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1112 Vascular Reactivity During the Progression of Atherosclerotic Plaque A Study in Watanabe Heritable Hyperlipidemic Rabbits Frank D. Kolodgie, Renu Virmani, Holly E. Rice, and Wolfgang J. Mergner The effects of varying degrees of atherosclerotic plaque on vascular responsiveness in aortas of Watanabe heritable hyperlipidemic (WHHL) rabbits and New Zealand White (normal choles- terolemic) rabbits were studied. Ring segments from the aortic arch and thoracic aorta were mounted in organ chambers for isometric tension recording and measurement of endothelium- derived relaxing factor. WHHL rabbits were divided into three groups according to age: group 1, 3-5 months; group 2, 6-9 months; and group 3, 12-14 months. Atherosclerotic changes (expressed as a percent of total surface area) in the aortic arches in groups 1, 2, and 3 were 11±3% (mild), 28±6% (moderate), and 54±8% (severe) respectively; only occasional plaques were present in the thoracic aorta in all groups. Maximal contractions elicited with phenyl- ephrine progressively decreased with increasing degrees of atherosclerotic plaque. Contractions evoked by histamine were augmented in all groups of WHHL rabbits when compared with controls, whereas those to serotonin were augmented only in vessels with mild atherosclerosis. As the severity of the intimal lesions increased, endothelium-dependent relaxations to acetylcholine, ATP, and calcium ionophore A23187 progressively decreased. Endothelium- independent relaxation to nitroglycerin was virtually complete in all segments. However, vessels with severe atherosclerosis were less sensitive to this agent as illustrated by a significant increase in the ED50 value. Scanning electron microscopy revealed a predominant loss of endothelial cells in the central regions of fibrous plaques. Thus, in WHHL rabbits, hypercho- lesterolemia and atherosclerosis result in an increased responsiveness of vascular smooth muscle to histamine and serotonin. Endothelium-mediated relaxation of vascular smooth muscle is reduced with the progression of atherosclerosis primarily due to a loss of endothelial cells. (Circulation Research 1990;66:1112-1126) C oronary spasm plays an essential role in the pathophysiology of ischemic heart disease.1 In patients with variant angina, coronary spasm has been observed angiographically after administration of certain pharmacological agents.2-4 Coronary vasospasm usually occurs at the sites of varying degrees of atherosclerosis.5 Although the clin- ical presentation is well defined, the mechanism underlying vasospasm is not well understood. Recently, it has become evident that endothelial cells selectively modulate vascular smooth muscle tone by From the Department of Cardiovascular Pathology, Armed Forces Institute of Pathology, Washington, DC. Presented in part in abstract form at the Eighth International Symposium on Atherosclerosis, Rome, Italy, October 1988. Supported in part by a grant from the American Registry of Pathology and the Armed Forces Institute of Pathology. Address for reprints: Renu Virmani, MD, Dept. of Cardiovas- cular Pathology, Armed Forces Institute of Pathology, Washing- ton, DC 20306-6000. Received September 6, 1988; accepted November 21, 1989. releasing a potent substance, endothelium-derived relaxing factor.6-8 Endothelial cell function may become impaired in atherosclerotic vessels, and the absence of vasodilatory responses may render the vessel defenseless against certain vasoconstrictor stimuli.9 Numerous animal models of diet-induced hypercholesterolemia with and without balloon injury have demonstrated attenuation of endothelium-medi- ated relaxation10-12 and also have augmented constric- tor responses to various neurohumoral agents both in vivo1314 and in vitro.15 Similar findings have been reported in isolated human coronary arteries.16-'9 The purpose of this investigation was to evaluate the effects of the progression of atherosclerosis on vascular reactivity in the aortas of Watanabe herita- ble hyperlipidemic (WHHL) rabbits and to deter- mine if impairment or loss of endothelial cells is the mechanism underlying altered vascular responsive- ness. This model was chosen because it exhibits vascular lesions morphologically comparable with human atherosclerosis.20 by guest on February 18, 2018 http://circres.ahajournals.org/ Downloaded from

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1112

Vascular Reactivity During the Progression ofAtherosclerotic Plaque

A Study in Watanabe Heritable Hyperlipidemic RabbitsFrank D. Kolodgie, Renu Virmani, Holly E. Rice, and Wolfgang J. Mergner

The effects of varying degrees of atherosclerotic plaque on vascular responsiveness in aortas ofWatanabe heritable hyperlipidemic (WHHL) rabbits and New Zealand White (normal choles-terolemic) rabbits were studied. Ring segments from the aortic arch and thoracic aorta weremounted in organ chambers for isometric tension recording and measurement of endothelium-derived relaxing factor. WHHL rabbits were divided into three groups according to age: group1, 3-5 months; group 2, 6-9 months; and group 3, 12-14 months. Atherosclerotic changes(expressed as a percent of total surface area) in the aortic arches in groups 1, 2, and 3 were11±3% (mild), 28±6% (moderate), and 54±8% (severe) respectively; only occasional plaqueswere present in the thoracic aorta in all groups. Maximal contractions elicited with phenyl-ephrine progressively decreased with increasing degrees of atherosclerotic plaque. Contractionsevoked by histamine were augmented in all groups of WHHL rabbits when compared withcontrols, whereas those to serotonin were augmented only in vessels with mild atherosclerosis.As the severity of the intimal lesions increased, endothelium-dependent relaxations toacetylcholine, ATP, and calcium ionophore A23187 progressively decreased. Endothelium-independent relaxation to nitroglycerin was virtually complete in all segments. However, vesselswith severe atherosclerosis were less sensitive to this agent as illustrated by a significantincrease in the ED50 value. Scanning electron microscopy revealed a predominant loss ofendothelial cells in the central regions of fibrous plaques. Thus, in WHHL rabbits, hypercho-lesterolemia and atherosclerosis result in an increased responsiveness of vascular smoothmuscle to histamine and serotonin. Endothelium-mediated relaxation of vascular smoothmuscle is reduced with the progression of atherosclerosis primarily due to a loss of endothelialcells. (Circulation Research 1990;66:1112-1126)

C oronary spasm plays an essential role in thepathophysiology of ischemic heart disease.1In patients with variant angina, coronary

spasm has been observed angiographically afteradministration of certain pharmacological agents.2-4Coronary vasospasm usually occurs at the sites ofvarying degrees of atherosclerosis.5 Although the clin-ical presentation is well defined, the mechanismunderlying vasospasm is not well understood.Recently, it has become evident that endothelial cellsselectively modulate vascular smooth muscle tone by

From the Department of Cardiovascular Pathology, ArmedForces Institute of Pathology, Washington, DC.

