Aortic Constriction Exacerbates Atherosclerosis and InducesCardiac Dysfunction in Mice Lacking Apolipoprotein E
Jiao-Hui Wu, John Hagaman, Shinja Kim, Robert L. Reddick, Nobuyo Maeda
Abstract—Despite considerable evidence suggesting that hypertension contributes to the development and progression ofatherosclerosis, the causative links remain unclear. We have tested the effects of chronic hypertension induced bysuprarenal aortic constriction on the development of atherosclerosis in apolipoprotein E–deficient (Apoe�/�) mice.Compared with a sham operation, narrowing the aortic luminal diameter by 33% increased blood pressure proximal tothe constriction by �15 mm Hg, but the pressures distal to the constriction were unchanged. Kidney renin mRNA andplasma renin activity were also unaffected. Compared with plaque size after the sham operation, atherosclerotic plaquesize in the aortic root 8 weeks after coarctation was increased to 245% and 152% in males and females, respectively.Aortic segments at the constriction were free of atherosclerotic deposits, but segments proximal to the constriction weredilated and had atherosclerotic lesions. Thrombi were present immediately below the constriction in Apoe�/� andwild-type vessels. Surprisingly, compared with wild-type mice, the Apoe�/� mice were more susceptible to the cardiachypertrophy and dysfunction induced by pressure overload. Thus, aortic coarctation exacerbates atherosclerosis invessels proximal to the constriction without a concomitant increase in the renin-angiotensin system. Our study alsosuggests that apolipoprotein E plays an important role in modulating cardiac hypertrophy. (Arterioscler Thromb VascBiol. 2002;22:469-475.)
Hypertension and atherosclerosis are 2 of the most im-portant causes of morbidity and mortality in humans.
Numerous clinical and epidemiological studies have identi-fied systemic arterial hypertension as an independent andpotent risk factor for the development of atheroscleroticdisease. For example, the Framingham study found thatdiastolic blood pressure (BP), systolic BP, and pulse pressureare predictors of coronary heart disease risk.1,2 The extent ofatherosclerosis in the aorta and coronary arteries of subjectsin the Pathobiological Determinants of Atherosclerosis inYouth (PDAY) study3 was greater in hypertensive comparedwith normotensive individuals. Similarly, an association be-tween ultrasound-measured carotid artery intima/media thick-ness and systolic BP was demonstrated in the AtherosclerosisRisk in Communities (ARIC) Study.4
Animal models with combined genetic risks for atheroscle-rosis and hypertension have also shown that elevated BPaccelerates atherogenesis. Thus, lack of endothelial NOsynthase increases BP and exacerbates atherosclerosis inmice lacking apoE (Apoe�/� mice).5 Similarly, Dahl salt-sensitive hypertensive rats that overexpress the human cho-lesteryl ester transfer protein develop severe hyperlipidemiaand atherosclerosis and show decreased survival.6 Neverthe-less, current data do not allow an unequivocal distinctionbetween the physical effects on the atherogenic process of the
increased systemic BP and the effects on vasculature causedby concurrent changes in circulating vasoactive mediators,and causative links between hypertension and atherosclerosishave yet to be defined.
Surgical coarctation of the aorta in combination with highdietary cholesterol has been shown to markedly increaseatherosclerosis in rabbits and monkeys.7–9 In the presentstudy, we have applied aortic coarctation to alter the BPs ofApoe�/� mice with the aim of understanding the interactionbetween BP changes and a genetic form of hyperlipidemia.We find that aortic coarctation in mice accelerates atheroscle-rosis in the aorta proximal to the constriction. Organizedthrombi are found immediately distal to the constrictionindependent of hyperlipidemia. A surprising new finding isthat compared with wild-type mice, Apoe�/� mice are signif-icantly more susceptible to the cardiac hypertrophy inducedby chronic coarctation of the aorta.
