Elena Chiarpotto

The lipid peroxidation end product 4-hydroxy-2,3-nonenal up-regulates transforming growth factor beta1 expression in the macrophage lineage: a link between oxidative injury and fibrosclerosis.

An increasing number of reports underscore the frequent association of fibrosclerotic diseases of lung, liver, arterial wall, brain, etc., with the accumulation of oxidatively modified lipids and proteins. A cause‐and‐effect relationship has been proposed between cellular oxidative damage and increased fibrogenesis based on the fact that experimental treatment with antioxidants either prevents or quenches the fibrotic process. With some peculiarities in the different organs, fibrosclerosis is essentially the result of the interaction of macrophages and extracellular matrix‐producing cells. The cross‐talk is mediated by fibrogenic cytokines, among which the most important appears to be transforming growth factor β1 (TGF‐β1). This report describes treatment of different types of macrophage, of both human and murine origin, with 4‐hydroxy‐2,3‐nonenal (HNE) a major aldehyde end product of membrane lipid oxidation found consistently to induce both mRNA expression and synthesis of TGF‐β1. Since increased HNE levels have been demostrated in the cirrhotic liver and in the oxidatively modified low‐density human lipoproteins associated with atherosclerosis, the up‐regulation of macrophage TGF‐β1 by HNE appears to be involved in the pathogenesis of these and similar diseases characterized by fibrosclerosis.—Leonarduzzi, G., Scavazza, A., Biasi, F., Ghiarpotto, E., Camandola, S., Vogl, S., Dargel, R., Poli, G. The lipid peroxidation end product 4‐hydroxy‐2,3‐nonenal up‐regulates transforming growth factor β1 expression in the macrophage lineage: a link between oxidative injury and fibrosclerosis. FASEB J. 11, 851–857 (1997)

Lipid oxidation products in cell signaling

The recent research on the impact that oxidative changes of biolipids could have in pathophysiology serves to explain how free radical-driven reactions not only are considered as mere toxicologic events, but also modulators of cell activity and function. Oxidatively modified low-density lipoproteins are known to affect various cellular processes by modulating various molecular pathways and signaling nuclear transcription. Among the lipid oxidation products detectable in ox-LDLs, and also in the atherosclerotic plaques, 4-hydroxynonenal has been widely investigated. This aldehyde was shown to upregulate AP-1 transcription factor, signaling through the MAP kinase pathway, with eventual nuclear localization and induction of a series of genes. Further, oxidation products of cholesterol and cholesterol esters, in ox-LDL are of similar interest, especially in relation to the pathogenesis of fibrosclerotic lesions of the arterial wall.

Autophagic Degradation Contributes to Muscle Wasting in Cancer Cachexia

Muscle protein wasting in cancer cachexia is a critical problem. The underlying mechanisms are still unclear, although the ubiquitin-proteasome system has been involved in the degradation of bulk myofibrillar proteins. The present work has been aimed to investigate whether autophagic degradation also plays a role in the onset of muscle depletion in cancer-bearing animals and in glucocorticoid-induced atrophy and sarcopenia of aging. The results show that autophagy is induced in muscle in three different models of cancer cachexia and in glucocorticoid-treated mice. In contrast, autophagic degradation in the muscle of sarcopenic animals is impaired but can be reactivated by calorie restriction. These results further demonstrate that different mechanisms are involved in pathologic muscle wasting and that autophagy, either excessive or defective, contributes to the complicated network that leads to muscle atrophy. In this regard, particularly intriguing is the observation that in cancer hosts and tumor necrosis factor α-treated C2C12 myotubes, insulin can only partially blunt autophagy induction. This finding suggests that autophagy is triggered through mechanisms that cannot be circumvented by using classic upstream modulators, prompting us to identify more effective approaches to target this proteolytic system.