Presented in part in abstract form at the Eighth InternationalSymposium on Atherosclerosis, Rome, Italy, October 1988.

Supported in part by a grant from the American Registry ofPathology and the Armed Forces Institute of Pathology.Address for reprints: Renu Virmani, MD, Dept. of Cardiovas-

cular Pathology, Armed Forces Institute of Pathology, Washing-ton, DC 20306-6000.Received September 6, 1988; accepted November 21, 1989.

releasing a potent substance, endothelium-derivedrelaxing factor.6-8 Endothelial cell function maybecome impaired in atherosclerotic vessels, and theabsence of vasodilatory responses may render thevessel defenseless against certain vasoconstrictorstimuli.9 Numerous animal models of diet-inducedhypercholesterolemia with and without balloon injuryhave demonstrated attenuation of endothelium-medi-ated relaxation10-12 and also have augmented constric-tor responses to various neurohumoral agents both invivo1314 and in vitro.15 Similar findings have beenreported in isolated human coronary arteries.16-'9The purpose of this investigation was to evaluate

the effects of the progression of atherosclerosis onvascular reactivity in the aortas of Watanabe herita-ble hyperlipidemic (WHHL) rabbits and to deter-mine if impairment or loss of endothelial cells is themechanism underlying altered vascular responsive-ness. This model was chosen because it exhibitsvascular lesions morphologically comparable withhuman atherosclerosis.20

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Kolodgie et al Vascular Reactivity in WHHL Rabbits 1113

Materials and MethodsAnimal Model ofAtherosclerosis

Homozygous WHHL rabbits were obtained fromour breeding colony at the Armed Forces Institute ofPathology, Washington, DC. These animals exhibitconsistent hereditary hyperlipidemia as a result ofinbreeding.21 WHHL rabbits of either sex, aged 3-5months (group 1, n=5), 6-9 months (group 2, n=6),and 12-14 months (group 3, n=5) and weighingapproximately 2-3.2 kg, were studied. New ZealandWhite rabbits, aged 5-6 months (n=5) and 10-14months (n=4), were studied as controls. In thehomozygous WHHL rabbit model, the aortic arch ismore susceptible to atherosclerotic plaque formationthan the thoracic aorta. All animals were fed stan-dard rabbit chow (Purina, St. Louis, Missouri). Thecare of rabbits used in the study complied with theguidelines of the National Institutes of Health onanimal experimentation.

Measurement of Serum Cholesterol and TriglyceridesSerum cholesterol and triglycerides were measured

enzymatically with an automated method.22,23

Vascular Reactivity StudiesBefore study, rabbits were fasted for 12 hours.

Animals were anesthetized with sodium pentobar-bital (25-30 mg/kg i.v.). A blood sample wasobtained for determination of serum cholesterol andtriglyceride levels. Rabbits were then killed by exsan-guination over a 5-10-minute period. The aortic archand thoracic aorta were carefully removed and imme-diately placed in modified Krebs-Henseleit buffer(KHB) containing (mM) NaCl 118.3, KCL 4.7, CaCl22.5, KH2PO4 1.2, NaHCO3 25.0, calcium EDTA0.026, and glucose 11.1 at 370 C and bubbled with amixture of 95% 02-5% CO2.

Vessels were cleaned of adherent fat and connec-tive tissue, cut into 3-5-mm ring segments, andsuspended vertically with suture in an organ bathwith 30 ml oxygenated KHB maintained at 370 C. Theinterval between the removal of the aorta from theanimal to the suspension of the aortic rings in organbaths did not exceed 20 minutes. One additional ringfrom the aortic arch and thoracic aorta of each rabbitwas immediately fixed in McDowell-Trump (formal-dehyde/glutaraldehyde) solution (for details, seebelow) for later assessment of intimal surfaces byscanning electron microscopy to determine if theexperimental time course affected vessel morphology.

All studies were performed in the presence ofpropranolol (0.1 uM). Tension was monitored con-tinuously with a linear force transducer (model B-25,Gould, Cleveland, Ohio). Aortic segments were pas-sively stretched to a resting tension of 2 g over a90-minute period. In some segments of the arch andthoracic aorta, the endothelium was removed bygently rubbing the intimal surface with a cotton swab.Four WHHL rabbits, aged 10-14 months, were

perfusion-fixed with McDowell-Trump fixative. The

method for perfusion fixation was similar to thatdescribed by Rosenfeld et al,24 with minor modifica-tions. The carotid artery was cannulated and perfusedwith lactated Ringer's solution at 100 mm Hg pres-sure. The inferior vena cava was exposed by midlineabdominal incision, and a surgical incision was madein the anterior wall of the vena cava. This allowed aflow rate of approximately 60-80 ml/min. LactatedRinger's solution was infused until the runoff was freeof blood. This was followed by perfusion-fixation withMcDowell-Trump solution for 20 minutes. The aortawas then carefully removed, and 3-5-mm rings werecut from the ascending and thoracic aorta for scanningelectron microscopy.

ProtocolsResponses to vasoactive stimuli were examined in

six rings from each animal (aortic arch, n =3; thoracicaorta, n=3). One ring from the aortic arch and onefrom the thoracic aorta of each animal underwentmechanical endothelial denudation.

Vasoconstrictor responses to phenylephrine (10-9-10-5 M), histamine (10-6_10-4 M), and serotonin(10-8_10-5 M) were determined in all groups.

