the 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm

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966 VOLUME 10 | NUMBER 9 | SEPTEMBER 2004 NATURE MEDICINE

Atherosclerosis, a chronic inflammatory disorder of the vascular wall,is characterized by the progressive formation of fatty streak lesions,stable plaques and unstable or ruptured plaques, which trigger clinicalcomplications1. Hypercholesterolemia and inflammation are thoughtto represent distinct elements that converge on a common pathogenicpathway in atherosclerosis2. Recognition of the hallmarks of inflam-matory pathology in atherosclerosis has opened up therapeutic oppor-tunities3,4.

5-LO is the key enzyme in leukotriene biosynthesis and catalyzesinitial steps in the conversion of arachidonic acid to these biologicallyactive lipid mediators, which are known to exert proinflammatoryeffects in vivo5,6. Emerging data implicate 5-LO in cardiovascular dis-ease (CVD). Products of the 5-LO pathway, LTB4 and LTE4, aredetectable in atherosclerotic lesions7,8, and expression of 5-LO in ath-erosclerotic lesions has been reported9,10. In human genetic studies,polymorphisms in the 5-LO gene promoter and certain 5-LO-activat-ing protein (FLAP) haplotypes have been linked to CVD susceptibility,namely, the risk of myocardial infarction and stroke11,12.

Data from mice that are null with respect to apolipoprotein E (Apoe–/–) and low-density lipoprotein receptor (Ldlr–/–) indicatethat LTB4 contributes to lesion formation13,14, and it has been sug-gested that 5-LO participates in the atherogenesis of Ldlr–/– mice10. Adefined locus on mouse chromosome 6, where the 5-LO gene (Alox5)is located15, has been identified to confer strong resistance to atheroge-

nesis in the resistant strain CAST/Ei16. Thus, both human and mousestudies raise the possibility that 5-LO may have a proatherogenic role.

Here we examined the expression and functional roles of 5-LO invascular inflammation and CVD pathogenesis in mice and the induc-tion of gene expression by LTD4 in human cells associated with ather-osclerosis. Our data indicate that 5-LO has a role in promoting theformation of aneurysms induced by an atherosclerotic diet throughpotential MIP-1α and MIP-2 chemokine-dependent inflammatorycircuits involving both myeloid and endothelial cells.

RESULTS5-LO expression in aortic lamina adventitia in Apoe–/– miceWe determined the expression of 5-LO in the arterial wall of Apoe–/–

mice. 5-LO was barely detectable in foam cells in intimal lesions ofApoe–/– mice at various ages (Fig. 1a). By contrast, adventitialmacrophages were strongly positive for 5-LO (Fig. 1a). CD11c, amarker of distinct activated macrophage populations and dendriticcells17, was highly expressed in intimal foam cells but not in adventitialCD68+ macrophages (Fig. 1b). Morphometric analyses showed that atboth 8 months and 1 year more than 95% of 5-LO+ cells per mm2 werein the lamina adventitia and the remainder were in the lamina intima;5-LO+ cells were rarely present in the lamina media (Fig. 1c).

Expression of 5-LO in the adventitial macrophages was accompa-nied by a significant increase in adventitial CD3+5-LO– T cells as the

1Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6160, USA. 2Institute for Vascular Medicine, Friedrich-SchillerUniversity of Jena, Bachstrasse 18, 07743 Jena, Germany. 3Departments of Physiology and Biochemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada.4Department of Cardiovascular Diseases, Merck & Company, Rahway, New Jersey 07065, USA. Departments of 5Medicine and 6Pharmacology, University ofPennsylvania, Philadelphia, Pennsylvania 19104-6160, USA. Correspondence should be addressed C.D.F. ([email protected]).

Published online 22 August 2004; doi:10.1038/nm1099

The 5-lipoxygenase pathway promotes pathogenesis ofhyperlipidemia-dependent aortic aneurysmLei Zhao1, Michael P W Moos2, Rolf Gräbner2, Frédérique Pédrono1,3, Jinjin Fan1, Brigitte Kaiser2, Nicole John2,Sandra Schmidt2, Rainer Spanbroek2, Katharina Lötzer2, Li Huang4, Jisong Cui4, Daniel J Rader1,5, Jilly F Evans4,Andreas J R Habenicht2 & Colin D Funk1,3,5,6

Activation of the 5-lipoxygenase (5-LO) pathway leads to the biosynthesis of proinflammatory leukotriene lipid mediators.Genetic studies have associated 5-LO and its accessory protein, 5-LO-activating protein, with cardiovascular disease, myocardialinfarction and stroke. Here we show that 5-LO-positive macrophages localize to the adventitia of diseased mouse and humanarteries in areas of neoangiogenesis and that these cells constitute a main component of aortic aneurysms induced by anatherogenic diet containing cholate in mice deficient in apolipoprotein E. 5-LO deficiency markedly attenuates the formation ofthese aneurysms and is associated with reduced matrix metalloproteinase-2 activity and diminished plasma macrophageinflammatory protein-1α (MIP-1α; also called CCL3), but only minimally affects the formation of lipid-rich lesions. Theleukotriene LTD4 strongly stimulates expression of MIP-1α in macrophages and MIP-2 (also called CXCL2) in endothelial cells.These data link the 5-LO pathway to hyperlipidemia-dependent inflammation of the arterial wall and to pathogenesis of aorticaneurysms through a potential chemokine intermediary route.

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NATURE MEDICINE VOLUME 10 | NUMBER 9 | SEPTEMBER 2004 967

mice aged (Fig. 1d). Two additional gene products of the 5-LO cascade, FLAP and the cysteinyl leukotriene 1 receptor (CysLT1R),were upregulated in Apoe–/– mice maintained on normal mouse chowas the development of atherosclerosis proceeded (Fig. 1e), andleukotriene products were detected in aorta extracts from these mice(Supplementary Table 1 online).

