In thc coming decades tbegenesis and treatment ofatherosclrosis wil continue to puzzk and chalknge us. Raman spectroscopy is an eiting new technique abk to inform usabout the composition of the atberosckrotic plaque and ako allowing us to study how the plaque is affected by interventions such as

antiatherosclrotic drugs. Teb experiments performed by Drs Van der Poll and Van der Laarse indicate to us that over time it is likely

thatther$inementsin cateertedhnology will make itposibk to sy tuo effeetsin the intact human heart.

N. Bom and H.J.J. Wellens Directors ofICIN.

EvaluatiOn Of -an erosclmuc dro by Rmans Rm speoopy allows q non-destructive evaluation of intact atherosclerotic plaques. A transgenic mouse strin that expresses a human dysfunctional apolipoprotein variant (APOE*3-ILiden) in combination with Raman spectroscopic ia g of the aortic arch has been used for the integral, quantitative study of pathogenesis and drug treatment of atherosclerosis. This mouse model of hyperlipidaemia and atherosclerosis has been shown to be responsive to lipid-lowering and antiatherosclerotic therapy. The strong atheroprote potential ofatorvastatin is evident in new lesions and existing lesions. The calcium channel blocker amlodipine reduces mineralisation in new lesions. The combination of amlodipe and atorvastin has no apparent synergistic effects on plaque development. By performing intravascular catheter-based Raman spectroscopy, similar cvaluations ofantiatherosderotc herapy may be possible in lavrr animals and humans in vivo. Net


dtat can provide this ldnd ofinformation.6 It provides quanttatve information about the molecular composition of a sample and enables the non-destructive examination of small volumes of tissue, without the need for tissue sectioning, thereby leaving the morphology of the plaque intact. A Raman method to Curretly, evaluation ofchemical changes quantiy ithe relative amounts ofprotein, in atherosclertic plaque compositaon in cholesterol, adventitial fit and calcium huumns in a clinical sig is not possible, salts in human coronary artery wall has as currently available diagnostic tech- been developed previously.7 We have niques such as angiography and intra- extended t model for the study ofdiet vascular ultrasound provide only scarce and lipid-lowering therapy on atheroinformation about the composition of sclerotic plaque fornation in transgenic the atherosclerotic lesion.'*3 Even post mice that were fed a high cholesterol mortem cheical evaluaton of athero- diet. These mice, carrying the dyssclerotic plaque in (small) animals is functional apoE variant from patients with a dominantly inherited form of laborious and tiMe-consuming.4 Most small-animal studies have familial dysbetalipoproteinaemia tracked the progression ofatherosclerosis (APOE*3-Leiden), develop severe by histlogic valuation, therebyvisual- hyperlipidaemia that results in rapid ising the morphological structures of atherosclerotic plaque formation when atherosclerotic lesions5 but providing f&ed a diet enriched in saturated fit and onlylimited information on the chemical cholesterol.5 This paper describes how Raman composition. Moreover, quanficaton of atheroslerosis remains difficult. For sposcopy cnhances the applicability instance, computer-assisted quanti- ofthis athrsclerotic mouse model for fication of positively stained structures the study of progression of atheroin histological sections can be obscured sclerosis and for the evaluation of antiby the nonspecific staining of adventitial atherosclerotic treatment with drugs. fit and by overlying tsue that hinders staining ofstructures in deeper layers. In Of U by addition, decalcification before sectiontn-w ing and staining may lead to inaccurate Raman spectroscopy is an optical techvisuaisaton and quantficaton of calci- nique that can quantify the amount of fications that are present in the plaque. protein, cldksterol and cholesteiyl esters, Likise, quantificain with other tech- calcification and adventital fit from an niques, such as sing intact plaques intact arty, simpFly by analingscattered with Oil red O orvisualising calcification laser light that is reflected from the with von Kossa stain, is of limited value intimal surface ofthe artery. Fora detailed betause lipids and calcium are usually description of the technique and its stained superficially. Clearly, novel possible applications in atherosclerosis, techniques tt can quantify the amount refcr to Van de Poll et al8 oflipids, proteins and calcificaton in an We used Raman spectroscopy for quantification of the amounts of cholatherosclerotic plaque are needecd. Raman spectroscopy is a technique esterol and calcification in the aorta of

