RESUMO
The prostanoid G protein-coupled receptor (GPCR) EP2 is widely expressed and implicated in endometriosis, osteoporosis, obesity, pre-term labour and cancer. Internalisation and intracellular trafficking are critical for shaping GPCR activity, yet little is known regarding the spatial programming of EP2 signalling and whether this can be exploited pharmacologically. Using three EP2-selective ligands that favour activation of different EP2 pathways, we show that EP2 undergoes limited agonist-driven internalisation but is constitutively internalised via dynamin-dependent, ß-arrestin-independent pathways. EP2 was constitutively trafficked to early and very early endosomes (VEE), which was not altered by ligand activation. APPL1, a key adaptor and regulatory protein of the VEE, did not impact EP2 agonist-mediated cAMP. Internalisation was required for ~70% of the acute butaprost- and AH13205-mediated cAMP signalling, yet PGN9856i, a Gαs-biased agonist, was less dependent on receptor internalisation for its cAMP signalling, particularly in human term pregnant myometrial cells that endogenously express EP2. Inhibition of EP2 internalisation partially reduced calcium signalling activated by butaprost or AH13205 and had no effect on PGE2 secretion. This indicates an agonist-dependent differential spatial requirement for Gαs and Gαq/11 signalling and a role for plasma membrane-initiated Gαq/11-Ca2+-mediated PGE2 secretion. These findings reveal a key role for EP2 constitutive internalisation in its signalling and potential spatial bias in mediating its downstream functions. This, in turn, could highlight important considerations for future selective targeting of EP2 signalling pathways.
Assuntos
Proteínas de Ligação ao GTP , Receptores de Prostaglandina E Subtipo EP2 , Transdução de Sinais , Feminino , Humanos , Gravidez , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Alprostadil/metabolismo , AMP Cíclico/metabolismo , Endossomos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Miométrio/metabolismo , Transporte Proteico , Receptores de Prostaglandina E Subtipo EP2/metabolismoRESUMO
Gene therapies provide treatment options for many diseases, but the safe and long-term control of therapeutic transgene expression remains a primary issue for clinical applications. Here, we develop a muscone-induced transgene system packaged into adeno-associated virus (AAV) vectors (AAVMUSE) based on a G protein-coupled murine olfactory receptor (MOR215-1) and a synthetic cAMP-responsive promoter (PCRE). Upon exposure to the trigger, muscone binds to MOR215-1 and activates the cAMP signaling pathway to initiate transgene expression. AAVMUSE enables remote, muscone dose- and exposure-time-dependent control of luciferase expression in the livers or lungs of mice for at least 20 weeks. Moreover, we apply this AAVMUSE to treat two chronic inflammatory diseases: nonalcoholic fatty liver disease (NAFLD) and allergic asthma, showing that inhalation of muscone-after only one injection of AAVMUSE-can achieve long-term controllable expression of therapeutic proteins (ΔhFGF21 or ΔmIL-4). Our odorant-molecule-controlled system can advance gene-based precision therapies for human diseases.
Assuntos
Alprostadil , Cicloparafinas , Camundongos , Humanos , Animais , Alprostadil/metabolismo , Transgenes , Cicloparafinas/metabolismo , Odorantes , Receptores Acoplados a Proteínas G/metabolismo , Dependovirus/genética , Vetores GenéticosRESUMO
ABSTRACT: As the pathogenesis of arterial thrombosis often includes platelet adhesion and aggregation, antiplatelet agents are commonly used to prevent thromboembolic events. Here, a new microfluidic method without additional adhesion protein modification was developed to quantify the inhibitory effect of antiplatelet drugs on the adhesion and aggregation behavior of platelets on glass surfaces under physiological flow conditions. Polydimethylsiloxane-glass microfluidic chips were fabricated by soft photolithography. Blood samples from healthy volunteers or patients before and after taking antiplatelet drugs flowed through the microchannels at wall shear rates of 300 and 1500 second -1 , respectively. The time to reach 2.5% platelet aggregation surface coverage (Ti), surface coverage (A 150s ), and mean fluorescence intensity (F 150s ) were used as quantitative indicators. Aspirin (80 µM) prolonged Ti and reduced F 150s . Alprostadil, ticagrelor, eptifibatide, and tirofiban prolonged Ti and reduced A 150s and F 150s in a concentration-dependent manner, whereas high concentrations of alprostadil did not completely inhibit platelet aggregation. Aspirin combined with ticagrelor synergistically inhibited platelet adhesion and aggregation; GPIb-IX-von Willebrand factor inhibitors partially inhibited platelet aggregation, and the inhibition was more pronounced at 1500 than at 300 second -1 . Patient administration of aspirin or (and) clopidogrel inhibited platelet adhesion and aggregation on the glass surface under flow conditions. This technology is capable of distinguishing the pharmacological effects of various antiplatelet drugs on inhibition of platelet adhesion aggregation on glass surface under physiological flow conditions, which providing a new way to develop microfluidic platelet function detection method without additional adhesive protein modification for determining the inhibitory effects of antiplatelet drugs in the clinical setting.
