Impaired Endothelium-Dependent Relaxation to Aggregating Platelets and Related Vasoactive Substances...

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Impaired Endothelium-Dependent Relaxationto Aggregating Platelets and Related Vasoactive

Substances in Porcine Coronary Arteriesin Hypercholesterolemia and Atherosclerosis

Hiroaki Shimokawa and Paul M. Vanhoutte

Vasoconstrictor responses are augmented in porcine coronary arteries in hypercholesterolemiaand atherosclerosis, leading to an occurrence of coronary vasospasm in the latter condition. Therole of the endothelium in the vascular hyperreactivity in hypercholesterolemic and atheroscle-rotic coronary arteries was examined, particularly in response to aggregating platelets and relatedvasoactive substances. Male Yorkshire pigs underwent balloon endothelial denudation of the leftanterior descending coronary artery (LAD) and 2% high-cholesterol feeding for 10 weeks.Electron microscopic examination demonstrated a full lining of endothelial cells in the LAD andthe left circumflex coronary artery (LCX). Endothelium-dependent responses were examined invitro. In cholesterol-fed animals, endothelium-dependent relaxations to aggregating platelets,serotonin, ADP, bradykinin, thrombin, and the calcium ionophore A23187 were depressed inLAD (atherosclerosis), while the relaxations to aggregating platelets, serotonin and ADP weredepressed in LCX (hypercholesterolemia). Serotonin-induced contractions were endothelium-dependently augmented in atherosclerotic LAD; tbe endothelium-dependent component of thecontractions was inhibited by blockers of cydooxygenase. Bioassay studies demonstrated adepressed rdease of endothelium-derived relaxing factors) from the atherosclerotic LAD hiresponse to serotonin. These experiments indicate that the endothelium-dependent relaxations toaggregating platelets and related vasoactive substances are severely impaired in atherosclerosisand moderately impaired hi hypercholesterolemia. Since coronary atherosderosis was induced bya combination of balloon endothelial injury (and regeneration) and high-cholesterol feeding in thisstudy, the combined effects of those factors must account for the severely impaired responses inatherosclerosis. The depressed release of the endothelium-derived relaxing factoids) and theconcomitant release of vasoconstrictor product(s) of cydooxygenase appear to be responsible forthe unpaired relaxations. (Circulation Research 1989;64:900-914)

The endothelial cells play an important role inmodulating the responsiveness of underlyingvascular smooth muscle. '~3 Among the pos-

sible pathophysiological roles of the endothelium,its protective action against platelet aggregationappears to be important.4-7 Impaired endothelium-dependent relaxations in atherosclerosis have been

From the Department of Physiology and Biophysics, MayoClinic and Mayo Foundation, Rochester, Minnesota.

Presented in part at the 37th Annual Scientific Session of theAmerican College of Cardiology, Atlanta, Georgia. Appearedpreviously in abstract form (J Am Coll Cardiol 1988;! 1:2A).

Supported in part by National Institutes of Health Grants HL-31183 and HL-31547 by a Grant-in-Aid Research Award ofAmerican Heart Association Minnesota Affiliate.

Address for correspondence: Paul M. Vanhoutte, MD, Depart-ment of Physiology and Biophysics, Mayo Clinic and MayoFoundation, Rochester, MN 55905.

Received February 22, 1988; accepted October 14, 1988.

reported in the rabbit aorta,8-10 monkey iliac artery,''pig coronary artery,12 and human coronary artery invitro9 and in vivo.13 In contrast, no information isavailable concerning the endothelium-dependentrelaxations to aggregating platelets in atherosclero-sis. Augmented vasoconstrictor responses havebeen reported in hypercholesterolemia14 andatherosclerosis.15-19 In particular, in a pig model ofcoronary atherosclerosis induced by a combinationof balloon endothelial injury and high-cholesterolfeeding, coronary vasospasm with myocardial isch-emia can be provoked in vivo.151619 Aggregatingplatelets are an important source of vasoconstrictorsubstances and have been regarded as one of themost possible causes for coronary vasospasm.6 Thepresent experiments were designed to examinewhether or not the protective role of the endothe-lium against aggregating platelets is impaired in

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Shimokawa and Vanhoutte Endothelium in Atherosclerosis 901

hypercholesterolemic and atherosclerotic porcinecoronary arteries. When it appeared that this wasthe case, we attempted to determine the mecha-nism^) involved.

Materials and MethodsFifty-four male Yorkshire pigs, 6-8 weeks of age

(18.2±0.6 kg), were randomly divided into twogroups. The control group (n=23) was fed a regularchow (Hog Finisher, Bedtke Brothers Feed andSeed Co, Dover, Minnesota) with 0.09% choles-terol; the cholesterol group («=31) was fed a dietwith 19% lard and 2% cholesterol (TD86019, Tek-lad, Madison, Wisconsin). After 2 weeks of feeding,pigs in the cholesterol group underwent coronaryendothelial denudation of the left anterior descend-ing coronary artery (LAD) to allow the rapid devel-opment of atherosclerosis.151618-20 Animals in thecontrol group underwent coronary angiogram only.After this, they were fed for 8 more weeks. In thismodel, the effects of atherosclerosis in the LAD andhypercholesterolemia in the left circumflex coro-nary artery (LCX) can be evaluated in the sameanimals in the cholesterol group by comparing thedata with those from the control group.151619 Theserum concentrations of lipids (enzymatic method),21

heart rate, and blood pressure (Mayo Medical Engi-neering, Rochester, Minnesota) were determinedbefore and 10 weeks after the feeding. The pigswere housed individually in temperature-controlledanimal quarters. To prevent excessive weight gain,the daily food intake was limited to an amount equalto 3% of the body weight per day.16-22 In vitroexperiments were performed after 10 weeks offeeding, using 42 pigs (n=18 in the control groupand «=24 in the cholesterol group). Two pigs fromthe cholesterol group, in which small apical myo-cardial infarction had occurred, were excluded fromthe study in order to avoid any possible influence ofthe infarction on vascular reactivity. The other 10pigs (n=5 in each group) were used for scanning andtransmission electron microscopic examinations.

