Downregulation of NOX4 Expression by Roflumilast N-Oxide Reduces Markers of Fibrosis in Lung Fibroblasts

The phosphodiesterase 4 inhibitor roflumilast prevents bleomycin- (BLM-) induced lung fibrosis in animal models. However, its mechanism of action remains unknown. We investigated whether roflumilast N-oxide (RNO), the active metabolite of roflumilast, can modulate in vitro the oxidative effects of BLM on human lung fibroblasts (HLF). In addition, since BLM increases the production of F2-isoprostanes that have per se fibrogenic activity, the effect of RNO on oxidative stress and fibrogenesis induced by the F2-isoprostane 8-epi-PGF2α was investigated. HLF were preincubated either with the vehicle or with RNO and exposed to either BLM or 8-epi-PGF2α. Proliferation and collagen synthesis were assessed as [3H]-thymidine and [3H]-proline incorporation. Reactive oxygen species (ROS) and F2-isoprostanes were measured. NADPH oxidase 4 (NOX4) protein and mRNA were also evaluated. BLM increased both cell proliferation and collagen synthesis and enhanced ROS and F2-isoprostane production. These effects were significantly prevented by RNO. Also, RNO significantly reduced the increase in both NOX4 mRNA and protein, induced by BLM. Finally, 8-epi-PGF2α per se stimulated HLF proliferation, collagen synthesis, and NOX4 expression and ROS generation, and RNO prevented these effects. Thus, the antifibrotic effect of RNO observed in vivo may be related to its ability to mitigate ROS generation via downregulation of NOX4

F2-isoprostanes can mediate bleomycin-induced lung fibrosis

F2-isoprostanes (F2-IsoPs) have been considered markers of oxidative stress in various pulmonary diseases, but little is known about their possible role in pulmonary fibrosis. In this study, we have investigated the potential key role of F2-IsoPs as markers and mediators of bleomycin (BLM)-induced pulmonary fibrosis in rats. During the in vivo study, plasma F2-IsoPs showed a peak at 7 days and remained elevated for the entire experimental period. Lung F2-IsoP content nearly tripled 7 days following the intratracheal instillation of BLM, and by 28 days, the value increased about fivefold compared to the controls. Collagen deposition correlated with F2-IsoP content in the lung. Furthermore, from day 21 onwards, lung sections from BLM-treated animals showed α-smooth muscle actin (α-SMA) positive cells, which were mostly evident at 28 days. In vitro studies performed in rat lung fibroblasts (RLF) demonstrated that either BLM or F2-IsoPs stimulated both cell proliferation and collagen synthesis. Moreover, RLF treated with F2-IsoPs showed a significant increase of α-SMA expression compared to control, indicating that F2-IsoPs can readily activate fibroblasts to myofibroblasts. Our data demonstrated that F2-IsoPs can be mediators of key events for the onset and development of lung fibrosis, such as cell proliferation, collagen synthesis and fibroblast activation. Immunocytochemistry analysis, inhibition and binding studies demonstrated the presence of the thromboxane A2 receptor (TP receptor) on lung fibroblasts and suggested that the observed effects may be elicited through the binding to this receptor. Our data added a new perspective on the role of F2-IsoPs in lung fibrosis by providing evidence of a profibrotic role for these mediators in the pathogenesis of pulmonary fibrosis

Cigarette Smoke Affects Keratinocytes SRB1 Expression and Localization via H2O2 Production and HNE Protein Adducts Formation

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H2O2) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H2O2, induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake

F2-Isoprostanes as markers of oxidative stress and mediators of CCl4-induced liver fibrosis

INTRODUCTION: F2-isoprostanes are considered as the most reliable markers of OS in vivo and mediators of some important biological effects, such as the vasoconstriction of kidney glomerular arterioles. It is known that hepatic fibrosis induced by CCl4 may be linked to oxidative stress (OS). In previous studies, we demonstrated the role of isoprostanes as possible mediators of CCl4-induced hepatic fibrosis. Plasma levels of isoprostanes were found to be elevated in rat chronically treated with CCl4 and correlated with hepatic collagen content. We also demonstrated that in vitro isoprostanes induced a marked increase in DNA and collagen synthesis in cultured hepatic stellate cells (HSC), in the concentration range found in the in vivo studies (1-10 nM). It has been suggested that isoprostanes act through the activation of receptors analogous to those for tromboxane A2 (TxA2r). Thus, the possible occurrence of TxA2r on HSC was investigated. Binding studies with [3H]SQ29548 (a specific antagonist of TxA2r) and competition binding experiments with I-BOP (a specific agonist of TxA2r) demonstrated the existence of TxA2r in HSC and the involvement of TxA2r in Iso-evoked responses. We then examined which signal transduction pathways are set into motion by isoprostanes to exert their fibrogenic effects. METHODS: HSC were isolated from the rat liver and treated with 8-epi-PGE2the most represented isomer). Ins(1,4,5)P3 (IP3) and cAMP levels were determined by commercial kits. Activation of mitogen-activated protein kinases (MAPK) and cyclin 1 was assessed by Western blotting. Cell proliferation and collagen production were assessed by measuring the incorporation of 3H-thymidine and 3H-proline, respectively. RESULTS: 8-epi-PGE2 increased 4 times IP3 and affected cAMP production. The expression of cyclin D1, a molecule involved in cell proliferation, is also increased. Furthermore, 8-epi-PGE2 activated two classes of MAPK: extracellular signal-regulated kinase (ERK) and p38, which have been shown to influence cell proliferation and collagen gene expression. CONCLUSION: On the basis of these data, it is possible to hypothesize that one of the pathways activated by 8-epi-PGF2a is that of Gq/PKC. The binding of isoprostanes to TxA2r could stimulate downstream MAPK activation, via PKC. In particular, p38 is known to increase in HSC collagen production, while ERK is able to increase cyclin D1 expression and then cell proliferation. Thus, fibrogenic effects of isoprostanes in HSC are mediated through TxA2r binding by specific activation of these transduction pathways

