Milagros Rocha

Oxidative stress, endothelial dysfunction and atherosclerosis.

This review focuses on the role of oxidative processes in atherosclerosis and the cardiovascular diseases (CVD) that can arise as a result. Atherosclerosis represents a state of heightened oxidative stress characterized by lipid and protein oxidation in the vascular wall. Overproduction of reactive oxygen species (ROS) under pathophysiologic conditions forms an integral part of the development of CVD, and in particular atherosclerosis. Endothelial dysfunction, characterized by a loss of nitric oxide (NO) bioactivity, occurs early on in the development of atherosclerosis, and determines future vascular complications. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are not clear, oxidative stress seems to play an important role. In general, ROS are essential to the functions of cells, but adequate levels of antioxidant defenses are required in order to avoid the harmful effects of excessive ROS production. In this review, we will provide a summary of the cellular metabolism of reactive oxygen species (ROS) and its role in pathophysiological processes such as atherosclerosis; and currently available antioxidants and possible reasons for their efficacy and inefficacy in ameliorating oxidative stress-mediated diseases.

Role of Free Radicals in Sepsis: Antioxidant Therapy

Severe sepsis leading to shock is the principal cause of death in intensive care units. It is a systemic inflammatory response caused by excessive secretion of pro-inflammatory mediators, such as tumor necrosis factor-alpha (TNFalpha) and reactive oxygen species (ROS), mainly induced by endotoxin (a major component of the Gram-negative bacterial outer membrane). Immune cells use ROS in order to support their functions and need adequate levels of antioxidant defenses to avoid harmful effects of an excessive ROS production. In addition, nitric oxide (NO) is thought to play a key role in the pathogenesis of sepsis and in the development of multiple organ failure. This article discusses the toxic effects of endotoxin, paying particular attention to cardiovascular damage. It continues by analysing the mechanism by which endotoxin is recognized by specific cells of the immune system, and the pathway leading to nuclear factor-kappaB (NF-kappaB) activation and pro-inflammatory gene transcription. In relation to this process, this review focuses on the involvement of reactive oxygen and nitrogen species. Finally, the protective role of antioxidants against homeostatic disturbances such as those caused by endotoxin toxicity, their potential clinical use and the effects on the redox state of the immune cells is discussed.

Oxidative Stress and Mitochondrial Dysfunction in Atherosclerosis: Mitochondria-Targeted Antioxidants as Potential Therapy

Chronic and acute overproduction of reactive oxygen species (ROS) under pathophysiologic conditions forms an integral part of the development of cardiovascular diseases (CVD), and in particular atherosclerosis. These ROS are released from different sources, such as xanthine oxidase, lipoxygenase, nicotinamide adenine dinucleotide phosphate oxidase, the uncoupling of nitric oxide synthase and, in particular, mitochondria. Endothelial dysfunction, characterized by a loss of nitric oxide (NO) bioactivity, occurs early on in the development of atherosclerosis, and determines future vascular complications. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are not clear, oxidative stress seems to play an important role. In general, ROS are essential to cell function, but adequate levels of antioxidant defenses are required in order to avoid the harmful effects of excessive ROS production. Mitochondrial oxidative stress damage and dysfunction contribute to a number of cell pathologies that manifest themselves through a range of conditions. This review considers the process of atherosclerosis from a mitochondrial perspective, and assesses strategies for the targeted delivery of antioxidants to mitochondria that are currently under development. We will provide a summary of the following areas: the cellular metabolism of reactive oxygen species (ROS) and its role in pathophysiological processes such as atherosclerosis; currently available antioxidants and possible reasons for their efficacy and inefficacy in ameliorating oxidative stress-mediated diseases; and recent developments in mitochondrially-targeted antioxidants that concentrate on the matrix-facing surface of the inner mitochondrial membrane in order to protect against mitochondrial oxidative damage, and their therapeutic potential as a treatment for atherosclerosis.

Complex I Dysfunction and Tolerance to Nitroglycerin: An Approach Based on Mitochondrial-Targeted Antioxidants

Nitroglycerin (GTN) tolerance was induced in vivo (rats) and in vitro (rat and human vessels). Electrochemical detection revealed that the incubation dose of GTN (5×10−6 mol/L) did not release NO or modify O2 consumption when administered acutely. However, development of tolerance produced a decrease in both mitochondrial O2 consumption and the Km for O2 in animal and human vessels and endothelial cells in a noncompetitive action. GTN tolerance has been associated with impairment of GTN biotransformation through inhibition of aldehyde dehydrogenase (ALDH)-2, and with uncoupling of mitochondrial respiration. Feeding rats with mitochondrial-targeted antioxidants (mitoquinone [MQ]) and in vitro coincubation with MQ (10−6 mol/L) or glutathione (GSH) ester (10−4 mol/L) prevented tolerance and the effects of GTN on mitochondrial respiration and ALDH-2 activity. Biotransformation of GTN requires functionally active mitochondria and induces reactive oxygen species production and oxidative stress within this organelle, as it is inhibited by mitochondrial-targeted antioxidants and is absent in HUVEC&rgr;0 cells. Experiments analyzing complex I–dependent respiration demonstrate that its inhibition by GTN is prevented by mitochondrial-targeted antioxidants. Furthermore, in presence of succinate (10×10−3 mol/L), a complex II electron donor added to bypass complex I–dependent respiration, GTN-treated cells exhibited O2 consumption rates similar to those of controls, thus suggesting that complex I was affected by GTN. We propose that, following prolonged treatment with GTN in addition to ALDH-2, complex I is a target for mitochondrially generated reactive oxygen species. Our data also suggest a role for mitochondrial-targeted antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use.

