Inhibition of Geranylgeranyltransferase I Decreases Generation of Vascular Reactive Oxygen Species and Increases Vascular Nitric Oxide Production 1 Brian S. Zuckerbraun,* ,2 Joel E. Barbato,* Andrew Hamilton,† Said Sebti,‡ and Edith Tzeng* *Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; †Department of Chemistry, Yale University, New Haven, Connecticut; and ‡Drug Discovery Program, H. Lee Moffit Cancer Center and Research Institute, Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida Submitted for publication June 26, 2004 Background. Vascular injury with endothelial dys- function results in an imbalance between the produc- tion of vasoprotective molecules such as nitric oxide (NO) and deleterious reactive oxygen species (ROS). The purpose of this work was to test the hypothesis that inhibition of geranylgeranyltransferase I (GG Tase I) reduces vascular injury by increasing vascular NO production while decreasing ROS generation. Methods and results. GGTI-298 decreased the forma- tion of intimal hyperplasia at 14 days following bal- loon injury. GGTI-298 (10 M) inhibited activation of RhoA and Rac1 as well as inhibited SMC proliferation. GGTI increased SMC-inducible NO synthase (iNOS) levels and NO production in vitro. Additionally, the activation of NAD(P)H oxidase subunits was de- creased by GGTI in vitro. This correlated with a de- crease in TNF-- or angiotensin-II-induced ROS pro- duction assayed by DCF fluorescence. In vivo, GGTI treatment increased endothelial NOS (eNOS) expres- sion in uninjured arteries and iNOS expression in balloon-injured arteries. Furthermore, GGTI treat- ment attenuated balloon-injury-induced superoxide generation assayed by MCLA luminescence. Conclusions. GGTI decreases the production of ROS and increases the production of NO both in vitro and in vivo. These effects may be mediated via the inhibi- tion of activation of the small GTPases Rac1 and RhoA. Pharmacological inhibition of GGTase I may prove to be a useful clinical adjunct in the treatment of cardio- vascular diseases. © 2005 Elsevier Inc. All rights reserved. Key Words: geranylgeranyltransferase inhibitors; smooth muscle cells; nitric oxide; reactive oxygen species. INTRODUCTION Vascular diseases contribute significantly to morbid- ity and mortality in the United States and Europe. Endothelial disruption and/or dysfunction are central to the pathogenesis of vascular injury, whether as a result of natural disease processes such as hyperten- sion and atherosclerosis or from mechanical interven- tions such as angioplasty or bypass grafting. Damage to the endothelium interrupts the natural homeostasis that occurs within the vessel wall, leading to increased elaboration of cytokines, growth factors, formation of reactive oxygen species, and conversely decreased va- soregulatory nitric oxide (NO) production [1, 2]. The consequence of this disrupted balance is increased smooth muscle cell (SMC) proliferation and migration resulting in the formation of hemodynamically signifi- cant flow-limiting lesions. Augmented production of vascular reactive oxygen species (ROS), mainly through the actions of NAD(P)H oxidase, is thought to contribute to the pathogenesis of vascular diseases [3– 6]. Increased release of ROS has been shown to increase SMC proliferation as well as induce apoptosis [7–9]. NAD(P)H oxidase is a complex of multiple subunits. The activated enzyme consists of cytochrome b558, made up of p22phox and gp91phox subunits, as well as p40phox, p47phox, p67phox, and the small GTPase rac1 [5, 10 –12]. Endothelial dysfunction also results in decreased en- dothelial NO synthase (eNOS) protein levels and di- minished production of NO [2, 13–15]. Additionally, 1 Support provided by NIH R01-HL5785405 (E.T.). 2 To whom correspondence and reprint requests should be ad- dressed at Department of Surgery, NW 607, MUH 3459 Fifth Ave- nue, Pittsburgh, PA 15213. E-mail: zuckerbraunbs@msx.upmc.edu. Journal of Surgical Research 124, 256 –263 (2005) doi:10.1016/j.jss.2004.10.022 256 0022-4804/05 $30.00 © 2005 Elsevier Inc. All rights reserved.