Methods
AnimalsApoe�/� mice10 were backcrossed at least 7 generations to a C57BL/6genetic background. C57BL/6 mice were used as the wild-typecontrol group. Care and experimental procedures were in compliancewith the Principles of Laboratory and Animal Care established by the
Received November 9, 2001; revision accepted December 31, 2001.From the Department of Pathology and Laboratory Medicine (J-H.W., J.H., S.K., N.M.), University of North Carolina, Chapel Hill, and the Department
of Pathology (R.L.R.), University of Texas Health Sciences Center, San Antonio.Correspondence to Dr Nobuyo Maeda, Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599-7525.
National Society for Medical Research and were approved by theinstitutional committee.
Aortic ConstrictionMice were anesthetized with isoflurane and placed in the supineposition on warm pads under an operating microscope. The abdomenwas opened, and the abdominal aorta at the suprarenal level wasfreed of the surrounding adventitial adipose tissue by gentle dissec-tion. The aorta between the celiac and superior mesenteric arterieswas constricted by tying a 6-0 silk suture ligature against a 28-gaugeneedle for males and 30-gauge needle for females to yield an �33%narrowing of the luminal diameter when the needle was removed.For the sham operations, 18-gauge needles and 20-gauge needleswere used for males and females, respectively. Coarctation wasapplied when the mice were 2 months of age, and they wereeuthanized at 4 months of age for evaluation.
EchocardiographyEchocardiograms were obtained on conscious mice with gentlerestraint at 2, 4, 6, and 8 weeks after surgery. Two-dimensionalguided M-mode echocardiography was performed by using HDI5000 echocardiograph equipment (ATL) and a 7.5-MHz transduc-er.11 Heart rate was calculated by using the period between 2consecutive end-diastolic points from pulsed Doppler echocardiog-raphy. Left ventricular (LV) mass was calculated as described.12
Intra-arterial BP MeasurementMice were anesthetized with isoflurane, and the right carotid andright femoral artery were cannulated with flame-stretched PE 20tubing. Catheters were connected to MLT1050 precision transducersand Power-Laboratory recording equipment (ADI Instruments).Approximately 90 minutes after the mice had recovered from theanesthesia, intracarotid and femoral artery BPs were measuredsimultaneously. An average of 10 measurements was taken as amean value for each animal.
Histology and Lesion MeasurementsMice were euthanized at 4 months of age. Blood was collected fromthe heart into a tube containing EDTA (final concentration5 mmol/L) for plasma lipid analysis.13 The heart and vascular treewere perfused by intracardiac infusion with 10 mL of 4% parafor-maldehyde in PBS under physiological pressure. Heart segments thatcontain the aortic sinus were serially sectioned for morphologicalevaluation of atherosclerotic lesions.13 Thoracic aortas were excised,freed of adventitial fat, and examined under microscopy. Specimenswere subsequently dried and weighed, and lipids were extracted bymethanol/chloroform (1:1 [vol/vol]). Serial 10-�m-thick sections ofthe contiguous abdominal aorta were cut every 100-�m distancefrom 5 mm above the suture level to 5 mm below for evaluation.Paraffin-embedded tissue sections were stained with hematoxylinand eosin (H&E), with periodic acid-Schiff stain (PAS), or with
trichrome. Aortic segments were also embedded in epoxy resin, andsections were stained with toluidine blue.
Renin mRNA and Plasma ActivityTotal RNA was prepared from homogenized kidney by using ABI 6700(ABI). Renin mRNA levels were determined by real-time quantitativereverse transcription–polymerase chain reaction with ABI 7700 (ABI) withthe use of 5�-ACAGTATCCCAACAGGAGAGACAAG-3� and 5�-GCACCCAGGACCCAGACA-3� as primers and 5�-FAM-TGGCTCTC-CATGCCATGGACATCC-Tamra-3� as the detection probe. Amplifica-tion of �-actin mRNA was used as an internal standard with 5�-CTGCCTGACGGCCAAGTC-3� and 5�-CAAGAAGGAAGGCTGG-AAAAGA-3� as primers and 5�-TET-CACTATTGGCAACGAGCGGT-TCCG-Tamra-3� as the detection probe. Plasma concentration of activerenin was determined by radioimmunoassay as described.14
Statistical AnalysisData were analyzed by using a JMP software package (SAS InstituteInc). Means of different groups were compared by ANOVA.Time-dependent changes were analyzed by MANOVA with repeatedmeasures.
