RESUMO
We addressed how advanced glycation (AGE) affects the ability of apoA-IV to impair inflammation and restore the expression of genes involved in cholesterol efflux in lipopolysaccharide- (LPS-) treated macrophages. Recombinant human apoA-IV was nonenzymatically glycated by incubation with glycolaldehyde (GAD), incubated with cholesterol-loaded bone marrow-derived macrophages (BMDMs), and then stimulated with LPS prior to measurement of proinflammatory cytokines by ELISA. Genes involved in cholesterol efflux were quantified by RT-qPCR, and cholesterol efflux was measured by liquid scintillation counting. Carboxymethyllysine (CML) and pyrraline (PYR) levels, determined by Liquid Chromatography-Mass Spectrometry (LC-MS/MS), were greater in AGE-modified apoA-IV (AGE-apoA-IV) compared to unmodified-apoA-IV. AGE-apoA-IV inhibited expression of interleukin 6 (Il6), TNF-alpha (Tnf), IL-1 beta (Il1b), toll-like receptor 4 (Tlr4), tumor necrosis factor receptor-associated factor 6 (Traf6), Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/Stat3), nuclear factor kappa B (Nfkb), and AGE receptor 1 (Ddost) as well as IL-6 and TNF-alpha secretion. AGE-apoA-IV alone did not change cholesterol efflux or ABCA-1 levels but was unable to restore the LPS-induced reduction in expression of Abca1 and Abcg1. AGE-apoA-IV inhibited inflammation but lost its ability to counteract the LPS-induced changes in expression of genes involved in macrophage cholesterol efflux that may contribute to atherosclerosis.
Assuntos
Apolipoproteínas A/metabolismo , Colesterol/metabolismo , Produtos Finais de Glicação Avançada , Lipopolissacarídeos/química , Macrófagos/metabolismo , Acetaldeído/análogos & derivados , Acetaldeído/química , Animais , Apolipoproteínas A/química , Células da Medula Óssea/citologia , Cromatografia Líquida , Perfilação da Expressão Gênica , Humanos , Inflamação , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Proteínas Recombinantes/químicaRESUMO
AIMS: Advanced glycated albumin (AGE-albumin) adversely impairs macrophage lipid homeostasis in vitro, which may be prevented by angiotensin receptor blockers. In vivo studies are inconclusive whether AGE-albumin itself plays important role in early-stage atherogenesis. We aimed at investigating how AGE-albumin by itself drives atherosclerosis development in dyslipidemic non-diabetic mice and if its effects are due to the activation of renin-angiotensin system in the arterial wall and the expression of genes and proteins involved in lipid flux. METHODS AND RESULTS: Murine albumin glycation was induced by incubation with 10mM glycolaldehyde and C-albumin with PBS alone. Twelve-week-old-male apoE knockout mice were submitted to a daily IP injection of control (C) or AGE-albumin (2mg/mL) during 30days with or without losartan (LOS: 100mg/L; C+LOS and AGE+LOS). Aortic arch was removed, and gene expression was determined by RT-PCR and protein content by immunofluorescence. Plasma lipid and glucose levels were similar among groups. Systolic blood pressure was similarly reduced in both groups treated with LOS. In comparison to C-albumin, aortic lipid infiltration was 5.3 times increased by AGE-albumin, which was avoided by LOS. LOS prevented the enhancement induced by AGE-albumin in Ager, Tnf and Cybb mRNA levels but did not reduce Olr1. Nfkb and Agt mRNA levels were unchanged by AGE-albumin. LOS similarly reduced Agtr1a mRNA level in both C and AGE-albumin groups. In AGE-albumin-treated mice, immunofluorescence for carboxymethyl-lysine, 4-hydroxynonenal and RAGE was respectively, 4.8, 2.6 and 1.7 times enhanced in comparison to C-albumin. These increases were all avoided by LOS. CONCLUSIONS: AGE-albumin evokes a pre-stage of atherogenesis in dyslipidemic mice independently of the presence of diabetes mellitus or modulation in the RAS in part by the induction of lipid peroxidation and inflammation.