Atorvastatin reduces the expression of cyclooxygenase-2 in a rabbit model of atherosclerosis and in...

Atherosclerosis 160 (2002) 49–58

Atorvastatin reduces the expression of cyclooxygenase-2 in arabbit model of atherosclerosis and in cultured vascular smooth

muscle cells

Miguel Angel Hernandez-Presa a, Jose Luis Martın-Ventura a, Monica Ortego a,Almudena Gomez-Hernandez a, Jose Tunon b, Purificacion Hernandez-Vargas a,Luis Miguel Blanco-Colio a, Sebastian Mas c, Cesar Aparicio a, Luis Ortega d,

Fernando Vivanco c, Juan Gomez Gerique e, Cristina Dıaz f, Gonzalo Hernandez f,J. Egido a,*

a Vascular Research Laboratory, Fundacion Jimenez Dıaz, Autonoma Uni�ersity, A�enida Reyes Catolicos 2, 28040 Madrid, Spainb Department of Cardiology, Fundacion Jimenez Dıaz, Autonoma Uni�ersity, Madrid, Spain

c Department of Immunology, Fundacion Jimenez Dıaz, Autonoma Uni�ersity, Madrid, Spaind Department of Pathology, Hospital Clınico San Carlos, Madrid, Spain

e Department of Biochemistry, Fundacion Jimenez Dıaz, Autonoma Uni�ersity, Madrid, Spainf Pfizer Spain, Madrid, Spain

Received 15 December 2000; received in revised form 4 April 2001; accepted 17 April 2001

Abstract

Inflammation is involved in the genesis and rupture of atherosclerotic plaques. We assessed the effect of atorvastatin (ATV) onthe expression of cyclooxygenase-2 (COX-2) and other proinflammatory molecules in a rabbit model of atherosclerosis. Fourteenanimals underwent injury of femoral arteries and 2 weeks of atherogenic diet. Afterwards, they were randomized to receive either5 mg/kg per day of ATV (n=8) or no treatment (NT, n=6) during 4 weeks, and were finally killed. ATV reduced lipid levels,neointimal size (0.13 (0.03–0.29) mm2 vs 0.65 (0.14–1.81) mm2, P=0.005) and the percentage of neointimal area positive formacrophages (1% (0–3) vs 19% (5–32), P=0.001), COX-2 (32% (23–39) vs 60% (37–81) P=0.019), interleukin-8 (IL-8) (23%(3–63) vs 63% (25–88) P=0.015), and metalloproteinase-3 (19% (12–34) vs 42% (27–93), P=0.010), without significantdifferences in COX-1 expression (immunohistochemistry). In situ hybridization confirmed a decreased expression of COX-2mRNA (22% (5–40) vs 43% (34–59) P=0.038). The activity of nuclear factor-�B, which controls many proinflammatory genesincluding COX-2, was reduced in atherosclerotic lesions (3538 (2663–5094) vs 8696 (5429–11 312)) positive nuclei per mm2,P=0.001) and circulating mononuclear cells (2966 vs 17 130 arbitrary units). In cultured vascular smooth muscle cells, ATVreduced the expression of COX-2 mRNA induced by IL-1� and TNF-� without affecting COX-1 expression. In conclusion, ATV,besides decreasing a number of inflammatory mediators in the atherosclerotic lesion, significantly downregulates COX-2 both invivo and in vitro. These anti-inflammatory actions could partially account for the reduction of acute coronary events achieved bystatins. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Atherosclerosis; Macrophages; Cyclooxygenase-2; Interleukin-8; Atorvastatin

www.elsevier.com/locate/atherosclerosis

1. Introduction

Inflammation is involved in the genesis, rupture andthrombosis of atherosclerotic plaques [1,2]. Inflamma-tory cells release metalloproteinases (MMP) and otherenzymes that degrade matrix proteins and weaken thefibrous cap of atherosclerotic lesions making them moreprone to rupture and subsequent thrombosis [3–7].

* Corresponding author. Tel.: +34-91-550-4800x3362; fax: +34-91-549-4764.