Coronary Endothelial DenudationThe details were reported previously.22 The pigs

were anesthetized with 300 mg i.m. ketamine hydro-chloride (Bristol Laboratories, Syracuse, NewYork), followed by 12.5 mg/kg i.v. sodium pento-barbital (Fort Dodge Laboratories, Inc, Fort Dodge,Iowa). Using aseptic surgical techniques, the rightcarotid artery was dissected free and a speciallydesigned coronary guide catheter was inserted downto the left coronary ostium under fluoroscopic guid-ance. The electrocardiogram (lead II) and the arte-rial pressure were monitored continuously. After 20mg i.v. lidocaine HC1 (Astra, Santa Clara, Califor-nia) was administered, a 3-mm balloon dilationcatheter (USCI, Glenn Falls, New York) 20 mm inlength was advanced through the guide catheter tothe LAD and gently rubbed against the first 4 cm ofthe proximal portion of that artery. The accuracy of

this technique for coronary endothelial denudationhas been demonstrated previously.22 Before andimmediately after the denudation, left coronary angio-grams were obtained during injection of 5 ml meglu-mine diatrizoate (Renographin 76, Squibb, Prince-ton, New Jersey) into the left main coronary artery,at end diastole with an R-wave-triggered x-rayswitch. Images were recorded on x-ray film using acassette-type film holder. To quantitate the epicar-dial coronary arterial response, angiograms wereanalyzed for coronary dimensions along the proxi-mal 4 cm of the artery at every 0.1 mm, using acomputer-based quantitative angiographicsystem.22-23

Organ Chamber ExperimentsThe pigs were anesthetized with 300 mg i.m.

ketamine hydrochloride and 12.5 mg/kg i.v. sodiumpentobarbital. After collecting antologous blood (300ml) from the left carotid artery, they were exsan-guinated and the heart was removed. The coronaryarteries were removed and immersed in cold mod-ified Krebs-Ringer bicarbonate solution of the fol-lowing millimolar composition: NaCl 118.3, KG4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, NaHCO325.0, CaEDTA 0.016, and glucose 11.1 (controlsolution). The experiments were performed on 3- to4-mm long rings of proximal LAD and LCX. Sinceendothelial denudation was performed in the first 4cm of the proximal portion of the LAD from the leftcoronary orifice, this portion of the artery was usedfor organ chamber experiments. The comparableportion of the LCX was used as control. The ringswere numbered from proximal to distal, and ringswith the same number taken from the LAD andLCX were studied in parallel. The rings were cleanedof loose connective tissue, with special care takennot to touch the luminal surface. In some of therings, the endothelium was removed deliberately byrubbing the luminal surface gently with a cottonswab wetted with control solution.22

The rings were mounted horizontally in organchambers filled with 25 ml of control solution (37° C,pH 7.4) gassed with 95% O r 5% CO2. The prepara-tions were attached to a strain gauge (model UC2,Gould Statham, Oxnard, California), and isometrictension was recorded. The rings were progressivelystretched until the contractile response evoked by20 mM potassium chloride was maximal (optimaltension).22 They were allowed to equilibrate for 30minutes before the experiments.

ProtocolAfter 30 minutes of equilibration, bradykinin (con-

centration response curve [10"'° to 10 M] or onedose [10~7 M]) was given to all rings during acontraction evoked by prostaglandin F2,, (2x 10"6 M)to confirm the presence or absence of functionalendothelial cells. After this, relaxations were exam-ined during a contraction caused by prostaglandin



902 Circulation Research Vol 64, No 5, May 1989

F ^ (2x 10 6 M) and contractions were examined inquiescent rings in the following order:

Set A (relaxation): 1) serotonin (l(T9-3xl(r6 M), 2)platelets (70,000/̂ ,1), and 3) thrombin (0.1 units/ml)

Set B (relaxation): 1) adenosine diphosphate (ADP)(10-8-10-4 M), 2) sodium nitropmsside (lO-'-lO"6 M),and 3) the calcium ionophore A23187 (10"9-10"* M)

Set C (contraction): 1) serotonin (1O~9-1O"3 M), 2)platelets (70,000/̂ 1), and 3) KC1 (5-100 mM)

In organ chamber experiments with isolated ringsof blood vessels, unlike under physiological condi-tions, or when examining perfused segments of arter-ies,24 the administered drugs can reach directly thevascular smooth muscle from the adventitial sides andthe cut surfaces. It is appropriate to inhibit the directeflFects of the drugs on the vascular smooth muscle toexamine their effects on the endothelium. Therefore,when determining relaxations to aggregating plateletsand serotonin, the rings first were incubated with10"* M ketanserin for 40 minutes. Previous experi-ments demonstrated that the S2-serotonergic antag-onist unmasks the endothelium-dependent responsesto serotonin22-25 and platelets22 by inhibiting theS2-serotonergic activation of vascular smooth mus-cle cells. Likewise, the rings were treated with theP,-purinergic blocker theophylline (10~* M) whenstudying relaxations to ADP since previous obser-vations demonstrated that this drug inhibits therelaxing effect of the adenine nucleotide on smoothmuscle cells without affecting the endothelium-dependent component of the action of ADP.22

In most experiments of endothelium-dependentrelaxations, all rings were treated with 10~5 Mindomethacin for 40 minutes before inducing con-traction with 2x 10"6 M prostaglandin F^ to preventthe synthesis of endogenous prostanoids. To exam-ine the eflFects of indomethacin on the relaxations,rings were treated with either 10~5 M indomethacinor 10"5 M Na2CO3 (solvent of indomethacin) onlyand were examined in parallel.

Since endothelium-dependent contractions werenoted in response to serotonin in the atheroscleroticLAD from cholesterol-fed pigs, the eflFects of block-ers of cylooxygenase (indomethacin and meclophen-amate), thromboxane synthetase (dazoxiben), throm-boxane receptor (SQ 29548), and lipoxygenase(nordihydroguaiaretic acid and BAY G6575) onthose contractions were examined. In addition, insome cases 2.0-ml samples of fluid were withdrawnfrom the chambers before and 5 minutes after thecompletion of cumulative concentrations of seroto-nin (10~9-10~3 M) and frozen for later analysis. Theconcentrations of prostaglandins in the bath solutionwere determined by radioimmunoassay (AdvancedMagnetics Inc, Cambridge, Massachusetts)26; thedetection level of prostaglandins in the present studywas 20 pg/ml for 6-keto-prostaglandin F,a and throm-boxane B2 and 5 pg/ml for prostaglandin Fz,, andprostaglandin F^. Cross-reactivity of the antisera for6-keto-prostaglandin F,o to prostaglandin F^ was

2.2%, that for prostaglandin Ej to prostaglandin F^was 1.3%, and those the rest of the combinations ofthe four prostaglandins were less than 1%.

BioassayThe bioassay method used has been described in

detail previously.27 After removal from the hearts,side branches of 4-cm segments of the LAD weretied. The segments were treated gently to minimizedamage to the endothelium. The segments were tiedto stainless steel canulas and placed into an organchamber maintained at 37° C and filled with 12 mlaerated (95% O2-5% COJ control solution. Thesegments were perfused at constant flow (2 ml/min)by means of a multichannel roller pump (Minipuls 2,Gilson Medical Electronics, Inc, Middleton, Wis-consin) with control solution maintained at 37° C. Astainless steel tube was also placed in the organchamber, through which control solution waspumped at the same rate. A ring of the LCX fromthe same animal, from which the endothelium hadbeen removed by rubbing the intimal surface (bio-assay ring), was suspended directly below the organchamber by means of two stainless steel stirrupspassed through its lumen. One stirrup was fixed andthe other connected to an isometric force trans-ducer (model FT03C, Grass Instrument Co, Quincy,Massachusetts), and changes in isometric tensionwere recorded. The assembly of bioassay ring,stirrups, and force transducer could be moved freelybelow the organ chamber, allowing the preparationto be superfused with the perfusate either from thecoronary segment with endothelium (endothelialsuperfusion, endothelial line) or from the stainlesssteel tube (direct superfusion, direct line); there wasno difference in the wet weight of the bioassay ringbetween the two groups (14±1 mg in both).