Oxysterol mixtures prevent proapoptotic effects of 7-ketocholesterol in macrophages: implications for proatherogenic gene modulation

Oxysterols are common components of oxidized low‐density lipoprotein and accumulate in the core of fibrotic plaques as a mixture of cholesterol and cholesteryl ester oxidation products. The proapoptotic effects of a biologically representative mixture of oxysterols was compared with equimolar amounts of 7‐ketocholesterol and unoxidized cholesterol. The oxysterol mixture in a concentration range actually detectable in hypercholesterolemic patients did not stimulate programmed cell death in cultivated murine macrophages. Unoxidized cholesterol also produced no effect. By contrast, when given alone, 7‐ketocholesterol strongly stimulated the mitochondrial pathway of apoptosis with cytochrome c release, caspase‐9 activation, and eventually caspase‐3 activation. Subsequent experiments showed that when 7‐ketocholesterol was administered to cells together with another oxysterol, namely 7βOH‐cholesterol, the strong proapoptotic effect of 7‐ketocholesterol was markedly attenuated. As regards the mechanism underlying this quenching, we found that the combined oxysterol treatment counteracted the ability of 7‐ ketocholesterol, when administered alone, to strongly up‐regulate the steady‐state levels of reactive oxygen species (ROS) without interfering with sterol uptake. Furthermore, this increase in intracellular ROS appeared to be responsible for the up‐regulation of proapoptotic factor, p21, after treatment with 7‐ketocholesterol but not in cells challenged with the oxysterol mixture. Competition among oxysterols, apparently at the level of NADPH oxidase, diminishes the ROS induction and direct toxicity that is evoked by specific oxysterols. As a consequence, a more subtle gene modulation by oxysterols becomes facilitated in vascular cells.

Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes

Treatment of isolated hepatocytes from 3-methylcholanthrene induced rats with 1 mM paracetamol has been found to greatly decrease cellular reduced glutathione (GSH) content and to promote lipid peroxidation, evaluated as malonaldehyde (MDA) production and conjugated diene absorbance. A similar dosing of hepatocytes from phenobarbital-induced or normal rats is ineffective in that respect. On the other hand, the aspecific stimulation of the cytochrome P-450-mediated paracetamol activation due to acetone addition further increases GSH depletion as well as MDA production. Isolated hepatocytes with basal low GSH content are also more susceptible to paracetamol-induced lipid peroxidation, indicating that the rate of the drug metabolism and the cellular GSH content are critical factors in the determination of such peroxidative attack. In isolated mouse liver cells paracetamol does not require preliminary cytochrome P-450 induction to stimulate MDA formation, even at concentrations ineffective in rat cells. However, 5 mM paracetamol, despite a great depletion of cellular GSH content, does not promote MDA formation either in the rat or in the mouse hepatocytes. This effect may be due to the ability of paracetamol to scavenge lipid peroxides under defined conditions, as tested in various lipid peroxidizing systems. Membrane leakage of lactate dehydrogenase (LDH) is evident in paracetamol treated cells undergoing lipid peroxidation, but not when MDA formation is inhibited by high doses of the drug or by addition of anti-oxidants such as alpha-tocopherol and diphenylphenylenediamine (DPPD). Nevertheless in these conditions the covalent binding of activated paracetamol metabolites is not affected, suggesting that lipid peroxidation might play a role in the pathogenesis of liver damage following paracetamol overdose.

Up-regulation of the fibrogenic cytokine TGF-beta1 by oxysterols: a mechanistic link between cholesterol and atherosclerosis.

Deposition of blood cholesterol in the subendothelial space of major arteries is a main feature of the fibrotic plaque as well as the key event in the progression of atherosclerosis. However, the mechanisms by which cholesterol triggers and sustains the fibrotic degeneration of blood arteries remain undefined. The results reported here indicate that a biologically representative mixture of oxysterols, 27‐carbon products of cholesterol oxidation, rather than an individual oxysterol at the same concentration, exerts a strong profibrogenic effect when taken up by macrophages. Incubation of murine and human macrophages with such oxysterol mixture markedly promotes both expression and synthesis of one of the most potent proinflammatory and fibrogenic cytokines, transforming growth factor β1 (TGF‐β1). By contrast, no effect on TGF‐β1 expression and synthesis was found with unoxidized cholesterol. Macrophage uptake of cholesterol and conversion to foam cells is a hallmark of early atherogenesis, but it appears that cholesterol oxidation products, as well as other low‐density lipoprotein oxidation products, are required to generate a proper fibrogenic stimulus that is not fulfilled by cholesterol deposition alone.

