Bruno Paim

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Mild Mitochondrial Uncoupling Decreases Experimental Atherosclerosis, A Proof of Concept

Journal of Atherosclerosis and Thrombosis, 2021

The official journal of the Japan Atherosclerosis Society and the Asian Pacific Society of Athero... more The official journal of the Japan Atherosclerosis Society and the Asian Pacific Society of Atherosclerosis and Vascular Diseases Original Article 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 dietinduced 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. duration) to influence key cells, cell compartments and pathways leading to atherosclerosis 3, 4). Furthermore, the treatment with antioxidants can disturb certain basic aspects of cell functionality since physiological levels of the oxidants are important for signaling proliferation, differentiation, migration, angiogenesis, and other processes 5). Mitochondrial respiration is an important source of intracellular oxidants 6). Mitochondria present an efficient antioxidant enzyme system and may decrease the generation of the oxidants through an increase in the respiration rate due to mild uncoupling 7, 8). Mild Copyright©2022 Japan Atherosclerosis Society This article is distributed under the terms of the latest version of CC BY-NC-SA defined by the Creative Commons Attribution License.

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$Research paper thumbnail of {"__content__"=>"Mangifera indica L. extract (Vimang) reduces plasma and liver cholesterol and leucocyte oxidative stress in hypercholesterolemic LDL receptor deficient mice.", "sup"=>{"__content__"=>"®"}}$

{"__content__"=>"Mangifera indica L. extract (Vimang) reduces plasma and liver cholesterol and leucocyte oxidative stress in hypercholesterolemic LDL receptor deficient mice.", "sup"=>{"__content__"=>"®"}}

Cell biology international, Jan 10, 2018

Cardiovascular diseases are major causes of death worldwide. Beyond the classical cholesterol ris... more 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 two 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 chan...

Th-P15:71 Reduced generation of reactive oxygen species by circulating lymphomononuclear cells and endothelial cellular model in post-prandial state

Atherosclerosis Supplements, 2006

Spontaneous experimental atherosclerosis in hypercholesterolemic mice advances with ageing and correlates with mitochondrial reactive oxygen species

Experimental gerontology, Jan 14, 2017

Ageing and atherosclerosis are associated with oxidative stress. Mitochondrial redox function dec... more 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.

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Correlation between Mitochondrial Reactive Oxygen and Severity of Atherosclerosis

Oxidative medicine and cellular longevity, 2016

Atherosclerosis has been associated with mitochondria dysfunction and damage. Our group demonstra... more 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...

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Abstract: P278 ACTIVATED MITOCHONDRIAL ATP-SENSITIVE K+ CHANNELS IN HYPERTRIGLYCERIDEMIC MICE PROMOTES CHANGES IN LIVER REDOX STATE AND BODY METABOLIC RATES

Atherosclerosis Supplements, 2009

L 016 Positive Correlation between Severity of Atherosclerosis and Liver Mitochondrial Oxidative Stress

Atherosclerosis Supplements, 2009

156 the Reduction in HDL Receptor Levels in Macrophages is Related to Ros Generation Induced by Advanced Glycated Albumin

Atherosclerosis Supplements, 2011

Atherosclerosis Supplements 12, no. 1 (2011) 13-184 35 3. Silencing of nampt markedly increased l... more Atherosclerosis Supplements 12, no. 1 (2011) 13-184 35 3. Silencing of nampt markedly increased lipid accumulation in macrophages as shown by increased protein levels of the lipid droplet marker adipose differentiation-related protein (ADRP). A specific inhibitor of nampt enzymatic activity, increased ADRP and cholesterol levels in oxLDL stimulated macrophages, and enhanced the binding of acetylated LDL in these cells. 4. Inhibition of nampt down-regulated the relative amount of cholesterol efflux in oxLDL exposed macrophages. Our data suggest that the regulation of nampt in macrophages is closely linked to inflammatory and pro-atherogenic stimuli, potentially mediating counteracting effects on lipid accumulation and foam cell formation. These findings suggest a rather complex role of nampt in atherogenesis, potentially mediating both adaptive and maladaptive responses.