Inhibition of Mitochondrial Function by Efavirenz Increases Lipid Content in Hepatic Cells

Efavirenz (EFV) is a non‐nucleoside reverse transcriptase inhibitor (NNRTI) widely used in human immunodeficiency virus (HIV) infection therapy. It has been associated with hepatotoxic effects and alterations in lipid and body fat composition. Given the importance of the liver in lipid regulation, we have evaluated the effects of clinically used concentrations of EFV on the mitochondria and lipid metabolism of human hepatic cells in vitro. Mitochondrial function was rapidly undermined by EFV to an extent that varied with the concentration employed; in particular, respiration and intracellular adenosine triphosphate (ATP) levels were reduced whereas reactive oxygen species (ROS) production increased. Results in isolated mitochondria suggest that the mechanism responsible for these actions was a specific inhibition of complex I of the respiratory chain. The reduction in energy production triggered a compensatory mechanism mediated by the enzyme adenosine monophosphate–activated protein kinase (AMPK), the master switch of cellular bioenergetics. Fluorescence and nuclear magnetic resonance demonstrated a rapid intracellular increase of neutral lipids, usually in the form of droplets. This was prevented by the AMPK inhibitor compound C and by removal of fatty acids from the culture medium. These effects were not reproduced by Nevirapine, another NNRTI. EFV is clinically coadministered with two nucleoside reverse transcriptase inhibitors. Evaluation of one of the most common combination, EFV/Lamivudine/Abacavir, revealed that the effects of EFV on ROS production were enhanced. Conclusion: Clinical concentrations of EFV induce bioenergetic stress in hepatic cells by acutely inhibiting mitochondrial function. This new mechanism of mitochondrial interference leads to an accumulation of lipids in the cytoplasm that is mediated by activation of AMPK. HEPATOLOGY 2010

N-acetylcysteine protects mice from lethal endotoxemia by regulating the redox state of immune cells.

The excessive production of reactive oxygen species (ROS) associated with inflammation leads to oxidative stress, which is involved with the high mortality from several diseases such as endotoxic shock and can be controlled to a certain degree by antioxidants. The immune cells use ROS in order to support their functions and, therefore, need adequate levels of antioxidant defenses in order to avoid the harmful effect of an excessive ROS production. In the present work, the effect of the administration of the antioxidant N-acetylcysteine (NAC) on the redox state of peritoneal macrophages and lymphocytes from mice with lethal endotoxic shock (100 mg/kg i.p. of lipopolysaccharide, LPS), was studied. In both types of immune cells at 0, 2, 4, 12 and 24 h after LPS injection, an increase of ROS, of the proinflammatory cytokine tumor necrosis factor alpha (TNFα), the lipid peroxidation (malonaldehyde levels, MDA), inducible nitric oxide synthase (iNOS) expression and the oxidized/reduced glutathione (GSSG/GSH) ratio, as well as a decrease of enzymatic antioxidant defenses, such as superoxide dismutase (SOD) and catalase (CAT) activity, was observed. The injection of NAC (150 mg/kg i.p. at 30 min after LPS injection) decreased the ROS, the TNFα the MDA levels, iNOS expression and the GSSG/GSH ratio, and increased the antioxidant defenses in both macrophages and lymphocytes. Moreover, the NAC treatment prevented the activation of nuclear translocation of the nuclear factor κB (NF-κB), which regulates ROS, inflammatory cytokines and antioxidant levels. Our present results provide evidence that both cell types have a relevant role in the pathogenesis of endotoxic shock, and that NAC, by improving the redox state of these immune cells, could increase mouse survival. Thus, antioxidants could offer an alternative treatment of human endotoxic shock.

Regulation of macrophage function by the antioxidant N-acetylcysteine in mouse-oxidative stress by endotoxin

Changes in several functions of peritoneal macrophages from mice with oxidative stress caused by intraperitoneal injection of endotoxin (Escherichia coli lipopolysaccharide, LPS) (100 mg/kg), and associated with a high production of reactive oxygen species (ROS), have been observed in our previous studies. Antioxidants such as N-acetylcysteine (NAC) are free radical scavengers that improve and modulate the immune response, especially in oxidative stress situations. Therefore, in the present work, we have studied the effects of the administration of NAC (150 mg/kg i.p.) on different functions of peritoneal macrophages from Swiss mice suffering that oxidative stress, caused by LPS (100 mg/kg). NAC was injected 30 min after LPS injection, and the peritoneal macrophages were obtained at 2, 4, 12, and 24 h after endotoxin injection. The following functions, key stages of the phagocytic process, were studied: adherence to substrate, chemotaxis, ingestion of particles, and production of ROS (reactive oxygen species), as well as tumor necrosis factor (TNFalpha) release. The decrease in chemotaxis and the increase in adherence, ingestion, superoxide anion production, and TNFalpha release shown by macrophages from animals with oxidative stress were counteracted by NAC injection. These data suggest that NAC administration may be useful for the treatment of oxidative stress-linked endotoxic shock, modulating the function of macrophages, specifically in decreasing the production of ROS and of inflammatory cytokines such as TNFalpha.

