RESUMEN
OBJECTIVE: Atherosclerosis is a chronic multifactorial and inflammatory disease of large and medium arteries and the leading cause of cardiovascular diseases worldwide. The aim of this study was to investigate whether and how the nSMase2 (type 2-neutral sphingomyelinase), a key enzyme of sphingolipid metabolism, may contribute to the development of atherosclerotic lesions. APPROACH AND RESULTS: The role of nSMase2 in atherosclerosis was investigated in Apoe-/-;Smpd3fro/fro mice, mutant for nSMase2, and in Apoe-/-;Smpd3+/+ mice intraperitoneally injected with GW4869, a pharmacological nSMase2 inhibitor. The defect or inhibition of nSMase2 resulted in a reduction of atherosclerotic lesions and a decrease in macrophage infiltration and lipid deposition, although cholesterolemia remained unchanged. nSMase2 inhibition decreased the inflammatory response of murine endothelial cells to oxLDL (oxidized low-density lipoprotein), as assessed by the significant reduction of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) mRNA expressions and macrophage recruitment. Likewise, in RAW264.7 or in macrophages isolated from Apoe-/-/Smpd3fro/fro or Apoe-/-/Smpd3+/+ mice stimulated by lipopolysaccharides, nSMase2 inhibition resulted in a decrease in the expression of inflammatory molecules. Mechanistically, the anti-inflammatory response resulting from nSMase2 inhibition involves Nrf2 (nuclear factor [erythroid-derived 2]-like 2 or NF-E2-related factor-2) activation in both endothelial cells and macrophages, as assessed by the lack of protective effect of GW4869 in endothelial cells silenced for Nrf2 by small interfering RNAs, and in lipopolysaccharide-stimulated macrophages issued from Nrf2-KO mice. CONCLUSIONS: The genetic deficiency or inhibition of nSMase2 strongly decreases the development of atherosclerotic lesions in Apoe-/- mice, by reducing inflammatory responses through a mechanism involving the Nrf2 pathway. Inhibitors of nSMase2 may, therefore, constitute a novel approach to slow down atherosclerosis progression.
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Compuestos de Anilina/farmacología , Antiinflamatorios/farmacología , Aorta/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Compuestos de Bencilideno/farmacología , Inhibidores Enzimáticos/farmacología , Inflamación/prevención & control , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/deficiencia , Animales , Aorta/enzimología , Aorta/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Inflamación/enzimología , Inflamación/genética , Inflamación/patología , Mediadores de Inflamación/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Placa Aterosclerótica , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Esfingomielina Fosfodiesterasa/genéticaRESUMEN
Stress-inducing agents, including oxidative stress, generate the sphingolipid mediators ceramide (Cer) and sphingosine-1-phosphate (S1P) that are involved in stress-induced cellular responses. The two redox-sensitive neutral sphingomyelinase-2 (nSMase2) and sphingosine kinase-1 (SK1) participate in transducing stress signaling to ceramide and S1P, respectively; however, whether these key enzymes are coordinately regulated is not known. We investigated whether a signaling link coordinates nSMase2 and SK1 activation by H2O2. In mesenchymal cells, H2O2 elicits a dose-dependent biphasic effect, mitogenic at low concentration (5µM), and anti-proliferative and toxic at high concentration (100µM). Low H2O2 concentration triggered activation of nSMase2 and SK1 through a nSMase2/Cer-dependent signaling pathway that acted upstream of activation of SK1. Further results implicated src and the trans-activation of PDGFRß, as supported by the blocking effect of specific siRNAs, pharmacological inhibitors, and genetically deficient cells for nSMase2, src and SK1. The H2O2-induced src/PDGFRß/SK1 signaling cascade was impaired in nSMase2-deficient fro/fro cells and was rescued by exogenous C2Cer that activated src/PDGFRß/SK1. Thus, the results define a nSMase2/SK1 signaling pathway implicated in the mitogenic response to low oxidative stress. On the other hand, high oxidative stress induced inhibition of SK1. The results also showed that the toxicity of high H2O2 concentration was comparable in control and nSMase2-deficient cells. Taken together the results identify a tightly coordinated nSMase2/SK1 pathway that mediates the mitogenic effects of H2O2 and may sense the degree of oxidative stress.
Asunto(s)
Ceramidas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/fisiología , Esfingomielina Fosfodiesterasa/metabolismo , Familia-src Quinasas/metabolismo , Animales , Línea Celular , Ceramidas/genética , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Lisofosfolípidos/genética , Lisofosfolípidos/metabolismo , Ratones , Ratones Mutantes , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Transducción de Señal/efectos de los fármacos , Esfingomielina Fosfodiesterasa/genética , Esfingosina/análogos & derivados , Esfingosina/genética , Esfingosina/metabolismo , Familia-src Quinasas/genéticaRESUMEN
The mouse mutation fragilitas ossium (fro) leads to a syndrome of severe osteogenesis and dentinogenesis imperfecta with no detectable collagen defect. Positional cloning of the locus identified a deletion in the gene encoding neutral sphingomyelin phosphodiesterase 3 (Smpd3) that led to complete loss of enzymatic activity. Our knowledge of SMPD3 function is consistent with the pathology observed in mutant mice and provides new insight into human pathologies.
