Radiation induced alterations in rabbit aortic prostacyclin formation

PROSTAGLANDINS

RADIATION

INDUCED

ALTERATIONS

IN

RABBIT

FORMATION

H.SINZINGER',

W.FIRBAS*

AORTIC

PROSTACYCLIN

“1

and M. CROMWELL I*)

Atherosclerosis Research Group (ASF) at the Department of Medical Physiology (1) and 1st Department of Anatomy (2), University of Vienna,Austria ABSTRACT Earlier studies have demonstrated very severe vascular lesions occuring after irradiation in human and experimental animals as well. We examined the rabbit aortic PGI,-formation using the platelet bioassay technique in one year aged rabbits after different irradiation doses and a time course. The findings demonstrate a significant increase in prostacyclin formation, which might be due to the damage of endothelial cells. This stage is followed by a long lasting severe depression. This behaviour in addition demonstrates a dose dependent manner, The findings of a long lasting decrease in vascular PGI,-formation might contribute to the understanding of the high incidence of vascular lesions being found at the site of irradiation. INTRODUCTION Some decades ago it has been reported (l-3) that at the site of irradiation very severe atherosclerotic lesions might occur as seen during human autopsy. Experimental studies (4-6) revealed more detailed information on this type of damage, mainly of the endothelial cells (7-9) and the elastic material (10). In an earlier investigation series, one of us (HS) had a look on morphological and cell-cinetic alterations after irradiation of rabbit abdominal aorta (ll13). Thus it was of interest, whether the very severe changes observed morphologically might be accompanied or probably preceeded by a change in local prostacyclin formation. MATERIAL

AND METHODS

One year aged rabbits were used for this study. The animals were divided into groups each containing 4 animals (experimental design see figure 1). The irradiation was done with a Gammatron (Fa. Siemens). The animals were irradiated with doses ranging from 1000 up to 5000 rad in an area of 10xlOcm in the region of abdominal aorta. The time intervals before killing the animals are given in figure 1. Fo the animals were anaesthetized with thiopcnta

*) MC is a Research

fellow

supported

by the ASF

") This study was granted by the Atherosclerosis and Thrombosis Research Group of the Austrian Academy of Sciences Reprint

requests

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to: HS, ASF, A-1090

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PROSTAGLANDINS

The same was performed with the control animals in order to exclude the drug effect. All animals received 100 units heparin 30 minutes prior killing to prevent thrombin induced PGI, -stimulation. One hour before killing 1 mCi 3H-thymidine (spec. activity 2,O Ci/mmol, 8,3 Ci/mg, radioact. cont. 1 mCi/ ml) was given into the ear vein. The animals were killed with a blow on their neck. After immediate laparatomy irradiated abdominal aortic segment and as control throacic aortic segments were stored in liquid nitrogen (- 70°C) until examination.

c

sham irradiation

f irradiation

1,2,... 1000, 2000 rad.... 4 animals each interval and group Fig.

1. Irradiation

$! killing (total 188)

scheme and number

of animals

studied

Prostacyclin estimation was performed using human platelet rich plasma anticoagulated with 3,8% sodium citrate. The aggregation induction was done with IMM ADP in 600~1 platelet rich plasma samples. The inhibition of ADP-induced aggregation was quantified by a standard (14) kindly supplied by Dr. John E. Pike, The Upjohn Company, Kalamazoo, Michigan, USA. The incubation of vascular tissue was done in 300~1 of tris-WC1 buffer (pH 7,4). The PGI,-synthesis is given in pg/mg tissue wet weight per minute. For light microscopical investigation the vascular tissue was immersion-fixed in buffered formaldehyde (phosphate buffer, pH 7,4) and embedded in paraffin. The 3H-thymidine uptake in the wall layers was examined using the dipping-method using a Kodak NTB 2 (Eastman Kodak Comp., USA) emulsion with an exposition period between 15 and 30 days. RESULTS Light microscopic investigation demonstrates a very severe damage of endothelial cells increasing with the radiation dose (especially during the first 2 hours),resulting in cellular detachment. In the media focal necrosis and destruction of elastic material can be found.

