THROMBOSIS RESEARCH 67; 81-94,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
EFFECTS OF ANTITHROMBOTIC DRUG,Y-20811 ON MURAL THROMBUS FORMATION AND INTIMALTHICKENING FOLLOWING INTIMALINJURY
Tohru HAYASHI,Yujiro ASADA,Atsushi KISANUKI,Akinobu SUMIYOSHI The First Department of Pathology, Miyazaki Medical College, Miyazaki 889-16, Japan
(Received 22.7.1991; accepted in revised form 22.5.1992 by Editor A. Takada)
ABSTRACT Inhibitory effect of Y-20811 on platelet thrombus formation induced by mechanical intimal injury and subsequent intimal fibrous thickening was studied. In the short term experiment, polyethylene tubing was inserted into the rabbit aorta and was drawn out one hour after with or without Y-20811 administration, then the rabbits were sacrificed. In the experiment for the quantitative analysis of platelet adhesion, ‘lCr-labeled platelets were used. Radioactivities of 2cm length of the injured segment of the thoracic aorta and the proximal 2cm of the normal segment were measured. Radioactivity of the injured segment was significantly lower in rabbits treated with Y-2081 1 than the control ones. The mean thickness(area of thrombi/length of injured intima) and the maximal thickness of the mural thrombi in the Y-2081 l-treated rabbits were significantly lower than those in control rabbits. De-endothelialized area showed raised platelet thrombi in the control group and diffuse thin-layered platelet sheets in Y-20811treated rabbits. In the long term experiment, polyethylene tubing was indwelled for 24 hours. Rabbits were sacrificed 10 days after drawing out the tubing. Through the experiment Y-20811 was injected intravenously every 24 hours. There was no significant difference between the control group and the Y-20811-treated one in both mean thickness and maximal thickness of the intimal f ibromuscular thickening. The experiments indicate that Y-20811 has an inhibitory effect on platelet thrombus formation following intimal injurv. but subsequent myointimal thickening is not inhibited by the drug:
Key words:
Platelet platelet
thrombus, damaged aorta, drug
81
intimal thickening,
anti-
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INTRODUCTION Although platelets have an important role in hemostatic process particularly in arterial injury, they are also one of the most important constituents of thrombi which cause ischemic troubles in various organs. In addition to these acute physiological or pathological phenomena platelets are considered to participate in a process of mesenchymal cell chemotaxis and proliferation(l,2). In recent years various growth factors: platelet-derived growth factor, epidermal growth factor, transforming growth factor beta and of so on, are shown to be involved in the biologic process of proliferation ?esenchymal ? cells which are the main constituents of fibromuscular lesion of intima. From these aspects in the platelet functions, various antiplatelet drugs are used experimentally and clinically for the treatment and proph;l;;;;,of ischemic diseases of various organs(3-8). sodium 4-[a-hydroxy-5-(l-imidazolyl)-2-methylbenzyl]-3,5-diis known to have a long-lasting and selective methylbenzoaie dihydrate, inhibitory effect on thromboxane AZ synthetase and brings about following : inhibition of platelet aggregation, antithrombotic biological effects effects of coronary artery(9-12). The present effect, and antivasospastic report shows the result of quantitative radioisotopic and morphometrical analyses of an antithrombotic effect of Y-20811, which was kindly supplied from Yoshitomi Pharmaceutical Industries, Ltd., Fukuoka, Japan and a minimizing effect of the drug on intimal fibrous thickening following aortic intimal injury. MATERIALS & METHODS QUANTITATIVE RADIOISOTOPIC ANALYSISOF AN ANTITHROMROTIC EFFECT: A preparation of slCr-labeled platelets was carried out according to the methods of Ardlie et a1.(13) and Groves et al.(l4) with a few modification. Rabbit platelets were separated from about 80 to 1Ooml of whole blood with 14% of citric acid as an anticoagulant. This was resuspended in the modified Tyrode’s solution lacking for calcium and containing 2.OmMMgC12, 0.2mM EDTA and 0.35% bovine albumin. Platelets were labeled in this suspension for 60 minutes at room temperature with 15OpCi Naas1Cr04(New England Nuclear, Canada Ltd., Quebec ;200-5OO#X/~g Cr). Labeled platelets were washed with calciumfree Tyrode’s solution containing 2.OmMMgClaand 0.35% albumin, and finally resuspended in lOm#of Tyrode’s solution containing 0.35% bovine albumin. 1.8x 10’ Labeled platelets( 1.8x lOloin a volume of 9.5ti : radioacitivity, cpm) were intravenously injected into rabbits before intimal injury. The animals were anesthetized with intravenous administration of sodium pentobarbital 60 minutes after injection of labeled platelets. Intimal injuries were induced in the aorta by insertion of polyethylene tubing(PE50, Intramedic, Parsippany, U.S.A.) via the femoral artery as previously described (15). Evans blue dye solution(4.5% in saline, O.SmP/kg of body weight) was given intravenously 45 minutes after insertion of polyethylene tubing to visualize the injured sites. Sixty minutes after insertion of tubing, animals were heparinized with a dose of 5OOWkg and the polyethylene tubing was drawn out immediately. Subsequently rabbits were perfused with O.lM phosphate buffered saline, pH7.4. The aortas were then perfused by a combination fixative of 4% neutralized formaldehyde and 1% glutaraldehyde in a 200-mOsmphosphate buffer, pH7.4 for 1 to 2 minutes. After that the aortas were dissected out and opened lengthwise, pinned out, and fixed by immersion in the same fixative at 4“C for 12 hours. The thoracic aorta which showed intimal injury was dissected out in 2cm length downstream from the cardiac
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63
side-margin of the injured site(injured segment). Non-injured part of the aorta was also dissected out in 2cm length upstream from that margin (normal segmentj(Fig.1).
