RESUMO
AIM: Atherosclerosis is responsible for high morbidity and mortality rates around the world. Local arterial oxidative stress is involved in all phases of atherosclerosis development. Mitochondria is a relevant source of the oxidants, particularly under certain risky conditions, such as hypercholesterolemia. The aim of this study was to test whether lowering the production of mitochondrial oxidants by induction of a mild uncoupling can reduce atherosclerosis in hypercholesterolemic LDL receptor knockout mice. METHODS: The mice were chronically treated with very low doses of DNP (2,4-dinitrophenol) and metabolic, inflammatory and redox state markers and atherosclerotic lesion sizes were determined. RESULTS: The DNP treatment did not change the classical atherosclerotic risk markers, such as plasma lipids, glucose homeostasis, and fat mass, as well as systemic inflammatory markers. However, the DNP treatment diminished the production of mitochondrial oxidants, systemic and tissue oxidative damage markers, peritoneal macrophages and aortic rings oxidants generation. Most importantly, development of spontaneous and diet-induced atherosclerosis (lipid and macrophage content) were significantly decreased in the DNP-treated mice. In vitro, DNP treated peritoneal macrophages showed decreased H2O2 production, increased anti-inflammatory cytokines gene expression and secretion, increased phagocytic activity, and decreased LDL-cholesterol uptake. CONCLUSIONS: These findings are a proof of concept that activation of mild mitochondrial uncoupling is sufficient to delay the development of atherosclerosis under the conditions of hypercholesterolemia and oxidative stress. These results promote future approaches targeting mitochondria for the prevention or treatment of atherosclerosis.
Assuntos
Aterosclerose , Hipercolesterolemia , Animais , Aterosclerose/metabolismo , Humanos , Peróxido de Hidrogênio , Hipercolesterolemia/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Oxidantes/metabolismoRESUMO
Cardiovascular diseases are major causes of death worldwide. Beyond the classical cholesterol risk factor, other conditions such as oxidative stress are well documented to promote atherosclerosis. The Mangifera indica L. extract (Vimang®) was reported to present antioxidant and hypocholesterolemic properties. Thus, here we evaluate the effects of Vimang treatment on risk factors of the atherosclerosis prone model of familial hypercholesterolemia, the LDL receptor knockout mice. Mice were treated with Vimang during 2 weeks and were fed a cholesterol-enriched diet during the second week. The Vimang treated mice presented significantly reduced levels of plasma (15%) and liver (20%) cholesterol, increased plasma total antioxidant capacity (10%) and decreased reactive oxygen species (ROS) production by spleen mononuclear cells (50%), P < 0.05 for all. In spite of these benefits, the average size of aortic atherosclerotic lesions stablished in this short experimental period did not change significantly in Vimang treated mice. Therefore, in this study we demonstrated that Vimang has protective effects on systemic and tissue-specific risk factors, but it is not sufficient to promote a reduction in the initial steps of atherosclerosis development. In addition, we disclosed a new antioxidant target of Vimang, the spleen mononuclear cells that might be relevant for more advanced stages of atherosclerosis.
Assuntos
Colesterol/sangue , Mangifera/química , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Receptores de LDL/genética , Animais , Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/veterinária , Colesterol/análise , Dieta Hiperlipídica , Leucócitos/citologia , Leucócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Mangifera/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , NADP/química , NADP/metabolismo , Extratos Vegetais/química , Espécies Reativas de Oxigênio/metabolismo , Receptores de LDL/deficiência , Triglicerídeos/análise , Triglicerídeos/sangueRESUMO
Ageing and atherosclerosis are associated with oxidative stress. Mitochondrial redox function declines with ageing. Here we tested whether ageing LDL receptor knockout mice (LDLr-/-) develop spontaneous atherosclerosis and whether mitochondrial reactive oxygen species (mtROS) correlate with atherosclerosis. Compared with young mice, aged LDLr-/- mice exhibited 20-fold larger aortic lesion size, although the plasma cholesterol levels did not vary between age groups. The lesion sizes increased exponentially from 3 to 24months of age (r=0.92, p=0.0001) and were correlated with mtROS across the age range (r=0.81, p=0.0001). Thus, LDLr-/- mice develop spontaneous diet-independent atherosclerosis, that advances exponentially with ageing. We propose that age related increases in mtROS contribute to accelerate atherosclerosis development in hypercholesterolemic mice.