Calorie Restriction and Dietary Restriction Mimetics: A Strategy for Improving Healthy Aging and Longevity

Improvements in health care have increased human life expectancy in recent decades, and the elderly population is thus increasing in most developed countries. Unfortunately this still means increased years of poor health or disability. Since it is not yet possible to modify our genetic background, the best anti-aging strategy is currently to intervene on environmental factors, aiming to reduce the incidence of risk factors of poor health. Calorie restriction (CR) with adequate nutrition is the only non-genetic, and the most consistent non-pharmacological intervention that extends lifespan in model organisms from yeast to mammals, and protects against the deterioration of biological functions, delaying or reducing the risk of many age-related diseases. The biological mechanisms of CRs beneficial effects include modifications in energy metabolism, oxidative stress, insulin sensitivity, inflammation, autophagy, neuroendocrine function and induction of hormesis/xenohormesis response. The molecular signalling pathways mediating the anti-aging effect of CR include sirtuins, peroxisome proliferator activated receptor G coactivator-1α, AMP-activated protein kinase, insulin/insulin growth factor-1, and target of rapamycin, which form a pretty interacting network. However, most people would not comply with such a rigorous dietary program; research is thus increasingly aimed at determining the feasibility and efficacy of natural and/or pharmacological CR mimetic molecules/ treatments without lowering food intake, particularly in mid- to late-life periods. Likely candidates act on the same signalling pathways as CR, and include resveratrol and other polyphenols, rapamycin, 2-deoxy-D-glucose and other glycolytic inhibitors, insulin pathway and AMP-activated protein kinase activators, autophagy stimulators, alpha-lipoic acid, and other antioxidants.

Glutathione depletion induces apoptosis of rat hepatocytes through activation of protein kinase C novel isoforms and dependent increase in AP-1 nuclear binding

Treatment of isolated rat hepatocytes with the glutathione depleting agents L-buthionine-S,R-sulfoximine or diethylmaleate reproduced various cellular conditions of glutathione depletion, from moderate to severe, similar to those occurring in a wide spectrum of human liver diseases. To evaluate molecular changes and possible cellular dysfunction and damage consequent to a pathophysiologic level of GSH depletion, the effects of this condition on protein kinase C (PKC) isoforms were investigated, since these are involved in the intracellular specific regulatory processes and are potentially sensitive to redox changes. Moreover, a moderate perturbation of cellular redox state was found to activate novel PKC isoforms, and a clear relationship was shown between novel kinase activation and nuclear binding of the redox-sensitive transcription factor, activator protein-1 (AP-1). Apoptotic death of a significant number of cells, confirmed in terms of internucleosomal DNA fragmentation was a possible effect of these molecular reactions, and was triggered by a condition of glutathione depletion usually detected in human liver diseases. Finally, the inhibition of novel PKC enzymatic activity in cells co-treated with rottlerin, a selective novel kinase inhibitor, prevented glutathione-dependent novel PKC up-regulation, markedly moderated AP-1 activation, and protected cells against apoptotic death. Taken together, these findings indicate the existence of an apoptotic pathway dependent on glutathione depletion, which occurs through the up-regulation of novel PKCs and AP-1.

Oxidative damage in human liver transplantation

The aim of this study was to evaluate oxygen-dependent hepatic reperfusion injury in humans following orthotopic liver transplantation. To this end, a number of blood indices of impaired tissue redox balance were monitored in 19 adult patients for 3 weeks after liver transplantation. Both red cell malonaldehyde and plasma lipid peroxides increased significantly soon after organ reperfusion. This finding was consistently accompanied by decreased plasma vitamin E and red cell total glutathione. A peak of oxidative stress, as measured by the parameters monitored, was evident within 24 h after reperfusion, together with a maximum expression of cytolysis, as measured by plasma alanine aminotransferase. The occurrence of redox imbalance after hepatic reperfusion was shown to be linearly related to irreversible cell damage. As regards the low plasma levels of the two antioxidants after reperfusion, only that of vitamin E appeared statistically related to oxidative stress. With the background of an increasing body of proof, mainly from animal models, the involvement of toxic oxygen metabolites in hepatic cytolysis following orthotopic liver transplantation appears likely. The statistical correlation among the markers of redox imbalance monitored indicates their combined use in further investigation.

Lipid peroxidation in human diseases: Evidence of red cell oxidative stress after circulatory shock

Erythrocytes obtained from human patients with circulatory shock of different aetiology consistently showed a strong increase in lipid peroxidation-derived aldehydes in comparison with red cells of normal adults. The highly toxic compound 4-hydroxynonenal has been recovered exclusively in the erythrocytes of the patients.