injured segment
normal segment
Fig. 1
Sampling method for the measurement of radioactivity
of damaged aorta.
The radioactivity of each segment was measured in a well type scintillation autogamma counter(Aloka ARC 360, Japan) after removing the adventitia. The area of each segment(cm*) was also measured morphometrically by using a Parsonal Image Analyse System, PIAS II(PIAS KK, Japan). Radioactivity of damaged aorta
of each rabbit
Radioactivity of damaged aorta = ( cpm/ cm*>
was calculated
Actual radioactivity of injured segment Area of the segment
as follows : Actual radioactivity of normal segment Area of the segment
The drug, Y-2081 1, was injected intravenously via the marginal ear vein the insertion in a dose of 3 .Omg/kg two times, 24 hours and 1 hour, before was injected in of the tubing in the experimental group. Placebo solution the same manner in the control group. This experiment was done with seven rabbits in each group.
MORPHOLOGICAL ANDMORPHOMETRICAL ANALYSES OF MURALTHROMRI: Rabbits were anesthetized with pentobarbital(25mg/kg, intravenously). One hour after the second administration of the drug or placebo solution, intimal injury was induced in the same manner as the experiment of the radioisotopic analysis. Sacrifice and fixation of the aorta were done in the same manner also. Five cross sectional tissue blocks at regular intervals of about lcm were obtained equally from each rabbit as shown in Fig.2, postfixed with 2% 0904 The in O.lM phosphate buffer, pH7.4, and embedded in Epon 812 as usual. residual aortic tissues were also post-fixed with 0~04, dehydrated with ethanol, critical-point-dried using liquid C02, sputter-coated with platinum and examined with a scanning electron microscope, Hitachi Sand palladium, 800(Hitachi, Japan).
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Fig.2
r.8. s.m.0. 1 \
u
-
- II 11 II (1 11 T5 T4T3 T2Tl
Samplingmethod for morphologicaland morphometricalanalyses of platelet thrombion the de-endothelialized intima(r.a.:renal artery, s.m.a.: superior mesentericartery).
One fl thick sections of Epon blocks were cut on a Reichert Ultracut microtome and stained with toluidineblue. All intimal lesions,mural platelet thombi, were photographedseriallyon color reversalfilm at a 33 magnifying power. These intimal lesions in photographswere further magnified 20 times on a screen and traced on papers. Area, basal length and maximal thickness of mural thrombi were measured morphometricallyby using PIAS II. Mean thickness of thrombus in each section was calculated by a following formula(l6): Mean thickness(@) =
total area of thrombi(@*) basal length of thrombi(&
Ultrathin sectionsof some blocks were stained with uranium and lead as usual and were examinedwith a JEOL-100stransmissionelectronmicroscope. The experimentwas done with 10 rabbits in each group, the experimental and control ones. MORPHOLOGICAL AND MORPHOMETRICAL ANALYSES OF INTIMAL THICKENING : The methods of inductionof intimal injury, administrationof drug and preparations of materials were the same as the former experiment.The drug and the placebo were injected two times, 24 hrs and 1 hr, before the intimal injury. In this experimentthe tubing was indwelledfor 24 hrs, and the animals were sacrificed 10 days after drawing out the tubing. The drug and the placebo were injected with 24 hrs interval through the experiment. Injured areas were still colored blue being rimmed by faintly colored and slightly elevated lesions. Area, basal length and maximal thickness of the thickened intima were measured by the same method as the former experiment.The mean thicknessof thickenedintima were calculatedby the same formula. In this experiment,10 rabbits in each group were used. Male New Zealand white rabbits weighing 2.0 to 2.8 kg were used in all these experiments. All these data were expressed as the mean + S.D. Student's t-test was used for statisticalanalysis. RESULTS QUANTITATIVE RADIOISOTOPICANALYSIS : Platelets labeled with '%r in this method retained the good function of aggregationwith an aggregometer in which 17.5/rg/ml(final concentration)of collagen was used as an agonist. More than 50% of the radioactivityof the blood taken 45 minutes after the injection of labeled platelets was observed in a platelet fraction. The pattern and extent of intimal injury estimated by discolorationof Evans blue dye were roughly similar in all animals (Fig.1).
