C. J. Little1, A. Kent1, B. Wang1, M. A. Chaudhary1, K. Kent1, L. Guo1 1University Of Wisconsin School Of Medicine And Public Health,Department Of Surgery,Madison, WI, USA
Introduction: Intimal hyperplasia leads to failure of 20-50% of vascular reconstructions. The underlying cause is smooth muscle cell (SMC) pathophysiology characterized by proliferation, migration and dedifferentiation. Apicidin, a class I histone deacetylase (HDAC3) inhibitor, has been shown to have a potent anti-proliferative effect in a number of cancers. The mechanism of its effect appears to be through activation of the tumor suppressor factor P53. The purpose of this study is to evaluate the effectiveness of Apicidin in preventing recurrent stenosis after vascular reconstruction.
Methods: In vitro assays included proliferation (CellTiter-Glo), migration (scratch assay), and western blotting. In vivo analysis was conducted using a rat model of carotid balloon angioplasty.
Results: Rat SMCs were pretreated (2 hours) with Apicidin and then stimulated with 10% Fetal Bovine Serum (FBS) for 72 hours followed by measurement of proliferation. Based upon an initial dose response curve, we found the optimal concentration of Apicidin to be 500nM, which reduced proliferation by 72.9% ± 5.3% (p < 0.05). Pretreatment of SMCs with Apicidin also reduced SMC migration by 77.1% ± 2.0% (p < 0.01) at 24 hours in response to 10% FBS. We have previously shown that the combination of TGF-β/Smad3 is a potent stimulant of SMC dedifferentiation. To evaluate whether Apicidin can reverse this effect and promote differentiation, SMCs transfected with Smad3 were pretreated for 2 hours with Apicidin and then stimulated with TGF-β (10ng/mL) for 48 hours. Reduction of several proteins indicative of SMC differentiation by TGF-β/Smad3 was rescued by Apicidin by the following percentages: Calponin (22.3%), Myosin Heavy Chain (108.9%), and Smooth Muscle Actin (30.0%). Blots were re-probed for levels of acetylated histone, which were increased 14.3 ± 2.1 fold (p < 0.05), confirming the expected function of the HDAC inhibitor. Finally, adult rats underwent carotid balloon angioplasty with periadventitial application of Apicidin (500µg) in 23% pluronic gel. Cross-sectional carotid samples were harvested at 21 days, yielding preliminary results indicating that Apicidin reduces neointimal area by 55% while preserving luminal and vessel integrity.
Conclusion: The HDAC3 inhibitor, Apicidin, can reduce SMC proliferation and migration, promote SMC differentiation, and subsequently lead to a significant reduction in intimal hyperplasia. Inhibition of HDAC3 could be an effective therapeutic intervention for the prevention of recurrent stenosis leading to improved long-term outcomes in patients treated for vascular disease.