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Dentinogénesis Imperfecta/genética , Eliminación de Gen , Osteogénesis Imperfecta/genética , Animales , Dentinogénesis Imperfecta/enzimología , Ratones , Ratones Mutantes , Mutación , Osteogénesis Imperfecta/enzimología , Esfingomielina FosfodiesterasaRESUMEN
BACKGROUND: Outcomes for organ transplantation are constantly improving because of advances in organ preservation, surgical techniques, immune clinical monitoring, and immunosuppressive treatment preventing acute transplant rejection. However, chronic rejection including transplant vasculopathy still limits long-term patient survival. Transplant vasculopathy is characterized by progressive neointimal hyperplasia leading to arterial stenosis and ischemic failure of the allograft. This work sought to decipher the manner in which the humoral immune response, mimicked by W6/32 anti-HLA antibody, contributes to transplant vasculopathy. METHODS AND RESULTS: Studies were performed in vitro on cultured human smooth muscle cells, ex vivo on human arterial segments, and in vivo in a model consisting of human arterial segments grafted into severe combined immunodeficiency/beige mice injected weekly with anti-HLA antibodies. We report that anti-HLA antibodies are mitogenic for smooth muscle cells through a signaling mechanism implicating matrix metalloproteinases (MMPs) (membrane type 1 MMP and MMP2) and neutral sphingomyelinase-2. This mitogenic signaling and subsequent DNA synthesis are blocked in smooth muscle cells silenced for MMP2 or for neutral sphingomyelinase-2 by small interfering RNAs, in smooth muscle cells transfected with a vector coding for a dominant-negative form of membrane type 1 MMP, and after treatment by pharmacological inhibitors of MMPs (Ro28-2653) or neutral sphingomyelinase-2 (GW4869). In vivo, Ro28-2653 and GW4869 reduced the intimal thickening induced by anti-HLA antibodies in human mesenteric arteries grafted into severe combined immunodeficiency/beige mice. CONCLUSIONS: These data highlight a crucial role for MMP2 and neutral sphingomyelinase-2 in vasculopathy triggered by a humoral immune response and open new perspectives for preventing transplant vasculopathy with the use of MMP and neutral sphingomyelinase inhibitors, in addition to conventional immunosuppression.
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Anticuerpos Antiidiotipos/farmacología , Arterias/trasplante , Antígenos HLA/inmunología , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Enfermedades Vasculares/fisiopatología , Compuestos de Anilina/farmacología , Animales , Anticuerpos Antiidiotipos/efectos adversos , Arterias/patología , Arterias/fisiopatología , Compuestos de Bencilideno/farmacología , Células Cultivadas , Constricción Patológica/etiología , Constricción Patológica/fisiopatología , Modelos Animales de Enfermedad , Humanos , Hiperplasia/etiología , Hiperplasia/fisiopatología , Técnicas In Vitro , Metaloproteinasa 14 de la Matriz/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones SCID , Modelos Animales , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Neointima/patología , Neointima/fisiopatología , Piperazinas/farmacología , Pirimidinas/farmacología , ARN Interferente Pequeño/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/efectos de los fármacos , Enfermedades Vasculares/etiología , Injerto VascularRESUMEN
Oxidized low-density lipoproteins (oxLDLs) trigger various biological responses potentially involved in atherogenesis. Disturbing endoplasmic reticulum (ER) function results in ER stress and unfolded protein response, which tends to restore ER homeostasis but switches to apoptosis when ER stress is prolonged. We aimed to investigate whether ER stress is induced by oxLDLs and can be prevented by the ER-associated chaperone ORP150 (150-kDa oxygen-regulated protein). oxLDLs and the lipid oxidation products 7-ketocholesterol and 4-hydroxynonenal induce ER stress in human endothelial cells (HMEC-1), characterized by the activation of ER stress sensors (phosphorylation of Ire1alpha and PERK, nuclear translocation of ATF6) and of their subsequent pathways (eukaryotic initiation factor 2alpha phosphorylation, expression of XBP1/spliced XBP1, CHOP, and KDEL chaperones GRP78, GRP94, ORP150). ER stress was inhibited by the antioxidant N-acetylcysteine. In advanced atherosclerotic lesions, phospho-Ire1alpha, KDEL, and ORP150 staining were localized in lipid-rich areas with 4-hydroxynonenal adducts and CD68-positive macrophagic cells. By comparison, staining for 4-hydroxynonenal, phospho-Ire1alpha, KDEL, and ORP were faint and more diffuse in intimal hyperplasia. ER stress takes part in the apoptotic effect of oxLDLs, through the Ire1alpha/c-Jun N-terminal kinase pathway, as assessed by the protective effect of specific small interfering RNAs and c-Jun N-terminal kinase inhibitor. Forced expression of the chaperone ORP150 reduced both oxLDL-induced ER stress and apoptosis. ER stress markers and ORP150 chaperone are expressed in areas containing oxLDLs in atherosclerotic lesions and are induced by oxLDLs and oxidized lipids in cultured cells. The forced expression of ORP150 highlights its new protective role against oxLDL-induced ER stress and subsequent apoptosis.
