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1.
Front Immunol ; 15: 1365946, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39131155

RESUMEN

Introduction: Humanized mouse models to recapitulate human biological systems still have limitations, such as the onset of lethal graft-versus-host disease (GvHD), a variable success rate, and the low accessibility of total body irradiation (TBI). Recently, mice modified with the CD47-SIRPA axis have been studied to improve humanized mouse models. However, such trials have been rarely applied in NOD mice. In this study, we created a novel mouse strain, NOD-CD47nullRag2nullIL-2rγnull (RTKO) mice, and applied it to generate humanized mice. Methods: Four-week-old female NOD-Rag2nullIL-2rγnull (RID) and RTKO mice pre-conditioned with TBI or busulfan (BSF) injection were used for generating human CD34+ hematopoietic stem cell (HSC) engrafted humanized mice. Clinical signs were observed twice a week, and body weight was measured once a week. Flow cytometry for human leukocyte antigens was performed at intervals of four weeks or two weeks, and mice were sacrificed at 48 weeks after HSC injection. Results: For a long period from 16 to 40 weeks post transplantation, the percentage of hCD45 was mostly maintained above 25% in all groups, and it was sustained the longest and highest in the RTKO BSF group. Reconstruction of human leukocytes, including hCD3, was also most prominent in the RTKO BSF group. Only two mice died before 40 weeks post transplantation in all groups, and there were no life-threatening GvHD lesions except in the dead mice. The occurrence of GvHD has been identified as mainly due to human T cells infiltrating tissues and their related cytokines. Discussion: Humanized mouse models under all conditions applied in this study are considered suitable models for long-term experiments based on the improvement of human leukocytes reconstruction and the stable animal health. Especially, RTKO mice pretreated with BSF are expected to be a valuable platform not only for generating humanized mice but also for various immune research fields.


Asunto(s)
Busulfano , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas , Ratones Endogámicos NOD , Ratones Noqueados , Acondicionamiento Pretrasplante , Animales , Busulfano/farmacología , Humanos , Ratones , Trasplante de Células Madre Hematopoyéticas/métodos , Acondicionamiento Pretrasplante/métodos , Células Madre Hematopoyéticas/metabolismo , Femenino , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Enfermedad Injerto contra Huésped/prevención & control , Enfermedad Injerto contra Huésped/inmunología , Modelos Animales de Enfermedad , Irradiación Corporal Total
2.
Biomater Res ; 27(1): 134, 2023 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-38102691

RESUMEN

BACKGROUND: Tumor-derived exosomes are critical elements of the cell-cell communication response to various stimuli. This study aims to reveal that the histone deacetylase 5 (HDAC5) and p53 interaction upon radiation in hepatocellular carcinoma intricately regulates the secretion and composition of exosomes. METHODS: We observed that HDAC5 and p53 expression were significantly increased by 2 Gy and 4 Gy radiation exposure in HCC. Normal- and radiation-derived exosomes released by HepG2 were purified to investigate the exosomal components. RESULTS: We found that in the radiation-derived exosome, exosomal Maspin was notably increased. Maspin is known as an anti-angiogenic gene. The expression of Maspin was regulated at the cellular level by HDAC5, and it was elaborately regulated and released in the exosome. Radiation-derived exosome treatment caused significant inhibition of angiogenesis in HUVECs and mouse aortic tissues. Meanwhile, we confirmed that miR-151a-3p was significantly reduced in the radiation-derived exosome through exosomal miRNA sequencing, and three HCC-specific exosomal miRNAs were also decreased. In particular, miR-151a-3p induced an anti-apoptotic response by inhibiting p53, and it was shown to induce EMT and promote tumor growth by regulating p53-related tumor progression genes. In the HCC xenograft model, radiation-induced exosome injection significantly reduced angiogenesis and tumor size. CONCLUSIONS: Our present findings demonstrated HDAC5 is a vital gene of the p53-mediated release of exosomes resulting in tumor suppression through anti-cancer exosomal components in response to radiation. Finally, we highlight the important role of exosomal Maspin and mi-151a-3p as a biomarker in enhancing radiation treatment sensitivity. Therapeutic potential of HDAC5 through p53-mediated exosome modulation in radiation treatment of hepatocellular carcinoma.

