RESUMO
BACKGROUND: (18)F-Fluorodeoxyglucose (FDG)-positron emission tomography (PET) imaging of atherosclerosis in the clinic is based on preferential accumulation of radioactive glucose analog in atherosclerotic plaques. FDG-PET is challenging in mouse models due to limited resolution and high cost. We aimed to quantify accumulation of nonradioactive glucose metabolite, FDG-6-phosphate, in the mouse atherosclerotic plaques as a simple alternative to PET imaging. METHODOLOGY/PRINCIPAL FINDINGS: Nonradioactive FDG was injected 30 minutes before euthanasia. Arteries were dissected, and lipids were extracted. The arteries were re-extracted with 50% acetonitrile-50% methanol-0.1% formic acid. A daughter ion of FDG-6-phosphate was quantified using liquid chromatography and mass spectrometry (LC/MS/MS). Thus, both traditional (cholesterol) and novel (FDG-6-phosphate) markers were assayed in the same tissue. FDG-6-phosphate was accumulated in atherosclerotic lesions associated with carotid ligation of the Western diet fed ApoE knockout mice (5.9 times increase compare to unligated carotids, p<0.001). Treatment with the liver X receptor agonist T0901317 significantly (2.1 times, p<0.01) reduced FDG-6-phosphate accumulation 2 weeks after surgery. Anti-atherosclerotic effects were independently confirmed by reduction in lesion size, macrophage number, cholesterol ester accumulation, and macrophage proteolytic activity. CONCLUSIONS/SIGNIFICANCE: Mass spectrometry of FDG-6-phosphate in experimental atherosclerosis is consistent with plaque inflammation and provides potential translational link to the clinical studies utilizing FDG-PET imaging.
Assuntos
Artérias/metabolismo , Química Farmacêutica/métodos , Glucose-6-Fosfato/análogos & derivados , Glucose/metabolismo , Placa Aterosclerótica/metabolismo , Animais , Apolipoproteínas E/genética , Aterosclerose/terapia , Artérias Carótidas/metabolismo , Linhagem Celular , Colesterol/metabolismo , Cromatografia Líquida/métodos , Diagnóstico por Imagem/métodos , Modelos Animais de Doenças , Desenho de Fármacos , Glucose/análogos & derivados , Glucose-6-Fosfato/metabolismo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Íons , Receptores X do Fígado , Espectrometria de Massas/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Nucleares Órfãos/antagonistas & inibidores , Tomografia por Emissão de Pósitrons/métodos , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed. Mouse arteries were dissected and placed in chloroform-methanol without tissue grinding. Extracts underwent hydrolysis of cholesteryl esters and derivatization of cholesterol followed by liquid chromatography/mass spectrometry (LC/MS/MS) analysis. We demonstrated that FC and CE could be quantitatively extracted without tissue grinding and that lipid extraction simultaneously worked for tissue fixation. Delipidated tissues can be embedded in paraffin, sectioned, and stained. Microscopic images obtained from delipidated arteries have not revealed any structural alterations. Delipidation was associated with excellent antigen preservation compatible with traditional immunohistochemical procedures. In ApoE(-/-) mice, LC/MS/MS revealed early antiatherosclerotic effects of dual PPARalpha,gamma agonist LY465606 in brachiocephalic arteries of mice treated for 4 weeks and in ligated carotid arteries of animals treated for 2 weeks. Reduction in CE and FC accumulation in atherosclerotic lesions was associated with the reduction of lesion size. Thus, a combination of LC/MS/MS measurements of CE and FC followed by histology and immunohistochemistry of the same tissue provides novel methodology for sensitive and comprehensive analysis of experimental atherosclerotic lesions.