ResultsAortic Coarctation and Survival of AnimalsSurgical coarctation (banding) of the abdominal aorta of2-month-old Apoe�/� or wild-type mice yielded an approxi-mately one third narrowing of the luminal diameter (seebelow). Aortas of the sham-operated mice had no luminalnarrowing. All mice recovered from anesthesia, but �20% ofsham-operated mice and 40% of the banded mice died withina week after surgery (most died within 1 or 2 days).Postmortem examination of some banded mice that died after24 hours showed thrombi at or near the banded area. Noadditional losses of mice were seen 1 week after banding,except in the banded Apoe�/� group. Thus, 34 of 86 bandedApoe�/� mice died during the first week, and 9 died between6 and 8 weeks after surgery. Some of the later deaths were byaortic dissection, and some mice showed signs of heartfailure. All the mice included in the present study that werealive at 8 weeks after surgery appeared healthy. There was nosignificant difference in weight gain between the sham-operated and banded groups (Table 1). Compared withsham-operated hearts, the hearts of male and female Apoe�/�
mice were significantly enlarged after banding and showed an�50% increase in the heart-to-body weight (HW/BW) ratio(P�0.0001). In contrast, the increase of the HW/BW ratio
TABLE 1. Effects of 8-wk Aortic Banding on Body and Organ Weights and Plasma Lipid Levels
Mice Sex Treatment n
BW, gHW/BW,
mg/gKW/BW,
mg/gCH,
mg/dLTGs,
mg/dL2 mo 4 mo
Apoe�/� M Sham 34 24.3�0.7 28.7�0.6 6.5�0.2 8.9�0.2 331�37 38�6
Apoe�/� M Banding 21 23.7�0.7 26.6�0.9 9.5�0.8* 8.6�0.3 324�26 48�9
Apoe�/� F Sham 25 19.1�0.5 22.9�0.3 6.6�0.2 8.1�0.2 286�24 31�6
Apoe�/� F Banding 22 18.6�0.5 21.8�0.8 10.1�0.9* 8.1�0.2 301�18 38�4
WT M Sham 5 24.5�0.7 28.4�0.7 5.8�0.4 7.6�0.1 56�3 63�14
WT M Banding 6 24.4�0.8 29.9�1.3 6.9�0.4† 7.7�0.4 54�6 63�20
WT F Sham 4 17.3�0.5 21.0�0.5 6.1�0.1 7.9�0.4 ND ND
WT F Banding 10 17.6�0.3 20.7�0.5 7.4�0.5 8.2�0.3 ND ND
BW indicates body weight before (2 mo) the surgery and at euthanasia (4 mo); HW, heart weight; KW, kidney weight; CH, plasmatotal cholesterol; TGs, plasma triglycerides; WT, wild type; and ND, not determined. Values are mean�SE.
*P�0.0001 compared with sham; †0.01�P�0.05 compared with sham.
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was 20% in the banded wild-type mice compared with thesham-operated mice (P�0.01, in genotype effects). Totalplasma cholesterol and triglyceride levels were not affectedby banding.
Increased BP Proximal to the CoarctationAortic banding significantly increased BP proximal to theconstriction (Table 2). The differences between the carotidand femoral artery BPs of individual animals averaged25�2 mm Hg (n�52) in the banded group versus 10�1 mm Hg(n�32) in the sham-operated group (P�0.0001). The increasedpressure differentials resulted mainly from an increased carotidBP in the banded mice (126�3 mm Hg in banded micecompared with 111�2 mm Hg in sham-operated mice,P�0.001). The difference in the femoral artery BP betweenbanded and sham-operated mice was not significant (100�2versus 101�2 mm Hg, P�0.3). There were no statisticallysignificant differences in BPs (carotid or femoral) between thewild-type and Apoe�/� mice or between the sexes. The HW/BWratio of individual Apoe�/� animals was positively correlatedwith the differential BPs between their carotid and femoralarteries (R2�0.31, P�0.0001; n�68).
