Abstract 364: Pioglitazone Inhibition of Vascular Smooth Muscle Cell Proliferation is Associated with Activation of AMP-activated Protein Kinase but not Peroxisome Proliferator-Activated Receptor Gamma

Abstract

Atherosclerosis and restenosis after angioplasty are associated with exaggerated proliferation of vascular smooth muscle cells (VSMCs). Pioglitazone (PIO), an antidiabetic drug that belongs to the family of peroxisome proliferator-activated receptor gamma (PPARg) agonists, reduces intimal hyperplasia after coronary angioplasty in diabetic and nondiabetic subjects. However, the molecular mechanisms by which PIO regulates VSMC phenotype remain unclear. The objective of the current study is to determine the role of AMP-activated protein kinase (AMPK, a cellular energy sensor) versus PPARg in PIO-induced changes in VSMC phenotype. Exposure of human aortic VSMCs to PIO followed by immunoblot analysis revealed a robust increase in the phosphorylation of AMPK (Thr172) and its downstream effector, Acetyl-CoA carboxylase (p < 0.05, n = 3), as a function of concentration (3 to 30 μM) and time (1 to 48 hr). Notably, PIO (30 μM, 48 hr) activation of AMPK remained essentially the same after PPARg downregulation (~90%) by target-specific siRNA (500 pmoles), suggesting lack of dependency on PPARg. In addition, PIO activation of AMPK was accompanied by induction of mitochondrial membrane depolarization with a trend toward an increase in AMP/ATP ratio (~40%), as revealed by tetramethylrhodamine methyl ester (TMRM) fluorescent redistribution & LC-MS/MS MRM analysis, respectively. Importantly, PIO pretreatment (30 μM, 30 min) resulted in significant inhibition of PDGF (30 ng/ml, 48 hr)-induced VSMC proliferation by ~85% (cell count) and the associated increases in proliferative markers (cyclin D1 and pRb) by > 95% (p < 0.05, n = 3). Downregulation of AMPKa1 (~80%) by target-specific AMPKa1 siRNA (500 pmoles) revealed that PIO-mediated AMPK activation inhibited mTORC1 signaling by > 95% & upregulated cell cycle inhibitor p27Kip1 by ~40%, thus accounting for PIO-mediated changes in VSMC phenotype. In vivo, PIO administration (10 mg/Kg/d, oral gavage) for 3 weeks in CJ57BL6 mice mitigated neointimal growth by ~60% in guide wire-injured femoral artery (p < 0.05, n = 8). In conclusion, the present study provides the first direct evidence for VSMC-specific AMPK activation by PIO, which may in part contribute to its vasoprotective effects in nondiabetic state.