After precontraction with a concentration of phen-ylephrine equivalent to the ED30-ED50 of this agent,endothelium-dependent relaxation responses to ATP(10-6_10-4 M), acetylcholine (10-9-10-5 M), and cal-cium ionophore A23187 (10`1o10-5 M) and endothe-lium-independent relaxation responses to nitroglyc-erin (10-9-10-5 M) were studied. Dose-responsecurves to the various drugs were randomized. Thishelped minimize the possibility that time in the organbath was a factor when the tissue was exposed to anagent or that previous exposure to a drug interferedwith the subsequent pharmacological response of theagonist tested. After each concentration-responsecurve, vessels were washed repeatedly with KHB andallowed to equilibrate for a minimum of 30 minutes.

Gross Morphological ExaminationAt the completion of each experiment, the rings

were washed with KHB and allowed to equilibrate tobaseline resting tension. Vascular segments were thenremoved from the apparatus, cut open longitudinally,and pinned stretched on a tongue depressor with theendothelial surface exposed. Specimens were fixedovernight in McDowell-Trump solution at 40 C. Thespecimens were then photographed under fixative.Photographic slides were projected (x 10), and thetotal surface and atherosclerotic plaque areas werequantitated by using computerized planimetry.

Histological ExaminationA 1-2-mm longitudinal section was cut from each

ring segment for light microscopy. The remainingtissue was submitted for scanning electron micros-copy, and a few samples were selected for transmis-sion electron microscopy.

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1114 Circulation Research Vol 66, No 4, April 1990

TABLE 1. Serum Cholesterol and Triglyceride Levels

Control Group 1 Group 2 Group 3(n=5) (n=5) (n=6) (n=5)

Total cholesterol(mg/dl) 30.3+0.8 722.4±34.6 750.0±92.3 494.6±135.4

Triglyceride(mg/dl) 51.7±2.3 619.4±222.0 456.6±65.6 425.4±63.7Values are expressed as mean±SEM. Control, New Zealand White rabbits; group 1, Watanabe heritable

hyperlipidemic (WHHL) rabbits aged 3-5 months; group 2, W IHL rabbits aged 6-9 months; group 3, WHHL rabbitsaged 12-14 months.

Light MicroscopyTissue samples were dehydrated and embedded

in paraffin on edge (cross section), cut into 4-,umsections, and stained with hematoxylin-eosin andMovat pentachrome stain. Histological sectionswere projected (x25), and the intima-media index(intima/media area times 100) was determined bycomputerized planimetry.

Scanning Electron MicroscopyTissue samples were dehydrated and critical-

point-dried with liquid carbon dioxide, mounted on

stubs, coated with gold palladium, and visualized on

a scanning electron microscope (model S-570, Hita-chi Instruments, Danbury, Connecticut). Each vascu-

lar segment was examined for the presence or

absence of endothelial cells, mononuclear adher-ence, and the extent of atherosclerosis. The percentsurface area covered by endothelium was estimatedvisually at x 150 magnification. Areas where thevessel was in contact with the suture were not eval-uated for endothelial cell loss.

Drug PreparationThe pharmacological agents used were acetylcho-

line chloride (Sigma Chemical, St. Louis, Missouri),ATP (Sigma Chemical), calcium ionophore A23187(Sigma Chemical), histamine (Sigma Chemical),nitroglycerin (Marion Laboratories, Kansas City,Missouri), phenylephrine (Sigma Chemical), pro-

pranolol HCL (Sigma Chemical), and 5-hydroxy-tryptamine creatinine sulfate (serotonin, SigmaChemical). All drugs were freshly prepared usingdistilled water except for A23187, which was dis-solved in dimethyl sulfoxide (1%). Agents were kepton ice during the experiments.

Statistical MethodsData are expressed as mean±SEM. Relaxation

responses are expressed as the percent relaxationfrom the amount of preconstriction produced byphenylephrine. ED50 was defined as the concentra-tion of agonist at which 50% of the maximal response

was obtained. Statistical evaluation of the data was

performed by Student's t test for unpaired observa-tions. A value ofp<0.05 was considered significant.

ResultsSerum Cholesterol and Triglyceride Levels

Table 1 is a comparison of the mean distribution oftotal serum cholesterol and triglycerides in controland WHHL rabbits. Total serum cholesterol andtriglyceride levels were markedly elevated in WHHLrabbits as compared with control rabbits. Serumcholesterol and triglyceride concentrations in WV HLrabbits were maximal in the 3-5-month-old groupand decreased with the age of the animal.

Vascular Responses in Control(New Zealand White) RabbitsPhenylephrine (10-9-10-5 M), histamine (10 '-

i0` M), and serotonin (10-8_10-5 M) elicitedconcentration-dependent contractions in control seg-ments of the aortic arch and thoracic aorta (Figure 1,Table 2). Mechanical removal of endothelial cells didnot significantly alter the maximal response or ED50value to these agonists.

During sustained contractions evoked by phenyl-ephrine (ED30-ED50 range), acetylcholine (109-106M), ATP (10-7-_0-4 M), calcium ionophore A23187(10-1Q-106 M), and nitroglycerin (10-9-10-6 M)induced concentration-dependent relaxations in allcontrol vessels (Figures 2 and 3, Table 3). Aftermechanical removal of the endothelial cell layer,relaxation to acetylcholine, ATP, and A23187 wasvirtually abolished (data not shown). In contrast,vascular relaxation in response to nitroglycerin wasunaffected in vessels denuded of endothelium (datanot shown). There were no statistical differences inany vascular reactivity responses between the 5-6-month-old and 10-14-month-old New ZealandWhite rabbits (Tables 2 and 3).

Vascular Responses in WHHL RabbitsIncreasing concentrations of phenylephrine, hista-

mine, and serotonin in WHHL rabbits, which wereidentical with concentrations in control animals,induced contractions in segments of the aortic archand thoracic aortas (Figure 1, Table 2). The maximalcontractile response caused by phenylephrine wassignificantly decreased in vessels with moderate andsevere atherosclerosis when compared with vessels ofcontrol tissues with intact endothelium (Figure 1,Table 2). Maximal contractions produced by hista-mine were significantly augmented in both the aortic

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Kolodgie et al Vascular Reactivity in WHHL Rabbits 1115

Aortic Arch

Serotonin

8 7 6 5

-Log Molor Concentration

Thoracic Aorta

Serotonin

FIGURE 1. Cumulative concentration-responsecurves tophenylephrine, histamine, and serotonin onunrubbed ring segments from the aortic arch andthoracic aorta of control rabbits (v) and Watanabeheritable hyperlipidemic rabbits aged 3-5 months(r1), 6-9 months (A), and 12-14 months (o). Theresults are shown as mean±SEM. Filled symbols(m,A,.) indicate statistically significant differences(p<O.05) compared with control rings.

