RESUMO
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.
Assuntos
Canais Iônicos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Humanos , CamundongosRESUMO
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.
RESUMO
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.
Assuntos
Glutationa/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/etiologia , Pré-Eclâmpsia/metabolismo , Adulto , Estudos de Casos e Controles , Linhagem Celular , Suscetibilidade a Doenças , Endotélio Vascular/metabolismo , Feminino , Imunofluorescência , Humanos , Óxido Nítrico/metabolismo , Oxirredução , Estresse Oxidativo , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/fisiopatologia , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Trofoblastos/metabolismoRESUMO
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.
Assuntos
Compostos de Anilina/farmacologia , Anti-Inflamatórios/farmacologia , Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Compostos de Benzilideno/farmacologia , Inibidores Enzimáticos/farmacologia , Inflamação/prevenção & controle , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/deficiência , Animais , Aorta/enzimologia , Aorta/patologia , Doenças da Aorta/enzimologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Placa Aterosclerótica , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Esfingomielina Fosfodiesterase/genéticaRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antioxidantes/farmacologia , Diarileptanoides/farmacologia , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Antioxidantes/síntese química , Antioxidantes/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Diarileptanoides/síntese química , Diarileptanoides/química , Relação Dose-Resposta a Droga , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Estrutura Molecular , Células RAW 264.7 , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Aldeídos/toxicidade , Células Endoteliais/metabolismo , Hidralazina , Neovascularização Patológica , Transdução de Sinais/efeitos dos fármacos , Esfingolipídeos/metabolismo , Linhagem Celular , Células Endoteliais/patologia , Humanos , Hidralazina/análogos & derivados , Hidralazina/farmacologia , Neovascularização Patológica/induzido quimicamente , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Patológica/prevenção & controle , Oxirredução/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingomielina Fosfodiesterase/metabolismoRESUMO
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.
Assuntos
Aterosclerose/metabolismo , Peroxidação de Lipídeos/genética , Lipoproteínas LDL/metabolismo , Estresse Oxidativo/genética , Apoptose/genética , Aterosclerose/genética , Aterosclerose/fisiopatologia , Autofagia/genética , Humanos , Lipoproteínas LDL/genética , Macrófagos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Capillaries of the external part of the normal arterial wall constitute the vasa vasorum network. In atherosclerotic lesions, neovascularization occurs in areas of intimal hyperplasia where it may promote plaque expansion, and intraplaque hemorrhage. Oxidized LDL that are present in atherosclerotic areas activate various angiogenic signaling pathways, including reactive oxygen species and the sphingosine kinase/sphingosine-1-phosphate pathway. We aimed to investigate whether oxidized LDL-induced angiogenesis requires neutral sphingomyelinase-2 activation and the neutral sphingomyelinase-2/sphingosine kinase-1 pathway. The role of neutral sphingomyelinase-2 in angiogenic signaling was investigated in Human Microvascular Endothelial Cells (HMEC-1) forming capillary tube on Matrigel and in vivo in the Matrigel plug assay in C57BL/6 mice and in the chicken chorioallantoic membrane model. Low concentration of human oxidized LDL elicits HMEC-1 capillary tube formation and neutral sphingomyelinase-2 activation, which were blocked by neutral sphingomyelinase-2 inhibitors, GW4869 and specific siRNA. This angiogenic effect was mimicked by low concentration of C6-Ceramide and was inhibited by sphingosine kinase-1 inhibitors. Upstream of neutral sphingomyelinase-2, oxidized LDL-induced activation required LOX-1, reactive oxygen species generation by NADPH oxidase and p38-MAPK activation. Inhibition of sphingosine kinase-1 blocked the angiogenic response and triggered HMEC-1 apoptosis. Low concentration of oxidized LDL was angiogenic in vivo, both in the Matrigel plug assay in mice and in the chorioallantoic membrane model, and was blocked by GW4869. In conclusion, low oxLDL concentration triggers sprouting angiogenesis that involves ROS-induced activation of the neutral sphingomyelinase-2/sphingosine kinase-1 pathway, and is effectively inhibited by GW4869.
Assuntos
Lipoproteínas LDL/metabolismo , Neovascularização Patológica/genética , Estresse Oxidativo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Esfingomielina Fosfodiesterase/biossíntese , Compostos de Anilina/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Compostos de Benzilideno/administração & dosagem , Ceramidas/metabolismo , Células Endoteliais/metabolismo , Humanos , Lipoproteínas LDL/genética , Lisofosfolipídeos/metabolismo , Camundongos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , NADPH Oxidases/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Ativação Transcricional/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The neutral type 2 sphingomyelinase (nSMase2) hydrolyzes sphingomyelin and generates ceramide, a major bioactive sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (oxidized LDL, hydrogen peroxide and nutrient starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient starvation (fetal calf serum/glucose/pyruvate-free DMEM), whereas wt fibroblasts die after 48h incubation in this medium. In contrast, oxidized LDL and hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor GW4869 were more resistant to starvation-induced apoptosis. The resistance of fro/fro cells to starvation-induced apoptosis is associated with an increased expression of hyaluronan synthase 2 (HAS2) mRNAs and protein, which is inhibited by ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against starvation-induced apoptosis. The protective mechanism of HAS2 involves an increased expression of the heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient starvation that could contribute to the delayed apoptosis of hypertrophic chondrocytes in the growth plate, and emphasize the antiapoptotic properties of HAS2.