Oxidative Stress and Mitochondrial Dysfunction in Sepsis: A Potential Therapy with Mitochondria-Targeted Antioxidants

Sepsis and septic shock are the major causes of death in intensive care units. The prevalent hypothesis regarding the mechanisms of sepsis and septic shock indicates that this syndrome is caused by an excessive defensive and inflammatory response characterised by massive increases in reactive oxygen species (ROS), nitric oxide (NO) and inflammatory cytokines. The consequences of these syndromes are systemic damage to the vascular endothelium, impaired tissue and a compromised whole body respiration, glutathione depletion and mitochondrial respiratory dysfunction with diminished levels of ATP and O(2) consumption. In general, ROS are essential to the functions of cells and particularly immune cells, but adequate levels of antioxidant defenses are required to protect against the harmful effects of excessive ROS production. Mitochondrial oxidative stress damage and dysfunction contribute to a number of cell pathologies that manifest themselves in a range of conditions, including sepsis. This review considers the process of sepsis from a mitochondrial perspective, discussing strategies for the targeted delivery of antioxidants to mitochondria currently under development. We will provide a summary of the following areas: the cellular metabolism of ROS and its role in pathophysiological processes such as sepsis; currently available antioxidants and possible reasons for their efficacy and inefficacy in ameliorating oxidative stress-mediated diseases; and recent developments in antioxidants that target the matrix-facing surface of the inner mitochondrial membrane in order to protect against mitochondrial oxidative damage, and their therapeutic potential as a treatment for sepsis.

Oxidative Stress and Mitochondrial Dysfunction in Type 2 Diabetes

Diabetes is a chronic disease and as a consequence of the overproduction of reactive oxygen species (ROS), is related with oxidative stress. There are different sources of ROS, of which mitochondria is the main one. Oxidative stress seems to play an important role in mitochondria- mediated disease processes, though the exact molecular mechanisms responsible remain elusive. There are evidences which supports the idea that impaired mitochondrial function is a cause of the insulin insensitivity in different type of cells that arised as a result of an insufficient supply of energy or defects in the insulin signaling pathway. ROS are generally necessary for the proper functioning of the cell, but excessive ROS production can be harmful, which makes antioxidant defenses essential. Moreover, some substances with antioxidant properties, such as vitamin C or vitamin E, erradicate the oxidative stress associated with diabetes. The results of clinical trials employing anti-oxidative stress reagents in patients with diabetes are contradictory, which may be a result of inadequate study design or selected targets. This review considers the process of diabetes from a mitochondrial perspective, and describes the role of autophagy in the development of diabetes. Furthermore, we discuss the possible beneficial effects of selectively targeting antioxidants to mitochondria as a strategy for modulating mitochondrial function in diabetes.

Mitochondrial Complex I Impairment in Leukocytes from Polycystic Ovary Syndrome Patients with Insulin Resistance

CONTEXT Insulin resistance is a feature of polycystic ovary syndrome (PCOS) and is related to mitochondrial function. OBJECTIVE Our objective was to assess mitochondrial function by evaluating mitochondrial oxygen (O(2)) consumption, reactive oxygen species (ROS) production, levels of glutathione (GSH), the oxidized glutathione/GSH ratio, TNFalpha levels, and membrane potential. Additionally, we have evaluated mitochondrial complex I as a target of the oxidative stress responsible for PCOS in polymorphonuclear cells. DESIGN AND SETTING This was a prospective controlled study conducted in an academic medical center. PATIENTS The study population consisted of 20 lean reproductive-age women with PCOS and 20 body composition-matched controls. MAIN OUTCOME MEASURES We evaluated mitochondrial O(2) consumption using the Clark-type O(2) electrode; levels of ROS, GSH, and membrane potential by means of fluorescence microscopy; TNFalpha levels by ELISA; and complex I activity by spectrophotometric assay. RESULTS An impairment in mitochondrial function was observed in PCOS patients, evident by a decrease in mitochondrial O(2) consumption; an increase in ROS production, oxidized glutathione/GSH ratio, and TNFalpha levels; a drop in GSH levels; and an undermining of membrane potential. Furthermore, an impairment of mitochondrial complex I was identified. CONCLUSION This study supports the hypothesis of an association between insulin resistance and an impaired mitochondrial oxidative metabolism. We also propose that the oxidative stress responsible for PCOS takes place at complex I. These abnormalities may contribute to the increased risk of type 2 diabetes among women with PCOS.