3.
J Cell Mol Med ; 26(7): 2104-2118, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35178859

RESUMEN

Damage to normal tissue can occur over a long period after cancer radiotherapy. Free radical by radiation can initiate or accelerate chronic inflammation, which can lead to atherosclerosis. However, the underlying mechanisms remain unclear. Vascular smooth muscle cells (VSMCs) proliferate in response to JAK/STAT3 signalling. C-reactive protein (CRP) can induce VSMCs apoptosis via triggering NADPH oxidase (NOX). Apoptotic VSMCs promote instability and inflammation of atherosclerotic lesions. Herein, we identified a VSMCs that switched from proliferation to apoptosis through was enhanced by radiation-induced CRP. NOX inhibition using lentiviral sh-p22phox prevented apoptosis upon radiation-induced CRP. CRP overexpression reduced the amount of STAT3/Ref-1 complex, decreased JAK/STAT phosphorylation and formed a new complex of Ref-1/CRP in VSMC. Apoptosis of VSMCs was further increased by CRP co-overexpressed with Ref-1. Functional inhibition of NOX or p53 also prevented apoptotic activity of the CRP-Ref-1 complex. Immunofluorescence showed co-localization of CRP, Ref-1 and p53 with α-actin-positive VSMC in human atherosclerotic plaques. In conclusion, radiation-induced CRP increased the VSMCs apoptosis through Ref-1, which dissociated the STAT3/Ref-1 complex, interfered with JAK/STAT3 activity, and interacted with CRP-Ref-1, thus resulting in transcription-independent cell death via p53. Targeting CRP as a vascular side effect of radiotherapy could be exploited to improve curability.


Asunto(s)
Proteína C-Reactiva , Músculo Liso Vascular , Apoptosis , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Células Cultivadas , Humanos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/metabolismo
4.
Breast Cancer Res Treat ; 182(3): 591-600, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32529408

RESUMEN

PURPOSE: This study evaluates the oncogenic role of PIBF1 in triple-negative breast cancer (TNBC). TNBC is considered to have a poorer prognosis than other types of breast cancer and is associated with high risk of recurrence and distant metastasis. Currently, there are no effective therapies for the TNBC patients with distant metastasis due to the lack of targeted therapeutic options. METHODS: The effects of PIBF1 knockdown on the cell viability and motility of TNBC cell lines were investigated. Effects of PIBF1 overexpression on tumorigenicity and cell motility were confirmed using Ba/F3 cell line and xenograft study on BALB/c nude mice. RESULTS: In TNBC cell lines that highly express PIBF1, knockdown of PIBF1 induces apoptosis and suppresses cell viability and motility with activation of the ATR/CHK1 signaling pathway. Moreover, the oncogenic function of PIBF1 was confirmed using the Ba/F3 cell line. CONCLUSION: For the first time, these findings clarify the role of PIBF1 in regulating ATR/CHK1 signaling pathway and inhibiting the proliferation and migration of TNBC cell lines. These results demonstrate the oncogenic roles of PIBF1 and provide new insights into the function and the molecular mechanism of PIBF1 in malignant TNBC.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Proteínas Gestacionales/metabolismo , Factores Supresores Inmunológicos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Apoptosis/fisiología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Gestacionales/biosíntesis , Proteínas Gestacionales/genética , Transducción de Señal , Factores Supresores Inmunológicos/biosíntesis , Factores Supresores Inmunológicos/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas
5.
Radiat Res ; 191(3): 262-270, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30702968

RESUMEN

In the event of a mass casualty radiation scenario, biodosimetry has the potential to quantify individual exposures for triaging and providing dose-appropriate medical intervention. Structural maintenance of chromosomes 1 (SMC1) is phosphorylated in response to ionizing radiation. The goal of this study was to develop a new biodosimetry method using SMC1 phosphorylation as a measure of exposure to radiation. In the initial experiments, two normal human cell lines (WI-38VA-13 and HaCaT) and four lymphoblastoid cell lines were irradiated, and the levels of SMC1 phosphorylation at Ser-360 and Ser-957 were assessed using Western blotting. Subsequently, similar experiments were performed using peripheral blood mononuclear cells (PBMCs) obtained from 20 healthy adults. Phosphorylation of SMC1 at Ser-957 and Ser-360 was increased by exposure in a dose-dependent manner, peaked at 1-3 h postirradiation and then decreased gradually. Ser-360 was identified as a new phosphorylation site and was more sensitive to radiation than Ser-957, especially at doses below 1 Gy. Our results demonstrate a robust ex vivo response of phospho-SMC1-(Ser-360) to ionizing radiation in human PBMCs. Detection of phosphorylation at Ser-360 in SMC1 could be used as a marker of radiation exposure. Our findings suggest that it is feasible to measure blood cell-based changes in the phosphorylation level of a protein as an ex vivo radiation exposure detection method, even after low-dose exposure.


Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/efectos de la radiación , Serina/metabolismo , Línea Celular , Cromatina/metabolismo , Relación Dosis-Respuesta en la Radiación , Humanos , Fosforilación/efectos de la radiación , Factores de Tiempo
6.
Anticancer Res ; 37(2): 607-614, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-28179307

RESUMEN

AIM: We investigated the therapeutic effects of a mitogen-activated protein (MEK) inhibitor, selumetinib, in a hepatic melanoma metastasis model and studied its possible mechanism of action. MATERIALS AND METHODS: Melanoma cell lines were exposed to selumetinib under different experimental conditions. We established a mouse model of liver metastasis and treated mice orally with vehicle or selumetinib and then evaluated metastasis progress. RESULTS: Growth inhibition was observed in melanoma cells as a consequence of G1-phase cell-cycle arrest and the subsequent induction of apoptosis in a dose- and time-dependent manner. Mice with established liver metastases that were treated with selumetinib exhibited significantly less tumor progression than vehicle-treated mice. c-Myc expression in metastasized liver tissues were suppressed by selumetinib. Moreover, oral treatment with selumetinib modulated expression of epithelial-to-mesenchymal transition- and metastasis-related genes, including integrin alpha-5 (ITGA5), jagged 1 (JAG1), zinc finger E-box-binding homeobox 1 (ZEB1), NOTCH, and serpin peptidase inhibitor clade E (SERPINE1). CONCLUSION: We established a mouse model of hepatic metastasis using a human melanoma cell line, such models are essential in elucidating the therapeutic effects of anti-metastatic drugs. Our data suggest the possibility that selumetinib presents a new strategy to treat liver metastasis in patients with melanoma by suppressing epithelial-to-mesenchymal transition-related genes.


Asunto(s)
Bencimidazoles/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Hepáticas/prevención & control , Melanoma/tratamiento farmacológico , Administración Oral , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Bencimidazoles/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Integrina alfa5/genética , Integrina alfa5/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Masculino , Melanoma/genética , Melanoma/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Oncotarget ; 8(3): 4181-4195, 2017 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-27935858

RESUMEN

Epidermal growth factor (EGF) signaling promotes cell proliferation and survival in several types of cancer. Here, however, we showed that EGF inhibits proliferation and promotes programmed cell death in non-small cell lung cancer (NSCLC) cells. In A549 cells, EGF increased redox factor-1 (Ref-1) expression and the association of Ref-1 with zinc finger-containing transcriptional regulator (EGR1) via activation of p22phox, RAC1, and an NADPH oxidase subunit. EGF increased p22phox and RAC1 expression through activation of purinergic receptors (P2Y). Elevated Ref-1/EGR1 levels increased phosphatase and tensin homolog (PTEN) levels, leading to inhibition of the Akt pathway. EGF-induced PTEN upregulation increased apoptosis and autophagy-induced damage in A549 cells, whereas Ref-1 knockdown blocked EGF-induced PTEN upregulation in an NADPH oxidase p22phox subunit-independent manner. In addition, p22phox knockdown restored EGF-induced effects, implying that changes in P2Y activity caused by EGF, which activates NADPH oxidase via RAC1, influenced Ref-1-mediated redox regulation. Finally, EGF similarly attenuated cell proliferation and promoted autophagy and apoptosis in vivo in a xenograft model using A549 cells. These findings reveal that EGF-induced redox signaling is linked to Ref-1-induced death in NSCLC cells.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Factor de Crecimiento Epidérmico/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Fosfohidrolasa PTEN/metabolismo , Regulación hacia Arriba , Células A549 , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Factor de Crecimiento Epidérmico/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fosfohidrolasa PTEN/genética , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Diabetes ; 65(9): 2624-38, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27284106

RESUMEN

Glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway, plays important roles in redox regulation and de novo lipogenesis. It was recently demonstrated that aberrant upregulation of G6PD in obese adipose tissue mediates insulin resistance as a result of imbalanced energy metabolism and oxidative stress. It remains elusive, however, whether inhibition of G6PD in vivo may relieve obesity-induced insulin resistance. In this study we showed that a hematopoietic G6PD defect alleviates insulin resistance in obesity, accompanied by reduced adipose tissue inflammation. Compared with wild-type littermates, G6PD-deficient mutant (G6PD(mut)) mice were glucose tolerant upon high-fat-diet (HFD) feeding. Intriguingly, the expression of NADPH oxidase genes to produce reactive oxygen species was alleviated, whereas that of antioxidant genes was enhanced in the adipose tissue of HFD-fed G6PD(mut) mice. In diet-induced obesity (DIO), the adipose tissue of G6PD(mut) mice decreased the expression of inflammatory cytokines, accompanied by downregulated proinflammatory macrophages. Accordingly, macrophages from G6PD(mut) mice greatly suppressed lipopolysaccharide-induced proinflammatory signaling cascades, leading to enhanced insulin sensitivity in adipocytes and hepatocytes. Furthermore, adoptive transfer of G6PD(mut) bone marrow to wild-type mice attenuated adipose tissue inflammation and improved glucose tolerance in DIO. Collectively, these data suggest that inhibition of macrophage G6PD would ameliorate insulin resistance in obesity through suppression of proinflammatory responses.