Heart Joura Voh= 11, Number 1, Jmnay 20034


the APOE*3-Lciden transgenic mice. In brief, the first 5 to 8 mm of each aorta just distally from the aortic valve was cut open through the outer aortic curvamtum. The samples were positioned undernath a computer-controlled scanning microscope that was equipped for Raman spectroscopic ging. 8 About 300 near infrared Raman spectra were obtained in 0.25 x 0.25 mm steps over the entire length and full width of the opened aorta. Total scanning time of the aorta was approximately 40 minutes. For each location of the grid, the relative contributions ofprotein, total cholesterol (TC, cholesterol plus cholesteryl esters) and calcium salts (CS) in the arterial waliwere derived from the Raman spectra. Maps reflecting the relative distribution of

these components in the aortic samples could then be reconstructed (figure 1). From these Ranan maps, the first 5 mm of the aortic arch were selected for fiurther evaluation. In a first validation study, 3-monthold male mice (n=20) were used to evaluate plaque progression in time. These male mice were randomly assigned to fbur subgroups, which were fed the same high fat/ high cholesterol diet for 0, 2, 4, or 6 months. From the 10


results of this Raman experiment, it appears that the rate of cholesterol accumulation in the atherosclerotic plaque in the period studied (O to 6 months) increased with cholesterol exposure (plasma cholesterol x time) in a semi-logarithmic fashion (figure 2, r=0.87, p1 0% colesrol coxtriution in th/ Raman signal fior mak mice fed a bigb cbolestrol/hi,gb fat (HFC/0.5% choIc) diet fur 0, 2, 4, and 6 Errorb st sr or of mean mobr. wis >O colrol (SEM). 71 cowreatio betwanp accumuation (in mm) andcoeo (CH) pore (in wek) issbown in ti inse Nott larithmc yaxis. (add romb Vasc Biol from: Van de Poll et al. Artrioscler ara




Neterands Hear




Nunmber 1, January 2003

amount of cholesterol quantfied by high

thin layer chromatography (r-0.86, pcO.OOl), or quantified by staining the plaque with Oil red 0

(r=0.88, pc0.001).10 Thus, Raman spectroscopy appears to be an accurate tool for quantification of cholesterol and calcification in the atheoscerotic plaque. The approach has many advantages over conventional techniques, such as the non-destructive and quantitative nature ofthe technique. In addition, the apoE*3-Leiden mouse model shows excellent correspondence with human atherosclerosis, including the development of a lipid core and calcification. It is a valuable model for studying hyperlipidaeniua and atherosclerosis and has shown to be responsive to lipid-lowering and antiatheroslerotic therapy." -13

Therefore, we further investigated the potential ofthe combination Raman spectroscopy andAPOE*3-Leiden mice for the study of the ef&cts of drugs on the progession and regression ofatherosClerosis.10"14 of unipine, atowastati atln Of both on wids Sid li st From a retrospective analysis of the REGRESS trial, it was hypothesised that

the combination ofa statin (pravastatin) and any calcum channe blocker might be more potent in retarding the progression of atherosclerosis than pravastatin alone.5 Accordingly we designed e





and specf the potentally synergistic effect of the HMG-CoA reductase inhibitor atorvastatin with the dihydropyridine CCB amlodipine, as compared with single drug treatment and no drug tatment, on the development and chemical composition of atherosclerotic plaques in APOE*3 Leiden transgenic mice. These studies were named CASSIA (Calcium Antagonist and Statin Synergy in Atherosdcerosis). In these studies, we investigated the eflcts of the dmgs both on early developing atherosclerosis (CASSIA-1, for new lesions) and on advanced atherosclerotic plaque (CASSIA-2, for exsingesions), thereby trying to mimic the human situation. In the CASSIA-1 study, 28 APOE*3 Leiden transgenic mice were fed a high to


fiLt/high cholesterol diet for 28 weeks.'0 These mice were assigned to a control grciup receiving the diet alone or to groups that reccived the diet with either 0.01% w/w atorvastatin, 0.002% w/w amlodipine, or the combination. When mice had been treated with atorvastatin, contents ofcholesterol and calcification in the aortic arch were reduced by 91% and 98%, respectively, (both pc0.001). Amlodipine did not reduce the cholesterol content but reduced calcification of the aorta by 69% (p10% cholesterol or >10% calcification in its Reman spectrumY.14 # p

Evaluation of antiatherosclerotic drugs by Raman spectroscopy.

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