5-LO+ macrophages are abundant in aortic aneurysmsThe cholate-containing atherogenic (Ath) diet has been shown toinduce aneurysms in Apoe–/– mice18. To explore the participation of5-LO in aneurysm formation, we examined expression of 5-LO inaneurysms induced by feeding Apoe–/– mice the Ath diet. We observeda main subpopulation of 5-LO+ macrophages among CD68+ cells andfew 5-LO– T or 5-LO– B lymphocytes in adventitial granulomas thathad formed around aneurysmal arteries19 (Fig. 2a,b and data notshown). Notably, the aneurysmal granuloma macrophages, unlike theadventitial macrophages of mice fed on chow (Fig. 1a), accumulatedlipid (Fig. 2c). Aneurysmal granulomas contained many vasa vasorum(Fig. 2d), and 5-LO+ macrophages preferentially accumulated in theproximity of these blood vessels. These data indicate that 5-LO+

macrophages are a main cellular constituent of aneurysmal granulomatissue and that the macrophages may enter the adventitia through vasa vasorum.

5-LO deficiency attenuates aortic aneurysms in Apoe–/– miceAfter 8 weeks on the Ath diet, Apoe–/– Alox5–/– mice, in which no 5-LOexpression was detected in macrophages or bone marrow cells(Supplementary Fig. 1 online), showed significantly less incidence (2 of 17 mice aneurysm positive) and advanced stages of aneurysmsthan did Apoe–/– mice (16 of 34 mice; Fig. 3a,b). The aneurysms werepresent most often at the abdominal–thoracic diaphragmatic border(Fig. 3c). Histological analysis showed complete disruption of mediaand elastin fibers in Apoe–/– mice, but the extent of media disruptionwas reduced in Apoe–/– Alox5–/– mice (Fig. 3d). Despite these marked

changes in the incidence and severity of aneurysms, en face and aorticroot analyses showed no or only a minor effect of 5-LO on the forma-tion of lipid-rich lesions in several hyperlipidemic mouse coloniesunder different dietary regimens (Supplementary Table 2 online); inaddition, plasma total cholesterol (Supplementary Table 3 online),triglyceride (data not shown), lipoprotein profiles (data not shown)and insulin (Supplementary Table 4 online) were not affected by alack of 5-LO.

5-LO deficiency reduces aortic MMP-2 activity in Apoe–/– miceThe metalloproteinases MMP-2 and MMP-9 have been reported toparticipate in the development of aneurysms20,21. To explore potentialmechanisms underlying the reduced formation of aneurysms result-ing from 5-LO deficiency, we determined the aortic activities ofMMP-2 and MMP-9 in Apoe–/– and Apoe–/– Alox5–/– mice fed on eitherchow or the Ath diet by using a zymogram assay.

Gelatinolytic activity was low in mice fed chow, whereas twoprincipal bands with apparent sizes corresponding to MMP-9 (92 kDa) and MMP-2 (62 kDa) were detected in aortic lysates frommice fed the Ath diet. 5-LO deficiency attenuated aortic MMP-2and MMP-9 activities in Ath-fed Apoe–/– mice; however, only thedecrease in MMP-2 activity was significant (P < 0.05; Fig. 3e,f).Thus, 5-LO deficiency is associated with a reduction in the inci-dence of diet-induced aneurysm formation and a decrease in aorticMMP-2 activity.

5-LO pathway mediates mouse MIP-1α productionWe examined circulating levels of several inflammatory cytokinesand chemokines in Apoe–/– and Ldlr–/– mice. Of 18 cytokine andchemokines examined, one (MIP-1α) was consistently affected byhyperlipidemia (Fig. 4 and Supplementary Table 5 online).Significantly increased concentrations of MIP-1α were observed inall hyperlipidemic mice on various diets (Fig. 4). The increase inMIP-1α was strongly reduced in a gene dosage–dependent manner

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Figure 1 Constituents of the 5-LO pathway inApoe–/– mouse aortas. (a) Immunofluorescencestaining. 5-LO (top left, Cy3, red); whitearrowheads indicate 5-LO+ cells in the intima.CD68 (top middle, Cy2, green). DNA (bottomleft, DAPI, blue). Phase contrast to delineatetissue structure (bottom middle). Merge of 5-LO, CD68, DNA and phase contrast images (right); yellow arrowheads indicateCD68+5-LO+ adventitial macrophages, redarrowheads indicate intimal 5-LO+ cells,broken lines indicate external elastic lamina,unbroken lines delineate internal elasticlamina. (b) CD11c (Cy3, red) is apparent inintimal lesion macrophages but not inadventitial macrophages. (c) 5-LO+ cellsdetermined by morphometry in the laminaadventitia and intima of Apoe–/– mice at 8 (n = 8) and 12 (n = 13) months. (d) Accumulation of T cells in the laminaadventitia of C57BL/6 and Apoe–/– mice.(e) mRNA levels (shown as a percentage ofwild-type mRNA expression) of aortic FLAPand CysLT1R transcripts are significantlyhigher in Apoe–/– mice than in wild-typeC57BL/6 mice. *P < 0.005 versus C57BL/6.Data are the mean ± s.e.m. Scale bars, 100 µm.

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in 5-LO-deficient mice (Fig. 4b–d). These data establish that hyper-lipidemia and its associated vascular inflammation induce systemicconcentrations of MIP-1α and that Alox5 gene disruption interruptsthis linkage.

To address the possibility that leukotrienes directly affect MIP-1αexpression in macrophages, we examined the response of culturedmouse macrophages (p388D1 cell line) to LTD4 challenge. LTD4 ledto a significant induction of MIP-1α mRNA compared to vehicle con-trol (Fig. 4e). The extent of LTD4 stimulation was similar to that oflipopolysaccharide (LPS; data not shown). Future studies using micedeficient in MIP-1α and its chemokine receptor will determinewhether this chemokine is required for the pathogenesis of diet-induced aneurysms.

LTD4 enhances CC and CXC chemokine gene expression in vitroTo explore the relevance of our findings in mice to human CVD, weexamined human coronary and carotid arteries and abdominalaneurysm tissues. In a carotid endarteriectomy specimen, 5-LO+

macrophages localized in close proximity (mean distance <20 µm) toendothelial cells of intimal lesions (Fig. 5a). In a study of 29 individu-als affected with coronary artery disease (CAD), the numbers of 5-LO+

macrophages and 5-LO− T cells in the adventitial or perivascular spacewere strongly correlated (Fig. 5b), indicating a possible functional linkbetween the accumulation of 5-LO+ macrophages and 5-LO− T cells inthe adventitia.