Asunto(s)
Tejido Adiposo/metabolismo , Deficiencia de Glucosafosfato Deshidrogenasa/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Resistencia a la Insulina/fisiología , Obesidad/inmunología , Obesidad/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Tejido Adiposo/inmunología , Animales , Western Blotting , Medios de Cultivo Condicionados , Dieta Alta en Grasa/efectos adversos , Ayuno/sangre , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Inmunohistoquímica , Insulina/sangre , Resistencia a la Insulina/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Mutantes , Obesidad/genética , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
9.
Oncotarget ; 7(13): 15554-65, 2016 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-26799284

RESUMEN

Radiation-induced fibrosis (RIF) is one of the most common late complications of radiation therapy. We found that α-lipoic acid (α-LA) effectively prevents RIF. In RIF a mouse model, leg contracture assay was used to test the in vivo efficacy of α-LA. α-LA suppressed the expression of pro-fibrotic genes after irradiation, both in vivo and in vitro, and inhibited the up-regulation of TGF-ß1-mediated p300/CBP activity. Thus, α-LA prevents radiation-induced fibrosis (RIF) by inhibiting the transcriptional activity of NF-κB through inhibition of histone acetyltransferase activity. α-LA is a new therapeutic methods that can be used in the prevention-treatment of RIF.


Asunto(s)
Fibrosis/prevención & control , Traumatismos por Radiación/prevención & control , Protectores contra Radiación/farmacología , Ácido Tióctico/farmacología , Animales , Antioxidantes/farmacología , Fibrosis/etiología , Ratones , Ratones Endogámicos BALB C
10.
FEBS Lett ; 588(4): 625-31, 2014 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-24440351

RESUMEN

We hypothesized that C-reactive protein (CRP) may affect the cell cycle and induce apoptotic changes of monocytes. CRP (∼25 µg/ml) significantly increased expressions of B-cell translocation gene 2 (BTG2) mRNA and protein in human monocytes through pathways involving CD32/NADPH oxidase 2/p53, which eventually induced G2/M phase arrest and apoptotic cell death. Such pro-apoptotic effect of CRP was not found in thioglycollate-elicited intraperitoneal monocytes/macrophages harvested from BTG2-knockout male C57BL/6 mice (n=5). Within atheromatous plaques obtained from CRP-transgenic male LDLR(-/-) C57BL/6 mice (n=5) and human coronary arteries, BTG2 co-localized with CRP, p53 and monocytes/macrophages. Therefore the pro-apoptotic pathway of CRP-CD32-Nox2-p53-BTG2 may contribute to the retardation of the atherogenic process.


Asunto(s)
Apoptosis , Proteína C-Reactiva/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Proteínas Inmediatas-Precoces/genética , Puntos de Control de la Fase M del Ciclo Celular , Monocitos/citología , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba , Animales , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
11.
J Inflamm (Lond) ; 9(1): 42, 2012 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-23114023

RESUMEN

RATIONALE: C-reactive protein (CRP) and lysophosphatidylcholine (LPC) are phosphorylcholine-(PC)-containing oxidized phospholipids (oxPLs) found in oxidized LDL (oxLDL), which trigger pro-atherogenic activities of macrophages during the process of atherosclerosis. It has been previously reported that CRP binds to the PC head group of oxLDL in a calcium-dependent manner. The aim of this study was to investigate the importance of binding between CRP and LPC to the pro-atherogenic activities of macrophages. OBJECTIVES AND FINDINGS: A chemiluminescent immunoassay and HPLC showed that human recombinant CRP formed a stable complex with LPC in the presence of calcium. The Kd value of the binding of the CRP-LPC complex to the receptors FcγRIA or FcγRIIA was 3-5 fold lower than that of CRP alone. The CRP-LPC complex triggered less potent generation of reactive oxygen species and less activation of the transcription factors AP-1 and NF-kB by human monocyte-derived macrophages in comparison to CRP or LPC alone. However, CRP did not affect activities driven by components of oxLDL lacking PC, such as upregulation of PPRE, ABCA1, CD36 and PPARγ and the enhancement of cholesterol efflux by human macrophages. The presence of CRP inhibited the association of Dil-labelled oxLDL to human macrophages. CONCLUSIONS: The formation of complexes between CRP and PC-containing oxPLs, such as LPC, suppresses the pro-atherogenic effects of CRP and LPC on macrophages. This effect may in part retard the progression of atherosclerosis.