8 7 6 5

.Log Molar Concentrtion

arch and thoracic aorta and were independent ofatherosclerotic plaque progression (Figure 1, Table 2).For serotonin, the concentration-response relation invessels with minimal atherosclerosis exhibited a

marked leftward shift with a significantly increasedmaximum constrictor response and a lower ED50 valuewhen compared with control rings; however, responseof vessels with moderate and severe atherosclerosiswas equal to control vessels (Figure 1, Table 2).

Responses to the endothelium-dependent vasodi-lators acetylcholine, ATP, and A23187 were similarin vessels from groups 1 and 2 WHHL rabbits whencompared with control rabbits (Figure 2, Table 3). Incontrast, relaxations to these agonists in the aorticarch and thoracic aorta obtained from group 3WHHL rabbits were markedly attenuated comparedwith the relaxations observed in control rabbits (Fig-ure 2, Table 3); these differences were not illustratedby a significantly decreased ED50 value. The magni-tude of the maximal relaxations (percent of initialphenylephrine-induced contractions) elicited by ace-

tylcholine in aortic arch vessels from WHHL rabbits

correlated significantly with the degree of atheroscle-rotic plaque and endothelial cell loss (r2=0.65 andr2=0.73, respectively; Figure 3) and to a lesser degreewith intimal medial index (r2=0.47). The maximalrelaxation with high concentrations of nitroglycerin(>10-6 M) was not different among all vessels stud-ied (Figure 4, Table 3). However, at lower concen-

trations, group 1 WHHL tissues were more sensitiveto nitroglycerin than moderate to severely atheroscle-rotic vessels (Figure 4, Table 3).

Morphological Evaluation of Ring Segments FromControl and WHHL AnimalsNo visible atherosclerotic lesions were detected in

any vessels removed from New Zealand White rab-bits (Table 4). Of the vessels from WHHL rabbits,the aortic arch was more severely affected by athero-sclerosis than was the thoracic aorta, and plaqueprogression increased with the age of the animal.Fatty streaks were the predominant lesion in youngeranimals, whereas fibrous plaques were more exten-sive in older animals (Figure 5 A-C). The fatty streak

Phonyblphrine Histamine

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1116 Circulation Research Vol 66, No 4, April 1990

TABLE 2. Evaluation of Contractile Responses in Control and Watanabe Heritable Hyperlipidemic Rabbits

Control 1 Control 2 Group 1 Group 2 Group 3

AA TA AA TA AA TA AA TA AA TA

PhenylephrineEndothelial cells present (n=20)Max (g) 4.5±0.4 3.9±0.3 4.7±0.5 4.50±0.3 3.9±0.4 3.3±0.3 3.4±0.4* 3.7±0.6 3.1±0.3* 2.8±0.4*ED50 (xlO-7 M) 5.8± 1.3 5.2+0.9 4.5± 1.1 3.8±0.5 4.3±0.6 3.4±0.3 5.4±1.7 3.2+0.9 5.2±0.6 4.4+0.7

Endothelial cells absent (n=10)Max (g) 4.0±0.2 4.3±0.7 4.4+0.6 4.3±0.2 3.0+0.4 3.3±0.3 2.3±0.1 2.0±0.2 2.1±0.4 2.1±0.5ED50 (xlO-7 M) 3.6±1.2 1.9±0.3 2.2±0.2 1.5±0.1 2.6±0.6 1.5+0.2 2.1±0.7 1.8±0.5 3.5±0.8 3.1+0.8

HistamineEndothelial cells present (n=20)Max (g) 3.4±0.8 4.3±0.8 5.1±0.6 4.7±0.6 6.1±0.5* 5.7±0.2* 5.4±0.2* 6.6±0.3* 5.5 ±0.3* 6.1±0.5*ED50 (xlO-5 M) 2.6±0.4 2.6±0.5 2.0±0.7 1.6±0.5 1.3±0.2 1.0±0.08 1.6±0.2 0.7±0.08* 1.2±0.8 0.8±0.1*

Endothelial cells absent (n = 10)Max (g) 3.6±0.8 4.2±1.4 5.4±1.1 4.8±0.7 6.0±0.4 6.1±0.4 5.2±1.0 5.4±0.7 4.5±0.7 5.6±0.7ED50 (xlO-5 M) 2.2±0.6 2.3±0.8 2.8±1.2 0.9±0.4 0.7±0.08 0.9±0.12 0.7±0.09 0.4±0.01 1.9±1.0 0.5±0.06

Serotonin (5-HT)Endothelial cells present (n=20)Max (g) 3.0±0.5 2.0±0.4 1.6±0.4 1.2±0.6 4.8±0.3* 4.2±0.3* 2.7±0.2 2.3±0.2 2.7±0.4 0.9±0.3ED50 (xlO-6 M) 1.4±0.2 1.6±0.3 1.1±0.4 1.6±0.5 0.3±0.02* 0.2±0.02* 1.5±0.6 1.2±0.2 2.1±0.5 3.4±1.2

Endothelial cells absent (n=10)Max (g) 2.7±0.6 3.4±0.6 3.3±0.3 2.0±0.6 4.8±0.4 4.6±0.2 2.4±0.6 2.3+0.5 2.0±+1.3 1.1±0.4ED50 (xlO-6 M) 1.2±0.4 1.4±0.5 1.3±0.5 1.8±0.8 0.2±0.03 0.4±0.09 1.0±0.3 0.9±0.3 1.0±0.4 3.2±0.5

Values are reported as mean±SEM. Control 1, New Zealand White rabbits aged 5-6 months; control 2, New Zealand White rabbits aged10-14 months; group 1, Watanabe heritable hyperlipidemic (WHHL) rabbits aged 3-5 months; group 2, WHHL rabbits aged 6-9 months;group 3, WVHL rabbits aged 12-14 months; AA, aortic arch; TA, thoracic aorta; Max, maximum contractile response; n, number of ringsegments in each group.

