RESUMEN
Arteriovenous fistula (AVF) failure often involves venous neointimal hyperplasia (VNH) driven by elevated hypoxia-inducible factor-1 alpha (HIF-1α) in the venous wall. Omentin, known for its anti-inflammatory and anti-hyperplasia properties, has an uncertain role in early AVF failure. This study investigates omentin's impact on VNH using a chronic renal failure (CRF) rabbit model. The CRF rabbit model of AVF received omentin-expressing adenoviral vector or control ß-gal vector to assess omentin's effects on VNH. Human vascular smooth muscle cells (HVSMCs), stimulated with tumor necrosis factor-α (TNF-α), were exposed to recombinant human omentin (Rh-OMT) to study its influence on cell proliferation and migration. The AMP-activated protein kinase (AMPK) inhibitor compound C and the mammalian target of rapamycin (mTOR) activator MHY1485 were employed to explore omentin's mechanisms in VNH reduction through HIF-1α inhibition. Omentin treatment reduced VNH in CRF rabbits, concomitant with HIF-1α down-regulation and the suppression of downstream factors, including vascular endothelial growth factor and matrix metalloproteinases. Rh-OMT inhibited TNF-α-induced HVSMC proliferation and migration by modulating both cell cycle and cell adhesion proteins. Additionally, omentin reduced HIF-1α expression through the AMPK/mTOR pathway activation. Notably, the blockade of AMPK/mTOR signaling reversed omentin-mediated inhibition of VNH, cell proliferation, and migration, both in vivo and in vitro. In conclusion, omentin mitigates VNH post-AVF creation by restraining HIF-1α via AMPK/mTOR signaling. Strategies boosting circulating omentin levels may offer promise in averting AVF failure.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Derivación Arteriovenosa Quirúrgica , Movimiento Celular , Proliferación Celular , Citocinas , Modelos Animales de Enfermedad , Proteínas Ligadas a GPI , Hiperplasia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Lectinas , Músculo Liso Vascular , Miocitos del Músculo Liso , Neointima , Transducción de Señal , Animales , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Citocinas/metabolismo , Conejos , Humanos , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/farmacología , Proteínas Ligadas a GPI/genética , Proliferación Celular/efectos de los fármacos , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Lectinas/farmacología , Lectinas/metabolismo , Movimiento Celular/efectos de los fármacos , Músculo Liso Vascular/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Células Cultivadas , Derivación Arteriovenosa Quirúrgica/efectos adversos , Masculino , Fallo Renal Crónico/patología , Serina-Treonina Quinasas TOR/metabolismo , Oclusión de Injerto Vascular/patología , Oclusión de Injerto Vascular/prevención & control , Oclusión de Injerto Vascular/metabolismo , Oclusión de Injerto Vascular/fisiopatología , Venas Yugulares/patología , Venas Yugulares/metabolismo , Venas Yugulares/trasplanteRESUMEN
More than 250 million people in the world are chronically infected with hepatitis B virus (HBV), which causes serious complications. Host genetic susceptibility is essential for chronic hepatitis B (CHB), and our previous genome-wide association study identified a single-nucleotide polymorphism (SNP), rs1883832, in the 5' untranslated region of CD40 predisposing to chronic HBV infection, but the underlying mechanism remains undefined. This study aimed to investigate whether rs1883832 was the real functional SNP (fSNP) of CD40 and how it modulated HBV clearance in hepatocytes. We determined the fSNP of CD40 and its regulatory protein(s) using luciferase reporter assays, electrophoretic mobility shift assay, flanking restriction enhanced pulldown and chromatin immunoprecipitation. The potential anti-HBV activity of CD40 and its downstream molecule BST2 was assessed in HBV-transfected and HBV-infected hepatoma cells and HBV-infected primary human hepatocytes. Moreover, the mechanism of CD40 was investigated by mRNA sequencing, quantitative real-time polymerase chain reaction, immunofluorescence and western blot. We revealed rs1883832 as the true fSNP of CD40 and identified ANXA2 as a negative regulatory protein that preferentially bound to the risk allele T of rs1883832 and hence reduced CD40 expression. Furthermore, CD40 suppressed HBV replication and transcription in hepatocytes via activating the JAK-STAT pathway. BST2 was identified to be the key IFN-stimulated gene regulated by CD40 after activating JAK-STAT pathway. Inhibition of JAK/STAT/BST2 axis attenuated CD40-induced antiviral effect. In conclusion, a functional variant of CD40 modulates HBV clearance via regulation of the ANXA2/CD40/BST2 axis, which may shed new light on HBV personalized therapy.