The bioassay ring was first superfused (for 60minutes) with control solution passing through thestainless steel canula. During this interval it wasstretched in a stepwise manner with repetitive expo-sure to 20 mM KC1 until the basal tension reachedapproximately 8-9 g, the optimal tension for activecontraction of rings of isolated porcine coronaryarteries, as determined in preliminary organ cham-ber studies. The bioassay ring was also treated with10~6 M ketanserin throughout the experiments byadding the antagonist directly to the ring using aseparate infusion line located below the donor seg-ment. To detect the basal release of endothelium-derived relaxing factor, the bioassay ring was con-tracted with prostaglandin F^ (2xlO~6 M) underdirect superfusion and then positioned beneath theendothelial line (containing 2x 10~6 M prostaglandinFjJ. Serotonin was chosen as the agonist in thebioassay experiment for the detection not only ofendothelium-derived relaxing factor(s) but also ofpossible release of indomethacin-sensitive,endothelium-derived contracting factoids) as sug-gested by the organ chamber experiments. Serotonin(3x 10~6 M) was infused by means of infusion pumps



Shimokawa and Vanhoutte Endothetium in Atherosclerosis 903

(model 901, Harvard Apparatus, South Natick, Mas-sachusetts) into the perfusate in either the absence orthe presence of 10~5 M indomethacin. Preliminaryexperiments demonstrated that this concentration(3x 10~6 M) of the monoamine causes maximal relax-ation of the bioassay preparations.

Drugs and PlateletsThe following drugs were used: ADP, bovine throm-

bin, bradykinin, calcium ionophore A23187, indometh-acin, nordihydroguaiaretic acid (NDGA), potassiumchloride, prostaglandin F^, 5-hydroxytryptaminecreatinine sulfate (serotonin), sodium nitroprusside,theophylline (all from Sigma Chemical Co, St. Louis,Missouri), dazoxiben HC1 (Pfizer, Groton, Connect-icut); ketanserin tartrate (Janssen Pharmaceutica,Beerse, Belgium), sodium meclofenamate (ParkeDavis, Detroit, Michigan), nafazatrom (Bay G6575,Miles Pharmaceutical, West Haven, Connecticut),and {lS-[la, 2/3 (5Z), 30, 4a]-7-[3-[[2-(phenylamino)carbonyl] hydrazino] methyl]-7 oxabicyclo [2.2.1]hept-2-yl}-5-heptenoic acid (SQ 29548, Squibb andSons, Princeton, New Jersey). Unless otherwisespecified, drugs were prepared daily in distilledwater, kept on ice, and added to the organ baths involumes less than 250 /ul. Indomethacin was dis-solved in equal molar concentrations of 10"5 MNa2CO3 and Bay G6575 and NDGA in dimethylsul-foxide (1%). SQ 29548 was dissolved in ethanol(final bath concentration, 4x 10~4 M) and diluted in2 mM Na2CO3 (final concentration, 4.6x 10"7 M) andthen in distilled water.7 Inhibitors were added to thebath 40 minutes before experiments; the only excep-tion was NDGA, which was present in the bath for20 minutes and then washed in order to avoid itsdirect inhibitory effects on vascular smooth muscle.28

In contrast, its inhibitory effects on lipoxygenasepersist for at least 2 hours after washing.28 Drugconcentrations are reported as the final molar con-centration in the bath solution.

A platelet-rich solution was prepared from autol-ogous blood by centrifugation as described previ-ously.22 Briefly, the autologous blood with citrateanticoagulant was centrifuged for 40 minutes at 500rpm and the platelet-rich plasma was pipetted off.An equal volume of citrate anticoagulant solutionwas added to the platelet-rich plasma, and themixture was centrifuged for 20 minutes at 1,600 rpm.The supernatant was discarded, and the remainingplatelet pellet was resuspended in a small volume ofthe citrate anticoagulant mixture. A platelet countof this suspension was then obtained (Coulter Elec-tronics, Inc, Hialeah, Florida). The platelet suspen-sion was kept at room temperature. Samples of fluid(1.5 ml) were withdrawn from the chambers 7minutes after the addition of platelets, divided forthe determination of the concentrations of throm-boxane B2 and serotonin, and frozen until analysis.22

The concentrations of serotonin and thromboxane B2in the bath solution after the addition of plateletswere determined by reverse-phase high pressure

liquid chromatography29 and by radioimmunoassay,26

respectively.22

MorphologyThe hearts were divided into five horizontal blocks

and examined macroscopically for the presence orabsence of myocardial infarction.

The rings used in the organ chamber study wereexamined histologically by hematoxylin-eosin stain-ing for determination of endothelial lining and gen-eral observation, by van Gieson's elastic stainingfor determination of the thickness of the intima andthe media,22 and by Sudan IV staining for confirma-tion of lipid deposition in the blood vessel wall.Morphometric determination was performed with acomputer-assisted image analyzer (IBAS 2000, Kon-tron Electronics, Munich, West Germany) to eval-uate cross-sectional area of the intima and themedia.30

Scanning and transmission electron microscopicexaminations were performed in five pigs in eachgroup. The hearts of these pigs were fixed in situwith 2% glutaraldehyde and 1% paraformaldehydein 0.1 M cacodylate (pH 7.25) (fixation solution) ata perfusion pressure of 110 mm Hg for 15 minutes.The coronary arteries were then removed and keptin the fixation solution until analysis. Longitudinalspecimens were cut from each coronary artery,coated with carbon and gold-palladium alloy, andexamined with a scanning electron microscope(ETEC Autoscan, Hayward, California).22 To avoidpossible anatomical differences in numbers of endo-thelial cells, 20 scanning electron micrographs, (mag-nification, x 1,000) were taken randomly from eachcoronary specimen (Figure 1), and numbers of thecells were counted without knowledge and correctedfor per square millimeter.22 Selected specimens werethin-sectioned (600-700 A), mounted on a 200-meshcopper grid, and stained with uranyl acetate and leadcitrate. Sections were examined with a Philips 201transmission electron microscope (Philips ElectronicInstruments, Inc, Mahwah, New Jersey).22