E-mail address: [email protected] (J. Egido).

0021-9150/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.PII: S 0 0 2 1 -9150 (02 )00547 -0

M.A. Hernandez-Presa et al. / Atherosclerosis 160 (2002) 49–5850

In a rabbit model of atherosclerosis, we demon-strated previously that lipid reduction with the 3-hy-droxy-3-methylglutaryl coenzyme A (HMG-CoA)reductase inhibitor atorvastatin (ATV) decreasesneointima formation and macrophage infiltration [8].This last finding was attributed to a reduction in theexpression of the monocyte chemoattractant protein-1(MCP-1) and in the activation of nuclear factor-kappaB (NF-�B), the transcription factor that controls MCP-1 expression. However, there are a number of moleculesinvolved in cell recruitment, such as cyclooxygenase-2(COX-2). This proinflammatory enzyme is being thefocus of increasing research in rheumatic diseases butits modulation by statins in atherosclerosis has not yetbeen explored.

Cyclooxygenase is an enzyme involved in arachidonicacid conversion into prostaglandins (PG). Two iso-forms of this enzyme have been described [9,10]: COX-1 and COX-2. COX-1 is expressed constitutively inalmost all tissues in basal conditions and has homeo-static effects [11]. COX-2 is an inducible enzyme that isexpressed in response to inflammatory cytokines and isabsent in most normal tissues [12]. It is responsible forthe synthesis of proinflammatory PGs such as PGE2,which is chemoattractant for leukocytes [12], and itsinhibition could fully account for the beneficial effectsof non-steroidal anti-inflammatory drugs in arthritis[13–15]. Its expression has been found increased inrheumatic inflammatory disorders [11] and, more re-cently, in the atherosclerotic lesions [16,17].

Given that COX-2 expression is controlled by NF-�B[18], it is conceivable that the reduction of the activityof this transcription factor by statins may lead to adecrease in COX-2 expression. Then, in a rabbit modelof atherosclerosis, we have explored the effects of theHMG-CoA reductase inhibitor ATV on the expressionof COX-2, as well as on macrophage infiltration, andinterleukin-8 (IL-8) and MMP expression. Concomi-tantly, we have assessed the effect of the statin onNF-�B activity in atherosclerotic lesions and in circu-lating mononuclear cells. Finally, we have studied theeffect of ATV on COX-2 expression in cultured vascu-lar smooth muscle cells (VSMC).

2. Methods

2.1. Studies in the rabbit model of atherosclerosis

2.1.1. Experimental modelFourteen New Zealand male rabbits (3.5–4 kg) were

used. The animals were housed in individual cages andquarantined for 7 days before use. On day 0, a 2%cholesterol and 6% peanut oil diet (Letica, Barcelona,Spain) was started. One week later, vascular injury wasinduced in the femoral arteries using nitrogen as previ-

ously described [19]. After 1 week, the animals wererandomized to receive either 5 mg/kg per day ATVmixed with chow (ATV, n=8) or no treatment (NT,n=6), keeping the atherogenic diet in all cases. Fourweeks later, they were killed. Five rabbits fed standardchow and with no intervention were included in thestudy as healthy controls.

At sacrifice, the animals were anesthetized and thefemoral arteries exposed. Then, they were killed with anoverdose of pentobarbital (Abbot, Madrid, Spain) andthe femoral arteries were fixed by perfusion of thedescending aorta with 4% buffered formaldehyde at 100mmHg. The femoral arteries were removed and keptfor 24 h in 4% buffered formaldehyde and afterwards in70% ethanol until paraffin embedded.

2.1.2. Serum chemistryRabbits were bled from a marginal ear vein 24 h

post-meal on day 0 and at the end of weeks 1, 2(randomization) and 6 (sacrifice) of the study. Totalcholesterol, chylomicrons, low-density lipoproteins(LDL), high-density lipoproteins (HDL), very low-den-sity lipoproteins (VLDL), intermediate-density lipo-proteins (IDL) and triglycerides were measured byenzymatic assays (Sigma Diagnostics, Madrid, Spain).