The inflammatory microenvironment in the lamina adventitiaconceivably facilitates the paracrine action of leukotrienes on neigh-boring endothelial cells and T cells and their autocrine action onmacrophages. To study such activations at the molecular level, we car-ried out in vitro microarray analyses. Because we had previouslyobserved potent Ca2+ signals in monocytes/macrophages and umbil-ical vein endothelial cells (HUVECs)22 in response to LTD4 but not inresponse to LTB4, we focused on CysLTR-dependent rather than onLTB4 receptor (BLTR)-mediated gene activation. For this purpose andto test the possibility that leukotrienes produced by macrophages maytarget myeloid cells and endothelial cells, we used human MonoMac6(MM6) cells and HUVECs22.

In response to LTD4, the gene encoding MIP-1α was the mostrobustly upregulated out of 14,000 genes in MM6 cells (Fig. 5c).LTD4 increased the expression of MIP-1α protein from unde-tectable amounts to 66 pg per 106 cells at 4 h after LTD4 addition(data not shown). Similar effects were observed in another humanmyeloid/macrophage cell line, THP-1 (data not shown). The effectof LTD4 (but not that of the control LPS) was clearly mediated byCysLT1R, because chemokine upregulation was completelyblocked by montelukast, a specific CysLT1R antagonist (data notshown).

In HUVECs, LTD4 did not affect MIP-1α , but induced the CXCchemoattractant MIP-2 up to 20-fold. Induction of the geneencoding MIP-2 ranked the seventh highest out of 14,000 genes(Fig. 5d), also indicating a strong upregulation. Both sets of arraydata were confirmed by real-time RT-PCR analyses (Fig. 5c,d), anddetermination of transcript kinetics showed that the transcriptswere upregulated within 20–30 min of cell challenge (data notshown). In contrast to LTD4, thrombin—the powerful prototypevasoactive mediator on endothelial cells—induced the expressionof MIP-2 mRNA only threefold in microarray analyses, indicatingthat the effect of LTD4 was pronounced and selective (data notshown). Unlike the effect of LTD4 on MIP-1α in myeloid cells,the effect of LTD4 on MIP-2 in HUVECs was not mediated byCysLT1R because montelukast, a highly selective antagonist forthis receptor, did not block the induction (data not shown),supporting previous conclusions that the actions of LTD4 onmyeloid cells and HUVECs are mediated through CysLT1R andCysLT2R, respectively22.

The effect of LTD4 on MMPs and TIMP family members wasexamined in p338D1 and MM6 macrophages, as well as HUVECs,using microarrays. LTD4 did not affect these transcript levels, indi-cating that the effect observed in vivo in mice was indirect, occurredat a post-transcriptional level or was mediated by 5-LO pathwayproducts other than LTD4. Overall, these results provide in vitro evi-dence for links between the 5-LO–leukotriene pathway and potent T cell and monocyte/macrophage chemoattractants in humanmyeloid and vascular cells.

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Figure 2 5-LO in aneurysmal tissue. (a) Immunofluorescence staining of a type I aneurysm. 5-LO (Cy3, red), CD68 (Cy2, green) and DNA (DAPI, blue).Phase contrast to delineate tissue structure. (b) Merge of 5-LO, CD68, DNA and phase contrast images; broken lines indicate external elastic lamina, whitearrowhead indicates area of elastic lamina dissolution and media destruction. (c) Area within aneurysmal granuloma; specimen was stained withhematoxylin. Oil red O stains lipid, broken line represents external elastic lamina. (d) Section of granuloma tissue of a type I aneurysm. MECA-32 (Cy5,white) indicates endothelial cells, DNA was stained with DAPI (blue). Scale bars, 100 µm.

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DISCUSSIONOur studies support the following conclusions: 5-LO contributes tothe formation of aortic aneurysms induced by an atherosclerotic dietin Apoe–/– mice; and 5-LO links hyperlipidemia and the productionof systemic inflammatory chemokines in several mouse models ofhyperlipidemia. The functional relevance of our in vivo mouse datato humans is supported by the expression pattern of 5-LO inaneurysms and the ability of leukotrienes to promote proinflamma-tory CC and CXC chemokine expression in mouse and humanmonocyte/macrophages and endothelial cells.

A salient finding of our work is that there is a connection between 5-LO and aneurysm pathogenesis: disruption of the gene encoding 5-LO reduces both the incidence and the extent of aortic aneurysmdevelopment. This finding is consistent with expression of 5-LO in thelamina adventitia rather than in the lamina intima. Aortic aneurysm ischaracterized by medial degeneration and involves several processes,including inflammation, immunological cell infiltration and proteoly-sis23–26. Indeed, the Ath diet has been shown to alter the immuneresponse in Apoe–/– mice27. Thus, in a setting of inflammation andmodest liver injury (L.Z., R.G. and C.D.F., unpublished observations),

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Figure 3 5-LO deficiency reduces aortic aneurysm formation and MMP-2 activity in Apoe–/– mice on Ath diet. (a) Reduced incidence of aneurysms inApoe–/– Alox5–/– mice (n = 17) as compared with Apoe–/– mice (n = 34). *P < 0.05. (b) Number of aneurysms per mouse in Apoe–/– and Apoe–/– Alox5–/–

mice on Ath diet. None of the aneurysms was type II or III, whereas there were seven type IV aneurysms in the 5-LO+ group and none in the 5-LO– group(see ref. 19 for aneurysm classification). (c) Representative aortic aneurysms in Apoe–/– and Apoe–/– Alox5–/– mice on Ath diet. (d) Characterization ofaortic aneurysm tissue in Apoe–/– and Apoe–/– Alox5–/– mice on Ath diet. Aneurysmal serial sections from Apoe–/– and Apoe–/– Alox5–/– mice are stained withhematoxylin, Verhoeff’s stain for elastin and Picro Sirius red for collagen, as indicated. Complete disruption of the elastin layer of the aorta is observed inApoe–/– mice but not in Apoe–/– Alox5–/– mice. Original magnification, ×100. (e) Representative zymogram analysis of MMP-2 and MMP-9 activities inaortas from Apoe–/– and Apoe–/– Alox5–/– mice on chow (19 weeks) and Ath (chow 11 weeks plus Ath 8 weeks) diets (top). Equal protein loading wasverified by western blot analysis of β-actin (bottom). (f) Quantitative analysis of MMP-2 and MMP-9 activities normalized to β-actin band density in Apoe–/–

and Apoe–/– Alox5–/– mice. Data are the mean ± s.e.m. *P < 0.05 versus Apoe–/– group.