Calculations and Statistical AnalysisResults are expressed as means±SEM. Unless

otherwise specified, n refers to the number ofanimals. In rings contracted with prostaglandin F^,dilator responses are expressed as percent changesfrom the contracted levels, and in quiescent ringsconstrictor responses are expressed as percent ofthe maximal response to KCI (100 mM). For relax-ations, the negative logarithm of the effective con-centration of agonist causing 50% inhibition [IC*,]of the contractions to prostaglandin F^ was calcu-lated for each concentration-response curve and themeans of these values are presented. For contrac-tions evoked by serotonin, the effective concentra-tion producing 40% of the maximal response to KCI(ED^ was calculated. The correlations between thecross-sectional area of the intima or the media andendothelium-dependent relaxations were examined




A: B:

Control ( LCX ) Control ( LAD )

FIGURE 1. Scanning electron micro-graphs of the endothelium of proximalcoronary artery of the pig in control (A,B)and the cholesterol (C,D) groups (originalmagnification, x 1,000). LCX, left circum-flex coronary artery; LAD, left anteriordescending artery. Direction of flow is fromtop to bottom. In control group, in both leftcircumflex (A) and left anterior descendingcoronary arteries (B), endothelial cells wereflat and arranged parallel to direction ofblood flow. In cholesterol group, endothe-lial cells were elongated and the number ofthe cells had increased in both left circum-flex (C) and left anterior descending coro-nary arteries (D). In the latter, size andshape of the nuclei were irregular, and inseveral regions adhesion of platelets andother blood cells were noted.

Cholesterol ( LCX ) Cholesterol ( LAD )

by linear regression analysis. Statistical evaluationof the data was performed by Student's / test foreither paired or unpaired observations. When morethan two means were compared, a two-way analysisof variance was used. If a significant value wasfound, Scheffe's test for multiple comparisons wasused to identify differences among groups. Differ-ences were considered to be significant at p<0.05.

ResultsBaseline Data

Body weight increased significantly in both groups(48.6±1.3 kg in the control and 50.3±1.2 kg in thecholesterol group) after 10 weeks of feeding. Heartrate and blood pressure were unchanged in bothgroups during the experimental period (data notshown). The serum concentration of cholesterolsignificantly increased in the cholesterol (604±40mg/dl) but not in the control group (105±4 mg/dl);mainly the fractions of low density and very lowdensity lipoproteins increased (data not shown). Incontrast, the concentration of triglyceride wasunchanged in both groups (36±4 mg/dl in the con-trol and 48±5 mg/dl in the cholesterol group).

Changes During Denudation ProcedureImmediately after the denudation, the mean diam-

eter of the denuded LAD was significantly reduced inthe cholesterol group (3.10±0.07 mm before and2.68±0.08 mm after denudation, n=29). In contrast,the mean diameter of the control, nondenuded LCXwas unchanged (3.05±0.07 mm before and 3.08±0.08after denudation, n=29). Heart rate and arterial bloodpressure were not significantly different before andafter the denudation (data not shown). The animals inthe control group (n=23) had the same size coronaryarteries as the cholesterol group (3.07±0.08 mm in theLADs and 3.02±0.06 mm in the LCXs).

MorphologyMacroscopically visible small regions of myocar-

dial infarction, which were limited to the apex, werepresent in two of the 31 hearts in the cholesterolgroup and in none of the 23 hearts in the controlgroup.

Under light microscopy, in all rings used for organchamber experiments, the presence or absence of theendothelium was confirmed histologjcally. In the con-trol group, no intimal thickening was noted and there



Shimokawa and Vanhoutte Endothetimn in Atherosclerosis 905

TABLE 1. Data FromCoronary Arteries

Intima (mm2)

Media (mm2)

Light Microscopic Examination of Cross-Sectional

LCX

0.03±0.011.21+0.06

Control

(40)(40)

group

0.1

i

LAD

.02+0.01

.20+0.06(40)

(40)

Area of the

LCX

0.04+0.011.28+0.05

Intimn and Media of Porcine

Cholesterol group

(47)(47)

LAD

0.48+0.05*1.86+0.08*

(62)(62)

Data are expressed as means±SEM.LCX, left circumflex coronary artery; LAD, left anterior descending coronary artery. Numbers in parentheses are

the numbers of rings with endothelium tested in each group.*p<0.05 compared with LAD in the control group or p><0.05 with LCX in the cholesterol group.

was no difference in cross-sectional area of the mediabetween the LAD and the LCX (Table 1). In contrast,in the cholesterol group eccentric myointimal thick-ening with lipid deposition (atheroma) had occurredalong the denuded LAD with concomitant medialthickening, whereas no intimal or medial thickeningwas noted in the LCX (Table 1). The endothelium

covered the inner surface of the blood vessel includ-ing the atheromatous plaque of the LADs, in whichno thrombus or hemorrhage was observed.1516

Scanning electron microscopy showed that in thecontrol group (n=5) the endothelial cells were flatand arranged parallel to the direction of blood flowin both the LCXs and the LADs (Figures 1A and

IEL

SMC

FIGURE 2. Transmission electron micrographs of the endothelium of the proximal coronary artery of the pig in thecontrol (A,B) and the cholesterol (C,D) groups (original magnification, x2,500). E, endothelial cells; IEL, internal elasticlamina; SMC, smooth muscle cells. In control group, endothelial cells were flat with well-formed junctional complexesin both left circumflex (A) and left anterior descending coronary arteries (B). In contrast, in left circumflex coronaryarteries of the cholesterol group (C), endothelial cells had become rounded or cuboidal with lipid deposition. Note theabsence of intimal thickening in this region. In left anterior descending coronary arteries of the cholesterol group (D),in addition to the same appearance of endothelial cells as in (C), some of them are under degeneration, suggesting theprocess of cell death. Multiple layers of modified smooth muscle cells with lipid deposition and interstitial tissue formedunderlying intimal thickening (atheroma).




TABLE 2. Characteristics of Porcine Coronary Smooth Muscle From Organ Chamber Experiments

Optimal tension (g)Developed tension to

2xlO-6M PGFi,(g)Relaxation to sodium

nitroprusside (n=6)IQo (-log M)Max. relaxation (%)

Contraction to KC1 (fl=6)EDJO (mM)

Max. contraction (g)

Control

LCX

8.6+0.2 (86)

5.9±0.3 (86)

7.67±0.09122±3

9.72+0.759.6±0.6

group

LAD

8.8±0.2 (86)

6.1+0.3(86)

7.70±0.10123+5

9.56±0.759.1 ±0.5

Cholesterol

LCX

8.8±0.2 (80)

6.7+0.5 (80)

7.76+0.09119±5

9.74±0.7710.2±1.7

group

LAD

8.7+0.2 (154)

6.3±0.4(154)

7.73±0.lI13±4

10.42±0.479.9±1.2

Data are expressed as means±SEM.LCX, left circumflex coronary artery; LAD, left anterior descending coronary artery, PGF^, prostaglandin F^,;

ICjo, effective concentration causing 50% inhibition of the contractions to prostaglandin F^ (2x10"* M); Max.relaxation, maximal relaxation in percent of the response to prostaglandin F^ (2x10"' M); EDM, effectiveconcentration producing 50% of the maximal response to KG; Max. contraction, maximal contraction to KG.