Assuntos
Microfluídica , Inibidores da Agregação Plaquetária , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Ticagrelor/farmacologia , Alprostadil/metabolismo , Alprostadil/farmacologia , Fator de von Willebrand/metabolismo , Fator de von Willebrand/farmacologia , Plaquetas , Agregação Plaquetária , Aspirina/farmacologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/farmacologiaRESUMO
We evaluated the suitability of Komagataeibacter europaeus, a vinegar production organism adept at synthetic media growth, as a host for heterologous gene expression. Cryptic plasmids (pGE1 and pGE2 derivatives) from K. europaeus strain KGMA0119 were employed as vectors for heterologous gene expression. The focus was placed on the groES promoter as a potential inducible switch. The groES promoter was fused with the EGFP gene and introduced into a pGE1 derivative to assess its suitability. Ethanol, acetic acid, and heat stresses were examined under various conditions for induction. EGFP transcription surged 600-fold when late logarithmic phase K. europaeus cells, cultured at 30 °C, endured heat stress at 40 °C, coupled with 20% acetic acid and 30% ethanol stress after an additional 6-hour cultivation. This robust induction system was then applied to express two proteins, Tth pol from the thermophilic bacterium Thermus thermophilus strain M1 and UPV230, a restriction enzyme from the acid-tolerant microorganism Ureaplasma parvum, known to cause vaginal infections and miscarriages. Both Tth pol and UPV230 were successfully expressed in K. europaeus cells and purified. The recovery of Tth pol from K. europaeus cells (480⯵g protein per liter culture) was approximately half that from E. coli (960⯵g protein per liter culture). In contrast, UPV230 recovery from K. europaeus cells (640⯵g protein per liter culture) was nearly 10 times higher than that from Escherichia coli (66⯵g protein per liter). The data highlights the potential of acetic acid bacteria as a host for producing acidophilic proteins. The shift in recognition from a 6-base sequence to a 4-base sequence of UPV230 was observed, accompanied by a change in structure as the pH transitioned from acidic pH to near-neutral pH.
Assuntos
Ácido Acético , Escherichia coli , Ácido Acético/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Alprostadil/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Etanol/metabolismoRESUMO
OBJECTIVE: To observe the morphological characteristics of clusters of Muse cells from normal human dermal fibroblasts (NHDFs) under different culture conditions. METHODS: Muse cells were sorted by magnetic activated cell sorting (MACS) from NHDFs, and were evaluated by flow cytometry. Muse cells were cultured in suspension and in adherent conditions to obtain Muse cell clusters (M-clusters), which were further characterized by alkaline phosphatase (AP) staining, immunofluorescence (IF) staining and transmission electron microscopy (TEM). The M-clusters were further cultured on Lando artificial dermal regeneration matrix (LADRM) for analysis by scanning electron microscopy (SEM) and IF staining of frozen sections. RESULTS: The proportion of SSEA3 and CD105 double-positive cells obtained by MACS was 87.4%. The sorted cells rapidly formed M-clusters after suspension culture, and showed internal characteristics of stem cells under TEM. After adherent culture, M-clusters stained positively for AP, SSEA-3 and OCT-4. Each M-cluster on the surface of the LADRM displayed an outer membrane of amorphous materials under SEM. Frozen sections and fluorescence staining of LADRM loaded with M-clusters showed an uneven fluorescence intensity of SSEA-3 within the clusters. CONCLUSIONS: Muse cells sorted by MACS from NHDFs could generate M-clusters, which included cells of different stemness and are wrapped in membrane-like structures.
