Flufenamic Acid: Growth Modulating Effects on Human Aortic Smooth Muscle Cells In Vitro Wolfgang Schöber, MD, Jakub Wiskirchen, MD, Rainer Kehlbach, Regina Gebert, Enno Rodegerdts, MD, Angelika Betsch, MD, Ursula Johst, MD, Claus D. Claussen, MD, and Stephan H. Duda, MD PURPOSE: The aim of the study was to examine the effects of flufenamic acid on proliferation, clonogenic activity, migratory ability, cell-cycle distribution, and p44/42-mitogen–activated protein kinase (MAPK) expression on serum- stimulated human aortic smooth muscle cells (haSMCs) in vitro. MATERIALS AND METHODS: HaSMCs were treated with flufenamic acid in three different doses (40 mol/L, 200 mol/L, 400 mol/L) for 4 days, and then flufenamic-acid–free culture medium was supplemented every 4 days until day 20 after initial treatment. The growth kinetics were assessed. Cell-cycle analysis was performed by flow cytometry. The clonogenic activity was evaluated with use of colony formation assays. The migratory ability was investigated by stimulation with platelet derived growth factor (PDGF-BB) in 24 well plates with 8-m pore membrane inserts. The p44/42 MAPK was detected by Western blot technique. RESULTS: Flufenamic acid inhibited the proliferation (400 mol/L treatment over 4 d; 179,700  49,800 vs 747,900  144,000; P < .001), clonogenic activity (400 mol/L treatment over 4 d; 1  0.3 vs 50  1.4; P < .001) and migratory ability (400 mol/L treatment over 4 d; 8 cells  2 vs 48 cells  15; P < .001) of haSMCs in a dose-dependent manner. Cell-cycle analysis revealed a G2/M-phase block (400 mol/L treatment over 4 d; 28.9  1.5 vs 9.5  3.2; P < .001). The expression of p44/42 MAPK was reduced for a treatment with 400 mol/L flufenamic acid (controls, 427 BLU  0.305 vs treatment group, 190 BLU  106; P < .05) CONCLUSION: Flufenamic acid inhibits the proliferation and migration of haSMCs. Further experiments with animal models concerning stenosis and restenosis are necessary to evaluate the potential of this promising drug. Index terms: Blood vessels, stenosis or obstruction • Flufenamic acid • Restenosis • Smooth muscle cells J Vasc Interv Radiol 2002; 13:89 –96 Abbreviations: BLU  Boehringer light units, haSMC  human aortic smooth muscle cell, MAPK  mitogen activated protein kinase, PDGF  platelet de- rived growth factor, PTA  percutaneous transluminal angioplasty, SMC  smooth muscle cell CHARLES Dotter first described the original technique of percutaneous stent placement for revascularization of peripheral arterial occlusive disease (1). Restenosis after primary success- ful percutaneous transluminal angio- plasty (PTA) remains the major prob- lem, limiting the long-term efficacy of the procedure in as many as 30%–50% of cases within the first 6 months (2,3). A hyperplastic intimal process is mainly responsible for restenosis, in- volving smooth muscle cell (SMC) proliferation, migration, and the pro- duction of extracellular matrix (4). This cascade occurs after PTA with an inflatable balloon because this proce- dure is associated with endothelial de- nudation and early accumulation of platelets and fibrin (5,6) after splitting of the intima and media, stretching the media, and overdistending the adven- titia (7,8). Because of the endothelial damage and inflammatory processes a release of cytokines is induced, fol- lowed by aggregation and thrombus formation of platelets (9,10). Above all, platelet-derived growth factor (PDGF), released from platelets, SMCs, and en- dothelial cells, is known to play a piv- otal role in the further processes in neo- intimal formation, such as proliferation and migration of SMCs (11). This phase decreases 2– 4 weeks after PTA and any further luminal narrowing is produced by the extracellular matrix. Therapeutic aims against pathogenesis of restenosis are application of antithrombotic agents and antiplatelet agents for preventing mural thrombus formation. To reduce the neointimal prolifera- tion, antiproliferative agents, either systemically or locally applied, have been investigated so far (12). Most of the compounds failed to show benefi- cial effects in reducing restenosis in clinical trials. The aim of this study is to investi- gate the antiproliferative, antimigra- tory potential in vitro of flufenamic acid, a compound already on the phar- From the Department of Diagnostic Radiology, Eberhard-Karls-Universität, Hoppe-Seyler-Str. 3, 72076 Tübingen, Germany. Received June 1, 2001; revision requested July 16; revision received and accepted August 29. Address correspondence to W.S., E-mail: schoeber.wogi@t-online.de © SCVIR, 2002 Laboratory Investigations 89