|Allen AM, Graham A (2010) Acute mitochondrial dysfunction inhibits macrophage cholesterol efflux to apolipoprotein AI.|
Link: Abstracts Session 1
Mitochondrial cholesterol trafficking, from the outer mitochondrial membrane to sterol 27-hydroxylase located on the inner mitochondrial membrane, facilitates generation of endogenous oxysterol ligands, capable of activating Liver X receptor (LXR) responsive genes such as ATP binding cassette transporters (ABCA1, ABCG1, ABCG4) which orchestrate cholesterol efflux from cells (1,2).
Mitochondrial cholesterol trafficking, from the outer mitochondrial membrane to sterol 27-hydroxylase located on the inner mitochondrial membrane, facilitates generation of endogenous oxysterol ligands, capable of activating Liver X receptor (LXR) responsive genes such as ATP binding cassette transporters (ABCA1, ABCG1, ABCG4) which orchestrate cholesterol efflux from cells (1,2). The first step in this pathway is critically regulated by ABCA1, in combination with lipid-poor acceptor apolipoproteins such as apoAI or apoE, while ABCG1 and ABCG4 transfer cholesterol to nascent HDL, so that these transporters act in concert to generate cholesterol-rich HDL. In this study, we explore the hypothesis that mitochondrial dysfunction, hallmark of developing atherosclerotic lesions, contributes to the accumulation of excess cholesterol within arterial macrophage ‘foam’ cells, by negatively impacting on the athero-protective cholesterol efflux pathway.
Macrophage (RAW264.7) mitochondrial function was pharmacologically modified, in acute incubations (3 h) to avoid overt toxicity, using antimycin A, which inhibits Complex III (CIII), dinitrophenol, which dissipates mitochondrial membrane potential ΔΨmt, nigericin, which alters mitochondrial ΔpH, and oligomycin, which inhibits CV. Cell viability was assessed by lactate dehydogenase (LDH) release, mitochondrial function assessed by dimethylthiazolyl diphenyltetrazolium bromide (MTT) conversion to formazan, total cellular ATP levels measured using CellTitre-Glo Bioluminescent assay and ΔΨmt measured via tetramethyl rhodamine ethyl ester (TMRE) binding. Quantitative PCR used to measure expression of key enzymes and transporters linked with cholesterol homeostasis, and measurement of (3H)cholesterol efflux to apoAI (20 µg∙ml-1) or HDL (20 µg∙ml-1) were performed as previously described (3).
Stimulation of murine RAW 264.7 macrophages with dibutyryl cyclic AMP (0.3mM) induced (3H) cholesterol efflux to apoAI by up to 5-fold (P<0.001) and to HDL by up to 3.5-fold (P<0.05), as previously (3). Acute incubation with oligomycin (3 h) significantly reduced (53%; P<0.01) cholesterol efflux to apoAI (right), whereas efflux to HDL remained consistently unaffected by this inhibitor. Oligomycin also induced a reduction in ΔΨmt (39%; P<0.05), while total cellular levels of ATP and release of cytosolic LDH remained unchanged during this period. Cholesterol efflux to both apoAI and HDL appeared inhibited by nigericin, although this proved significant only for apoAI (39%; P<0.01); however, this compound also reduced cellular levels of ATP (28%; P<0.01) and ΔΨmt (18%, P<0.5) during the incubation period. By contrast, cholesterol efflux to either apoAI or HDL remained unchanged following addition of dinitrophenol (10 µM) or antimycin A (10 µM) to the culture medium; these compounds did not alter ΔΨmt, total cellular ATP levels or LDH release during this short incubation period, although loss of mitochondrial function was evident after 24 h, or at higher concentrations of drug. Notably, acute treatment with oligomycin (10 µM) altered the expression of genes implicated in cholesterol homeostasis: expression of Hmgcr (3.9 fold; P<0.05) and Srebpf2 (3.4-fold; P<0.05) increased under basal conditions, while paradoxically both Abca1 and Ldlr expression increased by 16.6-fold (P<0.05) and 3.4-fold (P<0.05) respectively, following cholesterol efflux to apoAI; together, these data suggest integration of mitochondrial function with macrophage cholesterol homeostasis mechanisms.
In summary, changes in ΔΨmt, or intra-mitochondrial ATP levels, may be key factors regulating macrophage cholesterol homeostasis and efflux via ABCA1 to apoAI. The mechanisms involved remain unexplored, at present, but may provide targets for novel therapeutic strategies.
1. Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC, Edwards PA, Tontonoz P. (2000) Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXRalpha. Proc. Natl Acad. Sci USA 97: 12097-12102.
2. Oram JF, Vaughan AM (2006) ATP-binding cassette cholesterol transporters and cardiovascular disease. Circ. Res. 99: 1031-1043.
3. Taylor JM, Borthwick F, Bartholomew C, Graham A (2010) Over-expression of steroidogenic acute regulatory protein (StAR) increases macrophage cholesterol efflux to apolipoprotein AI. Cardiovasc. Res. cvq015.