RESUMO
Objective. Carboxypeptidase E (CPE) plays an important role in the biosynthesis of neurotransmitters and peptide hormones including insulin. It also promotes cell proliferation, survival, and invasion of tumor cells. The endoplasmic reticulum stress, hypoxia, and nutrient supply are significant factors of malignant tumor growth including glioblastoma. There are data indicating that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) suppressed glioblastoma cell proliferation and increased invasiveness of these cells. The present study aims to investigate the regulation of the CPE gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in the regulation of this gene expression and function in tumorigenesis. Methods. Human glioblastoma cells U87MG (transfected by an empty vector; control) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine; for glucose and glutamine deprivations, the cells were cultured in DMEM medium without glucose or glutamine for 16 h, respectively. The expression level of the CPE gene was studied by quantitative RT-PCR and normalized to ACTB. Results. It was found that inhibition of endoribonuclease and protein kinase activities of ERN1 led to a strong up-regulation of CPE gene expression in glioblastoma cells. The expression of this gene also increased in glioblastoma cells after silencing ERN1. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease only. The expression of the CPE gene was resistant to hypoxia in control U87MG cells, but increased in cells with ERN1 knockdown. The expression of this gene was up-regulated under glutamine deprivation in control glioblastoma cells, but decreased upon ERN1 knockdown. However, glucose deprivation decreased the expression of CPE gene in both types of used cells, but ERN1 inhibition enhanced this effect. Conclusion. The results of the present study demonstrate that inhibition of ERN1 strongly up-regulated the expression of pro-oncogenic CPE gene through protein kinase activity of ERN1 and that increased CPE gene expression possibly participates in ERN1 knockdown-mediated invasiveness of glioblastoma cells.
Assuntos
Carboxipeptidase H , Estresse do Retículo Endoplasmático , Endorribonucleases , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Proteínas Serina-Treonina Quinases , Humanos , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/patologia , Carboxipeptidase H/metabolismo , Carboxipeptidase H/genética , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Endorribonucleases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Glucose/metabolismo , Técnicas de Silenciamento de Genes , Hipóxia Celular/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Background: Hypoxic pulmonary hypertension (HPH) is one of the important pathophysiological changes in chronic pulmonary heart disease. Hypoxia promotes the phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs). Extracellular exosomes regulate vascular smooth muscle cell (VSMC) phenotypic switch. Aim: Given the importance of exosomes and alveolar epithelial cells (AECs) in HPH, the present study aimed to address the issue of whether AEC-derived exosomes promote HPH by triggering PASMC phenotypic switch. Methods: Cell Counting Kit-8 (CCK-8), TRITC-phalloidin staining, and Western blotting were used to examine the effects of AEC-derived exosomes on cell proliferation, intracellular actin backbone distribution, and expression of phenotypic marker proteins in PASMCs. Transcriptomics sequencing was used to analyze differentially expressed genes (DEGs) between groups. Results: Hypoxia-induced exosomes (H-exos) could promote the proliferation of PASMCs, cause the reduction of cellular actin microfilaments, promote the expression of synthetic marker proteins (ELN and OPN), reduce the expression of contractile phenotypic marker proteins (SM22-α and α-SMA), and induce the phenotypic transformation of PASMCs. Transcriptomics sequencing analysis showed that the Rap1 signaling pathway was involved in the phenotypic transformation of PASMCs induced by H-exos. Conclusion: The present study identified that hypoxia-induced AEC-derived exosomes promote the phenotypic transformation of PASMCs and its mechanism is related to the Rap1 signaling pathway.
Assuntos
Proliferação de Células , Exossomos , Miócitos de Músculo Liso , Fenótipo , Artéria Pulmonar , Transdução de Sinais , Exossomos/metabolismo , Artéria Pulmonar/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Células Epiteliais Alveolares/metabolismo , Ratos , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Músculo Liso Vascular/metabolismo , Hipertensão Pulmonar/metabolismo , Ratos Sprague-Dawley , Células Cultivadas , Hipóxia/metabolismo , Hipóxia Celular/fisiologiaRESUMO
The experimental study and transplantation of pancreatic islets requires their isolation from the surrounding tissue, and therefore, from the vasculature. Under these conditions, avascular islets rely on the diffusion of peripheral oxygen and nutrients to comply with the requirements of islet cells while responding to changes in body glucose. As a complement to the experimental work, computational models have been widely used to estimate how avascular islets would be affected by the hypoxic conditions found both in culture and transplant sites. However, previous models have been based on simplified representations of pancreatic islets which has limited the reach of the simulations performed. Aiming to contribute with a more realistic model of avascular human islets, in this work we used architectures of human islets reconstructed from experimental data to simulate the availability of oxygen for α, ß and δ-cells, emulating culture and transplant conditions at different glucose concentrations. The modeling approach proposed allowed us to quantitatively estimate how the loss of cells due to severe hypoxia would impact interactions between islet cells, ultimately segregating the islet into disconnected subnetworks. According to the simulations performed, islet encapsulation, by reducing the oxygen available within the islets, could severely compromise cell viability. Moreover, our model suggests that even without encapsulation, only microislets composed of less than 100 cells would remain viable in oxygenation conditions found in transplant sites. Overall, in this article we delineate a novel modeling methodology to simulate detailed avascular islets in experimental and transplant conditions with potential applications in the field of islet encapsulation.