Assuntos
Alprostadil , Fibroblastos , Humanos , Diferenciação Celular , Células Cultivadas , Alprostadil/metabolismo , PeleRESUMO
BACKGROUND: Human multilineage-differentiating stress enduring (Muse) cells are nontumorigenic endogenous pluripotent-like stem cells that can be easily obtained from various adult or fetal tissues. Regenerative effects of Muse cells have been shown in some disease models. Muse cells specifically home in damaged tissues where they exert pleiotropic effects. Exposition of the small intestine to high doses of irradiation (IR) delivered after radiotherapy or nuclear accident results in a lethal gastrointestinal syndrome (GIS) characterized by acute loss of intestinal stem cells, impaired epithelial regeneration and subsequent loss of the mucosal barrier resulting in sepsis and death. To date, there is no effective medical treatment for GIS. Here, we investigate whether Muse cells can prevent lethal GIS and study how they act on intestinal stem cell microenvironment to promote intestinal regeneration. METHODS: Human Muse cells from Wharton's jelly matrix of umbilical cord (WJ-Muse) were sorted by flow cytometry using the SSEA-3 marker, characterized and compared to bone-marrow derived Muse cells (BM-Muse). Under gas anesthesia, GIS mice were treated or not through an intravenous retro-orbital injection of 50,000 WJ-Muse, freshly isolated or cryopreserved, shortly after an 18 Gy-abdominal IR. No immunosuppressant was delivered to the mice. Mice were euthanized either 24 h post-IR to assess early small intestine tissue response, or 7 days post-IR to assess any regenerative response. Mouse survival, histological stainings, apoptosis and cell proliferation were studied and measurement of cytokines, recruitment of immune cells and barrier functional assay were performed. RESULTS: Injection of WJ-Muse shortly after abdominal IR highly improved mouse survival as a result of a rapid regeneration of intestinal epithelium with the rescue of the impaired epithelial barrier. In small intestine of Muse-treated mice, an early enhanced secretion of IL-6 and MCP-1 cytokines was observed associated with (1) recruitment of monocytes/M2-like macrophages and (2) proliferation of Paneth cells through activation of the IL-6/Stat3 pathway. CONCLUSION: Our findings indicate that a single injection of a small quantity of WJ-Muse may be a new and easy therapeutic strategy for treating lethal GIS.
Assuntos
Alprostadil , Células-Tronco Mesenquimais , Adulto , Camundongos , Humanos , Animais , Diferenciação Celular/fisiologia , Alprostadil/metabolismo , Células-Tronco Mesenquimais/metabolismo , Interleucina-6/metabolismo , IntestinosRESUMO
Stem cell transplantation has recently demonstrated a significant therapeutic efficacy in various diseases. Multilineage-differentiating stress-enduring (Muse) cells are stress-tolerant endogenous pluripotent stem cells that were first reported in 2010. Muse cells can be found in the peripheral blood, bone marrow and connective tissue of nearly all body organs. Under basal conditions, they constantly move from the bone marrow to peripheral blood to supply various body organs. However, this rate greatly changes even within the same individual based on physical status and the presence of injury or illness. Muse cells can differentiate into all three-germ-layers, producing tissue-compatible cells with few errors, minimal immune rejection and without forming teratomas. They can also endure hostile environments, supporting their survival in damaged/injured tissues. Additionally, Muse cells express receptors for sphingosine-1-phosphate (S1P), which is a protein produced by damaged/injured tissues. Through the S1P-S1PR2 axis, circulating Muse cells can preferentially migrate to damaged sites following transplantation. In addition, Muse cells possess a unique immune privilege system, facilitating their use without the need for long-term immunosuppressant treatment or human leucocyte antigen matching. Moreover, they exhibit anti-inflammatory, anti-apoptotic and tissue-protective effects. These characteristics circumvent all challenges experienced with mesenchymal stem cells and induced pluripotent stem cells and encourage the wide application of Muse cells in clinical practice. Indeed, Muse cells have the potential to break through the limitations of current cell-based therapies, and many clinical trials have been conducted, applying intravenously administered Muse cells in stroke, myocardial infarction, neurological disorders and acute respiratory distress syndrome (ARDS) related to novel coronavirus (SARS-CoV-2) infection. Herein, we aim to highlight the unique biological properties of Muse cells and to elucidate the advantageous difference between Muse cells and other types of stem cells. Finally, we shed light on their current therapeutic applications and the major obstacles to their clinical implementation from laboratory to clinic.
Assuntos
COVID-19 , Células-Tronco Pluripotentes , Humanos , Diferenciação Celular , Alprostadil/metabolismo , COVID-19/metabolismo , SARS-CoV-2 , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-TroncoRESUMO
BACKGROUND: Recent preclinical studies have demonstrated that bone marrow (BM)-derived Muse cells have a homing mechanism to reach damaged cardiac tissue while also being able to reduce myocardial infarct size and improve cardiac function; however, the potential of BM-Muse cells to foster new blood-vessel formation has not been fully assessed. Up to date, adipose tissue (AT)-derived Muse cells remain to be studied in acute myocardial infarction (AMI). The aim of the present study was to analyze in vitro and in vivo the neovascularization capacity of AT-Muse cells while exploring their biodistribution and differentiation potential in a translational ovine model of AMI. METHODS AND RESULTS: AT-Muse cells were successfully isolated from ovine adipose tissue. In adult sheep, one or more diagonal branches of the left anterior descending coronary artery were permanently ligated for thirty minutes. Sheep were randomized in two groups and treated with intramyocardial injections: Vehicle (PBS, n = 4) and AT-Muse (2x107 AT-Muse cells labeled with PKH26 Red Fluorescent Dye, n = 4). Molecular characterization showed higher expression of angiogenic genes (VEGF, PGF and ANG) and increased number of tube formation in AT-Muse cells group compared to Adipose-derived mesenchymal stromal cells (ASCs) group. At 7 days post-IAM, the AT-Muse group showed significantly more arterioles and capillaries than the Vehicle group. Co-localization of PKH26+ cells with desmin, sarcomeric actin and troponin T implied the differentiation of Muse cells to a cardiac fate; moreover, PKH26+ cells also co-localized with a lectin marker, suggesting a possible differentiation to a vascular lineage. CONCLUSION: Intramyocardially administered AT-Muse cells displayed a significant neovascularization activity and survival capacity in an ovine model of AMI.