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Fig.3 Radioactivity
Control
of damaged aorta.
Y-20811
Fig.4 Representative microscopic features control group(A) and Y-20811-treated toluidine blue, x 125)
of a mural platelet thrombus in the group(B)(Thick sections stained with
88
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The actual radioactivities of normal and injured aortic segments in the control group were 31f5 and 105f54cpm/cm2 with a significant difference between them. Those in the experimental group were 29 f 2 and 47 f 14 cpm/cm* The radioactivity of damaged aorta in also with a significant difference. the control group was 74+54cpm/cm2 and that in the experimental one was
Fig.5
Repr‘esentative scanning electron microscopic features of de-endothelialixed areaL in the control group(A,C) and in Y-20811-treated group(B,D)(A,B :x240, C,D :x2,000).
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18+14cpm/cm2 which were also
significantly
different
87
from each other(Fig.3).
MORPHOLOGICAL AND MORPHOMETRICAL ANALYSESOF MURALTHROMBI: The extent and shape of injured intima which was discolored by Evans blue dye were similar to those in the experiment of radioisotopic analysis(Fig.1). The internal elastic lamina was intact by light and electron microscopic examination. The injured area showing desquamation of endothelial cells was accompanied by different sizes of mural platelet thrombi. Platelets were never found to adhere to the intact endothelium. Platelets showed varying degrees of pseudopod extensions conglomerating with each other, adherence to the subendothelial tissue and degranulation(Figs.4,5,6). Although the amount and extent of raised platelet thrombi varied widely from segment to segment, the largest thrombi were observed at the areas where the tip of tubing had
Fig.6 transmission electron microscopic features of a mural Repre sentative group(A) and in Y-20811-treated group(B)(X7,2( throw lbus in the control lamina. Inter nal elastic
30).
E:
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stayed in the thoracic aortas in the control group. With the scanning electron microscope there were observed raised thrombi showing aggregation of abundant platelets in the control group(Fig.5). In the experimental group, the injured area was covered by thin carpet-like sheets of platelets intermingled with a few white blood cells. Some aortas showed raised thrombi, but their sixes were not so large as those seen in the control group. De-endothelialized area without a platelet covering was also seen in the experimental group. With the transmission electron microscope varying degrees of shape change, pseudopod formation and degranulation were observed in the basal part of raised thrombi and fibrin thread formation was observed among aggregated platelets in the control group. In the experimental group, the thickness of mural thrombi was only one to several layers of platelets showing a rather well preservation of its shape and with granules in the cytoplasm and fibrin threads were observed only in part(Fig.6). The data of morphometrical analysis of mural thrombi are shown in Fig. 7. The maximal thickness of mural thrombi in the experimental group were significantly smaller than that in the control group in T2 segment and the total of Tl plus T2 segments(P< 0.05). No significant difference was observed among Tl , T3, T4 and T5. The difference was the same in the mean thickness(Fig.7).
i
0
Control
=
Experiment
F-L 12
T3
14
TS
Fig.7
Maximal and mean thickness of thrombi measured by the morphometrical method.
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MORPHOLOGICALAND MORPHOMETRICAL ANALYSES OF INTIMAL THICKENING : The injured intima at 10 days after removing the tubing no longer had thrombi and showed intimal fibrous thickening composed of spindle-shaped cells probably derived from medial smooth muscle cells. They showed parallel arrangement to the internal elastic lamina in upper two thirds of the layer With the transmission electron and vertical arrangement in a lower third. various amounts of collagen and elastic fibers were observed microscope, The luminal surface was covered with endothelium-like among the cells. flattened cells without de-endothelialized lesion or platelet accumulation. The intimal thickening was most prominent at the periphery of the lesions in which more than ten layers of smooth muscle cells were observed. The lesion showed gradual thinning to only two or three layers of the spindle-shaped There was no morphological cells at the central part of thickened areas. difference in the intimal thickening between two groups, the control and experimental ones. The data of morphometrical analysis of intimal thickening are shown in Fig.8. There was no significant difference in the degree of intimal thickening both in the maximal and the mean thickness even in the T2 segment at which mural thrombi showed the most significant difference between the two The mean thickness of the intima was groups in the short term experiment. greater than that of mural thrombi in each segment and also in combined segments.
0
control
I
Experiment
Fig.8 Maximal and mean thickness method.