Numbers in parenthesis are the number of rings tested in each group. Since there was no difference in optimaltension or developed tension to PGF^ between rings with and without endothclium in both control and cholesterolgroups, the combined data are presented. For the relaxation to sodium nitroprusside or contraction to KG, the datawere obtained in rings without endothclium.

IB). In the cholesterol group (n=5), the endothelialcells were elongated and irregularly oriented in theLCXs (Figure 1C), and these tendencies were morepronounced in the LADs (Figure ID). The numberof the endothelial cells (xiOVmm2) in the controlgroup was 4.2 (±0.5) in the LCXs and 4.1 (±0.4) inthe LADs. In the cholesterol group, the number had

increased significantly in both the LCXs (6.9±0.4)and the LADs (8.0±0.4).

Transmission electron micrography showed thatin the control group endothelial cells were flat withwell-formed junctional complexes in both LCXsand LADs (Figure 2A and B). In contrast, in thecholesterol group, endothelial cells were rounded to

TABLE 3. Responses to Various Agonists in EndotheUmn-Dependent Relaxations of Porcine Coronary Arteries

Agonist

BradykininLCXLAD

SerotoninLCXLAD

ADPLCXLAD

A23I87LCXLAD

Thrombin (0.1 units/ml)LCXLAD

1C

Controlgroup

8.78±0.158.85±0.14

7.14±0.257.38±0.30

6.12±0.066.15+0.08

7.78±0.067.75±0.02

» (-log M)

Cholesterolgroup

8.58±0.07

8.15±0.12*t

5.85±0.14*

5.08±0.28*t

7.74+0.077.51±0.08*t

Max.

Controlgroup

IIO.8±2.4II6.3±3.6

76.3±5.677.8±6.6

104.9±2.51O8.3±3.3

I2O.9±4.O121.4±3.2

105.6±3.7106.9±2.5

relaxation (%)

Cholesterolgroup

I17.9±2.5104.1±6.7

55.3± 10.9*24.7±8.9*t

99.0+4.067.6+5.8*t

121.6±4.9116.7±3.3

98.5±4.445.7±12.0*t

Data are expressed as means±SEM.Each group consists of six experiments on rings with endothelium except for bradykinin (/i=12, control group;

n=14, cholesterol group). All rings were treated with indomethacin (10~3 M). In the experiments with serotonin andADP, rings were treated with ketanserin (10~6 M) and theophylline (10~4 M), respectively, to inhibit the direct effectson vascular smooth muscle.

IC50, effective concentration causing 50% inhibition of the contractions to prostaglandin F^, (2xl0~* M); Max.relaxation, maximal relaxation in percent of the response to prostaglandin F*, (2X10""6 M); ADP, adenosinediphosphate; A23187, Ca2+-ionophore; LCX, left circumflex coronary artery; LAD, left anterior descending coronaryartery. ICW was not calculated for serotonin in the cholesterol group because the response did not attain the ICm level.

•p<0.05 compared with control.tp<0.05 compared with LCX.




LCX LAD

6 0 8

Serotonin, -tog M

FIGURE 3. Cumulative concentration-response curves to serotonin during a con-traction evoked by prostaglandin F2a Q

x lO'6

M) in presence of IO~6 M ketanserin and 10~!

M indomethacin. Relaxations are expressedas percent decrease in tension of contractionevoked by prostaglandin F2a. Data shown asmeans±SEM. Left: Relaxations in the leftcircumflex coronary artery (LCX) in the con-trol group with (O) and without (•) endothe-lium and in the cholesterol group with (D) andwithout (•) endothelium. Right: Relaxationsin the left anterior descending coronary artery(LAD) in the two groups.

cuboidal, containing lipid granules in their bodies inboth coronary arteries (Figure 2C and D). Nointimal thickening was noted in the LCXs in thecholesterol-fed group (Figure 2C), while in theLADs in the same group multiple layers of trans-formed cells with lipid deposition, rounded smoothmuscle cells, and interstitial tissue formed the ather-omatous plaque (Figure 2D).

Organ Chamber ExperimentsThere were no significant differences in optimal

tension or contractions evoked in the smooth mus-cle by prostaglandin F^ (2xl(T6 M) between theLADs and LCXs in both groups (Table 2). Sodiumnitroprusside (10~9-10~6 M) caused comparableconcentration-dependent relaxations in rings with-out endothelium taken from the LCXs and LADs inboth groups. Similarly, KC1 (5-100 mM) causedcomparable concentration-dependent contractionsin rings without endothelium from the LCXs andLADs in both groups (Table 2).

In the first series of endothelium-dependent relax-ations, the responses to various agonists were exam-ined in the presence of 10"3 M indomethacin. Asshown in the Table 3 and Figures 3 and 4, in thecontrol group, there was no difference in theendothelium-dependent relaxations to bradykinin,serotonin, ADP, the calcium ionophore A23187, andthrombin between the LCXs and LADs. In contrast,

in the cholesterol-fed group, all of these relaxationswere impaired in the LAD, while relaxations toserotonin and ADP were depressed in the LCX,compared with those obtained in the control group.

Aggregating platelets (70,000/̂ .1) caused compara-ble, endothelium-dependent relaxations in the LCXsand LADs in the control group (Figure 5). In thecholesterol group these relaxations were depressed inthe LCXs and impaired further in the LADs (Figure5). The amount of serotonin released from aggregatingplatelets was not significantly different in the twogroups (217±18 ng/ml in the control and 186±21 ng/ml in the cholesterol group). Similarly, there was nostatistically significant difference in thromboxane B2levels between the cholesterol group (179±32 pg/ml)and the control group (356±84 pg/rnl).

In quiescent rings of control LCXs or LADs,aggregating platelets (70,000//xl) caused comparable,large contractions in the absence but comparable,small contractions in the presence of endothelium(Figure 6). In the cholesterol group, platelets causedsignificantly greater contractions in rings of bothLCXs and LADs compared with blood vessels fromthe control group (Figure 6). Especially in rings withendothelium of the LAD, the induced contractionswere comparable to those in rings without endothe-lium (Figure 6). In the cholesterol group, rings with-out endothelium showed contractions comparable tothose in the control group (Figure 6).

LCX LAO

6 S 4 8 7 8

Adenostna dJphosphate, -log M

FIGURE 4. Cumulative concentration-response curves to adenosine diphosphate(ADP) during a contraction evoked by pros-taglandin F^ (2 x W* M) in presence oflOr4 Mtheophylline and 1O~S M indomethacin. Relax-ations are expressed as percent decrease intension of contraction evoked by prostaglandinFfr. Data shown as means±SEM. Left: Relax-ations in the left circumflex coronary arteryLCX in the control group with (O) and without(•) endothelium and in the cholesterol groupwith (Q) and without (•) endothelium. Right:Relaxations in the left anterior descending cor-onary (LAD) artery in the two groups.