*Significantly different from control rings (endothelial cells present) atp<0.05 by Student's t test for unpaired observations.

Aortic Arch

9 a 7 6 7 6 5

Mobar Concentroatn

107

Thoracic Aorta

10 9 A 7 67 o 5 4

-Lag Molar Concentraton

FIGURE 2. Graphs showing effect ofacetylcholine,ATP, and Ca21 ionophore A23187 (endothelium-dependent relaxation) on unrubbed ring segments

6 from the aortic arch and thoracic aorta of controlrabbits (v) and Watanabe heritable hyperlipidemicrabbits aged 3-5 months (o), 6-9 months (A), and12-14 months (o) during contractions tophenyleph-

A23167 rine (PE) in the ED3o-ED50 range of this agonist.The results are mean+±SEM. Filled symbols (-,-)indicate statistically significant differences (p<0.05)compared with control rings.

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Kolodgie et al Vascular Reactivity in WHHL Rabbits 1117

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FIGURE 3. Graphs showing correlationbetween the maximum relaxation evoked byacetylcholine (Ach on figure) and the percentatherosclerotic plaque (left panel) and per-

cent endothelial loss (right panel) in un-

rubbed ring segments from the aortic arch ofWatanabe heritable hyperlipidemic rabbitsaged 3-5 months (o), 6-9 months (A), and12-14 months (o). The response to Ach isexpressed as a percent of the initial contrac-tion evoked by phenylephrine (PE) (ED30-ED50 range of this agonist).

lesions were formed mainly by foam cells, smoothmuscle cells, and connective tissue. Fibrous athero-sclerotic plaques consisted of central areas of pulta-ceous debris with surrounding foam cells, muco-polysaccharides, smooth muscle cells, and collagen(Figure 5C). Focal areas of calcification were seen inthe oldest animals with fibrous plaques. The thoracicaorta showed focal lesions (predominantly fattyplaques) around vertebral artery branch points (Fig-ure 5D).

Scanning electron microscopy in vessels withoutatherosclerosis revealed intact endothelial cells thatwere well aligned (Figure 6). Early atheroscleroticlesions consisted of focal monocytes adhering toendothelial cells at various stages of spreading andinvagination (Figure 7); older lesions showed numer-ous monocytes adhering and spreading over theendothelial surface (Figure 8). Plaque progressionshowed lobulated lesions with stretched endotheliumoverlying subendothelialized monocytes. Theseraised lesions were often disrupted with focal loss of

endothelium exposing underlying monocytes (Figure9). Fibrous plaques showed central regions of focalloss of endothelium or large deendothelialized sur-faces (Figures 9 and 10). All ring segments thatunderwent mechanical denudation demonstrated anear total loss of endothelium as confirmed by scan-ning electron microscopy (Figure 11). No differencesin endothelial cell preservation were noted betweenvessels in the bath versus those fixed immediatelyafter the animal was killed or when aortas wereperfusion-fixed in situ.The two 10-month-old perfusion-fixed rabbits

showed raised lesions with monocyte adherence andoccasional endothelial retraction in areas of fattystreaks. The two 14-month-old rabbits showed raisedatherosclerotic plaques with areas of focal endothelialloss with monocyte, platelet, and occasional red celladherence. At the edges of the raised atheroscleroticplaques, monocyte adherence was extensive. Focally,the raised fatty plaques showed endothelial retractionexposing the underlying foam cells. Also, fibrous

TABLE 3. Evaluation of Relaxation Responses in Control and Watanabe Heritable Hyperlipidemic Rabbits

Control 1 Control 2 Group 1 Group 2 Group 3

AA TA AA TA AA TA AA TA AA TA

Acetylcholine (n=20)Max (%)ED50 (xlO-8 M)

CA2' ionophore A23187 (n=20)Max (%)ED50 (xlO-8 M)

ATP (n=20)Max (%)ED5o (xlO-5 M)

Nitroglycerin (n=20)Max (%)EDso (xlO-8 M)

72+6 92+5 85±4 87±4 73±7 82±3 65±10

4.8±1.3 6.2±1.8 3.8±1.1 5.7±1.8 10.5±1.6* 8.5±1.4 6.4±1.3

84±4 40±6* 86±58.4±1.7 6.2±1.2 6.9±1.0

75±3 90±3 89±3 92+3 74+3 89±3 74±7 88±4 48+7* 81±5

2.8±0.5 4.2±1.2 2.3±0.2 4.5±1.9 4.6±1.2 5.0±1.1 5.2±1.9 3.8±1.1 4.7±2.9 5.4±3.6

62±5 60±6 67±7 65±7 55±8 43±7 59±8 48±7 44±7* 55±4

0.7±0.2 2.1±0.5 1.8±0.8 0.8±0.2 1.3±0.2 2.2±0.3 0.8±0.2 2.0±0.5 0.7±0.2 1.8±0.5

99±4 97±4 84±5 91±4 100 100 98±2

2.6±0.7 2.0±0.3 3.6±0.9 3.7±0.8 2.1±0.4 1.7±0.4 4.2±1.8

100 96±3 99±12.8±0.8 10.8±3.1* 5.1±1.8*

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Values are mean+SEM. Control 1, New Zealand White rabbits aged 5-6 months; control 2, New Zealand White rabbits aged 10-14months; group 1, Watanabe heritable hyperlipidemic (WHHL) rabbits aged 3-5 months; group 2, WHHL rabbits aged 6-9 months; group3, WHHL rabbits aged 12-14 months; AA, aortic arch; TA, thoracic aorta; Max, maximum relaxation (percent of phenylephrine-inducedcontraction; n, number of ring segments for each group.

*Significantly different from control rings at p<0.05 by Student's t test for unpaired observations.