12.
Magn Reson Med ; 64(1): 72-9, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20572147

RESUMEN

The CCR2 antagonist is a receptor antagonist for monocyte chemoattractant protein-1 and is known to be a potential anti-inflammatory therapeutic agent. Recently used optimized labeling techniques for superparamagnetic iron oxide, macrophage homing, and recruitment toward the infection site can be observed on in vivo MRI. This study details the effect of the CCR2 antagonist on the macrophage migration and the feasibility of in vivo MRI for assessing the inhibition of chemotactic activity by the CCR2 antagonist. On binding assay, the CCR2 antagonist inhibits the binding affinity of monocyte chemoattractant protein-1 to CCR2. Increased expression of messenger ribonucleic acid (mRNA) and expression of CCR2 and CD11b on the cellular surface, as induced by monocyte chemoattractant protein-1, was shown, and the effect of monocyte chemoattractant protein-1 on CCR2 and CD11b was restricted by the CCR2 antagonist. In a migration test using the transwell system, macrophages treated with the CCR2 antagonist showed significantly decreased chemotactic migration compared to that of wild-type macrophages. MR images of infected left calf muscles in 12 mice were obtained 24 h after administration of macrophages labeled with superparamagnetic iron oxide. MRI successfully demonstrated the effect of the CCR2 antagonist on the directional migration of macrophages.


Asunto(s)
Macrófagos/efectos de los fármacos , Imagen por Resonancia Magnética , Receptores CCR2 , Animales , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Movimiento Celular , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/metabolismo , Regulación hacia Abajo , Macrófagos/diagnóstico por imagen , Masculino , Ratones , Microscopía Confocal , Piperidinas/farmacología , ARN Mensajero/metabolismo , Radiografía , Receptores CCR2/antagonistas & inhibidores , Receptores CCR2/genética , Receptores CCR2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Compuestos de Espiro/farmacología
13.
Arterioscler Thromb Vasc Biol ; 29(12): 2138-45, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19745197

RESUMEN

OBJECTIVE: The serum level of tumor necrosis factor-alpha (TNF-alpha) is in the picomolar range under inflammatory conditions. We investigated whether these picomolar levels of TNF-alpha directly modulate the functional activities of circulating monocytes. METHODS AND RESULTS: In THP-1 monocytes treated with TNF-alpha (1 to 100 pmol/L/30 minutes), cytosolic RhoA small GTPase rapidly translocated to the plasma membrane via functionally active ezrin/radixin/moesin (ERM) complex, a cytoskeletal linker, and subsequent actin polymerization through NF-kappaB activation. The threonine phosphorylation of ERM was accomplished by the activation of TNF receptor type I (TNFRI) and signaling pathways involving PI3K and an atypical PKC; ie, PKCzeta. The TNF-alpha-treated monocytes (10 pmol/L) displayed more potent and prolonged generation of GTP-bound RhoA in response to secondary stimulation with RhoA-activating monocyte chemoattractant protein-1 (MCP-1). Clearly, human circulating monocytes preconditioned by 10 pmol/L TNF-alpha augmented MCP-1-mediated chemotaxis and firm adhesion on VCAM-1 and ICAM-1 in vitro and ex vivo. The elevation of serum TNF-alpha (>5 pmol/L within 16 hours), which was introduced by intraperitoneal injection of mouse-specific TNF-alpha to C57/BL6 mice, enhanced the number of CD80+ monocytes transmigrating to the JE/MCP-1-injected intraperitoneal space. CONCLUSIONS: Picomolar concentrations of TNF-alpha in the bloodstream may prime the RhoA-dependent activities of circulating monocytes to enhance recruitment to active inflammatory foci.


Asunto(s)
Monocitos/efectos de los fármacos , Monocitos/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Proteínas de Unión al GTP rho/fisiología , Proteína de Unión al GTP rhoA/fisiología , Animales , Transporte Biológico Activo/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Quimiocina CCL2/farmacología , Proteínas del Citoesqueleto/metabolismo , Humanos , Técnicas In Vitro , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/fisiología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , FN-kappa B/metabolismo , Cavidad Peritoneal/citología , Fosforilación , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/administración & dosificación
14.
Cardiovasc Res ; 84(3): 378-86, 2009 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-19596672