Asunto(s)
Anexina A2 , Hepatitis B Crónica , Hepatitis B , Humanos , Virus de la Hepatitis B/genética , Quinasas Janus/metabolismo , Estudio de Asociación del Genoma Completo , Transducción de Señal , Factores de Transcripción STAT/metabolismo , Hepatocitos/metabolismo , Hepatitis B Crónica/genética , Hepatitis B Crónica/metabolismo , Factores de Transcripción/genética , Hepatitis B/metabolismo , Antígenos CD/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/farmacología , Anexina A2/genéticaRESUMEN
OBJECTIVE: Omentin-1 is a newly discovered metabolic regulatory adipokine. Studies have shown that omentin-1 possesses pleiotropic effects in different types of cells. This study aims to investigate the regulation by omentin-1 on mitochondrial biogenesis in chondrocytes. METHODOLOGY: C-28/I2 chondrocytes were treated with omentin-1 (150 and 300 ng/ml) for 24 h. The expression of mitochondrial regulators, markers and the DNA copy was assessed. The mitochondrial morphology was observed by electron microscopy. The mitochondrial respiratory rate and ATP production in chondrocytes were measured by cell lysates. RESULTS: Omentin-1 treatment up-regulated PGC-1α, NRF-1 and mitochondrial transcription factor A (TFAM) in cultured chondrocytes, indicating that omentin-1 could be involved in the regulation of mitochondrial function. Omentin-1 promoted mtDNA/nDNA and four mitochondrial genes (Tomm20, Tomm40, Timm9 and Atp5c1), mRNA transcripts as well as two mitochondrial protein expressions (SDHB and MTCO1). At a cellular level, omentin-1 enhanced the mitochondrial respiratory rate and ATP production. Mechanistically, we proved that omentin-1 increased AMPKα activation, and the blockage of AMPKα by its inhibitor compound C abolished the inductive effect of omentin-1 on PGC1α expression and mtDNA/nDNA ratio, indicating that the effect of omentin-1 is dependent on AMPKα activation. CONCLUSION: Omentin-1 is a positive regulator of mitochondrial biogenesis in chondrocytes, and its action is dependent on the AMPK-PGC1α pathway. This study, therefore, implies that omentin-1 has the potential to remedy chondrocyte damage in the prevention and treatment of osteoarthritis.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Condrocitos , Adenosina Trifosfato/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Condrocitos/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas Mitocondriales/genética , Biogénesis de Organelos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Lectinas/farmacología , Citocinas/farmacología , Proteínas Ligadas a GPI/farmacologíaRESUMEN
OBJECTIVES: The aim of this study was to investigate the effects of curcumin on the viability, migration, and apoptosis of A549 lung cancer cells. Furthermore, RECK/MMPs axis as a probable regulator of cancer cell migration was assessed. METHODS: In this study, effect of curcumin on viability changes, cell migration, and percentage of apoptosis of A549 non-small cell lung carcinoma was examined. The methylation status of RECK gene was investigated using MS-HRM technique. Moreover, expression changes of genes involved in apoptosis and migration (including CASP3, CASP8, CASP9, BAX, BCL2, MMP9, MMP2, and RECK) were investigated by quantitative Real-Time PCR. RESULTS: The results of MTT assay showed that the cytotoxic effect of curcumin was in a dose dependent manner. Flow cytometry results demonstrated a significant increase in the percentage of apoptotic cells in curcumin treated group. In addition, curcumin inhibited migration rate in lung cancer cells. qRT-PCR revealed that expression of the candidate genes was in line with suppressed growth and migration. This could be due to, decreased methylation of the RECK gene promoter after curcumin treatment. CONCLUSIONS: Curcumin inhibited lung cancer cells through various molecular pathways. RECK/MMPs axis as a regulator of cancer cell migration was modulated after curcumin treatment and invasion of lung cancer cells was decreased.
Asunto(s)
Curcumina , Neoplasias Pulmonares , Humanos , Curcumina/farmacología , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Movimiento Celular/genética , Línea Celular Tumoral , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz/farmacología , Epigénesis Genética , Proliferación CelularRESUMEN
HIV-1 virus release from infected cells is blocked by human BST-2, but HIV-1 Vpu efficiently antagonises BST-2 due to direct transmembrane domain interactions that occur between each protein. Targeting the interaction between these two proteins is seen as viable for HIV-1 antiviral intervention. This study describes the successful over-expression and purification of a recombinant full-length human BST-2 from inclusion bodies using affinity and anion exchange chromatography. Two milligrams of purified full-length BST-2 were produced per litre of BL21 (DE3) T7 Express® pLysY E. coli culture. Far-UV circular dichroism validated the renaturing of the recombinant protein and retention of its secondary structure. Furthermore, through ELISA, a known human BST-2 binding partner, HIV-1 Vpu, was shown to bind to the renatured and purified protein, further validating its folding. To our knowledge this is the first report of the purification of a wild-type, full-length human BST-2 from Escherichia coli.
Asunto(s)
Antígenos CD/genética , VIH-1/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Viroporinas/metabolismo , Secuencia de Aminoácidos , Antígenos CD/biosíntesis , Antígenos CD/aislamiento & purificación , Antígenos CD/farmacología , Secuencia de Bases , Cromatografía de Afinidad/métodos , Cromatografía por Intercambio Iónico/métodos , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/aislamiento & purificación , Proteínas Ligadas a GPI/farmacología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , VIH-1/patogenicidad , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Cuerpos de Inclusión/química , Unión Proteica , Replegamiento Proteico , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Viroporinas/genéticaRESUMEN
Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.
Asunto(s)
5'-Nucleotidasa/farmacología , Antiinflamatorios/farmacología , Apirasa/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Ingeniería de Proteínas , 5'-Nucleotidasa/química , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Nucleótidos de Adenina/metabolismo , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Apirasa/química , Apirasa/genética , Apirasa/metabolismo , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/farmacología , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
OBJECTIVES: We aimed to describe mesothelin (MSLN) and programmed cell death 1 ligand 1 (PD-L1) tumour overexpression amongst patients with malignant mesothelioma (MM), and their associations with survival, amongst a cohort of patients with MM in Finland. METHODS: Between 2004 and 2017, 91 adults with histologically confirmed MM were identified from the Auria Biobank in Finland and followed-up using linked data from electronic health records and national statistics. Biomarker content in tumour cell membranes was determined using automated Immunohistochemistry on histological sections. Stained tumour sections were scored for MSLN and PD-L1 intensity. Adjusted associations between MSLN/PD-L1 co-expression and mortality were evaluated by estimating hazard ratios (HRs) with 95% confidence intervals (CIs) using Cox regression. RESULTS: Biomarker overexpression occurred in 52 patients for MSLN and 34 patients for PD-L1 and was associated with tumour histology and certain comorbidities. Fifteen per cent of patients had a tumour that overexpressed both biomarkers; r =-0.244, p-value: 0.02. Compared with MSLN+/PD-L1+ patients, HRs (95% CIs) for death were 4.18 (1.71-10.23) for MSLN-/PD-L1+ patients, 3.03 (1.35-6.77) for MSLN-/PD-L1- patients, and 2.13 (0.97-4.67) for MSLN+/PD-L1- patients. CONCLUSIONS: Both MSLN and PD-L1 markers were independent prognostic indicators in patients with MM. Overexpression of MSLN was associated with longer survival; yet their combined expression gave a better indication of survival. The risk of death was four times higher amongst MSLN-/PD-L1+ patients than in MSLN+/PD-L1+ patients.
Asunto(s)
Antígenos de Neoplasias/uso terapéutico , Antígeno B7-H1/metabolismo , Proteínas Ligadas a GPI/uso terapéutico , Mesotelioma Maligno/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/farmacología , Estudios de Cohortes , Femenino , Finlandia , Proteínas Ligadas a GPI/farmacología , Humanos , Masculino , Mesotelina , PronósticoRESUMEN
Omentin-1 is an adipokine expressed by the adipose tissue and is reduced in obesity. This study was designed to calculate the protective efficiency and mechanism of omentin-1 against inflammation of the adipose tissue in obese mice. A transgenic mouse model with omentin-1 protein overexpression was established by crossing omentin-1 transgenic mice with Fabp4-Cre mice. Obesity was induced in the mice by feeding them a high-fat diet for 10 weeks. Fabp4-Cre-mediated overexpression of omentin-1 significantly increased serum omentin-1 level, serum anti-inflammatory factor levels, and expression of M2-specific mRNAs; inhibited body weight and adipose tissue weight gain; improved glucose tolerance, insulin tolerance, and insulin sensitivity; decreased serum levels of insulin and proinflammatory factors, adipocyte size, and expression of M1-specific mRNAs; suppressed macrophage infiltration; downregulated expression of proinflammatory factors; upregulated expression of anti-inflammatory factors; and inhibited thioredoxin-interacting protein (TXNIP)/NOD-like receptor 3 (NLRP3) signaling in the adipose tissue of obese mice. An NLRP3 inhibitor (20 mg/kg MCC950) exhibited the same effects as overexpression of omentin-1. Pretreatment with omentin-1 inhibited lipopolysaccharide-induced inflammation via TXNIP/NLRP3 signaling in RAW 264.7 macrophages. These findings suggest that omentin-1 suppresses adipose tissue inflammation in obese mice, at least partly, via inhibiting the TXNIP/NLRP3 signaling pathway.
