Purpose of Study Methotrexate (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 Used THP-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 Results Following 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).
Conclusions This 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.
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