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1.
SLAS Discov ; 26(9): 1079-1090, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34269109

RESUMEN

The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.


Asunto(s)
COVID-19/diagnóstico por imagen , Ensayos Analíticos de Alto Rendimiento/métodos , Citometría de Imagen/métodos , Síndrome de Dificultad Respiratoria/diagnóstico por imagen , COVID-19/diagnóstico , COVID-19/virología , Permeabilidad de la Membrana Celular/genética , Descubrimiento de Drogas , Células Endoteliales/ultraestructura , Células Endoteliales/virología , Humanos , Procesamiento de Imagen Asistido por Computador , Pandemias/prevención & control , Fenotipo , Arteria Pulmonar/diagnóstico por imagen , Arteria Pulmonar/patología , Arteria Pulmonar/virología , Edema Pulmonar/diagnóstico , Edema Pulmonar/diagnóstico por imagen , Edema Pulmonar/virología , Síndrome de Dificultad Respiratoria/diagnóstico , Síndrome de Dificultad Respiratoria/virología , Insuficiencia Respiratoria/diagnóstico , Insuficiencia Respiratoria/diagnóstico por imagen , Insuficiencia Respiratoria/virología , SARS-CoV-2/patogenicidad , Trombina/farmacología , Factor de Necrosis Tumoral alfa/farmacología
2.
Am J Respir Cell Mol Biol ; 65(3): 300-308, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34003736

RESUMEN

Endothelial dysfunction is implicated in the thrombotic events reported in patients with coronavirus disease (COVID-19), but the underlying molecular mechanisms are unknown. Circulating levels of the coagulation cascade activator PAI-1 are substantially higher in patients with COVID-19 with severe respiratory dysfunction than in patients with bacterial sepsis and acute respiratory distress syndrome. Indeed, the elevation of PAI-1 is recognized as an early marker of endothelial dysfunction. Here, we report that the rSARS-CoV-2-S1 (recombinant severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] viral envelope spike) glycoprotein stimulated robust production of PAI-1 by human pulmonary microvascular endothelial cells (HPMECs). We examined the role of protein degradation in this SARS-CoV-2-S1 induction of PAI-1 and found that the proteasomal degradation inhibitor bortezomib inhibited SARS-CoV-2-S1-mediated changes in PAI-1. Our data further show that bortezomib upregulated KLF2, a shear-stress-regulated transcription factor that suppresses PAI-1 expression. Aging and metabolic disorders are known to increase mortality and morbidity in patients with COVID-19. We therefore examined the role of ZMPSTE24 (zinc metallopeptidase STE24), a metalloprotease with a demonstrated role in host defense against RNA viruses that is decreased in older individuals and in metabolic syndrome, in the induction of PAI-1 in HPMECs by SARS-CoV-2-S1. Indeed, overexpression of ZMPSTE24 blunted enhancement of PAI-1 production in spike protein-exposed HPMECs. In addition, we found that membrane expression of the SARS-CoV-2 entry receptor ACE2 was reduced by ZMPSTE24-mediated cleavage and shedding of the ACE2 ectodomain, leading to accumulation of ACE2 decoy fragments that may bind SARS-CoV-2. These data indicate that decreases in ZMPSTE24 with age and comorbidities may increase vulnerability to vascular endothelial injury by SARS-CoV-2 viruses and that enhanced production of endothelial PAI-1 might play role in prothrombotic events in patients with COVID-19.


Asunto(s)
COVID-19/virología , Células Endoteliales/patología , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Arteria Pulmonar/patología , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/metabolismo , Envejecimiento , COVID-19/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/virología , Humanos , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Inhibidor 1 de Activador Plasminogénico/genética , Proteolisis , Arteria Pulmonar/metabolismo , Arteria Pulmonar/virología , Glicoproteína de la Espiga del Coronavirus/genética
3.
Biomed Res Int ; 2021: 8851736, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33778084