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FIGURE 4. Graphs showing effect of nitro-glycerin (endothelium-independent relax-ation) on unrubbed ring segments from theaortic arch and thoracic aorta of controlrabbits (v) and Watanabe heritable hyperlip-idemic rabbits aged 3-5 months (o), 6-9months (A), and 12-14 months (o) duringcontractions to phenylephrine (PE) in theED30-ED50 range of this agonist. The resultsare mean +SEM. Filled symbol (a) indicatesstatistically significant differences (p<0.05)compared with control rings.

9 8 7 6 5 9 8 7 6 5

-Log Molor Concentrotion

plaques showed central regions of focal loss of endo-thelium. These were seen only in the ascending aorta.

DiscussionPresent StudyWHHL rabbits were grouped according to age and

compared with New Zealand White rabbits (con-trols). Mean plaque area in the aortic arch andthoracic aorta increased with age progression. Max-imal contractile responses to phenylephrine weredecreased in vessels obtained from WHHL rabbits(groups 2 and 3) compared with control rings. Vaso-constrictor effects of serotonin were augmented inWHHL rabbits aged 3-5 months in both the aorticarch and thoracic aorta as compared with controlrings, whereas responses in vessels from olderWHHL rabbits with moderate and severe atheroscle-rosis (groups 2 and 3, respectively) were similar tocontrols. In contrast, constrictor responses to hista-mine were augmented in all groups of WHHL rabbitswhen compared with controls.

In the aortic arch from WHHL rabbits with mod-erate and severe atherosclerosis, there was a gradualattenuation of endothelium-dependent relaxation tothe endothelium-dependent vasodilators. Endothe-lial surfaces outside the regions of fatty streaks werenormal in appearance. Endothelial disruption in theaortic arch of groups 1 and 2 was primarily focal(2+1% and 12+4%, respectively), whereas in group3, there was significant loss of endothelial cells in the

central regions of fibrous plaques (36 +4%). Themagnitude of maximal relaxation, by acetylcholine, inaortic arch vessels from WHHL rabbits correlatedsignificantly with the percent of atheroscleroticplaque (r2=0.65) and percent loss of endothelium(r2=0.73) and not with intimal medial index(r2=0.47). The correlation between amount ofplaque, to loss of endothelium, intimal medial index,and maximal relaxation to calcium ionophore A23187was less significant (r2=0.42, 0.46, 0.42, respectively).These data suggest that the defect in endothelium-mediated relaxation is primarily due to the destruc-tion of endothelial cells in vessels with severe athero-sclerosis, but dysfunction of endothelium may alsoplay a role in impaired relaxation. Endothelium-dependent relaxation appeared to decrease withplaque progression, which may be related to hyper-lipidemia and/or atherosclerosis in vessels with mildfocal endothelial cell disruption. However, no statis-tically significant differences were noted betweengroups 1 and 2. Increased sensitivity to histamine andserotonin in WHHL rabbits cannot be accounted forby the loss of endothelial cells during atheroscleroticprogression because no differences were seen indenuded vessels.Other factors in addition to endothelial cell loss,

such as activation of resident leukocytes, may con-tribute to alterations in vascular reactivity. Accumu-lation of monocytes/macrophages is a common occur-rence in atherosclerotic lesions, and recent evidence

TABLE 4. Morphological Evaluation of Vascular Ring Segments in Control and Watanabe Heritable Hyperlipidemic Rabbits

Control (n=5) Group 1 (n=5) Group 2 (n=6) Group 3 (n=5)AA TA AA TA AA TA AA TA

Plaque (%) 0 0 11+3 2+1 28+6 7±2 54±8* 10±4Endothelium loss(%) 0 0 1±2 0 12±4 3±1 36±4* 5±1I/M index 0 0 12±4 1±1 47±9 5±3 60±12* 10±3

Values are expressed as mean±SEM. Control, New Zealand White rabbits; group 1, Watanabe heritable hyperlipidemic (WHHL) rabbitsaged 3-5 months; group 2, WHHL rabbits aged 6-9 months; group 3, WHHL rabbits aged 12-14 months; AA, aortic arch; TA, thoracicaorta; I/M, intima/media area times 100.

*p<0.05 compared with AA in control and in groups 1 and 2.

Aortic Arch

o-

.2o 20-C0U0U. 40-0za-CL

.2a

o 80-0

100-

Thoracic Aorta

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Kolodgie et al Vascular Reactivity in WHHL Rabbits 1119

'A¾ iI

6'

Ai B , D

FIGURE 5. Representative photomicrographs of ring segments from the aortic arch and thoracic aorta of Watanabe heritablehyperlipidemic rabbits. Panel A: Group 1 (3-5 months) had occasionalfoam cell deposition. Panel B: Group 2 (6-9 months) hadpredominantly fatty lesions consisting offoam cells. Panel C: Group 3 (12-14 months) showed extensive thickening of the intimaby fibrous plaque with central areas of necrosis (pultaceous debris) and focal calcification. Surrounding the areas of necrosis arefoam cells, smooth muscle cells, and collagen. Also, the underlying media is focally involved with atherosclerosis. Panel D:Localized fatty plaque surrounding a vertebral branch from the thoracic aorta of a group 3 rabbit and consisting offoam cells,smooth muscle cells, and mucopolysaccharides. Movatpentachrome stain; magnification, x 75 (panelA) and x 150 (panels B-D).

suggests that a vasoactive product released by thesecells may contribute to vasospasm in atherosclerosis.Infusion of fmet-leu-phe (a known activator of mono-cytes/macrophages) into perfused hindlimbs of ath-erosclerotic monkeys produces an increase in vascularresistance when compared with that in nonathero-sclerotic animals.25 This vasoconstrictor responsemay selectively involve release of prostaglandin E2.Infusion of leukotriene D4 had minimal effects inboth atherosclerotic and nonatherosclerotic animals,whereas prostaglandin E2 produced a greater than10-fold increase in atherosclerotic vessels.25 Otherendogenous products of monocytes/macrophages,such as superoxide anions, may also contribute toaltered vascular tone. Release of superoxide anions(in vitro) depresses endothelium-mediated relax-ations to acetylcholine.26

Previous StudiesContractile responses. Contraction elicited by phen-

ylephrine is caused by activation of a1-adrenergicreceptors located on smooth muscle cells. In thepresent study, the contractile responses to phenyl-ephrine are decreased in aortas obtained fromWIHL rabbits with moderate and severe atheroscle-rosis as compared with control vessels. This reduc-tion, however, was not reflected by a significantlydecreased ED50 value. These results are differentfrom previous reports in cholesterol-fed and WHHL

rabbits in which contractile responsiveness to theadrenergic agonist phenylephrine was unaltered.13,27In rabbits fed a cholesterol-rich diet, an increasednumber of a-adrenergic receptors have beendetected.28 Despite the increase in receptor number,the results of our study and previous studies incholesterol-fed and WHHL rabbits do not demon-strate augmented contraction to phenylephrine.

