Biphasic response of intimal prostacyclin production during the development of experimental atherosclerosis

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BIPHASIC RESPONSE OF INTINAL PROSTACVCLIN PRODUCTION DURING THE DEVELOPMENT OF EXPERIMENTAL ATHEROSCLEROSIS J.R. Beetensl, M.2.

Coenel, A. Verheyenz, L. Zonnekeyn and A.G. Herman

Faculty of Medicine, Division of Pharmacology, University of Antwerp, B2610 Wilrijk, Belgium, and 2Laboratory of Cell Biology, Dept. of Life Sciences, Janssen Pharmaceutics Research Laboratories, 82340 Beerse, Belgium.

ABSTRACT Feeding a cholesterol rich diet (0.3 %l to rabbits for up to 10 weeks resulted in morphological changes of the vascular wall. Microscopic evaluation of the aorta revealed a lipid infiltration and an intimal thickening containing foam cells, which both became more pronounced as the cholesterol feeding was more prolonged. The intimal prostacyclin production showed a transient increase after 2 weeks, but was significantly decreased after 6 weeks of diet and remained at this low level during the rest of the experiment. No significant changes in formation of thromboxane B2 by the platelets could be observed, whereas the production of 12-HETE was enhanced. INTRODUCTION The "response-to-injury" hypothesis of atherosclerosis postulates that endothelial injury may be the first step in the development of atherosclerotic lesions, followed by a platelet/vessel wall interaction (1, 2). Arachidonic acid is considered to play an important role in this platelet/vessel wall interaction (3) and, several investigators have emphasized the importance of prostacyclin (PGI2) in maintaining the integrity of the vascular wall, more in particular the endothelial cells (4). The metabolism of arachidonic acid has been reported to be altered in atherosclerosis : thrombocytes from humans or animals with atheromatous arteries produce more thromboxane A2 (TXA2) (5-81, whereas the formation of prostacyclin by the atherosclerotic vascular wall is diminished (9-14). However, also an increased release of PG12 by the vascular tissue has been observed in minipigs and rabbits, fed a hypercholesterolaemic diet for 4 weeks (12, 15). In the present study, the metabolism of arachidonic acid in the vascular wall and in platelets was investigated during the early stages of experimentally induced atherosclerosis in rabbits.

I Present address : Dept. of Life Sciences, Janssen Pharmaceutics Research Laboratories, B2340 Beerse, Belgium.

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MATERIAL AM) METHODS Animal treatment Sixty albino rabbits (males, f 2 kg) were divided in two groups. During 10 weeks group A received a commercial standard chow whereas group B was fed the standard chow to which 0.33 % cholesterol was added. All animals received 150 g chow a day and tap water ad libitum. Every two weeks 6 animals from each group were killed and the following parameters were evaluated : - macroscopic and microscopic evaluation of the aortic lesions. - ex vivo production of prostacyclin by aortic endothelium and by rings of the abdominal aorta, arteria coeliaca and mesenterica. - arachidonic acid metabolism in washed platelets. - serum cholesterol and triglyceride levels. Evaluation of the atherosclerotic lesions in the aorta The macroscopic lesions were evaluated visually and planimetricallY* Visually, the lesions were scored on a scale from 0 (no visible lesions) to 5 (the whole intima covered with atherosclerotic fatty streaks). After the incubations, the aorta was rinsed in running tap water and stained during 15' in a Fat red solution (0.5 % in 70 % ethanol). Lipid loaden lesions became visible as darkred spots on a pink background. The aorta was photographed and the aortic lesions were quantified planimetrically by measuring the square area they occupied and expressed as a percentage of the surface of the total aorta. One segment of the aortic samples was rapidly frozen in isopentane, cooled with liquid nitrogen, and kept at -70°C until further proThe segments were then brought to -25°C in a cryostat cessing. whereafter 7 micron thick frozen sections were prepared and stained Counter-staining was perwith oil red 0 for lipid localization. formed with 1 % toluidine blue. The tissue segments for light microscopical and ultrastructural examination were immersed in 3 % glutaraldehyde in O.lM sodium cacodylate buffer (pH 7.4) for 1 to 3 days, rinsed overnight in O.lM sodium cacodylate buffer (pH 7.4) supplemented with 7.5 % sucrose at 4"C, postfixed in 2 % 0~04 in 0.05M Michaelis buffer (pH 7.4) containing 3.2 % sucrose for lh at 4°C and impregnated with 0.5 % uraniumacetate in 0.05M Michaelis buffer (pH 5.21 for 40 min at 4°C in the dark. The segments were then dehydrated in graded series of ethanol and routinely embedded in epon. Semi-thick sections (2 miThe ultracrons) were prepared and stained with toluidine blue. thin sections were examined in a Philips EM 201 electron microscope. Production of prostacyclin by vascular tissue ex vivo The production of endogenous prostacyclin was measured in the inti-