RESUMEN

PURPOSE: This study is aimed at assessing the prevalence of pulmonary artery filling defects (PAFDs) consistent with pulmonary artery embolism (PAE) in patients with SARS-CoV-2 infection and at investigating possible radiological or clinical predictors. MATERIALS AND METHODS: Computed Tomography Pulmonary Angiographies (CTPAs) from 43 consecutive patients with a confirmed COVID-19 infection were retrospectively reviewed, taking into consideration the revised Geneva score and the D-dimer value for each patient. Filling defects within the pulmonary arteries were recorded along with pleural and parenchymal findings such as ground glass opacities, consolidation, crazy paving, linear consolidation, and pleural effusion. All these variables were compared between patients with and without PAFD. The predictive performance of statistically different parameters was investigated using the receiver operating characteristics (ROC). RESULTS: Pulmonary embolism was diagnosed in 15/43 patients (35%), whereas CTPA and parenchymal changes related to pulmonary COVID-19 disease were evident in 39/43 patients (91%). The revised Geneva score and the mean D-dimer value obtained using two consecutive measurements were significantly higher in patients with PAFD. The ROC analysis demonstrated that a mean D-dimer value is the parameter with the higher predictivity (AUC 0.831) that is a cut-off value > 1800 µg/l which predicts the probability of PAFD with a sensitivity and specificity of 70% and 78%, respectively. CONCLUSIONS: This single centre retrospective report shows a high prevalence of pulmonary artery filling defects revealed using CTPA in COVID-19 patients and demonstrates that the mean value of multiple D-dimer measurements may represent a predicting factor of this complication.


Asunto(s)
COVID-19/diagnóstico por imagen , Angiografía por Tomografía Computarizada/métodos , Arteria Pulmonar/diagnóstico por imagen , Adulto , Anciano , COVID-19/metabolismo , COVID-19/virología , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Humanos , Pulmón/diagnóstico por imagen , Pulmón/patología , Masculino , Persona de Mediana Edad , Prevalencia , Arteria Pulmonar/patología , Arteria Pulmonar/virología , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/diagnóstico por imagen , Embolia Pulmonar/virología , Curva ROC , Estudios Retrospectivos , SARS-CoV-2/aislamiento & purificación , Sensibilidad y Especificidad
4.
Vascul Pharmacol ; 137: 106823, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33232769

RESUMEN

Currently, the world is suffering from the pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the host cells. So far, 60 million people have been infected with SARS-CoV-2, and 1.4 million people have died because of COVID-19 worldwide, causing serious health, economical, and sociological problems. However, the mechanism of the effect of SARS-CoV-2 on human host cells has not been defined. The present study reports that the SARS-CoV-2 spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in lung vascular cells. The treatment of human pulmonary artery smooth muscle cells or human pulmonary artery endothelial cells with recombinant SARS-CoV-2 spike protein S1 subunit (Val16 - Gln690) at 10 ng/ml (0.13 nM) caused an activation of MEK phosphorylation. The activation kinetics was transient with a peak at 10 min. The recombinant protein that contains only the ACE2 receptor-binding domain of the SARS-CoV-2 spike protein S1 subunit (Arg319 - Phe541), on the other hand, did not cause this activation. Consistent with the activation of cell growth signaling in lung vascular cells by the SARS-CoV-2 spike protein, pulmonary vascular walls were found to be thickened in COVID-19 patients. Thus, SARS-CoV-2 spike protein-mediated cell growth signaling may participate in adverse cardiovascular/pulmonary outcomes, and this mechanism may provide new therapeutic targets to combat COVID-19.