2.1.3. Histological analysis

2.1.3.1. Morphometric analysis. The morphometricanalysis was performed on hematoxilin stained arterialpreparations. Sections with the maximal lesion werechosen for quantification.

2.1.3.2. Immunohistochemistry. Paraffin-embedded ar-teries were cross-sectioned into 4 �m-thick pieces at5-mm intervals, dewaxed and rehydrated. Formacrophage and VSMC identification, a monoclonalanti-rabbit macrophage antibody (RAM11, DAKO,Denmark) and �-actin antibody (HHF-35, Sigma) wereapplied as described previously [20]. COX-1, COX-2and IL-8 were detected with polyclonal goat anti-hu-man antibodies (Santa Cruz Biotechnology, SantaCruz, CA). MMP-1, MMP-2 and MMP-3 proteinswere immunolocalized by using monoclonal mouseanti-human antibodies (Oncogene, Cambridge, UK) (5�g/ml). As secondary antibody, a donkey anti-goatperoxidase (The Binding Site, Birmingham, UK) wasused for COX-2, a donkey anti-goat IgG biotin labeled(Amersham Iberica S.A, Spain) for IL-8 and COX-1,and a goat anti-mouse IgG biotin labeled (DAKO) forRAM11, HHF-35 and MMP-1, MMP-2 and MMP-3.Anti-COX-1 secondary antibodies were applied for 1 hat 1:100 dilution and the remaining, for 30 min at 1:200dilution. Then, ABComplex/HRP (DAKO) was addedfor a further 30-min period. The sections were stainedfor 10 min at room temperature with 3,3�-diaminoben-

M.A. Hernandez-Presa et al. / Atherosclerosis 160 (2002) 49–58 51

zidine (DAKO) and were finally counterstained withhematoxylin and mounted in Pertex (Medite, Ger-many). In each experiment, negative controls withoutthe primary antibody or using a non-related antibodywere included to check for non-specific staining. Forquantification by image analysis, the sections with themost severe lesion in each animal were chosen. Resultswere expressed as immunostained area.

2.1.3.3. In situ hybridization. Hybridization probes wereobtained from reverse transcriptase-polymerase chainreaction (RT-PCR) products at 35 cycles and 58 °C.Primer sequences were: (anti-sense) 5�CCATTCAGG-ATGCTCCTGTT3� and (sense) 5�TCTTTGCCCA-AGCACTTCAC3�. The RT-PCR products were in-serted in the P-gem T-easy vector and transfected to E.coli, according to the manufacturer’s instructions(Promega, Madison, WI). In order to assure the properprobe and the correct sense of the insert, sequentiationof vector was performed. The vector was linearized andsimultaneously transcribed to RNA and labeled withdigoxigenin with the DIG RNA labeling kit.

The arterial sections were treated as previously de-scribed [21] and hybridization was conducted at 55 °Cwith 3 ng/�l denatured digoxigenin-labeled riboprobe.Negative controls consisted of matched serial sectionshybridized with sense probe or pretreated with 25 �g/mlRNAase A.

2.1.3.4. Southwestern histochemistry. The distributionand DNA-binding activity of NF-�B in situ was de-tected as described in our laboratory [22], using adigoxigenin labeled double stranded DNA probe with aspecific consensus sequence which binds to NF-�B (5�-AGTTGAGGGGACTTTCCCAGGC-3�). Preparationswithout probe were used as a negative control and totest the specificity of the technique a mutant NF-�Bprobe was used (5�-AGTTGAGGCTCCTTTCCC-AGGC-3�).

2.1.3.5. Image analysis. Preparations were digitized viaan Olympus microscope (BH-2) connected to a CCDvideo camera as previously described [8]. Image analysiswas performed using the Olympus software. For mor-phometric analysis, intimal area was measured. In im-munohistochemistry and in situ hybridizationspecimens, the percentage of neointima staining positivewas evaluated. In southwestern preparations, nucleistaining positive per mm2 were assessed.