Asunto(s)
Adipoquinas/farmacología , Tejido Adiposo/efectos de los fármacos , Citocinas/farmacología , Proteínas Ligadas a GPI/farmacología , Lectinas/farmacología , Adipoquinas/metabolismo , Tejido Adiposo/metabolismo , Animales , Proteínas Portadoras/metabolismo , Citocinas/metabolismo , Dieta Alta en Grasa , Proteínas Ligadas a GPI/metabolismo , Resistencia a la Insulina , Lectinas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Obesos , Ratones Transgénicos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal/efectos de los fármacos , Tiorredoxinas/metabolismoRESUMEN
Endothelial cell injury caused by reactive oxygen species (ROS) plays a critical role in the pathogenesis of cardiovascular diseases. Omentin, an adipocytokine that is abundantly expressed in visceral fat tissue, has been reported to possess anti-inflammatory and antidiabetic properties. However, endothelial protective effects of omentin against oxidative stress remain unclear. This study aimed to evaluate the protective effect of omentin against hydrogen peroxide (H2O2)-induced cell injury in human umbilical vein endothelial cells (HUVECs). Cytotoxicity and cytoprotective effects of omentin were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic activity of HUVECs was detected using Annexin-V/PI and Hoechst 33258 staining methods. Antioxidant activity of omentin was evaluated by measuring both reactive oxygen species (ROS) levels and glutathione peroxidase (GPx) activity. No cytotoxicity effect was observed in HUVECs treated with omentin alone at concentrations of 150 to 450 ng/ml. MTT assay showed that omentin significantly prevented the cell death induced by H2O2 (p < 0.001). Hoechst staining and flow cytometry also revealed that omentin markedly prevented H2O2-induced apoptosis. Moreover, omentin not only significantly inhibited ROS production (p < 0.01) but also significantly (p < 0.01) increased GPx activity in HUVECs. In conclusion, our data suggest that omentin may protect HUVECs from injury induced by H2O2.
Asunto(s)
Citocinas/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Lectinas/farmacología , Estrés Oxidativo , Adipoquinas/farmacología , Antioxidantes/farmacología , Apoptosis , Supervivencia Celular , Proteínas Ligadas a GPI/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Sustancias Protectoras/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
OBJECTIVES: To investigate the impact of omentin on the release of inflammation-related biomarkers and inflammatory pathways in primary human adipocytes. METHODS: Adipocytes were treated with or without omentin (500 and 2,000 ng/mL), and the supernatants were analyzed for inflammation-related biomarkers using proximity extension assay technology. Potential upstream regulators of the omentin-stimulated proteins were identified using Ingenuity Pathway Analysis. Protein levels of components of inflammatory pathways were measured using Western blotting. RESULTS: 2,000 ng/mL omentin induced the release of 30 biomarkers 97.1 ± 31.1-fold in the supernatants (all p < 0.05). Most biomarkers were proin-flammatory chemokines and cytokines. We identified the transcription factor nuclear factor "kappa-light-chain-enhancer" of activated B cells (NFĸB) and the kinases p38 and extracellular signal-regulated kinase (ERK)1/2 as potential upstream regulators in silico. On the cellular level, treatment with 2,000 ng/mL omentin for 24 h enhanced the phosphorylation levels of NFĸB 2.1 ± 0.3-fold (p < 0.05), of p38 2.6 ± 0.4-fold (p < 0.05), and of ERK1/2 1.8 ± 0.2-fold (p < 0.05). CONCLUSIONS: These data argue that omentin exerts proinflammatory effects through the activation of the inflammatory NFĸB, p38, and ERK1/2 pathways in cultured primary adipocytes.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Citocinas/farmacología , Mediadores de Inflamación/metabolismo , Lectinas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Adipocitos/citología , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas Ligadas a GPI/farmacología , Humanos , FN-kappa B/metabolismo , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
High glucose-induced endothelial dysfunction is a critical initiating factor in the development of diabetic vascular complications. Omentin-1 has been regarded as a novel biomarker of endothelial function in subjects with type-2 diabetes (T2D); however, it is unclear whether omentin-1 has any direct effect in ameliorating high glucose-induced endothelial dysfunction. In the present study, we analyzed the effect of omentin-1 on high glucose-induced endothelial dysfunction in isolated mouse aortas and mouse aortic endothelial cells (MAECs). Vascular reactivity in aortas was measured using wire myography. The expression levels of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor δ (PPARδ), Akt, endothelial nitric-oxide synthase (eNOS), and endoplasmic reticulum (ER)-stress markers in MAECs were determined by Western blotting. The production of reactive oxygen species (ROS) and nitric oxide (NO) was assessed by diluted fluoroprobe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA), respectively. We found that ex vivo treatment with omentin-1 reversed impaired endothelial-dependent relaxations (EDR) in