Assuntos
Sobrevivência Celular , Simulação por Computador , Glucose , Ilhotas Pancreáticas , Modelos Biológicos , Oxigênio , Humanos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/irrigação sanguínea , Oxigênio/metabolismo , Glucose/metabolismo , Sobrevivência Celular/fisiologia , Biologia Computacional , Transplante das Ilhotas Pancreáticas/métodos , Hipóxia Celular/fisiologiaRESUMO
INTRODUCTION: Pericytes wrap microvessels and interact with endothelial cells to regulate vascular growth. Though pericyte dropout has been reported in pathological human placentae and mouse models of placental pathology, there has been limited investigation of the role and function of placental pericytes in vascular health and pathology. This study aimed to investigate the angiogenic potential of human placental pericytes relative to other villous cell populations. METHODS: Primary human placental pericytes, human umbilical vein endothelial cells (HUVEC), and BeWo cells ( ± 20 µM forskolin) were cultured in 1 % O2 or ambient air, followed by analysis of secreted angiogenic factors (ELISA). Additionally, the placental pericytes and HUVECs were co-cultured in a 3D sprouting assay to assess the capacity of pericytes to contribute to vascular sprouts. RESULTS: 1 % O2 affected secretion of angiogenic factors in placental pericytes, HUVECs, and syncytialized BeWo cells. Specifically, in placental pericytes, angiopoietin-1 (ANG1) and soluble fms-like tyrosine kinase-1 (sFLT1) were decreased, while vascular endothelial growth factor (VEGF) was increased. In HUVECS, matrix metalloproteinase-2 (MMP2), VEGF, angiopoietin-2 (ANG2), platelet-derived growth factor beta (PDGFB), placental growth factor (PlGF), and sFLT1 were increased. In syncytialized BeWo cells, VEGF, MMP2, PDGFB, PlGF, and sFLT1 secretion were increased. Placental pericytes and HUVECS colocalized to vessel sprouts in the 3-D sprouting assay. DISCUSSION: Hypoxic conditions altered placental pericyte, endothelial, and syncytialized BeWo secretion of angiogenic factors. We speculate that pericyte dropout and, by extension, the loss of pericyte-derived angiogenic factors in hypoxic conditions may contribute to compromised fetal vascular development observed in placental pathologies.
Assuntos
Células Endoteliais da Veia Umbilical Humana , Pericitos , Placenta , Pericitos/metabolismo , Humanos , Feminino , Gravidez , Placenta/metabolismo , Placenta/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Hipóxia Celular/fisiologia , Técnicas de Cocultura , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Cultivadas , Hipóxia/metabolismoRESUMO
Hypoxia, as a prominent feature of the tumor microenvironment, has a profound impact on the multicomponent changes within this environment. Under hypoxic conditions, the malignant phenotype of tumor cells, the variety of cell types within the tumor microenvironment, as well as intercellular communication and material exchange, undergo complex alterations. These changes provide significant prospects for exploring the mechanisms of tumor development under different microenvironmental conditions and for devising therapeutic strategies. Exosomes secreted by tumor cells and stromal cells are integral components of the tumor microenvironment, serving as crucial mediators of intercellular communication and material exchange, and have consequently garnered increasing attention from researchers. This review focuses on the mechanisms by which hypoxic conditions promote the release of exosomes by tumor cells and alter their encapsulated contents. It also examines the effects of exosomes derived from tumor cells, immune cells, and other cell types under hypoxic conditions on the tumor microenvironment. Additionally, we summarize current research progress on the potential clinical applications of exosomes under hypoxic conditions and propose future research directions in this field.