Several studies have described augmented con-strictor responses to serotonin in animal models ofatherosclerosis. Yokoyama et all5 have reported anincreased responsiveness to serotonin in atheroscle-rotic aortas from WHHL rabbits aged 8-12 months.More recently, they observed augmented contractionto serotonin in ring segments of WHHL rabbits aged10-22 months with moderate atherosclerosis com-pared with ring segments of rabbits with mild or severeatherosclerosis.29 We have demonstrated that vaso-constrictor responses elicited by serotonin were mark-edly increased in aortas only during the early stages ofhypercholesterolemia and/or atherosclerosis. Aug-mented constrictor responses to serotonin have beendemonstrated in aortas from rabbits fed a 2% and0.3% cholesterol-rich diet for 10 weeks and 16 weeks,respectively,12 and in cholesterol-fed cynomolgusmonkeys.30 Hypersensitivity to serotonin in atheroscle-rotic vessels may be due to an increased number ofserotonergic receptors.28 Results from the present

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FIGURE 6. Scanning electron micro-graph of the endothelium from the tho-racic aorta (unrubbed segment) of agroup 2 rabbit showing intact, well-aligned endothelial cells with promi-nent intercellular junctions (arrows).

study suggest that an increase in serotonin receptordensity is an early phenomenon in atherosclerosis.

Histamine causes vasoconstriction as a result ofstimulating the H1-receptor subtype.31 To our knowl-edge, there are no previous vascular reactivity studiesevaluating the effect of histamine on atheroscleroticvessels in the rabbit model although an increasedsensitivity to histamine has been reported in athero-sclerotic vessels from miniature pigs and humancoronary arteries.'8'19,32 Vascular constriction in theaortas of all groups of WHHL rabbits showed anenhanced responsiveness to histamine. Augmentedconstrictor responses to histamine may be due to anincrease in H1-receptor density as previously de-scribed in aortas of rabbits that were fed choles-terol.33 Other mechanisms may involve increasedsensitivity of the receptor and/or effector units afterstimulation by histamine in the presence ofatherosclerosis. 18

In the present study, contractile responses to phen-ylephrine, histamine, and serotonin were unchangedby mechanical removal of endothelium. Increasedcontractile responses to these agents have beenreported in denuded vessels of rat, dog, andswine.34-36 However, Furchgott6 and Verbeuren eta112 have demonstrated that removal of endothelialcells in the rabbit aorta does not cause enhancedvasoconstriction. Therefore, augmented contractionsto histamine and serotonin are most likely due toalterations in smooth muscle cell functions ratherthan loss of endothelial cells in this model. Previousstudies demonstrate that vasoconstrictor responses toKCI in normal and atherosclerotic vessels are similar;therefore, it appears that the contractile machineryin atherosclerotic vessels is not altered.15,27

In the present study, vasoconstrictor responses toserotonin were augmented in vessels with mild ath-erosclerosis, whereas hyperresponsiveness to hista-

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FIGURE 7. Scanning electron micro-graph of the endothelial surface fromthe aortic arch of a group 1 rabbitshowing monocytes focally adhering tothe endothelium (panel A) and a highpower view of a focal collection ofmonocvtes (panel B).

mine was noted independent of the severity of thelesion. Ginsburg et all8 demonstrated that the phar-macological sensitivity of certain vasoconstrictor sub-stances differs in the ability to mediate contractionand is dependent on calcium pools. They showed thatcoronary arteries use both "intracellular" and "extra-cellular" calcium in response to various vasoconstric-tor agents and that the use of these calcium pools isspecific for each receptor pathway. Increases in cho-lesterol content of membranes in early atherosclero-sis may cause an increased permeability to calcium.37In contrast to histamine, contractile responses toserotonin in human coronary arteries are moredependent on extracellular calcium.'8 Therefore, ifcalcium permeability is reduced in later stages ofatherosclerosis, contractile responses to serotoninmay be decreased.

Relaxation responses. Numerous studies have con-firmed that vascular endothelium selectively modu-lates smooth muscle contraction indirectly by the

release of an endothelium-derived relaxing factor.6-8Endothelium-derived relaxing factor, now thought tobe nitric oxide,38 is suggested to have a mechanism ofaction on vascular smooth muscle similar to nitroglyc-erin. Both compounds activate soluble guanylatecyclase, which leads to increased levels of cyclic GMPand subsequent relaxation.3940 In addition to pro-moting vasodilation, the endothelium attenuates vaso-constrictor effects of several agents, such as platelets,serotonin, and norepinephrine.3441 It is speculatedthat impairment or loss of endothelial cell functionmay predispose atherosclerotic vessels to vasospasm.