RESUMEN

AIMS: We investigated the mechanism by which cannabinoid receptors-1 (CB1) and -2 (CB2) modulate inflammatory activities of macrophages. METHODS AND RESULTS: Real-time polymerase chain reaction showed the predominant CB2 expression in freshly isolated human monocytes. PMA, a potent inducer of differentiation, upregulated CB1 and increased CB1:CB2 transcript ratio from 1:17.5 to 1:3 in 5 days of culture. Immunohistochemistry showed that CB1 protein was colocalized in CD68- and CD36-positive macrophages in human atheroma. Through selective expression of CB1 or CB2 to thioglycollate-elicited peritoneal macrophages, we proved that CB1 and CB2 mediate opposing influences on the production of reactive oxygen species (ROS). Flow cytometry showed that cannabinoid-induced ROS production by macrophages was CB1-dependent. Immunoblotting assays confirmed that macrophage CB1, not CB2, induced phosphorylation of p38-mitogen-activated protein kinase, which modulated ROS production and the subsequent synthesis of tumour necrosis factor-alpha and monocyte chemoattractant protein-1. Pull-down assays showed that the Ras family small G protein, Rap1 was activated by CB2. Dominant-negative Rap1 profoundly enhanced CB1-dependent ROS production by macrophages, suggesting CB2 Rap1-dependently inhibits CB1-stimulated ROS production. CONCLUSION: CB1 promotes pro-inflammatory responses of macrophages through ROS production, which is negatively regulated by CB2 through Rap1 activation. Blocking CB1 together with selective activation of CB2 may suppress pro-inflammatory responses of macrophages.


Asunto(s)
Macrófagos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Animales , Línea Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Citocinas/metabolismo , Humanos , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB2/agonistas , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas de Unión al GTP rap1/metabolismo
15.
Cardiovasc Res ; 78(2): 333-40, 2008 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-18006432

RESUMEN

AIMS: The present study investigated the detailed mechanism by which fractalkine (Fkn), a CX3C chemokine, induces angiogenesis and its functional implication in alleviating ischaemia in vivo. METHODS AND RESULTS: Fkn induced new vessel formation on the excised rat aorta and chick chorioallantoic membrane (CAM) through CX3CR1 activation. Immunoblotting analysis, promoter assay and electrophoretic mobility shift assay showed that Fkn upregulated hypoxia-inducible factor-1 alpha (HIF-1alpha) by cultured human aortic endothelial cells (ECs), which in turn induced mRNA and protein levels of vascular endothelial growth factor (VEGF)-A through a p42/44 mitogen-activated protein kinase pathway. In vivo Fkn-induced angiogenesis on CAM was completely blocked by functional inhibition of VEGF receptor 2 kinase insert domain-containing receptor (KDR) and Rho GTPase. C57/BL6 mice with CX3CR1(-/-) bone marrow-derived cells developed angiogenesis in the implanted Fkn-mixed Matrigel plug, suggesting CX3CR1 activation in vascular ECs is sufficient for Fkn-induced angiogenesis in vivo. The condition of rat hindlimb ischaemia, which rapidly stimulated mRNA expression of both Fkn and VEGF-A, was significantly alleviated by the injection of whole-length Fkn protein. CONCLUSION: Fkn-induced activation of CX3CR1 by ECs leads to in vivo angiogenesis through two sequential steps: the induction of HIF-1alpha and VEGF-A gene expression by CX3CR1 activation and the subsequent VEGF-A/KDR-induced angiogenesis. The potent induction of angiogenesis by Fkn can be used as a therapeutic strategy for alleviating peripheral ischaemia.


Asunto(s)
Proteínas Angiogénicas/metabolismo , Quimiocina CX3CL1/metabolismo , Células Endoteliales/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Receptores CXCR/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Angiogénicas/farmacología , Animales , Receptor 1 de Quimiocinas CX3C , Línea Celular , Células Cultivadas , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/farmacología , Embrión de Pollo , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Miembro Posterior , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia/fisiopatología , Isquemia/terapia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores CXCR/genética , Receptores de Quimiocina/metabolismo , Proteínas Recombinantes/metabolismo , Flujo Sanguíneo Regional , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/genética , Proteínas de Unión al GTP rho/metabolismo
16.
Cardiovasc Res ; 75(3): 555-65, 2007 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-17531211

RESUMEN

OBJECTIVES: We investigated the mechanism by which C-reactive protein (CRP) affects pro-inflammatory activities of vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: RT-PCR, flow cytometry, and immunoblotting assays consistently showed the expression of FcgammaRIIa by cultured VSMCs isolated from human coronary arteries. Immunofluorescence staining of human coronary artery plaque showed the co-localization of FcgammaRIIa with alpha-actin(+) VSMCs in atheromatous regions. Confocal microscopic image analysis of H(2)DCFDA-labeled cells showed that CRP induced intracellular reactive oxygen species (ROS) generation by FcgammaRIIa(+) HEK293T cells. Moreover, CRP time- and dose-dependently generated ROS in VSMCs through FcgammaRIIa activation. VSMCs mainly express NADPH oxidase 4 isoform (Nox4), the suppression of which using a specific siRNA completely abolished CRP-induced ROS generation by VSMCs. The downregulation of p22(phox), a component of the active Nox4 complex, by transfecting with specific decoy oligomers and functional blocking of FcgammaRIIa not only inhibited the CRP-induced ROS generation but also reduced the degree of AP-1 and NF-kappaB activation, the production of MCP-1, IL-6, and ET-1, and the apoptotic changes of VSMCs in response to CRP. CONCLUSIONS: CRP-induced ROS generation by VSMCs, which requires functional activation of FcgammaRIIa and NADPH oxidase 4, orchestrates pro-inflammatory activities of VSMCs and may eventually promote atherogenesis and plaque rupture.