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ma1 layer of the thoracic aorta and in segments of whole arteries. The intimal PG12 production was measured with the well technique as previously described (16). The thoracic aorta was opened longitudinally, thereby avoiding damage to the endothelium, and placed between two lucite plates. The upper plate contained several holes (0 7 mm), which served as incubation chambers in which the aortic The incubation chambers luminal surface formed the chamber base. were filled with 0.1 ml physiological Hepes buffer (pH 7.4) and the aorta was allowed to equilibrate for 40 min (pre-incubation period). Thereafter, the buffer was discarded and the chambers were refilled with 100 yl buffer. After 15 min incubation time at room temperature, 95 yl of the incubation fluid was transferred to a test tube containing indomethacin (28 yM final concentration) and Prostacyclin the volume was adjusted to 1 ml with Hepes buffer. production by the intimal layer was measured as 6-oxo-PGFI, , its stable metabolite, by a specific radioimmunoassay (17). Rings of the arteria mesenterica (from the aorta to the first bifurcation), of the arteria coeliaca (from the aorta to the second bifurcation) and of the abdominal aorta (between the a. coeliaca and a. nlesenterica), were incubated in Hepes buffer as previously described (18). Rings were pre-incubated for 15 min at 37"C, and thereafter incubated for 30 min at 37"C, in 1 ml physiological Hepes buffer. The incubation was stopped by cooling the test tubes on ice, addition of indomethacin (28 yM final concentration) and removal of the tissue. The PG12 released during the incubation was measured by a specific radioimmunoassay for 6-oxo-PGFIa . Arachidonic acid metabolism in washed platelets Rabbit platelets were washed following methods previously described Washed platelets were incubated with exogenous I4C-AA (10 (191. uM; specific activity : 10 mCi/mmol) for 30 min at 37°C. The reaction was stopped by acidification of the reaction mixture to pH 3.0. The reaction products were extracted twice with 2 volumes of ethyl acetate and separated on TLC in the solvent mixture chloroform:methanol:water:acetic acid (90:6:0.6:1). Radioactive metabolites (TXB2, HHT, PGF20, 12-HETE and AA) were located by radiochromatogram scanning, scraped off and quantified by liquid scintillation counting (19). Results were expressed nmol metabolite formed per mg platelet protein. The absolute amount of the metabolites were calculated from the relative distribution of the radioactivity on the plate. Endogenous TXB2 production by washed platelets after thrombin stimulation was measured by a specific radioimmunoassay (201. Other determinations Serum cholesterol was enzymatically determined following routine methods using the catalase method (Boehringer Mannheim, Diagnostica). Serum triglycerides were determined using the enzymatic test combination (Boehringer Mannheim, Diagnostica).

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MATERIALS Radioactive

(l-14C)-arachidonic acid (56 mCi/mmol), (5,8 9,11,12,Flcl and (5,8,9,11,12,14,15-3H)-thromboxane B2 were purchased from New England Nuclear (NEN, Boston). The animal chow was provided by Animalabo (Brussels, Belgium). TLC analysis was performed on silica plates 6OF254 (Merck, Darmstadt, F.R.G.). Arachidonic acid and Hepes (N-2-2_hydroxyethylpiperazine-N'-2 ethanesulfonic acid) were obtained from Sigma. All solvents used were of analytical grade (Merck). Fat red was purchased :;T GB.)D.H. (Poole, U.K.). All other stains were from Merck