of the intima measured by the morphometrical
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DISCUSSION In the pathogenesis of acute ?yocardial ? infarction, coronary thrombosis with or without plaque fissuring or rupture is considered to be important (17). This is verified by various antithrombotic therapy. Not only for the treatment or the prevention of the disease, anti-thrombotic therapy has also been challenged for the prevention of restenosis of affected coronary arteries after percutaneous transluminal coronary angioplasty(PTCA). This comes from the consideration that platelets are the most important blood constituents in the initiation and promotion of atherosclerosis(l8). Intimal injury is followed by varying degrees of localized intimal fibrous thickening and sometimes by characteristic atherosclerotic lesions seen in human(19,20). There are several reports which reveal that the inhibitory effect on platelets or the reduction of their number results in the reduction of the degree of intimal thickening(21,22). However, there are also several reports showing the contradictory results(28,24) Although various clinical trials have demonstrated a protective effect of antiplatelet agents on acute events complicating PTCA(5,25), a reduced incidence of restenosis has not been documented so far. There are only a few studies which precisely evaluate the grade or the amount of mural thrombi and the degree of intimal thickening referring to their relationship in the same experimental model. In this study we investigated the efficacy of an antiplatelet drug, Y-2081 1, in the prophylaxis of thrombosis and subsequent intimal hyperplasia in the same experimental model and with the same morphometrical method. Many procedures are employed to introduce intimal injury for production of mural thrombi and subsequent intimal fibromuscular thickening, for example, balloon catheter, air drying(20), administration of homocystine(26) or immune complexes, cuff methods, and so on. We used here the method of indwelling of polyethylene tubing in the thoracic aorta to introduce intimal in jury, because this procedure can produce fairly well-controlled intimal lesions on the well-controlled site of the aortic intima without interruption of the internal elastic lamina(27). The short term experiments revealed the efficacy of Y-20811 in the inhibition of raised mural thrombus formation following mechanical intimal injury. The agent has an inhibitory effect on rabbit platelet aggregation induced by arachidonic acid(AA) in vitro(g). The efficacy comes from its inhibitory effect on thromboxane synthetase, which is known from the in vivo experiment showing the reduction of serum TXB2 levels with increasing 6-keto PGFla. The dose of the agent is the same as in our experiment, lmg/kg/day, with which the biological effects are not discontinued. The long term experiment revealed that Y-20811 has no significant inhibitory effect on the intimal fibromuscular thickening after the mechanical injury. Even in the T2 segment and in Tl+T2 segments which showed a significant reduction of thrombus formation in the short term experiment, the myointimal thickening was observed without a significant difference from the control group. In experiments reported here, we measured the size of platelet thrombi using histometrical methods. Light- and electron-microscopical examinations revealed that the mural thrombi formed in this experimental model were al?OSt ? exclusively composed of platelets associated with polymerized fibrin in part. Therefore, the size of mural thrombi may roughly represent the number of platelets adhered and aggregated to the injured intima. These indicate that the grade of intimal fibrous thickening is not directly proportional to the number of platelets accumulated onto the injured intima.
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Platelet derived growth factor(PDGF), which is released from activated platelets, is considered to be one of the most important factors for vascular smooth muscle cell proliferation in in vitro experiment, but the amount The least of PDGFrequired for the proliferation in vivo is not hown(28). amount of PDGF supplied from only one layer of adhered platelets may be sufficient to stimulate the migration and the proliferation of smooth muscle cells. Although there were observed de-endothelialized areas without platelet adhesion, the most part showed thin-layered platelet adhesion intermingled with a few white blood cells in the experimental group. The drug does not seem to inhibit platelet adhesion to the subendothelial tissue. This was the same as that in the experiments with dilazep(l5,16,27) and with aspirin(29). Alternatively, medial smooth muscle cells may not require the adhesion of platelets to the injured site for their migration into and proliferation in the intima in in vivo experimental model. Guyton et al. referred to the possibility of participation of products of inflammatory cells in smooth muscle cell proliferation(24). In the pathogenesis of atherosclerosis various growth factors are considered to participate in smooth muscle cell proliferation with either paracrine or autocrine manner. The most important factor is PDGF, the synthesis of which by various cells is confirmed, in platelets, macrophages(30,31), endothelial cells(32,33) and smooth muscle cells themselves(34,35). Monocytes/macrophages may be more important cells as a source of PDGFthan platelets because activated macrophages may have a continuous production and secretion of growth factor locally in the injured site over a prolonged time course in contrast to the bursting release of PDGFby platelets(36). Regenerative endothelial cells covering the injured site may participate in the smooth muscle cell proliferation in the intimal layer. Minick et al. stated that the intimal thickening following the removal of endothelium from the aorta with a balloon catheter is more prominent in re-endothelialized areas than in the adjacent intima lacking the endothelial lining in both hyper- and normocholesterolemic rabbits (37). Factors other than cellular elements may influence smooth muscle cell proliferation, such as fibrin or fibrin degradation products which show either a stimulatory or an inhibitory effect(38). These multiple factors are considered to blur the distinction between the control and experimental groups in our long term experiment. Thus, the actual mechanisms of growth regulation of cells in the blood vessel wall are undoubtedly much more complex in vivo. From these considerations inhibition of mural platelet thrombi is considered not to be sufficient to prevent the intimal thickening after the intimal injury and thrombosis. In conclusion, Y-20811, an inhibitor of TXA2 synthetase, showed an anti-platelet effect on its aggregation in vivo. Myointimal thickening following intimal injury, however, could not be inhibited significantly in this experimental model. In the preventive treatment against myointimal thickening and atherosclerosis associated with various troubles such as restenosis after PTCA, other approaches should be taken in consideration together with the antiplatelet therapy.