Asunto(s)
Aterosclerosis/metabolismo , Retículo Endoplásmico/metabolismo , Células Endoteliales/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas/metabolismo , Estrés Fisiológico/fisiología , Acetilcisteína/farmacología , Aldehídos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Aterosclerosis/patología , Biomarcadores/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Células Endoteliales/citología , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Proteínas HSP70 de Choque Térmico , Humanos , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Cetocolesteroles/farmacología , Lipoproteínas LDL/farmacología , Oxígeno/metabolismo , Estrés Fisiológico/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: High-density lipoprotein (HDL) levels are inversely associated with stroke incidence, suggesting a protective effect. Using a rat model, we tested the hypothesis that HDL exerts direct vasculo-/neuroprotective effects when administered during the acute phase of embolic stroke. METHODS: After embolic occlusion, Sprague-Dawley rats were randomly treated intravenously with purified HDL versus saline immediately (2, 10 mg/kg) or 3 or 5 hours (10 mg/kg) after stroke. The effects of HDL were assessed blindly 24 hours later by evaluating neurological deficit score and measuring the infarct volume and blood-brain barrier breakdown. Protease activities and neutrophil infiltration were also evaluated. RESULTS: HDL injection immediately after stroke (10 mg/kg) reduced by 68% the mortality at 24 hours (P=0.015). HDL administration immediately or at 3 or 5 hours after stroke also reduced cerebral infarct volume by 74%, 68%, and 70.7%, respectively (P=0.0003, P=0.011, and P=0.019; n=17 per group). The neurological deficit at 24 hours in the HDL-treated group was decreased versus the saline-treated group (P=0.015). Ischemia-induced blood-brain barrier breakdown was significantly reduced in HDL-treated rats versus controls (P=0.0045). Neuroprotective effects of HDL were associated with decreased neutrophil recruitment in the infarct area (P=0.0027) accompanied by reduced matrix metalloproteinase gelatinase activity. Immunostaining showed that HDL was associated with endothelial and glial cells, and also that intercellular adhesion molecule-1 expression was decreased in vessels within the infarct area. CONCLUSIONS: Administration of HDL is neuroprotective when performed up to 5 hours after experimental stroke. This effect may be attributed to the ability of HDL to protect the blood-brain barrier and limit neutrophil recruitment.
Asunto(s)
Modelos Animales de Enfermedad , Embolia Intracraneal/prevención & control , Lipoproteínas HDL/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Animales , Embolia Intracraneal/sangre , Embolia Intracraneal/patología , Masculino , Ratas , Ratas Sprague-Dawley , Accidente CerebrovascularRESUMEN
Recent studies showed that mesenchymal stem cells (MSCs) transplantation significantly decreased cardiac fibrosis; however, the mechanisms involved in these effects are still poorly understood. In this work, we investigated whether the antifibrotic properties of MSCs involve the regulation of matrix metalloproteinases (MMPs) and matrix metalloproteinase endogenous inhibitor (TIMP) production by cardiac fibroblasts. In vitro experiments showed that conditioned medium from MSCs decreased viability, alpha-smooth muscle actin expression, and collagen secretion of cardiac fibroblasts. These effects were concomitant with the stimulation of MMP-2/MMP-9 activities and membrane type 1 MMP expression. Experiments performed with fibroblasts from MMP2-knockout mice demonstrated that MMP-2 plays a preponderant role in preventing collagen accumulation upon incubation with conditioned medium from MSCs. We found that MSC-conditioned medium also decreased the expression of TIMP2 in cardiac fibroblasts. In vivo studies showed that intracardiac injection of MSCs in a rat model of postischemic heart failure induced a significant decrease in ventricular fibrosis. This effect was associated with the improvement of morphological and functional cardiac parameters. In conclusion, we showed that MSCs modulate the phenotype of cardiac fibroblasts and their ability to degrade extracellular matrix. These properties of MSCs open new perspectives for understanding the mechanisms of action of MSCs and anticipate their potential therapeutic or side effects.