Assuntos
Comunicação Celular , Exossomos , Neoplasias , Microambiente Tumoral , Exossomos/metabolismo , Humanos , Comunicação Celular/fisiologia , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Hipóxia Celular/fisiologia , Hipóxia Tumoral , Hipóxia/metabolismoRESUMO
Inflammatory response mediated by M1 microglia is a crucial factor leading to the exacerbation of brain injury after ischemic stroke (IS). Under the stimulation of IS, vascular smooth muscle cells (VSMCs) switch to the synthetic phenotype characterized by exosome secretion. Previous studies have shown that exosomes play an important role in the regulation of microglial polarization. We reported that exosomes derived from primary human brain VSMCs under hypoxia (HExos), but not those under normoxia (Exos), significantly promoted primary human microglia (HM1900) shift to M1 phenotype. Proteomic analysis showed that the Src protein enriched in HExos was a potential pro-inflammatory mediator. In vitro experiments showed that the expression of Src and M1 markers were upregulated in HM1900 co-incubated with HExos. However, the Src inhibitor dasatinib (DAS) significantly promoted the transformation of HM1900 phenotype from M1 to M2. In vivo experiments of pMCAO mice also revealed that DAS could effectively inhibit the activation of M1 microglia/macrophages, protect neurons from apoptosis, and improve neuronal function. These data suggested that hypoxic-VSMCs-derived exosomes were involved in post-IS inflammation by promoting M1 microglial polarization through Src transmission. Targeting inhibition of Src potentially acts as an effective strategy for treating brain injury after IS.
Assuntos
Exossomos , Microglia , Músculo Liso Vascular , Animais , Exossomos/metabolismo , Microglia/metabolismo , Microglia/efeitos dos fármacos , Humanos , Camundongos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Masculino , Quinases da Família src/metabolismo , Quinases da Família src/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Hipóxia Celular/fisiologia , Hipóxia Celular/efeitos dos fármacos , Polaridade Celular/fisiologia , Polaridade Celular/efeitos dos fármacos , Células CultivadasRESUMO
Carnitine palmitoyltransferase 1C (CPT1C) is an enzyme that regulates tumor cell proliferation and metabolism by modulating mitochondrial function and lipid metabolism. Hypoxia, commonly observed in solid tumors, promotes the proliferation and progression of pancreatic cancer by regulating the metabolic reprogramming of tumor cells. So far, the metabolic regulation of hypoxic tumor cells by CPT1C and the upstream mechanisms of CPT1C remain poorly understood. Yin Yang 1 (YY1) is a crucial oncogene for pancreatic tumorigenesis and acts as a transcription factor that is involved in multiple metabolic processes. This study aimed to elucidate the relationship between YY1 and CPT1C under hypoxic conditions and explore their roles in hypoxia-induced proliferation and metabolic alterations of tumor cells. The results showed enhancements in the proliferation and metabolism of PANC-1 cells under hypoxia, as evidenced by increased cell growth, cellular ATP levels, up-regulation of mitochondrial membrane potential, and decreased lipid content. Interestingly, knockdown of YY1 or CPT1C inhibited hypoxia-induced rapid cell proliferation and vigorous cell metabolism. Importantly, for the first time, we reported that YY1 directly activated the transcription of CPT1C and clarified that CPT1C was a novel target gene of YY1. Moreover, the YY1 and CPT1C were found to synergistically regulate the proliferation and metabolism of hypoxic cells through transfection with YY1 siRNA to CRISPR/Cas9-CPT1C knockout PANC-1 cells. Taken together, these results indicated that the YY1-CPT1C axis could be a new target for the intervention of pancreatic cancer proliferation and metabolism.
Assuntos
Carnitina O-Palmitoiltransferase , Proliferação de Células , Neoplasias Pancreáticas , Transdução de Sinais , Fator de Transcrição YY1 , Fator de Transcrição YY1/metabolismo , Fator de Transcrição YY1/genética , Carnitina O-Palmitoiltransferase/metabolismo , Carnitina O-Palmitoiltransferase/genética , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Proliferação de Células/fisiologia , Linhagem Celular Tumoral , Transdução de Sinais/fisiologia , Hipóxia Celular/fisiologiaRESUMO
The placenta experiences a low-oxygen stage during early pregnancy. Aspirin is an effective preventative treatment for preeclampsia if applied early in pregnancy. Elevation of fibronectin (FN) level has been reported to be associated with preeclampsia; however, the role of FN in the physiological hypoxic phase and whether aspirin exerts its effect on FN at this hypoxic stage remain unknown. We determined pregnancy outcomes by injecting saline or recombinant FN protein into C57BL/6 pregnant mice and one group of FN-injected mice was fed aspirin. The effects of FN, the underlying pathways on trophoblast biology, and cilia formation under hypoxia were investigated in FN-pretreated or FN-knockdown HTR-8/SVneo cells in a hypoxic chamber (0.1 % O2). Preeclampsia-like phenotypes, including blood pressure elevation and proteinuria, developed in FN-injected pregnant mice. The fetal weight of FN-injected mice was significantly lower than that of non-FN-injected mice (p < 0.005). Trophoblast FN expression was upregulated under hypoxia, which could be suppressed by aspirin treatment. FN inhibited trophoblast invasion and migration under hypoxia, and this inhibitory effect occurred through downregulating ZEB1/2, MMP 9 and the Akt and MAPK signaling pathways. Ciliogenesis of trophoblasts was stimulated under hypoxia but was inhibited by FN treatment. Aspirin was shown to reverse the FN-mediated inhibitory effect on trophoblast invasion/migration and ciliogenesis. In conclusion, FN overexpression induces preeclampsia-like symptoms and impairs fetal growth in mice. Aspirin may exert its suppressive effect on FN upregulation and FN-mediated cell function in the hypoxic stage of pregnancy and therefore provides a preventative effect on preeclampsia development.