Contraction, %20

20

40

Relaxation, % 60

80-

100-

120

Platelets ( 70.000/jd )

LCX LADn-6

• control0 cholesterol

FIGURE 5. Relaxations to aggregating plate-lets (70,000l\il) during a contraction evoked byprostaglandin F2a (2x/0~6 M) in control andcholesterol groups with (+) and without (—)endothelium. Left: Relaxations in the left cir-cumflex coronary artery (LCX). Right: Relax-ations in the left anterior descending coronaryartery (LAD). The responses are expressed aspercent change in tension from the contractionlevel evoked by prostaglandin F2a. Data shownas means±SEM. *Significant difference(p<0.05) compared with control; t, significantdifference (p<0.05) compared with LCX.

In quiescent control rings of both LCXs andLADs, serotonin (10~9-10~5 M) caused comparablecontractions, which were significantly smaller inpreparations with endothelium than in those with-out endothelium (Figure 7). In the cholesterol group,the contractions in rings without endothelium inboth arteries were comparable to those in the con-trol group. However, serotonin caused significantlylarger contractions in rings with endothelium ofboth LADs and LCXs than in those of the normalgroup (Figure 7). At higher concentrations, thesecontractions were larger in rings with endotheliumof LAD than those obtained in acutely denudedrings of the same artery (Figure 7). These aug-mented contractions were inhibited by 10~5 M indo-methacin or 10~6 M meclofenamate but not by 10~4

M dazoxiben (a thromboxane synthetase blocker),10~6 M SQ 29548 (a thromboxane receptor blocker),2x 10"3 M NDGA, or 1(T5 M BAY G 6575 (bothlipoxygenase blockers) (Table 4). The contractileproperties of the smooth muscle (rings withoutendothelium) were unchanged by these blockers(Table 4). In the cholesterol group, a decreasedrelease of prostacyclin in response to 10~5 M sero-tonin (from both the endothelium and the smoothmuscle) was noted, while the amount of prostaglan-din F^ or prostaglandin E2 released was not statis-tically different between the two groups (Table 5).The levels of 6-keto-prostaglandin Fla measured byradioimmunoassay may be slightly different fromthe values that would have been obtained by gaschromatography/mass spectrometry.31 However, thisdifference does not affect the direction of thereported change or its level of significance. Therelease of thromboxane B2 could not be detected. Incontrast, in the LCXs no endothelium-dependentcontractions were observed even though the con-tractions to the monoamine in rings with endothe-lium also were significantly augmented compared tothe control group (Figure 7).

Since indomethacin-sensitive, endothelium-dependent contractions were observed in responseto serotonin in the atherosclerotic LAD of thecholesterol group, endothelium-dependent relax-ations to serotonin and ADP were reexamined in

both groups in either the absence or the presence ofindomethacin (10~5 M). The relaxations were signif-icantly larger in the atherosclerotic LADs in thepresence than in the absence of indomethacin,while they were comparable in both arteries in thecontrol group or in the LCX in the cholesterol group(Figures 8 and 9). In contrast, the relaxations tosodium nitroprusside in rings without endotheliumwere comparable in the absence or presence ofindomethacin in both groups (data not shown).

Bioassay ExperimentsThe basal release of endothelium-derived relax-

ing factor(s), which could be evaluated by measur-ing the degree of relaxations that occurred whenendothelial superfusion was initiated, was signifi-cantly less in the cholesterol group than in thecontrol group (Figure 10). Stimulated release of

o

O control0 cholesterol

FIGURE 6. Contractions evoked by aggregating plate-lets (70,0001'(il) in quiescent rings from control and cho-lesterol groups, with (+) and without (—) endothelium.Left: Contractions in the left circumflex coronary artery(LCX). Right: Contractions in the left anterior descend-ing coronary artery (LAD). Responses are expressed aspercent increase in tension from resting level comparedwith maximal contractions to KCI (100 mM). ^Significantdifference (p<0.05) compared with control; ^significantdifference (p<0.05) compared with LCX.




LCX LAD

-20. 6 5 9 8Serotonin, - log M

FIGURE 7. Cumulative concentration-response curves to serotonin in quiescentrings in control and cholesterol groups. Left:Contractions in the left circumflex coronaryartery (LCX) in the central group with (O)and without (•) endothelium and in the cho-lesterol group with (n) and without (•) endo-thelium. Right: Contractions in the left ante-rior descending coronary artery (LAD) in thetwo groups. Contractions are expressed aspercent increase in tension compared withmaximal contractions to KCl (100 mM).*Significant difference (p<0.05) between ringswith and without endothelium in cholesterolgroup.

endothelium-derived relaxing factors) in responseto serotonin also was significantly depressed in thecholesterol group compared with the normal group(Figure 10). Treatment of the coronary segmentswith indomethacin did not affect the basal releaseof endothelium-derived relaxing factors) in bothgroups but significantly augmented the relaxationsevoked by serotonin in the cholesterol group(Figure 10). Infusion of the monoamine underdirect superfusion did not relax bioassay ringsfrom both groups contracted by prostaglandin F^(N=5 each, data not shown).

CorrelationsIn rings with endothelium from the atheroscle-

rotic LADs, a significant correlation was notedbetween the cross-sectional area of the intima andthe IC,o values to bradykinin (r=0.72, «=62), ADP(r=0.79, n=\2), and the calcium ionophore A23187(r=0.75, n=6). A similar significant correlation alsowas noted between the cross-sectional area of theintima and the percent maximal relaxations to ser-otonin (r=-0.82, n=12) or the percent relaxationsto thrombin (0.1 units/ml) (r=-0.94, n=6) and

aggregating platelets (70,000/^1) (r= -0.91, n=6). Incontrast, there was no significant correlation betweenthe cross-sectional area of the media and the IC50values or percent maximal relaxations to thoseagonist (r<±0.2 for any agonist).

DiscussionThe major findings in the present study were 1)

the endothelium-dependent relaxations to bradyki-nin, serotonin, ADP, thrombin, and the calciumionophore A23187 are impaired in coronary athero-sclerosis, while the relaxations to serotonin andADP are depressed in hypercholesterolemia, 2) theendothelium-dependent relaxations to aggregatingplatelets also are depressed in hypercholester-olemia and more severely impaired in atherosclero-sis, 3) these impairments occur at a time that theability of the underlying smooth muscle to relax orcontract is unchanged, 4) the impaired endothelium-dependent relaxations in atherosclerosis are partlydue to the depressed release of endothelium-derived relaxing factors) from the endothelium,and 5) concomitant release of vasoconstrictor prod-

TABLE 4. Effects of Various Btockers on Contractions of the Atherosclerotic Porcine Left Anterior DescendingCoronary Artery Evoked by Serotonin

Blockers

No treatmentIndomethacinMeclofenamateDazoxibenSQ 29548NDGABAY G6575

n

8

66

4

4

4

4

ED«

Withendothelium

6.61+0.106.11 ±0.10*6.25±0.10*6.53+0.146.41+0.176.40±0.166.50+0.17

Withoutendothelium

6.69±O.IO6.52±0.106.53±0.156.7l±0.166.5l±0.216.51±0.166.65±0.17

Max.

