Abstract
BackgroundMitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis. This study investigated the role of mitochondrial function in regulation of macrophage cholesterol efflux to apolipoprotein A-I, by the addition of established pharmacological modulators of mitochondrial function.MethodsMurine RAW 264.7 macrophages were treated with a range of concentrations of resveratrol, antimycin, dinitrophenol, nigericin and oligomycin, and changes in viability, cytotoxicity, membrane potential and ATP, compared with efflux of [3H]cholesterol to apolipoprotein (apo) A-I. The effect of oligomycin treatment on expression of genes implicated in macrophage cholesterol homeostasis were determined by quantitative polymerase chain reaction, and immunoblotting, relative to the housekeeping enzyme, Gapdh, and combined with studies of this molecule on cholesterol esterification, de novo lipid biosynthesis, and induction of apoptosis. Significant differences were determined using analysis of variance, and Dunnett’s or Bonferroni post t-tests, as appropriate.ResultsThe positive control, resveratrol (24 h), significantly enhanced cholesterol efflux to apoA-I at concentrations ≥30 μM. By contrast, cholesterol efflux to apoA-I was significantly inhibited by nigericin (45%; p<0.01) and oligomycin (55%; p<0.01), under conditions (10 μM, 3 h) which did not induce cellular toxicity or deplete total cellular ATP content. Levels of ATP binding cassette transporter A1 (ABCA1) protein were repressed by oligomycin under optimal efflux conditions, despite paradoxical increases in Abca1 mRNA. Oligomycin treatment did not affect cholesterol biosynthesis, but significantly inhibited cholesterol esterification following exposure to acetylated LDL, and induced apoptosis at ≥30 μM. Finally, oligomycin induced the expression of genes implicated in both cholesterol efflux (Abca1, Abcg4, Stard1) and cholesterol biosynthesis (Hmgr, Mvk, Scap, Srebf2), indicating profound dysregulation of cholesterol homeostasis.ConclusionsAcute loss of mitochondrial function, and in particular Δψm, reduces cholesterol efflux to apoA-I and dysregulates macrophage cholesterol homeostasis mechanisms. Bioavailable antioxidants, targeted to mitochondria and capable of sustaining effective mitochondrial function, may therefore prove effective in maintenance of arterial health.
Highlights
Mitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis
Increased oxidative stress within the artery wall modifies low-density lipoprotein (LDL) to a form recognised by macrophage scavenger receptors, resulting in the unregulated uptake of excess cholesterol and cholesteryl esters within macrophage ‘foam cells’, a hallmark of early and developing atheroma [1,2,3,4,5]
Regression and stabilisation of atherosclerotic lesions requires the efficient removal of cholesterol from macrophage ‘foam cells’ to apolipoproteinA-I or apoE, allowing nascent high density lipoprotein (HDL) particles to enter and mature within the reverse cholesterol transport (RCT) pathway in the bloodstream, which delivers excess cholesterol to the liver for excretion as bile and bile acids [6]
Summary
Mitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis. Sterol 27-hydroxylase generates oxysterol ligands for Liver X Receptors (LXRα/β); nuclear transcription factors which act as master regulators of genes encoding proteins involved in the cholesterol efflux pathway [8]. These include ATP binding cassette (ABC) transporters, such as ABCA1 and ABCG1/ABCG4, which work in concert to transfer cholesterol across the plasma membrane to apoA-I and HDL, respectively [9,10]
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