accumulation of i-tyramine cellobiose-labeled low density

446

Accumulation of 125I-TyramineCellobiose-Labeled Low Density Lipoprotein IsGreater in the Atherosclerosis-Susceptible Region

of White Carneau Pigeon Aorta and FurtherEnhanced Once Atherosclerotic Lesions Develop

Dawn C. Schwenke and Richard W. St. Clair

Previous studies have suggested that greater arterial concentrations of undegraded low density lipoprotein(LDL) and/or greater arterial rates of LDL degradation may play role(s) in determining regionaldifferences in arterial susceptibility to atherosclerosis in rabbits (Schwenke and Carew, Arteriosclerosis1989;9:895-918). The White Carneau (WC) pigeon is also useful for investigating potential mechanism(s)that might account for regional variation in arterial susceptibility to atherosclerosis because atheroscle-rosis develops predictably in the aorta at the level of the cellac bifurcation (atherosclerosis-susceptibleceliac site). In this study we sought to determine whether the 123I-tyramine cellobiose (125I-TC) content 1day after injecting '"I-TC-LDL ("^I-TC-LDL accumulation") would be greater in the celiac site in arteriesof WC pigeons and whether ^I-TC-LDL accumulation would be exaggerated by cholesterol feeding.Because '"I-TC remains trapped in cells after cellular degradation, arterial sites that either degrade LDLat higher rates or contain higher concentrations of undegraded LDL or both will demonstrate greater"'I-TC-LDL accumulation. Young WC pigeons were studied while consuming a cholesterol-free diet andafter consuming a cholesterol-containing diet for 1,2, 4, and 8 weeks. In pigeons fed a cholesterol-free diet,•"I-TC-LDL accumulation in the celiac site was equivalent to 0.24 ±0.02 u% LDL cholesterol/cm2 aorticsurface/day compared with only 0.14±0.02 /ig LDL cholesterol/cm1/day for the adjacent aorta, which isresistant to atherosclerosis (atherosclerosis-resistant site) (p<0.025). In atherosclerotic lesions excisedfrom the celiac site, 115I-TC-LDL accumulation was equivalent to 21±10 fig LDL cholesterol/cm1 aorticsurface/day compared with 0.66±0.17 fig LDL cholesterol/cm1 /day for the adjacent atherosclerosis-resistantsite (p<0.001). During cholesterol feeding, I25I-TC-LDL cholesterol accumulation in the celiac site as awhole increased 30-fold compared with a fivefold increase in plasma LDL cholesterol. In comparison,'"I-TC-LDL cholesterol accumulation in the adjacent atherosclerosis-resistant arterial site increased at thesame rate as the plasma LDL cholesterol, while 125I-TC-LDL cholesterol accumulation in two otherrelatively atherosclerosis-resistant arterial sites that we studied increased relatively little during cholesterolfeeding. The results of this study suggest that differences in arterial '"I-TC-LDL accumulation, both thosepresent in normal animals and those induced by cholesterol feeding, may contribute to the characteristicregional variation in arterial susceptibility to atherosclerosis in WC pigeons. Additional studies will beneeded to determine whether this difference reflects differences in arterial permeability to LDL, retention ofundegraded LDL within the arterial extracellular matrix, or the cellular metabolism of LDL. (Arteriosclerosisand Thrombosis 1992;12:446-460)

KEYWORDS • pigeons • atherosclerosis • low density lipoproteins • atherosclerosis susceptibility• cholesterol feeding • arterial wall metabolism • atherogenesis mechanisms

The clinical sequelae of atherosclerosis, coronaryheart disease, stroke, and peripheral vasculardisease, are the leading causes of morbidity and

mortality in the United States.1 Evidence from severalsources indicates that elevated levels of cholesterol inplasma, particularly that in the low density lipoprotein(LDL) fraction, are causally related to atherosclerosis.2-4

From the Department of Pathology, Arteriosclerosis ResearchCenter, The Bowman Gray School of Medicine of Wake ForestUniversity, Winston-Salem, N.C.

A portion of these data were presented at the 62nd ScientificSessions of the American Heart Association, November 1989.

Supported by US Public Health Service grant HL-14164, Be-

Because most of the cholesterol in atherosclerotic lesionsis derived from plasma lipoproteins,5-7 it seems likely thatincreased concentrations of LDL cholesterol in plasmapromote atherosclerosis by directly or indirectly increasingdelivery of cholesterol to cells within the arterial wall.

Consideration of all factors known to influence ath-erogenesis, including the LDL cholesterol concentra-

thesda, Md., and by the North Carolina United Way and TheBowman Gray School of Medicine, Winston-Salem, N.C.

Address for correspondence: Dawn C. Schwenke, PhD, Depart-ment of Pathology, The Bowman Gray School of Medicine,Medical Center Boulevard, Winston-Salem, NC 27157.

Received September 27, 1991; revision accepted January 6, 1992.

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Schwenke and St. Clair LDL Accumulation in Pigeon Artery 447

tion, can only explain part of the risk of atherosclerosisin human beings.8 In addition, in humans9'10 and exper-imental animals,11"13 atherosclerosis develops preferen-tially at certain susceptible arterial sites, whereas otheradjacent sites are resistant to atherosclerosis. Theseobservations suggest that other yet-to-be-identified"risk factors" acting at the level of the arterial wall maydetermine the regional differences in susceptibility toatherosclerosis within an individual and may also beresponsible for some of the unexplained variability inthe extent of atherosclerosis among different individu-als. Differences in hemodynamics may play a role in theregional variation in the development of atherosclerosis.However, it seems equally likely that localized alter-ation^) in the interaction of lipoproteins with thearterial wall (permeability, cellular metabolism, and/orretention within the arterial wall) may also play a role inthe preferential development of atherosclerosis at char-acteristic sites.

Previous studies of rabbits showed that arterial con-centrations of undegraded LDL and rates of LDLdegradation were enhanced in atherosclerosis-suscepti-ble aortic sites14 and that these parameters, most nota-bly the arterial concentration of undegraded LDL, wereenhanced during 16 days of cholesterol feeding.15 Thelocalization of these changes to the most atherosclero-sis-susceptible aortic sites and the fact that thesechanges were observed before the appearance of fattystreak lesions or large accumulations of foam cellswithin the intima15 suggest that such changes are anearly manifestation of the pathogenesis of atherosclero-sis in the rabbit.

The White Carneau (WC) pigeon is another well-characterized model of human atherosclerosis. Thisspecies develops atherosclerosis that shares several fea-tures of the human disease, including accumulation ofcholesterol esters, macrophages, and smooth musclecells, and, unique among animal models of atheroscle-rosis, the development of complicated plaques includingmineralization, ulceration, and thrombosis.1316"19 InWC pigeons, atherosclerosis develops spontaneously inthe aorta near the site of bifurcation of the celiac artery(celiac site), while the immediately proximal part of theaorta is highly resistant to atherosclerosis.13-16"19 Cho-lesterol feeding accelerates the development of athero-sclerosis at the susceptible celiac site.13-17

In this study we considered whether the total arterial125I-tyramine cellobiose (TC) content, reflecting thesum of arterial undegraded LDL and products of arte-rial LDL degradation ("125I-TC-LDL accumulation"),might be enhanced at the atherosclerosis-susceptibleceliac site of WC pigeons and thus suggest potentialmechanism(s) to account for the characteristic develop-ment of atherosclerosis at this site. In this report weshow that 123I-TC-LDL accumulation is greater in theceliac site than in the adjacent atherosclerosis-resistantsite in the same pigeons. 125I-TC-LDL accumulation wasfurther increased in the celiac site when atheroscleroticlesions were present after cholesterol feeding.

MethodsAnimals and Diets

Fifteen random-bred WC pigeons were obtainedfrom our breeding colony, where they were fed a

cholesterol-free grain diet. Young birds (3.5-7 monthsof age) were chosen for this study so that little if anyspontaneous atherosclerosis would be present. Thebirds were divided into five groups of three each so thatthe average age of the pigeons in each group wassimilar. One group was retained on the grain diet as acontrol. The other four groups were fed a grain dietcontaining 0.5% (wt/wt) cholesterol and 10% (wt/wt)lard beginning at appropriate intervals so that all cho-lesterol-fed birds could be studied during the same3-day interval and after the four groups of pigeons hadconsumed the atherogenic diet for 1, 2, 4, and 8 weeks,respectively. The control pigeons were studied duringthis same 3-day interval.

Plasma Lipids and Lipoproteins

Plasma and lipoprotein lipids were determined in4-5-ml blood samples collected just before the pigeonswere killed. The blood samples, which were collectedfrom an alar vein, were immediately mixed withNa2EDTA at a final concentration of 2.7 mM. Plasmawas obtained after low-speed centrifugation. The con-centrations of cholesterol and triglyceride in plasmawere measured by enzymatic methods.20 Very low den-sity lipoproteins (VLDLs) were isolated from the topsof the tubes after ultracentrifuging 1.5-ml plasma sam-ples at a density of 1.006 g/ml for 4.38xl08g- min at4°C in an SW 55 rotor.21 High density lipoproteins(HDLs) were obtained from the d> 1.006 g/ml fractionafter precipitating LDL with heparin-manganese.22 Thecholesterol concentrations in VLDL, the d> 1.006 g/mlfraction, and HDL were corrected for recovery ofcholesterol from ultracentrifugation, which averaged94.9±1.4% (mean±SEM, n = 14). The LDL cholesterolwas calculated as the difference between the d> 1.006g/ml cholesterol and the HDL cholesterol.