mouse aortas after high-glucose insult. Elevated ER-stress markers, oxidative stress, and reduction of NO production induced by high glucose in MAECs were reversed by omentin-1 treatment. Omentin-1 also effectively reversed tunicamycin-induced ER stress responses in MAECs, as well as ameliorated impairment of endothelial-dependent relaxation in mouse aortas. Moreover, omentin-1 increased AMPK phosphorylation with a subsequent increase in PPARδ expression, while also restoring the decreased phosphorylation of Akt and eNOS. The effects of omentin-1 were abolished by cotreatment of compound C (AMPK inhibitor) and GSK0660 (PPARδ antagonist). These data indicate that omentin-1 protects against high glucose-induced vascular-endothelial dysfunction through inhibiting ER stress and oxidative stress and increasing NO production via activation of AMPK/PPARδ pathway.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Citocinas/farmacología , Endotelio Vascular/metabolismo , Proteínas Ligadas a GPI/farmacología , Glucosa/toxicidad , Lectinas/farmacología , PPAR delta/metabolismo , Transducción de Señal/fisiología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Transducción de Señal/efectos de los fármacosRESUMEN
Over 90% of pancreatic ductal adenocarcinomas (PDAC) express mesothelin (MSLN). Overexpression or knockdown of MSLN has been implicated in PDAC aggressiveness. This activity has been ascribed to MSLN-induced activation of MAPK or NF-κB signaling pathways and to interaction of MSLN with its only known binding partner, MUC16. Here, we used CRISPR/Cas9 gene editing to delete MSLN from PDAC, then restored expression of wild-type (WT) or Y318A mutant MSLN by viral transduction. We found that MSLN KO cells grew in culture and as subcutaneous tumors in mouse xenografts at the same rate as WT cells but formed intraperitoneal metastases poorly. Complementation with WT MSLN restored intraperitoneal growth, whereas complementation with Y318A mutant MSLN, which does not bind MUC16, was ineffective at enhancing growth in both MUC16(+) and MUC16(-) models. Restoration of WT MSLN did enhance growth but did not affect cell-to-cell binding, cell viability in suspension or signaling pathways previously identified as contributing to the protumorigenic effect of MSLN. RNA deep sequencing of tumor cells identified no changes in transcriptional profile that could explain the observed phenotype. Furthermore, no histologic changes in tumor cell proliferation or morphology were observed in mature tumors. Examination of nascent MSLN KO tumors revealed decreased microvascular density as intraperitoneal tumors were forming, followed by decreased proliferation, which resolved by 2 weeks postimplantation. These data support a model whereby MSLN expression by tumor cells contributes to metastatic colonization. IMPLICATIONS: MSLN confers a growth advantage to tumor cells during colonization of peritoneal metastasis. Therapeutic blockade of MSLN might limit peritoneal spread.
Asunto(s)
Antígenos de Neoplasias/uso terapéutico , Carcinoma Ductal Pancreático/complicaciones , Proteínas Ligadas a GPI/uso terapéutico , Neoplasias Peritoneales/secundario , Animales , Antígenos de Neoplasias/farmacología , Línea Celular Tumoral , Femenino , Proteínas Ligadas a GPI/farmacología , Humanos , Mesotelina , Ratones , Ratones Desnudos , Metástasis de la NeoplasiaRESUMEN
BACKGROUND: We have previously demonstrated in vitro cytotoxicity of mesothelin-chimeric antigen receptor autologous T cells against pancreatic cancer cells using lentiviral vectors, but these vectors pose safety concerns. Here, we incorporated Sleeping Beauty and minicircle design enhancements into interleukin-2-secreting natural NK-92MI cells to eliminate both bacterial and viral components and address inhibition by the tumor microenvironment. METHODS: Parental (conventional deoxyribonucleic acid)-mesothelin-chimeric antigen receptor and minicircle-mesothelin-chimeric antigen receptor vectors were electroporated into NK-92MI cells and engraftment was visualized by immunofluorescence analysis with protein-L staining. Interferon-γ and granzyme B secretion were measured by enzyme-linked immunosorbent assay from cocultures of parental-mesothelin-chimeric antigen receptors and minicircle-mesothelin-chimeric antigen receptors with human pancreatic cancer cells, and cytotoxicity of chimeric antigen receptor NK-92MI cells was tested against three pancreatic cancer cell lines. RESULTS: Cloning of mesothelin-chimeric antigen receptor Sleeping Beauty into a minicircle vector removed its bacterial backbone and reduced its size with improved electroporation efficiency. Chimeric antigen receptor engraftment, Interferon-γ and granzyme B secretion, and specific lysis against all three pancreatic cancer lines were significantly increased with minicircle-mesothelin-chimeric antigen receptor versus parental-mesothelin-chimeric antigen receptor NK-92MI cells. CONCLUSION: We provide proof of concept that allogeneic mesothelin-chimeric antigen receptor NK-92MI cells with hybrid Sleeping Beauty and minicircle technologies provide increased engraftment and cytotoxicity in vitro with potential safety benefits when translated to the clinical arena.