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Colagenasas/metabolismo , Fibroblastos/metabolismo , Fibrosis/prevención & control , Células Madre Mesenquimatosas/fisiología , Infarto del Miocardio/patología , Actinas/metabolismo , Animales , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Medios de Cultivo Condicionados/farmacología , Ecocardiografía , Fibroblastos/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Inmunohistoquímica , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados , Infarto del Miocardio/metabolismo , Infarto del Miocardio/terapia , Reacción en Cadena de la Polimerasa , Puntos Cuánticos , Ratas , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
Neutral sphingomyelinase (nSMase), the initial enzyme of the sphingolipid signaling pathway, is thought to play a key role in cellular responses to tumor necrosis factor alpha (TNF-alpha), such as inflammation, proliferation, and apoptosis. The mechanism of TNF-alpha-induced nSMase activation is only partly understood. Using biochemical, molecular, and pharmacological approaches, we found that nSMase activation triggered by TNF-alpha is required for TNF-alpha-induced proliferation and in turn requires a proteolytic cascade involving furin, membrane type 1 matrix metalloproteinase (MT1-MMP), and MMP2, and leading finally to extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and DNA synthesis, in smooth muscle cells (SMC) and fibroblasts. Pharmacological and molecular inhibitors of MMPs (batimastat), furin (alpha1-PDX inhibitor-transfected SMC), MT1-MMP (SMC overexpressing a catalytically inactive MT1-MMP), MMP2 (fibroblasts from MMP2(-/-) mice), and small interfering RNA (siRNA) strategies (siRNAs targeting furin, MT1-MMP, MMP2, and nSMase) resulted in near-complete inhibition of the activation of nSMase, sphingosine kinase-1, and ERK1/2 and of subsequent DNA synthesis. Exogenous MT1-MMP activated nSMase and SMC proliferation in normal but not in MMP2(-/-) fibroblasts, whereas exogenous MMP2 was active on both normal and MMP2(-/-) fibroblasts. Altogether these findings highlight a pivotal role for furin, MT1-MMP, and MMP2 in TNF-alpha-induced sphingolipid signaling, and they identify this system as a possible target to inhibit SMC proliferation in vascular diseases.
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Furina/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Mitógenos/metabolismo , Esfingolípidos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Brefeldino A/farmacología , Proliferación Celular/efectos de los fármacos , Ceramidas/metabolismo , Activación Enzimática/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Lisofosfolípidos/metabolismo , Ratones , Modelos Biológicos , Monensina/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Conejos , Transducción de Señal/efectos de los fármacos , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Red trans-Golgi/efectos de los fármacosRESUMEN
OBJECTIVE: Transplantation of adipose-derived stroma cells (ADSCs) stimulates neovascularization after experimental ischemic injury. ADSC proangiogenic potential is likely mediated by their ability to differentiate into endothelial cells and produce a wide array of angiogenic and antiapoptotic factors. Mitochondrial reactive oxygen species (ROS) have been shown to control ADSC differentiation. We therefore hypothesized that mitochondrial ROS production may change the ADSC proangiogenic properties. METHODS AND RESULTS: The use of pharmacological strategies (mitochondrial inhibitors, antimycin, and rotenone, with or without antioxidants) allowed us to specifically and precisely modulate mitochondrial ROS generation in ADSCs. We showed that transient stimulation of mitochondrial ROS generation in ADSCs before their injection in ischemic hindlimb strongly improved revascularization and the number of ADSC-derived CD31-positive cells in ischemic area. Mitochondrial ROS generation increased the secretion of the proangiogenic and antiapoptotic factors, VEGF and HGF, but did not affect ADSC ability to differentiate into endothelial cells, in vitro. Moreover, mitochondrial ROS-induced ADSC preconditioning greatly protect ADSCs against oxidative stress-induced cell death. CONCLUSIONS: Our study demonstrates that in vitro preconditioning by moderate mitochondrial ROS generation strongly increases in vivo ADSC proangiogenic properties and emphasizes the crucial role of mitochondrial ROS in ADSC fate.
Asunto(s)
Diferenciación Celular/fisiología , Células Endoteliales/citología , Células Endoteliales/fisiología , Mitocondrias/metabolismo , Neovascularización Fisiológica/fisiología , Especies Reactivas de Oxígeno/metabolismo , Adipocitos , Animales , Células Cultivadas , Masculino , Ratones , Daño por Reperfusión/fisiopatología , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
Endogenous PIEZO1 channels of native endothelium lack the hallmark inactivation often seen when these channels are overexpressed in cell lines. Because prior work showed that the force of shear stress activates sphingomyelinase in endothelium, we considered if sphingomyelinase is relevant to endogenous PIEZO1. Patch clamping was used to quantify PIEZO1-mediated signals in freshly isolated murine endothelium exposed to the mechanical forces caused by shear stress and membrane stretch. Neutral sphingomyelinase inhibitors and genetic disruption of sphingomyelin phosphodiesterase 3 (SMPD3) cause PIEZO1 to switch to profoundly inactivating behavior. Ceramide (a key product of SMPD3) rescues non-inactivating channel behavior. Its co-product, phosphoryl choline, has no effect. In contrast to ceramide, sphingomyelin (the SMPD3 substrate) does not affect inactivation but alters channel force sensitivity. The data suggest that sphingomyelinase activity, ceramide, and sphingomyelin are determinants of native PIEZO gating that enable sustained activity.