14,15-3H) 6-oxo-prostaglandin

. . . . RESULTS Diet-induced histological changes of the aorta Feeding the animals a cholesterol-rich diet resulted in histological changes and formation of "fatty streaks" in the aorta. These changes became macroscopically visible after 4 weeks feeding and the severity of the lesions increased during the length of the experiment. The scores are summarized in Table 1. The lesions were most pronounced in the aortic arch and the severity decreased towards the abdominal part of the aorta. TABLE

1 : Severity of the atherosclerotic lesions visual and planimetrical methods. ----____________-_-_-------------______-__----__________ -_-CHOLESTEROL Visual score Planimetry ==============================================~

as

scored

by

;5 f 3 i.8 + 0.7 3.0 f 0.6 57 -f 9 3.6 -f 0.4 70 + 7 85 f 6 4.6 ? 0.6 1oW _-_________-_____-_--------------------------_-__________________~~~~~~----~---_---_-----The visual score is evaluated on a scale from 0 to 5. Planimetry is expressed as the percentage of the aortic surface which is covered by atherosclerotic lesions. A linear relationship between both methods is found (r : 0.87; P cO.05; Spearman's rang correlation). z 6w &I

Cross-sections of the thoracic aorta were examinated with light miIn the control animals, no intimal thickening nor fat croscopy. After 2 accumulation was observed at any time of the experiment. weeks of cholesterol rich diet 1/3rd to 1/4th of the media (from the luminal side on) was infiltrated with fat and a small intimal After thickening with sporadic foam cells became already visible.

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Figure 1. Intimal surface of thoracic aortas of control rabbits and of animals on a cholesterol enriched diet. Endothelium (END); foam cell (FC); endothelial lipid inclusions (b). a. Control animal. Normal endothelium adjacent to the internal elastic lamina (IEL). b. fOUr Weeks of diet. Disruption of an endothelial junction is indicated by the arrow; cytoplasmic densification is observed in another endothelial cell (*I and a foam cell is bulging through the endothelial layer (arrowhead). These individual changes were more frequently observed after 8 and 10 weeks of diet. C. Eight weeks of diet. In these endothelial cells several lipid inclusions can be observed and dilatation of the rough endoplasmic reticulum (rer) is evident. d. Ten weeks of diet. Severely altered, edematic endothelial cells are visible.

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4 weeks, d clear intimal thickening containing foam cells was observed. During the experiment, the intimal thickening as well as the fat infiltration increased, resulting after 10 weeks in an intima twice as thick as the media. Electron microscopic examinations revealed no changes of the aortic wall in the control animals during the entire experiment (Fig. 1). Feeding the animals a cholesterol rich diet for 2 weeks resulted already in morphological changes of the vascular wall. The aortic endothelium contained lipid inclusions and often showed dilatation of the rouch endoplasmic reticulum which enclosed flocculent material. An intimal thickening became visible and was infiltrated with monocytes as well as with smooth muscle cells. Both celltypes contained lipid droplets. After 4 weeks of diet, the intimal thickening was more elaborated; more smooth muscle cells than monocytes had occupied the intima and the lipid infiltration in both celltypes was more pronounced resulting in the establishment of foam cells. Endothelial lipid inclusions and dilatation of rough endoplasmic reticulum were more frequently observed than after 2 weeks of diet. Additionally, some cells showed either disruption of interendothelial junctions, (and) or cytoplasmic densification or severe edema; rare death cells were apparently desquamating. These endothelial changes were particularly present over intimal plaques containing foam cells. Some of these foam cells were bulging through the endothelial layer. The severe endothelial alterations were more frequently seen in animals put on a diet for 8 and Monocytes and smooth muscle cells in the intima were 10 weeks. mostly transformed into foam cells. Intimal prostacyclin formation The intimal PG12 production during the various stages of the experiment is summarized in Fig. 2. The PG12 production in the control group remained fairly constant during the whole experiment, with In the cholesterol treated animals, a only minor fductuations. transient increase in the intimal PG12 formation was observed after After 4 weeks, the production returned to control 2 weeks diet. values and continued to decline to values which were, from the sixth week on, significantly lower than the control levels. ?roduction of prostacyclin by vascular

SegmentS

After 4 to 8 weeks diet, vascular rings from cholesterol treated animals released slightly higer amounts of PG12 as compared to control rings. After 10 weeks, the PG12 production was diminished in However, vascular segments of the cholesterol-treated animals. none of these changes were statistically significant, probably due to the large variation.

