The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway

1 Department of Pharmacology and Toxicology, Dartmouth Medical School; and 2 Department of Surgery, Section of Vascular Surgery, Dartmouth-Hitchcock Medical Center; and 3 Department of Medicine, Section of Cardiology, Dartmouth Medical School, Lebanon, New Hampshire Submitted 1 September 2005 ; accepted in final form 14 November 2005 Recent studies of cyclooxygenase-2 (COX-2) inhibitors suggest that the balance between thromboxane and prostacyclin is a critical factor in cardiovascular homeostasis. Disruption of prostacyclin signaling by genetic deletion of the receptor or by pharmacological inhibition of COX-2 is associated with increased atherosclerosis and restenosis after injury in animal models and adverse cardiovascular events in clinical trials (Vioxx). Human vascular smooth muscle cells (VSMC) in culture exhibit a dedifferentiated, migratory, proliferative phenotype, similar to what occurs after arterial injury. We report that the prostacyclin analog iloprost induces differentiation of VSMC from this synthetic, proliferative phenotype to a quiescent, contractile phenotype. Iloprost induced expression of smooth muscle (SM)-specific differentiation markers, including SM-myosin heavy chain, calponin, h-caldesmon, and SM -actin, as determined by Western blotting and RT-PCR analysis. Iloprost activated cAMP/protein kinase A (PKA) signaling in human VSMC, and the cell-permeable cAMP analog 8-bromo-cAMP mimicked the iloprost-induced differentiation. Both myristoylated PKA inhibitor amide-(14–22) (PKI, specific PKA inhibitor), as well as ablation of the catalytic subunits of PKA by small interfering RNA, opposed the upregulation of contractile markers induced by iloprost. These data suggest that iloprost modulates VSMC phenotype via G s activation of the cAMP/PKA pathway. These studies reveal regulation of VSMC differentiation as a potential mechanism for the cardiovascular protective effects of prostacyclin. This provides important mechanistic insights into the induction of cardiovascular events with the use of selective COX-2 inhibitors. iloprost; smooth muscle-specific differentiation markers; phenotypic modulation; signal transduction; intimal hyperplasia Address for reprint requests and other correspondence: K. A. Martin, Section of Vascular Surgery, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, 1 Medical Center Dr., Lebanon, NH 03756 (e-mail: kathleen.a.martin{at}dartmouth.edu )