Oxidized low-density lipoprotein (oxLDL) is a well-recognized proatherogenic particle that functions in atherosclerosis. In this study, we established conditions to generate human oxLDL, characterized according to the grade of lipid and protein oxidation, particle size and oxylipin content. The induction effect of the cellular proatherogenic response was assessed in foam cells by using an oxLDL-macrophage interaction model. Uptake of oxLDL, reactive oxygen species production and expression of oxLDL receptors (CD36, SR-A and LOX-1) were significantly increased in THP-1 macrophages. Analyses of 35 oxylipins revealed that isoprostanes (IsoP) and prostaglandins (PGs) derived from the oxidation of arachidonic, dihomo gamma-linolenic and eicosapentaenoic acids were strongly and significantly induced in macrophages stimulated with oxLDL. Importantly, the main metabolites responsible for the THP1-macrophage response to oxLDL exposure were the oxidative stress markers 5-epi-5-F2t-IsoP, 15-E1t-IsoP, 8-F3t-IsoP and 15-keto-15-F2t-IsoP as well as inflammatory markers PGDM, 17-trans-PGF3α, and 11β-PGF2α, all of which are reported here, for the first time, to function in the interaction of oxLDL with THP-1 macrophages. By contrast, a salvage pathway mediated by anti-inflammatory PGs (PGE1 and 17-trans-PGF3α) was also identified, suggesting a response to oxLDL-induced injury. In conclusion, when THP-1 macrophages were treated with oxLDL, a specific induction of biomarkers related to oxidative stress and inflammation was triggered. This work contributes to our understanding of initial atherogenic events mediated by oxLDL-macrophage interactions and helps to generate new approaches for their modulation.

Rett syndrome (RTT) is a rare neurodevelopmental disorder affecting almost exclusively females, caused in the overwhelming majority of the cases by loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). High circulating levels of oxidative stress (OS) markers in patients suggest the involvement of OS in the RTT pathogenesis. To investigate the occurrence of oxidative brain damage in Mecp2 mutant mouse models, several OS markers were evaluated in whole brains of Mecp2-null (pre-symptomatic, symptomatic, and rescued) and Mecp2-308 mutated (pre-symptomatic and symptomatic) mice, and compared to those of wild type littermates. Selected OS markers included non-protein-bound iron, isoprostanes (F2-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes) and 4-hydroxy-2-nonenal protein adducts. Our findings indicate that oxidative brain damage 1) occurs in both Mecp2-null (both -/y and stop/y) and Mecp2-308 (both 308/y males and 308/+ females) mouse models of RTT; 2) precedes the onset of symptoms in both Mecp2-null and Mecp2-308 models; and 3) is rescued by Mecp2 brain specific gene reactivation. Our data provide direct evidence of the link between Mecp2 deficiency, oxidative stress and RTT pathology, as demonstrated by the rescue of the brain oxidative homeostasis following brain-specifically Mecp2-reactivated mice. The present study indicates that oxidative brain damage is a previously unrecognized hallmark feature of murine RTT, and suggests that Mecp2 is involved in the protection of the brain from oxidative stress.

Oxidative damage has been reported in Rett syndrome (RTT), a pervasive developmental disorder caused in up to 95% of cases by mutations in the X-linked methyl-CpG binding protein 2 gene. Herein, we have synthesized F(2)-dihomo-isoprostanes (F(2)-dihomo-IsoPs), peroxidation products from adrenic acid (22:4 n-6), a known component of myelin, and tested the potential value of F(2)-dihomo-IsoPs as a novel disease marker and its relationship with clinical presentation and disease progression. F(2)-dihomo-IsoPs were determined by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Newly synthesized F(2)-dihomo-IsoP isomers [ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP] were used as reference standards. The measured ions were the product ions at m/z 327 derived from the [M-181](-) precursor ions (m/z 597) produced from both the derivatized ent-7(RS)-F(2t)-dihomo-IsoP and 17-F(2t)-dihomo-IsoP. Average plasma F(2)-dihomo-IsoP levels in RTT were about one order of magnitude higher than those in healthy controls, being higher in typical RTT as compared with RTT variants, with a remarkable increase of about two orders of magnitude in patients at the earliest stage of the disease followed by a steady decrease during the natural clinical progression. hese data indicate for the first time that quantification of F(2)-dihomo-IsoPs in plasma represents an early marker of the disease and may provide a better understanding of the pathogenic mechanisms behind the neurological regression in patients with RTT.