Assuntos
Alprostadil , Infarto do Miocárdio , Animais , Ovinos , Alprostadil/metabolismo , Distribuição Tecidual , Infarto do Miocárdio/terapia , Infarto do Miocárdio/metabolismo , Adipócitos/metabolismoRESUMO
Mesenchymal stem cells (MSCs) are increasingly used as regenerative therapies for patients in the preclinical and clinical phases of various diseases. However, the main limitations of such therapies include functional heterogeneity and the lack of appropriate quality control (QC) methods for functional screening of MSC lines; thus, clinical outcomes are inconsistent. Recently, machine learning (ML)-based methods, in conjunction with single-cell morphological profiling, have been proposed as alternatives to conventional in vitro/vivo assays that evaluate MSC functions. Such methods perform in silico analyses of MSC functions by training ML algorithms to find highly nonlinear connections between MSC functions and morphology. Although such approaches are promising, they are limited in that extensive, high-content single-cell imaging is required; moreover, manually identified morphological features cannot be generalized to other experimental settings. To address these limitations, we propose an end-to-end deep learning (DL) framework for functional screening of MSC lines using live-cell microscopic images of MSC populations. We quantitatively evaluate various convolutional neural network (CNN) models and demonstrate that our method accurately classifies in vitro MSC lines to high/low multilineage differentiating stress-enduring (MUSE) cells markers from multiple donors. A total of 6,120 cell images were obtained from 8 MSC lines, and they were classified into two groups according to MUSE cell markers analyzed by immunofluorescence staining and FACS. The optimized DenseNet121 model showed area under the curve (AUC) 0.975, accuracy 0.922, F1 0.922, sensitivity 0.905, specificity 0.942, positive predictive value 0.940, and negative predictive value 0.908. Therefore, our DL-based framework is a convenient high-throughput method that could serve as an effective QC strategy in future clinical biomanufacturing processes.
Assuntos
Aprendizado Profundo , Células-Tronco Mesenquimais , Humanos , Ensaios de Triagem em Larga Escala , Alprostadil/metabolismo , Aprendizado de MáquinaRESUMO
Gap junctions (GJ) are suggested to support stem cell differentiation. The Muse cells that are applied in clinical trials are non-tumorigenic pluripotent-like endogenous stem cells, can be collected as stage-specific embryonic antigen 3 (SSEA-3+) positive cells from multiple tissues, and show triploblastic differentiation and self-renewability at a single cell level. They were reported to up-regulate pluripotency gene expression in suspension. We examined how GJ inhibition affected pluripotency gene expression in adherent cultured-Muse cells. Muse cells, mainly expressing gap junction alpha-1 protein (GJA1), reduced GJ intercellular communication from ~85% to 5-8% after 24 h incubation with 120 µM 18α-glycyrrhetinic acid, 400 nM 12-O-tetradecanoylphorbol-13-acetate, and 90 µM dichlorodiphenyltrichloroethane, as confirmed by a dye-transfer assay. Following inhibition, NANOG, OCT3/4, and SOX2 were up-regulated 2-4.5 times more; other pluripotency-related genes, such as KLF4, CBX7, and SPRY2 were elevated; lineage-specific differentiation-related genes were down-regulated in quantitative-PCR and RNA-sequencing. Connexin43-siRNA introduction also confirmed the up-regulation of NANOG, OCT3/4, and SOX2. YAP, a co-transcriptional factor in the Hippo signaling pathway that regulates pluripotency gene expression, co-localized with GJA1 (also known as Cx43) in the cell membrane and was translocated to the nucleus after GJ inhibition. Adherent culture is usually more suitable for the stable expansion of cells than is a suspension culture. GJ inhibition is suggested to be a simple method to up-regulate pluripotency in an adherent culture that involves a Cx43-YAP axis in pluripotent stem cells, such as Muse cells.