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Canales Iónicos/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Humanos , RatonesRESUMEN
Inflammation is a well-known pathophysiological factor of atherosclerosis but its therapeutic targeting has long been ignored. However, recent advances in the understanding of the immune mechanisms implicated in atherosclerosis have unveiled several therapeutic targets currently undergoing clinical trials. These studies have also shed light on a dialogue between the immune compartment and vascular smooth muscle cells (VSMCs) that plays a critical role in atherosclerotic disease initiation, progression, and stabilization. Our review focuses on the link between cellular and soluble immune effectors and VSMC behavior at different phases of the pathology. Furthermore, we discuss the potential targeting of these interactions to efficiently prevent cardiovascular diseases.
RESUMEN
Decreased nitric oxide (NO) bioavailability plays a critical role in the pathophysiology of preeclampsia (PE). Recent evidence indicates that S-glutathionylation may occur on the endothelial nitric oxide synthase (eNOS), leading to eNOS uncoupling, characterized by a decreased NO production and an increased generation of superoxide anion (O2â¢-). We hypothesized that eNOS glutathionylation may occur in PE placentas and participate in eNOS dysfunction. The glutathionylation of eNOS was investigated in thirteen PE-affected patients and in nine normal pregnancies. Immunofluorescence, confocal microscopy and western-blot experiments carried out on eNOS immunoprecipitates, revealed a high level of eNOS glutathionylation in PE placentas, mostly reversed by dithiotreitol (DTT), thus indicative of S-glutathionylation. In order to investigate whether eNOS glutathionylation may alter trophoblast migration, an important event occurring during early placentation, cultured HTR-8/SVneo human trophoblasts (HTR8) were exposed either to low pO2 (O2 1%) or to pO2 changes (O2 1-20%), in order to generate oxidative stress. Trophoblasts exposed to low pO2, did not undergo oxidative stress nor eNOS S-glutathionylation, and were able to generate NO and migrate in a wound closure model. In contrast, trophoblasts submitted to low/high pO2 changes, exhibited oxidative stress and a (DTT reversible) S-glutathionylation of eNOS, associated with reduced NO production and migration. The autonomous production of NO seemed necessary for the migratory potential of HTR8, as suggested by the inhibitory effect of eNOS silencing by small interfering RNAs, and the eNOS inhibitor L-NAME, in low pO2 conditions. Finally, the addition of the NO donor, NOC-18 (5⯵M), restored in part the migration of HTR8, thereby emphasizing the role of NO in trophoblast homeostasis. In conclusion, the high level of eNOS S-glutathionylation in PE placentas provides new insights in the mechanism of eNOS dysfunction in this disease.
Asunto(s)
Glutatión/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Placenta/metabolismo , Preeclampsia/etiología , Preeclampsia/metabolismo , Adulto , Estudios de Casos y Controles , Línea Celular , Susceptibilidad a Enfermedades , Endotelio Vascular/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Óxido Nítrico/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Preeclampsia/diagnóstico , Preeclampsia/fisiopatología , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Trofoblastos/metabolismoRESUMEN
PURPOSE: Low molecular weight carbonyl compounds, such as the alpha-ketoaldehydes methylglyoxal (MGO) and glyoxal (GO), are formed under hyperglycemic conditions and behave as advanced glycation end product (AGE) precursors. They form adducts on proteins, thereby inducing cellular dysfunctions involved in chronic complications of diabetes. METHODS AND MAIN FINDINGS: Nontoxic concentrations of GO or MGO altered the PDGF-induced PDGFRbeta-phosphorylation, ERK1/2-activation, and nuclear translocation, and the subsequent proliferation of mesenchymal cells (smooth muscle cells and skin fibroblasts). This resulted mainly from inhibition of the intrinsic tyrosine kinase of PDGFRbeta and in part from altered PDGF-BB binding to PDGFRbeta. Concomitantly, the formation of AGE adducts (N(epsilon)carboxymethyl-lysine and N(epsilon)carboxyethyl-lysine) was observed on immunoprecipitated PDGFRbeta. Arginine and aminoguanidine, used as carbonyl scavengers, reversed the inhibitory effect and the formation of AGE adducts on PDGFRbeta. AGE-PDGFRbeta adducts were also detected by anti-AGE antibodies in PDGFRbeta immunopurified from aortas of diabetic (streptozotocin-treated) compared to nondiabetic apolipoprotein E-null mice. Mass spectrometry analysis of aortas demonstrated increased AGE formation in diabetic specimens. CONCLUSIONS: These data indicate that MGO and GO induce desensitization of PDGFRbeta that helps to reduce mesenchymal cell proliferation.