Isolation and Labeling of Low Density Lipoproteinfor Reinjection Studies

LDLs for labeling and reinjection were isolated frompooled plasma obtained from donor WC pigeons fed theatherogenic diet. The mean plasma cholesterol concen-tration of these plasma donors was 33.0 mM (SEM=3.6,n = ll). Blood collected from these donor birds wasimmediately mixed with Na2EDTA and the lecithin:cholesterol acyltransferase inhibitor 5,5'-dithio-6£s(2-nitrobenzoic acid) at final concentrations of 2.7 mM and1.0 mM, respectively, and placed on ice. The VLDLfraction was removed by ultracentrifugation at d=1.006g/ml for 2.5 x 10s g • min at 4°C in an SW 40 rotor. Theinfranatant was adjusted to d= 1.080 g/ml with solidKBr and overlayered with a NaCl/KBr solution ofdensity 1.063 g/ml containing 2.7 mM EDTA. Afterultracentrifuging for S^xl^g-min at 4°C in an SW 40rotor, the 1.006<d< 1.063 g/ml fraction, which we de-note here as LDL, was isolated from the top of the tubesusing a tube slicer. The isolated LDL was dialyzedagainst five changes of 500 volumes of 0.15 M NaCl, 2mM EDTA, pH 7.4 (buffer A). After dialysis, proteincontent was determined by the method of Lowry et al,23

with bovine serum albumin as a standard, and incorpo-rated extraction with chloroform to eliminate turbiditydue to lipids. Three aliquots of LDL protein (8 mg each)were separately labeled with U1I (0.6 mCi/mg protein)



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448 Arteriosclerosis and Thrombosis Vol 12, No 4 April 1992

using l,3,4,6-tetrachloro-3a,6a-diphenylglycouril (Io-dogen)24 as described.14-15 After dialysis overnightagainst buffer A, each of these three U1I-LDL prepara-tions was separately coupled to 125I-TC. TC was labeledwith ^ I using Iodogen (0.1 mCi I25I/nmol TC), and theÎ-TC was covalently linked to the U1I-LDL (6.2 nmolTC/mg LDL protein) after activating the Î-TC withcyanuric chloride.1415-25-26 Afterward, this mI,125I-TC-LDL (doubly labeled LDL) was diatyzed overnightagainst 500 volumes of buffer A.

Depletion of Non-Apolipoprotein B Radiolabel FromDoubly Labeled Low Density Lipoprotein byExchange With High Density Lipoprotein

To deplete doubly labeled LDL of radiolabel presentin small apoproteins and in lipid, each doubly labeledLDL preparation was separately incubated with thed> 1.063 g/ml plasma fraction of WC pigeons fed theatherogenic diet as described in detail in the footnote toTable 1. After incubation the mixtures were transferredto separate ultracentrifuge tubes, and solid KBr wasadded to increase the density to 1.080 g/ml. Eachmixture was overlayered with a 1.063 g/ml densitysolution containing 2.7 mM EDTA, and doubly labeledLDL was reisolated from the tops of the tubes afterultracentrifugation for S^xlCr'g-min at 4°C in an SW40 rotor. The reisolated doubly labeled LDL was dia-lyzed against two changes of 600 volumes of buffer Aand sterilized by passing it through a 0.45 -^m filterbefore injection into the pigeons.1415

Characterization of Doubly Labeled LowDensity Lipoprotein

Both before and after incubation with the d> 1.063g/ml plasma fraction, doubly labeled LDLs were char-acterized by agarose gel electrophoresis,27 by precipita-tion with 10% (vol/vol, final concentration) trichloro-acetic acid (TCA), and by extraction with chloroform/methanol.28 The integrity of apoprotein B (apo B) andthe distribution of radioactivity between apo B andother apoproteins was evaluated by electrophoresis ofdelipidated28 LDL on 4-12% gradient polyacrylamidegels under denaturing conditions.29

Animal Studies

To determine the arterial degradation rate and theintra-arterial concentration of undegraded LDL, thepigeons were injected intravenously with doubly labeledLDL (9.07±0.20xlOs cpm 125I and 1.35+0.06x10" cpmulI/kg body wt). To avoid potential confounding ofcomparisons among data for different times of choles-terol feeding by differences in LDL preparations, eachof the three doubly labeled LDL preparations was givento one untreated pigeon and one pigeon fed cholesterolfor each of the four different intervals. One of thepigeons fed cholesterol for 2 weeks died shortly afterinjection of doubly labeled LDL. Thus, data for 14pigeons are reported here. To follow the decline ofradioactivity in plasma, 0.5-ml blood samples werecollected at 2, 10, 25, 45, and 80 minutes, at 2, 4, 8, and12-15 hours, and in some cases at 20 hours afterinjecting doubly labeled LDL. Just before terminatingthe metabolic study at 25-29 hours after injectingdoubly labeled LDL, a 4-5-ml blood sample was col-

TABLEI. Characterization of Doubly Labeled Low Density Lipopro-tein Before and After Exchange With High Density lipoprotein andDistribution of Low Density Lipoprotein Label in Plasma of Pigeons*

Before exchangePercent of cpm soluble in

10% TCA 3.0±0.2 6.1±0.5Percent of TCA-precipitatable cpm

Comigrating withtProtein of -66 kd 2.96±0.29 9.95±0.21Apoprotein of ~45 kd 6.44±0.02 8.60±0.40Apo A-I 12.22±0.45 16J4±1.14Apoproteins < 14 kd 5.24+0.26 4.92+0.27

In lipid (soluble inchloroform/raethanol) 27.9±1.1 4.05+0.14

After exchange with d> 1.063 g/mlplasma fraction and reisolation ofLDL by ultracentrifugationPercent of cpm soluble in

10% TCAPercent of TCA-precipitatable cpm

Comigrating withfProtein of «66 kdApoprotein of »45 kdApo A-IApoproteins <14 kd

In lipidSpecific activity (cpm/ng)

Percent distribution of labels inplasma 25-29 hours after injectionNormal birds («=3)

VLDLLDL

HDL

Cholesterol-fed birds (TJ = 11)VLDLLDL

HDL

1.20±0.04

4.19±0.668.58±0.505.73 ±0.474.01 ±0.10

13.16±0.4441.3±2.7

10.4±5.349.3±2.840.2±3.9

15.5±4J61.4±2.023.1±2.8

0.85±0.06

9.82±0.518.68±0.48

10.44±0.644.11±0.152.63±0.14354 ±27

8.0±4.172.7±5.219.3±1.0

15.2±3.677.1±3.57.7±1.0

TC, tyramine cellobiose; TCA, trichloroacetic acid; apo,apoprotein.

*LDL was isolated from pooled plasma from cholesterol-fed WhiteCameau pigeons. Three aliquots of the LDL preparation (8 mg each)were labeled separately with 131I and coupled to Î-TC as describedin "Methods." Each doubly labeled LDL was incubated at 37°C for18 hours with the d> 1.063 gAnl plasma fraction at a d> 1.063 to LDLratio 12 times that present in plasma from which the LDL wasisolated. This corresponds to about a 5:1 ratio of HDL protein/LDLprotein.38 The ratio of total d>1.063 protein to LDL protein was403±l-5:1. The incubation mixtures contained 0.15 M Nad, 5.4 mMEDTA, 2.0 mM sodium phosphate, pH 7.4, and 1.0 mM 5,5'-dithio-fcis(2-nitrobenzoic acid) to inhibit lecithin:cholesterol acyltransfer-ase. After incubation, LDLs were reisolated by ultracentrifugation inthe d< 1.063 g/ml fraction and dialyzed against buffer A.

•Determined by radioassay of slices of lanes from 4-12%sodium dodecyl sulfate-polyacrylamide gels. Values aremean±SEM for the three doubly labeled LDL preparations.

lected for measurement of radioactivity and cholesterolcontent of plasma and lipoprotein fractions. Immediatelyafterward, the birds were anticoagulated with heparin(400IU) and anesthetized with the inhalant methoxyflu-



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Schwenke and St Clair LDL Accumulation in Pigeon Artery 449

ProximalResistant

Sites

abdominalaorta

ResistantAorticSite

Susceptible8-9 mm CeRacI Site

celiacartery

FIGURE 1. Illustration of the arterial sites studied. Charac-teristic atherosclerosis-susceptible (S) and atherosclerosis-re-sistant (R) sites in the thoracic distal aorta. P, and P2 areproximal atherosclerosis-resistant parts of the arterial tree. P2

includes the aortic arch and proximal parts of the innominatearteries.

rane (Metofane, Pitman-Moore, Inc., Washington Cross-ing, NJ.). The birds were exsanguinated via the rightatrium while being perfused at physiological pressure(120 mm Hg) with about 1 1 buffer A via the ventricleuntil the perfusate exiting the right atrium was color-less.14-15 These procedures were approved by the WakeForest University Animal Care and Use Committee andconformed to guidelines established by that committee.