2.1.4. Studies in circulating mononuclear cells

2.1.4.1. Isolation of peripheral blood mononuclear cells.Thirty milliliter of blood were drawn from the ear vein

at sacrifice. Blood samples were diluted in phosphatebuffered saline (PBS) 1:1 and the cells were separated in5 ml ficoll gradient (lymphocyte isolation solution,Rafer, Spain) by centrifugation at 2000×g for 30 min.Peripheral blood mononuclear cells were collected,washed twice with cold PBS and resuspended in bufferA for later process.

2.1.4.2. Electrophoretic mobility shift assay. Protein ex-tracts from mononuclear cells were prepared as previ-ously described [19]. Protein concentration wasquantified by the BCA method (Pierce, Rockford, IL).NF-�B consensus oligonucleotide (5�-AGTT-GAGGGGACTTTCCCAGGC-3�) was 32P-end-labeledwith 10 U of T4 polynucleotide kinase (Promega). Fortesting of specificity, binding antibodies against the p65and p50 subunits of NF-�B (Santa Cruz Biotechnol-ogy) were incubated for 1 h, or a 100-fold excess ofunlabeled probe was added to the binding reaction,leading to a decrease in NF-�B binding to the labeledprobe. Quantification was performed by densitometricanalysis, yielding the results in arbitrary units.

2.2. In �itro studies

2.2.1. Cell cultureRat VSMC were isolated and cultured as previously

described [19]. Cells were growth-arrested by incubationin free serum medium for 48 h, and then incubated withthe corresponding stimuli. For the inhibition experi-ments, ATV was added to the culture medium 1 h priorto the stimuli.

2.2.2. RNA extraction, re�erse transcriptase-polymerasechain reaction

Total RNA from different experimental conditionswas obtained by Trizol method (Life technologies) andquantified by absorbance at 260 nm in duplicate. ForRT-PCR, 100 ng of RNA from different experimentalconditions were applied to the access RT-PCR System(Promega). The following primers were used for COX-2: anti-sense: 5�-CCATTCAGGATGCTCCTGTT-3�and sense: 5�-TCTTTGCCCAGCACTTCAC-3�, whichyielded products of 371 bp (1 min at 58 °C for anneal-ing of the primers, 32 cycles), COX-1: anti-sense: 5�-CTTCAGCAGGTCACACACAC-3� and sense:5�-GTTTGCCTTCTTTGCACAACAC-3� (1 min at59 °C for annealing primers, 30 cycles), which yieldedproducts of 340 bp and G3PDH: anti-sense: 5�-AATG-CATCCTGCACCACCAA-3� and sense: 5�-ATACT-GTTACTTATACCGATG-3�, which yielded productsof 515 bp (1 min at 58 °C for annealing primers, 25cycles). The DNA products from RT-PCR reactionswere analyzed on a 4% polyacrylamide/urea gel in the


same buffer. The polyacrylamide gels were dried, ex-posed to X-ray film, and scanned using the ImageQuantdensitometer.

2.3. Statistical analysis

Statistical analysis was performed with GraphPADInStat (GraphPAD Software). Lipid values, morpho-metric analysis, immunohistochemistry, in situ hy-bridization, and PCR data are presented asmean�S.D. and range and were analyzed by the UMann–Whitney test. When multiple comparisons wereneeded, the Kruskal–Wallis test was used. Elec-trophoretic mobility shift assay (EMSA) data for NF-�B activity in blood mononuclear cells were obtainedfrom the analysis of pools of proteins from cellularextracts of every group of animals, thus, yielding nu-merical values without S.D. where no statistical test canbe performed.

3. Results

3.1. Plasma lipid le�els and morphometric analysis of thelesions

At the moment of randomization (second week), theatherogenic diet caused a marked increment in all lipidparameters, except in HDL levels (Table 1). At the endof the study, the ATV group showed significantly lowerlevels of total cholesterol, VLDL and chylomicronsthan in the NT group (Table 1). There were no signifi-cant differences in triglycerides, IDL, LDL and HDLlevels between both groups. It is important to remem-ber that in rabbits most of atherogenic lipid fractionsare associated with VLDL.