Asunto(s)
Aterosclerosis , Complicaciones de la Diabetes , Productos Finales de Glicación Avanzada/metabolismo , Piruvaldehído/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Arginina/metabolismo , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Becaplermina , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Diabetes Mellitus Experimental , Glioxal/metabolismo , Guanidinas/metabolismo , Humanos , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-sis , Conejos , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
Reactive oxygen species (ROS) are key signaling molecules and their overproduction plays an important role in the inflammation process, the secretion of inflammatory cytokines such as IL-1ß and IL-6 and the progression of inflammatory disorders. Decreasing oxidative stress represents a promising challenge in the design of antioxidant and anti-inflammatory agents. In the present study, a series of new diarylheptanoids containing allylic alcohol, amide, hydantoin or triazole fragments were synthesized and fully characterized. We evaluated the ability of these agents to block the production of intracellular ROS and the subsequent inflammatory events exerted by lipopolysaccharide (LPS) on murine macrophage RAW 264.7. Five diarylheptanoids were found to exhibit the dual required properties.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/farmacología , Diarilheptanoides/farmacología , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Antioxidantes/síntesis química , Antioxidantes/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Diarilheptanoides/síntesis química , Diarilheptanoides/química , Relación Dosis-Respuesta a Droga , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Estructura Molecular , Células RAW 264.7 , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-ActividadRESUMEN
The degradation of biogenic amines by monoamine oxidase A (MAO-A) generates reactive oxygen species (ROS) which participate in serotonin and tyramine signaling. This study aimed to investigate the role of ROS in the mitogenic signaling activated during tyramine and serotonin oxidation by MAO-A in smooth muscle cells (SMC). Incubation of SMC with serotonin or tyramine induced intracellular ROS generation, and a signaling cascade involving metalloproteases and the neutral sphingomyelinase-2 (nSMase2, the initial step of the sphingolipid pathway), ERK1/2 phosphorylation, and DNA synthesis. Silencing MAO-A by siRNA, pharmacological MAO-A inhibitors (pargyline and Ro41-1049), and the antioxidant/ROS scavenger butylated hydroxytoluene (BHT) inhibited the signaling cascade, suggesting that ROS generated during tyramine oxidation by MAO-A are required. The MMP inhibitor Batimastat, MMP2-specific siRNA, and MMP2 deletion (MMP2(-/-) fibroblasts) blocked nSMase activation and SMC proliferation, suggesting a role for MMP2 in this signaling pathway. Silencing nSMase2 by siRNA did not inhibit ROS generation and MMP2 activation, but blocked SMC proliferation induced by tyramine, suggesting that nSMase2 is downstream MMP2. These findings demonstrate that H(2)O(2)-generated during tyramine oxidation by MAO-A triggers a stress-induced mitogenic signaling via the MMP2/sphingolipid pathway, which could participate in excessive remodeling and alteration of the vascular wall.
Asunto(s)
Aminas Biogénicas/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Mitógenos/metabolismo , Monoaminooxidasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Aminas Biogénicas/farmacología , Vasos Sanguíneos/metabolismo , Hidroxitolueno Butilado/farmacología , Proliferación Celular , Depuradores de Radicales Libres/farmacología , Peróxido de Hidrógeno/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Mutantes , Mitógenos/farmacología , Monoaminooxidasa/efectos de los fármacos , Monoaminooxidasa/genética , Inhibidores de la Monoaminooxidasa/farmacología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Oxidación-Reducción , Pargilina/farmacología , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , ARN Interferente Pequeño/farmacología , Conejos , Serotonina/metabolismo , Serotonina/farmacología , Transducción de Señal , Esfingolípidos/metabolismo , Tiazoles/farmacología , Tiofenos/farmacología , Tiramina/metabolismo , Tiramina/farmacologíaRESUMEN
Besides its involvement in clot lysis, the plasminogen activator (PA) system elicits various cellular responses involved in cell migration, adhesion, and proliferation and plays a key role in the progression of cancers. beta-Catenin interacts with E-cadherins and functions as transcriptional coactivator of the Wnt-signaling pathway, which is implicated in tumor formation when aberrantly activated. We report that tissue-type plasminogen activator (tPA) elicited tyrosine phosphorylation and cytosolic accumulation of an active (non-serine-threonin phosphorylated, nonubiquitinated) form of beta-catenin in ECV304 carcinoma cells. tPA-dependent beta-catenin activation is mediated through epidermal growth factor receptor (EGFR) transactivation (via Src), suggested by the inhibitory effects of AG1478 and PP2 (specific inhibitors of EGFR and Src, respectively) and by the lack of beta-catenin activation in EGFR-negative B82 fibroblasts. EGFR phosphorylation and beta-catenin activation were inhibited by plasminogen activator inhibitor 1 and pertussis toxin, two inhibitors of the urokinase-type plasminogen activator (uPA)/uPA receptor system. beta-Catenin activation was correlated with the phosphorylation of glycogen synthase kinase-3beta through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. Gel shift experiments revealed the activation of beta-catenin/T-cell-specific transcription factor (Tcf)/lymphoid enhancer factor-1 (Lef) transcriptional complex, evidenced by an increased binding of nuclear extracts to oligonucleotides containing the cyclin D1 Lef/Tcf site. beta-Catenin silencing through small interfering RNA and antisense oligonucleotides inhibited both the tPA-mediated cyclin D1 expression and cell proliferation. A similar activation of the beta-catenin pathway was triggered by amino-terminal fragment, the NH(2)-terminal catalytically inactive fragment of tPA, thus suggesting that this effect was independent of the proteolytic activity of plasminogen activators. In conclusion, the beta-catenin/Lef/Tcf pathway is activated by tPA and is involved in cell cycle progression and proliferation.