Arterial Samples

After perfusing the vascular system, the portion ofthe arterial tree including the aorta from the aortic valveto 2-3 mm below the celiac bifurcation and the innom-inate arteries extending from the aortic arch to 2 mmbeyond the origins of the subclavian and commoncarotid arteries were removed en bloc. Adhering adven-titial tissue was removed. The arterial samples weretrimmed just distal to the origins of the common carotid,subclavian, and celiac arteries. These samples weredivided into two segments, the descending thoracicaorta (segments S+R+P]j see Figure 1) and the rightand left innominate arteries with the aortic arch (P2).Both arterial segments were opened longitudinally andfixed flat in half-strength Karnovsky's solution for 24hours.1415-25 Fixation in this solution preserves the125I-TC and 131I present on undegraded LDL and the123I-TC representing products of arterial LDL degrada-tion trapped within cells. Arterial TCA-soluble 131Iradioactivity is leached out by this process.25

After fixing the arterial segments, the descendingthoracic aorta was divided into the most proximal10-11-mm portion (Pi, Figure 1) and the distal thoracicaorta (R+S, Figure 1). The distal thoracic aorta wasdivided into the most distal 8-9 mm containing theceliac axis (atherosclerosis-susceptible celiac site S, Fig-ure 1) and the adjacent proximal 9-10-mm portion(atherosclerosis-resistant site R, Figure 1). When pres-ent, areas of macroscopic atherosclerotic lesions weredissected from within each of the four arterial segmentsand analyzed separately. Each sample was weighed and

its perimeter outlined on paper. The surface areas of allarterial samples were determined by planimetry facili-tated by a Hipad Digitizer (Houston Instruments, Hous-ton) and software on an Apple lie computer. Surfaceareas of each arterial sample were determined at leastthree times. Coefficients of variation for replicate mea-surements were typically less than 3%.

To determine if the more luminal portion of pigeonartery degrades LDL at a higher rate than the moreabluminal portion, as has been reported for the thoracicaorta of normal rabbits,25 atherosclerosis-free areas ofthe proximal atherosclerosis-resistant artery (Pl and P2,Figure 1) were divided with forceps into inner and outerlayers that were then weighed. The inner layers repre-sented 45.2± 1.0% and 39.7± 1.4% (mean±SEM, n = 14)of the weight (and probably also the thickness) of theatherosclerosis-free portions of segments P! and P2,respectively. Susceptible and resistant areas of the distalthoracic aorta were too thin to permit their separationinto inner and outer layers in this manner.

Radioassay

Total and TCA-soluble 125I-TC and 131I radioactivity inplasma, lipoprotein fractions, and slices from sodiumdodecyl sulfate (SDS)-pofyacrylamide gels and ^I-TCand 131I radioactivity in arterial samples after fixationwere determined simultaneously in a two-channelgamma counter equipped with a 7.6-cm crystal (Gamma5500B, Beckman Instruments, Inc., Norcross, Ga.). Ra-dioactivity in all samples was corrected for backgroundradioactivity, overlap of the energy spectra of 125I and U1I,and isotopic decay. Aliquots of plasma and lipoproteinfractions and slices from SDS-poryacrylamide gels werecounted for sufficient time so that the standard deviationof the observed net counting rate was <,\%. Arterialsamples were counted for 40 minutes. Standard devia-tions of observed net counting rates for 125I in arterialsamples were typically £ 1%. Arterial 131I data will not bereported here (see below). The procedures for radioio-dination, use, and disposal of labeled LDLs were ap-proved by the Bowman Gray School of Medicine Officeof Health Protection.

Arterial Cholesterol Content

After radioassay arterial samples were extracted with2:1 (vol/vol) chloroform/methanol, and the resultingextracts were washed with water.30 [3H]cholesterol (99%pure by thin-layer chromatography [TLC] on silica gel60 [EM Science, Cherry Hill, N.J.] with development in70:30:1 [vol/vol/vol] hexane/diethyl ether/acetic acid)was added as an internal standard to correct for proce-dural losses. The total cholesterol content was deter-mined31 after saponification32 of an aliquot of the lipidextract. Nonesterified and esterified cholesterol wereseparated from another aliquot of the lipid extract byTLC as described above. Cholesterol present in thenonesterified form was determined31 and corrected forrecovery from TLC. The esterified cholesterol contentof each arterial sample was calculated as the differencebetween the measured total and nonesterified choles-terol contents. For a few samples the measured valuefor nonesterified cholesterol content was slightly greaterthan that for the total cholesterol content (esterified



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cholesterol not detectable). For these samples the es-terified cholesterol content was considered to be zero.

Arterial Accumulation of 125I-TyramineCellobiose-Low Density Lipoprotein

In these studies we set out to independently deter-mine the arterial content of undegraded LDL (usingdirectly incorporated m I ) and the sum of the arterialcontent of undegraded LDL and products of arterialLDL degradation (using the 12iI-TC label). Those datawould have then allowed calculation of both arterialconcentrations of undegraded LDL and arterial rates ofLDL degradation. However, for reasons described inthe "Results" section, we report here only data for thetotal arterial I25I-TC content after fixation. We denotethe total arterial 125I-TC content after fixation as arterial125I-TC-LDL accumulation. The arterial 125I-TC-LDLaccumulation was first expressed in terms of equivalentvolumes of plasma (microliters plasma per gram or squarecentimeter artery per day). This provides a measure of thesum of the arterial undegraded LDL and products ofarterial LDL degradation that is independent of theplasma LDL concentration. This was calculated by divid-ing the ^I-TC present in each arterial sample afterfixation (counts per minute per gram or square centime-ter) by the area under the curve for the decline ofprotein-bound 125I-TC in plasma after injecting 131I,125I-TC-LDL [counts per minute per microliter • day].33 Pro-tein-bound radioactivity in plasma was calculated by sub-tracting the radioactivity soluble in 10% (vol/vol, finalconcentration) TCA from the total plasma radioactivity.The area under each of these plasma 125I-TC radioac-tivity curves was determined by integrating a biexpo-nential equation fitted to the protein-bound 125I-TCradioactivity in the plasma from each bird using theSimulation Analysis and Modeling (SAAM) program.34

The arterial 123I-TC accumulation was converted toamounts of LDL cholesterol by multiplying the I25I-TCaccumulation by arterial samples of individual birds,expressed in plasma equivalents, by the concentration ofLDL cholesterol in their own plasma.

Whole-Body Metabolism of Low Density Lipoprotein

The fractional catabolic rate (FCR) of LDL by thewhole body, a measure of whole-body LDL metabo-lism, was calculated35 from coefficients and exponentsdetermined by the biexponential equation fitted toprotein-bound 125I-TC radioactivity in the plasma fromeach bird (see above).

Statistical Analysis

Comparisons of 125I-TC accumulation and cholesterolconcentrations between arterial sites were made eitherwith paired t tests36 (two arterial sites) or analysis ofvariance (ANOVA) with multiple measures on thearterial site (three or more arterial sites).37 The Scheff6criterion36 was used for unplanned comparisons be-tween the atherosclerosis-susceptible celiac site and theproximal atherosclerosis-resistant artery (P! and P2).For the ANOVAs comparing the three atherosclerosis-resistant arterial sites (R, P,, P2, Figure 1), the twodegrees of freedom for the arterial site were partitionedinto a comparison of the distal atherosclerosis-resistantsite with the proximal atherosclerosis-resistant arterial

sites (R versus P, and P2 combined) and a comparison ofthe two proximal atherosclerosis-resistant sites (P, ver-sus P2). Data for each gTOup of birds were consideredalone to investigate differences between these arterialsites under normal conditions and at each time ofcholesterol feeding.

To investigate the effect of cholesterol feeding, datafor all birds were considered together by ANOVA (asingle arterial site)36 or ANOVA with multiple mea-sures (two or more arterial sites)37 and included thegrouping factor, time of cholesterol feeding (0 [control],1, 2, 4, or 8 weeks) or diet (cholesterol free versuscholesterol containing).

Regression analyses36 were used to consider whetherthe characteristics of arterial sites changed in consistentmanners during cholesterol feeding and whether corre-sponding characteristics changed at different rates inadjacent arterial sites. The effect of cholesterol feedingon plasma lipids, lipoproteins, and whole-body LDLcatabolism was evaluated using linear regression.36

ANOVAs and regression analyses were done with BMDPstatistical programs (BMDP Statistical Software, Inc.,Los Angeles). When coefficients of variation of mea-sured or calculated values were proportional to individ-ual mean values, ANOVAs and t tests were performedon the logarithms of data or logarithms of (data+1) formeasurements with small values (arterial esterified cho-lesterol concentration).36 A probability value of lessthan 0.05 was considered significant.