Maximal intimal areas were reduced in ATV withrespect to NT animals: 0.13�0.12 (range 0.03–0.29) vs0.65�0.60 (0.14–1.81) mm2 (P=0.005).

3.2. Expression of cyclooxygenase isoforms in the lesions

The percentage of neointimal area with positive im-munostaining for COX-2 was reduced in ATV withrespect to NT rabbits (32�6% (23–39) vs 60�22%(37–81), P=0.019) (Fig. 1). Positive immunostainingfor COX-2 colocalized both with macrophages andsmooth muscle cells (Fig. 2). In situ hybridization confi-rmed a significant diminution in COX-2 mRNA inATV animals (22�12% (5–40) vs 43�11% (34–59),P=0.038) (Fig. 1). Healthy rabbits were negative forthe presence of COX-2 (protein and mRNA).

COX-1 immunohistochemistry was positive in theintima and media of the arteries of healthy animals(Fig. 1). Its expression was unchanged in the atheroscle-rotic lesions of NT animals and ATV did not affect it(healthy rabbits=39�22% (12–63), NT=35�8%(25–44), and ATV=29�20% (11–57), P=0.673).

3.3. Expression of interleukin-8, macrophage infiltrationand expression of metalloproteinases in the plaques

Positive immunostaining for IL-8 was markedly re-duced in ATV animals when compared with NT ani-mals (23�25% (3–63) vs 63�22% (25–88) ofneointimal area; P=0.015) (Fig. 3). Control animalsdid not show positive staining for IL-8.

Positive staining with RAM11 for macrophages wasstrongly reduced in the ATV group when comparedwith NT animals (1�1% (0–3) vs 19�12% (5–32) ofthe neointimal area respectively; P=0.001) (notshown).

Positive staining for IL-8 colocalized both withmacrophages and smooth muscle cells, although it wasmore marked in macrophage-positive areas (Fig. 2).

MMP-3 expression was significantly lower in ATVthan in NT animals (19�7% (12–34) vs 42�28%(27–93), P=0.010) (Fig. 4). There was a trend towards

Table 1Lipid values (mg/dl)

Day 0 1 Week 2 Weeks 6 Weeks

NT ATV

4140�479 (3450–4719)2303�10571670�66985�17 2730�690* (1700–3680)TC14�2VLDL 562�279 826�395 2006�422 (1551–2789) 1317�502** (380–1932)

TG 107�38 135�76 183�116 201�66 (93–290) 159�65 (63–245)Chylom 0 625�343 924�551 1430�582 (884–2336) 548�373*** (155–1119)

10�6 208�85IDL 293�82 438�175 (271–729) 405�152 (183–581)220�76 310�97 314�96 (183–444)LDL 386�113 (216–492)67�12

37�8 34�11 39�10 44�9 (33–56) 44�9 (35–62)HDL

Data are displayed as mean�S.D. (range). ATV, atorvastatin-treated animals; Chylom, chylomicrons; HDL, high-density lipoproteins; IDL,intermediate-density lipoproteins; LDL, low-density lipoproteins; NT, non-treated animals; TC, total cholesterol; TG, triglycerides; VLDL, verylow-density lipoproteins.

* P=0.001 vs NT; **P=0.020 vs NT; ***P=0.022 vs NT.


Fig. 1. Effect of ATV on COX-1 and COX-2 expression in the atherosclerotic lesions. (A) Representative examples of COX-1 and COX-2immunohistochemistry (IH) and COX-2 in situ hybridization (ISH) in non-treated (NT), ATV and healthy animals. Positivity for COX-2 andCOX-1 IH is displayed in brown and for ISH in violet (magnification, 400× ). (B) Graph bar. Arteries of healthy animals do not display COX-2expression, which is strongly increased in the lesions of NT atherosclerotic group (IH and ISH). ATV significantly reduced protein and mRNAexpression of COX-2 in atherosclerotic animals (mean�S.D.; (*)P=0.038 and (**)P=0.019). COX-1 protein is present in the intima and mediaof healthy arteries and is not modified in NT and ATV animals.

a reduction in the expression of MMP-1 and MMP-2 inthe ATV animals with respect to NT animals, but it didnot reach statistical significance (43�24% (0–72) vs56�7% (45–64) and 14�13% (2–35) vs 32�20%(7–59), respectively). Control animals did not showpositivity for either macrophages or MMPs.