Short-term aortic constriction above the renal arteries canincrease renin production in the kidneys, which can sense thereduced BP distal to the constriction.15 However, we found nodifference in renin mRNA levels in the kidneys or in plasmarenin activity of banded and sham-operated mice at 8 weeksafter banding (Table 2), which is consistent with the absenceof significant differences between the femoral arterial BPs ofthe banded and the sham-operated animals.
Vessels in the Vicinity of ConstrictionsCross sections of the abdominal aorta of the banded area wereexamined by light microscopy (Figure 1). The position of thebanding applied to the aorta was identified by the presence ofthe suture (Figure 1D and 1E). The banding narrowed theaortic luminal area in the Apoe�/� and wild-type mice to39�4% (n�11) relative to that in the sham-operated mice(100�5%, n�5). The intima contained 2 or 3 layers of smallcuboidal cells, most likely representing proliferation ofsmooth muscle cells to accommodate the increased pressure(Figure 1E and 1F). Lipids were absent in the intima.
The aorta proximal to the banding site was dilated with aluminal diameter �30% larger than the diameter of the
TABLE 2. Increased BPs of Apoe�/� Mice Proximal to the Aortic Constriction
BP indicates mean intra-arterial BP; �BP, BP differences between carotid and femoral arteries; and HR, heart rate. Values are mean�SE. Thenumbers of animals are in parentheses. BP was measured at 8 wk after banding. The amount of renin message is expressed as nanograms permilligram of the total cellular RNA. HR was measured by echocardiogram. BPs of WT sham males were not determined.
*P�0.01 vs sham; †0.01�P�0.05 vs sham.
Figure 1. Histological evaluation of changes inApoe�/� aorta immediately proximal to thecoarctation (A through C), at the coarctation (Dthrough F), and distal to the coarctation (Gthrough I). Cross sections were stained withH&E (A, D, E, and G), with Sudan IVB andhematoxylin (B, C, and H), and with toluidineblue (F and I). A, Original magnification �35. B,i indicates intima; m, media; and a, adventitia.Original magnification �175. C, Black arrowsindicate the complete break of the elastica.Original magnification �27. D, s indicatessuture. Original magnification �35. E, Originalmagnification �167. F, Original magnification�580. G, th indicates organized thrombus.Original magnification �35. H, White arrow indi-cates lipids in media (m). Original magnification�100. I, White arrow indicates small aggre-gates of platelets. Original magnification �315.
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sham-operated aorta (Figure 1A). In 10 of the 12 bandedApoe�/� mice examined, the aortas immediately above theconstriction contained atherosclerotic lesions of various sizes,ranging from foam cell accumulations to well-developedplaques (Figure 1B). In 4 mice, the medial layer wasdisrupted, and plaque material was deposited outside thevessel wall (Figure 1C). Plaques were not present furtherupstream (500 �m) from the banding site. The presence ofabdominal aortic lesions is rare in Apoe�/� mice at 4 monthsof age, and none of the 6 sham-operated Apoe�/� mice thatwere examined had plaques in this area.
Vessels immediately distal to the site of banding wereslightly dilated, with the luminal diameter increased by�10% relative to the sham-operated vessels. In all 6 Apoe�/�
vessels and in the 3 wild-type vessels evaluated, there werelarge thrombi attached at multiple locations to the aortic wall(Figure 1G). Smooth muscle cells within the medial layersnear the thrombi were elongated and contained lipids, butfoamy macrophages were not present (Figure 1H). Most ofthe thrombi were well organized and composed of fibrousmaterials, but occasionally, small aggregates of platelets werepresent on the surface (Figure 1I), suggesting that thrombusformation at this location was an ongoing process. However,even in the hyperlipidemic environment of Apoe�/�, lipidswere not identified as a component of these thrombi. Thrombiin the same area were also found in the vessels of 3 of the 6Apoe�/� mice 4 weeks after the banding but in none of thevessels of 9 mice 2 weeks after banding (not shown). None ofthe sham-operated mice developed thrombi near the site ofbanding.