Withendothelium

75.8±2.260.1 ±5.0*57.8+3.8*72.3±4.976.3±6.275.2±2.074.4±3.3

contraction (%)

Withoutendothelium

63.1±2.767.2±5.461.8±5.062.6±2.762.4±5.060.2±2.062.6±3.7

Data are expressed as means±SEM.ED*), effective concentration of serotonin (-log M producing 40% of the maximal response to KCl; Max.

contraction, maximal contraction to serotonin as expressed in percent of the maximal contraction to KCl in the samering. Concentrations: indomethacin (I0"5 M), meclofenamate (10~6 M), dazoxiben (10~4 M), SQ 29548 (10~6 M),nordihydroguaiaretic acid (NDGA) (2xl0~3 M), and BAY G6575 (IO~3 M).

*p<0.05 compared with no treatment.




TABLE 5. Concentrations (pg/ml) of Prostaglandins Released From the Porcine Left Anterior DescendingCoronary Artery

Controlgroup

Cholesterolgroup

Endothelium

WithWithoutWithWithout

n

5566

6-keto-PGF,,,

Basal

36±935±847±1035±7

Stimulated

79±2033±827±9*11±3*

Basal

20±8I5±321±1014±7

PGFi,

Stimulated

17±84±2

53+205±3

Basal

15±218±214±513±3

PGE2

Stimulated

16±107±4

11±54±3

Data are expressed as means±SEM.6 keto PGF,a, 6 keto-prostaglandin F,o; PGFa,, prostaglandin F^; PGE2, prostaglandin E2; Basal, basal release

of prostaglandins (during 40-minute incubation). Stimulated release of prostaglandins evoked by serotonin (10~5 M).The concentrations of released thromboxane B̂ were below the detection level (20 pg/ml) in both groups.*p<0.05 compared with control group.

uct(s) of cyclooxygenase may depress further theimpaired relaxations in atherosclerosis.

In the present model, coronary atherosclerosiswas induced in the LADs by a combination ofballoon endothelial denudation and hypercholester-olemia12-15-'6.i8-2o; the LCXs were affected only byhypercholesterolemia. Therefore, it is reasonable toconsider that both endothelial recovery (regenera-tion) after the injury and hypercholesterolemia playcausative roles in the impaired endothelial functions.Under atherogenic conditions, such as hyperlipid-emia,32 hypertension,33 and increases in shear stress,34

the endothelial turnover rate is accelerated in responseto the continuous injury by these factors. Underthese conditions, regenerated endothelial cells seemto have an important role in mediating the interactionbetween blood components and vascular smoothmuscle cells.3536 In fact, the morphological appear-ances of endothelial cells in both LCXs and LADsfrom cholesterol animals are consistent with those ofregenerated endothelial cells reported in previousstudies.3738 In porcine coronary arteries with regen-erated endothelial cells (4 weeks after endotheliumremoval), endothelium-dependent relaxations to ser-otonin and aggregating platelets are reduced.22 Thisfinding could explain partly the augmented vasocon-strictor responses to serotonin that have been

described in animals with hypercholesterolemia,14

hypertension,39 and atherosclerosis.1214-'8 In recentstudy, the time course of the impairment ofendothelium-dependent relaxations was examinedup to 24 weeks after the balloon endotheliumremoval.40 After 4 weeks, the relaxations to seroto-nin and aggregating platelets were impaired asreported previously22 and remained so throughoutthe follow-up period. After 8 weeks the relaxationsto thrombin and ADP were impaired, and after 16weeks those to bradykinin were, while those to thecalcium ionophore A23187 were well maintained.40

Another important factor is hypercholesterolemiaitself. Low-density lipoproteins, when administeredacutely, inhibit endothelium-dependent relaxations toacetylcholine, adenosine triphosphate, and the cal-cium ionophore A23187 in the rabbit aorta.41 In addi-tion, lipid deposition was noted in the thickenedintima, including endothelial cells. Thus, the above-mentioned two factors should be involved in theimpaired endothelial functions. However, the endo-thelial denudation and repair alone cannot explain theimpaired relaxations to bradykinin and the calciumionophore A23187 or the severe impairments of therelaxations to thrombin and ADP observed in athero-sclerotic coronary arteries in the present study. Sim-ilarly, the increase in low-density lipoproteins fails to

LCX LAD

xao

CE

0

20

40

60

80

100n=5

9 6 9Serotonin, -log M

FIGURE 8. Cumulative concentration-response curves to serotonin in rings withendothelium during a contraction evokedby prostaglandin F2a (2xlO~6 M) in pres-ence of IO~6 M ketanserin. Relaxationresponses are expressed as percentdecrease in tension of contraction evokedby prostaglandin F2a. Data shown asmeans±SEM. Left: Relaxations in the leftcircumflex coronary artery (LCX). Right:Relaxations in the left anterior descendingcoronary artery (LAD). Control rings, with-out indomethacin (•), were treated with10~5 M Na2CO3 (solvent for indomethacin)only. O, control rings with indomethacin.*Significant difference (p<0.05) betweenrings with (a) and without (•) indometh-acin in the cholesterol group.




LCX LAD

5 4 8 7Adenosine diphospriate, -log M

explain why the relaxations to bradykinin and thecalcium ionophore A23187 were well maintained inthe hypercholesterolemic porcine coronary arteries.

The depressed endothelium-dependent relax-ations in atherosclerosis could be due to a numberof factors.

First, the production and/or release of theendothelium-derived relaxing factor(s) may bedecreased. This possibility is most likely becausethe depressed release of the factors) (in both basaland stimulated conditions) was confirmed in bioas-say experiments. Similar observations have beenreported in the atherosclerotic rabbit aorta.4243

Second, since the half-life of the endothelium-derived relaxing factor(s) is extremely short,44 thethickened intima with lipid deposition may reducethe diffusion of the factors). In fact, there is asignificant correlation between the impairment ofendothelium-dependent relaxation and the degreeof intimal thickening. However, the intraluminalrelease of the factors) in response to serotonin isdecreased (bioassay experiment) and endothelium-dependent relaxations also are depressed in thehypercholesterolemic LCX that does not exhibit

Or1*5

10

20

| 30eg

3 40oX

50

60

70

FIGURE 9. Cumulative concentration-response curves to adenosine diphosphate(ADP) in rings with endothelium during a con-traction evoked by prostaglandin F^ (2x70"*M) in the presence of IO~* M theophylline.Relaxations are expressed as percent decreasein tension of contraction evoked by prostaglan-din F2a. Data shown as means±SEM. Left:Relaxations in the left circumflex coronaryartery (LCX) in control and cholesterol groups.Right: Relaxations in the left anterior descend-ing coronary artery (LAD) in the two groups.Control rings, without indomethacin (•), weretreated with Na2CO3 (1(T5 M, solvent for indo-methacin) only. O, control rings with indometh-acin. 'Significant difference (p<0.05) betweenrings with (a) and without (•) indomethacin inthe cholesterol group.

intimal thickening. The intraluminal release of thefactor(s) in response to bradykinin also is depressedin the atherosclerotic porcine coronary arteries inthe same model as used in the present study.45

Dietary treatment of atherosclerosis in monkeysrestores endothelium-dependent relaxations to ace-tylcholine and thrombin even in the presence ofintimal thickening.46 The thickening of the intimaafter balloon injury may have other consequences,such as inflammation47 and lipid deposition (in thepresence of hyperlipidemia), which also may alterblood-vessel wall interactions. Therefore, the roleof the atheroma, including possibly to constitute a"functional" (but not "distance") barrier, remainsto be examined in further studies.