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EFFECTS OF ANTITHROMBOTIC DRUG,Y-20811 ON MURAL THROMBUS FORMATION AND INTIMALTHICKENING FOLLOWING INTIMALINJURY
Tohru HAYASHI,Yujiro ASADA,Atsushi KISANUKI,Akinobu SUMIYOSHI The First Department of Pathology, Miyazaki Medical College, Miyazaki 889-16, Japan
(Received 22.7.1991; accepted in revised form 22.5.1992 by Editor A. Takada)
ABSTRACT Inhibitory effect of Y-20811 on platelet thrombus formation induced by mechanical intimal injury and subsequent intimal fibrous thickening was studied. In the short term experiment, polyethylene tubing was inserted into the rabbit aorta and was drawn out one hour after with or without Y-20811 administration, then the rabbits were sacrificed. In the experiment for the quantitative analysis of platelet adhesion, ‘lCr-labeled platelets were used. Radioactivities of 2cm length of the injured segment of the thoracic aorta and the proximal 2cm of the normal segment were measured. Radioactivity of the injured segment was significantly lower in rabbits treated with Y-2081 1 than the control ones. The mean thickness(area of thrombi/length of injured intima) and the maximal thickness of the mural thrombi in the Y-2081 l-treated rabbits were significantly lower than those in control rabbits. De-endothelialized area showed raised platelet thrombi in the control group and diffuse thin-layered platelet sheets in Y-20811treated rabbits. In the long term experiment, polyethylene tubing was indwelled for 24 hours. Rabbits were sacrificed 10 days after drawing out the tubing. Through the experiment Y-20811 was injected intravenously every 24 hours. There was no significant difference between the control group and the Y-20811-treated one in both mean thickness and maximal thickness of the intimal f ibromuscular thickening. The experiments indicate that Y-20811 has an inhibitory effect on platelet thrombus formation following intimal injurv. but subsequent myointimal thickening is not inhibited by the drug:
Key words:
Platelet platelet
thrombus, damaged aorta, drug
81
intimal thickening,
anti-
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INTRODUCTION Although platelets have an important role in hemostatic process particularly in arterial injury, they are also one of the most important constituents of thrombi which cause ischemic troubles in various organs. In addition to these acute physiological or pathological phenomena platelets are considered to participate in a process of mesenchymal cell chemotaxis and proliferation(l,2). In recent years various growth factors: platelet-derived growth factor, epidermal growth factor, transforming growth factor beta and of so on, are shown to be involved in the biologic process of proliferation ?esenchymal ? cells which are the main constituents of fibromuscular lesion of intima. From these aspects in the platelet functions, various antiplatelet drugs are used experimentally and clinically for the treatment and proph;l;;;;,of ischemic diseases of various organs(3-8). sodium 4-[a-hydroxy-5-(l-imidazolyl)-2-methylbenzyl]-3,5-diis known to have a long-lasting and selective methylbenzoaie dihydrate, inhibitory effect on thromboxane AZ synthetase and brings about following : inhibition of platelet aggregation, antithrombotic biological effects effects of coronary artery(9-12). The present effect, and antivasospastic report shows the result of quantitative radioisotopic and morphometrical analyses of an antithrombotic effect of Y-20811, which was kindly supplied from Yoshitomi Pharmaceutical Industries, Ltd., Fukuoka, Japan and a minimizing effect of the drug on intimal fibrous thickening following aortic intimal injury. MATERIALS & METHODS QUANTITATIVE RADIOISOTOPIC ANALYSISOF AN ANTITHROMROTIC EFFECT: A preparation of slCr-labeled platelets was carried out according to the methods of Ardlie et a1.(13) and Groves et al.(l4) with a few modification. Rabbit platelets were separated from about 80 to 1Ooml of whole blood with 14% of citric acid as an anticoagulant. This was resuspended in the modified Tyrode’s solution lacking for calcium and containing 2.OmMMgC12, 0.2mM EDTA and 0.35% bovine albumin. Platelets were labeled in this suspension for 60 minutes at room temperature with 15OpCi Naas1Cr04(New England Nuclear, Canada Ltd., Quebec ;200-5OO#X/~g Cr). Labeled platelets were washed with calciumfree Tyrode’s solution containing 2.OmMMgClaand 0.35% albumin, and finally resuspended in lOm#of Tyrode’s solution containing 0.35% bovine albumin. 1.8x 10’ Labeled platelets( 1.8x lOloin a volume of 9.5ti : radioacitivity, cpm) were intravenously injected into rabbits before intimal injury. The animals were anesthetized with intravenous administration of sodium pentobarbital 60 minutes after injection of labeled platelets. Intimal injuries were induced in the aorta by insertion of polyethylene tubing(PE50, Intramedic, Parsippany, U.S.A.) via the femoral artery as previously described (15). Evans blue dye solution(4.5% in saline, O.SmP/kg of body weight) was given intravenously 45 minutes after insertion of polyethylene tubing to visualize the injured sites. Sixty minutes after insertion of tubing, animals were heparinized with a dose of 5OOWkg and the polyethylene tubing was drawn out immediately. Subsequently rabbits were perfused with O.lM phosphate buffered saline, pH7.4. The aortas were then perfused by a combination fixative of 4% neutralized formaldehyde and 1% glutaraldehyde in a 200-mOsmphosphate buffer, pH7.4 for 1 to 2 minutes. After that the aortas were dissected out and opened lengthwise, pinned out, and fixed by immersion in the same fixative at 4“C for 12 hours. The thoracic aorta which showed intimal injury was dissected out in 2cm length downstream from the cardiac
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63
side-margin of the injured site(injured segment). Non-injured part of the aorta was also dissected out in 2cm length upstream from that margin (normal segmentj(Fig.1).