3.4. NF-�B acti�ation in the lesions and in circulatingmononuclear cells

The number of nuclei staining positive per mm2 forNF-�B activation with southwestern histochemistrywas markedly reduced by ATV (3538�802 (2663–


Fig. 2. Colocalization by immunohistochemistry for smooth muscle cells (HHF-35), macrophages (RAM-11), COX-2 and IL-8. Contiguoussections show that COX-2 colocalizes with smooth muscle cells and macrophages. IL-8 is also localized in regions staining positive for both celltypes, although this is more marked in macrophage area (magnification, 200× ).Fig. 3. Effect of ATV on IL-8 expression in the atherosclerotic lesions (immunohistochemistry). ATV reduces the percentage of neointimal areapositive for IL-8 ((*)P=0.015). Representative examples show intense IL-8 staining (brown) in the neointima of a non-treated (NT) animal, whichwas markedly decreased with ATV. Vessels from healthy animals did not stain positive for IL-8 (magnification, 400× ).


5094) vs 8696�2305 (5429–11 312), P=0.001) (Fig.5(A)). Control animals did not show NF-�B activation.

For the assessment of NF-�B activation in circulat-ing mononuclear cells, and due to the limited amountof protein extracted, two pools were formed with theprotein extracts of six animals of NT and six animals ofthe ATV groups. The EMSA assay was performed by

duplicate with each pool. Densitometry of EMSA yieldeda marked lower activation in the ATV than in the NTgroup (2966 vs 17 130 arbitrary units) (Fig. 5(B)).

3.5. Effect of ator�astatin on the expression ofcyclooxygenase isoforms in cultured �ascular smoothmuscle cells

To determine whether COX-2 diminution in theatherosclerotic plaque could be due in part to a directeffect of ATV, we performed in vitro experiments. RatVSMC were stimulated with a standard cocktail ofcytokines (10 U/ml IL-1� and 100 U/ml TNF-�), fordifferent periods of time. We observed a marked increaseof COX-2 at 3 h (3.2�1.2-fold; n=5; P�0.05), dimin-ishing at 6 h, increasing again at 8 h (1.8�0.8-fold; n=9;P�0.05) and keeping constant until 24 h (1.8�1; n=3;P�0.05) (Fig. 6). Preincubation for 1 h with ATV (10−5

M) reduced significantly COX-2 expression at 3 h (0.7�0.3-fold; n=3; P�0.05) and at 8 h (1.0�0.8-fold; n=5;P�0.05). COX-1 mRNA expression was no changedsignificantly along the time: 1.1�0.5-fold at 3 h (n=3)and 1.2�1-fold at 8 h (n=7) (Fig. 6). ATV did notmodify COX-1 expression neither at 3 h (1.2�0.7-fold;n=3) nor at 8 h (0.8�0.1-fold; n=6) (not shown).

Preincubation with acetylsalicylic acid (100 �M) ordexametasone (1 �M) diminished the increase of COX-2at 3 h (0.4- and 0.6-fold; n=2 respectively). The NF-�Binhibitors pyrrolidine dithiocarbamate (200 �M) andparthenolide (0.1 �M) also reduced COX-2 expression(1.1- and 0.6-fold, respectively; n=2) (not shown).