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1 pmol

PGI2 0

*

CONTROL CHOLESTEROL

10

2

weeks

The influence of a cholesterol diet on the intimal PG12 Fig. 2. production. Results are ex ressed as pmol PG12 released in 15 min PG12 was measured as 6-0x0per incubation well (38 mm ! aorta). PGFI by a specific radioimmunoassay. * : significantly different from the control (P < 0.05, Student's t-test). Arachidonic acid metabolism in washed platelets The influence of the different diets on the metabolism of exogenous No significant 14C-arachidonic acid is summarized in Table 2. changes in the formation of TXB2 or in the total production of cycle-oxygenase derived metabolites (TXB2, PGE2, PGD2 and PGF2a and HHT) could be observed. However, in the platelets from the cholesterol supplemented animals, the formation of the 12-lipoxygenase product 12-HETE was significantly increased. The production of TXB2 from endogenous arachidonic acid in washed platelets stimulated with thrombin, was not significantly increased in the cholesterol treated animals (Table 3).

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TABLE

2 : Metabolism of exogenous rabbit platelets. ------_______I_____-___I =======z==~~

23 TXB 4:

8w lh

CONTROL =----------__-=

3.93 7.62 7.74 7.57

+ f f +

0.42 0.41 0.28 0.56

14C-arachidonic acid by washed

== CHOLESTEROL

4.74 6.76 8.01 7.75

f f + f

0.55 0.53 0.66 1.30

C.O. ii

14.8 10.20 f+ 0.65 0.88

16.1 12.56 + f 1.32 0.80

&

18.7 19.2

21.2 20.6

12-HETE 2w ii

f 0.95 + 1.90

6.49 f 1.11 4.43 5.10 k If 0.54 1.31

f 1.14 0.90

10.40 f 1.13* 8.42 8.86 tf 1.22* 1.68*

1OW 6.50 + 3.81 9.30 f 0.90* ~=--------~ =========~-=~ Results are expressed as nmol product formed per mg protein (mean f s.e.m.; n = 61. C.O. : total of cycle-oxygenase products (TXB2, HHT, PGE2 & PGF2o I. * - significantly different from the control (P < 0.05, Student ‘Is t-test).

TABLE 3 : Release of endogenous TXB2 from washed platelets after thrombin stimulation. ----------------------~===--_--~CONTROL =====------~----------I--_---~--~

CHOLESTEROL

0.65 f 0.13 1.10 4w 1.14 f 0.24 1.44 & 1.00 + 0.24 1.53 1OW 1.28 + 0.24 1.95 =---------------====2=================_-----2w

+ 0.28 + 0.18 + 0.16 f 0.33 _------

Results are expressed as nmol TXB2 released per mg platelet protein (mean f s.e.m., n = 6).

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Cholesterol and triglyceride levels Feeding the rabbits a cholesterol rich diet resulted in significantly elevated serum cholesterol and triglycerides levels which increased throughout the entire experiments (Table 4).

TABLE 4a : Serlpncholesterol levels.

-~~~--~--~--~-~_-~_-~-~___ -~~~-~~-----~~_-~_-_____

---cc--- -_-

CONTROL CHOLESTEROL ______-_-_--_-_--_----~-~~----~-~_-_ ______--_---_-_--__---~~-~-~-~ 2w 48 f 7 519 + 52* 4w 42 f 7 690 t 52* 6w 41 f 4 8 8w 35 + 1oW 37 + 6 ____-_____-__-__-_-_--___________ ~_-___-_-__-_______-____

::; : 22* 1269 ? 36:: ____ Results are expressed as mg cholesterol per 100 ml serum (mean f s.e.m., n = 6). *. significantly different from control (Student's t-test, P< '0.05).