Asunto(s)
Antígenos CD/fisiología , Proteína C-Reactiva/farmacología , Miocitos del Músculo Liso/inmunología , NADPH Oxidasas/metabolismo , Receptores de IgG/fisiología , Anciano , Antígenos CD/genética , Apoptosis , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Línea Celular , Células Cultivadas , Quimiocina CCL2/genética , Enfermedad Coronaria/metabolismo , Enfermedad Coronaria/patología , Vasos Coronarios , Endotelina-1/genética , Activación Enzimática , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/genética , Masculino , Glicoproteínas de Membrana/genética , Microscopía Confocal , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/análisis , NADPH Oxidasas/genética , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/genética , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción CHOP/genética , Transfección/métodos
17.
Circulation ; 111(11): 1439-47, 2005 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-15781755

RESUMEN

BACKGROUND: The migration of circulating monocytes to the arterial wall during atherogenesis is largely modulated by activation of the CC chemokine receptor 2 (CCR2), a dominant monocyte chemotaxis receptor. The present study investigated whether 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition affects CCR2 gene expression and CCR2-dependent monocyte recruitment. METHODS AND RESULTS: Competitive reverse transcription-polymerase chain reaction analysis and flow cytometry showed that simvastatin, an HMG-CoA reductase inhibitor, dose-dependently reduced monocyte CCR2 mRNA and protein expression. Treatment of 21 normocholesterolemic men with simvastatin (20 mg/d for 2 weeks) decreased CCR2 protein and mRNA expression in circulating monocytes. Promoter and electrophoretic mobility shift assays showed that simvastatin activated a peroxisome proliferator response element in THP-1 monocytes. Moreover, simvastatin-induced CCR2 downregulation was completely reversed by the synthetic peroxisome proliferator-activated receptor-gamma antagonist GW9662. Simvastatin-treated monocytes showed little chemotaxis movement in response to monocyte chemoattractant protein-1 (MCP-1), a specific CCR2 ligand. Treatment of C57/BL6 mice with simvastatin (0.2 microg/g body weight IP, daily for 1 week) inhibited transmigration of CD80+ monocytes to the MCP-1-injected intraperitoneal space. Moreover, few circulating inflammatory cells from simvastatin-treated Sprague-Dawley rats (0.2 microg/g body weight IP, daily for 2 weeks) were recruited to the aortic wall of hypercholesterolemic littermates. CONCLUSIONS: The inhibition of CCR2/MCP-1-dependent monocyte recruitment by simvastatin may prevent excessive accumulation of monocytes in the arterial wall during atherogenesis.


Asunto(s)
Quimiocina CCL2/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Monocitos/efectos de los fármacos , Receptores de Quimiocina/biosíntesis , Simvastatina/farmacología , Anilidas/farmacología , Animales , Aorta/metabolismo , Aorta/patología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Depresión Química , Dieta Aterogénica , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patología , Masculino , Ácido Mevalónico/análisis , Ratones , Ratones Endogámicos C57BL , Monocitos/química , PPAR gamma/antagonistas & inhibidores , PPAR gamma/fisiología , Fosfatos de Poliisoprenilo/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores CCR2 , Receptores de Quimiocina/genética , Rosiglitazona , Sesquiterpenos , Tiazolidinedionas/farmacología
18.
Blood ; 105(4): 1405-7, 2005 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-15498848

RESUMEN

Monocyte chemoattractant protein-1 (MCP-1) has been recognized as an angiogenic chemokine. In the present study, we investigated the detailed mechanism by which MCP-1 induces angiogenesis. We found that MCP-1 up-regulated hypoxia-inducible factor 1 alpha (HIF-1 alpha) gene expression in human aortic endothelial cells (HAECs), which induced vascular endothelial growth factor-A(165) (VEGF-A(165)) expression in the aortic wall and HAECs through activation of p42/44 mitogen-activated protein kinase (MAPK). In vivo angiogenesis assay using chick chorioallantoic membrane (CAM) showed that MCP-1-induced angiogenesis was as potent as that induced by VEGF-A(165) and completely inhibited by a VEGF inhibitor, Flt(2-11). The inhibition of RhoA small G protein did not affect MCP-1-induced VEGF-A(165) production and secretion but completely blocked both MCP-1- and VEGF-A-induced new vessel formation, as determined by CAM assay. These results suggest that MCP-1-induced angiogenesis is composed largely of 2 sequential steps: the induction of VEGF-A gene expression by MCP-1 and the subsequent VEGF-A-induced angiogenesis.