injured segment
normal segment
Fig. 1
Sampling method for the measurement of radioactivity
of damaged aorta.
The radioactivity of each segment was measured in a well type scintillation autogamma counter(Aloka ARC 360, Japan) after removing the adventitia. The area of each segment(cm*) was also measured morphometrically by using a Parsonal Image Analyse System, PIAS II(PIAS KK, Japan). Radioactivity of damaged aorta
of each rabbit
Radioactivity of damaged aorta = ( cpm/ cm*>
was calculated
Actual radioactivity of injured segment Area of the segment
as follows : Actual radioactivity of normal segment Area of the segment
The drug, Y-2081 1, was injected intravenously via the marginal ear vein the insertion in a dose of 3 .Omg/kg two times, 24 hours and 1 hour, before was injected in of the tubing in the experimental group. Placebo solution the same manner in the control group. This experiment was done with seven rabbits in each group.
MORPHOLOGICAL ANDMORPHOMETRICAL ANALYSES OF MURALTHROMRI: Rabbits were anesthetized with pentobarbital(25mg/kg, intravenously). One hour after the second administration of the drug or placebo solution, intimal injury was induced in the same manner as the experiment of the radioisotopic analysis. Sacrifice and fixation of the aorta were done in the same manner also. Five cross sectional tissue blocks at regular intervals of about lcm were obtained equally from each rabbit as shown in Fig.2, postfixed with 2% 0904 The in O.lM phosphate buffer, pH7.4, and embedded in Epon 812 as usual. residual aortic tissues were also post-fixed with 0~04, dehydrated with ethanol, critical-point-dried using liquid C02, sputter-coated with platinum and examined with a scanning electron microscope, Hitachi Sand palladium, 800(Hitachi, Japan).
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Fig.2
r.8. s.m.0. 1 \
u
-
- II 11 II (1 11 T5 T4T3 T2Tl
Samplingmethod for morphologicaland morphometricalanalyses of platelet thrombion the de-endothelialized intima(r.a.:renal artery, s.m.a.: superior mesentericartery).
One fl thick sections of Epon blocks were cut on a Reichert Ultracut microtome and stained with toluidineblue. All intimal lesions,mural platelet thombi, were photographedseriallyon color reversalfilm at a 33 magnifying power. These intimal lesions in photographswere further magnified 20 times on a screen and traced on papers. Area, basal length and maximal thickness of mural thrombi were measured morphometricallyby using PIAS II. Mean thickness of thrombus in each section was calculated by a following formula(l6): Mean thickness(@) =
total area of thrombi(@*) basal length of thrombi(&
Ultrathin sectionsof some blocks were stained with uranium and lead as usual and were examinedwith a JEOL-100stransmissionelectronmicroscope. The experimentwas done with 10 rabbits in each group, the experimental and control ones. MORPHOLOGICAL AND MORPHOMETRICAL ANALYSES OF INTIMAL THICKENING : The methods of inductionof intimal injury, administrationof drug and preparations of materials were the same as the former experiment.The drug and the placebo were injected two times, 24 hrs and 1 hr, before the intimal injury. In this experimentthe tubing was indwelledfor 24 hrs, and the animals were sacrificed 10 days after drawing out the tubing. The drug and the placebo were injected with 24 hrs interval through the experiment. Injured areas were still colored blue being rimmed by faintly colored and slightly elevated lesions. Area, basal length and maximal thickness of the thickened intima were measured by the same method as the former experiment.The mean thicknessof thickenedintima were calculatedby the same formula. In this experiment,10 rabbits in each group were used. Male New Zealand white rabbits weighing 2.0 to 2.8 kg were used in all these experiments. All these data were expressed as the mean + S.D. Student's t-test was used for statisticalanalysis. RESULTS QUANTITATIVE RADIOISOTOPICANALYSIS : Platelets labeled with '%r in this method retained the good function of aggregationwith an aggregometer in which 17.5/rg/ml(final concentration)of collagen was used as an agonist. More than 50% of the radioactivityof the blood taken 45 minutes after the injection of labeled platelets was observed in a platelet fraction. The pattern and extent of intimal injury estimated by discolorationof Evans blue dye were roughly similar in all animals (Fig.1).
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Fig.3 Radioactivity
Control
of damaged aorta.