Asunto(s)
Carcinoma/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Ciclo Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Ciclina D1/antagonistas & inhibidores , Ciclina D1/biosíntesis , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Citosol/metabolismo , Activación Enzimática , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Factor de Unión 1 al Potenciador Linfoide , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , ARN Interferente Pequeño , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , beta CateninaRESUMEN
The oxidative theory of atherosclerosis relies on the modification of low density lipoproteins (LDLs) in the vascular wall by reactive oxygen species. Modified LDLs, such as oxidized LDLs, are thought to participate in the formation of early atherosclerotic lesions (accumulation of foam cells and fatty streaks), whereas their role in advanced lesions and atherothrombotic events is more debated, because antioxidant supplementation failed to prevent coronary disease events and mortality in intervention randomized trials. As oxidized LDLs and oxidized lipids are present in atherosclerotic lesions and are able to trigger cell signaling on cultured vascular cells and macrophages, it has been proposed that they could play a role in atherogenesis and atherosclerotic vascular remodeling. Oxidized LDLs exhibit dual biological effects, which are dependent on extent of lipid peroxidation, nature of oxidized lipids (oxidized phospholipids, oxysterols, malondialdehyde, α,ß-unsaturated hydroxyalkenals), concentration of oxidized LDLs and uptake by scavenger receptors (e.g. CD36, LOX-1, SRA) that signal through different transduction pathways. Moderate concentrations of mildly oxidized LDLs are proinflammatory and trigger cell migration and proliferation, whereas higher concentrations induce cell growth arrest and apoptosis. The balance between survival and apoptotic responses evoked by oxidized LDLs depends on cellular systems that regulate the cell fate, such as ceramide/sphingosine-1-phosphate rheostat, endoplasmic reticulum stress, autophagy and expression of pro/antiapoptotic proteins. In vivo, the intimal concentration of oxidized LDLs depends on the influx (hypercholesterolemia, endothelial permeability), residence time and lipid composition of LDLs, oxidative stress intensity, induction of defense mechanisms (antioxidant systems, heat shock proteins). As a consequence, the local cellular responses to oxidized LDLs may stimulate inflammatory or anti-inflammatory pathways, angiogenic or antiangiogenic responses, survival or apoptosis, thereby contributing to plaque growth, instability, complication (intraplaque hemorrhage, proteolysis, calcification, apoptosis) and rupture. Finally, these dual properties suggest that oxLDLs could be implicated at each step of atherosclerosis development, from early fatty streaks to advanced lesions, depending on the nature and concentration of their oxidized lipid content.
Asunto(s)
Aterosclerosis/metabolismo , Peroxidación de Lípido/genética , Lipoproteínas LDL/metabolismo , Estrés Oxidativo/genética , Apoptosis/genética , Aterosclerosis/genética , Aterosclerosis/fisiopatología , Autofagia/genética , Humanos , Lipoproteínas LDL/genética , Macrófagos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
The neovascularization of atherosclerotic lesions is involved in plaque development and may contribute to intraplaque hemorrhage and plaque fragilization and rupture. Among the various proangiogenic agents involved in the neovascularization process, proatherogenic oxidized LDLs (oxLDLs) contribute to the formation of tubes via the generation of sphingosine 1-phosphate (S1P), a major mitogenic and proangiogenic sphingolipid mediator. In this study, we investigated whether 4-hydroxynonenal (4-HNE), an aldehydic lipid oxidation product abundantly present in oxLDLs, contributes to their proangiogenic properties. Immunofluorescence analysis of human atherosclerotic lesions from carotid endarterectomy showed the colocalization of HNE-adducts with CD31, a marker of endothelial cells, suggesting a close relationship between 4-HNE and neovessel formation. In vitro, low 4-HNE concentration (0.5-1 µM) elicited the formation of tubes by human microvascular endothelial cells (HMEC-1), whereas higher concentrations were not angiogenic. The formation of tubes by 4-HNE involved the generation of reactive oxygen species and the activation of the sphingolipid pathway, namely, the neutral type 2 sphingomyelinase and sphingosine kinase-1 (nSMase2/SK-1) pathway, indicating a role for S1P in the angiogenic signaling of 4-HNE. Carbonyl scavengers hydralazine and bisvanillyl-hydralazone inhibited the nSMase2/SK1 pathway activation and the formation of tubes on Matrigel® evoked by 4-HNE. Altogether, these results emphasize the role of 4-HNE in the angiogenic effect of oxLDLs and point out the potential interest of pharmacological carbonyl scavengers to prevent the neovascularization process.
