RESUMEN
Vasohibin is thought to be an important negative feedback regulator of angiogenesis that is selectively induced in endothelial cells by VEGF. Here, we assessed the role of vasohibin on HIF-1α expression under oxidative stress induced by hydrogen peroxide (H2O2) in HUVEC. VEGF induced significant cell growth that was associated with an increase in vasohibin expression. Following H2O2-pretreatment, VEGF further increased cell growth but this was contrastingly associated with a decrease in vasohibin expression when compared with VEGF alone. Interestingly, vasohibin inhibited cell proliferation through degradation of HIF-1α expression during H2O2-pretreatment. Furthermore, vasohibin elevated the expression of prolyl hydroxylase (PHD). These results suggest that vasohibin plays crucial roles as a negative feedback regulator of angiogenesis through HIF-1α degradation via PHD.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/enzimología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Estrés Oxidativo , Procolágeno-Prolina Dioxigenasa/metabolismo , Transporte Biológico/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Retroalimentación Fisiológica , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Isoenzimas/metabolismo , Neovascularización Patológica/prevención & control , Oxidantes/farmacología , Proteolisis/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In this study, we improved a method for rapid determination of viral RNA sequences (RDV) to overcome the limitations of previous versions. The RDV ver4.0 method can detect RNA sequences with at least 1,000 copies as starting material. A novel virus, which was isolated from field-collected Aedes aegypti larvae in the Phasi Charoen district of Thailand using C6/36 cells, was identified using the RDV ver4.0 protocol. The virus was named Phasi Charoen virus (PhaV). We used a high-throughput pyrosequencing approach to obtain more information about the genome sequence of PhaV. Analysis of a phylogenic tree based on amino acid sequences strongly suggested that PhaV belongs to the family Bunyaviridae.
Asunto(s)
Aedes/virología , Bunyaviridae/genética , Bunyaviridae/aislamiento & purificación , ARN Viral/genética , Virología/métodos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bunyaviridae/clasificación , Chlorocebus aethiops , ADN Complementario/química , Larva/virología , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico/métodos , Filogenia , ARN Viral/química , Células VeroRESUMEN
A method for rapid determination of viral RNA sequences (RDV) was applied to homogenates of Aedes aegypti collected in Thailand in an area in which dengue fever (dengue hemorrhagic fever) is endemic, using the mosquito cell line C6/36. Nucleic acid sequences of dengue virus type 4 and cell fusing agent virus were detected. This RDV method has the potential to become a standard method for detection of both known and newly emerging, unknown mosquito-borne viruses.
Asunto(s)
Aedes/virología , Virus del Dengue/aislamiento & purificación , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Línea Celular , Virus del Dengue/genética , Femenino , Dengue Grave/virología , TailandiaRESUMEN
Lipopolysaccharide (LPS) and inflammatory cytokines cause activation of sphingomyelinases (SMases) and subsequent hydrolysis of sphingomyelin (SM) to produce a lipid messenger ceramide. The use of SMase inhibitors may offer new therapies for the treatment of the LPS- and cytokines-related inflammatory bowel disease (IBD). We synthesized a series of difluoromethylene analogues of SM (SMAs). Here, we show that LPS efficiently increases the release of IL-8 from HT-29 intestinal epithelial cells by activating both neutral SMase and nuclear factor (NF)-kappaB in the cells. The addition of SMA-7 suppressed neutral SMase-catalyzed ceramide production, NF-kappaB activation, and IL-8 release from HT-29 cells caused by LPS. The results suggest that activation of neutral SMase is an underlying mechanism of LPS-induced release of IL-8 from the intestinal epithelial cells. Ceramide production following LPS-induced SM hydrolysis may trigger the activation of NF-kappaB in nuclei. Oral administration of SMA-7 (60 mg/kg) to mice with 2% dextran sulfate sodium (DSS) in their drinking water, for 21 consecutive days, reduced significantly the severity of colonic injury. This finding suggests a central role for SMase/ceramide signaling in the pathology of DSS-induced colitis in mice. The therapeutic effect of SMA-7 observed in mice may involve the suppression of IL-8 production from intestinal epithelial cells by LPS or other inflammatory cytokines.
Asunto(s)
Colitis/tratamiento farmacológico , Interleucina-8/metabolismo , Mucosa Intestinal/inmunología , Lipopolisacáridos/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielinas/farmacología , Esfingomielinas/uso terapéutico , Enfermedad Aguda , Administración Oral , Animales , Línea Celular , Ceramidas/metabolismo , Colitis/inducido químicamente , Sulfato de Dextran/efectos adversos , Interleucina-8/antagonistas & inhibidores , Mucosa Intestinal/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Esfingomielinas/administración & dosificación , Esfingomielinas/químicaRESUMEN
Lipopolysaccharide (LPS) and inflammatory cytokines cause activation of sphingomyelinases (SMases) and subsequent hydrolysis of sphingomyelin (SM) to produce a lipid messenger ceramide. The design of SMase inhibitors may offer new therapies for the treatment of LPS- and cytokine-related inflammatory bowel disease. We synthesized a series of difluoromethylene analogues of SM (SMAs). We report here the effects of the most potent SMase inhibitor, SMA-7, on the LPS-mediated release of tumour necrosis factor-alpha, interleukin-1beta and interleukin-6 from THP-1 macrophages and the pathology of dextran sulphate sodium (DSS)-induced colitis in mice. SMA-7 suppressed the LPS-induced cytokine release and nuclear factor-kappaB activation. LPS stimulation caused a four-fold increase in acid SMase activation, but little increase in neutral SMase activity. The presence of 10 microm SMA-7 caused acid SMase to remain at the control levels and reduced the formation of ceramide. HT-29 cells had significantly decreased cell viability when incubated with media from LPS-stimulated THP-1 macrophages. However, incubating the colon cells in media from both SMA-7 and LPS-treated macrophages caused little decrease in viability, suggesting that ceramide has a role in the LPS-stimulated signalling that releases cytotoxic factors against colon cells. Oral administration of SMA-7 to mice with 2% DSS in the drinking water, for 10 or 21 consecutive days, reduced significantly the cytokine levels in the colon and the severity of colonic injury. These findings suggest a central role for acid SMase/ceramide signalling in the pathology of DSS-induced colitis in mice, indicating a possible preventive or therapeutic role for SMase inhibitor in inflammatory bowel disease.