Cardiac Hypertrophy and OtherPathological Complications.We used echocardiography to monitor changes in the heartinduced by aortic banding. Abdominal aortic banding ofApoe�/� mice induced a time-dependent increase in LVend-diastolic dimension (LVEDD; P�0.01 by MANOVAversus sham, Figure 2A) and LV mass (P�0.01, Figure 2B)and a trend toward a decrease in ejection fraction (P�0.066,Figure 2C). Hearts were hypertrophied by 4 weeks afterbanding and began to dilate significantly after 6 weeks. At 8weeks, 5 of 41 banded Apoe�/� mice had an ejection fractionof �30%, indicating heart failure. In contrast, neither the LVdiameter nor the heart mass was increased significantly inwild-type mice after banding. Thus, the Apoe�/� mice aremore prone to develop cardiomegaly and heart failure inresponse to chronic cardiac overload induced by aortic
constriction than are wild-type mice. Heart rate was notsignificantly influenced by sex, treatment, or genotype (Table 2).
Histological examination showed that the LVs and rightventricles were dilated in the 8-week banded Apoe�/� mice.Myocardial hypertrophy and cardiac damage, as evidenced bylarger myocytes with enlarged nuclei and extensive intersti-tial and perivascular fibrosis, were present in the hearts of thebanded Apoe�/� mice (Figure 3A and 3B). In addition, in 8 of37 Apoe�/� mice, the left atrium was enlarged and firm andcontained large organized thrombi (Figure 3C). Foam cellswere found on the left atrial wall adjacent to the thrombusand, presumably, were the consequence of a disturbed bloodflow in this area (Figure 3D). Most of the mice with atrialthrombi also showed evidence of pulmonary congestion(Figure 3E). The lungs contained intra-alveolar collections ofenlarged macrophages that were filled with acidophilicneedle-shaped crystals (Figure 3F) that are typical of acido-philic macrophage pneumonia. Acute inflammatory cellswere absent. Bronchi and alveolar spaces contained PASstaining of the proteinaceous materials (Figure 3E). Areas offibrosis were present in some animals. Neither the sham-operated Apoe�/� mice nor the banded wild-type mice hadatrial thrombi or pulmonary congestion, implicating thedetrimental effects of the Apoe mutation under thesecircumstances.
Increased Aortic Atherosclerosis Proximalto the ConstrictionLesions in the aortic arch were significantly increased inbanded Apoe�/� mice compared with sham-operated mice(Figure 4A through 4D). All the thoracic aortas from bandedmice (6 males and 5 females) contained at least 3 largeplaques in the aortic arch, whereas no aortas from thesham-operated mice (6 males and 5 females) had 1 plaque.The cholesterol ester content of the excised thoracic aortas ofbanded Apoe�/� mice was 244�38% that of the sham-operated vessels per aorta or 166�16% per dry weight (n�11and 9, respectively; P�0.001; effects of the sex of the animalwere not significant).
The mean size of lesions within the aortic root of bandedmales (86 000�8000 �m2) and females (128 000�9000�m2) were 245% and 152% those of sham-operated males(35 000�7000 �m2) and females (84 000�8000 �m2), re-spectively (Figure 4E, P�0.0001 for effects of banding). Noqualitative changes in the plaque morphology were notable inassociation with the banding (not shown). The lesion size wasnot correlated with either HW/BW ratio or ejection fraction
Figure 2. Cardiac hypertrophy and LV dilata-tion of Apoe�/� mice after aortic coarctation.LVEDD (A), LV mass (B), and ejection frac-tion (C) are shown. Solid triangles indicatebanded Apoe�/� (n�41); open triangles,sham-operated Apoe�/� (n�27); solid circles,banded wild-type mice (n�16); and opencircles, sham-operated wild-type mice (n�8).Females and males are approximatelyequally represented. The time-dependentchanges in LVEDD and LV mass of thebanded Apoe�/� mice were statistically sig-nificant (P�0.01) compared with those of theother groups. *P�0.05 compared with allother groups.