Assuntos
Conexina 43 , Células-Tronco Pluripotentes , Alprostadil/metabolismo , Comunicação Celular , Conexina 43/genética , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Expressão Gênica , Células-Tronco Pluripotentes/metabolismoRESUMO
Considering the rapidly increasing prevalence of obesity worldwide, the number of weight control drugs is very few. Incretin-based therapies are currently being developed to achieve weight control, and Glucagon-Like Peptide-1 Receptor Agonists (GLP-1RA) are used in incretin-based therapies. This study aimed to investigate the cytotoxicity of exenatide, a GLP-1A, on 3T3-L1 adipocytes and the effect of exenatide on the expression of adipogenesis-related genes, insulin and glucose levels, and apoptosis. Cytotoxic activity of exenatide on 3T3-L1 adipocytes was determined by MTT method. Gene expression levels were determined by qPCR. Apoptosis studies were performed on the Muse Cell Analyzer. C1q/TNF-related protein-3 (CTRP3) expression levels were found to be higher in exenatide treated adipocyte cells than in control cells (p < 0.001). Adipocyte cells treated with exenatide were found to have lower PPAR-γ gene expression levels when compared to control adipocyte cells (p < 0.001). Intracellular insulin (p < 0.001) and glucose levels were higher in 3T3-L1 adipocytes treated with exenatide compared to control adipocyte cells. Total apoptosis increased approximately 1.5 times as a result of exenatide administration. The increase in CTRP3 gene expression, which is thought to be a new biomarker for obesity, and the decrease in PPAR-γ gene expression indicate that exenatide is a promising new pharmacotherapeutic agent in the treatment of obesity by regulating the expression of genes related to adipogenesis and lipogenesis and inducing apoptosis.
Assuntos
Adipogenia , Incretinas , Camundongos , Animais , Exenatida/farmacologia , Exenatida/genética , Exenatida/uso terapêutico , Incretinas/metabolismo , Incretinas/farmacologia , Incretinas/uso terapêutico , Alprostadil/metabolismo , Alprostadil/farmacologia , Alprostadil/uso terapêutico , Complemento C1q/genética , Complemento C1q/metabolismo , Complemento C1q/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/uso terapêutico , Adipócitos , Células 3T3-L1 , PPAR gama/metabolismo , Obesidade/genética , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Insulina/farmacologia , Insulina/metabolismo , Glucose/metabolismo , Apoptose , Expressão Gênica , Diferenciação CelularRESUMO
Stem cell-based regenerative therapies have recently become promising and advanced for treating stroke. Mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) have received the most attention for treating stroke because of the outstanding paracrine function of MSCs and the three-germ-layer differentiation ability of iPSCs. However, the unsatisfactory homing ability, differentiation, integration, and survival time in vivo limit the effectiveness of MSCs in regenerative medicine. The inherent tumorigenic property of iPSCs renders complete differentiation necessary before transplantation, which is complicated and expensive and affects the consistency among cell batches. Multilineage differentiating stress-enduring (Muse) cells are natural pluripotent stem cells in the connective tissues of nearly every organ and thus are considered nontumorigenic. A single Muse cell can differentiate into all three-germ-layer, preferentially migrate to damaged sites after transplantation, survive in hostile environments, and spontaneously differentiate into tissue-compatible cells, all of which can compensate for the shortcomings of MSCs and iPSCs. This review summarizes the recent progress in understanding the biological properties of Muse cells and highlights the differences between Muse cells and other types of stem cells. Finally, we summarized the current research progress on the application of Muse cells on stroke and challenges from bench to bedside.
Assuntos
Células-Tronco Mesenquimais , Células-Tronco Pluripotentes , Acidente Vascular Cerebral , Alprostadil/metabolismo , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes/metabolismo , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/terapiaRESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is associated with oxidative stress and affects the survival and homing of transplanted mesenchymal stem cells (MSCs) as well as cytokine secretion by the MSCs, thereby altering their therapeutic potential. In this study, we preconditioned the MSCs with prostaglandin E1 (PGE1) and performed in vitro and in vivo cell experiments to evaluate the therapeutic effects of MSCs in rats with PAH. METHODS: We studied the relationship between PGE1 and vascular endothelial growth factor (VEGF) secretion, B-cell lymphoma 2 (Bcl-2) expression, and C-X-C chemokine receptor 4 (CXCR4) expression in MSCs and MSC apoptosis as well as migration through the hypoxia-inducible factor (HIF) pathway in vitro. The experimental rats were randomly divided into five groups: (I) control group, (II) monocrotaline (MCT) group, (III) MCT + non-preconditioned (Non-PC) MSC group, (IV) MCT + PGE1-preconditioned (PGE1-PC) MSC group, and (V) MCT+PGE1+YC-1-PCMSC group. We studied methane dicarboxylic aldehyde (MDA) levels, MSC homing to rat lungs, mean pulmonary artery pressure, pulmonary artery systolic pressure, right ventricular hypertrophy index, wall thickness index (%WT), and relative wall area index (%WA) of rat pulmonary arterioles. RESULTS: Preconditioning with PGE1 increased the protein levels of HIF-1 alpha (HIF-1α) in MSCs, which can reduce MSC apoptosis and increase the protein levels of CXCR4, MSC migration, and vascular endothelial growth factor secretion. Upon injection with PGE1-PCMSCs, the pulmonary artery systolic pressure, mean pulmonary artery pressure, right ventricular hypertrophy index, %WT, and %WA decreased in rats with PAH. PGE1-PCMSCs exhibited better therapeutic effects than non-PCMSCs. Interestingly, lificiguat (YC-1), an inhibitor of the HIF pathway, blocked the effects of PGE1 preconditioning. CONCLUSIONS: Our findings indicate that PGE1 modulates the properties of MSCs by regulating the HIF pathway, providing insights into the mechanism by which PGE1 preconditioning can be used to improve the therapeutic potential of MSCs in PAH.