Asunto(s)
Aldehídos/toxicidad , Células Endoteliales/metabolismo , Hidralazina , Neovascularización Patológica , Transducción de Señal/efectos de los fármacos , Esfingolípidos/metabolismo , Línea Celular , Células Endoteliales/patología , Humanos , Hidralazina/análogos & derivados , Hidralazina/farmacología , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neovascularización Patológica/prevención & control , Oxidación-Reducción/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
The grape-derived polyphenol resveratrol is anti-proliferative for human liver myofibroblasts, which may be beneficial for the treatment of liver fibrosis. However, its mechanism of action is ill understood. Here, we have studied how resveratrol interfered with signaling pathways used by epidermal or platelet-derived growth factors to induce the proliferation of these cells. We found that resveratrol inhibited epidermal growth factor or platelet-derived growth factor-induced DNA synthesis. Resveratrol did not, however, decrease epidermal growth factor receptor autophosphorylation or activation of extracellular regulated kinases, but strongly inhibited the phosphorylation of Akt and of its substrate forkhead related transcription factor. This suggested that resveratrol inhibited epidermal growth factor-induced mitogenic signaling through inhibition of the phosphatidylinositol 3-kinase /Akt pathway. The phosphatidylinositol 3-kinase inhibitor LY 294002, also, inhibited epidermal growth factor-dependent DNA synthesis and Akt phosphorylation but did not decrease extracellular regulated kinases phosphorylation. In contrast, resveratrol inhibited platelet-derived growth factor-stimulated receptor autophosphorylation and every subsequent signaling step. Resveratrol did not directly inhibit phosphatidylinositol 3-kinase activity measured on immunoprecipitates from epidermal growth factor-stimulated myofibroblasts, but it strongly reduced the autophosphorylation of the phosphatidylinositol 3-kinase downstream target phospho-inositide-dependent kinase-1 that phosphorylates Akt. We, thus, show that resveratrol has growth factor-specific effects: it inhibits platelet-derived growth factor signaling via reduced receptor activation, whereas it reduces epidermal growth factor-dependent DNA synthesis via inhibition of the phosphatidylinositol 3-kinase/Akt pathway, possibly through inhibition of phospho-inositide-dependent kinase-1 activity.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Hígado/metabolismo , Mioblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Estilbenos/farmacología , Vitis/química , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Factores de Crecimiento de Fibroblastos/farmacología , Fibroblastos/citología , Flavonoides/química , Flavonoides/farmacología , Flavonoides/uso terapéutico , Humanos , Hígado/citología , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/metabolismo , Mioblastos/citología , Fenoles/química , Fenoles/farmacología , Fenoles/uso terapéutico , Factor de Crecimiento Derivado de Plaquetas/farmacología , Polifenoles , Resveratrol , Estilbenos/química , Estilbenos/uso terapéuticoRESUMEN
OBJECTIVE: The atherogenic oxidized low-density lipoprotein (oxLDL) induces the formation of carbonyl-protein adducts and activates the epidermal [corrected] growth factor receptor (EGFR) signaling pathway, which is now regarded as a central element for signal transduction. We aimed to investigate whether and by which mechanism the anti-atherogenic high-density lipoprotein (HDL) prevents these effects of oxLDL. METHODS AND RESULTS: In vascular cultured cells, HDL and apolipoprotein A-I inhibit oxLDL-induced EGFR activation and subsequent signaling by acting through 2 separate mechanisms. First, HDL, like the aldehyde scavenger dinitrophenyl hydrazine, prevented the formation of oxLDL-induced carbonyl-protein adducts and 4-hydroxynonenal (HNE)-EGFR adducts. Secondly, HDL enhanced the cellular antioxidant defenses by preventing (through a scavenger receptor class B-1 (SR-BI)-dependent mechanism) the increase of intracellular reactive oxygen species (ROS) and subsequent EGFR activation triggered by oxLDL or H2O2. A pharmacological approach suggests that this protective effect of HDL is independent of cellular glutathione level and glutathione peroxidase activity, but it requires catalase activity. Finally, we report that oxLDL upregulates both membrane type 1 (MT1)-matrix metalloproteinase-1 (MT1-MMP) and MMP-2 through an EGFR-dependent mechanism and that HDL inhibits these events. CONCLUSIONS: HDLs block in vitro oxLDL-induced EGFR signaling and subsequent MMP-2 activation by inhibiting carbonyl adducts formation and cellular oxidative stress. These effects of HDL may participate to reduce cell activation, excessive remodeling, and alteration of the vascular wall.