Asunto(s)
Muerte Celular/inmunología , Proteínas Ligadas a GPI/farmacología , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/inmunología , Neoplasias Pancreáticas/patología , Receptores Quiméricos de Antígenos/inmunología , Línea Celular Tumoral , Electroporación/métodos , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas In Vitro , Células Asesinas Naturales/efectos de los fármacos , Mesotelina , Neoplasias Pancreáticas/terapia , Sensibilidad y Especificidad , Microambiente TumoralRESUMEN
PURPOSE: Targeted thorium-227 conjugates (TTC) represent a new class of molecules for targeted alpha therapy (TAT). Covalent attachment of a 3,2-HOPO chelator to an antibody enables specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. Because of the high energy and short penetration range, TAT efficiently induces double-strand DNA breaks (DSB) preferentially in the tumor cell with limited damage to the surrounding tissue. We present herein the preclinical evaluation of a mesothelin (MSLN)-targeted thorium-227 conjugate, BAY 2287411. MSLN is a GPI-anchored membrane glycoprotein overexpressed in mesothelioma, ovarian, pancreatic, lung, and breast cancers with limited expression in healthy tissue. EXPERIMENTAL DESIGN: The binding activity and radiostability of BAY 2287411 were confirmed bioanalytically. The mode-of-action and antitumor potency of BAY 2287411 were investigated in vitro and in vivo in cell line and patient-derived xenograft models of breast, colorectal, lung, ovarian, and pancreatic cancer. RESULTS: BAY 2287411 induced DSBs, apoptotic markers, and oxidative stress, leading to reduced cellular viability. Furthermore, upregulation of immunogenic cell death markers was observed. BAY 2287411 was well-tolerated and demonstrated significant antitumor efficacy when administered via single or multiple dosing regimens in vivo. In addition, significant survival benefit was observed in a disseminated lung cancer model. Biodistribution studies showed specific uptake and retention of BAY 2287411 in tumors and enabled the development of a mechanistic pharmacokinetic/pharmacodynamic model to describe the preclinical data. CONCLUSIONS: These promising preclinical results supported the transition of BAY 2287411 into a clinical phase I program in mesothelioma and ovarian cancer patients (NCT03507452).
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Partículas alfa/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/farmacología , Neoplasias/tratamiento farmacológico , Radiofármacos/farmacología , Torio/farmacología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular , Femenino , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/farmacocinética , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Mesotelina , Mesotelioma/tratamiento farmacológico , Mesotelioma/metabolismo , Mesotelioma/patología , Mesotelioma Maligno , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Radiofármacos/farmacocinética , Torio/administración & dosificación , Torio/química , Torio/farmacocinética , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeted 227Th conjugates (TTCs) represent a new class of therapeutic radiopharmaceuticals for targeted α-therapy. They comprise the α-emitter 227Th complexed to a 3,2-hydroxypyridinone chelator conjugated to a tumor-targeting monoclonal antibody. The high energy and short range of the α-particles induce antitumor activity, driven by the induction of complex DNA double-strand breaks. We hypothesized that blocking the DNA damage response (DDR) pathway should further sensitize cancer cells by inhibiting DNA repair, thereby increasing the response to TTCs. Methods: This article reports the evaluation of the mesothelin (MSLN)-TTC conjugate (BAY 2287411) in combination with several DDR inhibitors, each of them blocking different DDR pathway enzymes. MSLN is a validated cancer target known to be overexpressed in mesothelioma, ovarian, lung, breast, and pancreatic cancer, with low expression in normal tissue. In vitro cytotoxicity experiments were performed on cancer cell lines by combining the MSLN-TTC with inhibitors of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related (ATR), DNA-dependent protein kinase, and poly[adenosine diphosphate ribose] polymerase (PARP) 1/2. Further, we evaluated the antitumor efficacy of the MSLN-TTC in combination with DDR inhibitors in human ovarian cancer xenograft models. Results: Synergistic activity was observed in vitro for all tested inhibitors (inhibitors are denoted herein by the suffix "i") when combined with MSLN-TTC. ATRi and PARPi appeared to induce the strongest increase in potency. Further, in vivo antitumor efficacy of the MSLN-TTC in combination with ATRi or PARPi was investigated in the OVCAR-3 and OVCAR-8 xenograft models in nude mice, demonstrating synergistic antitumor activity for the ATRi combination at doses demonstrated to be nonefficacious when administered as monotherapy. Conclusion: The presented data support the mechanism-based rationale for combining the MSLN-TTC with DDR inhibitors as new treatment strategies in MSLN-positive ovarian cancer.