Y-20811
Fig.4 Representative microscopic features control group(A) and Y-20811-treated toluidine blue, x 125)
of a mural platelet thrombus in the group(B)(Thick sections stained with
88
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The actual radioactivities of normal and injured aortic segments in the control group were 31f5 and 105f54cpm/cm2 with a significant difference between them. Those in the experimental group were 29 f 2 and 47 f 14 cpm/cm* The radioactivity of damaged aorta in also with a significant difference. the control group was 74+54cpm/cm2 and that in the experimental one was
Fig.5
Repr‘esentative scanning electron microscopic features of de-endothelialixed areaL in the control group(A,C) and in Y-20811-treated group(B,D)(A,B :x240, C,D :x2,000).
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18+14cpm/cm2 which were also
significantly
different
87
from each other(Fig.3).
MORPHOLOGICAL AND MORPHOMETRICAL ANALYSESOF MURALTHROMBI: The extent and shape of injured intima which was discolored by Evans blue dye were similar to those in the experiment of radioisotopic analysis(Fig.1). The internal elastic lamina was intact by light and electron microscopic examination. The injured area showing desquamation of endothelial cells was accompanied by different sizes of mural platelet thrombi. Platelets were never found to adhere to the intact endothelium. Platelets showed varying degrees of pseudopod extensions conglomerating with each other, adherence to the subendothelial tissue and degranulation(Figs.4,5,6). Although the amount and extent of raised platelet thrombi varied widely from segment to segment, the largest thrombi were observed at the areas where the tip of tubing had
Fig.6 transmission electron microscopic features of a mural Repre sentative group(A) and in Y-20811-treated group(B)(X7,2( throw lbus in the control lamina. Inter nal elastic
30).
E:
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stayed in the thoracic aortas in the control group. With the scanning electron microscope there were observed raised thrombi showing aggregation of abundant platelets in the control group(Fig.5). In the experimental group, the injured area was covered by thin carpet-like sheets of platelets intermingled with a few white blood cells. Some aortas showed raised thrombi, but their sixes were not so large as those seen in the control group. De-endothelialized area without a platelet covering was also seen in the experimental group. With the transmission electron microscope varying degrees of shape change, pseudopod formation and degranulation were observed in the basal part of raised thrombi and fibrin thread formation was observed among aggregated platelets in the control group. In the experimental group, the thickness of mural thrombi was only one to several layers of platelets showing a rather well preservation of its shape and with granules in the cytoplasm and fibrin threads were observed only in part(Fig.6). The data of morphometrical analysis of mural thrombi are shown in Fig. 7. The maximal thickness of mural thrombi in the experimental group were significantly smaller than that in the control group in T2 segment and the total of Tl plus T2 segments(P< 0.05). No significant difference was observed among Tl , T3, T4 and T5. The difference was the same in the mean thickness(Fig.7).
i
0
Control
=
Experiment
F-L 12
T3
14
TS
Fig.7
Maximal and mean thickness of thrombi measured by the morphometrical method.
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MORPHOLOGICALAND MORPHOMETRICAL ANALYSES OF INTIMAL THICKENING : The injured intima at 10 days after removing the tubing no longer had thrombi and showed intimal fibrous thickening composed of spindle-shaped cells probably derived from medial smooth muscle cells. They showed parallel arrangement to the internal elastic lamina in upper two thirds of the layer With the transmission electron and vertical arrangement in a lower third. various amounts of collagen and elastic fibers were observed microscope, The luminal surface was covered with endothelium-like among the cells. flattened cells without de-endothelialized lesion or platelet accumulation. The intimal thickening was most prominent at the periphery of the lesions in which more than ten layers of smooth muscle cells were observed. The lesion showed gradual thinning to only two or three layers of the spindle-shaped There was no morphological cells at the central part of thickened areas. difference in the intimal thickening between two groups, the control and experimental ones. The data of morphometrical analysis of intimal thickening are shown in Fig.8. There was no significant difference in the degree of intimal thickening both in the maximal and the mean thickness even in the T2 segment at which mural thrombi showed the most significant difference between the two The mean thickness of the intima was groups in the short term experiment. greater than that of mural thrombi in each segment and also in combined segments.
0
control
I
Experiment
Fig.8 Maximal and mean thickness method.
