Adenosine, Via A 2B Receptors, Inhibits Human (P-SMC) Progenitor Smooth Muscle Cell Growth

Abstract

c-Kit+ progenitor smooth muscle cells (P-SMCs) can develop into SMCs that contribute to injury-induced neointimal thickening. Here, we investigated whether adenosine reduces P-SMC migration and proliferation and whether this contributes to adenosine’s inhibitory actions on neointima formation. In human P-SMCs, 2-chloroadenosine (stable adenosine analogue) and BAY60-6583 (A 2B agonist) inhibited P-SMC proliferation and migration. Likewise, increasing endogenous adenosine by blocking adenosine metabolism with erythro-9-(2-hydroxy-3-nonyl) adenine (inhibits adenosine deaminase) and 5-iodotubercidin (inhibits adenosine kinase) attenuated P-SMC proliferation and migration. Neither N 6 -cyclopentyladenosine (A 1 agonist), CGS21680 (A 2A agonist), nor N 6 -(3-iodobenzyl)-adenosine-5’-N-methyluronamide (A 3 agonist) affected P-SMC proliferation or migration. 2-Chloroadenosine increased cyclic AMP, reduced Akt phosphorylation (activates cyclin D expression), and reduced levels of cyclin D1 (promotes cell-cycle progression). Moreover, 2-chloroadenosine inhibited expression of Skp2 (promotes proteolysis of p27 Kip1 ) and upregulated levels of p27 Kip1 (negative cell-cycle regulator). A 2B receptor knockdown prevented the effects of 2-chloroadenosine on cyclic AMP production and P-SMC proliferation and migration. Likewise, inhibition of adenylyl cyclase and protein kinase A rescued P-SMCs from the inhibitory effects of 2-chloroadenosine. The inhibitory effects of adenosine were similar in male and female P-SMCs. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 μmol/L for 7 days) reduced neointimal hyperplasia by 64.5% ( P <0.05; intima/media ratio: control, 1.4±0.02; treated, 0.53±0.012) and reduced neointimal c-Kit+ cells. Adenosine inhibits P-SMC migration and proliferation via the A 2B receptor/cyclic AMP/protein kinase A axis, which reduces cyclin D1 expression and activity via inhibiting Akt phosphorylation and Skp2 expression and upregulating p27 kip1 levels. Adenosine attenuates neointima formation in part by inhibiting infiltration and proliferation of c-Kit+ P-SMCs.