Loss of mitochondrial function, a key element of atherogenesis, is caused by an increase in reactive oxygen species, resulting in accumulation of mitochondrial DNA damage, and respiratory chain deficiencies, apoptosis and cell death, favouring plaque formation and instability[1–5]. This study argues that dysregulation of macrophage cholesterol homeostasis should also join this list, apparently driven by the specific loss of macrophage cholesterol efflux to apoA-I.
Notably, resveratrol, a natural phytoalexin antioxidant found in grapes and red wine, known to induce mitochondrial biogenesis and protect against atherosclerosis in animal models[23, 25], increased cholesterol efflux to apoA-I, at concentrations around 20-30 μM, as previously described[22, 24]. However, it is evident that concentrations of resveratrol (30-100 μM) which increase cholesterol efflux (Figure1), and indeed those reported to activate LXRα, are associated with loss of mitochondrial membrane potential and cell viability and induction of cytotoxicity in RAW 264.7 macrophages. Moreover, since resveratrol is characterised by high absorption but very low bioavailability, coupled with extensive and rapid metabolism to conjugates of this compound, it seems unlikely that monocyte-macrophages would be exposed to the concentrations of resveratrol needed to stimulate the cholesterol efflux process. For example, absorption (70%) of a dietary relevant 25 mg oral dose generated peak plasma levels of around 2 μM resveratrol metabolites, with only trace amounts of unchanged resveratrol detected in the plasma (<5 ng ml-1). However, it may be possible that the more potent antioxidant function of resveratrol (≤5 μM), or the ability of this compound to amplify SIRT-1 dependent biogenesis of mitochondria (≤10 μM), might help to sustain mitochondrial function in vivo, particularly if local depots of resveratrol accumulate within arterial cells. If so, macrophages may have to be oxidatively challenged, or subject to chronic incubation with resveratrol, for the protective impact of this compound on the cholesterol efflux pathway to become more (patho)physiologically relevant.
By contrast, inhibitors of differing aspects of mitochondrial function (ΔpH, Δψm complex III or complex V) exerted convincing, if selective, effects on the cholesterol efflux pathway over the acute time period tested here. Notably, the uncoupling agent, dinitrophenol, and complex III inhibitor, antimycin, had negligible impact on efflux to apoA-I, while nigericin and oligomycin both reduced efflux to apoA-I. While nigericin (10 μM) decreased total cellular ATP levels, which could impact on the activity of ABC transporters, ABCA1 and ABCG1/G4, oligomycin did not alter cell viability or total ATP levels, but did significantly reduce Δψm, unlike the other inhibitors tested. In turn, loss of Δψm due to oligomycin was associated with inhibition of cholesterol esterification and a trend towards induction of apoptosis, together with increased expression of an array of genes involved in cholesterol homestasis, arguing a profound dysregulation of cholesterol homeostasis as sequelae of acute loss of mitochondrial function. Importantly, the coordinated response which should link impaired cholesterol efflux with SREBP-2 repression of cholesterol biosynthesis and LDL uptake, and with increased cholesterol esterification, seems to be lost.
While this is the first study to focus on the importance of mitochondria in regulation of macrophage cholesterol efflux, related studies have been performed in steroidogenic cells [16–19; 26]. As previously introduced, the rate-limiting step in the generation of steroid hormones, is the transfer of cholesterol into mitochondria, to the CYP11A1 protein which resides on the inner mitochondrial membrane. Dissipation of Δψmwith CCCP, inhibition of electron transport using antimycin A, disruption of pH using nigericin, and inhibition of F0/F1 ATP synthase using oligomycin, all inhibited progesterone synthesis in Leydig cells, indicating that altered mitochondrial function regulates steroid biosynthesis [16–19; 26]. However, in RAW 264.7 macrophages, only nigericin and oligomycin regulated macrophage cholesterol efflux to apoA-I, and at concentrations 10-fold higher than those required to inhibit steroidogenesis[16–19, 26]. It is noteworthy that cellular ATP content was more sensitive to depletion by mitochondrial disruption in Leydig cells than in RAW 264.7 macrophages, as only oligomycin (≥30 μM) and nigericin (≥10 μM) reduced cellular ATP levels over the same time scale in the latter; equally, Δψm was markedly reduced by antimycin (1 μM) and oligomycin (1 μM) in Leydig cells, while only oligomycin (≥10 μM) affected this parameter in the current study. This may explain the apparent selectivity for nigericin and oligomycin in repression of cholesterol efflux to apoA-I; certainly, loss of either Δψm or cellular ATP seems sufficient to negatively affect macrophage cholesterol efflux when cell viability is sustained (Figures2 and3).
Oligomycin treatment, by limiting cholesterol efflux (Figure2A) and reducing cholesterol esterification (Figure4B), without impacting on cholesterol biosynthesis (Figure4C), should therefore lead to accumulation of sterol at the endoplasmic reticulum (ER). This should trigger a protective cholesterol homeostasis response, sequestering SREBPs at the ER, and providing oxysterol ligands for Liver X Receptors (LXRs) complexed with retinoid X receptors (RXR) at the LXR response element within genes involved in the cholesterol efflux pathway. Sterol-dependent sequestration of SREBPs at the ER prevents proteolytic processing of this transcription factor to the nuclear SREBP fragment capable of inducing the transactivation of genes involved in cholesterol biosynthesis and uptake. However, instead of these correctly orchestrated events, treatment with oligomycin resulted in upregulation of genes involved in both cholesterol biosynthesis (Hmgr, Mvk, Scap, Srebf1, Srebf2) and efflux (Abca1, Abcg4, StarD1) pathways, arguing a profound dysregulation of this response. The mechanism involved was not investigated here, but it is possible to speculate that loss of mitochondrial production of oxysterol LXR ligands may release SREBP2 from the ER, explaining the increased expression of genes involved in increasing cholesterol biosynthesis. This could then result in increased production of 24(S),25-epoxycholesterol via the cholesterol biosynthetic pathway, facilitating LXR activation and induction of genes involved in the efflux process.
One further outcome predicted by the macrophage lipid phenotype observed following oligomycin treatment is toxic overaccumulation of cholesterol at the ER, which can trigger oxidative stress and proteasomal degradation of existing ABCA1 protein and ultimately trigger apoptosis, observed at higher concentrations of oligomycin (Figure4D). While it is clear that oligomycin induction of Abca1 mRNA may explain the modest increase in ABCA1 protein under basal conditions, a clear reduction in ABCA1 is noted when macrophages are treated with this inhibitor under optimal efflux conditions (Figure3B), which agrees well with the observed loss of 3H]cholesterol efflux to apoA-I under this condition (Figure3A). The dissociation between expression of ABCA1 mRNA and protein in oligomycin-treated macrophages under this condition reflects that observed in carotid atherosclerotic lesions, where elevated Abca1 mRNA is associated with reduced expression of ABCA1 protein.