Assuntos
Envelhecimento , Aterosclerose/etiologia , Hipercolesterolemia/complicações , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Masculino , Camundongos , Camundongos Knockout , Receptores de LDL/fisiologiaRESUMO
Atherosclerosis has been associated with mitochondria dysfunction and damage. Our group demonstrated previously that hypercholesterolemic mice present increased mitochondrial reactive oxygen (mtROS) generation in several tissues and low NADPH/NADP+ ratio. Here, we investigated whether spontaneous atherosclerosis in these mice could be modulated by treatments that replenish or spare mitochondrial NADPH, named citrate supplementation, cholesterol synthesis inhibition, or both treatments simultaneously. Robust statistical analyses in pooled group data were performed in order to explain the variation of atherosclerosis lesion areas as related to the classic atherosclerosis risk factors such as plasma lipids, obesity, and oxidative stress, including liver mtROS. Using three distinct statistical tools (univariate correlation, adjusted correlation, and multiple regression) with increasing levels of stringency, we identified a novel significant association and a model that reliably predicts the extent of atherosclerosis due to variations in mtROS. Thus, results show that atherosclerosis lesion area is positively and independently correlated with liver mtROS production rates. Based on these findings, we propose that modulation of mitochondrial redox state influences the atherosclerosis extent.
Assuntos
Aterosclerose/metabolismo , Colesterol/metabolismo , Mitocôndrias Hepáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Colesterol/genética , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/patologia , NADP/genética , NADP/metabolismoRESUMO
Different regimens of food restriction have been associated with protection against obesity, diabetes and CVD. In the present study, we hypothesised that food restriction would bring benefits to atherosclerosis- and diabetes-prone hypercholesterolaemic LDL-receptor knockout mice. For this purpose, 2-month-old mice were submitted to an intermittent fasting (IF) regimen (fasting every other day) over a 3-month period, which resulted in an overall 20 % reduction in food intake. Contrary to our expectation, epididymal and carcass fat depots and adipocyte size were significantly enlarged by 15, 72 and 68 %, respectively, in the IF mice compared with the ad libitum-fed mice. Accordingly, plasma levels of leptin were 50 % higher in the IF mice than in the ad libitum-fed mice. In addition, the IF mice showed increased plasma levels of total cholesterol (37 %), VLDL-cholesterol (195 %) and LDL-cholesterol (50 %). As expected, in wild-type mice, the IF regimen decreased plasma cholesterol levels and epididymal fat mass. Glucose homeostasis was also disturbed by the IF regimen in LDL-receptor knockout mice. Elevated levels of glycaemia (40 %), insulinaemia (50 %), glucose intolerance and insulin resistance were observed in the IF mice. Systemic inflammatory markers, TNF-α and C-reactive protein, were significantly increased and spontaneous atherosclerosis development were markedly increased (3-fold) in the IF mice. In conclusion, the IF regimen induced obesity and diabetes and worsened the development of spontaneous atherosclerosis in LDL-receptor knockout mice. Although being efficient in a wild-type background, this type of food restriction is not beneficial in the context of genetic hypercholesterolaemia.
Assuntos
Aterosclerose/etiologia , Diabetes Mellitus/etiologia , Jejum/efeitos adversos , Hipercolesterolemia/complicações , Obesidade/etiologia , Adipócitos/patologia , Animais , Glicemia/análise , Composição Corporal , Tamanho Celular , Colesterol/sangue , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Epididimo , Privação de Alimentos , Intolerância à Glucose/etiologia , Hipercolesterolemia/genética , Inflamação/etiologia , Insulina/sangue , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/deficiênciaRESUMO
BACKGROUND: We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoK(ATP) protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoK(ATP) activity and evaluate the influence of this trait on cell redox state and viability. METHODS: Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. RESULTS: HTG mice mononuclear cells displayed increased mitoK(ATP) activity, as evidenced by higher resting respiration rates that were sensitive to mitoK(ATP) antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoK(ATP) further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. CONCLUSIONS: These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoK(ATP) channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoK(ATP) channels. Thus, mitoK(ATP) opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.
Assuntos
Hiperlipidemias/metabolismo , Mitocôndrias/metabolismo , Canais de Potássio/metabolismo , Superóxidos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/genética , Hiperlipidemias/genética , Hiperlipidemias/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Camundongos , Mitocôndrias/patologia , Estresse Oxidativo , Consumo de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Baço/citologiaRESUMO
We have recently demonstrated that hypertriglyceridemic (HTG) mice present both elevated body metabolic rates and mild mitochondrial uncoupling in the liver owing to stimulated activity of the ATP-sensitive potassium channel (mitoK(ATP)). Because lipid excess normally leads to cell redox imbalance, we examined the hepatic oxidative status in this model. Cell redox imbalance was evidenced by increased total levels of carbonylated proteins, malondialdehydes, and GSSG/GSH ratios in HTG livers compared to wild type. In addition, the activities of the extramitochondrial enzymes NADPH oxidase and xanthine oxidase were elevated in HTG livers. In contrast, Mn-superoxide dismutase activity and content, a mitochondrial matrix marker, were significantly decreased in HTG livers. Isolated HTG liver mitochondria presented lower rates of H(2)O(2) production, which were reversed by mitoK(ATP) antagonists. In vivo antioxidant treatment with N-acetylcysteine decreased both mitoK(ATP) activity and metabolic rates in HTG mice. These data indicate that high levels of triglycerides increase reactive oxygen generation by extramitochondrial enzymes that promote mitoK(ATP) activation. The mild uncoupling mediated by mitoK(ATP) increases metabolic rates and protects mitochondria against oxidative damage. Therefore, a biological role for mitoK(ATP) as a redox sensor is shown here for the first time in an in vivo model of systemic and cellular lipid excess.
