disease-relevant targets simultaneously. Huang-Lian-Jie-Du decoction is a remedy made of four herbs that are widely used to treat oral ulcers, gingivitis, and periodontitis. However, the active ingredients and underlying mechanisms are not clear. To address these questions, we prepared a water extract solution of Huang-Lian-Jie-Du decoction (HLJDD), called it as WEH (Water Extract Solution of HLJDD), and used it to treat LPS-induced systemic inflammation in mice. We observed that WEH attenuated inflammatory responses including reducing production of cytokines, chemokines and interferons (IFNs), further attenuating emergency myelopoiesis, and preventing mice septic lethality. Upon LPS stimulation, mice pretreated with WEH increased circulating Ly6C- patrolling and splenic Ly6C+ inflammatory monocytes. The acute myelopoiesis related transcriptional factor profile was rearranged by WEH. Mechanistically we confirmed that WEH interrupted LPS/TLR4/CD14 signaling-mediated downstream signaling pathways through its nine principal ingredients, which blocked LPS stimulated divergent signaling cascades, such as activation of NF-κB, p38 MAPK, and ERK1/2. We conclude that the old remedy blunts LPS-induced "danger" signal recognition and transduction process at multiple sites. To translate our findings into clinical applications, we refined the crude extract into a pure multicomponent drug by directly mixing these nine chemical entities, which completely reproduced the effect of protecting mice from lethal septic shock. Finally, we reduced a large number of compounds within a multi-herb water extract to seven-chemical combination that exhibited superior therapeutic efficacy compared with WEH.


Subject(s)
Drugs, Chinese Herbal/therapeutic use , Inflammation/drug therapy , Monocytes/drug effects , Plant Extracts/therapeutic use , Transcription Factors/drug effects , Animals , Cellular Reprogramming/drug effects , Coptis chinensis , Drugs, Chinese Herbal/administration & dosage , Flow Cytometry , Gene Expression Regulation/drug effects , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Plant Extracts/administration & dosage , RAW 264.7 Cells/drug effects , Transcription Factors/metabolism
15.
Am J Physiol Lung Cell Mol Physiol ; 320(5): L770-L784, 2021 05 01.
Article in English | MEDLINE | ID: mdl-33624555

ABSTRACT

Gestational long-term hypoxia increases the risk of myriad diseases in infants including persistent pulmonary hypertension. Similar to humans, fetal lamb lung development is susceptible to long-term intrauterine hypoxia, with structural and functional changes associated with the development of pulmonary hypertension including pulmonary arterial medial wall thickening and dysregulation of arterial reactivity, which culminates in decreased right ventricular output. To further explore the mechanisms associated with hypoxia-induced aberrations in the fetal sheep lung, we examined the premise that metabolomic changes and functional phenotypic transformations occur due to intrauterine, long-term hypoxia. To address this, we performed electron microscopy, Western immunoblotting, calcium imaging, and metabolomic analyses on pulmonary arteries isolated from near-term fetal lambs that had been exposed to low- or high-altitude (3,801 m) hypoxia for the latter 110+ days of gestation. Our results demonstrate that the sarcoplasmic reticulum was swollen with high luminal width and distances to the plasma membrane in the hypoxic group. Hypoxic animals were presented with higher endoplasmic reticulum stress and suppressed calcium storage. Metabolically, hypoxia was associated with lower levels of multiple omega-3 polyunsaturated fatty acids and derived lipid mediators (e.g., eicosapentaenoic acid, docosahexaenoic acid, α-linolenic acid, 5-hydroxyeicosapentaenoic acid (5-HEPE), 12-HEPE, 15-HEPE, prostaglandin E3, and 19(20)-epoxy docosapentaenoic acid) and higher levels of some omega-6 metabolites (P < 0.02) including 15-keto prostaglandin E2 and linoleoylglycerol. Collectively, the results reveal broad evidence for long-term hypoxia-induced metabolic reprogramming and phenotypic transformations in the pulmonary arteries of fetal sheep, conditions that likely contribute to the development of persistent pulmonary hypertension.


Subject(s)
Cellular Reprogramming , Fetal Hypoxia/physiopathology , Fetus/physiopathology , Hypoxia/physiopathology , Metabolome , Prenatal Exposure Delayed Effects/physiopathology , Pulmonary Artery/physiopathology , Altitude , Animals , Calcium , Female , Gestational Age , Pregnancy , Sheep
16.
Toxicol Appl Pharmacol ; 417: 115467, 2021 04 15.
Article in English | MEDLINE | ID: mdl-33631231

ABSTRACT

Owing to the technological advancements, including next generation sequencing, the significance of deregulated epigenetic mechanisms in cancer initiation, progression and treatment has become evident. The accumulating knowledge relating to the epigenetic markers viz. DNA methylation, Histone modifications and non-coding RNAs make them one of the most interesting candidates for developing anti-cancer therapies. The reversibility of deregulated epigenetic mechanisms through environmental and dietary factors opens numerous avenues in the field of chemoprevention and drug development. Recent studies have proven that plant-derived natural products encompass a great potential in targeting epigenetic signatures in cancer and numerous natural products are being explored for their possibility to be considered as "epi-drug". This review intends to highlight the major aberrant epigenetic mechanisms and summarizes the essential functions of natural products like Resveratrol, Quercetin, Genistein, EGCG, Curcumin, Sulforaphane, Apigenin, Parthenolide and Berberine in modulating these aberrations. This knowledge along with the challenges and limitations in this field has potential and wider implications in developing novel and successful therapeutic strategies. The increased focus in the area will possibly provide a better understanding for the development of dietary supplements and/or drugs either alone or in combination. The interaction of epigenetics with different hallmarks of cancer and how natural products can be utilized to target them will also be interesting in the future therapeutic approaches.


Subject(s)
Anticarcinogenic Agents/therapeutic use , Antineoplastic Agents, Phytogenic/therapeutic use , Cellular Reprogramming/drug effects , Diet , Epigenesis, Genetic/drug effects , Neoplasms/drug therapy , Neoplasms/prevention & control , Animals , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Chromatin Assembly and Disassembly/drug effects , DNA Methylation/drug effects , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/genetics , Neoplasms/pathology
17.
Int J Mol Sci ; 22(3)2021 Jan 25.
Article in English | MEDLINE | ID: mdl-33503959

ABSTRACT

Pyruvate kinase is a key regulator in glycolysis through the conversion of phosphoenolpyruvate (PEP) into pyruvate. Pyruvate kinase exists in various isoforms that can exhibit diverse biological functions and outcomes. The pyruvate kinase isoenzyme type M2 (PKM2) controls cell progression and survival through the regulation of key signaling pathways. In cancer cells, the dimer form of PKM2 predominates and plays an integral role in cancer metabolism. This predominance of the inactive dimeric form promotes the accumulation of phosphometabolites, allowing cancer cells to engage in high levels of synthetic processing to enhance their proliferative capacity. PKM2 has been recognized for its role in regulating gene expression and transcription factors critical for health and disease. This role enables PKM2 to exert profound regulatory effects that promote cancer cell metabolism, proliferation, and migration. In addition to its role in cancer, PKM2 regulates aspects essential to cellular homeostasis in non-cancer tissues and, in some cases, promotes tissue-specific pathways in health and diseases. In pursuit of understanding the diverse tissue-specific roles of PKM2, investigations targeting tissues such as the kidney, liver, adipose, and pancreas have been conducted. Findings from these studies enhance our understanding of PKM2 functions in various diseases beyond cancer. Therefore, there is substantial interest in PKM2 modulation as a potential therapeutic target for the treatment of multiple conditions. Indeed, a vast plethora of research has focused on identifying therapeutic strategies for targeting PKM2. Recently, targeting PKM2 through its regulatory microRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) has gathered increasing interest. Thus, the goal of this review is to highlight recent advancements in PKM2 research, with a focus on PKM2 regulatory microRNAs and lncRNAs and their subsequent physiological significance.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Cellular Reprogramming , Energy Metabolism , Gene Expression Regulation , Membrane Proteins/genetics , Membrane Proteins/metabolism , Thyroid Hormones/genetics , Thyroid Hormones/metabolism , Animals , Carrier Proteins/antagonists & inhibitors , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cellular Reprogramming/genetics , Disease Susceptibility , Drug Development , Drug Evaluation, Preclinical , Energy Metabolism/genetics , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Homeostasis , Humans , Membrane Proteins/antagonists & inhibitors , Mutation , Protein Transport , Pyruvate Kinase/genetics , Pyruvate Kinase/metabolism , RNA Interference , RNA, Long Noncoding/genetics , Research , Thyroid Hormone-Binding Proteins
18.
J Cell Physiol ; 236(2): 1545-1558, 2021 02.
Article in English | MEDLINE | ID: mdl-33000501

ABSTRACT

The therapeutic potential of α,ß-thujone, a functional compound found in many medicinal plants of the Cupressaceae, Asteraceae, and Lamiaceae families, has been demonstrated, including in inflammation and cancers. However, its pharmacological functions and mechanisms of action in ovarian cancer remain unclear. We investigated the anticancer properties of α,ß-thujone in ES2 and OV90 human ovarian cancer cells and its effect on sensitization to cisplatin. α,ß-thujone inhibited cancer cell proliferation and induced cell death through caspase-dependent intrinsic apoptotic pathways. Moreover, α,ß-thujone-mediated endoplasmic reticulum stress was associated with the loss of mitochondrial functions and altered metabolic landscape of ovarian cancer cells. α,ß-Thujone attenuated blood vessel formation in transgenic zebrafish, implying it has significant antiangiogenic potential. In addition, α,ß-thujone sensitized ovarian cancer cells to cisplatin, causing synergistic pharmacological effects. Collectively, our results suggest that α,ß-thujone has therapeutic potential in human ovarian cancer and functions via regulating multiple intracellular stress-associated metabolic reprogramming and caspase-dependent apoptotic pathways.