Assuntos
Hipertensão Pulmonar , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Hipertensão Arterial Pulmonar , Alprostadil/metabolismo , Animais , Apoptose , Hipertensão Pulmonar/patologia , Hipertrofia Ventricular Direita/patologia , Células-Tronco Mesenquimais/metabolismo , Monocrotalina , Ratos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The aim of this study is to characterize the molecular properties of multilineage differentiating stress-enduring (Muse) cells compared with dermal fibroblasts (FBs) and to characterize differences in their transcriptomes and open chromatin regions that are involved in cellular plasticity. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) analyses was then performed on FBs and Muse cells. Subsequently, cell type-selective gene regulatory regions were identified by coalition analysis. Expression patterns of transcription factors (TFs) and signaling pathways intermediates were verified using quantitative real-time polymerase chain reaction and Western blot analyses. RNA-seq identified 2355 significantly differentially expressed genes (DEGs) that regulate the transcriptome, including 1222 upregulated and 1133 downregulated DEGs. The general panorama of RNA-seq and ATAC-seq analyses confirmed the differences in TFs and open chromatin regions between FBs and Muse cells. ATAC-seq analysis showed that Muse cells had more reproducible and meaningful peaks than FBs, and the peak signals were concentrated near promoter-transcription start site areas. In genomic regions that can be preferentially accessed in FBs and Muse cells, more than 200 TFs had binding motif sequences. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and coalition analyses identified differences in factors involved in the cell cycle and the protein kinase B (AKT) signaling pathway of FBs and Muse cells. The results of RNA-seq and ATAC-seq analyses clarified the genetic basis of the different biological properties of Muse cells and FBs. These results suggest that the cell cycle transition and the AKT signaling pathway may affect the morphology and biological characteristics of Muse cells.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Proteínas Proto-Oncogênicas c-akt , Alprostadil/metabolismo , Cromatina/metabolismo , Fibroblastos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA-Seq , Análise de Sequência de RNARESUMO
Obesity is a chronic disease associated with increased morbidity and mortality. The rapidly increasing prevalence of obesity makes it a global health problem, while treatment options remain limited. Given the potential of boron in the treatment of obesity, the aim of this study is to investigate the anti-adipogenic activity of the newly synthesised boron glycine monoester compound (BGM) using 3T3-L1 adipocytes by analysing lipid accumulation, CTRP3 and PPARy gene expression, oxidative stress and apoptotic effects. 3T3-L1 fibroblast cells (ATCC® CL-173) were transformed into adipocyte cells in vitro. Fat accumulation in the 3T3-L1 adipocyte cells was detected by Oil Red O staining. Gene expression levels were determined with qPCR. Biochemical analyzes were performed using spectrophotometric method (CAT, ALP and ACP) and ELISA kit (TAS, TOS, NADP-IDH). Apoptosis studies were performed on the muse cell nalyser using the Muse Annexin V & Dead Cell Assay Kit. When BGM-treated cells were compared to control adipocyte cells, lipid accumulation decreased in a dose-dependent manner. BGM-treated adipocyte cells had higher CTRP3 expression levels and lower PPAR-γ gene expression levels compared to control adipocyte cells (p < 0.001). While BGM application increased the TAS level, it showed an antioxidant effect by regulating the activity of oxidative metabolism enzymes (p < 0.001). BGM application increased total apoptosis by 1.5-fold. These results show that BGM is a potential therapeutic agent for obesity by regulating the expression of genes related to adipogenesis and lipogenesis in adipocyte cells and by affecting the activity of enzymes of oxidative metabolism and apoptosis.