Asunto(s)
COVID-19/metabolismo , Células Endoteliales/metabolismo , Pulmón/irrigación sanguínea , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/patología , COVID-19/virología , Células Cultivadas , Células Endoteliales/patología , Células Endoteliales/virología , Interacciones Huésped-Patógeno , Humanos , Cinética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/patología , Músculo Liso Vascular/virología , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/virología , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arteria Pulmonar/virología , Receptores Virales/metabolismo , SARS-CoV-2/patogenicidad , Transducción de Señal
5.
Clin Appl Thromb Hemost ; 26: 1076029620936772, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32726134

RESUMEN

The aim of this study was to describe clinical, imaging, and laboratory features of acute pulmonary embolism (APE) in patients with COVID-19 associated pneumonia. Patients with COVID-19 associated pneumonia who underwent a computed tomography pulmonary artery (CTPA) scan for suspected APE were retrospectively studied. Laboratory data and CTPA images were collected. Imaging characteristics were analyzed descriptively. Laboratory data were analyzed and compared between patients with and without APE. A series of 25 COVID-19 patients who underwent CTPA between January 2020 and February 2020 were enrolled. The median D-dimer level founded in these 25 patients was 6.06 µg/mL (interquartile range [IQR] 1.90-14.31 µg/mL). Ten (40%) patients with APE had a significantly higher level of D-dimer (median, 11.07 µg/mL; IQR, 7.12-21.66 vs median, 2.44 µg/mL; IQR, 1.68-8.34, respectively, P = .003), compared with the 15 (60%) patients without APE. No significant differences in other laboratory data were found between patients with and without APE. Among the 10 patients with APE, 6 (60%) had a bilateral pulmonary embolism, while 4 had a unilateral embolism. The thrombus-prone sites were the right lower lobe (70%), the left upper lobe (60%), both upper lobe (40%) and the right middle lobe (20%). The thrombus was partially or completely absorbed after anticoagulant therapy in 3 patients who underwent a follow-up CTPA. Patients with COVID-19 associated pneumonia have a risk of developing APE during the disease. When the D-dimer level abnormally increases in patients with COVID-19 pneumonia, CTPA should be performed to detect and assess the severity of APE.


Asunto(s)
Betacoronavirus , Angiografía por Tomografía Computarizada/métodos , Infecciones por Coronavirus/diagnóstico por imagen , Neumonía Viral/diagnóstico por imagen , Embolia Pulmonar/diagnóstico por imagen , Enfermedad Aguda , Adulto , Anciano , Anticoagulantes/uso terapéutico , Betacoronavirus/patogenicidad , COVID-19 , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/complicaciones , Infecciones por Coronavirus/virología , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/sangre , Neumonía Viral/complicaciones , Neumonía Viral/virología , Arteria Pulmonar/anatomía & histología , Arteria Pulmonar/diagnóstico por imagen , Arteria Pulmonar/virología , Embolia Pulmonar/sangre , Embolia Pulmonar/etiología , Embolia Pulmonar/virología , Estudios Retrospectivos , SARS-CoV-2 , Trombosis/tratamiento farmacológico , Tomografía Computarizada por Rayos X/métodos
6.
Adv Clin Exp Med ; 26(3): 475-481, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28791823

RESUMEN

BACKGROUND: The prevalence of primitive pulmonary arterial hypertension (PAH) in patients with human immunodeficiency virus infection (HIV) is estimated at approximately 0.5%, significantly higher than in the general population. OBJECTIVES: This study aimed to assess the echocardiographic modifications in HIV-associated pulmonary arterial hypertension (PAH). MATERIAL AND METHODS: A group of 117 patients, aged under 16, with horizontally transmitted HIV staged according to the U.S. Center for Disease Control and Prevention criteria, were included in this prospective study, with echocardiographic abnormalities in 79 children. The study group consisted of 27 HIV-infected patients with PAH, while the control group consisted of 38 patients with normal ultrasound features. The diagnostic criterion for PAH was the presence of a mean pulmonary artery pressure above 25 mm Hg, determined at 2 consecutive measurements having at least 6 months distance between them. All subjects underwent a complex echocardiographic assessment, including assessment of left and right ventricular hypertrophy and evaluation of left ventricular function, associated with determination of the immunological stage. RESULTS: We recorded the presence of PAH in 27 patients (23.08%), in whom an average value of 31.48 mm Hg was recorded for pulmonary artery pressure. All patients had mild forms of PAH. Age, gender and immunological stage showed no significant differences in the PAH group compared to patients in the control group. Right ventricular hypertrophy was encountered in 95.23% and left ventricular hypertrophy in 88.88% of the patients with PAH. Left ventricular dysfunction, a complication of pulmonary hypertension, was relatively rare (11.11%). CONCLUSIONS: In children with HIV infection, PAH is present in a relatively mild form and does not correlate with the clinical and immunological stage of HIV infection, evolving as a seemingly primitive condition.