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P390 Mitochondrial Independent Apoptosis is Stimulated in Mononuclear Cells from Hypertriglyceridemic Mice

Atherosclerosis Supplements, 2010

L 007 Aging Enhances the Mitochondrial Oxidative Stress in Atherosclerosis-Prone, Hypercholesterolemic LDL Receptor Knockout Mice

Atherosclerosis Supplements, 2007

Food restriction by intermittent fasting induces diabetes and obesity and aggravates spontaneous atherosclerosis development in hypercholesterolaemic mice

British Journal of Nutrition, 2013

Different regimens of food restriction have been associated with protection against obesity, diab... more 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 thead libitum-fed mice. Accordingly, plasma levels of leptin were 50 % higher in the IF mice than in thead 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 level...

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Mangifera indica L. extract (Vimang®) and its main polyphenol mangiferin prevent mitochondrial oxidative stress in atherosclerosis-prone hypercholesterolemic mouse

Pharmacological Research, 2008

Atherosclerosis is linked to a number of oxidative events ranging from low-density lipoprotein (L... more 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.

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Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats

Molecular and Cellular Biochemistry, 2010

This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as ev... more This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca 2? levels, and interleukin (IL)-1b expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 lg ml-1 single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca 2? , and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca 2? levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1b but IL-6 and-10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

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Inhibition of Macrophage Oxidative Stress Prevents the Reduction of ABCA-1 Transporter Induced by Advanced Glycated Albumin

Lipids, 2012

We investigated the role of aminoguanidine and benfotiamine on the inhibition of reactive oxygen ... more We investigated the role of aminoguanidine and benfotiamine on the inhibition of reactive oxygen species (ROS) generation in macrophages induced by advanced glycated albumin (AGE-albumin) and its relationship with cell cholesterol homeostasis, emphasizing the expression of the ATP binding cassette transporter A-1 (ABCA-1). AGE-albumin was made by incubating fatty acid-free albumin with 10 mM glycolaldehyde. ROS production and ABCA-1 protein level were determined by flow cytometry in J774 macrophages treated along time with control (C) or AGE-albumin alone or in the presence of aminoguanidine or benfotiamine. Mitochondrial function was evaluated by oxygraphy. Compared to C-albumin, AGE-albumin increased ROS production in macrophages, which was ascribed to the activities of NADPH oxidase and of the mitochondrial system. Mitochondrial respiratory chain activity was reduced in cells incubated with AGE-albumin. ROS generation along time was associated with the reduction in macrophage ABCA-1 protein level. Aminoguanidine prevented ROS elevation and restored the ABCA-1 content in macrophages; on the other hand, benfotiamine that promoted a lesser reduction in ROS generation was not able to restore ABCA-1 levels. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus.

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Mitochondrial ATP-sensitive K+ channels as redox signals to liver mitochondria in response to hypertriglyceridemia

Free Radical Biology and Medicine, 2009

We have recently demonstrated that hypertriglyceridemic (HTG) mice present both elevated body met... more 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.

Oxidative stress in hypercholesterolemic LDL (low-density lipoprotein) receptor knockout mice is associated with low content of mitochondrial NADP-linked substrates and is partially reversed by citrate replacement

Free Radical Biology and Medicine, 2008

We have previously proposed that hypercholesterolemic LDL receptor knockout (k/o) mice mitochondr... more 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.

S12.36 Oxidative stress in hypercholesterolemic ldl receptor knockout mice: Role of mitochondrial nadp-linked substrates and intracellular calcium levels

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008

IF 1 inhibits the reverse activity of the ATP synthase, limiting mitochondrial ATP consumption du... more IF 1 inhibits the reverse activity of the ATP synthase, limiting mitochondrial ATP consumption during pathological states (e.g. ischaemia). IF 1 expression has also been reported to be upregulated in neoplastic cell lines. Thus, IF 1 may play a fundamental role in the S84 ABSTRACTS / Biochimica et Biophysica Acta 1777 (2008) S2-S111

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Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane

Cytometry Part A, 2006

Background:Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is us... more 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...

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