Activation of Adenosine A1 Receptor Induces Coronary Smooth Muscle Cell Proliferation in an in vitro Model of Atherosclerosis:

PURPOSE: Ossabaw swine are an excellent large animal model of the cardiometabolic risk. Compared to other breeds (e.g. Yucatan), lean Ossabaws are insulin-resistant and are highly prone to develop obesity and cardiometabolic risk. Restenosis after coronary artery stent placement is a continuing clinical problem that is more severe in patients with cardiometabolic risk. However, the mechanisms underlying increased restenosis in cardiometabolic risk are not clear. Our lab previously showed that activation of adenosine A1 receptors induces proliferation of coronary smooth muscle cell in monolayer cultures. Therefore, derangements in adenosine A1 receptor signaling could contribute to excessive cell proliferation in restenosis. Our previous work showed that the placement of bare metal coronary stents induced adenosine A1 receptor gene expression in Ossabaws. Hypothesis: Activation of the adenosine A1 receptor will elicit coronary smooth muscle cell proliferation in in vitro organ cultured arterial rings. METHODS: We put Ossabaw coronary arterial rings in Petri dish with RPMI media at 37 degree C for 24 hours, which we define as organ culture. Organ cultured rings were treated with either the A1R-selective agonist CCPA (10−6M) or the A1R-selective antagonist DPA (10−6M) or both CCPA (10−6M) and DPA (10−6M). Two groups of untreated rings at either 37 degree C (organ culture) or 4 degree C (cold store) served as controls. After 24 hour treatment, coronary smooth muscle cells were dispersed from the rings and stained with BrdU. The BrdU incorporation (DNA synthesis) was measured using confocal microscopy to determine the degree of cell proliferation. RESULTS: Organ culture increased BrdU incorporation in coronary smooth muscle cells by 5 fold over cold store (p < 0.05), demonstrating that organ culture is a useful in vitro model of SMC proliferation and stenosis. CCPA further increased BrdU incorporation by ∼4 fold over untreated organ culture control (p < 0.05), whereas DPA abolished the increase in DNA synthesis observed in response to CCPA treatment (p < 0.05). CONCLUSION: A1 receptor mediates CASMC proliferation in the in vitro organ culture model in the setting of cardiometabolic risk. Support: NIH RR13223, HL62552, ADA.