Oxidative stress, isoprostanes and hepatic fibrosis

An introduction to oxidative stress enlightening the spreading of interest in lipid peroxidation in the 60's and in the identification of cytotoxic aldehydes originating from it is given. The discovery of F2 -isoprostanes as specific markers of oxidative stress is described. Isoprostanes are also agonists of important biological effects. Since a relationship between oxidative stress and collagen hyperproduction has been previously suggested, and since lipid peroxidation products (aldehydes) have been proposed as possible mediators of liver fibrosis, we investigated whether collagen synthesis is induced by F2 -isoprostanes, which can posses receptors for signal transduction pathways. In a rat model of carbon tetrachloride-induced hepatic fibrosis, plasma isoprostanes were markedly elevated for the entire experimental period and hepatic collagen content was also increased. Moreover, when hepatic stellate cells (HSC) isolated from normal livers were cultured up to activation and then treated with F2 -isoprostanes (8-epiPGF2α ) in the concentration range found in the in vivo studies (10-9 to 10-8 M), a striking increase in DNA synthesis, in cell proliferation and in collagen synthesis was observed. F2 -isoprostanes also increased the production of transforming growth factor-ß1 by U937 cells, assumed as a model of Kupffer cells or liver macrophages. The hypothesis that F2 -isoprostanes generated by lipid peroxidation in hepatocytes mediate HSC proliferation and collagen hyperproduction, seen in this experimental hepatic fibrosis, was reinforced by the demonstration, by using immunoblot analysis, that isoprostane receptors identical or analogous to those for thromboxane A2 (TxA2 r) are present in HSC. Immunocytochemical studies showed the major localization of TxA2 r in the perinuclear site and its colocalization with α-smooth muscle actin

Cigarette smoke affects SRB1 levels in human keratynocites via H202 production

The pathological effects of cigarette smoke (CS) have been extensively documented and many diseases such as emphysema and lung cancer are directly linked to the consequences of chronic smoke exposure. The combustion of CS produces over 4,000 different compounds in gaseous and particulate states that are able to induce oxidative stress to the cells exposed to the tobacco smoke. In addition, side stream smoke has been shown to be nearly as toxic as direct stream, therefore CS present two-fold heath problem to both passive and active smokers. Skin acts as a first line of defense against environmental trauma. The upper layer of the skin, the stratum corneum (SC), is composed by lipid barrier that contains unsaturated lipids that are susceptible to oxidation. The alteration of the skin lipids composition by the exposure to environmental stressors such as CS can affect the capacity of the SC to protect us from dehydration and external dangers. Scavenger Receptor B1 (SR-B1), one of the large family of scavenger receptors, has been shown to be involved in the uptake of cholesterol from HDL to peripheral tissues via apo A-1 mediated processes and also to be important in the delivery of tocopherol to the cells, therefore its role in skin homeostasis is crucial. In the present studies the effects of CS and of products related to CS such as acrolein, 4HNE and H2O2 on SR-B1 expression in human keratinocytes were assessed. CS exposure induced a significant decrease of SR-B1 expression and the formation of acrolein and 4NHE protein adducts. On the other hand, cells treated with several doses of acrolein or 4HNE or H2O2 did not affect SR-B1 levels. The treatment with glucose oxidase induces the same effect of CS as to concern SR-B1 levels and this was reversed when cells were treated with catalase. In addition, CS induced the activation of NADPH oxidase measured as p67 translocation from the cytoplasm to the membrane. The data from this study show that CS decreases the levels of SR-B1 in human keratinocytes and this effect is mainly driven by the production of H2O2 (exogenous and endogenous). The decrease of SR-B1 could lead to the disturbance of the skin lipid barrier affecting skin physiology and be a possible cause to skin disorders such as skin aging and wound healing linked to CS exposure