Third, the increase in the amount of the medialsmooth muscle may result in relative shortage of theendothelium-derived relaxing factor(s) to cause relax-ations. This is unlikely to be the cause because nosignificant correlation exists between the degree ofmedial mass and the impairment of the relaxations.

Fourth, the characteristics of smooth muscle cellsand, in particular, their sensitivity to endothelium-derived relaxing factors) may be changed. This

FIGURE 10. Bioassay experiments using the leftanterior descending coronary artery as a donor andthe left circumflex coronary artery as a bioassay ringin the control (O, with indomethacin; • , without) andcholesterol (a, with indomethacin; • , without) groups.Relaxations are expressed as percent decrease intension of contraction evoked by prostaglandin F2a

(2XW6 M). Data shown as means±SEM. 'Signifi-cant difference (p<0.05) between control and cho-lesterol groups. ^Significant difference (p<0.05)between with and without indomethacin.

Direct Basal Stimulated releasesuperfuslon release ( 3x1 O^M serotonin )




possibility seems less likely because in rings with-out endothelium the relaxations to sodium nitroprus-side (which induces relaxations through activationof guanylate cyclase as does endothelium-derivedrelaxing factoids)2), and the contractions to potas-sium chloride (activating voltage-dependent Ca2+-channels) and serotonin (receptor-operated Ca2+-channels) were unaltered. Whether or not therelaxations to nitrovasodilators are impaired in ath-erosclerosis is controversial; it is impaired in theaorta of the rabbit fed a 0.3% cholesterol diet for 16weeks8 but not in the same species fed a 1%cholesterol diet for 6 weeks9 or in the iliac artery ofthe monkey fed a 0.74% cholesterol diet for 18months." These discrepancies could be due to thedegree of atherosclerosis (early or advanced) and/orthe anatomical type of blood vessels, that is, elastic(aorta) or muscular (femoral and coronary).

Finally, in coronary atherosclerosis, vasoconstric-tor product(s) of cyclooxygenase may be releasedtogether with a smaller amount of endothelium-derived relaxing factoids).48"51 This interpretation issupported by the following observations: 1) higherconcentrations of serotonin induced endothelium-dependent contractions, which were inhibited byblockers of cyclooxygenase but not of lipoxygenaseand 2) the endothelium-dependent relaxations toserotonin (organ chamber and bioassay experi-ments) and ADP (organ chamber experiments) weregreater in the presence than in the absence ofindomethacin. The release of the indomethacin-sensitive, endothelium-derived contracting factor(s)have been reported in the saphenous vein48 and thebasilar artery51 of the dog and in the aorta of thespontaneously hypertensive rat.50 Since inhibitionof thromboxane receptors (SQ 29548) or thrombox-ane synthetase (dazoxiben) did not antagonize theendothelium-dependent contractions, a prostanoidother than thromboxane A2 or an endoperoxideintermediate of cyclooxygenase pathway may beresponsible for the contractions. The same conclu-sions were reached for the endothelium-dependentcontractions to arachidonic acid in canine saphen-ous veins.48 The pathological importance of thesignificant decrease in prostacyclin release duringendothelium-dependent responses is unknown.

The mechanisms of the depressed endothelium-dependent relaxations in hypercholesterolemiaappears easier to understand. Since no intimal ormedial thickening, no impairments in the relax-ations to sodium nitroprusside, or no endothelium-dependent contractions were observed in the LCX,the depressed production and/or release of theendothelium-derived relaxing factor(s) is the mostlikely explanation.

Endothelium-dependent responses to aggregatingplatelets are the global expression of the responsesto several released platelet products and their inter-actions.6 In porcine coronary arteries the platelet-induced relaxations are achieved by both S,-serotonergic and purinergic (ADP) mechanisms and

the platelet-induced contractions are achieved by anSrserotonergic mechanism with little contribution ofthromboxanes.22 Therefore, the observed impairedrelaxations of porcine coronary arteries to aggregat-ing platelets in atherosclerosis and hyperlipidemiacan be explained by the impaired endothelium-dependent responses to serotonin and ADP.

Among the endothelium-dependent relaxations tovarious factors derived from platelets and the coag-ulation system, those to serotonin are impaired inthe chronic regenerated state (a very early stage ofatherosclerosis).22 Combining this finding togetherwith the present experiments, it is tempting tosuggest that during the process of atherosclerosis,endothelium-dependent responses are impaired firstto serotonin, then to ADP, and finally to othervasoactive agents. Since both serotonin and ADPare the major platelet-derived vasoactive sub-stances, the endothelium-dependent responses toaggregating platelets are impaired, beginning fromthe early stage of atherosclerosis and progressivelyworsening during the process.

Endothelium-derived relaxing factor inhibits plate-let aggregation.52-'3 In human coronary arteries,aggregating platelets cause endotheliumndependentrelaxations.56 If the endothelial dysfunction to aggre-gating platelets and their products observed in thepresent study also occurs during coronary athero-sclerosis in humans, the impaired interactionsbetween platelets and the blood vessel wall wouldfavor the occurrence of platelet aggregation andplatelet-induced contractions of coronary smoothmuscle, leading to ischemic events such as coro-nary vasospasm and coronary thrombosis.

AcknowledgmentsThe authors wish to thank Dr. G.M. Tyce for the

serotonin determinations, Dr. J.C. Romero for theprostaglandin assay, and Dr. P.E. Zollman at MayoInstitute Hills Farm for valuable help. They alsothank Mr. K.S. Rud for technical assistance, Ms.H. Hendrickson and Mr. R.R. Lorenz for preparingthe figures, and Ms. C. Camrud and Ms. K. Krosfor secretarial assistance.

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KEY WORDS • endothelium-derived relaxing factor • serotonin• adenosine diphosphate • thrombin



H Shimokawa and P M Vanhouttesubstances in porcine coronary arteries in hypercholesterolemia and atherosclerosis.

Impaired endothelium-dependent relaxation to aggregating platelets and related vasoactive

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