4. Discussion

Inflammation plays a role in the rupture of atheroscle-rotic plaques [1,2]. Inflammatory cells, mainlymacrophages, are present more frequently in the lesions

Fig. 4. Effect of ATV on MMP expression. The expression of MMP-3was reduced by ATV ((*)P=0.010), while the expression of MMP-1and MMP-2 was not significantly affected. The representative examplesshow strong MMP-3 staining in a non-treated (NT) animal (brown),which was decreased in an ATV rabbit. Vessels from healthy animalsshowed no staining (magnification, 400× ).Fig. 5. Activity of the transcription factor NF-�B. (A) Representativeexamples of southwestern histochemistry of NF-�B activity in thelesions, showing a great number of nuclei staining positive in anon-treated (NT) animal, which was reduced in a rabbit treated withATV, and absent in a healthy animal (magnification, 400× ). A detailof nuclei positive for NF-�B activity in the NT animal is given(magnification, 1000× ). (B) NF-�B activity was increased in circulat-ing mononuclear cells of NT animals and decreased by ATV (EMSA).Due to the limited amount of protein extracted, two pools were formedwith the proteins obtained from six NT and six ATV animals. Therewas no activated NF-�B in healthy animals. Competition assay (comp)performed with cold probe in 100-fold excess resulted in disappearanceof positivity, thus, demonstrating the specificity of the experiment.Incubation with antibodies against p50 and p65 caused a supershiftedband, confirming that the observed NF-�B activity is composed of thesesubunits. HELA cells extract was used as a positive control.


responsible for acute coronary syndromes than in stableones [6], infiltrating the neighborhood of cap rupture[5]. Several works have suggested that these cells couldmake the cap more prone to rupture by releasingMMPs and other enzymes that degrade its extracellularmatrix [3,4,7]. In accordance with this, it has beenobserved that lipid rich plaques with thin fibrous capsdisplay greater MMP content than those with thickcaps [23].

4.1. Lipid lowering and plaque stability

HMG-CoA reductase inhibitors have been shown toreduce the incidence of acute coronary events [24,25],suggesting a decrease in the incidence of plaque throm-bosis. In addition to a reduction of blood thrombo-genicity in humans [26], these drugs possessanti-inflammatory effects. In a rabbit model ofatherosclerosis, lipid lowering with diet manipulationlead to a diminution in macrophage infiltration andMMP activity [27]. In a previous work, we havedemonstrated that lipid reduction with the HMG-CoAreductase inhibitor ATV decreased neointimalmacrophage infiltration in a rabbit model of atheroscle-rosis [8]. This finding was attributed to a decrease inMCP-1 expression and a diminution in the activation of

NF-�B, a transcription factor that regulates MCP-1gene [8,28]. However, NF-�B is a major player ininflammation and controls the expression of othermolecules involved in monocyte recruitment [18,29,30].Then, if statins are able to reduce NF-�B activation, abroader effect on the molecules controlled by this tran-scription factor would be expected, rather than a merereduction of MCP-1 expression.

4.2. Present findings

In this study, we demonstrate that ATV significantlyreduces the expression of COX-2 in atheroscleroticlesions in rabbits, along with a decrease in macrophageinfiltration, IL-8 and MMP-3 expression and NF-�Bactivation. In addition, the activity of this transcriptionfactor was also markedly reduced in blood mononu-clear cells.

COX-2 is expressed in human atherosclerotic arteries[16,17] colocalizing with MMP-9 and membrane type-1MMP [17], while normal arteries express only COX-1[16]. In this work, we have also evidenced COX-2 andMMP-3 expression in a rabbit model of atherosclerosiswithout changes in the expression of COX-1 with re-spect to healthy vessels. Treatment with ATV reducedthe amount of COX-2 (protein and gene) in the lesions,

Fig. 6. Effect of ATV on the expression of COX-1 and COX-2 in cultured VSMC (RT-PCR). (A) Densitometric analysis: IL-1� (10 U/ml) andTNF-� (100 U/ml) induce an increase in COX-2 expression with peaks at 3 and 8 h. COX-1 expression is not modified. Preincubation with ATV10−5 M reduced COX-2 expression without affecting COX-1. (B) Representative gel. t, time; G3PDH expression was used as control, I, IL-1�,and T, TNF-�.