Assuntos
Boro , PPAR gama , Células 3T3-L1 , Adipócitos , Adipogenia , Alprostadil/metabolismo , Alprostadil/farmacologia , Alprostadil/uso terapêutico , Animais , Anexina A5/metabolismo , Anexina A5/farmacologia , Anexina A5/uso terapêutico , Antioxidantes/metabolismo , Apoptose , Boro/farmacologia , Diferenciação Celular , Glicina/farmacologia , Lipogênese , Camundongos , NADP/metabolismo , NADP/farmacologia , NADP/uso terapêutico , Obesidade/metabolismo , Estresse Oxidativo , PPAR gama/genética , PPAR gama/metabolismoRESUMO
Cyclooxygenase metabolizes dihomo-γ-linolenic acid and arachidonic acid to form prostaglandin (PG) E, including PGE1 and PGE2, respectively. Although PGE2 is well known to play an important role in the development and maintenance of hyperalgesia and allodynia, the role of PGE1 in pain is unknown. We confirm whether PGE1 induced pain using orofacial pain behavioral test in mice and determine the target molecule of PGE1 in TG neurons with whole-cell patch-clamp and immunohistochemistry. Intradermal injection of PGE1 to the whisker pads of mice induced a reduced threshold, enhancing the excitability of HCN channel-expressing trigeminal ganglion (TG) neurons. The HCN channel-generated inward current (Ih) was increased by 135.3 ± 4.8% at 100 nM of PGE1 in small- or medium-sized TG, and the action of PGE1 on Ih showed a concentration-dependent effect, with a median effective dose (ED50) of 29.3 nM. Adenylyl cyclase inhibitor (MDL12330A), 8-bromo-cAMP, and the EP2 receptor antagonist AH6809 inhibited PGE1-induced Ih. Additionally, PGE1-induced mechanical allodynia was blocked by CsCl and AH6809. PGE1 plays a role in mechanical allodynia through HCN2 channel facilitation via the EP2 receptor in nociceptive neurons, suggesting a potential therapeutic target in that PGE1 could be involved in pain as endogenous substances under inflammatory conditions.
Assuntos
Alprostadil/metabolismo , Gânglios Espinais/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Dor/metabolismo , Canais de Potássio/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Gânglio Trigeminal/metabolismo , Potenciais de Ação/fisiologia , Animais , Hiperalgesia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuralgia/metabolismo , Nociceptores/metabolismo , Medição da Dor/métodosRESUMO
This study aimed to investigate the stress tolerance mechanism of multilineage-differentiating stress enduring (Muse) cells and elucidate the means to improve the stress tolerance of mesenchymal stem cells. Cell viability, apoptosis, and senescence-related protein expression were detected under H2O2 stress by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium reduction assay, flow cytometry in combination with Annexin V-FITC/PI staining, and western blotting analysis, respectively. A significant increase in the CCNA2 gene level within Muse cells relative to adipose stem cells (ASCs) was observed. In the H2O2 stress environment in vitro, the survival rate of Muse cells remarkably increased compared with the survival rate of the ASCs. In addition, a reduced level of apoptosis and senescence-related protein expression of Muse cells relative to ASCs was documented. The miR-29b-3p-induced negative regulation of CCNA2 gene expression was confirmed by in vitro luciferase assay. A significant upregulation of CCNA2 gene expression in ASCs, transfected with antagomir-29b-3p, improved the survival rate of ASCs under H2O2 stress but dramatically reduced the apoptosis and expression of the senescence-related gene; agomir-29b-3p could partially reverse these effects. In conclusion, high expression of the CCNA2 gene is associated with an increased stress tolerance of Muse cells. Regulating the expression of CCNA2 by miR-29b-3p can alter the stress tolerance of ASCs.
Assuntos
Ciclina A2 , Células-Tronco Mesenquimais , MicroRNAs , Tecido Adiposo/metabolismo , Alprostadil/metabolismo , Alprostadil/farmacologia , Apoptose/genética , Ciclina A2/metabolismo , Peróxido de Hidrogênio/farmacologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismoRESUMO
Having played homeostatic role, the immune system maintains the integrity of the body. Such a characteristic makes immune system as an attractive candidate for resolution of inflammatory disease followed by tissue repair. As first responder cells, neutrophils direct immune response playing key role in tissue remodeling. Previous studies revealed that sperm attracts neutrophils and promotes uterine remodeling suitable for fetus growth. Accordingly, sperm and more efficiently sperm head had remodeling effects on damaged brain in Alzheimer's disease (AD) model. To further reveal the mechanism, two kinds of in vivo study, including kinetic study and inhibition of neutrophil phagocytosis on AD model, as well as in vitro study using co-culture of neutrophil and sperm head were performed. Kinetic study revealed that sperm head recruited neutrophil to nasal mucosa similar to that of uterus and sperm head-phagocytizing neutrophils acquired new activation status comparing to control. In vitro study also demonstrated that sperm head-phagocytizing neutrophils acquire new activation status and express coding RNAs of sperm head. Accordingly, inhibition of neutrophil phagocytic activity abrogated therapeutic effects of sperm head. Neutrophils activation status is important in the fate of inflammatory process. Modulation but not suppression of neutrophils helps remodeling and repair of damaged tissue. Sperm head is an intelligent cell and not just a simple particle to remove by phagocytosis but instead can program neutrophils and consequently immune response into reparative mode after phagocytosis.
