Adenosine Attenuates Aortic Smooth Muscle Cell Calcification through A₃ Adenosine Receptor

Abstract

Vascular calcification is a typical feature of atherosclerosis and is associated with adverse cardiovascular events such as myocardial infarction and stroke. Several studies have suggested that adenosine, an ATP metabolite may function as an endogenous regulator of arterial calcification. However, its effects on vascular smooth muscle cell calcification have not been clarified. In this study, we investigated the inhibitory effects of adenosine on vascular calcification in vitro by utilizing the culture of human aortic smooth muscle cells (HASMCs). Osteoblastic differentiation of HASMCs was induced by the treatment with oncostatin M and osteogenic differentiation medium. Adenosine and its metabolically stable analogue, 2-chloroadenosine (CADO) significantly reduced matrix mineralization and alkaline phosphatase (ALP) activities in HASMCs. The mRNA expression of tissue non-specific alkaline phosphatase (TNAP) was down-regulated by adenosine and CADO, but the mRNA expression of other osteoblastic differentiation markers, such as Runt-related transcription factor 2 (RUNX2) and bone sialoprotein (BSP)-II, was not significantly affected by these two reagents. Among the adenosine receptor (AR) subtype-selective agonists used, only IB-MECA (A₃ AR-selective agonist) significantly decreased in vitro mineralization and ALP activities in HASMCs, but not with CCPA (A₁ AR-selective agonist), CGS21680 (A_2a AR-selective agonist), or BAY60-6583 (A_2b AR-selective agonist). Importantly, IB-MECA also down-regulated expression of TNAP mRNA. Finally, knockdown of A₃ AR, but not A₁ AR, A_2a AR, or A_2b AR, significantly reversed the inhibitory actions of adenosine, CADO, or IB-MECA on in vitro calcification and ALP activities in HASMCs. These data suggest that adenosine attenuates HASMC calcification through A₃ AR.