Assuntos
Aspirina , Fibronectinas , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Pré-Eclâmpsia , Proteínas Proto-Oncogênicas c-akt , Trofoblastos , Animais , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/prevenção & controle , Pré-Eclâmpsia/patologia , Fibronectinas/metabolismo , Fibronectinas/genética , Feminino , Gravidez , Aspirina/farmacologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Humanos , Modelos Animais de Doenças , Cílios/efeitos dos fármacos , Cílios/metabolismo , Cílios/fisiologia , Fenótipo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Hipóxia/metabolismo , Linhagem CelularRESUMO
Cellular responses to hypoxia are crucial in various physiological and pathophysiological contexts and have thus been extensively studied. This has led to a comprehensive understanding of the transcriptional response to hypoxia, which is regulated by hypoxia-inducible factors (HIFs). However, the detailed molecular mechanisms of HIF regulation in hypoxia remain incompletely understood. In particular, there is controversy surrounding the production of mitochondrial reactive oxygen species (ROS) in hypoxia and how this affects the stabilization and activity of HIFs. This review examines this controversy and attempts to shed light on its origin. We discuss the role of physioxia versus normoxia as baseline conditions that can affect the subsequent cellular response to hypoxia and highlight the paucity of data on pericellular oxygen levels in most experiments, leading to variable levels of hypoxia that might progress to anoxia over time. We analyze the different outcomes reported in isolated mitochondria, versus intact cells or whole organisms, and evaluate the reliability of various ROS-detecting tools. Finally, we examine the cell-type and context specificity of oxygen's various effects. We conclude that while recent evidence suggests that the effect of hypoxia on ROS production is highly dependent on the cell type and the duration of exposure, efforts should be made to conduct experiments under carefully controlled, physiological microenvironmental conditions in order to rule out potential artifacts and improve reproducibility in research.
Assuntos
Mitocôndrias , Espécies Reativas de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Humanos , Animais , Mitocôndrias/metabolismo , Hipóxia/metabolismo , Hipóxia Celular/fisiologia , Oxigênio/metabolismoRESUMO
OBJECTIVE: We aimed to explore the influence of ferroptosis on an oxygen-glucose deprivation/reoxygenation (OGD/R) model in primary rat microglia. METHODS: Primary microglia were extracted from rats and cultured in vitro. The cells were subjected to a hypoxic environment for 6 h in a glucose-free medium, and then re-oxygenated for 24 h in DMEM/F12. Rat microglia were pretreated with the ferroptosis activator erastin and the ferroptosis inhibitor ferrostatin 1 for 24 h, followed by detection of cell cycle progression and apoptosis by flow cytometry. Intracellular total iron levels were measured. In addition, the relative levels of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were determined using enzyme-linked immunosorbent assay. The protein levels of 15-lox2, GPX4, SLC7A11, ACSL4, and TFR1 were examined by western blotting. RESULTS: Compared with rat microglia subjected to OGD/R, pretreatment with erastin did not influence cell apoptosis but significantly enhanced total iron levels, MDA, and ROS levels, whereas it reduced SOD levels. Moreover, it upregulated ACSL4, TFR1, and 15-lox2 and downregulated GPX4 and SLC7A11. Pretreatment with ferrostatin 1 significantly inhibited cell apoptosis and cell cycle arrest in the G0/G1 phase. It significantly reduced total iron levels, MDA, and ROS levels and enhanced SOD levels, which also downregulated ACSL4, TFR1, and 15-lox2, and upregulated GPX4 and SLC7A11. CONCLUSION: Our study showed that inhibition of ferroptosis is favorable against potential OGD/R-induced damage in rat microglia.