while COX-1 was not affected. Seemingly, the decreasein NF-�B activity found in the atherosclerotic lesionswas involved in the reduction of COX-2 expressionachieved by ATV. In fact, in in vitro experiments, theblockade of NF-�B activity reduced COX-2 expressionby VSMC. Finally, ATV also decreased COX-2 expres-sion in cultured VSMC. This suggests that the diminu-tion in COX-2 expression induced by this drug couldhave been due, at least in part, to a direct effect on thecells of atherosclerotic lesions. The mechanism underly-ing this effect was probably a decrease in NF-�B activ-ity secondary to a reduction of isoprenylation ofproteins involved in intracellular signal transductionnecessary for their correct function. In this sense, wehave previously reported a decrease in the activity ofthis transcription factor in cultured VSMC by ATVthat was reversed after treatment with the isoprenoidsnecessary for protein isoprenylation [28].

The reduction in the infiltration of macrophages wasprobably related to the decrease in COX-2 expression.In addition, there was also a reduction in the expressionof IL-8, a chemoattractant cytokine [31] that is presentin macrophage-rich areas of human atheroclerotic le-sions [32] and whose expression is also controlled byNF-�B [18]. Although statins have been found to re-duce IL-8 expression in cultured monocytes [33], thiseffect had not been confirmed in vivo. Finally, althoughunexplored in the present study, the expression of theMCP-1 has been previously reported to be reduced byATV in a similar model with certain differences in thecomposition and duration of the atherogenic diet [8].

Although the decrease in macrophage infiltrate couldaccount in part for the reduced presence of MMP-3,this enzyme, as well as MMP-1, has been shown re-cently to be regulated by NF-�B [34]. The lack ofstatistical significance for the reduced expression ofMMP-1 could have been related to other additionaland still unknown factors that play a role in itsregulation.

4.3. Effect of ator�astatin on NF-�B acti�ity in bloodmononuclear cells

Some serum inflammatory markers, mainly C-reac-tive protein, have been demonstrated to predict plaqueinstability [35–38]. Moreover, C-reactive protein levelsdecrease after treatment with statins [39]. NF-�B activ-ity in circulating monocytes has been found to beelevated in patients with unstable angina [40]. In thepresent study, we demonstrate for the first time thatATV decreases NF-�B activity in circulating mononu-clear cells in a model of atherosclerosis with hyperlipi-demia. This factor is not just a marker of inflammationbut, more important, a key mediator of inflammation.Then, these findings suggest that statin treatment mayaffect the inflammatory activity of monocytes before

they enter the vascular wall and differentiate tomacrophages.

4.4. Limitations

The findings reported here have been obtained in anexperimental model of accelerated atherosclerosis thatdiffers from the chronic lesions of human disease. Also,the dose of ATV is higher than that used in the clinicalpractice. Nevertheless, it must be emphasized that thehyperlipidemia obtained by diet manipulation was alsogreater than that usually seen in human beings. More-over, the fact that statins reduce the incidence of acutecoronary events and the observation that inflammationis related to plaque instability in humans fit with theeffects of ATV described in this study and make themvery probably to be responsible in part of the benefitsof these drugs in clinical practice.

4.5. Conclusions

These results demonstrate that ATV reduces inflam-mation by decreasing the activity of NF-�B and theexpression of the proinflammatory molecule COX-2,which is controlled by this transcription factor. Giventhe proven ability of statins to reduce the incidence ofacute ischemic events, and the role played by inflamma-tion in their pathogenesis, these data suggest that COX-2 could be a therapeutic target in atherosclerosis as it isin rheumatic diseases. Finally, the fact that NF-�Bactivity was also reduced in circulating mononuclearcells suggests that the effects of HMG-CoA reductaseinhibition could begin at the level of circulating leuko-cytes. Nevertheless, further work is needed to confirmthese issues.

Acknowledgements

This work was supported by Fundacion Ramon Are-ces, Fundacion Mapfre Medicina, Fondo de Investiga-ciones Sanitarias (98/1269) and Fundacion Renal InigoAlvarez de Toledo.

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