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Daño del ADN/efectos de los fármacos , Proteínas Ligadas a GPI/farmacología , Neoplasias Ováricas/diagnóstico por imagen , Radiofármacos/farmacología , Torio/farmacología , Partículas alfa , Animales , Antineoplásicos , Apoptosis , Línea Celular Tumoral , Quelantes/farmacología , Reparación del ADN , Femenino , Xenoinjertos , Humanos , Mesotelina , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Piridonas/farmacología , Distribución TisularRESUMEN
BACKGROUND: Gastric cancer (GC) is a common cancer in Asia and currently lacks a targeted therapy approach. Mesothelin (MSLN) has been reported to be expressed in GC tissue and could be targeted by chimeric antigen receptor (CAR) T cells. Mesothelin targeting CAR-T has been reported in mesothelioma, lung cancer, breast cancer, and pancreas cancer. However, the feasibility of using anti-MSLN CAR T cells to treat GC remains to be studied. METHODS: We verified MSLN expression in primary human GC tissues and GC cell lines and then redirected T cells with a CAR containing the MSLN scFv (single-chain variable fragment), CD3ζ, CD28, and DAP10 intracellular signaling domain (M28z10) to target MSLN. We evaluated the function of these CAR T cells in vitro in terms of cytotoxicity, cytokine secretion, and surface phenotype changes when they encountered MSLN+ GC cells. We also established four different xenograft GC mouse models to assess in vivo antitumor activity. RESULTS: M28z10 T cells exhibited strong cytotoxicity and cytokine-secreting ability against GC cells in vitro. In addition, cell surface phenotyping suggested significant activation of M28z10 T cells upon target cell stimulation. M28z10 T cells induced GC regression in different xenograft mouse models and prolonged the survival of these mice compared with GFP-transduced T cells in the intraperitoneal and pulmonary metastatic GC models. Importantly, peritumoral delivery strategy can lead to improved CAR-T cells infiltration into tumor tissue and significantly suppress the growth of GC in a subcutaneous GC model. CONCLUSION: These results demonstrate that M28z10 T cells possess strong antitumor activity and represent a promising therapeutic approach to GC.
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Antígenos de Neoplasias/uso terapéutico , Proteínas Ligadas a GPI/uso terapéutico , Receptores Quiméricos de Antígenos/inmunología , Neoplasias Gástricas/genética , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/farmacología , Modelos Animales de Enfermedad , Proteínas Ligadas a GPI/farmacología , Humanos , Mesotelina , Ratones , TransfecciónRESUMEN
AIMS: Experimental and epidemiological studies reported controversial data on the role of omentin in type 2 diabetes and cardiovascular diseases. This study aimed to characterise the impact of omentin on the secretome of human adipocytes to analyse the enrichment of these proteins in metabolic and cellular signalling pathways underlying its physiological function. MATERIAL/METHODS: Differentiated primary human adipocytes were treated without or with 500 or 2000 ng/mL omentin for 24 hours. The secretome was analysed by liquid chromatography coupled tandem-mass spectrometry. Differences in protein secretion between untreated and omentin-treated adipocytes were compared using a paired t-test. Other potential upstream regulators and the overrepresentation in canonical pathways of omentin-stimulated proteins were analysed using Ingenuity Pathway Analysis. RESULTS: The supernatant of adipocytes contained 3493 proteins, of which 140 were differentially secreted by both concentrations of omentin compared with untreated adipocytes. Among the most strongly increased proteins, tumour necrosis factor-inducible gene 6 protein (TNFAIP6) was increased by 140-fold in the supernatant. Omentin-regulated proteins were overrepresented in seven canonical pathways including eukaryotic initiation factor 2 signalling, complement system, and inhibition of matrix metalloproteases. We further identified 25 other potential upstream activators of omentin-regulated proteins, mainly pro-inflammatory cytokines and transcription regulators including NFκB. CONCLUSIONS: In differentiated human adipocytes, the release of the anti-inflammatory TNFAIP6 might be part of a counterregulatory response to the pro-inflammatory action of omentin. Omentin-regulated proteins were overrepresented in pathways indicating cellular stress, a pro-inflammatory environment and a crosstalk with other organs. Other potential activators of omentin-regulated proteins point towards a central role of NFκB activation in the omentin-induced secretory process.