Asunto(s)
Infecciones por VIH/fisiopatología , Hipertensión Pulmonar/fisiopatología , Arteria Pulmonar/fisiopatología , Adolescente , Ecocardiografía/métodos , Femenino , Humanos , Hipertensión Pulmonar/virología , Estudios Longitudinales , Masculino , Prevalencia , Estudios Prospectivos , Arteria Pulmonar/virología , Volumen Sistólico/fisiología , Disfunción Ventricular Izquierda/fisiopatología , Disfunción Ventricular Izquierda/virología , Función Ventricular Izquierda/fisiología
7.
Basic Res Cardiol ; 109(2): 401, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24442486

RESUMEN

Both human cytomegalovirus (HCMV) and arginase II (ARG II) have been implicated in the pathogenesis of cardiovascular diseases. The effects of HCMV on ARG II are unknown. The aim of this study was to investigate the effects of HCMV on ARG II expression in endothelial and vascular smooth muscle cells (SMC) both in vitro and ex vivo. Endothelial and SMC were infected with either HCMV or UV-irradiated HCMV. Expression of ARG II, endothelial or inducible nitric oxide synthase (eNOS and iNOS, respectively) and viral immediate early (IE) was quantified using quantitative PCR. Ganciclovir and short interfering RNA were used to determine the viral gene mediating the effects on ARG II. Detection of viral antigens and ARG II expression was performed by immunofluorescence or immunohistochemistry. HCMV infection increased both ARG II mRNA and protein levels in the examined cells; this effect was mediated by the HCMV IE2-p86 protein. The upregulation of ARG II was accompanied by a downregulation of eNOS but an induction of iNOS in HCMV-infected endothelial cells. Both eNOS and iNOS expressions were induced in HCMV-infected SMC. ARG II was abundantly expressed in endothelial cells, foam cells and SMC and was importantly significantly upregulated in HCMV-immunoreactive human carotid atherosclerotic plaques. HCMV IE2-p86 mediates ARG II upregulation in vitro and ARG II is co-expressed with HCMV antigens in human carotid atherosclerotic plaques. We speculate that HCMV may contribute to endothelial dysfunction via ARG II induction and reduced eNOS production.


Asunto(s)
Arginasa/genética , Enfermedades de las Arterias Carótidas/virología , Infecciones por Citomegalovirus/complicaciones , Citomegalovirus/genética , Vasculitis/enzimología , Vasculitis/virología , Antivirales/farmacología , Aorta/citología , Aorta/virología , Arginasa/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/patología , Células Endoteliales/citología , Células Endoteliales/virología , Ganciclovir/farmacología , Regulación Enzimológica de la Expresión Génica , Regulación Viral de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas Inmediatas-Precoces/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/virología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Arteria Pulmonar/citología , Arteria Pulmonar/virología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Transactivadores/genética , Regulación hacia Arriba/genética , Vasculitis/patología
8.
Arterioscler Thromb Vasc Biol ; 33(11): 2585-95, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24008158