Assuntos
Apoptose/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Glucuronosiltransferase/biossíntese , Proteínas de Choque Térmico HSP72/biossíntese , Esfingomielina Fosfodiesterase/genética , Animais , Apoptose/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/genética , Proteínas de Choque Térmico HSP72/metabolismo , Hialuronan Sintases , Peróxido de Hidrogênio/farmacologia , Lipoproteínas LDL/farmacologia , Camundongos , Transdução de Sinais , Esfingomielina Fosfodiesterase/metabolismo , Ativação TranscricionalRESUMO
Actin remodeling is a dynamic process associated with cell shape modification occurring during cell cycle and proliferation. Oxidative stress plays a role in actin reorganization via various systems including p38MAPK. Beside, the mitogenic response evoked by hydrogen peroxide (H2O2) in fibroblasts and smooth muscle cells (SMC) involves the metalloproteinase (MMPs)/sphingomyelinase 2 (nSMase2) signaling pathway. The aim of this work was to investigate whether this system plays a role in actin remodeling induced by H2O2. Low H2O2 dose (5µM) rapidly triggered a signaling cascade leading to nSMase2 activation, src and annexin 2 (AnxA2) phosphorylation, and actin remodeling, in fibroblasts and SMC. These events were blocked by pharmacological inhibitors of MMPs (Ro28-2653) and p38MAPK (SB203580), and were lacking in MMP2(-/-) and in nSMase2-mutant (fro) fibroblasts. Likewise, H2O2 was unable to induce actin remodeling in fro and MMP2(-/-) fibroblasts or in cells pretreated with p38MAPK, or MMP inhibitors. Finally we show that nSMase2 activation by H2O2, depends on MMP2 and p38MAPK, and is required for the src-dependent phosphorylation of AnxA2, and actin remodeling. Taken together, these findings indicate for the first time that AnxA2 phosphorylation and actin remodeling evoked by oxidative stress depend on the sphingolipid pathway, via MMP2 and p38MAPK.
Assuntos
Actinas/metabolismo , Anexina A2/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anexina A2/biossíntese , Proliferação de Células/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Metaloproteinase 2 da Matriz/biossíntese , Camundongos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Esfingolipídeos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/biossínteseRESUMO
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.
Assuntos
Ceramidas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Esfingomielina Fosfodiesterase/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Ceramidas/genética , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Lisofosfolipídeos/genética , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Mutantes , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/efeitos dos fármacos , Esfingomielina Fosfodiesterase/genética , Esfingosina/análogos & derivados , Esfingosina/genética , Esfingosina/metabolismo , Quinases da Família src/genéticaRESUMO
AIMS: Protein disulfide isomerase (PDI) is an abundant endoplasmic reticulum (ER)-resident chaperone and oxidoreductase that catalyzes formation and rearrangement (isomerization) of disulfide bonds, thereby participating in protein folding. PDI modification by nitrosative stress is known to increase protein misfolding, ER stress, and neuronal apoptosis. As LDL oxidation and ER stress may play a role in atherogenesis, this work was designed to investigate whether PDI was inactivated by oxLDLs, thereby participating in oxLDL-induced ER stress and apoptosis. RESULTS: Preincubation of human endothelial HMEC-1 and of macrophagic U937 cells with toxic concentration of oxLDLs induced PDI inhibition and modification, as assessed by 4-HNE-PDI adducts formation. PDI inhibition by bacitracin potentiated ER stress (increased mRNA expression of CHOP and sXBP1) and apoptosis induced by oxLDLs. In contrast, increased PDI activity by overexpression of an active wild-type PDI was associated with reduced oxLDL-induced ER stress and toxicity, whereas the overexpression of a mutant inactive form was not protective. These effects on PDI were mimicked by exogenous 4-HNE and prevented by the carbonyl-scavengers N-acetylcysteine and pyridoxamine, which reduced CHOP expression and toxicity by oxLDLs. Interestingly, 4-HNE-modified PDI was detected in the lipid-rich areas of human advanced atherosclerotic lesions. Innovation and CONCLUSIONS: PDI modification by oxLDLs or by reactive carbonyls inhibits its enzymatic activity and potentiates both ER stress and apoptosis by oxLDLs. PDI modification by lipid peroxidation products in atherosclerotic lesions suggests that a loss of function of PDI may occur in vivo, and may contribute to local ER stress, apoptosis, and plaque progression.
Assuntos
Apoptose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/patologia , Humanos , Lipoproteínas LDL/metabolismo , Oxirredução , Relação Estrutura-Atividade , Células U937RESUMO
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.
Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Artérias/transplante , Antígenos HLA/imunologia , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Doenças Vasculares/fisiopatologia , Compostos de Anilina/farmacologia , Animais , Anticorpos Anti-Idiotípicos/efeitos adversos , Artérias/patologia , Artérias/fisiopatologia , Compostos de Benzilideno/farmacologia , Células Cultivadas , Constrição Patológica/etiologia , Constrição Patológica/fisiopatologia , Modelos Animais de Doenças , Humanos , Hiperplasia/etiologia , Hiperplasia/fisiopatologia , Técnicas In Vitro , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos SCID , Modelos Animais , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Neointima/patologia , Neointima/fisiopatologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Doenças Vasculares/etiologia , Enxerto VascularRESUMO
Lipid peroxidation (LPO) product accumulation in human tissues is a major cause of tissular and cellular dysfunction that plays a major role in ageing and most age-related and oxidative stress-related diseases. The current evidence for the implication of LPO in pathological processes is discussed in this review. New data and literature review are provided evaluating the role of LPO in the pathophysiology of ageing and classically oxidative stress-linked diseases, such as neurodegenerative diseases, diabetes and atherosclerosis (the main cause of cardiovascular complications). Striking evidences implicating LPO in foetal vascular dysfunction occurring in pre-eclampsia, in renal and liver diseases, as well as their role as cause and consequence to cancer development are addressed.
Assuntos
Envelhecimento/fisiologia , Aterosclerose/fisiopatologia , Diabetes Mellitus/fisiopatologia , Peroxidação de Lipídeos/fisiologia , Doenças Neurodegenerativas/fisiopatologia , Animais , Humanos , Hepatopatias/fisiopatologia , Neoplasias/fisiopatologia , Estresse Oxidativo/fisiologiaRESUMO
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.
Assuntos
Modelos Animais de Doenças , Embolia Intracraniana/prevenção & controle , Lipoproteínas HDL/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Animais , Embolia Intracraniana/sangue , Embolia Intracraniana/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Acidente Vascular CerebralRESUMO
BACKGROUND: Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg(2+)-dependent neutral sphingomyelinase-2 (nSMase2) plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations. METHODOLOGY AND PRINCIPAL FINDINGS: For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium) mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice) does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum) requires nSMase2 activation. CONCLUSION AND SIGNIFICANCE: nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents.
Assuntos
Apoptose , Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Proliferação de Células , Fibroblastos/metabolismo , Vetores Genéticos , Humanos , Hidrólise , Lipoproteínas LDL/metabolismo , Fígado/patologia , Camundongos , Camundongos Transgênicos , Esfingomielina Fosfodiesterase/biossíntese , TransfecçãoRESUMO
Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin alpha(v)beta(3), evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the alpha(v)beta(3) blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin alpha(v)beta(3) interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin alpha(v)beta(3), uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin alpha(v)beta(3) and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.
Assuntos
Integrina alfaVbeta3/metabolismo , Metaloproteinases da Matriz/metabolismo , Mitógenos/metabolismo , Mitose , Esfingomielina Fosfodiesterase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Linhagem Celular Tumoral , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
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.
Assuntos
Colagenases/metabolismo , Fibroblastos/metabolismo , Fibrose/prevenção & controle , Células-Tronco Mesenquimais/fisiologia , Infarto do Miocárdio/patologia , Actinas/metabolismo , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Meios de Cultivo Condicionados/farmacologia , Ecocardiografia , Fibroblastos/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Imuno-Histoquímica , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/terapia , Reação em Cadeia da Polimerase , Pontos Quânticos , Ratos , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
Diabetes mellitus is a multifactorial disease, classically influenced by genetic determinants of individual susceptibility and by environmental accelerating factors, such as lifestyle. It is considered a major health concern,as its incidence is increasing at an alarming rate, and the high invalidating effects of its long-term complications affect macro- and microvasculature, heart, kidney, eye, and nerves. Increasing evidence indicates that hyperglycemia is the initiating cause of the tissue damage occurring in diabetes, either through repeated acute changes in cellular glucose metabolism, or through the long-term accumulation of glycated biomolecules and advanced glycation end products (AGEs). AGEs represent a heterogeneous group of chemical products resulting from a nonenzymatic reaction between reducing sugars and proteins, lipids, nucleic acids, or a combination of these.The glycation process (glucose fixation) affects circulating proteins (serum albumin, lipoprotein, insulin, hemoglobin),whereas the formation of AGEs implicates reactive intermediates such as methylglyoxal. AGEs form cross-links on long-lived extracellular matrix proteins or react with their specific receptor RAGE, resulting inoxidative stress and proinflammatory signaling implicated in endothelium dysfunction, arterial stiffening, and microvascular complications. This review summarizes the mechanism of glycation and of AGEs formation and the role of hyperglycemia, AGEs, and oxidative stress in the pathophysiology of diabetic complications.