Purpose of StudyMethotrexate (MTX) is an anti-rheumatic drug with atheroprotective properties mediated through adenosine release and activation of the adenosine A2A receptor (A2AR). A2AR ligation increases reverse cholesterol transport via upregulation of cholesterol efflux proteins ATP-binding cassette transporter (ABC)A1 and ABCG1, liver X receptor (LXR) and cholesterol 27-hydroxylase. MTX is non-specific and associated with adverse effects on liver and kidney. Therefore, this study examines the anti-atherogenic efficacy of a specific A2AR agonist, UK-432,097, a drug with an established safety profile in humans.Methods UsedTHP-1 human macrophages were incubated in the following conditions: (1) RPMI media (untreated control); (2) dimethyl sulfoxide vehicle control; (3) UK-432,097 (100 nM); (4) ZM-241385 (1 µM) (A2AR antagonist)+UK-432,097 (100 nM). Gene expression analysis was performed using QRT-PCR for cholesterol efflux genes, normalized to the housekeeping gene GAPDH. Western blotting was performed using specific antibodies. All data were analyzed by one-way ANOVA with P values <0.05 considered significant.Summary of ResultsFollowing 6 h exposure to UK-432,097, mRNA and protein levels of ABCA1 increased by 88.75±5.4% and by 56.34±12.4% above control (P<0.01), respectively. ABCG1 expression increased by 58.42±6.32% and 65.45±5.24% vs. control (P<0.01), respectively. Following 18 h incubation in UK-432,097, 27-hydroxylase mRNA and protein increased by 46.45±3.4% and 50.27±8.9% (P<0.01), respectively. Message and protein level of LXRα were upregulated to 155.80±4.9% and 157.98±12.9% (n=3, P<0.01), respectively. A2AR blockade with ZM-241385 negated the effect of UK-432,097. UK-432,097 decreased oxidized LDL uptake by 28.9% in THP-1 macrophages (P<0.01).ConclusionsThis study demonstrates that UK-432,097 increases anti-atherogenic reverse cholesterol transport proteins with concomitant reduction in oxidized lipid accumulation in THP-1 macrophages. Since MTX is already being used in clinical trials to reduce cardiovascular risk, our results encourage further studies of specific A2AR agonists as cardioprotective treatment in high risk individuals.
Localisation and regulation of cholesterol transporters in the human hair follicle: mapping changes across the hair cycle