Assuntos
Ferroptose , Glucose , Microglia , Ratos Sprague-Dawley , Animais , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Microglia/metabolismo , Microglia/efeitos dos fármacos , Glucose/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células Cultivadas , Ferro/metabolismo , Malondialdeído/metabolismo , Hipóxia Celular/fisiologia , Hipóxia Celular/efeitos dos fármacos , Oxigênio/metabolismo , Cicloexilaminas , FenilenodiaminasRESUMO
The hypoxic microenvironment in esophageal carcinoma is an important factor promoting the rapid progression of malignant tumor. This study was to investigate the lactylation of Axin1 on glycolysis in esophageal carcinoma cells under hypoxia exposure. Hypoxia treatment increases pan lysine lactylation (pan-kla) levels of both TE1 and EC109 cells. Meanwhile, ECAR, glucose consumption and lactate production were also upregulated in both TE1 and EC109 cells. The expression of embryonic stem cell transcription factors NANOG and SOX2 were enhanced in the hypoxia-treated cells. Axin1 overexpression partly reverses the induction effects of hypoxia treatment in TE1 and EC109 cells. Moreover, lactylation of Axin1 protein at K147 induced by hypoxia treatment promotes ubiquitination modification of Axin1 protein to promote glycolysis and cell stemness of TE1 and EC109 cells. Mutant Axin1 can inhibit ECAR, glucose uptake, lactate secretion, and cell stemness in TE1 and EC109 cells under normal or hypoxia conditions. Meanwhile, mutant Axin1 further enhanced the effects of 2-DG on inhibiting glycolysis and cell stemness. Overexpression of Axin1 also inhibited tumor growth in vivo, and was related to suppressing glycolysis. In conclusion, hypoxia treatment promoted the glycolysis and cell stemness of esophageal carcinoma cells, and increased the lactylation of Axin1 protein. Overexpression of Axin1 functioned as a glycolysis inhibitor, and suppressed the effects of hypoxia exposure in vitro and inhibited tumor growth in vivo. Mechanically, hypoxia induces the lactylation of Axin1 protein and promotes the ubiquitination of Axin1 to degrade the protein, thereby exercising its anti-glycolytic function.
Assuntos
Proteína Axina , Neoplasias Esofágicas , Glicólise , Camundongos Nus , Humanos , Proteína Axina/metabolismo , Proteína Axina/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Glicólise/fisiologia , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos BALB C , Hipóxia Celular/fisiologiaRESUMO
Glucagon-like peptide (GLP)-1 is a hormone released by enteroendocrine L-cells after food ingestion. L-cells express various receptors for nutrient sensing including G protein-coupled receptors (GPRs). Intestinal epithelial cells near the lumen have a lower O2 tension than at the base of the crypts, which leads to hypoxia in L-cells. We hypothesized that hypoxia affects nutrient-stimulated GLP-1 secretion from the enteroendocrine cell line STC-1, the most commonly used model. In this study, we investigated the effect of hypoxia (1% O2) on alpha-linolenic acid (αLA) stimulated GLP-1 secretion and their receptor expressions. STC-1 cells were incubated for 12 h under hypoxia (1% O2) and treated with αLA to stimulate GLP-1 secretion. 12 h of hypoxia did not change basal GLP-1 secretion, but significantly reduced nutrient (αLA) stimulated GLP-1 secretion. In normoxia, αLA (12.5 µM) significantly stimulated (~ 5 times) GLP-1 secretion compared to control, but under hypoxia, GLP-1 secretion was reduced by 45% compared to normoxia. αLA upregulated GPR120, also termed free fatty acid receptor 4 (FFAR4), expressions under normoxia as well as hypoxia. Hypoxia downregulated GPR120 and GPR40 expression by 50% and 60%, respectively, compared to normoxia. These findings demonstrate that hypoxia does not affect the basal GLP-1 secretion but decreases nutrient-stimulated GLP-1 secretion. The decrease in nutrient-stimulated GLP-1 secretion was due to decreased GPR120 and GPR40 receptors expression. Changes in the gut environment and inflammation might contribute to the hypoxia of the epithelial and L-cells.