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a situation that is likely to occur frequently in an elderly populationpredisposed to aneurysm development, this effect is observed and isstriking.

Adventitial infiltrations (periarteritis), immune reactions (attrac-tion of T cells) and neoangiogenesis have all been associated withadvanced experimental porcine, murine and human atheroscleroticplaques, as well as with aneurysm pathogenesis28,29, althoughaneurysms in mice, in contrast to those in humans, are associatedalmost exclusively with atherosclerotic lesions in mouse models ofhypercholesterolemia23. Our clinical studies of advanced lesions inhuman coronary arteries have shown that there are abundant 5-LO+

macrophages and 5-LO– T cells in the adventitia. 5-LO participates ininflammation and possibly in adaptive immune responses through thebiosynthesis of leukotrienes6,9, and we have verified leukotriene pro-duction in mouse aortas (Supplementary Table 1 online). We havealso documented a 4–5-fold upregulation of transcripts for FLAP and

CysLT1R, two important components of the5-LO cascade, in mouse aortas.

The lack of 5-LO expression in the intima ofApoe–/– mice may relate to our failure to con-firm the effects of 5-LO on aortic lipid accu-mulation found in a previous study10

(Supplementary Table 2 online). There arethree main differences between that study10

and our current study: first, the extent oflesion attenuation (∼ 95% in ref. 10 versus aminimal effect here); second, the dominanteffect of the 5-LO gene on atherosclerosis(observed with one 5-LO allele disrupted inref. 10 versus no 5-LO gene dosage effecthere); and last, variations in the numbers ofmice and the measurements made (four miceper group, one atherosclerotic strain (Ldlr–/–)and one diet (Ath) at one time point in themain experiment examined by one method(aortic root analysis) of lesion quantitation inref. 10 versus large numbers of mice, two ath-erosclerotic models, (Ldlr–/– and Apoe–/–),three different dietary regimens and multipletime points examined by two methods (en facesurface lesion and aortic root analyses) in twodistinct 5-LO-deficient mouse colonies here).

In addition, we did not observe 5-LO-dependent alterations ininsulin10. The reasons underlying these inconsistencies remain unclear.

MMPs have been implicated in the pathogenesis of aorticaneurysms20,21,25, and the reduced formation of aneurysms observedin 5-LO–/– mice is associated with a decrease in aortic MMP-2 activity.Thus, our data suggest that 5-LO can participate in aneurysm develop-ment through proinflammatory leukotrienes by indirectly affectingextracellular matrix (ECM)-degrading enzymes26,30, including MMP-2, as a potential pathway in pathogenesis. Studies in humanshave linked the pathogenesis of both atherosclerosis and aneurysms toinflammation. Coordinated changes in chemokines and cytokines areresponsible for inflammatory responses of atherogenesis in vivo3,4. Weobserved that MIP-1α was strongly increased in all hyperlipidemicmice as compared with their wild-type (C57BL/6) counterparts. Thisfinding links hyperlipidemia to a principal inflammation- andimmune response–regulating CC chemokine, MIP-1α.

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Figure 4 The 5-LO/leukotriene pathway mediates MIP-1α production in mouse. (a) Plasma MIP-1α isincreased in Ldlr–/– mice on Western diet and in Apoe–/– mice on chow or Ath diet as compared withC57BL/6 mice on chow diet. *P < 0.05 versus C57BL/6 on chow diet. (b) Plasma MIP-1α isdecreased in Apoe–/– Alox5+/– and Apoe–/– Alox5–/– mice on chow diet as compared with Apoe–/– miceon chow diet. (c) Plasma MIP-1α is reduced in Ldlr–/– Alox5+/– and Ldlr–/– Alox5–/– mice on Westerndiet as compared with Ldlr–/– mice on Western diet. (d) Plasma MIP-1α is reduced in Apoe–/– Alox5–/–

mice on Ath diet as compared with Apoe–/– mice on Ath diet. Data are the mean ± s.e.m. *P < 0.05versus control (b–d). (e) LTD4 (100 nM) induces mRNA expression of MIP-1α in mouse p388D1macrophages (*P < 0.01 versus control). Data are normalized to GAPDH transcripts.

Figure 5 5-LO+ macrophages in areas of neoangiogenesis and LTD4 activates chemoattractant gene expression. (a) Area of a capillary in intimal lesions (type V according to American Heart Association nomenclature) of a carotid endarteriectomy specimen. Endothelial cells are labeled by antisera to vWF (Cy2,green) and 5-LO (Cy3, red), DNA is labeled by DAPI blue, and phase contrast is shown to delineate tissue structure. Arrowheads designate 5-LO+

macrophages; arrows designate 5-LO– intimal cells. (b) Parallel thin sections of left descending coronary arteries of 29 individuals affected with CADexamined morphometrically with antisera to CD68 and CD3. 5-LO+ macrophages and T cells correlated at P < 0.0001 (r = 0.79). (c,d) MIP-1α (c) and MIP-2 (d) transcripts (left, microarray analyses; right, real-time RT-PCR analyses) in controls and in MM6 cells stimulated for 1 h with LTD4.

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5-LO may be crucially involved in MIP-1α expression, because 5-LOdeficiency induced a gene dosage–dependent reduction in plasma lev-els of this chemokine. Notably, the 5-LO–MIP-1α connection may bedirect rather than indirect, as indicated by the ability of LTD4 to acti-vate MIP-1α potently in both mouse and human myeloid cells in vitroin a CysLT1R-dependent way. Thus, our data reveal an associationbetween hyperlipidemia and chemokine production, and they furthersupport the conclusion that the 5-LO pathway, directly or indirectly,controls one or more linkage points between hyperlipidemia andchemokine production. MIP-1α has several activities including promoting the chemotaxis of T lymphocytes—a prominent leukocytelineage of both adventitial inflammation in mice and intimal lesions inhumans31,32.