Assuntos
Doença de Alzheimer/terapia , Neutrófilos/imunologia , Fagocitose/imunologia , Cabeça do Espermatozoide/transplante , Administração Intranasal , Alprostadil/metabolismo , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/toxicidade , Animais , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Humanos , Masculino , Mucosa Nasal , Ativação de Neutrófilo , Neutrófilos/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/toxicidade , Cultura Primária de Células , RNA/metabolismo , Ratos , Receptor Tipo 1 de Angiotensina/metabolismoRESUMO
Grape seed procyanidin extract (GSE) has been shown to exert antineoplastic properties in preclinical studies. Recently, we reported findings from a modified phase I, open-label, dose escalation clinical study conducted to evaluate the safety, tolerability, MTD, and potential chemopreventive effects of leucoselect phytosome, a standardized GSE complexed with soy phospholipids to enhance bioavailability, in heavy active and former smokers. Three months of leucoselect phytosome treatment significantly decreased bronchial Ki-67 labeling index (LI), a marker of cell proliferation on the bronchial epithelium. Because GSE is widely used as a supplement to support cardiovascular health, we evaluate the impact of oral leucoselect phytosome on the fasting serum complex lipid metabolomics profiles in our participants. One month of leucoselect phytosome treatment significantly increased eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the omega-3 polyunsaturated fatty acids (n-3 PUFA) with well-established anticancer properties. Leucoselect phytosome also significantly increased unsaturated phosphatidylcholines (PC), likely from soy phospolipids in the phytosome and functioning as transporters for these PUFAs. Furthermore, 3-month leucoselect phytosome treatment significantly increased serum prostaglandin (PG) E3 (PGE3), a metabolite of EPA with anti-inflammatory and antineoplastic properties. Such increases in PGE3 correlated with reductions of bronchial Ki-67 LI (r = -0.9; P = 0.0374). Moreover, posttreatment plasma samples from trial participants significantly inhibited proliferation of human lung cancer cell lines A549 (adenocarcinoma), H520 (squamous cell carcinoma), DMS114 (small cell carcinoma), and 1198 (preneoplastic cell line). Our findings further support the potential utility of leucoselect phytosome in reducing cardiovascular and neoplastic risks in heavy former and active smokers. PREVENTION RELEVANCE: In this correlative study of leucoselect phytosome for lung cancer chemoprevention in heavy active and former smokers, we demonstrate for the first time, favorable modulations of n-3PUFA and downstream PGE3 in fasting serum, further supporting the chemopreventive potential of leucoselect phytosome against lung cancer.
Assuntos
Extrato de Sementes de Uva/administração & dosagem , Neoplasias Pulmonares/prevenção & controle , Administração Oral , Alprostadil/análogos & derivados , Alprostadil/sangue , Alprostadil/metabolismo , Brônquios/patologia , Linhagem Celular Tumoral , Ácidos Docosa-Hexaenoicos/sangue , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/sangue , Ácido Eicosapentaenoico/metabolismo , Extrato de Sementes de Uva/efeitos adversos , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Resultado do TratamentoRESUMO
Objectives: Dual platelet inhibition is commonly used for prevention of cardiovascular events in patients undergoing neuroendovascular procedures. Non-responsiveness to platelet inhibitors may be associated with adverse outcomes. The aim of this study was to evaluate the reliability of the platelet function analyzer PFA-100® in comparison to light transmittance aggregometry (LTA) for monitoring clopidogrel and acetylsalicylic acid (ASA) non-responsiveness in a cohort of patients treated for intracranial aneurysm or cranial artery stenosis. Methods: Non-responsiveness to clopidogrel and ASA was assessed by LTA using adenosine diphosphate (ADP) and arachidonic acid and by PFA-100® with the ADP/prostaglandin E1 (PGE1) and collagen/epinephrine cartridges, respectively. Results: A total of 203 patients (145 females; median age, 57 years) were analyzed. Agreement between the two tests was poor for clopidogrel non-responsiveness (Æ=0.19) and not better than chance for ASA non-responsiveness (Æ=0.01). Clopidogrel non-responsiveness by LTA and PFA-100® was associated with higher von Willebrand factor antigen and activity levels. ADP-induced platelet disaggregation was lower in patients with clopidogrel non-responsiveness as assessed by PFA-100®. Clopidogrel non-responsiveness by LTA was associated with a higher prevalence of diabetes and a higher body mass index (BMI). Adverse outcomes (death, thromboembolism, or in-stent thrombosis) occurred in 13% (n=26) of all patients independently of ASA and clopidogrel non-responsiveness as assessed by both devices. Conclusions: Our results show that LTA and PFA-100® are not interchangeable in the assessment of ASA and clopidogrel non-responsiveness in patients undergoing neuroendovascular interventions.