ResultsLabeled Low Density Lipoprotein

As shown in Table 1, the methods used for labelingLDL resulted in significant incorporation of radioiodineinto LDL lipid and proteins other than apo B. Exchang-ing the doubly labeled LDL against a source of HDLremoved about 53% of the 13II associated with lipid andthat comigrating with apo A-I and about 35% of the125I-TC label associated with lipid and that comigratingwith apo A-I. About 20% of the 131I and 125I-TC labelscomigrating with apoproteins of less than 14 kd werealso removed by the exchange. The label comigratingwith an unidentified apoprotein of apparent molecularweight 45 kd and a protein of about 66 kd remained withthe doubly labeled LDL.

In these pigeons as in others described previously,39

more than 90% of the injected LDL was cleared fromplasma in 1 day. Despite prior exchange against HDL,some of the I31I label on the injected LDL was presenton small apoproteins and lipids, which not only ex-change between lipoprotein fractions40 but also arecleared from plasma more slowly than apo B.4142 Thus,it was not surprising to find that other lipoproteins(principally the HDL fraction; Table 1) contained sig-nificant portions of the 131I label remaining in plasma 1day after injection when the arterial samples wereanalyzed. Because albumin (an example of a smallprotein) equilibrates more rapidly with the artery thando lipoproteins43 and the concentration of albumin inthe artery of normal animals has been estimated to betwo to 10 times that of LDL,44-45 it seems likely that thepresence of a significant portion of the protein-bound

I radioactivity in the HDL-plasma protein fractionwould result in significant overestimation of the arterial



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TABLE 2. Plasma LJpids and Upoprotelns and Fractional Catabolic Rate of Low Density Lipoprotein*

Duration ofcholesterol feeding

0 (Control)1 Week2 Weeks4 Weeks8 Weeks

Plasma

6.0±0.615.1 ±0.717.1 ±0.827.3+3.533.7±6.8

Cholesterol

VLDL

0.31 ±0.103.28±1.014.58 ±0.70113+5.113.9±43

LDL

2.90±0.237.01±0.28834±0.4411.6±1.815^+3.0

HDL

2.82 ±0.494.78±0.623.96±0.364.32±0.474.27±0.21

Triglycerideplasma

1.46±0.141.71±0.341.27±0.091.38±0.782.75+0.98

FCR of LDL

0.188±0.0170.149±0.0160.085 ±0.0330.095 ±0.0100.093 ±0.006

VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL high density lipoprotein; FCR, fractional catabolic rate.'Values are mean±SEM. Lipid and lipoprotein concentrations are presented in mM, whereas LDL FCRs are presented in pools per hour,

«=3 birds at each time period except for the birds fed cholesterol for 2 weeks, for which n=2. To obtain cholesterol and triglycerideconcentrations in milligrams per deciliter, multiply values in mM by 38.7 and 88.5, respectively. The concentration of cholesterol in plasma,VLDL, and LDL increased progressively with length of cholesterol feeding, p<0.005 (regression analyses). Neither HDL cholesterol norplasma triglyceride concentrations were significantly affected by cholesterol feeding. FCR of LDL decreased during cholesterol feeding,p<0.02 (regression analysis).

content of undegraded LDL. In addition, about 23% ofthe 131I radioactivity remaining in plasma at the timethat the arteries were analyzed was accounted for bylabeled lipids. These labeled lipids could potentiallyexchange with arterial membrane lipids,4* causing thearterial 131I radioactivity to further overestimate thearterial concentration of undegraded LDL. Thus, we donot believe that it would be valid to estimate arterialconcentrations of undegraded LDL from the arterial 131Idata. In fact, for two of the 14 birds the estimate ofundegraded LDL provided by the U1I label exceeded theestimate for undegraded LDL plus products of LDLdegradation provided by the 125I-TC label.

At the end of the metabolic study when only 10% ofthe injected '"I-TC remained in the plasma, about 25%of the Î-TC label was not present in LDL comparedwith about 45% for 131I. However, for the followingreasons we believe that the arterial content of 125I-TCshould reflect primarily undegraded LDL and productsof arterial LDL degradation. First, the 123I-TC represent-ing products of lipoprotein degradation remains trappedwithin tysosomes after cellular degradation. Second, rel-atively more of the arterial 125I-TC content would haveentered the artery early after injection when the specificactivity of LDL in plasma was high and relatively more ofthe Î-TC label was present in the LDL fraction. Third,most of the 125I-TC label lost from LDL was transferredto VLDL, which at least in humans47 and rabbits,48"49

enters the artery less rapidly than LDL. Thus, whereasthe arterial content of 125I-TC after fixation, denoted hereas arterial I25I-TC-LDL accumulation, should reflectprimarily undegraded LDL and products of LDL degra-dation, the portion that represents undegraded LDL andthat which represents products of arterial LDL degrada-tion must await further study.

Plasma Lipids and Lipoproteins and Whole-BodyLow Density Lipoprotein Catabolism

Table 2 shows plasma lipids and lipoproteins justbefore euthanasia for the controls and the birds fedcholesterol for 1-8 weeks. The concentration of choles-terol in plasma and within the VLDL and LDL fractionsincreased progressively with the duration of cholesterolfeeding (for each,/?<0.005). The increase in the VLDLfraction reflects the appearance of cholesterol-enriched^migrating VLDL.38 The HDL cholesterol concentra-tion was unaffected by cholesterol feeding. Neither the

concentration of triglyceride in plasma nor the distribu-tion of triglyceride among lipoprotein fractions (notshown) was affected by cholesterol feeding. The whole-body FCR of LDL decreased with cholesterol feeding(/><0.02), reaching a plateau at about 50% of thenormal value by 2 weeks of cholesterol feeding.

Comparison of Atherosclerosis-Susceptible andAtherosclerosis-Resistant Arteries

Figure 2 shows 125I-TC accumulation by atherosclero-sis-susceptible and atherosclerosis-resistant arterial sitesin terms of equivalent volumes of plasma. In normalpigeons (0 weeks of cholesterol feeding), 125I-TC-LDLaccumulation in the atherosclerosis-susceptible celiacsite was about 80% greater per unit aortic surface areathan in the adjacent atherosclerosis-resistant site of thesame birds (0.215+0.005 versus 0.121±0.018 ftl plasma/cm2 aortic surface/day, mean±SEM,p<0.05; Figure 2A).Î-TC-LDL accumulation also tended to be higher inthe susceptible site when expressed per unit fixed weight(Figure 2B). Surprisingly, when expressed per unit sur-face area, 125I-TC-LDL accumulation in the proximalatherosclerosis-resistant artery (Pj and P2) of normalbirds was 3.3 times that of the atherosclerosis-susceptibleceliac site (S) of the same birds (/?<0.005).

Regression analysis indicated a significant increasingtrend for 125I-TC-LDL accumulation by the atheroscle-rosis-susceptible celiac site during cholesterol feedingboth per unit surface area and per unit fixed weight (foreach,p<0.01). As shown below (Table 4), this increasewas due to the development of atherosclerotic lesions inthe susceptible site. In contrast, 125I-TC-LDL accumu-lation (in plasma equivalents) by the adjacent athero-sclerosis-resistant site (R) was not influenced by choles-terol feeding. Surprisingly, expressed as plasmaequivalents, the 125I-TC-LDL accumulation by the twoproximal atherosclerosis-resistant sites (Pi and P2) wasdecreased about 50% by cholesterol feeding (/?<0.05 orbetter; Figure 2) with no difference among birds fedcholesterol for different times.

As indicated in Table 2, the cholesterol carried by theLDL in a unit volume of plasma increased about fivefoldduring cholesterol feeding. Figure 3 shows arterialI25I-TC-LDL accumulation expressed as amounts ofLDL cholesterol for normal birds and those fed choles-terol. Per unit surface area (Figure 3A), Î-TC-LDLaccumulation by the atherosclerosis-susceptible celiac



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1.75

1.60

1.25

1.00

0.75

0.50

0.25

0.00

. A

i

m

• 1 S loilul

ED PI

CDP2

I

I , ,Lrfltl 1

1

4

Length of Cholesterol Feeding Weeks)

FIGURE 2. Bar graphs showing l25I-tyramine cellobiose-lowdensity lipoprotein (TC-LDL) accumulation in arterial sitesof White Carneau pigeons. mI-TC-LDL accumulation isexpressed as equivalent volumes of plasma per square centi-meter of arterial surface area (panel A) and per gram fixedweight (panel B) per day. Data are mean±SEM for arterialsegments from three birds at each time point except at 2 weeksof cholesterol feeding, where n=2. Difference between sites Sand R of normal birds for 125I-TC-LDL accumulation persquare centimeter, p<0.05 (paired t test). Differences betweenproximal (Pj+P2) and distal (R) atherosclerosis-resistant ar-teries of normal birds for '"I-TC-LDL accumulation persquare centimeter and per gram, p<0.01 and p<0.02, respec-tively (analysis of variance [ANOVA] on data transformed tologarithms). Difference between I25I-TC-LDL accumulationper square centimeter in proximal (P1+P2) and distal (R),atherosclerosis-resistant arteries of birds fed cholesterol for 1week and 4 weeks, p<0.02 for each (ANOVA on datatransformed to logarithms). Difference between proximalatherosclerosis-resistant artery (P1+P2) and atherosclerosis-susceptible celiac site S of normal birds for data per squarecentimeter, p<0.005 (t test on data transformed to loga-rithms, Scheffe criterion). During cholesterol feeding 125I-TC-LDL accumulation showed a significant increasing trend insite S (data per square centimeter and per gram, p<0.01 foreach by regression analyses). Normal vs. all cholesterol-fedbirds for 125I-TC-LDL accumulation per square centimeter forsite Pi, p<0.001; for site P2, p<0.05; per gram for site P,,p<0.0001; for site P2, p<0.025 (ANOVA).

site of normal birds represented 0.24 ±0.02 /xg LDLcholesterol/day compared with only 0.14 ±0.02 fig LDLcholesterol/day by the adjacent atherosclerosis-resistantsite (R) (/7<0.025). During cholesterol feeding, 125I-TC-LDL accumulation increased most markedly in theatherosclerosis-susceptible celiac site (per square centi-meter and per gram; for each,p<0.005). 125I-TC-LDLaccumulation also increased in the adjacent atheroscle-rosis-resistant site (R) (per square centimeter and per

gram; for each,p<0.005). 125I-TC-LDL accumulation inthe two proximal atherosclerosis-resistant sites Pi and P2

also showed small increasing trends (p<0.05), but theserates of increase were much lower than the rate ofincrease of LDL cholesterol in plasma (Table 2).

