Fiorella Biasi

Oxidative Stress and Cell Signalling

An increasing body of evidence from animal models, human specimens and cell lines points to reactive oxygen species as likely involved in the pathways, which convey both extracellular and intracellular signals to the nucleus, under a variety of pathophysiological conditions. Indeed, reactive oxygen species (ROS), in a concentration compatible with that detectable in human pathophysiology, appear able to modulate a number of kinases and phosphatases, redox sensitive transcription factors and genes. This type of cell signalling consistently implies the additional involvement of other bioactive molecules that stem from ROS reaction with cell membrane lipids. The present review aims to comprehensively report on the most recent knowledge about the potential role of ROS and oxidised lipids in signal transduction processes in the major events of cell and tissue pathophysiology. Among the lipid oxidation products of ROS-dependent reactivity, which appear as candidates for a signalling role, there are molecules generated by oxidation of cholesterol, polyunsaturated fatty acids and phospholipids, as well as lysophosphatidic acid and lysophospholipids, platelet activating factor-like lipids, isoprostanes, sphingolipids and ceramide.

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)

Oxysterols in the pathogenesis of major chronic diseases.

Pathological accumulation of 27-carbon intermediates or end-products of cholesterol metabolism, named oxysterols, may contribute to the onset and especially to the development of major chronic diseases in which inflammation, but also oxidative damage and to a certain extent cell death, are hallmarks and primary mechanisms of progression. Indeed, certain oxysterols exercise strong pro-oxidant and pro-inflammatory effects at concentrations detectable in the lesions typical of atherosclerosis, neurodegenerative diseases, inflammatory bowel diseases, age-related macular degeneration, and other pathological conditions characterized by altered cholesterol uptake and/or metabolism.

Activation of chloroform and related trihalomethanes to free radical intermediates in isolated hepatocytes and in the rat in vivo as detected by the ESR-spin trapping technique

When hepatocytes isolated from phenobarbital-induced rats were incubated with chloroform and the spin trap phenyl-t-butyl nitrone (PBN) under anaerobic conditions, a free radical-spin trap adduct was detectable by ESR spectroscopy. A similar incubation of hepatocytes in the presence of air resulted in an ESR signal that was eight times less intense than that seen under anaerobic conditions; incubation mixtures exposed to pure oxygen had no detectable adduct signal. A significant reduction in the signal intensity was also produced by the addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone and carbon monoxide, indicating that free radical formation depended upon the reductive metabolism of chloroform mediated by the mixed oxidase system. The origin of the CHCl3-derived free radical has been confirmed by using [13C]CHCl3, while the comparison between the ESR spectra obtained in the presence of deuterated chloroform (CDCl3) and bromodichloro-methane (CHBrCl2) suggests that the free radical derived from CHCl3 may be CHCl2. Free radical intermediates were also detected during the aerobic and anaerobic incubation of isolated hepatocytes with bromoform (CHBr3), and iodoform (CHI3). The intensity of the ESR signal obtained with the various trihalomethanes increases in the order CHCl3 less than CHBrCl2 less than CHBr3 less than CHI3. The formation of PBN-free radical adducts has also been observed in phenobarbital-induced rats in vivo when intoxicated with chloroform, bromoform or iodoform, suggesting that the reductive metabolism of trihalomethanes might be of relevance to their established toxicity in the whole animal.

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.

Inflammation-related gene expression by lipid oxidation-derived products in the progression of atherosclerosis.

Vascular areas of atherosclerotic development persist in a state of inflammation, and any further inflammatory stimulus in the subintimal area elicits a proatherogenic response; this alters the behavior of the artery wall cells and recruits further inflammatory cells. In association with the inflammatory response, oxidative events are also involved in the development of atherosclerotic plaques. It is now unanimously recognized that lipid oxidation-derived products are key players in the initiation and progression of atherosclerotic lesions. Oxidized lipids, derived from oxidatively modified low-density lipoproteins (LDLs), which accumulate in the intima, strongly modulate inflammation-related gene expression, through involvement of various signaling pathways. In addition, considerable evidence supports a proatherogenic role of a large group of potent bioactive lipids called eicosanoids, which derive from oxidation of arachidonic acid, a component of membrane phospholipids. Of note, LDL lipid oxidation products might regulate eicosanoid production, modulating the enzymatic degradation of arachidonic acid by cyclooxygenases and lipoxygenases; these enzymes might also directly contribute to LDL oxidation. This review provides a comprehensive overview of current knowledge on signal transduction pathways and inflammatory gene expression, modulated by lipid oxidation-derived products, in the progression of atherosclerosis.

Polyphenol Supplementation as a Complementary Medicinal Approach to Treating Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) comprises a group of idiopathic chronic intestinal inflammation syndromes that are very common in developed countries. It is characterized by intermittent episodes of clinical remission and relapse, with recurrent inflammatory injury that can lead to structural damage of the intestine. The uncontrolled intestinal immune response to bacterial antigens leads to the production of abundant cytokines and chemokines, by activated leukocytes and epithelial cells, which trigger inflammatory and oxidative reactions. The current treatment of IBD consists in long-term anti-inflammatory therapy that, however, does not exclude relapses and side effects, frequently resulting in surgical intervention. Polyphenols have been acknowledged to be anti-oxidant and anti-inflammatory and therefore, have been proposed as an alternative natural approach to prevent or treat chronic inflammatory diseases. Most studies have been in animal models of colitis, using chemical inducers or mice defective in anti-inflammatory mediators and in intestinal cell lines treated with pro-inflammatory cytokines or lipid oxidation products. These studies provide evidence that polyphenols can effectively modulate intestinal inflammation. They exert their effects by modulating cell signaling pathways, mainly activated in response to oxidative and inflammatory stimuli, and NF-kB is the principal downstream effector. Polyphenols may thus be considered able to prevent or delay the progression of IBD, especially because they reach higher concentrations in the gut than in other tissues. However, knowledge of the use of polyphenols in managing human IBD is still scanty, and further clinical studies should afford more solid evidence of their beneficial effects.

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.