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 N⁶-cyclopentyladenosine (A₁ agonist), CGS21680 (A_2A agonist), nor N⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (A₃ 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% (<i>P</i><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.