Oxidative stress in scleroderma: maintenance of scleroderma fibroblast phenotype by the constitutive up-regulation of reactive oxygen species generation through the NADPH oxidase complex pathway.

OBJECTIVE: To explore the role of reactive oxygen species (ROS) in the in vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc). METHODS: Fibroblasts were obtained from involved skin of patients with limited or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels of O2- and H2O2 released from fibroblasts were estimated by the superoxide dismutase (SOD)-inhibitable cytochrome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted with an anti-p47phox-specific antibody. Fibroblasts were stimulated with various cytokines and growth factors to determine whether any of these factors modulate ROS generation. Cell proliferation was estimated by 3H-thymidine incorporation. Northern blot analysis was used to study alpha1 and alpha2 type I collagen gene expression. RESULTS: Unstimulated skin fibroblasts from SSc patients released more O2- and H2O2 in vitro through the NADPH oxidase complex pathway than did normal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodonium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppression was not seen with rotenone, a mitochondrial oxidase inhibitor, or allopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic component of NADPH oxidase, p47phox, was translocated to the plasma membrane of resting SSc fibroblasts. A transient increase in ROS production was induced in normal but not in SSc fibroblasts by interleukin-1beta (IL-1beta), platelet-derived growth factor type BB (PDGF-BB), transforming growth factor beta1 (TGFbeta1), and H2O2. Treatment of normal and SSc fibroblasts with tumor necrosis factor a (TNFalpha), IL-2, IL-4, IL-6, IL-10, interferon-alpha (IFNalpha), IFNgamma, granulocyte-macrophage colony-stimulating factor (GM-CSP), G-CSF, or connective tissue growth factor (CTGF) had no effect on ROS generation. Constitutive ROS production by SSc fibroblasts was not inhibited when these cells were treated with catalase, SOD, IL-1 receptor antagonist, or antibodies blocking the effect of TGFbeta1, PDGF-BB, and other agonists (IL-4, IL-6, TNFalpha, CTGF). In contrast, treatment of SSc fibroblasts with the membrane-permeant antioxidant N-acetyl-L-cysteine inhibited ROS production, and this was accompanied by decreased proliferation of these cells and down-regulation of alpha1(I) and alpha2(I) collagen messenger RNA. CONCLUSION: The constitutive intracellular production of ROS by SSc fibroblasts derives from the activation of an NADPH oxidase-like system and is essential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O2-, H2O2, IL-1beta, TGFbeta1, PDGF-BB, IL-4, IL-6, TNFalpha, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.

Solid monounsaturated diet lowers LDL unsaturation trait and oxidisability in hypercholesterolemic (type IIb) patients.

Lowering high cholesterol concentration decreases the probability of atherosclerotic-related pathology onset. MUFA and PUFA decrease total plasma and LDL cholesterol but PUFA may increase the susceptibility of LDL to undergo oxidative modifications thus becoming more atherogenetic. Olive oil, the predominant fat source in Mediterranean diet, may combine the advantages of both lowering cholesterol level and decreasing LDL susceptibility to oxidation. We studied the effects of feeding MUFA vs PUFA enriched diet on LDL composition and feature in hypercholesterolemic (IIb) patients. Antioxidant values remained constant during the study while LDL fatty acids composition reflected the dietary intake: MUFA concentration increased 11% whereas PUFA decreased 10% after olive oil diet (p < 0.05). PUFA/MUFA ratio and the unsaturation index were lower at the end of MUFA-enriched diet. The challenge, in vitro, of oleate-enriched LDL with Cu2- yielded to lower lag-phase (p < 0.05) in diene conjugated production; the same LDL gave lower lipid hydroperoxide contents after exposition to AAPH. We conclude that oleate-enriched LDL and with lower PUFA content were more resistant to oxidative modifications, as measured by different peroxidation indexes. This feature acquired with the diet may be an useful tool for lowering LDL oxidation and indirectly their atherogenicity.