Arterial Esterified and Nonesterified Cholesterol

The extent of atherosclerosis was assessed chemicallyby determining the arterial esterified and nonesterifiedcholesterol concentration. As shown in Figure 4A, theesterified cholesterol concentration of the atherosclero-sis-susceptible celiac site increased during cholesterolfeeding (p<0.005), with most of the increase occurringbetween weeks 2 and 8. The esterified cholesterolconcentration of the adjacent atherosclerosis-resistantsite (R) and the two proximal atherosclerosis-resistantarterial sites (Pj and P2) showed smaller increasingtrends during cholesterol feeding (site R, p<0.02; sitePi, p<0.001; site P2, p<0.05). Esterified cholesterolconcentrations were elevated in all arterial sites by 4weeks of cholesterol feeding but were increased most inthe susceptible site. After 8 weeks of cholesterol feed-ing, the esterified cholesterol concentration of the ath-erosclerosis-susceptible celiac site was eight times thatof the adjacent atherosclerosis-resistant site (R) and 27times as great as the atherosclerosis-susceptible celiacsite of untreated WC pigeons.

The nonesterified cholesterol concentration (Figure4B) of the atherosclerosis-susceptible celiac site in-creased during cholesterol feeding (p<0.001) but did somore slowly than the esterified cholesterol concentra-tion, only reaching twice the normal value by 8 weeks. Inthe adjacent atherosclerosis-resistant site (R) the non-esterified cholesterol concentration increased signifi-cantly (p<0.005) during cholesterol feeding but at amuch lower rate than that in the susceptible celiac site(/?<0.005).

To determine the extent to which any changes inarterial cholesterol concentration could be attributed tothe presence of atherosclerotic lesions, macroscopicraised atherosclerotic lesions, when present, were dis-sected from within each arterial site and analyzedseparately. Table 3 shows data for the concentration ofesterified and nonesterified cholesterol of atheroscle-rotic lesions and the adjacent macroscopically normalparts of the atherosclerosis-susceptible celiac site andfor the atherosclerosis-resistant site (R). A single smallraised lesion was detected in the atherosclerosis-suscep-tible celiac site of one bird fed cholesterol for only 1week. Single larger lesions were present in this arterialsite in three of three and two of three birds studied after4 and 8 weeks of cholesterol feeding, respectively.Lesions were not detected in atherosclerosis-resistantsite R, although single small lesions were found inproximal atherosclerosis-resistant sites P] and P2 of oneof three birds studied at each of 4 and 8 weeks ofcholesterol feeding.

When atherosclerotic lesions within the atherosclero-sis-susceptible celiac site were compared with the adja-cent macroscopically normal part of the same sitewithout regard to the length of cholesterol feeding, thelesions were found to contain 13.4±4.5 mg esterifiedcholesterol/g fixed wt compared with 0.92±0.26 mgesterified cholesterol/g for areas without lesions



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/ \ M S (c«H*o)

U*3 R

CD PI

dipt

I iII

FIGURE 3. Bar graphs of 125I-tyramine ceUobiose-low den-sity lipoprotein (TC-LDL) accumulation in arterial sites ofWhite Carneau pigeons. 125I-TC-LDL accumulation ex-pressed as amounts of LDL cholesterol are shown per unit ofarterial surface area (panel A) and per gram fixed weight(panel B) per day. Numbers of birds and identity of bars arethe same as in Figure 2. 12iI-TC-LDL accumulation persquare centimeter, site S vs. site R of normal birds, p<0.025(paired t test). Difference between proximal (Pj+PJ anddistal (R) atherosclerosis-resistant artery of normal birds formI-TC-LDL accumulation per square centimeter, p<0.01;per gram, p<0.02; for birds fed cholesterol for 1 week,125I-TC-LDL accumulation per square centimeter, p<0.02;for birds fed cholesterol for 4 weeks, 125I-TC-LDL accumu-lation per square centimeter, p<0.02. Difference between Pt

and P2 of birds fed cholesterol for 4 weeks, 125I-TC-LDLaccumulation per square centimeter, p<0.05 (all by analysisof variance on data transformed to logarithms). Differencebetween proximal atherosclerosis-resistant artery (Pj+PJ andatherosclerosis-susceptible celiac site S of normal birds,p<0.005 for 125I-TC-LDL accumulation per square centime-ter (t test on data transformed to logarithms, Scheffi criteri-on). Increasing trends for mI-TC-LDL accumulation duringcholesterol feeding (all regression analyses) for site S, persquare centimeter and per gram, each p<0.005; for site R persquare centimeter and per gram, each p<0.005; for site Pjper square centimeter, p<0.05; for site P2per square centime-ter and per gram, each p<0.05. Difference in rate of increasein 123I-TC-LDL accumulation during cholesterol feeding be-tween site S and site R per square centimeter and per gram,each p<0.005.

(p<0.005, n=6). These lesions also contained morenonesterified cholesterol per gram of artery than did themacroscopically normal part of the same arterial site(7.83±0.46 versus 2.74±0.17 mg/g fixed wt, /><0.001).The macroscopically normal areas of the atherosclerosis-susceptible celiac site surrounding lesions containedmore esterified cholesterol than did the adjacent athero-

B

- 1- i { I

L«ngtti ol Cho4««t«rol

FIGURE 4. Bar graphs showing cholesterol concentrations inarterial sites of normal and cholesterol-fed White Carneaupigeons. Panel A: Esterified cholesterol. Panel B: Nonesteri-fied cholesterol Numbers of birds and identity of bars are thesame as in Figures 2 and 3 except that n=2for site Rat 1 weekof cholesterol feeding (one sample lost). Influence of length ofcholesterol feeding on esterified cholesterol concentration forsiteS, p<0.005; for site R, p<0.02; for site P,, p<O.OOI; forsite P2, p<0.05; nonesterified cholesterol for site S, p<0.001;for site R, p<0.005; for site P2, p=0.051. Trend for differencebetween site S and site R during cholesterol feeding foresterified and nonesterified cholesterol, each p<0.005 (allregression analyses).

sclerosis-resistant site (R) (0.92±0.26 versus 0.59±0.27mg esterified cholesterol/g, p<0.01). Although not statis-tically significant, the esterified cholesterol concentrationof the atherosclerosis-susceptible celiac site tended to behigher than that of the atherosclerosis-resistant site Reven for birds without lesions. Nonesterified cholesterolcontents of the macroscopically normal atherosclerosis-susceptible site S also tended to be higher than those ofthe resistant site R regardless of the presence of athero-sclerotic lesions in the susceptible site, but the differencewas only statistically significant for the birds withoutatherosclerotic lesions.

