Gabriella Leonarduzzi

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.

HNE interacts directly with JNK isoforms in human hepatic stellate cells.

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.

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.

On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis

Previous studies have suggested a possible involvement of free radical reactions in the pathogenesis of cholestatic liver injury as well as in the modulation of hepatic fibrogenesis. In this study we investigated whether lipid peroxidation is involved in the development of chronic liver damage induced by long-standing cholestasis. For this purpose we have used the rat model of bile duct ligation (BDL), which leads to liver fibrosis and cirrhosis. Using this model we observed that the development of chronic liver damage was associated with the onset of lipid peroxidation, as pointed out by detection of carbonyl compounds, 4-hydroxynonenal (HNE) and malondialdehyde (MDA), in BDL livers and of fluorescent adducts between MDA and serum proteins. Lipid peroxidation was a relatively late event (starting after 1-2 weeks of BDL) and was unrelated to the early development of liver necrosis and cholestasis (already evident after 72 h after BDL). A positive significant linear correlation between the kinetic of infiltration of neutrophils and of a monocyte/macrophage population in BDL livers and MDA and HNE generation in the same organs is presented, indicating a close link between lipid peroxidation and the activation of inflammatory cells. We also observed that a positive linear correlation exists between collagen deposition in these livers and hepatic production of MDA and HNE. This event, which is accompanied by an increase in the number of fat storing cells (FSC, the cells that produce collagen in fibrotic liver), suggests that lipid peroxidation in this model may contribute to stimulate collagen synthesis by proliferating FSC.

Oxidized products of cholesterol: dietary and metabolic origin, and proatherosclerotic effects (review)

Cholesterol oxidation products, termed oxysterols, are increasingly considered of potential interest in the pathogenesis of atherosclerotic lesions. Of dietary or endogenous origin, oxysterols may occur in significant amounts in low density lipoprotein (LDL) particles, especially in hypercholesterolemic subjects. They likely contribute to the uptake of modified LDL by scavenger receptors and some of them finally accumulate in the subintimal space of major arteries; here cholesterol oxides may favor the perpetuation of a chronic inflammatory state, through their ability to trigger irreversible damage of vascular cells with consequent activation of phagocytes. Furthermore, practically all oxysterols of major pathophysiologic interest have been shown to markedly up-regulate expression and synthesis of adhesion molecules, inflammatory cytokines and chemokines. Cholesterol oxidation thus appears to be an important biochemical pathway through which it exerts toxic, inflammatory and finally atherogenic effects.

Vitamin E dietary supplementation inhibits transforming growth factor β1 gene expression in the rat liver

Overexpression of transforming growth factor β1 (TGFβ1) and increased transcription of pro‐collagen type I, are known to represent major events implicated in the development of liver fibrosis under either experimental or clinical conditions. Here we report that long‐term dietary vitamin E supplementation in animals undergoing an experimental model of liver fibrosis (induced by chronic treatment of rats with carbon tetrachloride) results in a net inhibition of both hepatic TGFβ1 and α2 (I) procollagen mRNA levels. Moreover, of striking interest is the observation that vitamin E supplementation per so down‐modulates basal levels of TGFβ1 mRNA in the liver of untreated animals, suggesting that a dietary regimen rich in vitamin E may potentially interfere with both the initiation and progression of the fibrosclerotic processes.

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.

Cholesterol oxidation products in the vascular remodeling due to atherosclerosis

Like the other oxidation products of the lipid moiety of plasma low density lipoproteins (LDL), cholesterol oxidation products are consistently found within the characteristic lesions of atherosclerosis, both in experimental animals and in man. A growing bulk of evidence suggests that oxysterols make a significant contribution to the vascular remodeling that occurs in atherosclerosis, being involved in various key steps of this complex process: endothelial cell dysfunction, adhesion of circulating blood cells, foam cell and fibrous cap formation, modulation of the extracellular matrix (ECM), vascular cell apoptosis and plaques instability. Moreover, oxysterols have been demonstrated to be at least one or two orders of magnitude more reactive than unoxidized cholesterol in exerting pro-inflammatory, pro-apoptotic, and pro-fibrogenic effects. Thus, a pathological level of cholesterol oxidation in the vasculature may be the missing molecular link between hypercholesterolemia and the formation of atherosclerotic lesions.

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.