of the intima measured by the morphometrical
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DISCUSSION In the pathogenesis of acute ?yocardial ? infarction, coronary thrombosis with or without plaque fissuring or rupture is considered to be important (17). This is verified by various antithrombotic therapy. Not only for the treatment or the prevention of the disease, anti-thrombotic therapy has also been challenged for the prevention of restenosis of affected coronary arteries after percutaneous transluminal coronary angioplasty(PTCA). This comes from the consideration that platelets are the most important blood constituents in the initiation and promotion of atherosclerosis(l8). Intimal injury is followed by varying degrees of localized intimal fibrous thickening and sometimes by characteristic atherosclerotic lesions seen in human(19,20). There are several reports which reveal that the inhibitory effect on platelets or the reduction of their number results in the reduction of the degree of intimal thickening(21,22). However, there are also several reports showing the contradictory results(28,24) Although various clinical trials have demonstrated a protective effect of antiplatelet agents on acute events complicating PTCA(5,25), a reduced incidence of restenosis has not been documented so far. There are only a few studies which precisely evaluate the grade or the amount of mural thrombi and the degree of intimal thickening referring to their relationship in the same experimental model. In this study we investigated the efficacy of an antiplatelet drug, Y-2081 1, in the prophylaxis of thrombosis and subsequent intimal hyperplasia in the same experimental model and with the same morphometrical method. Many procedures are employed to introduce intimal injury for production of mural thrombi and subsequent intimal fibromuscular thickening, for example, balloon catheter, air drying(20), administration of homocystine(26) or immune complexes, cuff methods, and so on. We used here the method of indwelling of polyethylene tubing in the thoracic aorta to introduce intimal in jury, because this procedure can produce fairly well-controlled intimal lesions on the well-controlled site of the aortic intima without interruption of the internal elastic lamina(27). The short term experiments revealed the efficacy of Y-20811 in the inhibition of raised mural thrombus formation following mechanical intimal injury. The agent has an inhibitory effect on rabbit platelet aggregation induced by arachidonic acid(AA) in vitro(g). The efficacy comes from its inhibitory effect on thromboxane synthetase, which is known from the in vivo experiment showing the reduction of serum TXB2 levels with increasing 6-keto PGFla. The dose of the agent is the same as in our experiment, lmg/kg/day, with which the biological effects are not discontinued. The long term experiment revealed that Y-20811 has no significant inhibitory effect on the intimal fibromuscular thickening after the mechanical injury. Even in the T2 segment and in Tl+T2 segments which showed a significant reduction of thrombus formation in the short term experiment, the myointimal thickening was observed without a significant difference from the control group. In experiments reported here, we measured the size of platelet thrombi using histometrical methods. Light- and electron-microscopical examinations revealed that the mural thrombi formed in this experimental model were al?OSt ? exclusively composed of platelets associated with polymerized fibrin in part. Therefore, the size of mural thrombi may roughly represent the number of platelets adhered and aggregated to the injured intima. These indicate that the grade of intimal fibrous thickening is not directly proportional to the number of platelets accumulated onto the injured intima.
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Platelet derived growth factor(PDGF), which is released from activated platelets, is considered to be one of the most important factors for vascular smooth muscle cell proliferation in in vitro experiment, but the amount The least of PDGFrequired for the proliferation in vivo is not hown(28). amount of PDGF supplied from only one layer of adhered platelets may be sufficient to stimulate the migration and the proliferation of smooth muscle cells. Although there were observed de-endothelialized areas without platelet adhesion, the most part showed thin-layered platelet adhesion intermingled with a few white blood cells in the experimental group. The drug does not seem to inhibit platelet adhesion to the subendothelial tissue. This was the same as that in the experiments with dilazep(l5,16,27) and with aspirin(29). Alternatively, medial smooth muscle cells may not require the adhesion of platelets to the injured site for their migration into and proliferation in the intima in in vivo experimental model. Guyton et al. referred to the possibility of participation of products of inflammatory cells in smooth muscle cell proliferation(24). In the pathogenesis of atherosclerosis various growth factors are considered to participate in smooth muscle cell proliferation with either paracrine or autocrine manner. The most important factor is PDGF, the synthesis of which by various cells is confirmed, in platelets, macrophages(30,31), endothelial cells(32,33) and smooth muscle cells themselves(34,35). Monocytes/macrophages may be more important cells as a source of PDGFthan platelets because activated macrophages may have a continuous production and secretion of growth factor locally in the injured site over a prolonged time course in contrast to the bursting release of PDGFby platelets(36). Regenerative endothelial cells covering the injured site may participate in the smooth muscle cell proliferation in the intimal layer. Minick et al. stated that the intimal thickening following the removal of endothelium from the aorta with a balloon catheter is more prominent in re-endothelialized areas than in the adjacent intima lacking the endothelial lining in both hyper- and normocholesterolemic rabbits (37). Factors other than cellular elements may influence smooth muscle cell proliferation, such as fibrin or fibrin degradation products which show either a stimulatory or an inhibitory effect(38). These multiple factors are considered to blur the distinction between the control and experimental groups in our long term experiment. Thus, the actual mechanisms of growth regulation of cells in the blood vessel wall are undoubtedly much more complex in vivo. From these considerations inhibition of mural platelet thrombi is considered not to be sufficient to prevent the intimal thickening after the intimal injury and thrombosis. In conclusion, Y-20811, an inhibitor of TXA2 synthetase, showed an anti-platelet effect on its aggregation in vivo. Myointimal thickening following intimal injury, however, could not be inhibited significantly in this experimental model. In the preventive treatment against myointimal thickening and atherosclerosis associated with various troubles such as restenosis after PTCA, other approaches should be taken in consideration together with the antiplatelet therapy.
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