Nitro-linoleic acid inhibits vascular smooth muscle cell proliferation via the Keap1/Nrf2 signaling pathway

1 Department of Internal Medicine, Cardiovascular Center, University of Michigan Medical Center, Ann Arbor, Michigan; 2 Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta; 3 Atherogenics Inc., Alpharetta, Georgia; and 4 Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Submitted 2 March 2007 ; accepted in final form 27 April 2007 Nitroalkenes, the nitration products of unsaturated fatty acids formed via NO-dependent oxidative reactions, have been demonstrated to exert strong biological actions in endothelial cells and monocytes/macrophages; however, little is known about their effects on vascular smooth muscle cells (VSMCs). The present study examined the role of nitro-linoleic acid (LNO 2 ) in the regulation of VSMC proliferation. We observed that LNO 2 inhibited VSMC proliferation in a dose-dependent manner. In addition, LNO 2 induced growth arrest of VSMCs in the G 1 /S phase of the cell cycle with an upregulation of the cyclin-dependent kinase inhibitor p27 kip1 . Furthermore, LNO 2 triggered nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and activation of the antioxidant-responsive element-driven transcriptional activity via impairing Kelch-like ECH-associating protein 1 (Keap1)-mediated negative control of Nrf2 activity in VSMCs. LNO 2 upregulated the expression of Nrf2 protein levels, but not mRNA levels, in VSMCs. A forced activation of Nrf2 led to an upregulation of p27 kip1 and growth inhibition of VSMCs. In contrast, knock down of Nrf2 using an Nrf2 siRNA approach reversed the LNO 2 -induced upregulation of p27 kip1 and inhibition of cellular proliferation in VSMCs. These studies provide the first evidence that nitroalkene LNO 2 inhibits VSMC proliferation through activation of the Keap1/Nrf2 signaling pathway, suggesting an important role of nitroalkenes in vascular biology. nitroalkenes; nuclear factor-erythroid 2-related factor 2; Kelch-like ECH-associating protein 1 Address for reprint requests and other correspondence: T. Cui, 1150 W. Medical Ctr. Dr., MSRB III 7200, Ann Arbor, MI 48109 (e-mail: tcui{at}umich.edu )