The pathophysiological processes governing aneurysm develop-ment and advanced atherosclerosis in humans and mice bear somerelationship to expansive arterial remodeling. Hyperlipidemia,other risk factors (such as smoking or oxidative stress) and distinctgenetic components23,33–36 differentially regulate these events in amanner that is not completely understood. The different geneticdeterminants for susceptibility to aneurysm development (in which5-LO has a role in the diet-induced context) and plaque formation(in which 5-LO did not seem to have a chief role in mice in our stud-ies, although it may have a role in humans9,11) necessitate the identi-fication of essential pathways involved in these cardiovascularevents. Alox5–/– mice may help to uncover additional mechanisms ofarterial wall remodeling processes that occur in response to hyper-lipidemia, for example, the development of angiotensin II–inducedaneurysms19,20.

Much evidence related to arterial wallpathology, the cellular composition ofinflammatory infiltrates, and the action ofleukotrienes in vivo and in vitro has estab-lished similarities between mice and humans,and mechanistic links between the 5-LOpathway and vascular inflammation in bothhumans and mice are beginning to emerge:5-LO+ cells localize in close proximity tonewly formed blood vessels in CVD adventi-tia, intima and aneurysmal granulomas inhumans, as they do in mice; macrophageaccumulation and T cell accumulation corre-late in the periarteritic tissues of individualsaffected with CVD and progressively accumu-late in the adventitia of Apoe–/– mice; and theCC-chemokine MIP-1α is upregulated inmacrophages in response to LTD4, possiblythrough the activation of CysLT1R, in mouseand human myeloid cells, and MIP-1αexpression is attenuated in 5-LO-deficientmice. MIP-2 is also strongly upregulated byLTD4 in HUVECs, although no evidence isavailable in mouse endothelial cells. We note,however, that products of the 5-LO pathwayother than LTD4, including LTB4, may con-ceivably participate in the pathogenesis ofaneurysms that we have observed in vivo.Indeed, our previous and unpublished studieshave indicated that the enzymes for both LTB4biosynthesis and their response system (thatis, both BLTRs) are expressed in mouse peritoneal macrophages, p388D1 and MM6

macrophages, and HUVECs, and are also expressed in human atherosclerotic lesions9,22.

Our data support the hypothesis that leukotrienes produced bymacrophages, in addition to targeting their own CysLT1R in an autocrinefashion22, may target T cells and endothelial cells in a paracrine manner,resulting in the recruitment of inflammatory cells in human diseased tissue through mechanisms that involve CC- and CXC-typechemokines37–42. Some of the effects of products of the 5-LO pathwaymay be direct, whereas others may be indirect. Further work will berequired to elucidate fully the action of 5-LO pathway products at themolecular and cellular levels. These 5-LO-rich inflammatory infiltrates,in which chemokines and their receptors are found, may conceivablypromote pathophysiological levels of ECM-degrading proteases. Indeed,a connection between MIP-2 and MMP-9 has been reported to promotethe release of hematopoietic stem cells from the bone marrow43.

Our preliminary data have further indicated that another CXCchemokine, interleukin-8 (IL-8), is also upregulated in HUVECs byLTD4 (A.J.R.H., unpublished data). Work by others42 has shown thatthis chemokine potently mediates firm monocyte adhesion toHUVECs under flow conditions. IL-8 is present in human atheroscle-rotic lesions and overexpressed in aneurysms, and its preferred recep-tor, CXCR2, is involved in atherosclerotic lesion formation in Ldlr–/–

mice41,42,44. The relationship between the 5-LO cascade andchemokines and their receptors is clearly complex; however, the rela-tionship between eicosanoid action and chemokines merits closeattention, because it suggests that there are previously unrecognizedlinks between the arachidonic acid cascade that generates lipid media-tors5,6 and the peptide chemokines45.

Figure 6 Model of 5-LO pathway participation in leukocyte recruitment and aneurysm formation. (1) LTD4 stimulates the production of MIP-1α in macrophages through CysLT1R in an autocrinefashion. (2) MIP-1α has a role in the recruitment of T cells in a paracrine fashion. (3) LTD4 stimulatesthe production of MIP-2 in endothelial cells of vasa vasorum. (4) MIP-2 participates in the recruitmentof leukocytes. (5) Cells in 5-LO-dependent granuloma tissue may release ECM-degrading factors (such as MMP-2), which break down the elastic lamina of the vascular wall and lead to the formation ofaneurysms. (6) Lipids accumulate in aneurysmal macrophages. Steps 1–6 do not necessarily representthe temporal sequence of events.

ECM-degrading factors(e.g. MMP-2)

Macrophage

Elastin

Aneurysmgranuloma

Adventitia

Smoothmuscle

cell

MIP-2

T cells

LTD4

Intima

5-LO

Endothelial cells ofvasa vasorum

Macrophage

MIP-1α

Media

Blood vessel lumen

Macrophage

5-LO

1 2

Monocyte

4

5

6

3

CysLT1R

LTD4

CysLT2R

Endothelial cell

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Taken together, our present and previous data9,22 make a strongcase for the existence of 5-LO-dependent inflammatory circuits inatherosclerosis (Fig. 6). The macrophage 5-LO cascade generatesleukotrienes, which act on neighboring endothelial cells and possi-bly T cells, as well as on macrophages themselves, thereby activatingthe release of CC and CXC chemokines and, through indirect mech-anisms, protease activities. Thus, amplifying signals are generated toinitiate cycles of inflammation and arterial wall remodeling inwhich 5-LO has a regulatory role. Our data identify potential thera-peutic target sites for pharmacological intervention in aorticaneurysm and possibly in additional complications of late-stageatherosclerosis.

METHODSMice, diet and mouse procedures. Alox5–/– mice (backcrossed more than ninetimes to the C57BL/6 background) from the Funk46 and Koller47 labs were usedto initiate the crossbreeding of separate mouse colonies in the United States andGermany, respectively. Apoe–/– and Ldlr–/– mice (each backcrossed ten times tothe C57BL/6 background) were from The Jackson Laboratory. Apoe–/– Alox5–/–

and Ldlr–/– Alox5–/– mice were genotyped individually by PCR analysis(Supplementary Fig. 2 online). All mouse experiments were approved by theInstitutional Animal Care and Use Committee of the University ofPennsylvania and done in accordance with the Association for the Assessmentand Accreditation of Laboratory Animal Care guidelines or were approved bythe Animal Ethics Committee of the Friedrich-Schiller University Jena. We fedApoe–/– and Apoe–/– Alox5–/– mice either on a mouse chow diet (RP5001; PMIFeeds) for 6, 8, 10 or 12 months, or on a chow diet for 9 or 11 weeks followed bya high-fat, high-cholesterol, cholic acid–containing diet (Ath diet; from eitherHarlan-Teklad or Altromin) for 8 weeks. Ldlr–/–, Ldlr–/– Alox5+/– and Ldlr–/–

Alox5–/– mice were fed on normal mouse chow until 11 weeks and then on aWestern diet (Harlan Teklad) for 12 weeks. Human tissues were used asdescribed9. Informed consent was obtained from each individual and the studywas approved by local ethical committees of the Universities of Jena, Münsterand Heidelberg.

Human samples. The origin of human samples used here has previously beendescribed9. All samples were derived from the PDAY (PathologicalDeterminants of Atherosclerosis in Youth) program or patients that wereafflicted with clinically significant atherosclerosis, that is, coronary heart dis-ease, carotid artery disease or aortic aneurysm. A carotid endarteriectomy spec-imen (AHA type V lesion) of a 73-year-old male patient was used for theexperiment shown in Figure 5a. The data shown in Figure 5b were derivedfrom the adventitia of 29 coronary heart disease patients: 11 from PDAY (5 type0 lesions, 5 type I lesions; 1 type II lesion; see ref. 9 for further information);9 from the University of Heidelberg, Department of Thoracic Surgery (1 type Ilesion, 3 type II lesions, 2 type III lesions, 1 type IV lesion, 2 type V lesions); and9 from the University of Münster, Department of Surgery (3 type II lesions,3 type III lesions, 1 type IV lesion, 2 type V lesions). Informed consent wasobtained from each individual and the study was approved by local ethicalcommittees of the Universities of Jena, Münster and Heidelberg. The left cir-cumflex coronary artery was analyzed for adventitial macrophages (CD68+

cells using clone EBM11; Dako) and T cells (CD3+ cells using clone UCHT1;Dako) by morphometry as described9.

Preparation of mouse aortas and atherosclerotic lesion analysis. Mice aortaswere prepared as described48. The extent of atherosclerosis was assessed by en face analysis. We analyzed serial aortic root sections for the area staining withOil red O (US colony), or for the plaque area and intima to media ratio(Germany colony). All samples were analyzed by an investigator kept blinded tothe genotypes.

Characterization of aortic aneurysms. Aneurysms were classified according toa published scale19.

Morphometry. Morphometric analyses of 5-LO+ cells and T cells were done asdescribed9.

Histology and immunohistochemistry. Paraffin aortic sections (8 µm) werestained with Gill-modified hematoxylin (EM Science), Picro Sirius red49 andVerhoeff ’s stain (Newcomer Supply). The cellular composition of Apoe–/–

aortas and aneurysmal tissue was analyzed on freshly frozen tissues by fluo-rescence immunohistochemistry as described9. We used three antisera to 5-LO (one a gift of O. Rådmark, Karolinska Institute, Stockholm), whichyielded similar results in human and mouse tissues9. Macrophages weredetected by antibody to CD68 (FA-11; Serotec); T and B lymphocytes byantibodies to CD3e (145-2C11; PharMingen) and CD45R/B220, respec-tively; dendritic cells or activated macrophages by hamster polyclonal anti-body to CD11c (N418; Serotec); endothelial cells by pan-endothelialmonoclonal rat antisera (MECA-32; PharMingen) or von Willebrand Factor(vWF) antisera (DakoCytomation); and DNA by 4′ ,6-diamidino-2-phenylindole dihydrochloride (DAPI). Secondary antibodies were Cy3-con-jugated donkey anti–rabbit IgG, Cy5-conjugated donkey anti–rat IgG, andCy2-conjugated donkey anti–hamster IgG, (Jackson ImmunoresearchLaboratories).

Plasma cytokine and chemokine analysis. Plasma MCP-1 concentrations weredetermined by Quantikine kits (R&D Systems). Optical densities were deter-mined on a Multiscan Spectrum (Thermo Labsystems) at 450 nm (sensitivity15.6 pg/ml). Plasma cytokine and chemokine concentrations for IL-4, IL-10,TNF-α, IL-17, MCP-1, MIP-1α and RANTES were determined with Bio-Plexcytokine assay kits using the Bio-Plex Protein Array System and Plex Manage 3software (Bio-Rad).

Zymography and western-blot analysis. Aortic MMP-2 and MMP-9 activi-ties were determined by zymography as described20. Equal loading of pro-tein in zymography was verified by western blot analysis of β-actin in thesame sample.

Cell culture, treatment and RNA extraction. We maintained p388D1 andHUVEC cells (American Type Culture Collection) in serum-free conditions for24 h and MM6 cells (German Collection of Microorganisms and Cell Culture)in serum-containing medium, stimulated them with 100 or 200 nM LTD4

(ref. 22) for 1 h and subjected them to RNA extraction.

Microarray and real-time RT-PCR. Total RNA was treated with RNase-freeDNase I and subjected to microarray analyses with Affymetrix HU133 arrays.The results were evaluated by Genespring software (Silicon Genetics). Real-time RT-PCR analyses were done as described9,50. The specificity of eachprimer pair was confirmed by melting curve analysis. We used the followingprimers: mGAPDH forward, 5′-GGGAAGCCCATCACCATCTTC-3′;mGAPDH reverse, 5′-GTTCTGGGCAGCCCCACGGCC-3′; mMIP-1αforward, 5′-CTGACAAGCTCACCCTCTGTC-3′; mMIP-1α reverse, 5′-GAAAATGACACCTGGCTGGG-3′; mCysLT1R forward, 5′-GGCAATAGCTTTGTGCTCTATGTC-3′ ; mCysLT1R reverse, 5′-GCATAGGTGGTGAGGCGGCAC-3′; mFLAP forward, 5′-GGCCCTTGTCACCCTCATCAGCG-3′;mFLAP reverse, 5′-CCGGCGAAGGACATGAGG AACAGG-3′; hGAPDH for-ward, 5′-TCGGAGTCAACGGATTTGGTCGTA-3′; hGAPDH reverse,5′-CTTCCTGA GTACTGGTGTCAGGTA-3′; hMIP-1α forward, 5′-TGA-CAC-TCGAGCCCACATTCC-3′; hMIP-1α reverse, 5′-GGTTCGGG-CCACAGTAGAG-3′; hMIP-2 forward, 5′-GAATCTACTTGCACACT-CTCCC-3′; and hMIP-2 reverse, 5′-GA AGCACTACTGTATAGTGTACAG-3′.mRNA expression was normalized to GAPDH transcripts as a control.

Statistical analysis. Initial analyses were done by Student’s t-test. If the datadid not fit the constraints of this parametric test, they were analyzed by one-way analysis of variance (ANOVA). A value of P < 0.05 was considered signif-icant. Aneurysm incidence was analyzed by Fisher’s exact test. Prism 3.0software (GraphPad) was used for all calculations. All data are presented asthe mean ± s.e.m.

GEO accession numbers. General accession number, GSE1644; individualHUVEC data sets, GSM27929 and GSM27930; individual MM6 cell data sets,GSM27931 and GSM27932.

Note: Supplementary information is available on the Nature Medicine website.

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ACKNOWLEDGMENTSWe thank J.A. Lawson for technical support with LC–MS/MS assays; A.J. Cucchiarafor assistance with statistical analysis; M. Hildner and G. Weber for technicalassistance with microarray analyses, real-time RT-PCR and immunohistochemicalmorphometry; D. Marchadier and S. Jahn for lipoprotein profile analyses; J. Ventre,T. Dobber and J. Menke for insulin measurements; and B. Koller for 5-LO–/– mice.This work was supported by grants from the National Institutes of Health(HL53558 to C.D.F.; HL70128 and HL55323 to D.J.R.), the Canadian Institutes ofHealth Research (MOP-67146 to C.D.F.), the Deutsche Forschungsgemeinschaft(Ha 1083/13-1/13-2/13-3/13-4/12-5/12-6), the European Union research network(QLG1-CT-2001-01521 to A.J.R.H.), the Interdisziplinäre Zentrum für KlinischeForschung Jena and the Singulair Medical School Program (to A.J.R.H.), and by anAmerican Heart Association postdoctoral fellowship (0225369U to L.Z.). C.D.F.holds a Canada Research Chair in Molecular, Cellular and Physiological Medicine.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 15 April; accepted 20 July 2004Published online at http://www.nature.com/naturemedicine/

1. Ross, R. Atherosclerosis—an inflammatory disease. N. Engl. J. Med. 340,115–126 (1999).

2. Steinberg, D. Atherogenesis in perspective: hypercholesterolemia and inflamma-tion as partners in crime. Nat. Med. 8, 1211–1217 (2002).

3. Libby, P. Inflammation in atherosclerosis. Nature 420, 868–874 (2002).4. Libby, P., Ridker, P.M. & Maseri, A. Inflammation and atherosclerosis. Circulation

105, 1135–1143 (2002).5. Samuelsson, B. Leukotrienes: mediators of immediate hypersensitivity actions

and inflammation. Science 220, 568–575 (1983).6. Funk, C.D. Prostaglandins and leukotrienes: advances in eicosanoid biology.

Science 294, 1871–1875 (2001).7. De Caterina, R. et al. Leukotriene B4 production in human atherosclerotic

plaques. Biomed. Biochim. Acta 47, S182–S185 (1988).8. Patrignani, P. et al. Release of contracting autacoids by aortae of normal and ath-

erosclerotic rabbits. J. Cardiovasc. Pharmacol. 20, S208–S210 (1992).9. Spanbroek, R. et al. Expanding expression of the 5-lipoxygenase pathway within

the arterial wall during human atherogenesis. Proc. Natl. Acad. Sci. USA 100,1238–1243 (2003).

10. Mehrabian, M. et al. Identification of 5-lipoxygenase as a major gene contributingto atherosclerosis susceptibility in mice. Circ. Res. 91, 120–126 (2002).

11. Dwyer, J.H. et al. Arachidonate 5-lipoxygenase promoter genotype, dietary arachi-donic acid, and atherosclerosis. N. Engl. J. Med. 350, 29–37 (2004).

12. Helgadottir, A. et al. The gene encoding 5-lipoxygenase activating protein confersrisk of myocardial infarction and stroke. Nat. Genet. 36, 233–239 (2004).

13. Aiello, R.J. et al. Leukotriene B4 receptor antagonism reduces monocytic foamcells in mice. Arterioscler. Thromb. Vasc. Biol. 22, 443–449 (2002).

14. Subbarao, K. et al. Role of leukotriene B4 receptors in the development of athero-sclerosis: potential mechanisms. Arterioscler. Thromb. Vasc. Biol. 24, 369–375(2004).

15. Chen, X.S. et al. cDNA cloning, expression, mutagenesis, intracellular localizationand gene chromosomal assignment of mouse 5-lipoxygenase. J. Biol. Chem. 270,17993–17999 (1995).

16. Mehrabian, M. et al. Genetic locus in mice that blocks development of atheroscle-rosis despite extreme hyperlipidemia. Circ. Res. 89, 125–130 (2001).

17. Hart, D.N. Dendritic cells: Unique leukocyte populations which control the pri-mary immune response. Blood 90, 3245–3287 (1997).

18. Silence, J., Lupu, F., Collen, D. & Lijnen, H.R. Persistence of atheroscleroticplaque but reduced aneurysm formation in mice with stromelysin-1 (MMP-3) geneinactivation. Arterioscler. Thromb. Vasc. Biol. 21, 1440–1445 (2001).

19. Daugherty, A., Manning, M.W. & Cassis, L.A. Antagonism of AT2 receptors aug-ments angiotensin II–induced abdominal aortic aneurysms and atherosclerosis.Br. J. Pharmacol. 134, 865–870 (2001).

20. Bruemmer, D. et al. Angiotensin II–accelerated atherosclerosis and aneurysm for-mation is attenuated in osteopontin-deficient mice. J. Clin. Invest. 112,1318–1331 (2003).

21. Longo, G.M. et al. Matrix metalloproteinases 2 and 9 work in concert to produceaortic aneurysms. J. Clin. Invest. 110, 625–632 (2002).

22. Lötzer, K. et al. Differential leukotriene receptor expression and calciumresponses in endothelial cells and macrophages indicate 5-lipoxygenase-depend-ent circuits of inflammation and atherogenesis. Arterioscler. Thromb. Vasc. Biol.23, e32–e36 (2003).

23. Daugherty, A. & Cassis, L.A. Mouse models of abdominal aortic aneurysms.Arterioscler. Thromb. Vasc. Biol. 24, 429–434 (2004).

24. Manning, M.W., Cassis, L.A., Huang. J., Szilvassy, S.J. & Daugherty, A. Abdominalaortic aneurysms: fresh insights from a novel animal model of the disease. Vasc.Med. 7, 45–54 (2002).

25. Sinha, S. & Frishman, W.H. Matrix metalloproteinases and abdominal aorticaneurysms: a potential therapeutic target. J. Clin. Pharmacol. 38, 1077–1088(1998).

26. Werb, Z. ECM and cell surface proteolysis: regulating cellular ecology. Cell 91,439–442 (1997).

27. Zhou, X., Paulsson, G., Stemme, S. & Hansson, G.K. Hypercholesterolemia isassociated with a T helper (Th) 1/Th2 switch of the autoimmune response in ath-erosclerotic apoE-knockout mice. J. Clin. Invest. 101, 1717–1725 (1998).

28. Houtkamp, M.A., de Boer, O.J., van der Loos, C.M., van der Wal, A.C. & Becker,A.E. Adventitial infiltrates associated with advanced atherosclerotic plaques:structural organization suggests generation of local humoral immune responses. J. Pathol. 193, 263–269 (2001).

29. Kumamoto, M., Nakashima, Y. & Sueishi, K. Intimal neovascularization in humancoronary atherosclerosis: its origin and pathophysiological significance. Hum.Pathol. 26, 450–456 (1995).

30. Dollery, C.M. et al. Neutrophil elastase in human atherosclerotic plaques: produc-tion by macrophages. Circulation 107, 2829–2836 (2003).

31. Hansson, G.K., Libby, P., Schonbeck, U. & Yan, Z.Q. Innate and adaptive immu-nity in the pathogenesis of atherosclerosis. Circ. Res. 91, 281–291 (2002).

32. Lusis, A.J. Atherosclerosis. Nature 407, 233–241 (2000).33. Kuhel, D.G., Zhu, B., Witte, D.P. & Hui, D.Y. Distinction in genetic determinants

for injury-induced neointimal hyperplasia and diet-induced atherosclerosis ininbred mice. Arterioscler. Thromb. Vasc. Biol. 22, 955–960 (2002).

34. Shi, W. et al. Genetic backgrounds but not sizes of atherosclerotic lesions deter-mine medial destruction in the aortic root of apolipoprotein E–deficient mice.Arterioscler. Thromb. Vasc. Biol. 23, 1901–1906 (2003).

35. Galis, Z.S. & Khatri, J.J. Matrix metalloproteinases in vascular remodeling andatherogenesis: the good, the bad, and the ugly. Circ. Res. 90, 251–262 (2002).

36. Ward, M.R., Pasterkamp, G., Yeung, A.C. & Borst, C. Arterial remodeling.Mechanisms and clinical implications. Circulation 102, 1186–1191 (2000).

37. Baggiolini, M. Chemokines and leukocyte traffic. Nature 392, 565–568 (1998).38. Springer, T.A. Traffic signals for lymphocyte recirculation and leukocyte emigra-

tion: the multistep paradigm. Cell 76, 301–314 (1994).39. Mach, F. et al. Differential expression of three T lymphocyte–activating CXC

chemokines by human atheroma-associated cells. J. Clin. Invest. 104,1041–1050 (1999).

40. Apostolopoulos, J., Davenport, P. & Tipping, P.G. Interleukin-8 production bymacrophages from atheromatous plaques. Arterioscler. Thromb. Vasc. Biol. 16,1007–1012 (1996).

41. Koch, A.E. et al. Enhanced production of the chemotactic cytokines interleukin-8and monocyte chemoattractant protein-1 in human abdominal aneurysms. Am. J.Pathol. 142, 1423–1431 (1993).

42. Gerszten R.E. et al. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascu-lar endothelium under flow conditions. Nature 22, 718–723 (1999).

43. Pelus, L.M., Bian, H., King, A.G. & Fukuda, S. Neutrophil-derived MMP-9 medi-ates synergistic mobilization of hematopoietic stem and progenitor cells by thecombination of G-CSF and the chemokines GROβ/CXCL2 and GROβT/CXCL2δ4.Blood 103, 110–119 (2004).

44. Boisvert, W.A., Santiago, R., Curtiss, L.K. & Terkeltaub, R.A. A leukocyte homo-logue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages inatherosclerotic lesions of LDL receptor-deficient mice. J. Clin. Invest. 101,353–363 (1998).

45. Luster, A.D. Chemokines—chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 338, 436–445 (1998).

46. Chen, X.S., Sheller, J.R., Johnson, E.N. & Funk, C.D. Role of leukotrienesrevealed by targeted disruption of the 5-lipoxygenase gene. Nature 372, 179–182(1994).

47. Goulet, J.L., Snouwaert, J.N., Latour, A.M., Coffman, T.M. & Koller, B.H. Alteredinflammatory responses in leukotriene-deficient mice. Proc. Natl. Acad. Sci. USA91, 12852–12856 (1994).

48. Tangirala, R.K., Rubin, E.M. & Palinski, W. Quantitation of atherosclerosis inmurine models: correlation between lesions in the aortic origin and in the entireaorta, and differences in the extent of lesions between sexes in LDL receptor–defi-cient and apolipoprotein E–deficient mice. J. Lipid Res. 36, 2320–2328 (1995).

49. Cyrus, T. et al. Effect of low-dose aspirin on vascular inflammation, plaque stabil-ity, and atherogenesis in low-density lipoprotein receptor-deficient mice.Circulation 106, 1282–1287 (2002).

50. Basso, K. et al. Gene expression profiling of hairy cell leukemia reveals a pheno-type related to memory B cells with altered expression of chemokine and adhesionreceptors. J. Exp. Med. 199, 59–68 (2004).

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the 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm

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