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of the heart of the individual animals, suggesting that cardiacdysfunction and atherosclerosis are independent processes inthis system.
DiscussionWe have used mechanical methods to increase the aortic BPin mice to study the relationships between hypertension,atherosclerosis, and Apoe genotype. Our data show that BPand atherosclerosis in the aorta proximal to the constrictionwere increased by banding the suprarenal abdominal aorta ofApoe�/� mice, whereas no changes occurred in the BP andatherosclerosis in the distal aorta. We also found that Apoe�/�
mice are significantly more susceptible than are wild-typemice to the cardiac hypertrophy induced by chronic constric-tion of the aorta.
The effects of chronic aortic coarctation on atherosclerosisin Apoe�/� mice confirm the effects observed in Watanabeheritable hyperlipidemic rabbits,7 in rabbits fed high choles-terol diets,8 and in cynomolgus monkeys.9 All these experi-ments demonstrate a marked increase in atherosclerotic le-sions in response to elevated BP. The suggested explanationsinclude the possibility that an increased intimal permeabilityinduced by hypertension might lead to an increased rate ofentry of lipoproteins into the intima16,17 or that the higherpressure could increase endothelial or medial damage,thereby triggering the initiation of plaque formation byrecruiting proinflammatory leukocytes to the sites.18
Angiotensin II is a potent vasoactive mediator that hasprofound effects on atherosclerosis. Thus, mice that overex-press a human renin transgene and a human angiotensinogentransgene are hypertensive and develop larger lesions than donontransgenic mice when these mice are fed a high choles-terol diet.19 Daugherty et al20 and Weiss et al21 have shown
that systemic infusion of angiotensin II at supraphysiologicallevels of �0.7 mg/kg per day dramatically increased athero-sclerosis in Apoe�/� mice in the relatively short time of 4weeks. Although these 2 studies differ (in that Daugherty et aldid not see any BP increase in their animals, whereas Weisset al reported that the treatment increased BP by�40 mm Hg), a marked infiltration of inflammatory cells inplaques and in the adventitia was noted by both studies,which agree that this is indicative of the proatherogeniceffects of angiotensin II.
One of the hypotheses explaining the mechanism by whichbanding of the abdominal aorta above the renal arteriesinduces hypertension postulates that the constriction causes areduction in renal blood flow, which in turn activates therenin-angiotensin system.15 In support of this idea, studieshave shown that circulating renin levels22 and renin mRNA inthe renal afferent arterioles23 increase 3- to 4-fold afterabdominal aortic coarctation. However, other studies haveshown that circulating renin levels return to normal withinseveral days after the surgery22 and that chronic coarctationdoes not alter mean BPs distal to the coarctation.24 Our dataagree with and extend the latter observations by showing thatrenin mRNA levels in the kidney, plasma renin activity, andthe femoral BP in Apoe�/� mice 8 weeks after banding are notdifferent from those in sham-operated mice. Thus, it is veryunlikely that systemic angiotensin II levels are chronicallyaltered in the banded animals compared with the sham-operated animals. Nevertheless, coarctation induced a sus-tained increase in BP and enhanced lesion development.
Although the increased atherosclerosis present in the prox-imal aorta and its branches is similar to that described in otheranimal models, the presence of organized thrombi in theportion of the aorta just distal to the coarctation has not, to our
Figure 3. Histological examination of heart (Athrough D) and lung (E and F) of Apoe�/� miceafter 8 weeks of aortic coarctation. A and B,Interstitial (A) and perivascular (B) fibrosis in themyocardium is illustrated as blue staining ofcollagens (Masson’s trichrome, original magnifi-cation �100). C and D, Organized thrombus(th) is present in left atrium (la) in panel C (origi-nal magnification �5) and panel D (originalmagnification �20). Arrow in panel D indicatesfoam cells on the wall of the left atrium (la). aoindicates aorta; rv, right ventricle, and ra, rightatrium. Stain was Sudan IVB. E, The bronchus(arrow) contains a cellular infiltrate of macro-phages and mononuclear cells that areadmixed with mucoid material (H&E stain, origi-nal magnification �150). F, Large histiocyticcells (arrows) are present within alveolar spaces(H&E stain, original magnification �500).