TABLE 4b : Serum triglyceride levels.

------I-_--==----_---_------_--==

CONTROL CHOLESTEROL =-------_--_-'------------------2w 4w

109 + 16 92 + 11

6w

98 f 15

153 + 15

157 f 36 139 + 31

rlw 97 84 fk 19 16 217 229 + 24* 45* Pm==a========-_-_=========_____ Results are expressed as mg triglyceride per 100 ml serum (mean f s.e.m.. n = 6). * : significantly different from control (Student's t-test, P < 0.05).

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DISCUSSION

The arachidonic acid metabolism in the vascular wall is altered during

atherosclerosis.

Our

results

demonstrate

that the

intimal

prostacyclin production showed a biphasic response to cholesterol feeding.

After an initial increase, the PG12 production declined

and was significantly below control values from the 6th week on. Our experiments could

offer an explanation

for the contradictory

reports on the vascular PG12 production during the development of atherosclerosis.

Indeed, a decreased production of PG12 by vascu-

lar tissue has been reported in experimental as well as in human atherosclerotic lesions (9, 10,

11,

14).

hand, an in-

On the other

creased release of PG12 was observed in minipigs and rabbits, who were fed a hypercholesterolaemic diet during 4 weeks (12, 15).

Our

experiments clearly demonstrate the time dependent changes of the intimal PG12 production sclerosis

during

the onset of experimental

in rabbits and confirm

the

suggestion of

al. (12) that the PGI2 production might

athero-

Sinzinger

et

show a biphasic response

during the development of atherosclerosis.

The initial increase of

PG12 production by the vascular wall, before macroscopic morphologic changes are observed, can be explained by a reaction of the tissue to exogenous noxious stimuli as also suggested by Voss et al. (21).

However, already at this stage of the experiment, micro-

scopic examination

revealed morphological

changes of the vascular

wall, with an oedematous endothelium and an increased amount of endoplasmic reticulum.

Such a state is accompanied by an increased

metabolic activity of the eildoplasmic membranes.

As the cyclooxy-

genase and the PG12 synthase are membrane bound enzymes we can assume that the initial increase of the intimal PG12 formation can be explained by an increased activity and/or increased amount of the enzymes. this

However no experimental

hypothesis

and

other

data

explanations

are available remain

to support

possible

such

e.g. an increased availability of the substrate arachidonic

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As atherosclerosis maged

progresses and the endothelium becomes more da-

(as shown by the microscopic

studies) the intimal PG12 pro-

duction declines and after 6 weeks of diet the formation of PG12 is significantly lower as compared

to the control

It has

animals.

been shown in cultures of smooth muscle cells from atherosclerotic animals that the decreased formation of PG12 was due to a decreased activity of the cyclooxygenase

and PG12 synthase, and not to a di-

minished availability of tl,e substrate arachidonic acid (22).

In

these cells, an increased lipoxygenase activity was observed, with the formation of monohydroperoxides of arachidonic acid, which correlated well with the decreased PG12 synthesis (231.

Such hydrope-

roxides of arachidonic acid are potent inhibitors of the PG12 synthase

(24, 25).

Lipid peroxides, which might

vascular wall during hyperlipidemia

accumulate

in the

accompanying atherosclerosis,

could also be responsible for the diminished activity of the PGI2forming enzymes (25, 26). and Sinzinger et al.

Contradictory to Gryglewski et al. (10)

(111, no significant changes in the PG12 pro-

duction by vascular segments could be observed.

This discrepancy

can be explained by the different assay methods used for the detection of PG12 : both authors used the bio-assay technique

(inhibi-

tion of platelet aggregation), whereas we estimated PG12 by radioimmunoassay of the stable hydrolysis product 6-oxo-PGFI

.

On the

other hand, the extent of the atherosclerotic lesions into the arterial wall might also have some importance.

Although, on weight

basis, the endothelial cells show the largest PG12 producing activity

(271, the underlying layer of smooth muscle cells contribute

substantially to the total PG12 production by the vascular rings. Furthermore, it has been shown that deendothelialized aortic rings produce

amounts of

PG12, comparable

with that of "intact" rings

(281, most likely due to the mechanical stimulation of the underlying layers during the manipulation revealed that the atherosclerotic

(29).