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Adipocitos/metabolismo , Citocinas/farmacología , Inflamación/metabolismo , Lectinas/farmacología , Adipocitos/efectos de los fármacos , Citocinas/metabolismo , Proteínas Ligadas a GPI/farmacología , Humanos , Fenotipo , Proteómica , Transducción de Señal/efectos de los fármacosRESUMEN
Acute lung injury (ALI) is characterized by inflammatory cell infiltration, macrophage activation, and excessive pro-inflammatory cytokine production. Bleomycin (BLM) is widely used to induce acute lung injury (ALI) and fibrosis in murine models. Intratracheally administration of BLM leads to the early stage of inflammatory response and the late stage of collagen deposition. Omentin-1 exerts an anti-inflammatory role in reducing tumor necrosis factor α (TNF-α)-induced cyclooxygenase-2 expression in endothelial cells and attenuating lipopolysaccharide (LPS)-induced ALI. However, the role of omentin-1 in BLM-induced ALI remains unclear. The aim of this study is to examine the effects of omentin-1 on BLM-induced ALI. We found that omentin-1 was decreased in lungs of BLM-induced ALI mice. Omentin-1 overexpression mediated by adenovirus alleviated lung injury and maintained the integrity of the alveolar septa. Omentin-1 overexpression also remarkably decreased the aggregation of neutrophil and macrophages activation, the expression of monocyte chemotactic protein 1 (MCP-1), and down-regulated expression of interleukin 1ß (IL-1ß) in lungs of BLM-induced ALI mice. Furthermore, we observed that omentin-1 reduced oxidative stress and suppressed the activation of NF-κB pathway in BLM-induced ALI and LPS-induced macrophages activation. Together, our findings indicated that omentin-1 protected mice from BLM-induced ALI may through reducing inflammatory cells recruitment and macrophages activation via alleviation of oxidative stress and NF-κB pathway. Thus, therapeutic strategies aiming to restore omentin-1 levels may be valuable for the prevention of BLM-induced ALI.
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Lesión Pulmonar Aguda/prevención & control , Citocinas/farmacología , Lectinas/farmacología , Pulmón/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Animales , Bleomicina , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/farmacología , Mediadores de Inflamación/metabolismo , Lectinas/genética , Lectinas/metabolismo , Lipopolisacáridos/farmacología , Pulmón/metabolismo , Pulmón/patología , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Sustancias Protectoras/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Omentin-1, a novel identified adipokine, always significantly decreases in patients with metabolic syndrome. However, the functional roles of omentin-1 in diabetic nephropathy (DN) remains largely unknown. In the present study, we found that omentin-1 treatment could improve renal function of type 2 diabetic db/db mice. ELISA assay and immunohistochemistry staining showed that omentin-1 reduced the productions of proinflammatory cytokines (IFN-γ, TNF-α, MCP-1 and IL-8), and improved oxidative stress level (CAT, MDA and SOD) in the kidney tissue, indicating omentin-1 could relieved the inflammatory response and suppressed oxidative stress. Mechanistic analysis demonstrated that omentin-1 down-regulated miR-27a expression, and subsequently inhibited oxidative stress and inflammation. Luciferase reporter assay and western blot further revealed that miR-27a directly targeted the 3' untranslated region (UTR) of nuclear factor erythroid 2-like 2 (Nrf2) and reduced its expression in type 2 DN. Taken together, these findings provide a new function of omentin-1 in renal protection and also delineate multiple potential targets for therapeutic intervention for type 2 DN.
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Citocinas/farmacología , Diabetes Mellitus Tipo 2/fisiopatología , Nefropatías Diabéticas/fisiopatología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Riñón/fisiopatología , Lectinas/farmacología , MicroARNs/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Sustancias Protectoras/farmacología , Animales , Secuencia de Bases , Peso Corporal/efectos de los fármacos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Proteínas Ligadas a GPI/farmacología , Humanos , Inflamación/patología , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Modelos Biológicos , Tamaño de los Órganos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Neuritin is a neurotrophic factor that is activated by neural activity and neurotrophins. Its major function is to promote neurite growth and branching; however, the underlying mechanisms are not fully understood. To address this issue, this study investigated the effects of neuritin on neurite and spine growth and intracellular Ca2+ concentration in rat cerebellar granule neurons (CGNs). Incubation of CGNs for 24 h with neuritin increased neurite length and spine density; this effect was mimicked by insulin and abolished by inhibiting insulin receptor (IR) or mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) activity. Calcium imaging and western blot analysis revealed that neuritin enhanced the increase in intracellular Ca2+ level induced by high K+ , and stimulated the cell surface expression of CaV 1.2 and CaV 1.3 α subunits of the L-type calcium channel, which was suppressed by inhibition of IR or mitogen-activated protein kinase kinase/ERK. Treatment with inhibitors of L-type calcium channels, calmodulin, and calcineurin (CaN) abrogated the effects of neuritin on neurite length and spine density. A similar result was obtained by silencing nuclear factor of activated T cells c4, which is known to be activated by neuritin in CGNs. These results indicate that IR and ERK signaling as well as the Ca2+ /CaN/nuclear factor of activated T cells c4 axis mediate the effects of neuritin on neurite and spine growth in CGNs. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14195.