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knowledge, been described. The thrombi were present atsimilar frequencies in banded wild-type and Apoe�/� mice,suggesting that disturbed blood flow due to the aortic con-striction, not hyperlipidemia, is the cause of the thrombusformation. Thromboembolism induced by the injection ofagents such as lipopolysaccharide, collagen, and thrombin orstasis induced by carotid artery ligation has been used as amodel of thrombosis in mice.25 Aortic coarctation offersanother reproducible system in which to study the process ofchronic thrombus formation in vessels in which blood flow isdisturbed. Whether the chronic and ongoing process ofthrombus formation distal to the coarctation is relevant to thedevelopment of clinical complications of atherosclerosis inhumans remains to be determined.
The second location at which we found organized thrombiin Apoe�/� mice but not in wild-type mice was the left atrium.The mice with atrial thrombi were also likely to havepulmonary acidophilic pneumonia. These complications in-dicate severe cardiac dysfunction and have been reported inrodents with hypertrophic cardiomyopathy26,27 or acute viralmyocarditis.28,29 There were occasional small atheroscleroticplaques in the small- to medium-sized vessels in some of thebanded Apoe�/� mice. However, cardiac changes in thesemice are unlikely to be attributable to changes in theirintracardiac vasculature, because the degree of atherosclero-sis in the smaller vessels of the banded mice was not differentfrom that of the sham-operated mice in the histologicalsections that we analyzed. Thus, perivascular fibrosis in thebanded mice appears to be independent of atherosclerosis inthe intracardiac vessels.
Cardiac dysfunction was not observed in any wild-typemice despite the fact that some of them had BP increases thatwere more than those seen in Apoe�/� mice with cardiacdysfunction. This suggests that a lack of apoE may exacerbatecardiac hypertrophy and subsequent dysfunction. Althoughlittle is known about the role of apoE in heart function, ourearlier work30 has shown that apoE is highly expressed in themouse heart, whereas its expression is low in skeletal mus-cles. In addition, Hartley et al31 have reported significantlyelevated HW/BW ratios of the 13-month-old Apoe�/� micecompared with the wild-type mice.31 Many circulatory prob-lems, including severe aortic atherosclerosis, can contributeto the hemodynamic changes in old Apoe�/� mice. However,our present study showed that the hearts of 2-month-oldApoe�/� mice, although normal, have a significantly largerLVEDD and a smaller ejection fraction than are seen inwild-type C57BL/6 mice (Figure 2). Furthermore, our unpub-lished data (J. Knowles, N. Maeda, 2001) show that theHW/BW ratios of Apoe�/� mice at 4 months of age are �15%larger than those of the wild-type mice with a similar geneticbackground (6 to 8 generations of backcrossing to C57BL/6).The degrees of atherosclerosis at these ages are still minimal.Although we cannot completely eliminate the possibility thatsome residual contribution of strain 129 derived genes linkedto the Apoe locus and that differing in the 2 strains isresponsible for this phenotype, a more conservative sugges-tion is that apoE is important for cardiac energy metabolismand has direct effects in modulating cardiac hypertrophy.Further studies are necessary to elucidate the mechanismswhereby apoE may exert this function.
AcknowledgmentsThis study was supported by National Institutes of Health grantsHL-62845 and HL-42630. The authors thank Dr H. Rockman foradvice on transverse aortic constriction and Drs H.-S. Kim, J.Knowles, L. James, and O. Smithies for useful discussions.
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Figure 4. Increased atherosclerosis in banded Apoe�/� mice. Athrough D, Representatives of aortic arch with plaques in sham-operated (A and B) and banded (C and D) mice. Arrows indicateplaques. Bars indicate 1 mm. E, Bar graph showing mean�SEMfor size of lesions in aortic sinus of sham-operated mice (openbars) and banded mice (solid bars). *P�0.0001; **P�0.005.
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