Synthesis and characterization of glucosyl-curcuminoids as Fe3+ suppliers in the treatment of iron deficiency

The Fe3+ chelating ability of some curcumin glucosyl derivatives (Glc-H; Glc-OH; Glc-OCH3) is tested by means of UV and NMR study. The pK a values of the ligands and the overall stability constants of Fe3+ and Ga3+ complexes are evaluated from UV spectra. The only metal binding site of the ligand is the β-diketo moiety in the keto-enolic form; the glucosyl moiety does not interact with metal ion but it contributes to the stability of metal/ligand 1:2 complexes by means of hydrophilic interactions. These glucosyl derivatives are able to bind Fe3+ in a wide pH rage, forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. In addition they demonstrate to have a poor affinity for competitive biological metal ions such as Ca2+. All ligands and their iron complexes have a good lypophilicity (log P > −0.7) suggesting an efficient gastrointestinal absorption in view of their possible use as iron supplements in oral therapy. The ligand molecules are also tested for their antioxidant properties in “ex vivo” biological system

Synthesis, chemical and biological studies on new Fe3þ-glycosilated b-diketo complexes for the treatment of iron deficiency

A simple synthetic pathway to obtain glycosilated b-diketo derivatives is proposed. These compounds show a good iron(III) affinity thereforewe may suggest the use of their Fe3þ-complexes as oral iron supplements in the treatment of anaemia. The glycosilated compounds (6-GlcH, 6-GlcOH and 6-GlcOCH3) are characterized by means of spectroscopic (UV, 1H and 13C NMR) and potentiometric techniques; they have a good water solubility, are kinetically stable in physiological condition (t1/2 > 100 h) and show a low cytotoxicity also in high concentrations (IC50 > 400 mM). They are able to bind Fe3þ ion in acid condition (pHw2) forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. The iron complexes show also a good kinetic stability both in acidic and physiological pH and have a good lypophilicity (log P > 0.7) that suggests an efficient gastrointestinal absorption in view of their possible use in oral therapy. In addition they demonstrate a poor affinity for competitive biological metal ion such as Ca2þ, and in particular 6-GlcOCH3 is able to inhibit lipid peroxidation

Ethanol-induced oxidative stress: basic knowledge

After a general introduction, the main pathways of ethanol metabolism (alcohol dehydrogenase, catalase, coupling of catalase with NADPH oxidase and microsomal ethanol-oxidizing system) are shortly reviewed. The cytochrome P450 isoform (CYP2E1) specifically involved in ethanol oxidation is discussed. The acetaldehyde metabolism and the shift of the NAD/NADH ratio in the cellular environment (reductive stress) are stressed. The toxic effects of acetaldehyde are mentioned. The ethanol-induced oxidative stress: the increased MDA formation by incubated liver preparations, the absorption of conjugated dienes in mitochondrial and microsomal lipids and the decrease in the most unsaturated fatty acids in liver cell membranes are discussed. The formation of carbon-centered (1-hydroxyethyl) and oxygen-centered (hydroxyl) radicals during the metabolism of ethanol is considered: the generation of hydroxyethyl radicals, which occurs likely during the process of univalent reduction of dioxygen, is highlighted and is carried out by ferric cytochrome P450 oxy-complex (P450–Fe3+O2·−) formed during the reduction of heme-oxygen. The ethanol-induced lipid peroxidation has been evaluated, and it has been shown that plasma F2-isoprostanes are increased in ethanol toxicity

Synthesis, chemical and biological study on new Fe3+-glycosilated β-diketo complexes for the treatment of iron deficiency

A simple synthetic pathway to obtain glycosilated β-diketo derivatives is proposed. These compounds show a good iron(III) affinity therefore we may suggest the use of their Fe3+-complexes as oral iron supplements in the treatment of anaemia. The glycosilated compounds (6-GlcH, 6-GlcOH and 6-GlcOCH3) are characterized by means of spectroscopic (UV, 1H and 13C NMR) and potentiometric techniques; they have a good water solubility, are kinetically stable in physiological condition (t1/2 > 100 h) and show a low cytotoxicity also in high concentrations (IC50 > 400 μM). They are able to bind Fe3+ ion in acid condition (pH ∼ 2) forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. The iron complexes show also a good kinetic stability both in acidic and physiological pH and have a good lypophilicity (log P > -0.7) that suggests an efficient gastrointestinal absorption in view of their possible use in oral therapy. In addition they demonstrate a poor affinity for competitive biological metal ion such as Ca2+, and in particular 6-GlcOCH3 is able to inhibit lipid peroxidation. © 2008 Elsevier Masson SAS. All rights reserved