Several recent studies indicate that cholesterolfeeding impairs endothelium-dependent relaxationin New Zealand White rabbits.12A4243 Verbeuren etal12 have shown that endothelium-dependent relax-ations to acetylcholine and ATP are impaired inrabbits as early as 8 weeks after diet-induced hyper-cholesterolemia (0.3% cholesterol). In addition,impairment of endothelium-dependent relaxation

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FIGURE 8. Scanning electron micro-graph of the endothelial surface fromthe aortic arch of a group 2 rabbitshowing numerous monocytes coveringthe endothelial cell surface (panel A)and a high power view of a monocytewith many cytoplasmic projections(panel B).

correlated with the degree of atherosclerotic plaque.Similarly, Jayakody et a142 have reported that feedingrabbits a diet high in cholesterol (2%) (cholesterollevels >2,000 mg/dl) for 4 weeks produced fattystreaks in the aorta and that these were associatedwith morphological alterations in endothelial cells,which correlated with a decrease in endothelium-dependent relaxation. Fatty streak lesions had swol-len endothelial cells with numerous cytoplasmic pro-

jections and early occasional irregularly shapedulcerations. Yokoyama et al,29 in a recent preliminaryreport, described progressive attenuation of endothe-lium-dependent relaxation in three age groups ofWHHL rabbits: less than 9 months, 10-22 months,and more than 23 months. However, a description ofendothelial cell morphology was lacking; thus, com-

parison with the present study was difficult.29

Impairment of endothelium-dependent relaxationin diet-induced hypercholesterolemia may be specificfor relaxations mediated by receptors. Bossaller eta144 have observed that other agents, in particular thecalcium ionophore A23187, which elicits responsesindependent of receptors, were effective in relaxingthe coronary artery and aorta obtained fromcholesterol-fed rabbits but that responses to acetyl-choline were significantly reduced. Similar findingshave been reported in atherosclerotic vesselsremoved from cholesterol-fed pigs, cynomolgus mon-keys, and humans with atherosclerotic coronaryarteries1617,3245 These data suggest that in athero-sclerotic vessels there are selective alterations ofmuscarinic receptors and that other features ofendothelium-dependent relaxation are preserved.Our studies of WHHL rabbits with hyperlipidemia

and/or mild atherosclerosis did not demonstrate

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Kolodgie et al Vascular Reactivity in WHHL Rabbits 1123

FIGURE 9. Scanning electron micro-graph of the arch of the aorta from agroup 3 Watanabe heritable hyperlipi-demic rabbit showing e-xtensive focaldisruption of the endothelium withplatelets and monocytes adhering inareas of endothelial cell loss. Arrowsindicate endothelial cells.

impairment of endothelium-dependent relaxation. Inpreparations of mild to moderate atherosclerotictissues with primarily intact endothelium (12+4%),relaxation responses to acetylcholine, ATP, and cal-cium ionophore A23187 were relatively maintained.However, in tissues with severe atherosclerosis(36±t4%), the loss of endothelial cells resulted insignificant attenuation of endothelium-dependentrelaxation in response to these agents. One mightargue that because blockers of prostaglandin synthe-sis were not present in our studies, maintenance ofendothelium-dependent relaxation in vessels withearly lesions may be the result of endogenous pros-tacyclin released from endothelial cells and wasindependent of EDRF mechanisms. However, otherstudies in normal and atherosclerotic vesselsremoved from cholesterol-fed rabbits have demon-strated a minimal role of prostacylin in promotingvascular relaxation elicited by acetylcholine.4246 In

addition, prostacyclin (prostaglandin I2) is sup-pressed in aortas from cholesterol-fed rabbits.47One possible explanation for the preservation of

endothelium-dependent relaxation in vessels withmild and moderate atherosclerosis is that WHHLrabbits are hyperlipidemic from birth. It is conceiv-able that endothelial cells from these animals, whichwere predominantly intact, may have adapted to ahigh cholesterol environment and, therefore, func-tion normally as opposed to animals with acutediet-induced hypercholesterolemia. In addition,endothelium-dependent responses to calcium iono-phore A23187 were similarly attenuated in vesselswith severe atherosclerosis in which there was asubstantial loss of endothelial cells. We believe thatdestruction of endothelial cells may be the dominantmechanism of impaired vasodilation in the WHHLrabbit model. However, endothelial cell dysfunctioncannot be excluded.

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FIGURE 10. Scanning electron micro-graph of the edge of a fibrous plaquefrom a group 3 Watanabe heritablehyperlipidemic rabbit showing V-shapedloss of endothelium (arrows) (panelA) and a high power view of the borderareas of a fibrous plaque (arrows)(panel B).

Impaired relaxation to acetylcholine in aortasfrom spontaneously hypertensive rats has beenshown, and this abnormality is associated with a

concomitant production of an endothelium-derivedconstricting factor.48 In the present study, in ath-erosclerotic and control vessels with and withoutendothelium, augmented vasoconstriction to acetyl-choline could not be demonstrated (data notshown). Therefore, a decreased endothelium-derived relaxing factor response to acetylcholinewas not related to concomitant production of an

endothelium-derived constricting factor by vesselswith severe atherosclerosis or enhanced activationof muscarinic receptors in smooth muscle cells ofatherosclerotic vessels.

In summary, vasoconstrictor responses to the a-

agonist phenylephrine progressively decreased withincreasing plaque formation, whereas responses tohistamine and serotonin were augmented by hyper-

lipidemia and/or atherosclerosis. Increased sensitiv-ity to histamine and serotonin was independent ofendothelial cell function in that removal of endothe-lial cells did not potentiate vasoconstriction. Thus,selective alteration(s) in smooth muscle cell functionin atherogenic vessels rather than loss of endothelialcells may precipitate hypersensitivity to histamineand serotonin in the WHHL rabbit model.Endothelium-mediated relaxation in the WHHL

rabbit is attenuated with atherosclerotic progres-sion. The mechanism of this defect in the WHHLrabbit model appears to be due to a significant lossof endothelial cells in plaque areas as opposed toalterations in endothelial cell function and/ormechanisms subsequent to the release of endothe-lium-derived relaxing factor. Loss of endothelialcells has been speculated to be of importance inatherosclerosis, and the findings of our study con-firm this hypothesis.

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FIGURE 11. Scanning electron micro-graph showing intimal surface from agroup 3 Watanabe hertable hyperlipi-demic rabbit with total destruction of theendothelium in an animal that hadundergone mechanical denudation(rubbed segment). Only subendothelialfibers are seen.

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KEY WORDS * atherosclerosis * endothelium-derivedrelaxing factor * endothelium * vascular reactivity * rabbits

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F D Kolodgie, R Virmani, H E Rice and W J Mergnerheritable hyperlipidemic rabbits.

Vascular reactivity during the progression of atherosclerotic plaque. A study in Watanabe

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1990 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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