Asunto(s)
Inductores de la Angiogénesis/administración & dosificación , Quimiocina CCL2/administración & dosificación , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Aorta Abdominal/fisiología , Aorta Torácica/fisiología , Células Cultivadas , Quimiocina CCL2/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Masculino , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genética
19.
Nat Med ; 10(7): 727-33, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15195087

RESUMEN

AMP-activated protein kinase (AMPK) functions as a fuel sensor in the cell and is activated when cellular energy is depleted. Here we report that alpha-lipoic acid (alpha-LA), a cofactor of mitochondrial enzymes, decreases hypothalamic AMPK activity and causes profound weight loss in rodents by reducing food intake and enhancing energy expenditure. Activation of hypothalamic AMPK reverses the effects of alpha-LA on food intake and energy expenditure. Intracerebroventricular (i.c.v.) administration of glucose decreases hypothalamic AMPK activity, whereas inhibition of intracellular glucose utilization through the administration of 2-deoxyglucose increases hypothalamic AMPK activity and food intake. The 2-deoxyglucose-induced hyperphagia is reversed by inhibiting hypothalamic AMPK. Our findings indicate that hypothalamic AMPK is important in the central regulation of food intake and energy expenditure and that alpha-LA exerts anti-obesity effects by suppressing hypothalamic AMPK activity.


Asunto(s)
Fármacos Antiobesidad/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Hipotálamo/efectos de los fármacos , Ácido Tióctico/farmacología , Animales , Peso Corporal/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Ingestión de Alimentos/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Hipotálamo/enzimología , Hipotálamo/fisiología , Leptina/fisiología , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Ratas
20.
Arterioscler Thromb Vasc Biol ; 24(5): 864-70, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15016641

RESUMEN

OBJECTIVE: Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and regulates production of reactive oxygen species in macrophages. Previous studies have shown that selective genetic disruption of UCP2 in bone marrow cells results in excess accumulation of monocytes/macrophages in the vascular wall of hypercholesterolemic low-density lipoprotein receptor-deficient (LDLR-/-) mice. Here we investigated whether UCP2 regulates expression of genes involved in monocyte recruitment. METHODS AND RESULTS: UCP2 overexpression in THP1 monocytes, which induced a 10-fold increase in mitochondrial UCP2 protein levels, reduced steady-state level of intracellular reactive oxygen species (ROS) and H2O2-induced ROS production. THP1 monocytes with UCP2 overexpression showed lower intracellular calcium levels and less H2O2-triggered intracellular calcium mobilization, and less protein and mRNA levels of beta2 integrins, most notably CD11b. UCP2 overexpression reduced beta2 integrin-mediated firm adhesion of monocytes to either tumor necrosis factor-alpha (TNF-alpha)-stimulated human aortic endothelial cell (HAEC) monolayers or to plates coated with intercellular adhesion molecule-1, not vascular cell adhesion molecule-1. UCP2 overexpression also inhibited cell spreading and actin polymerization in monocytes treated with TNF-alpha and monocyte chemoattractant protein-1 (MCP-1), and reduced MCP-1-induced transmigration of monocytes through HAEC monolayers. CONCLUSIONS: Mitochondrial UCP2 in circulating monocytes may prevent excessive accumulation of monocytes/macrophages in the arterial wall, thereby reducing atherosclerotic plaque formation.


Asunto(s)
Antígenos CD18/fisiología , Proteínas de Transporte de Membrana/fisiología , Proteínas Mitocondriales/fisiología , Monocitos/metabolismo , Actinas/metabolismo , Aorta/citología , Arteriosclerosis/metabolismo , Biopolímeros , Antígenos CD18/biosíntesis , Antígenos CD18/genética , Señalización del Calcio , Adhesión Celular , Moléculas de Adhesión Celular/fisiología , Línea Celular/citología , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Quimiocina CCL2/farmacología , Células Endoteliales/citología , Endotelio Vascular/citología , Expresión Génica , Humanos , Canales Iónicos , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Microscopía Confocal , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Monocitos/citología , Monocitos/efectos de los fármacos , ARN Mensajero/biosíntesis , Receptores CCR2 , Receptores de Quimiocina/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Proteína Desacopladora 2
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