RESUMEN

OBJECTIVE: Our previous findings support an additive effect of cocaine to HIV infection in the development of pulmonary arteriopathy through enhanced proliferation of human pulmonary smooth muscle cells. We now examined the role of antiproliferative bone morphogenetic protein receptor (BMPR) axis in HIV protein and cocaine-mediated pulmonary smooth muscle hyperplasia. APPROACH AND RESULTS: Stimulation of BMPR axis resulted in attenuation of synergistic increase in the proliferation of human pulmonary arterial smooth muscle cells in response to cocaine and HIV protein, transactivator of transcription (Tat). Interestingly, an increase in mRNA but decrease in protein levels of BMPR with correlated decrease in the activation of Sma- and MAD-related family protein 1/5/8 and Id1 gene expression was observed on combined treatment with cocaine and Tat when compared with the untreated cells at all time points tested. Although longer exposure to either cocaine or Tat alone also resulted in a significant decrease in the BMPR protein expression, the abrogation on combined treatment was still significantly more when compared with that of the monotreatments. Significant increase in mRNA but downmodulation of BMPR protein expression was also observed in the lung extracts from HIV-infected intravenous drug users (HIV+IVDU) when compared with that from HIV-infected non-IVDUs (HIV) or uninfected IVDUs (IVDU). Furthermore, significant decrease in BMPR protein expression was also observed in HIV or IVDUs when compared with normal controls that correlated with in vitro findings on chronic exposure to cocaine or HIV protein alone. CONCLUSIONS: Simultaneous exposure of pulmonary smooth muscle cells to viral protein(s) and cocaine exacerbates downregulation of BMPR axis that may result in enhanced pulmonary vasculature aberrations in HIV+IVDUs.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Cocaína/farmacología , Infecciones por VIH/complicaciones , VIH-1 , Hipertensión Pulmonar , Proteína Morfogenética Ósea 2/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Inhibidores de Captación de Dopamina/farmacología , Regulación hacia Abajo/fisiología , Hipertensión Pulmonar Primaria Familiar , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , Humanos , Hiperplasia/metabolismo , Hiperplasia/patología , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/virología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/virología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Arteria Pulmonar/virología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo
9.
PLoS Pathog ; 9(7): e1003470, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23874198

RESUMEN

Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC) display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE)-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF). To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC) to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS). We show that incubation of factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen (HK) plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL) and increased liberation of bradykinin (BK). Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS), we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation during hantavirus infection and could have profound implications for treatment of hantavirus infections.


Asunto(s)
Capilares/virología , Permeabilidad Capilar , Endotelio Vascular/virología , Activación Enzimática , Factor XII/metabolismo , Infecciones por Hantavirus/virología , Sistema Calicreína-Quinina , Bradiquinina/antagonistas & inhibidores , Bradiquinina/metabolismo , Capilares/efectos de los fármacos , Capilares/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Activación Enzimática/efectos de los fármacos , Factor XII/antagonistas & inhibidores , Orthohantavirus/fisiología , Infecciones por Hantavirus/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/virología , Humanos , Sistema Calicreína-Quinina/efectos de los fármacos , Quininógeno de Alto Peso Molecular/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/virología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/virología , Precalicreína/antagonistas & inhibidores , Precalicreína/metabolismo , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Arteria Pulmonar/virología , Propiedades de Superficie , Replicación Viral
10.
Respir Res ; 12: 103, 2011 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-21819559

RESUMEN

BACKGROUND: Human immunodeficiency virus (HIV) infected patients are at increased risk for the development of pulmonary arterial hypertension (PAH). Recent reports have demonstrated that HIV associated viral proteins induce reactive oxygen species (ROS) with resultant endothelial cell dysfunction and related vascular injury. In this study, we explored the impact of HIV protein induced oxidative stress on production of hypoxia inducible factor (HIF)-1α and platelet-derived growth factor (PDGF), critical mediators implicated in the pathogenesis of HIV-PAH. METHODS: The lungs from 4-5 months old HIV-1 transgenic (Tg) rats were assessed for the presence of pulmonary vascular remodeling and HIF-1α/PDGF-BB expression in comparison with wild type controls. Human primary pulmonary arterial endothelial cells (HPAEC) were treated with HIV-associated proteins in the presence or absence of pretreatment with antioxidants, for 24 hrs followed by estimation of ROS levels and western blot analysis of HIF-1α or PDGF-BB. RESULTS: HIV-Tg rats, a model with marked viral protein induced vascular oxidative stress in the absence of active HIV-1 replication demonstrated significant medial thickening of pulmonary vessels and increased right ventricular mass compared to wild-type controls, with increased expression of HIF-1α and PDGF-BB in HIV-Tg rats. The up-regulation of both HIF-1α and PDGF-B chain mRNA in each HIV-Tg rat was directly correlated with an increase in right ventricular/left ventricular+septum ratio. Supporting our in-vivo findings, HPAECs treated with HIV-proteins: Tat and gp120, demonstrated increased ROS and parallel increase of PDGF-BB expression with the maximum induction observed on treatment with R5 type gp-120CM. Pre-treatment of endothelial cells with antioxidants or transfection of cells with HIF-1α small interfering RNA resulted in abrogation of gp-120CM mediated induction of PDGF-BB, therefore, confirming that ROS generation and activation of HIF-1α plays critical role in gp120 mediated up-regulation of PDGF-BB. CONCLUSION: In summary, these findings indicate that viral protein induced oxidative stress results in HIF-1α dependent up-regulation of PDGF-BB and suggests the possible involvement of this pathway in the development of HIV-PAH.