125I-Tyramine Cellobiose-Low Density LipoproteinAccumulation in Atherosclerotic Lesions andMacroscopically Normal Distal Aorta

To consider whether any changes in the arterial125I-TC-LDL accumulation during cholesterol feedingcould be attributed to the development of atheroscle-rotic lesions, raised atherosclerotic lesions were sepa-rated from macroscopicalry normal parts of each arterialsite (the same areas for which cholesterol concentra-tions are presented in Table 3) before radioassay. Datafor ^I-TC-LDL accumulation are presented as equiv-alent volumes of plasma (Table 4) and as amounts ofLDL cholesterol (Table 5). For the six birds with lesions



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TABLE 3. Cholesterol Concentrations of Atherosclerotic Lesions and Macroscopically Normal* Distal Aorta

Length of cholesterol feeding(weeks)

0

1

2

4

8

All without lesionst

All with lesionst

0

1

2

4

8


All with lesionst

Atherosclerosis-susceptibleceliac site (""

Lesion

20.0(1/3)

4.81 ±1.70(3/3)

22.9±8.5||(2/3)

13.4±4.51#(6/14)

7.50(1/3)

8.03 ± 1.02ft(3/3)

7.70± 0.04ft(2/3)

7.83±0.465§|||(6/14)

Normal

Esterified cholesterol

0.243±0.1070.148±0.064

0.392±0.0580.694±0.193

1.303±0J75

0307±0.085(8/14)

0.916±0.263"(6/14)

Nonesterified cholesterol

2.04±0.132.51±0.14

2.08±0.032.39±0.05

3.08±0.16tt

2.22+0.10tt(8/14)

2.74±0.17(6/14)

Atherosclerosis-resistantsite (R) (normal)

0.014±0.0140J73±0.19O§

0.009+0.0090.234±0.060

0.817+0.410

0.128+0.069(8/14)

0.588±0.267(5/14)

2.00±0.151.88±0.08§

1.95±0.032.20+0.07

2.51±0.22

1.98±0.06(8/14)

2.40+0.14(5/14)

Values are in milligrams of cholesterol per gram fixed weight.'"Normal," macroscopically normal portions of these arterial sites. For normal areas of each arterial site, values are

mean±SEM for three birds at each of 0,1,4, and 8 weeks of cholesterol feeding and two birds at 2 weeks of cholesterolfeeding. The number of birds from which atherosclerotic lesions (lesions) were identified and analyzed separately areindicated in parentheses.

fAll birds without lesions in the atherosclerosis-susceptible site, regardless of the diet fed.£A11 birds with lesions in the atherosclerosis-susceptible site, irrespective of the length of cholesterol feeding.§n=2, one sample lost.Rp<0.025, H/><0.005, ttp<0.01, §§p<0.001 vs. adjacent normal artery, paired t test on data transformed to

logarithms.**p<0.01, $£p<0.05 vs. adjacent atherosclerosis-resistant site R, paired t test.#p<0.005, |||]/7<0.001 vs. atherosclerosis-resistant site R, paired t test on data transformed to logarithms.Influence of length of cholesterol feeding (regression analyses) for normal part of the atherosclerosis-susceptible

celiac site (site S): esterified cholesterol,/><0.001; nonesterified cholesterol,p<0.001; for the atherosclerosis-resistantsite R: esterified cholesterol, p<0.O2; nonesterified cholesterol, p<0.005; for the difference between trends in thenormal part of the atherosclerosis-susceptible celiac site and atherosclerosis-resistant site R: nonesterified cholesterol,p<0.05.

Overall difference between macroscopically normal atherosclerosis-susceptible site S and atherosclerosis-resistantsite R: esterified cholesterol, p<0.005 (analysis of variance on data+1 transformed to logarithms); nonesterifiedcholesterol, p<0.005 (analysis of variance on data transformed to logarithms).

in the atherosclerosis-susceptible celiac site, 125I-TC-LDL accumulation in excised lesions was equivalent to3.1±1.1 /il plasma/cm2/day compared with only0.309±0.090 (A plasma/cm2/day in the macroscopicallynormal part of the celiac site of the same birds(p<0.005). Qualitatively similar results were obtainedwhen 125I-TC-LDL accumulation was expressed per unitfixed weight. 125I-TC-LDL accumulation by the lesionsin the atherosclerosis-susceptible celiac site represented21 ± 10 fig LDL cholesterol/cm2 aortic surface comparedwith 1.6±0.5 fig LDL cholesterol/cm2 for the adjacentmacroscopically normal part of the same site and0.66±0.17 fig LDL cholesterol/cm2 for the adjacent

atherosclerosis-resistant site R (p<0.005 and/;<0.001,respectively; Table 5). Per unit weight, the results werequalitatively similar.

For birds without atherosclerotic lesions, 125I-TC-LDL accumulation in the atherosclerosis-susceptibleceliac site was 76% greater than in the adjacent athero-sclerosis-resistant site R of the same birds when ex-pressed per unit surface area (0.183±0.023 versus0.104±0.015 ftl plasma/cm2/day, p<0.005, n=8) and38% greater when expressed per unit fixed weight(7.6±0.8 versus 5.5±0.8 fi\ plasma/g/day,/><0.02; Table4). The percentage increases in these amounts of 125I-TC-LDL accumulation in the atherosclerosis-suscepti-



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TABLE 4. U5I-Tyramiiie Cellobiose-Low Density Iipoprotein Accumulation in Atherosclerotic and MacroscoplcallyNormal Distal Aorta Expressed as Plasma Equivalents*

Length of cholesterolfeeding (weeks)

0

1

2

4

8


All with lesionst

0

1

2

4

8


All with lesions}:

Atherosclerosis-susceptibleceliac site (S)

Lesion

1.77(1/3)

2.08±0J3HH

(3/3)5.9±2.8

3.1±l.l"tt(6/14)

56.4(1/3)

57.7±21.8(3/3)

135±51(2/3)

83.2±23.2"tt(6/14)

NormalAtherosclerosis-resistant

site (R) (normal)

MicToliters plasma/cm2 surface area/day

0.215±0.005§0.194±0.102

0.246±0.0130.183±0.047#

0350±0.180(2/3)

0.183±0.023#(8/14)

0JO9±0.0901(6/14)

0.121 ±0.0180.072±0.004

0.135±0.0450.110+0.024

0.130±0.042

0.104 ±0.015(8/14)

0.123+0.021(6/14)

Microliters plasma/g fixed wt/day

8.7+0.87.2±2.8

9.8±0.027.2±1.6tt

13.7±6.5

7.6±0.8§§(8/14)

11.7+3.2t4:(6/14)

6.3±0.84.1 ±0.4

7.0±2.45.5±1.0

6J±1.8

5.5±0.8(8/14)

6.0±0.9(6/14)

't+As in Table 3.§p<0.05, §§/?<0.02 compared with atherosclerosis-resistant site R, paired t test||p<0.05, **p<0.005 compared with normal area of the atherosclerosis-susceptible celiac site, paired / test on

log-transformed data.H/><0.025, #p<0.005, ttp<0.001, %%p<QS& compared with atherosclerosis-resistant site R, paired t test on log-

transformed data.Overall difference between macroscopically normal atherosclerosis-susceptible site S and atherosclerosis-resistant

site R, micTOliters per square centimeter, p<0.02; microliters per gram, p<0.025 (analysis of variance).

ble celiac site compared with the atherosclerosis-resis-tant site R for these birds without lesions were identicalto the percent enhancement of 125I-TC-LDL accumula-tion in the atherosclerosis-susceptible celiac site of birdsnot fed cholesterol. The 125I-TC-LDL accumulation inplasma equivalents either per unit surface area or perunit fixed weight was not influenced by the length ofcholesterol feeding in the macroscopically normal part ofthe atherosclerosis-susceptible celiac site or atheroscle-rosis-resistant site R (Table 4). However, these amountsof 125I-TC-LDL accumulation represented progressivelylarger amounts of LDL cholesterol during cholesterolfeeding (Table 5) due to the increase in concentration ofLDL cholesterol per unit volume of plasma.

121I-Tyramine Cellobiose-Low Density LipoproteinAccumulation by Inner and Outer Layers of ProximalAtherosclerosis-Resistant Artery

A previous study of rabbits25 has suggested that aftera 1-day metabolic study, the intima of the thoracic aortaaccounted for a disproportionately large fraction ofarterial LDL degradation. To investigate whether U 5I-

TC-LDL accumulation might be greater in the innerpart (intima plus some media) of the arteries of WCpigeons, the macroscopically normal part of the proxi-mal atherosclerosis-resistant artery (P! and P2) wasseparated into inner and outer layers. Figure 5 showsthe 125I-TC-LDL accumulation (plasma equivalents,Figure 5A; amounts of LDL cholesterol, Figure 5B) forinner and outer layers of macroscopically normal partsof the two proximal atherosclerosis-resistant arterialsites (P! and P2). Data for inner and outer layers do notinclude the tiny atherosclerotic lesions found in each ofthese sites for one bird at 4 and 8 weeks of cholesterolfeeding. The data shown above (Figures 2-4) for sitesP! and P2 did include these tiny lesions. For untreatedbirds 125I-TC-LDL accumulation was 146% and 118%greater for inner arterial layers of sites P, and P2 thanfor the outer layers of these sites (19.4±2.8 versus7.9±0.9 and 22.6±4.7 versus 10.4±1.4 /*1 plasma/g/day,respectively, eachp<0.025). 125I-TC-LDL accumulationexpressed as equivalent volumes of plasma was de-creased 69% and 65% in inner layers of sites P! and P2

of cholesterol-fed birds compared with birds not fed



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TABLE 5. ""I-Tyramine Celloblose-Low Density LJpoprotein Accumulation in Atherosclerotic and MacroscoplcallyNormal Distal Aorta Expressed as Amounts of Low Density Lipoprotein Cholesterol*

Length of cholesterolfeeding (weeks)

0

1

2

4

8


All with lesionst

0

1

2

4

8


All with lesionst

Atherosclerosis-susceptibleceliac site (S)

Lesion Normal

Micrograms LDL cholesterol/cm2 s

4.9(1/3)

9.7±3.0||U

45±26(2/3)

21±10**tt(6/14)

0.24±0.02§0.53±0.28

0.81 ±0.0030.84 ±0.311

(3/3)2.1±1.1

0.43±0.09#(8/14)

1.6±0^H(6/14)