Assuntos
Hipóxia Celular , Células Enteroendócrinas , Peptídeo 1 Semelhante ao Glucagon , Receptores Acoplados a Proteínas G , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Enteroendócrinas/metabolismo , Animais , Hipóxia Celular/fisiologia , Linhagem Celular , Camundongos , Ácido alfa-Linolênico/farmacologia , Ácido alfa-Linolênico/metabolismoRESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by chronic bronchitis, emphysema and vascular remodelling. The disease is associated with hypoxia, inflammation and oxidative stress. Lung fibroblasts are important cells in remodelling processes in COPD, as main producers of extracellular matrix proteins but also in synthesis of growth factors and inflammatory mediators. METHODS: In this study we aimed to investigate if there are differences in how primary distal lung fibroblasts obtained from COPD patients and healthy subjects respond to hypoxia (1% O2) and pro-fibrotic stimuli with TGF-ß1 (10 ng/mL). Genes and proteins associated with oxidative stress, endoplasmic reticulum stress, remodelling and inflammation were analysed with RT-qPCR and ELISA. RESULTS: Hypoxia induced differences in expression of genes involved in oxidative stress (SOD3 and HIF-1α), ER stress (IRE1, PARK and ATF6), apoptosis (c-Jun and Bcl2) and remodelling (5HTR2B, Collagen7 and VEGFR2) in lung fibroblasts from COPD subjects compared to control subjects, where COPD fibroblasts were in general less responsive. The release of VEGF-C was increased after hypoxia, whereas TGF-ß significantly reduced the VEGF response to hypoxia and the release of HGF. COPD fibroblasts had a higher release of IL-6, IL-8, MCP-1 and PGE2 compared to lung fibroblasts from control subjects. The release of inflammatory mediators was less affected by hypoxia, whereas TGFß1 induced differences in inflammatory profile between fibroblasts from COPD and control subjects. CONCLUSION: These results suggest that there is an alteration of gene regulation of various stress responses and remodelling associated mediator release that is related to COPD and hypoxia, where fibroblasts from COPD patients have a deficient response.
Assuntos
Fibroblastos , Pulmão , Doença Pulmonar Obstrutiva Crônica , Humanos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Masculino , Feminino , Pessoa de Meia-Idade , Células Cultivadas , Idoso , Pulmão/metabolismo , Pulmão/patologia , Hipóxia Celular/fisiologia , Estresse Oxidativo/fisiologia , Mediadores da Inflamação/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Inflamação/metabolismo , Inflamação/patologia , Hipóxia/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Estudos de Casos e ControlesRESUMO
Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating solid tumors remains challenging, as exemplified by the safety concerns that arise when CAR-T cells attack normal cells expressing the target antigens. Researchers have explored various approaches to enhance the tumor selectivity of CAR-T cell therapy. One representative strategy along this line is the construction of hypoxia-sensitive CAR-T cells, which are designed by fusing an oxygen-dependent degradation domain to the CAR moiety and are strategized to attain high CAR expression only in a hypoxic environment-the tumor microenvironment (TME). This paper presents a protocol for the generation of such CAR-T cells and their functional characterization, including methods to analyze the changes in CAR expression and killing capacity in response to different oxygen levels established by a mobile incubator chamber. The constructed CAR-T cells are anticipated to demonstrate CAR expression and cytotoxicity in an oxygen-sensitive manner, thus supporting their capability to distinguish between hypoxic TME and normoxic normal tissues for selective activation.
Assuntos
Receptores de Antígenos Quiméricos , Linfócitos T , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Humanos , Linfócitos T/imunologia , Imunoterapia Adotiva/métodos , Hipóxia Celular/fisiologia , Microambiente Tumoral/imunologiaRESUMO
CDK5RAP3 is a recognized tumor suppressor that inhibits Chk1 and Chk2 and activates p53, all of which are involved with mediating toxin-induced apoptosis of cancer cells. CDK5RAP3 also inhibits p38MAPK phosphorylation and activity via mediating a p38 interaction with wild-type p53-induced phosphatase 1. This study aimed to investigate the antiangiogenic activity of CDK5RAP3 and its molecular mechanisms in human umbilical vein endothelial cells (HUVECs) under conditions of hypoxic conditions. Angiogenesis was induced in HUVECs mainly by vascular endothelial growth factor (VEGF). The CDK5RAP3 levels of HUVECs were reduced in a time-dependent manner in response to hypoxic treatment at 2% O2. The reduction of CDK5RAP3 was accompanied with increased p38MAPK phosphorylation and activation. Moderate hypoxia was found to significantly increase secreted VEGF concentrations, and the hypoxic conditioned medium (HCM) markedly enhanced proliferation, migration, and tube formation. Our findings indicate that moderate hypoxia facilitates angiogenesis by inhibiting CDK5RAP3. CDK5RAP3 exhibits a clear regulatory role in vascular regeneration, as downregulating its expression in endothelial cells enhances VEGF synthesis and subsequently improves cell migration and lumen formation capability. This study presents evidence indicating that moderate hypoxia facilitates angiogenesis by inhibiting CDK5RAP3, demonstrating the potential for CKD5RAP3 to be a potent antiangiogenic agent in angiogenesis regulation of cancer, ischemic diseases, and wound healing.