Subject(s)
Bicyclic Monoterpenes/pharmacology , Cell Proliferation/drug effects , Cellular Reprogramming/genetics , Ovarian Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cisplatin/pharmacology , Endoplasmic Reticulum Stress/drug effects , Female , Humans , Mitochondria/drug effects , Mitochondria/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Ovary/drug effects , Signal Transduction/drug effects , Zebrafish/genetics
19.
Stem Cells Dev ; 30(2): 79-90, 2021 01 15.
Article in English | MEDLINE | ID: mdl-33256572

ABSTRACT

Forced coexpression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc reprograms somatic cells into pluripotent stem cells (PSCs). Such induced PSCs (iPSCs) can generate any cell type of the adult body or indefinitely proliferate without losing their potential. Accordingly, iPSCs can serve as an unlimited cell source for the development of various disease models and regenerative therapies for animals and humans. Although canine peripheral blood mononuclear cells (PBMCs) can be easily obtained, they have a very low iPSC reprogramming efficiency. In this study, we determined the reprogramming efficiency of canine PBMCs under several conditions involving three types of media supplemented with small-molecule compounds. We found that canine iPSCs (ciPSCs) could be efficiently generated from PBMCs using N2B27 medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and a small-molecule cocktail (Y-27632, PD0325901, CHIR99021, A-83-01, Forskolin, and l-ascorbic acid). We generated five ciPSC lines that could be maintained in StemFit® medium supplemented with LIF. The SeVdp(KOSM)302L vectors were appropriately silenced in four ciPSC lines. Of the two lines characterized, both were positive for alkaline phosphatase activity and expressed pluripotency markers, including the Oct3/4, Sox2, and Nanog transcripts, as well as the octamer-binding transcription factor (OCT) 3/4 and NANOG proteins, and the SSEA-1 carbohydrate antigen. The ciPSCs could form embryoid bodies and differentiate into the three germ layers, as indicated by marker gene and protein expression. Furthermore, one ciPSC line formed teratomas comprising several tissues from every germ layer. Our ciPSC lines maintained a normal karyotype even after multiple passages. Moreover, our new reprogramming method was able to generate ciPSCs from multiple donor PBMCs. In conclusion, we developed an easy and efficient strategy for the generation of footprint-free ciPSCs from PBMCs. We believe that this strategy can be useful for disease modeling and regenerative medicine in the veterinary field.


Subject(s)
Cell Differentiation/genetics , Cellular Reprogramming/genetics , Gene Expression/genetics , Induced Pluripotent Stem Cells/metabolism , Leukocytes, Mononuclear/metabolism , Animals , Cells, Cultured , Cellular Reprogramming Techniques/methods , Culture Media/chemistry , Culture Media/pharmacology , Dogs , Ectoderm/cytology , Ectoderm/metabolism , Endoderm/cytology , Endoderm/metabolism , Gene Expression/drug effects , Humans , Induced Pluripotent Stem Cells/cytology , Leukocytes, Mononuclear/cytology , Mesoderm/cytology , Mesoderm/metabolism , Mice, Inbred ICR , Nanog Homeobox Protein/genetics , Nanog Homeobox Protein/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Reproducibility of Results , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism
20.
Methods Mol Biol ; 2239: 47-59, 2021.
Article in English | MEDLINE | ID: mdl-33226612

ABSTRACT

There are currently no effective treatments to regenerate the heart after cardiac injury. Following cardiac injury, heart muscle cells, also known as cardiomyocytes, die in large numbers. The adult mammalian heart does not have the ability to replace these dead cardiomyocytes. In their place, fibroblasts invade the injury zone and generate a scar. The scar impairs cardiac function. An important approach to cardiac regeneration is direct cardiac reprogramming, whereby cardiac fibroblasts within the scar are directly converted into functional cardiomyocytes. Several laboratories have achieved direct cardiac reprogramming via overexpression of the cardiac transcription factors. In contrast, we utilize a combination of four miRNAs (miR-1, miR-133, miR-208, miR-499) that we call miR Combo. One common issue regarding direct cardiac reprogramming strategies is low efficiency. Recently, we have demonstrated that the efficiency of direct cardiac reprogramming is enhanced in the chemically defined reprogramming media.


Subject(s)
Cellular Reprogramming/genetics , Culture Media/chemistry , Fibroblasts/cytology , MicroRNAs/metabolism , Myocytes, Cardiac/cytology , Animals , Ascorbic Acid/chemistry , Cell Culture Techniques/methods , Cells, Cultured , Fibroblasts/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Mice , MicroRNAs/genetics , Myocytes, Cardiac/metabolism , Real-Time Polymerase Chain Reaction , Regeneration , Selenium/chemistry , Transfection/methods
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