Asunto(s)
Células Endoteliales/virología , Infecciones por VIH/virología , VIH-1/genética , Hipertensión Pulmonar/virología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/irrigación sanguínea , Estrés Oxidativo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Antioxidantes/farmacología , Becaplermina , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Hipertensión Pulmonar Primaria Familiar , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/complicaciones , Infecciones por VIH/enzimología , Infecciones por VIH/genética , VIH-1/metabolismo , VIH-1/patogenicidad , Humanos , Hipertensión Pulmonar/enzimología , Hipertensión Pulmonar/genética , Hipertrofia Ventricular Derecha/enzimología , Hipertrofia Ventricular Derecha/virología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Microvasos/enzimología , Microvasos/virología , Estrés Oxidativo/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Proto-Oncogénicas c-sis , Arteria Pulmonar/enzimología , Arteria Pulmonar/virología , Interferencia de ARN , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Regulación hacia Arriba , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo
11.
Vet Immunol Immunopathol ; 136(3-4): 292-6, 2010 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-20359752

RESUMEN

Bluetongue virus (BTV) is the cause of bluetongue (BT), an emerging, arthropod-transmitted disease of ungulates. The cellular tropism of BTV in ruminants includes macrophages, dendritic cells and endothelial cells (ECs), and fulminant infection is characterized by lesions consistent with those of so-called viral hemorrhagic fevers. Specifically, BT is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema. To further investigate the pathogenesis of vascular injury in BT, we evaluated the responses of cultured bovine pulmonary artery EC (bPAEC) and monocyte-derived macrophages (bMDM) to BTV infection by measuring transcript levels of genes encoding molecules important in mediating EC activation and/or endothelial barrier dysregulation. The data confirm that BTV infection of bPAEC resulted in increased transcription of genes encoding chemokine ligand 2 (CCL2) and E-selectin, and BTV infection of bMDM resulted in increased transcription of genes encoding TNF-alpha, IL-1beta, IL-8, and inducible nitric oxide synthase (iNOS). The data from these in vitro studies provide further evidence that cytokines and other vasoactive substances produced in macrophages potentially contribute to vascular injury in BTV-infected ruminants, along with direct effects of the virus itself on ECs.


Asunto(s)
Virus de la Lengua Azul/inmunología , Lengua Azul/virología , Enfermedades de los Bovinos/virología , Arteria Pulmonar/virología , Transcripción Genética/inmunología , Animales , Lengua Azul/inmunología , Bovinos , Enfermedades de los Bovinos/inmunología , Citocinas/genética , Citocinas/inmunología , Células Endoteliales/inmunología , Células Endoteliales/virología , Cinética , Macrófagos/inmunología , Macrófagos/virología , Microscopía Fluorescente , Arteria Pulmonar/citología , Arteria Pulmonar/inmunología , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria
12.
Am J Physiol Lung Cell Mol Physiol ; 294(2): L276-89, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18083765