Micrograms LDL cholesterol/g

156(1/3)

258±106(3/3)

997±510(2/3)

487±214"tt(6/14)

10.0±1.721.2±8.0

32J±1.6

33.7±11.8tt

83 ±38

18.0±3.6§§(8/14)

61±2Ott(6/14)

Atherosclerosis-resistantsite (R) (normal)

iurface area/day

0.14±0.020.19±0.02

0.45±0.170.51±0.17

0.82 ±0.29

0.24±0.06(8/14)

0.66±0.17(6/14)

fixed wt/day

7.1±1.111.2±1.4

23.4±9.125.5±8.1

39.5±13.4

12.9±3.0(8/14)

32.2±7.8(6/14)

LDL, low density lipoprotein.• t t As in Table 3.§p<0.025, §§p<0.05 compared with atherosclerosis-resistant site R, paired / test.l/xO.05, **p<0.005 compared with normal area of the atherosclerosis-susceptible celiac site, paired t test on data

transformed to logarithms.1/X0.025, #/><0.005, ttP<0.001, tt/><0.05 compared with atherosclerosis-resistant site R, paired f test on data

transformed to logarithms.Time trends for 125I-tyramine cellobiose-LDL accumulation during cholesterol feeding (all regression analyses): for

normal part of atherosclerosis-susceptible celiac site S, data per square centimeter, p<0.02; per gram, p<0.01; foratherosclerosis-resistant site R, data per square centimeter and per gram, eachp<0.005; difference between normalpart of the atherosclerosis-susceptible celiac site and atherosclerosis-resistant site R, per square centimeter and pergram, eachp<0.05.

Overall difference between normal part of the atherosclerosis-susceptible site S and atherosclerosis-resistant site R,micrograms per square centimeter, p<0.0005; micrograms per gram, p<0.005 (analysis of variance on logarithms ofdata).

cholesterol (19.4±2.8 versus 6.1 ±0.8 and 22.6±4.7 ver-sus 7.9±1.0 fi\ plasma/g/day, p<0.0001 and p<0.0005,respectively). Expressed as amounts of LDL cholesterol,125I-TC-LDL accumulation by inner and outer layers ofsites P, and P2 were influenced relatively little bycholesterol feeding.

DiscussionIn this study we asked whether the arterial content of

123I-TC 1 day after injecting 125I-TC-LDL (I25I-TC-LDLaccumulation) would be greater in the atherosclerosis-susceptible celiac site of WC pigeons before develop-ment of atherosclerosis and whether arterial 125I-TC-LDL accumulation would be exaggerated by cholesterolfeeding. For birds not fed cholesterol, the 125I-TC-LDLaccumulation per unit arterial surface area was greaterin the atherosclerosis-susceptible celiac site compared

with the adjacent atherosclerosis-resistant site (R). Ifwe assume that all of the arterial content of 125I-TCreflects products of arterial LDL degradation, the rateof LDL degradation in the celiac site of these normalpigeons (calculated as described in References 14 and15, except using the total arterial 125I-TC content afterfixation) would be 1.21±O.O8xlO"5 of the plasma LDLpool/cm2/day, about three times the value that Schwen-ke and Carew1415 have reported for the correspondingatherosclerosis-susceptible site of rabbit aorta (branchorifice regions of the abdominal aorta, 0.37±0.07 and0.47±0.11, each xl0~5 of the plasma LDL pool/cm2/day, from References 14 and 15, respectively). However,because the rate of whole-body catabolism of LDL innormal pigeons (4.5±0.4 pools/day, this study; 4.9±0.2pools/day, Reference 39) is about three times thatreported for normal rabbits (1.6 pools/day; References



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4 S

B

Length of Cholesterol Feeding (Weeks)

FIGURE 5. Bar graphs showing 12SI-tyramine ceUobiose-lowdensity lipoprotein (TC-LDL) accumulation per gram ofinner and outer layers of proximal arterial sites Pt and P2.Panel A: 125I-TC-LDL accumulation expressed as volumes ofplasma. For birds not fed cholesterol, inner layer vs. outerlayer, p<0.025 for both sites P, and P2 (paired t tests on datatransformed to logarithms). Comparison between normalbirds and all cholesterol-fed birds combined: for inner layers ofsites P, and P2, p<0.0001 and p<0.0005, respectively (analy-sis of variance [ANOVA]). Panel B: 125I-TC-LDL accumu-lation expressed as amounts of LDL cholesterol For normalbirds, inner layer vs. outer layer, p < ft 025 for both sites P, andP2 (paired t tests on data transformed to logarithms). Signif-icant increasing trend during cholesterol feeding for I25I-TC-LDL accumulation occurred only in the inner layer of site P2

(p<0.05, regression analysis). Comparison of 125I-TC-LDLaccumulation between normal birds and those fed cholesterolwas only significant for outer layer of site Pj (p < 0.05, ANOVAon data transformed to logarithms).

14 and 15), this suggests that arteries of normal rabbitsand normal WC pigeons each account for the samefraction of whole-body LDL metabolism. Our value ofÎ-TC-LDL accumulation (per unit surface area) wasabout 80% greater for the atherosclerosis-susceptibleceliac site of normal WC pigeons compared with theadjacent atherosclerosis-resistant site R, in good agree-ment with the 60% greater rate of LDL degradation perunit surface area at branch orifices of abdominal aortasof rabbits compared with the adjacent atherosclerosis-resistant aorta.14

In normal pigeons Î-TC-LDL accumulation in thearterial sites closest to the heart (sites P! and P2) wastwo to three times as great as that in the susceptibleceliac site of the distal aorta. In comparison, the aorticarch of normal rabbits degraded LDL at a rate twicethat of the abdominal branch orifice regions.14 Thedifference, however, is that the aortic arch of rabbits ishighly susceptible to atherosclerosis,11 whereas these

proximal sites in the pigeon artery are relatively resis-tant to atherosclerosis (References 16-19; Figure 4). Apossible explanation for this apparent discrepancy maybe that there is a difference in the part of the arterial125I-TC content that reflects undegraded LDL and thatrepresents products of arterial LDL degradation at theproximal sites compared with the susceptible site. Alter-natively, changes in arterial rates of LDL degradation orconcentrations of undegraded LDL may occur duringcholesterol feeding that protect the proximal arterialsites from the development of atherosclerosis.

During the first 16 days of cholesterol feeding whenarteries remained macroscopically normal, fractionalrates of LDL degradation decreased in all arterial sitesin the rabbit except the highly atherosclerosis-suscepti-ble abdominal branch orifices. However, the decreasesin fractional rates of arterial LDL degradation weresimilar to or less than the decrease in the whole-bodyFCR of LDL.15 In the pigeon, Î-TC-LDL accumula-tion expressed in terms of volumes of plasma did notchange significantly during cholesterol feeding in eitherthe macroscopically normal atherosclerosis-susceptibleceliac site or the adjacent atherosclerosis-resistant site(R) and increased in the celiac site as a whole becauseof the development of atherosclerotic lesions, eventhough the whole-body FCR of LDL was suppressed bycholesterol feeding. However, by 1 week of cholesterolfeeding, 123I-TC-LDL accumulation (microliters plasmaper gram or square centimeter per day) in the twoproximal atherosclerosis-resistant sites (p! and P2) wasdecreased by 50%, 2.5 times the decrease in the whole-body FCR of LDL at that time. After feeding rabbitscholesterol for 16 days, the fractional rate of LDLdegradation (per unit weight) in the atherosclerosis-susceptible aortic arch, although decreased comparedwith that in untreated rabbits, was 2.5 times as great asthat in the adjacent atherosclerosis-resistant descendingthoracic aorta and 1.5 times as great as that in theatherosclerosis-susceptible abdominal branch orifices.15

In contrast, after feeding WC pigeons cholesterol for 1week, 125I-TC-LDL accumulation (per unit fixed weight;Figure 2B) in the proximal arterial sites (P, and P2) haddecreased so that they were no greater than in theatherosclerosis-susceptible celiac site. Furthermore, by4 weeks of cholesterol feeding, 123I-TC-LDL accumula-tion in the atherosclerosis-susceptible celiac site ofpigeons was about twice that of the other arterial sitesthat we studied (see Figure 2B). Thus, it seems thatinherent differences in 'Î-TC-LDL accumulation, re-flecting the combination of arterial rates of LDL deg-radation and the arterial concentration of undegradedLDL, among arterial sites within normal WC pigeonsmay play a role in regional variation in susceptibility toatherosclerosis just as rates of LDL degradation andconcentrations of undegraded LDL appear to do innormal rabbits. The ability of arterial sites to regulatemetabolism of LDL (i.e., decrease fractional rates ofLDL degradation) may play a more significant role indetermining regional susceptibility to atherosclerosis inWC pigeons than in rabbits.

