Raman spectroscopic investigation of atorvastatin, amlodipine, and both on atherosclerotic plaque development in APOE*3 Leiden transgenic mice Sweder W.E. van de Poll a,b,1 , Dianne J.M. Delsing 1, c , J. Wouter Jukema a , Hans M.G. Princen c , Louis M. Havekes c , Gerwin J. Puppels d , Arnoud van der Laarse a,b, * a Department of Cardiology, C5-P, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands b Interuniversity Cardiology Institute of the Netherlands, Utrecht, The Netherlands c Gaubius Laboratory, TNO-PG, Leiden, The Netherlands d Laboratory for Intensive Care Research and Optical Spectroscopy, Erasmus University Rotterdam, Rotterdam, The Netherlands Received 13 February 2001; received in revised form 6 February 2002; accepted 15 February 2002 Abstract Raman spectroscopy allows quantitative, non-destructiveevaluation of entire, intact atherosclerotic plaques. We quantified the anti-atherosclerotic effects of atorvastatin and amlodipine on progression of atherosclerosis using post-mortem Raman spectro- scopic plaque imaging in 28 APOE*3 Leiden transgenic mice who were fed a high fat/high cholesterol diet for 28 weeks. Mice were assigned to a control group receiving the diet alone or to groups that received the diet with either 0.01% w/w atorvastatin, 0.002% w/ w amlodipine, or the combination. The entire excised aortic arch was scanned with Raman microspectroscopy for quantitation of the distribution of cholesterol and calcification content. When mice had been treated with atorvastatin, cholesterol accumulation and calcification in the aortic arch was reduced by 91 and 98%, respectively, (both P B/0.001). Amlodipine did not reduce the cholesterol content but reduced calcification of the aorta by 69% (P B/0.05). The combination of amlodipine and atorvastatin was as effective as atorvastatin alone. This study demonstrates the strong atheroprotective potential of atorvastatin. In addition it is demonstrated that amlodipine reduces mineralization of atherosclerotic plaque. No synergistic effect of the combination of amlodipine and atorvastatin on plaque development is demonstrated. This study encourages Raman spectroscopic evaluations of anti-atherosclerotic drugs in larger animals and humans in vivo. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Statins; Calcium channel inhibitors; Cholesterol; Calcification; Plaque; Raman spectroscopy 1. Introduction Clinical trials with 3-hydroxy-3-methylglutaryl coen- zyme A (HMG-CoA) reductase inhibitors (statins) have shown highly significant reductions in the risk of acute coronary events in both primary and secondary preven- tion. The beneficial effect of statin therapy is thought to be caused primarily by its ability to reduce circulating cholesterol levels, although its putative benefits may include additional cholesterol-independent effects lead- ing to plaque stabilization [1,2]. Apart from lowering plasma cholesterol levels with statins, many investigations have lent support to the view that a number of key processes in atherosclerosis are calcium dependent and may be influenced by calcium channel blockers (CCBs) [3  /9]. From these studies performed in animals and humans, we can conclude that some aspects of atherosclerosis are inhibited by CCBs. In patients allocated to CCB therapy (amlodipine), a recent prospective randomized trial [9] found no angiographic evidence of inhibition of plaque progression. However, in this trial, a 31% reduction of * Corresponding author. Tel.:  /31-71-526-2020; fax:  /31-71-526- 6809. E-mail address: a.van_der_laarse@lumc.nl (A. van der Laarse). 1 Both authors contributed equally to this work. Atherosclerosis 164 (2002) 65  /71 www.elsevier.com/locate/atherosclerosis 0021-9150/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII:S0021-9150(02)00055-2