Microscopic examination

lesions and lipid

infiltration

was only after 10 weeks deeply extended in the smooth muscle cell layer, time at which a tendency

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the vascular rings could be observed. Feeding a cholesterol-rich diet to rabbits hardly changed the arachidonic acid metabolism in blood platelets.

No differences could

be found in the production of thromboxane 62 or other cyclooxygenase products from exogenous arachidonic acid.

These experiments are

in agreement with the results of Gryglewski et al. (101, who found an increased TX82 formation only after three months of diet.

On

the other hand, a tendency was observed to increased TXB2 production from endogenous substrate by platelets stimulated with thrombin.

As the enzymes, which are involved in the transformation of

arachidonic acid to thromboxane, seem to display the same activity in the cholesterol supplemented as in the control animals, the increased TXB2 production might be explained by an increased liberation of the arachidonic acid. addition of cholesterol

Stuart et al. (30) showed that the

to platelet rich plasma in vitro resulted

in an increased capacity of thrombin to stimulate the release of arachidonic acid from the platelet phospholipids.

Furthermore, it

has recently been demonstrated that cholesterol-enriched

platelets

show an increased number of thrombin receptors, accompanied by an increased platelet responsiveness for thrombin (33). Although

the

formation

of

the

lipoxygenase

product

12-HETE

exogenous arachidonic acid is increased, its physiological

Several authors reported recently that 12-

tance remains unclear. HPETE

(the

precursor

from

impor-

of

12-HETE)

inhibits

the

thromboxane B2 formation in platelets (32-34).

aggregation

and

The increased acti-

vity of the lipoxygenase in our experiments, however, was not sufficient to influence the thromboxane production. been

reported

to be a

potent

chemoattractant

12-HETE has also for

aortic

smooth

muscle cells and to stimulate the migration of the smooth muscle cells in vitro (35, 36).

Therefore, the 12-HETE, released by pla-

telets adhering or aggregating on the damaged vessel wall, might attribute to the increased infiltration of smooth muscle Cells

in

the intima and exacerbate the atherosclerotic lesions.

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The

presently

described

experiments

demonstrate

that during

the

early stages of experimental atherosclerosis, besides morphological and histological changes in the cardiovascular system, the arachidonic acid metabolism is altered.

These changes are most prominent

at the site of the intimal prostacyclin production, whereas the metabolism of arachidonic acid in platelets is hardly changed.

ACKNOWLEDGEMENTS We thank Mrs. R. Van den Bossche, Ms. A. Van Hoydonck and Mr. L. Leijssen for their excellent technical assistance and Ms. L. Van den Eynde for typing the manuscript. We acknowledge the collaboration with Prof. Dr. L. Lepoutre (University of Anwerp) for the estimation of the levels of cholestrol and triglycerides and with Dr. G. Prop and Dr. H. Winkelmann (Nattermann GmbH, Kiiln, F.R.G.) This work was supported by a research for helpful discussion. grant 3.900.77 of the F.G.W.O. and by a postgraduate grant of the I.W.O.N.L. to J.R. Beetens and M.-C. Coene. REFERENCES

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34. Aharony, D., J.B. Smith, and M.J. Silver. 1982. Regulation of arachidonate induced platelet aggregation by the lipoxyenase product 12-hydroperoxy-eicosatetraenoic acid. Biochim. Biophys. Acta 718 : 193-200. 35. Nakao, J.,T, Ooyama, W.C. Chang, S. Murota and H. Orimo. 1982. Platelets stimulate aortic smooth muscle cell migration in vitro. Involvement of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid. Atherosclerosis, 43 : 143-150. 36. Nakao, J., T. Ooyama, H. Ito, KC. Chang and S. Murota. 1982. Comparative effect of lipoxygenase products of arachidonic acid Atherosclerosis on rat aortic smooth muscle cell miaration. 44 : 339-342. Editor: G. Kaley

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Received:4-22-85

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Accepted: 6-13-86

1986 VOL. 32 NO. 3