Assuntos
Proteínas de Ciclo Celular , Células Endoteliais da Veia Umbilical Humana , Neovascularização Fisiológica , Proteínas Quinases p38 Ativadas por Mitógeno , Humanos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Hipóxia Celular/fisiologia , Fosforilação , Angiogênese , Proteínas Supressoras de TumorRESUMO
BACKGROUND: Asthma's complexity, marked by airway inflammation and remodeling, is influenced by hypoxic conditions. This study focuses on the role of Hypoxia-Inducible Factor-1 Alpha (HIF-1α) and P53 ubiquitination in asthma exacerbation. METHODS: High-throughput sequencing and bioinformatics were used to identify genes associated with asthma progression, with an emphasis on GO and KEGG pathway analyses. An asthma mouse model was developed, and airway smooth muscle cells (ASMCs) were isolated to create an in vitro hypoxia model. Cell viability, proliferation, migration, and apoptosis were assessed, along with ELISA and Hematoxylin and Eosin (H&E) staining. RESULTS: A notable increase in HIF-1α was observed in both in vivo and in vitro asthma models. HIF-1α upregulation enhanced ASMCs' viability, proliferation, and migration, while reducing apoptosis, primarily via the promotion of P53 ubiquitination through MDM2. In vivo studies showed increased inflammatory cell infiltration and airway structural changes, which were mitigated by the inhibitor IDF-11,774. CONCLUSION: The study highlights the critical role of the HIF-1α-MDM2-P53 axis in asthma, suggesting its potential as a target for therapeutic interventions. The findings indicate that modulating this pathway could offer new avenues for treating the complex respiratory disorder of asthma.
Assuntos
Asma , Subunidade alfa do Fator 1 Induzível por Hipóxia , Miócitos de Músculo Liso , Proteína Supressora de Tumor p53 , Asma/metabolismo , Asma/patologia , Asma/genética , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Células Cultivadas , Camundongos Endogâmicos BALB C , Apoptose/fisiologia , Proliferação de Células/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Hipóxia/metabolismo , Hipóxia/patologia , Modelos Animais de Doenças , Hipóxia Celular/fisiologia , Feminino , Humanos , Movimento Celular/fisiologia , UbiquitinaçãoRESUMO
Objective. Serine hydroxymethyltransferase (SHMT2) plays a multifunctional role in mitochondria (folate-dependent tRNA methylation, translation, and thymidylate synthesis). The endoplasmic reticulum stress, hypoxia, and glucose and glutamine supply are significant factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) pathway of endoplasmic reticulum stress strongly suppressed glioblastoma cell proliferation and modified the sensitivity of these cells to hypoxia and glucose or glutamine deprivations. The present study aimed to investigate the regulation of the SHMT2 gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in sensitivity of this gene expression to hypoxia and nutrient supply. Methods. The control U87MG glioblastoma cells (transfected by an empty vector) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine (500 ng/ml for 4 h). For glucose and glutamine deprivations, cells were exposed in DMEM without glucose and glutamine, respectively for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of the SHMT2 gene was studied by real-time qPCR and normalized to ACTB. Results. It was found that inhibition of ERN1 endoribonuclease and protein kinase in glioblastoma cells led to a down-regulation of SHMT2 gene expression in U87MG cells. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease, but tunicamycin strongly increased its expression. Moreover, the expression of the SHMT2 gene was not affected in U87MG cells after silencing of XBP1. Hypoxia up-regulated the expression level of the SHMT2 gene in both control and ERN1 knockdown U87MG cells. The expression of this gene was significantly up-regulated in glioblastoma cells under glucose and glutamine deprivations and ERN1 knockdown significantly increased the sensitivity of the SHMT2 gene to these nutrient deprivation conditions. Conclusion. The results of the present study demonstrate that the expression of the SHMT2 gene responsible for serine metabolism and formation of folate one-carbon is controlled by ERN1 protein kinase and induced by hypoxia as well as glutamine and glucose deprivation conditions in glioblastoma cells and reflects the ERN1-mediated reprogramming of sensitivity this gene expression to nutrient deprivation.
Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Glicina Hidroximetiltransferase , Humanos , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Estresse do Retículo Endoplasmático/fisiologia , Estresse do Retículo Endoplasmático/genética , Linhagem Celular Tumoral , Endorribonucleases/genética , Endorribonucleases/metabolismo , Glucose/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Hipóxia Celular/fisiologia , Hipóxia Celular/genética , Glutamina/metabolismo , Técnicas de Silenciamento de GenesRESUMO
During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed toward the relevance of hypoxia as modulator of trophoblast cell death. Previous reports have shown that leptin, a placental cytokine, promotes cell survival in both cell culture and placental explant models. The aim of this work is to establish the role of leptin in apoptosis under hypoxic condition in trophoblast cells. In this study, we evaluated the effect of cobalt chloride, a hypoxia mimicking agent that stabilizes the expression of hypoxia-inducible factor-1 alpha, on Swan-71 and human placental explants. Hypoxia chamber was also used to generate 2% oxygen. Apoptosis was determined by the presence of apoptotic nucleus, fragmentation of DNA and Caspase-3 and PARP-1 cleavage. The pro-apoptotic proteins BAX, BID, BAD, and BAK and the anti-apoptotic effectors BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1 were also analyzed. We found that hypoxia-inducible factor-1 alpha stabilization increased the appearance of apoptotic nucleus, fragmentation of DNA, and Caspase-3 and PARP-1 cleavage. Hypoxia mimicking conditions enhanced the expression of pro-apoptotic effectors BAX, BID, BAD, and BAK. Hypoxia-inducible factor-1 alpha stabilization also downregulated the level of BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1. All these apoptotic parameters changes were reversed with leptin treatment. Moreover, we showed that leptin action on apoptosis modulation involves PI3K and MAPK signaling pathways. Obtained data demonstrate that hypoxia-inducible factor-1 alpha stabilization induces apoptosis in human placenta and leptin counteracts this effect, reinforcing its role as a survival cytokine.