Separate analysis of atherosclerotic lesions and mac-roscopically normal artery allowed us to considerwhether the marked increase in 125I-TC-LDL accumu-lation at the atherosclerosis-susceptible celiac site dur-ing cholesterol feeding (Figures 2 and 3) could be



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completely attributed to the presence of macroscopi-cally evident atherosclerotic lesions. 125I-TC-LDL accu-mulation in atherosclerotic lesions was much greaterthan in the macroscopicaUy normal part of the samearterial site. Furthermore, the greater 125I-TC-LDLaccumulation in the atherosclerotic lesions contributedsubstantially to the greater 125I-TC-LDL accumulationduring cholesterol feeding in the atherosclerosis-suscep-tible celiac site as a whole. Nonetheless, after 8 weeks ofcholesterol feeding, 125I-TC-LDL accumulation ex-pressed as amounts of LDL cholesterol increased abouteightfold and fivefold in the macroscopically normalareas of the atherosclerosis-susceptible celiac site andthe adjacent atherosclerosis-resistant site R, respec-tively. This compares with as much as a 170-fold in-crease in atherosclerotic lesions themselves. However, itshould be pointed out that the increase in the LDLcholesterol represented by the I25I-TC-LDL accumula-tion in the atherosclerosis-resistant site during choles-terol feeding was completely accounted for by the in-crease in the LDL concentration in the plasma, as125I-TC-LDL accumulation in this site was not altered bycholesterol feeding when expressed as volumes ofplasma. These data would suggest that intrinsically largeramounts of 125I-TC-LDL accumulation predispose sus-ceptible arterial sites to atherosclerosis and that latermarked increases in arterial Î-TC-LDL accumulationoccur coincident with the development of atheroscleroticlesions. These data agree qualitatively with previousreports for rates of LDL degradation and arterial con-centrations of undegraded LDL in rabbits.15-50'51

It was of interest to consider how the cumulativedelivery of cholesterol to arterial cells via estimatedcellular degradation of LDL, calculated as described inReferences 14 and 15 but using the total arterial 125I-TCcontent, would compare with the observed increase inarterial cholesterol concentration during cholesterolfeeding. We first estimated the cumulative degradationof LDL by the four arterial sites in normal animalsduring their average lifetimes of IVi months, assumingthat arterial rates of LDL degradation were constantover that interval at the values calculated from the totalarterial 125I-TC content (S, 11.3; R, 8.0; P^ 16.7; P2,18.8;all in micrograms LDL cholesterol per gram per day).For the atherosclerosis-susceptible celiac site and ath-erosclerosis-resistant site R, the estimated cumulativedegradation of LDL accounted for 112% and 89% ofthe observed arterial cholesterol concentration, respec-tively. For the two proximal atherosclerosis-resistantsites (Pj and P2), the estimated cumulative degradationof LDL accounted for 229% (P,) and 263% (P2) of theobserved arterial cholesterol concentration. These cal-culations suggest that cellular uptake and metabolism ofLDL alone may be sufficient to supply the cholesterolcontent of arterial sites in normal WC pigeons. Further-more, these calculations suggest that significant efflux ofcholesterol may occur from the proximal arterial sites(P! and P2) of normal WC pigeons. This could suggestan important independent role for cholesterol efflux inmediating susceptibility and resistance to atherosclero-sis of specific arterial sites.

Assuming that the rate of LDL degradation increasedin a linear fashion between the rates estimated fornormal birds and those estimated in the same way foreach time of cholesterol feeding, it was possible to

compute estimated cumulative degradation of LDL bythe arterial sites during each interval of cholesterolfeeding. By subtracting the cumulative rate of LDLdegradation estimated to have occurred over these sameintervals if the birds had not been fed cholesterol, it waspossible to estimate the incremental cumulative LDLdegradation associated with cholesterol feeding andcompare this increment with the observed increment inarterial cholesterol concentration (Figure 4 and Table3). The estimated cumulative incremental LDL degra-dation could account for 47% and 82% of the incrementin cholesterol concentration of atherosclerotic lesionsfound at the susceptible celiac site after 4 and 8 weeksof cholesterol feeding, respectively. For the macroscop-ically lesion-free part of the susceptible celiac site, theestimated cumulative incremental LDL degradationcould account for 55% and 89% of the observed in-crease in cholesterol concentration after 4 and 8 weeksof cholesterol feeding, respectively. The estimated cu-mulative incremental LDL degradation accounted for85 ±6% (mean±SEM, n=6) of the cholesterol incre-ment observed in the three atherosclerosis-resistantarterial sites (R, P b and P2) after 4-8 weeks of choles-terol feeding. On the other hand, the estimated incre-mental cumulative LDL degradation could account foronly 2.2% of the incremental cholesterol concentrationof the single small lesion found in the atherosclerosis-susceptible celiac site of one of the birds fed cholesterolfor 1 week, suggesting that that lesion had begundeveloping spontaneously before the onset of choles-terol feeding. These comparisons suggest that cumula-tive LDL degradation may be nearly sufficient to pro-vide the increase in arterial cholesterol concentrationobserved during cholesterol feeding. It is likely, how-ever, that other lipoproteins such as 0-VLDL alsocontribute cholesterol to the arterial wall. If this isadded to the amount estimated to be delivered by LDL,then it would seem that some of this lipoprotein-derivedcholesterol must subsequently be lost because of effluxprocesses.

In this study we have reported values for arterialÎ-TC-LDL accumulation, which we assume primarilyreflects products of arterial LDL degradation, both perunit surface area and per unit weight. This is so becausealthough all cells within an arterial site potentiallydegrade LDL (thus the reason for expressing 125I-TC-LDL accumulation per unit weight), data presented byCarew et al25 would suggest that after a 24-hour exper-iment, the intima of the thoracic aorta of rabbits ac-counted for a disproportionately large fraction of thearterial LDL degradation (providing a rationale forexpressing the arterial 125I-TC-LDL accumulation perunit surface area). Consistent with those reports, wefound that in WC pigeons not fed cholesterol, 125I-TC-LDL accumulation in the inner layers of the two prox-imal arterial sites (P, and P2) was more than twice asgreat (per unit weight) as that in the correspondingouter layers. The difference between the inner andouter layers was abolished by 1 week of cholesterolfeeding when the 125I-TC-LDL accumulation expressedas plasma equivalents decreased more in the innerlayers than in the outer layers. Because amounts of LDLcholesterol represented by the Î-TC-LDL accumula-tion were influenced relatively little during cholesterolfeeding in these atherosclerosis-resistant sites, it is



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tempting to speculate that the decrease in 125I-TC-LDLaccumulation when expressed as amounts of plasmareflects saturation of specific processes for degradationof LDL or its binding to the extracellular matrix at thesearterial sites.

In the course of these studies, we calculated thewhole-body FCR of LDL and determined that it de-creased to a plateau value that was 50% of normal by 2weeks of cholesterol feeding. This would appear tocontradict an earlier result obtained in this laboratorythat cholesterol feeding did not downregulate clearanceof LDL in WC pigeons.52 However, another study39

showed that whereas cholesterol feeding had almost noinfluence on clearance of LDL isolated from normocho-lesterolemic donor pigeons, clearance of LDL isolatedfrom hypercholesterolemic donor pigeons was de-creased 50% in cholesterol-fed recipient pigeons, inexcellent agreement with the results of this study.

In summary, we found differences in Î-TC-LDLaccumulation among arterial sites of normal and cho-lesterol-fed WC pigeons. The 125I-TC-LDL accumula-tion was greater in the atherosclerosis-susceptible celiacsite than in the adjacent atherosclerosis-resistant sitebefore the development of atherosclerosis. However,differences in 125I-TC-LDL accumulation among arte-rial sites of normal pigeons did not always predictsusceptibility to atherosclerosis. In normal pigeons tworelatively atherosclerosis-resistant arterial sites close tothe heart accumulated the most Î-TC-LDL. However,these arterial sites showed a striking ability to regulate125I-TC-LDL accumulation to levels that representednearly constant amounts of LDL cholesterol despitemarked increases in plasma LDL cholesterol duringcholesterol feeding. This was in contrast to the macro-scopically normal atherosclerosis-susceptible celiac siteand the adjacent atherosclerosis-resistant site, wherethe increase in '"I-TC-LDL accumulation paralleledthe increase in plasma LDL cholesterol during choles-terol feeding, and atherosclerotic lesions in the suscep-tible site, where "Î-TC-LDL accumulation increasedmuch more than the increase in plasma LDL choles-terol. Thus, arterial sites resistant to development ofatherosclerosis in WC pigeons differ from the suscepti-ble site by either accumulating less 125I-TC-LDL orhaving the capacity to limit increases in I2iI-TC-LDLaccumulation during exposure to hypercholesterolemia.These observations provide a potential mechanisticexplanation for the preferential development of athero-sclerosis at the susceptible celiac site. Further experi-ments will be required to determine whether thesedifferences in 125I-TC-LDL accumulation reflect differ-ences in intimal permeability to LDL, retention ofundegraded LDL within the extracellular matrix, and/orcellular degradation.

AcknowledgmentWe gratefully acknowledge Ms. Tricia Hester for her skillful

technical assistance with the analysis of plasma samples.

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D C Schwenke and R W St Clairatherosclerotic lesions develop.

onceatherosclerosis-susceptible region of White Carneau pigeon aorta and further enhanced Accumulation of 125I-tyramine cellobiose-labeled low density lipoprotein is greater in the

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accumulation of i-tyramine cellobiose-labeled low density

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