RESUMEN

S100A4/Mts-overexpressing mice have thick elastic laminae and mild pulmonary arterial hypertension (PAH), and the occasional older mouse develops occlusive neointimal lesions and perivascular inflammation. We hypothesized that a vasculotropic virus could induce neointimal lesions in the S100A4/Mts1 mouse by facilitating breakdown of elastin and migration and proliferation of smooth muscle cells. To test this hypothesis, we infected S100A4/Mts1 mice with gammaherpesvirus 68 (gammaHV68). We observed, 6 mo after gammaHV68 [4 x 10(3) plaque-forming units (PFU)], perivascular inflammation in 10/15 S100A4/Mts1 mice and occlusive neointimal formation in 3/10 mice, accompanied by striking degradation of elastin. We then compared the early response after high-dose gammaHV68 (4 x 10(6) PFU) in C57Bl/6 and S100A4/Mts1 mice. In S100A4/Mts1 mice only, significant PAH, muscularization of distal vessels, and elastase activity were observed 6 wk after gammaHV68. These features resolved by 3 mo without neointimal formation. We therefore infected mice with the M1-gammaHV68 strain that reactivates from latency with higher efficiency and observed neointimal lesions at 3 mo in 2/5 C57Bl/6 (5-9% of vessels) and in 5/5 S100A4/Mts1 mice (13-40% of vessels) accompanied by mild PAH, heightened lung elastase activity, and intravascular viral expression. This suggested that enhanced generation of elastin peptides in S100A4/Mts1 mice may promote increased viral entry in the vessel wall. Using S100A4/Mts1 PA organ culture, we showed, in response to elastase activity, heightened production of elastin peptides associated with invasion of inflammatory cells and intravascular viral antigen. We therefore propose that early viral access to the vessel wall may be a critical determinant of the extent of vascular pathology following reactivation.


Asunto(s)
Elastina/metabolismo , Gammaherpesvirinae/fisiología , Procesamiento Proteico-Postraduccional , Arteria Pulmonar/patología , Arteria Pulmonar/virología , Proteínas S100/genética , Activación Viral , Animales , Antígenos Virales/metabolismo , Presión Sanguínea , Infecciones por Herpesviridae/virología , Hipertensión Pulmonar/fisiopatología , Hipertensión Pulmonar/virología , Hipertrofia Ventricular Derecha/fisiopatología , Hipertrofia Ventricular Derecha/virología , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Mutación/genética , Péptidos/metabolismo , Proteína de Unión al Calcio S100A4 , Carga Viral , Internalización del Virus
13.
Eur J Cardiothorac Surg ; 24(3): 358-63, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12965305

RESUMEN

OBJECTIVE: Acellularised porcine scaffolds have been successfully used for cardiovascular tissue engineering. However, there is concern about the possibility of porcine endogenous retrovirus (PERV) transmission. In this study we developed an in vivo model for cross-species PERV transmission. METHODS: In vitro autologous repopulated porcine pulmonary arteries (n=6) were implanted in sheep in orthotopic position. Blood samples were collected regularly up to 6 months after implantation and tested for PERV by means of polymerase chain reaction and reverse transcriptase-polymerase chain reaction. Explanted tissue engineered pulmonary arteries were tested for PERV sequences. RESULTS: PERV DNA was detectable in acellularised porcine scaffolds. No PERV sequences were detectable 6 months after implantation of in vitro repopulated acellularised porcine pulmonary arteries and in all tested peripheral blood samples. CONCLUSIONS: Acellularised porcine matrix scaffolds can be used for cardiovascular tissue engineering of autologous grafts without risk of PERV transmission.