Assuntos
Hipertrigliceridemia/metabolismo , Mitocôndrias Hepáticas/metabolismo , Canais de Potássio/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
Atherosclerosis is linked to a number of oxidative events ranging from low-density lipoprotein (LDL) oxidation to the increased production of intracellular reactive oxygen species (ROS). We have recently demonstrated that liver mitochondria isolated from the atherosclerosis-prone hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice have lower content of NADP(H)-linked substrates than the controls and, as consequence, higher sensitivity to oxidative stress and mitochondrial membrane permeability transition (MPT). In the present work, we show that oral supplementation with the antioxidants Mangifera indica L. extract (Vimang) or its main polyphenol mangiferin shifted the sensitivity of LDLr(-/-) liver mitochondria to MPT to control levels. These in vivo treatments with Vimang and mangiferin also significantly reduced ROS generation by both isolated LDLr(-/-) liver mitochondria and spleen lymphocytes. In addition, these antioxidant treatments prevented mitochondrial NAD(P)H-linked substrates depletion and NADPH spontaneous oxidation. In summary, Vimang and mangiferin spared the endogenous reducing equivalents (NADPH) in LDLr(-/-) mice mitochondria correcting their lower antioxidant capacity and restoring the organelle redox homeostasis. The effective bioavailability of these compounds makes them suitable antioxidants with potential use in atherosclerosis susceptible conditions.
Assuntos
Aterosclerose/tratamento farmacológico , Hipercolesterolemia/tratamento farmacológico , Mangifera , Mitocôndrias Hepáticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Xantonas/farmacologia , Animais , Antioxidantes/farmacologia , Aterosclerose/etiologia , Aterosclerose/metabolismo , Feminino , Hipercolesterolemia/complicações , Hipercolesterolemia/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , NADP/metabolismo , Fitoterapia , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genéticaRESUMO
We have previously proposed that hypercholesterolemic LDL receptor knockout (k/o) mice mitochondria possess a lower antioxidant capacity due to a large consumption of reducing equivalents from NADPH to sustain high rates of lipogenesis. In this work, we tested the hypothesis that this k/o mice mitochondrial oxidative stress results from the depletion of NADPH-linked substrates. In addition, the oxidative stress was further characterized by showing a lower mitochondrial GSH/GSSG ratio and a higher liver content of protein carbonyls as compared to controls. The activity of the antioxidant enzyme system glutathione reductase/peroxidase did not differ in k/o and control mitochondria. The faster spontaneous oxidation of endogenous NADPH in the k/o mitochondria was prevented by the addition of exogenous catalase, indicating that this oxidation is mediated by mitochondrially generated H(2)O(2). The higher rate of H(2)O(2) production was also prevented by the addition of exogenous isocitrate that maintains NADP fully reduced. The hypothesis that high rates of lipogenesis in the k/o cells decrease mitochondrial NADPH/NADP(+) ratio due to consumption of NADPH-linked substrates was supported by two findings: (i) oxygen consumption supported by endogenous NAD(P)H-linked substrates was slower in k/o than in control mitochondria, but was similar in the presence of exogenous isocitrate; (ii) in vivo treatment of k/o mice with sodium citrate/citric acid drinking solution for 2 weeks partially restored both the rate of oxygen consumption supported by NAD(P)H-linked substrates and the mitochondrial capacity to sustain reduced NADPH. In conclusion, the data demonstrate that the mitochondrial oxidative stress in hypercholesterolemic LDL receptor knockout mice is the result of a low content of mitochondrial NADPH-linked substrates in the intact animal that can be, at least in part, replenished by oral administration of citrate.
Assuntos
Hipercolesterolemia/enzimologia , Mitocôndrias Hepáticas/enzimologia , NADP/metabolismo , Estresse Oxidativo/genética , Receptores de LDL/genética , Animais , Ácido Cítrico/administração & dosagem , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glutationa Redutase/metabolismo , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismoRESUMO
BACKGROUND: Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria-dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time-consuming and inaccurate, and the latter is still limited by sample size. METHODS: We developed a rapid and reliable technique to detect cytochrome c release during drug-induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL-60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells. RESULTS: The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4-8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c. CONCLUSIONS: This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose.