Age-Related Effects on Atherogenesis and Scavenger Enzymes of Intracranial and Extracranial Arteries...

Age-Related Effects on Atherogenesis and ScavengerEnzymes of Intracranial and Extracranial Arteries in Men

Without Classic Risk Factors for AtherosclerosisFrancesco P. D’Armiento, MD; Alfredo Bianchi, MD; Filomena de Nigris, PhD;

David M. Capuzzi, MD; Maria R. D’Armiento, MD; Giuseppe Crimi, MD; Pasquale Abete, MD;Wulf Palinski, MD; Mario Condorelli, MD; Claudio Napoli, MD

Background and Purpose—Atherosclerosis occurs later and is less extensive in intracranial arteries than in extracranialarteries. However, the mechanisms responsible are poorly understood. A previous study has suggested a betterantioxidant protection of intracranial arteries.

Methods—To assess the influence of age on arterial activity of antioxidant enzymes and atherogenesis, we comparedintracranial and extracranial arteries of humans of different ages who retrospectively lacked confounding classic riskfactors (48 premature fetuses aged 6.4�0.8 months [mean�SD], 58 children aged 7.9�3.8 years, 42 adults aged42.5�5.1 years, and 40 elderly subjects aged 71.8�3.4 years; all males). Lesions were quantified by computer-assistedimaging analysis of sections of the middle cerebral and basilar arteries, the left anterior descending coronary artery, thecommon carotid artery, and the abdominal aorta. Macrophages, apolipoprotein B, oxidized LDL, and matrixmetalloproteinase-9 in lesions were determined by immunocytochemistry. The effect of aging on atherogenesis was thencompared with that on the activity of 4 antioxidant enzymes in the arterial wall.

Results—Atherosclerosis was 6- to 19-fold greater (P�0.01) in extracranial arteries than in intracranial arteries, and itincreased linearly with age. Intracranial arteries showed significantly greater antioxidant enzyme activities than didextracranial arteries. However, the antioxidant protection of intracranial arteries decreased significantly in older age,coinciding with a marked acceleration of atherogenesis. An increase in matrix metalloproteinase-9 protein expressionand in gelatinolytic activity consistent with the degree of intracranial atherosclerosis was also observed.

Conclusions—These results suggest that a greater activity of antioxidant enzymes in intracranial arteries may contributeto their greater resistance to atherogenesis and that with increasing age intracranial arteries respond with acceleratedatherogenesis when their antioxidant protection decreases relatively more than that of extracranial arteries. (Stroke.2001;32:2472-2480.)

Key Words: atherosclerosis � cerebral arteries � lipoproteins, LDL � oxygen radical

Despite progress in prevention, ischemic stroke remainsthe third most common cause of death in the Western

world, after coronary heart disease and cancer.1 The role ofhypercholesterolemia in atherosclerotic cerebrovascular dis-ease is still unclear.2,3 Statins reduce the incidence of nonfatalstroke in coronary heart disease patients1–3 but are apparentlyless effective in reducing the mortality from ischemicstroke.1–3 Although intracranial arteries eventually developatherosclerotic lesions, the onset of atherogenesis occursmuch later in life, and the severity of the lesions at variousages is consistently less than that in extracranial arteries inhumans,2,4–9 nonhuman primates,10 rhesus and cynomolgusmonkeys,11 spontaneously hypertensive rats,12,13 cholesterol-

See Editorial Comment, page 2479fed rabbits,11 and Watanabe heritable hyperlipidemic rabbits.14

To date, it is unknown whether the difference in atherosclerosisis due to anatomic differences between intracranial and extracra-nial arteries, systemic differences (eg, lower local blood pres-sure), or other differences in atherogenic mechanisms. LDLoxidation is thought to affect many atherogenic mechanisms.15

In a preceding study,9 we reported that the activity of antioxidantenzymes, in particular the oxygen-radical scavenger manganesesuperoxide dismutase (Mn-SOD), tended to be consistentlygreater in intracranial arteries of premature human fetuses thanin extracranial arteries. However, it is not known whether this

Received July 4, 2001; final revision received August 8, 2001; accepted August 21, 2001.From the Departments of Medicine, Gerontology, and Human Pathology (F.P.D., F.d.N., M.R.D., P.A., M.C., C.N.), Federico II University of Naples,

Naples, Italy; the Departments of Medical Toxicology and Anesthesiology (A.B., G.C.), University of Catania, Catania, Italy; the Department of Medicine(W.P., C.N.), University of California San Diego, La Jolla, Calif; and the Department of Medicine (A.B., D.M.C.), Division of Cardiology, JeffersonMedical College, Thomas Jefferson University, Philadelphia, Pa.

Correspondence to Claudio Napoli, MD, Department of Medicine-0682, University of California, San Diego, La Jolla, CA 92093. [email protected]

© 2001 American Heart Association, Inc.

Stroke is available at http://www.strokeaha.org

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difference persists after birth. One of the mechanisms by whichenhanced lipid peroxidation could affect atherogenesis andplaque rupture would be by enhancing the expression of matrixmetalloproteinases (MMPs).16 Oxygen radicals17 and oxidizedLDLs (oxLDLs)18 have both been shown to enhance MMP-9activity, and human carotid unstable plaques undergoing spon-taneous embolization have increased MMP-9 activity.19 Toinvestigate whether differences in arterial antioxidant protectionmay contribute to the different susceptibility to atherogenesisand to assess the impact of aging on both parameters, wecompared the effect of age on human intracranial and extracra-nial arteries in subjects lacking classic risk factors of atheroscle-rosis as far as retrospective data could prove.

Subjects and MethodsHuman SubjectsArteries were obtained at autopsy from spontaneously abortedfetuses (fetal age 6.4�0.8 months, n�48),9,20 including 42 fetusesthat were part of our previous studies,9,20 58 children (aged 7.9�3.8years), including 21 that were part of the Fate of Early Lesions inChildren (FELIC) study,21 42 adults (aged 42.5�5.1 years), and 40elderly subjects (aged 71.8�3.4 years) who underwent routineautopsy at Federico II University of Naples. The study protocol wasapproved by the local human ethics committee.

We previously reported that maternal hypercholesterolemia duringpregnancy was associated with greatly increased lesions in the aortaof the fetus20 and faster progression of atherosclerosis in theirnormocholesterolemic children.21 Because the relative frequency ofmaternal hypercholesterolemia in adults and elderly subjects has notbeen established, we arbitrarily included an equal number fetusesfrom normocholesterolemic and hypercholesterolemic mothers(n�24 each). Similarly, we included equal numbers of male childrenof both groups of mothers (n�29 each). Note that the childrenthemselves were normocholesterolemic. Causes of death in adult andelderly men were trauma (n�57) or liver cirrhosis (n�25). Althoughdata were obtained retrospectively, detailed medical histories, clini-cal records, and blood samples obtained from all subjects shortlybefore death or at the time of autopsy permitted us to establish withsome degree of confidence that none of the subjects had the classicrisk factors for atherosclerosis (family history for coronary heartdisease, diabetes, smoking, hypertension, and hyperlipidemias) ormanifest atherosclerosis-related diseases. Plasma vitamin E wasmeasured by high-performance liquid chromatography, as previouslydescribed.22

Preparation of Arterial Sections, Histological andImmunohistochemical Analyses, and ZymographyRepresentative segments of the abdominal aorta and the entirecommon carotid, left anterior descending coronary (LAD), basilar,and middle cerebral arteries were dissected, cut open, washedthoroughly with cold sterile PBS, and placed in ice-cold PBScontaining 50 �mol/L butylated hydroxytoluene, 0.001% aprotinin,50 mmol/L EDTA, and 0.008% chloramphenicol, equilibrated withnitrogen, as described.9,20,21 For each subject, one segment of eachartery was immersed in optimal cutting temperature (OCT) com-pound and flash-frozen in liquid nitrogen, and 30 to 40 sections persegment (7 �m thick) were prepared for morphometry of thelesions.9,20,21 Cryosections were stained with oil red O and counter-stained with hematoxylin. The cumulative area of all lesions (oil redO–positive areas) per section was then determined by computer-assisted image analysis. To permit a direct comparison of lesionformation between arteries of different size, data were then correctedby dividing the cumulative lesion area by the average outer circum-ference of each artery. Another segment from the same artery of eachpatient was fixed in buffered 10% formalin and paraffin-embedded,and 12 to 15 serial sections (5 to 7 �m thick) were prepared forimmunocytochemistry, as described.9,20,21 Serial sections were

stained with the following: (1) MDA2, a murine monoclonal anti-body against malondialdehyde (MDA)-lysine epitopes of oxLDL; (2)NP1539, a mouse monoclonal antibody (IgG1) to human apolipopro-tein B (Boehringer-Mannheim Italia); and (3) HAM-56, a monoclo-nal antibody against human macrophage/foam cells (Axcel Accu-rate). MMP-9 was detected with a mouse monoclonal antibodyagainst the active form of MMP-9 (Oncogene Science) that was alsoused for Western blot analysis in arterial whole-cell extracts, aspreviously described.23 All antibodies were used at a dilution of1:500. Epitopes recognized by the primary antibody were detectedby an avidin-biotin-peroxidase method.9,20,21 MMP zymography(measuring gelatinolytic activity) was performed after homogeniza-tion of 4 to 9 �g arterial tissue, as described,24 and results werenormalized to total protein by Bradford assay (Bio-Rad Laborato-ries). Briefly, the composition of enzyme assay buffer for thedevelopment of enzyme activity bands was as follows: Tris (3.02g/L), CaCl2 (0.75 g/L), NaCl (0.9 g/L), and Na3N (0.5 g/L), at pH 7.5.After incubation, the gels were stained with Coomassie brilliant blue,and gelatinolytic activities were detected as transparent bands againstthe background of Coomassie-stained gelatin. The intensity of thezymogram bands was expressed as arbitrary units and analyzed bydensitometry.23

Determination of Antioxidant Enzymes in theArterial WallAdditional arterial segments were homogenized in potassium phos-phate buffer, pH 7.4, containing 10 �mol/L deferoxamine, 0.03%butylated hydroxytoluene, and 2% ethanol, equilibrated with nitro-gen (to reduce autoxidation), and centrifuged at 1000g for 15minutes at 4°C to remove nuclei and tissue debris. The supernatantwas centrifuged again at 3000g for 35 minutes at 4°C. Glutathioneperoxidase, catalase, copper-zinc superoxide dismutase (CuZn-SOD), and Mn-SOD tissue activities, normalized for the proteincontent, were determined spectrophotometrically, as described9.

Statistical AnalysisResults were analyzed by 1-way ANOVA followed by Bonferronicorrection. A value of P�0.05 was considered significant. Numeri-cal data obtained from immunohistochemistry were analyzed formean, variance, standard deviation, kurtosis, and skew. To control

Figure 1. Corrected cumulative lesion area in intracranial andextracranial arteries in humans of different age groups. Inset,Noncorrected cumulative lesion areas per section. MCA indi-cates middle cerebral artery; BAS, basilar artery; CC, commoncarotid artery; and AA, abdominal aorta. Results are expressedas the mean�SD. *P�0.001 compared with MCA and BAS;�P�0.01 compared with the same arteries of fetuses and withMCA and BAS; **P�0.001 compared with the same arteries ofchildren and fetuses; ***P�0.0001 compared with the samearteries of children and fetuses and with MCA and BAS; and��P�0.001 compared with the same arteries of adults, chil-dren, and fetuses. Note that noncorrected cumulative lesionareas in the inset are reported as mm2 and are on a logarithmicscale.

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for the effect of age and plasma cholesterol, glucose, and vitamin Econcentration on cumulative lesion areas, multiple regression anal-yses was performed, and � coefficients are presented. Correlationsbetween the results were also evaluated by linear regression analysis.All data were analyzed by SPSS statistical software (SPSS Inc).

ResultsExtent of AtherosclerosisMorphometric evaluation of atherosclerosis in extracranialand intracranial arteries revealed that lesions in each arteryincreased with increasing age. Figure 1 shows results ex-pressed as corrected cumulative lesion area per section, aparameter that allows comparison of arteries of differentcaliber. The noncorrected cumulative lesion sizes are indi-cated in the inset. At any age, lesions were significantlygreater in the abdominal aorta, carotid artery, and LAD thanin the basilar and middle cerebral arteries. Even in adults andelderly subjects, the majority of lesions in intracranial arteriesof adults and elderly groups were fatty streaks (class I lesions)or fibrous lesions (class II lesions). The mean intimal thick-ness in the middle cerebral and basilar arteries increased from4.2�1.1�10�2 and 6.3�1.1�10�2 mm in adults to 12.2�4.4and 14.0�5.6�10�2 mm in elderly subjects, respectively(P�0.001 for both arteries). The average extent of lesions inthe middle cerebral artery was 3.5�0.6% and 5.2�1.1% in

adult and elderly men, respectively (P�NS), and that in thebasilar artery was 6.2�1.4% and 9.1�2.5%, respectively(P�NS). In contrast, mostly class II and III lesions (fibroath-eromatous plaques) were seen in the LAD of adults andelderly subjects. The majority of lesions in the carotid arteryand abdominal aorta of adult and elderly men were offibroatheromatous and calcific stages (classes III and IV).The average of lesions in the carotid artery was 18�4% and26�6% in adult and elderly men, respectively (P�0.05). Asignificant increase of mean intimal thickness with advancingage was observed in the carotid arteries (P�0.05) but not inthe abdominal aorta (P�NS).As expected from the exclusion of dyslipidemic and diabeticsubjects, multiple regression analysis showed that plasmacholesterol and glucose of the adult and elderly groups didnot influence atherogenesis (Table 1). However, plasmacholesterol levels were an independent risk factor for LADlesions. More important, age was independently related toatherogenesis in both intracranial and extracranial arteries inthe adult group and in elderly men, except for the aorta (Table1). Plasma vitamin E concentrations were inversely correlatedwith atherogenesis of brain arteries in adult and elderlygroups; this effect was also seen in LAD and carotid arteriesbut not in the aorta (Table 1). A separate analysis of adult

TABLE 1. Multiple Regression Analysis Evaluating the Effect of Age, Plasma Cholesterol, Plasma Glucose,and Plasma Vitamin E on Lesion Sizes in Intracranial and Extracranial Arteries of Adults andElderly Subjects

MCA Basilar LAD Carotid AA

� P � P � P � P � P

No liver cirrhosis

Adults

Age 2.199 0.033 6.329 0.021 1.155 0.012 1.495 0.004 1.834 0.038

Glucose 4.898 0.456 5.111 0.723 4.106 0.543 1.145 0.123 3.157 0.654

Cholesterol 3.271 0.656 5.533 0.654 5.111 0.048 3.773 0.456 2.001 0.432

Vitamin E �2.222 0.013 �6.938 0.008 �9.849 0.010 �1.188 0.044 �1.916 0.118

Elderly

Age 0.169 0.020 1.192 0.009 1.900 0.034 1.418 0.043 2.050 0.543

Glucose 0.523 0.323 1.099 0.412 1.877 0.543 1.940 0.321 2.702 0.771

Cholesterol 2.918 0.258 1.757 0.342 2.678 0.042 1.329 0.218 1.548 0.421

Vitamin E �3.887 0.006 �5.786 0.001 �5.495 0.00002 �1.155 0.033 �0.757 0.328

Liver cirrhosis

Adults (n�16)

Age 2.044 0.038 4.785 0.036 2.342 0.041 2.041 0.016 2.321 0.026

Glucose 4.123 0.321 5.865 0.675 3.008 0.432 1.886 0.225 3.732 0.551

Cholesterol 4.004 0.512 4.876 0.554 3.777 0.073 2.987 0.321 2.657 0.471

Vitamin E �2.003 0.024 �6.003 0.012 �6.821 0.038 �1.654 0.031 �2.316 0.073

Elderly (n�9)

Age 0.204 0.013 1.876 0.010 2.006 0.042 1.654 0.048 2.851 0.421

Glucose 0.342 0.128 2.004 0.275 2.223 0.135 2.786 0.287 1.983 0.633

Cholesterol 2.453 0.321 1.998 0.288 3.234 0.068 1.972 0.183 1.744 0.509

Vitamin E �3.004 0.018 �4.112 0.021 �5.072 0.003 �1.437 0.028 �0.973 0.194

MCA indicates middle cerebral artery; AA, abdominal aorta. Data for subjects without and with cirrhosis are presented separately,because an influence of coagulopathies in cirrhotic patients on atherogenesis could not be ruled out a priori. Significant P values areindicated in boldface.

2474 Stroke November 2001



(n�16) and elderly men (n�9) who died of liver cirrhosisshowed similar results (Table 1).

ImmunohistochemistryParaffin-embedded serial sections of arteries from the studypopulation were immunostained and assessed for the intimalpresence of apolipoprotein B, oxLDL, macrophage-derivedfoam cells, and MMP-9. Results are shown in Figure 2. Theabdominal aorta, common carotid artery, and LAD showedsignificantly more staining for LDL, oxLDL, foam cells, andMMP-9 than did the middle cerebral and basilar arteries. Thiswas in agreement with the smaller numbers and sizes oflesions in intracranial arteries compared with extracranialarteries. In each artery, there was an age-related increase ofstaining for all epitopes. Only in the elderly group did brainarteries show a marked staining for oxLDL, MMP-9, andfoam cells. Staining of the same section with differentlylabeled detection antibodies indicated colocalization betweenoxLDL and MMP-9 in basilar and middle cerebral arteries ofadults (r�0.41 and 0.35, respectively; P�0.04) and elderlymen (r�0.48 and 0.41, respectively; P�0.01). Both endothe-lial and smooth muscle cells (Figure 3, top panel, arrow-heads) showed immunostaining for MMP-9.

MMP-9 ActivityEvidence of an age-related increase of MMP-9 activity inbrain arteries was also provided by Western blot analysis andby zymography in whole-cell extracts, which showed anincrease in MMP-9 protein expression and gelatinolyticactivity consistent with the degree (class I, II, or III lesions)of atherosclerotic lesions (Figure 3, bottom panel).

Tissue Scavenger EnzymesTo investigate whether differences in lipid peroxidation maycontribute to the greater resistance of intracranial arteries toatherogenesis, we determined the arterial activities of gluta-thione peroxidase, catalase, CuZn-SOD, and Mn-SOD. As

evident in Table 2, both intracranial arteries showed muchbetter antioxidant protection than did extracranial arteriesuntil the adult age. In contrast, the content of all antioxidantsin intracranial arteries significantly decreased in the elderlygroup. Glutathione peroxidase was inversely correlated withatherosclerotic lesion size in the middle cerebral artery(r��0.56, P�0.002) and basilar artery (r��0.61, P�0.001)in the elderly group. Mn-SOD activity was also correlatedwith lesion sizes in the middle cerebral artery (r��0.71,P�0.0008) and the basilar artery (r��0.77, P�0.0005).

Figure 2. Presence of oxLDL, apolipoproteinB, macrophage-derived foam cells, andMMP-9 in sections of intracranial and extra-cerebral arteries. Serial sections from eachartery were immunostained with monoclonalantibodies MDA2 (against epitopes of oxLDL),NP1539 (against apolipoprotein B), HAM-56(against macrophages), and �-MMP-9(against MMP-9), as described in Subjectsand Methods. Sections containing at least 1lesion showing substantial immunostainingwere counted as positive, and results areexpressed as percentage of all sections of thesame artery. Results are expressed as themean�SD. *P�0.05 compared with MCA andBAS; **P�0.001 compared with MCA andBAS; �P�0.01 vs fetuses; and ��P�0.01 vschildren and adults.

Figure 3. Western blot analysis (top) of MMP-9 protein expres-sion in arterial whole-cell extracts. The same amount of protein(100 �g) was loaded on each lane, and results were normalizedfor �-tubulin. In an aliquot of the same samples (bottom), theMMP-9 matrix-degrading activity was evaluated by gelatinzymography. Lanes are as follows: 1, MCA from a child; 2, MCAwith fatty streaks from an adult man; 3, MCA with extensivelesions from an elderly man; 4, MCA with moderate lesions froman elderly man; 5, BAS from a child; and 6, BAS with moderatelesions from an elderly man. Densitometric scan analysis of 4different Western blots showed that lane 2 was significantlyhigher compared with lane 1 (P�0.05). Similarly, lane 3 was sig-nificantly higher compared with lane 1 (P�0.00001) and lane 2(P�0.00004). P�0.0001 for lane 4 vs lane 1; P�0.001 for lane 4vs lane 2; and P�0.00003 for lane 4 vs lane 3. Finally, lane 6was significantly higher than lane 5 (P�0.001). Densitometricscan analysis of 3 different zymograms showed that lane 3 wassignificantly greater than lane 1 (P�0.004) and lane 2 (P�0.01).Lane 4 was greater than lane 1 (P�0.03), lane 2 (P�0.05), andlane 3 (P�0.05). Finally, lane 6 was significantly greater thanlane 5 (P�0.05).




To further examine the potential connection between anti-oxidant defenses in the arterial wall and lesion sizes, thevascular activity of Mn-SOD was plotted over age (Figure 4).Although in each age group data appeared to be clustered,

linear regression analysis indicated that there was a stronginverse correlation between Mn-SOD activity in the 2 intra-cranial arteries but not in the extracranial arteries (data for theLAD are not shown but closely resembled data for the carotid

TABLE 2. Comparison of the Total Activity of Scavenger Enzymes in Homogenates of Intracranial andExtracranial Arteries of Humans of Different Ages

MCA Basilar Artery LAD Carotid Artery AA

Fetuses

Glutathione peroxidase, mU/mg protein 84�16 80�11 72�12 68�14 70�11

Catalase, IU/mg protein 19�6 18�5 15�6 14�7 18�5

CuZn-SOD, IU/mg protein 6.5�0.9 6.6�0.9 6.2�0.8 7.2�1.0 6.8�0.9

Mn-SOD, IU/mg protein 3.1�0.6* 3.2�0.6* 1.5�0.4 1.6�0.4 2.0�0.4

Children

Glutathione peroxidase, mU/mg protein 90�12 91�14 85�15 80�16 81�15

Catalase, IU/mg protein 19�8 20�6 19�6 18�7 19�7

CuZn-SOD, IU/mg protein 7.5�1.1 7.0�1.1 6.5�0.9 7.5�1.4 7.0�1.2


Adults

Glutathione peroxidase, mU/mg protein 78�12 75�13 65�12‡ 61�10‡ 68�12

Catalase, IU/mg protein 14�3 15�4 12�4‡ 11�5‡ 13�4

CuZn-SOD, IU/mg protein 5.9�0.6 5.6�0.8 5.2�0.4‡ 5.0�0.8† 5.2�0.5


Elderly

Glutathione peroxidase, mU/mg protein 62�11† 60�10† 48�11‡ 57�12‡ 58�11‡

Catalase, IU/mg protein 13�5† 14�3† 11�3† 12�5† 14�4

CuZn-SOD, IU/mg protein 5.4�0.5† 5.6�0.8 4.8�0.6‡ 4.6�0.8‡ 4.8�0.7

Mn-SOD, IU/mg protein 1.8�0.4† 1.6�0.3† 1.0�0.3† 1.3�0.5 1.5�0.4

Values are mean�SD.*P�0.05 vs LAD, carotid artery, and AA; †P�0.05 vs fetuses and children; and ‡P�0.05 vs children.

Figure 4. Activity of Mn-SOD in intracra-nial and extracranial arteries of all 183subjects over age. For this purpose, thefetal ages (months of fetal age) wereconverted to negative fractions of ayear.




artery and abdominal aorta). Indeed, only a minor influenceof aging was evident in the extracranial arteries (r2�0.16 and0.37, respectively) compared with the intracranial arteries(r2�0.71 and 0.68).

Lesion Progression Over TimeWhen the size of atherosclerotic lesions of all subjects wassimilarly plotted over age (Figure 5), data of the carotid arteryand abdominal aorta were best fitted by a linear regressionline. This is consistent with the linear progression of overallaortic lesions during childhood and infancy reported by theFELIC study.21 In contrast, in both intracranial arteries, datafollowed an exponential curve, with the most apparentacceleration occurring in the elderly subjects.

DiscussionThe present study represents the first systematic comparison ofintracranial and extracranial atherogenesis in humans rangingfrom premature fetuses to elderly subjects. None of the studysubjects had confounding classic risk factors of the disease, asfar as could be ascertained retrospectively. The main results are(1) that intracranial arteries generally contain higher activities ofantioxidant (oxygen radical scavenger) enzymes than do ex-tracranial arteries, (2) that the antioxidant protection of intracra-nial arteries markedly decreases with increasing age, and (3) thatthis coincides with a rapid acceleration of atherosclerosis inintracranial arteries of elderly subjects, whereas the progressionof atherogenesis in extracranial arteries is linear over all ages.This suggests that the progression of atherosclerosis in intracra-nial arteries of older men may in part be due to reducedintracellular defenses against oxygen radical–mediated pro-cesses. Increased levels of in situ MMP-9 activity are consistentwith this assumption, because oxLDL is known to promoteMMP-9 expression. These findings are noteworthy, because

they indicate a potential role of lipid peroxidation even insubjects without conventional risk factors, in particular withouthypercholesterolemia or dyslipidemia.

Our findings are consistent with several previous observa-tions. For example, the presence of oxLDL in lesions signifi-cantly increases with advancing age, and plasma LDL alsobecomes more susceptible to oxidation.22,25 This may be aconsequence of the age-related reduced capability of intracellu-lar defenses against oxygen radical–mediated processes. As wenow show, glutathione peroxidase (one of the most importantantioxidative enzymes in the brain),26 Mn-SOD, CuZn-SOD,and catalase, were significantly decreased in intracranial arteriesof elderly men. Clearly, the relative contributions of thesepotential mechanisms to atherogenesis must be addressed inexperimental models of the disease rather than in postmortemtissues. However, in vitro exposure to oxLDL has been shown toresult in impaired vasodilatation of carotid arteries but not basilararteries,27 suggesting that the differences in arterial enzymeactivities may be functionally relevant. Recent data in stroke-prone hypertensive rats have demonstrated that exogenousadministration of the antioxidant, vitamin E, or calcium antag-onists with antioxidant properties reduces their long-term mor-tality.13 On the other hand, studies in humans have yieldedconflicting effects of vitamin E treatment on clinical end points(see review28). However, atherogenesis is a complex disease,29 andit is possible that the lower susceptibility of intracranial arteries tocholesterol-induced atherogenesis results mainly from the coinci-dence of lower blood pressure1 and decreased susceptibility toendothelial dysfunction.30 Reduced blood-brain barrier permeabilityto LDL and oxLDL may also account for the lesser atherogenicresponse of intracranial arteries to hypercholesterolemia.

The present study also demonstrates that MMP-9 expres-sion in brain arteries progressively increases with age.MMP-9 activity is correlated with human carotid plaque

Figure 5. Atherosclerosis (correctedcumulative lesion sizes per section) inintracranial and extracranial arteries of all183 subjects over age. Fetal ages wereconverted to negative fractions of a year.Best fitting regression models areshown.




instability.19 Because oxLDL upregulates MMP-9 expres-sion18 and because we found a colocalization of MMP-9 andoxLDL in atherosclerotic lesions and age-related increases inboth oxLDL and MMP-9 gelatinolytic activity, it is possiblethat age-related MMP-9 overexpression may play a role inplaque rupture of brain arteries. However, we cannot estab-lish whether increased in situ MMP-9 activity in brain arteriesis a primary event caused by weakening antioxidant defensesor whether it is a consequence of increased lesion formation.

Increased plasma oxidative stress31,32 and low plasma antiox-idant activity33 are seen during stroke in middle-aged men.Experiments evaluating whether interventions strengthening an-tioxidant defenses offer particular benefits to elderly subjectsmay resolve the question of whether increased oxidative stresscontributes to increased atherogenesis and/or stroke. Althoughthe present results support a causal role of oxidative stress inincreased atherogenesis, it cannot be ruled out that the increaseis the result rather than the cause of increased lesion formation.For example, it is conceivable that the progression of lesionsdisrupts protective anatomic features of intracranial arteries orthat the generation of antioxidant enzymes is affected oncelesions reach a certain stage.

AcknowledgmentsThis study is dedicated to the memory of Dr Fulvio Pinto (1916 to 1981)and was supported by grants 97/60% from the Ministero della Univer-sità e Ricerca Scientifica e Tecnologica (grant ISNIH, 33343/97) andfrom the National Heart, Lung, and Blood Institute (grant HL-56989).

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17. Rajagopalan S, Meng XP, Ramasamy S, Harrison DG, Galis ZS. Reactiveoxygen species produced by macrophage-derived foam cells regulate theactivity of vascular matrix metalloproteinases in vitro: implications foratherosclerotic plaque stability. J Clin Invest. 1996;98:2572–2579.

18. Xu XP, Meisel SR, Ong JM. Oxidized low-density lipoprotein regulatesmatrix metalloproteinase-9 and its tissue inhibitor in human monocyte-derived macrophages. Circulation. 1999;99:993–998.

19. Loftus IM, Naylor AR, Goodall S, Crowther M, Jones L, Bell PR,Thompson MM. Increased matrix metalloproteinase-9 activity in unstablecarotid plaques: a potential role in acute plaque disruption. Stroke. 2000;31:40–47.

20. Napoli C, D’Armiento FP, Mancini FP, Witztum JL, Palumbo G, PalinskiW. Fatty streak formation occurs in human fetal aortas and is greatlyenhanced by maternal hypercholesterolemia: intimal accumulation ofLDL and its oxidation precede monocyte recruitment into early athero-sclerotic lesions. J Clin Invest. 1997;100:2680–2690.

21. Napoli C, Glass CK, Witztum JL, Deutch R, D’Armiento FP, Palinski W.Influence of maternal hypercholesterolaemia during pregnancy on pro-gression of early atherosclerotic lesions in childhood: Fate of EarlyLesions in Children (FELIC) study. Lancet. 1999;354:1234–1241.

22. Napoli C, Abete P, Corso G, Malomi A, Postiglione A, Ambrosio G,Cacciatore F, Rengo F, Palumbo G. Increased low-density lipoproteinperoxidation in elderly men. Coron Artery Dis. 1997;8:129–136.

23. Napoli C, Cicala C, Wallace JL, de Nigris F, Santagada V, Caliendo G,Franconi F, Ignarro IJ, Cirino G. Protease-activated receptor-2 modulatesmyocardial ischemia-reperfusion injury in the rat heart. Proc Natl AcadSci U S A. 2000;97:3678–3683.

24. Fernandez-Patron C, Zhang Y, Radomski MW, Hollenberg MD, DavidgeST. Rapid release of matrix metalloproteinase by thrombin in the rataorta: modulation by protein tyrosine kinase/phosphatase. ThrombHaemost. 1999;82:1353–1357.

25. Reaven PD, Napoli C, Merat S, Witztum JL. Lipoprotein modificationand atherosclerosis in aging. Exp Gerontol. 1999;34:527–537.

26. Reiter RJ. Oxidative processes and antioxidative defense mechanisms inthe aging brain. FASEB J. 1995;9:526–533.

27. Napoli C, Paternò R, Faraci FM, Taguchi H, Postiglione A, Heistad DD.Mildly oxidized low-density lipoprotein impairs responses of carotid butnot basilar artery. Stroke. 1997;28:2266–2272.

28. Pryor WA. Vitamin E and heart disease: basic science to clinical inter-vention trials. Free Radic Biol Med. 2000;28:141–164.

29. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999;340:115–126.

30. Ignarro LJ, Cirino G, Casini A, Napoli C. Nitric oxide as a signalingmolecule in the vascular system: an overview. J Cardiovasc Pharmacol.1999;34:879–886.

31. Polidori MC, Frei B, Cherubini A, Nelles G, Rordorf G, Keaney JF Jr,Schwamm L, Mecocci P, Koroshetz WJ, Beal MF. Increased plasmalevels of lipid hydroperoxides in patients with ischemic stroke. FreeRadic Biol Med. 1998;25:561–567.

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33. Leinonen JS, Ahonen JP, Lonnrot K, Jehkonen M, Dastidar P, MolnarG, Alho H. Low plasma antioxidant activity is associated with highlesion volume and neurological impairment in stroke. Stroke. 2000;31:33–39.




Editorial Comment

Age, Antioxidants, and Atherogenesis

An increasing amount of evidence suggests that oxidativemechanisms, in particular oxidative modification of LDL, areclosely related to a number of inflammatory reactions, whichseem essential for both initiation and progression of athero-sclerosis.1 This process involves infiltration of inflammatorycells, intimal thickening, accumulation of extracellular ma-trix, fibrous cap formation, and angiogenesis.1 Whereasatherosclerotic plaques develop over decades, it is largelyunknown what suddenly triggers an unstable plaque torupture and cause clinical symptoms such as unstable angina,myocardial infarction, and stroke.2 The protective role ofantioxidants such as vitamin E on atherosclerotic disease orclinical event rate is still unclear, but research in this areacontinues.3 The article by D’Armiento et al is part of theintense search for links between age, antioxidants, andatherogenesis.

The initiating step of atherogenesis is thought to beendothelial dysfunction, which may be caused by one orseveral factors, including elevated and oxidized LDL as wellas free radicals caused by smoking, hypertension, and diabe-tes.1 These factors activate the endothelial cells, expressingadhesion molecules such as the vascular cell adhesion mole-cule (VCAM)-1 and promoting monocyte and T-lymphocyteinfiltration of the intima.1 To minimize the damage caused byoxidation, the oxidized LDL particles are internalized by theactivated monocytes, so-called macrophages, via surfacescavenger receptors, thereby transforming them into foamcells. Macrophages produce cytokines and growth factors andinduce smooth muscle cell proliferation, ultimately increasingatherosclerotic plaque size.4,5 The inflammatory process alsoinvolves upregulation and activation of matrix metallopro-teinases (MMPs) in endogenous smooth muscle cells.6 In-creased production of MMPs in the shoulder region of theplaque is thought to play a role in plaque instability andrupture through degradation of extracellular matrix and thefibrous cap, facilitation of migration through the endothelialcell layer and basement membrane,7 intimal thickening, andangiogenesis.

Supporting the oxidation theory outlined above, structur-ally different antioxidants such as vitamin E, probucol,butylated hydroxytoluene, and N,N�-diphenyl-phenylene dia-mine are known to both inhibit ex vivo LDL oxidation andreduce free radical formation by modified LDL and athero-sclerosis in animals.8 However, in the clinical trials reported,vitamin E intake/supplementation reduced the incidence ofmyocardial infarction in patients with established coronaryartery disease in the Cambridge Heart Antioxidant Study

(CHAOS) trial,9 but not in the Alpha-Tocopherol Beta-Carotene (ATBC), Heart Outcomes Prevention Evaluation(HOPE), and Gruppo Italiano per lo Studio della Sopravvi-venza nell’ Infarto Miocardico (GISSI) trials.10 Furthermore,the outcomes of intervention trials with vitamin C in coronaryheart disease are still disappointing. This indicates that ourknowledge about links between lipoprotein oxidation andatherogenesis is incomplete.

In this autopsy study by D’Armiento et al, the authorspursue their original hypothesis that intracranial arteriespossess a greater resistance than do extracranial arteries. Thiscould be due to a better antioxidant protection of theintracranial arteries, which might also explain the lateroccurrence of atherogenesis in these vessels. The authors alsodescribe the effect of aging on the extent of atherosclerosisand compare this to the activity of 4 important antioxidantenzymes (glutathione peroxidase, Mn and CuZn superoxidedismutase, and catalase) and the MMP-9 (gelatinase-2) in thewalls of intracranial and extracranial arteries. The histomor-phometric amount of atherosclerosis was found to increaselinearly with age in the 4 different male age groups (prema-ture fetuses, children, adults, and elderly) without known riskfactors for atherosclerosis. At any age, the amount of athero-sclerosis was 6- to 19-fold greater in extracranial than inintracranial arteries. Furthermore, the antioxidant activitydecreased with age in the intracranial arteries. Moreover,MMP-9 expression and gelatinolytic activity increased withage and correlated with severity of intracranial atherosclero-sis. The fact that levels of MMP-9 and LDL both increasedwith age and were colocalized in atherosclerotic lesionssuggests that age-related overexpression of MMP-9 may playa role in plaque rupture. Supporting this hypothesis, MMP-9activity was found to correlate with human carotid plaqueinstability.11 As these studies point out, the question ofwhether increased in situ MMP-9 activity and atherogenesisin cerebral arteries is a result of weakened antioxidantdefenses or a consequence of increased lesion formationremains unanswered.

When these findings are implemented in future research,the role of age must not be underestimated. Intervention trialswith antioxidants should include younger, less-diseased indi-viduals than previously studied, since the protective effectsagainst atherogenesis, cardiovascular disease, and events maybe most profound in individuals aged 20 to 40 years or mayneed a longer period of time to exert protection. Such data areneeded as valuable directions for targeting preventive therapyagainst complications to atherosclerosis, which remains theleading cause of death in the industrial world.

Marie-Louise M. Grønholdt, MD, PhD, Guest EditorDepartment of Vascular Surgery

RigshospitaletCopenhagen, Denmark




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340:115–126.2. Falk E. Why do plaques rupture? Circulation. 1992;86(suppl 6):III-

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11. Loftus IM, Naylor AR, Goodall S, Crowther M, Jones L, Bell PR,Thompson MM. Increased matrix metalloproteinase-9 activity in unstablecarotid plaques: a potential role in acute plaque disruption. Stroke. 2000;31:40–47.




NapoliD'Armiento, Giuseppe Crimi, Pasquale Abete, Wulf Palinski, Mario Condorelli and Claudio Francesco P. D'Armiento, Alfredo Bianchi, Filomena de Nigris, David M. Capuzzi, Maria R.

Extracranial Arteries in Men Without Classic Risk Factors for AtherosclerosisAge-Related Effects on Atherogenesis and Scavenger Enzymes of Intracranial and

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