Assuntos
Apoptose , Leptina , Placenta , Humanos , Feminino , Placenta/metabolismo , Placenta/efeitos dos fármacos , Gravidez , Leptina/metabolismo , Leptina/farmacologia , Apoptose/efeitos dos fármacos , Trofoblastos/metabolismo , Trofoblastos/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Cobalto/farmacologia , Hipóxia Celular/fisiologiaRESUMO
BACKGROUND: Tamoxifen (TAM) is a selective estrogen receptor modulator and has anti-estrogenic activity. Breast cancer cells acquire drug resistance to TAM as a consequence of long-term treatment. Lysophosphatidic acid (LPA) receptor-mediated signaling contributes to the promotion of tumor progression. This study aimed to evaluate the role of LPA receptors in the modulation of biological functions by long-term TAM treatment in breast cancer MCF-7 cells under hypoxic and estrogen-deprived conditions. METHODS: Long-term TAM treated (MCF-TAM) cells were generated from MCF-7 cells. Cells were cultured in estrogen-free medium at 1â¯% O2. LPA receptor expressions were measured by quantitative real-time RT-PCR analysis. Cell motile activity was investigated using Cell Culture Inserts. The CCK-8 kit was used to determine the cell proliferation rate. RESULTS: LPAR1 and LPAR3 expressions were elevated in MCF-TAM cells. MCF-TAM cell motility was enhanced by culturing at 1â¯% O2, compared with MCF-7 cells. When cells were cultured in estrogen-deprived medium at 1â¯% O2, the cell proliferation rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. CONCLUSION: These results suggest that LPA receptor-mediated signaling plays an important role in the acquisition of malignant properties in long-term TAM treated MCF-7 cells under hypoxic and estrogen-deprived conditions.
Assuntos
Antineoplásicos Hormonais , Neoplasias da Mama , Movimento Celular , Proliferação de Células , Receptores de Ácidos Lisofosfatídicos , Transdução de Sinais , Tamoxifeno , Humanos , Receptores de Ácidos Lisofosfatídicos/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Tamoxifeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células MCF-7 , Feminino , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Antineoplásicos Hormonais/farmacologia , Estrogênios/metabolismo , Estrogênios/farmacologia , Hipóxia Celular/fisiologia , Hipóxia Celular/efeitos dos fármacosRESUMO
p53 has diversity functions in regulation of transcription, cell proliferation, cancer metastasis, etc. Recent studies have shown that p53 and nuclear factor-κB (NF-κB) co-regulate proinflammatory responses in macrophages. However, the role of p53 lysine lactylation (p53Kla) in mediating proinflammatory phenotypes in microglia under hypoxic conditions remains unclear. In the current study, we investigated the proinflammatory activation exacerbated by hypoxia and the levels of p53Kla in microglial cells. BV2 cells, an immortalized mouse microglia cell line, were divided into control, lipopolysaccharide (LPS)-induced, hypoxia (Hy), and LPS-Hy groups. The protein expression levels of p53 and p53Kla and the activation of microglia were compared among the four groups. Sodium oxamate and mutant p53 plasmids were transfected into BV2 cells to detect the effect of p53Kla on microglial proinflammatory activation. LPS-Hy stimulation significantly upregulated p53Kla levels in both the nucleus and the cytoplasm of BV2 cells. In contrast, the p53 protein levels were downregulated. LPS-Hy stimulation upregulated phosphorylated p65 protein levels in nuclear and activated the NF-κB pathway in BV2 cells, resulting in increased expression of pro-inflammatory cytokines (iNOS, IL6, IL1ß, TNFα), enhanced cell viability, and concomitantly, increased cytotoxicity. In conclusion, p53 lysine-lactylated modification contributes to LPS-induced proinflammatory activation in BV2 cells under hypoxia through NF-κB pathway and inhibition of lactate production may alleviate neuroinflammatory injury.