Asunto(s)
Retrovirus Endógenos/aislamiento & purificación , Arteria Pulmonar/trasplante , Infecciones por Retroviridae/transmisión , Ingeniería de Tejidos/métodos , Trasplante Heterólogo , Animales , Bioprótesis , ADN Viral/análisis , Modelos Animales de Enfermedad , Masculino , Reacción en Cadena de la Polimerasa/métodos , Arteria Pulmonar/citología , Arteria Pulmonar/virología , Ovinos , Porcinos , Transfección
15.
J Gen Virol ; 82(Pt 4): 787-794, 2001 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11257183

RESUMEN

Bluetongue virus (BTV) infection causes a haemorrhagic disease in sheep, whereas BTV infection typically is asymptomatic in cattle. Injury to the endothelium of small blood vessels is responsible for the manifestations of disease in BTV-infected sheep. The lungs are central to the pathogenesis of BTV infection of ruminants; thus endothelial cells (ECs) cultured from the pulmonary artery and lung microvasculature of sheep and cattle were used to investigate the basis for the disparate expression of bluetongue disease in the two species. Ovine and bovine microvascular ECs infected at low multiplicity with partially purified BTV were equally susceptible to BTV-induced cell death, yet ovine microvascular ECs had a lower incidence of infection and produced significantly less virus than did bovine microvascular ECs. Importantly, the relative proportions of apoptotic and necrotic cells were significantly different in BTV-infected EC cultures depending on the species of EC origin and the presence of inflammatory mediators in the virus inoculum. Furthermore, BTV-infected ovine lung microvascular ECs released markedly less prostacyclin than the other types of ECs. Results of these in vitro studies are consistent with the marked pulmonary oedema and microvascular thrombosis that characterize bluetongue disease of sheep but which rarely, if ever, occur in BTV-infected cattle.


Asunto(s)
Apoptosis , Virus de la Lengua Azul/fisiología , Citocinas/fisiología , Endotelio Vascular/virología , Epoprostenol/metabolismo , Arteria Pulmonar/virología , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Mediadores de Inflamación/fisiología , Arteria Pulmonar/citología , Ovinos
16.
Circ Res ; 85(7): 614-22, 1999 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-10506486

RESUMEN

Angiotensin I-converting enzyme (ACE) inhibitors have been proven to be highly effective and are for the most part the drugs of choice in the treatment and control of hypertension, congestive heart failure, and left ventricular dysfunction. Despite this, questions regarding side effects and compliance with this traditional pharmacological strategy remain. In view of these observations, coupled with recent advances in gene-transfer technology, our objective in this study was to determine whether the expression of ACE could be controlled on a permanent basis at a genetic level. We argued that the introduction of ACE antisense to inhibit the enzyme would be a prerequisite in considering the antisense gene therapy for the control of hypertension and other related pathological states. Retroviral vectors (LNSV) containing ACE sense (LNSV-ACE-S) and ACE antisense (LNSV-ACE-AS) sequences were constructed and were used in rat pulmonary artery endothelial cells (RPAECs) to determine the feasibility of this approach. Infection of rat RPAECs with LNSV-ACE-S and LNSV-ACE-AS resulted in a robust expression of transcripts corresponding to ACE-S and ACE-AS, respectively, for the duration of these experiments, ie, 8 consecutive passages. The expression of ACE-AS but not of ACE-S was associated with a permanent decrease of approximately 70% to 75% in ACE expression and a 50% increase in the B(max) for the AT(1)s. Although angiotensin II caused a concentration-dependent stimulation of intracellular Ca(2+) levels in both ACE-S- and ACE-AS-expressing cells, the stimulation was significantly higher in ACE-AS-expressing RPAECs. In vivo experiments demonstrated a prolonged expression of ACE-AS transcripts in cardiovascularly relevant tissues of rats. This was associated with a long-term reduction in blood pressure by approximately 15 mm Hg, exclusively in the spontaneously hypertensive rat. These observations demonstrate that delivery of ACE-AS by retroviral vector results in a permanent inhibition of ACE and a long-term reduction in high blood pressure in the spontaneously hypertensive rat.


Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Antihipertensivos/farmacología , ADN Complementario/farmacología , Oligonucleótidos Antisentido/farmacología , Peptidil-Dipeptidasa A/genética , Angiotensina II/farmacología , Animales , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/virología , Vectores Genéticos , Hipertensión/genética , Hipertensión/fisiopatología , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/virología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores de Angiotensina/metabolismo , Retroviridae/genética , Virión/fisiología
17.
Vet Pathol ; 33(1): 95-9, 1996 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-8826015
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