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Studying the vascular PGI,-formation one hour after irradiation a significant increase to about the double of the control values can be found in the irradiated abdominal aorta. This increase was found to be in a dose dependent manner (table I). The highest actual values for PGI,-formation are observed between 2 and 4 hours after irradiation. In the first 2 hours the dose dependent increase in PGI,-synthesis is more pronounced, leading to a more severe exhaustion phenomenon in the high-dose groups in the longest observation periods. Thereafter, between 24 and 48 hours the PGI,-geneTABLE 1 hrs

3000

controls

4000

5000

46,824,l 55,3+5,4 57,1?4,7 *** *** *** 2a,5+4,0 3l,3+2,9 33,224,0

AA

31,3+2,6

AT

29,7?3,1

AA

32,4+1,9

AT

30,6+2,3

AA

31,5?2,6

AT

32,4+3,4

AA

32,4+3,1

AT

33,5+2,a

AA

31,6:2,4

AT

30,5+3,6

39,9+3,4 * 30,4+3,1

AA

30,9+3,7

34,7+3,6

AT

28,623,0

32,4+3,3

AA

31,523,2

AT

31,7+2,7

16,1+3,6 *** 28,453,6

AA

27,823,1

AT

29,0+4,1

I

5a,3+4,5 55,623,a 63,6+3,2 71,7+6,2 El4,7+4,5 *** *** *** *** *** 33,4+2,4 34,7+3,0 31,821,7 34,3+3,1 32,7+2,0 52,4+3,3 57,1+4,5 61,0+3,6 62,4+2,9 54,3+6,1 *** *** *** *** *** 30,622,a 32,5+l,a 33,9+3,9 34,722,a 30,6?3,0 I

44,2+3,3 46,3+5,1 50,3+4,7 53,1+4,0 55,a+4,4 *** *** *** ** * 29,3+3,6 29,6+2,8 33,123,5 36,3+4,6 30,7+3,3

24

48

168

336

17,3I3,3 22,9+3,1 20,7+2,5 15,3+2,7 l7,8+3,7 ** ** *** : 28,624,8 30,6?6,3 26,8-5,6 25,3+4,9 27,1+4,4

table 1: prostacyclin formation in rabbit abdominal (AA) and thoracic aorta (plT)after various time intervals (hours) in pg PGI,/mg/min in x + SEM; * plO,Ol; ** pCO,OO5 *** pLo,ool. Radiation dose in rad.

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ration in irradiated and non-irradiated vessels was nearly comparable. 168 - 336 hours after a single irradiation there occurs in most of the groups a very severe decrease in PGI,formation of the abdominal aorta, whereas nearly no change can be followed up in the thoracic segment serving as an internal control. Only 336 hours after irradiation there is also in the thoracic aortic segment (at higher radiation dose) a moderate trend towards a decrease in PGI,-synthesis, how-

TABLE hrs

Intima

co

1

Media

8

24

48

168

336

-

Adventitia

AA

0,47 + 0,21

1,20 + 0,63

1,43 + 0,57

AT

0,50 2 0,19

1,26 + 0,57

1,50 I 0,61

AA

1‘56 + 0,41

2,57 + 0‘64

3,74 z 1,36

AT

0,60 2 0,23

1,30 + 0,53

1,48 + 0157

AA

1,44 + 0,37

2,68 + 0,51

4,16 k 1,28

AT

0,66 + 0,24

I,34 + 0,67

1,70 + 1,12

2

4

2

AA

1,68 2 0,40

3,17 + 0,59

4,46 + 1,29

AT

0,71 + 0,31

1,44 + 0,47

1,60 + 1,34

AA

I,57 + 0,d'

3,31 + 0,86

5,87 + I,72

AT

0,67 + 0, ;(

I,67 + 0,58

1,65 + 0,62

AA

1,94 2 0,61

3,86 + I,27

6,97

AT

0,68 + 0,33

1,56 + 0,61

1,84 + 1.09 --

AA

2,06 + 0,69

4,03 + 1,17

6,83 + 1,74

AT

0,84 z 0,35

1,81 + 0,63

I,81 + 1,23

AA

2,41 2 0,79

4,67 + 1,33

7,23 + I,68

AT

0,78 + 0,34

1,56 + 0,56

1,83 + 1,42

AA

2,24 + 0,86

4,76 + I,14

6,83 + 1,84

AT

0,88 + 0,35

-

I

- i

1,71 2 0,59

-

1,68

1,81 + I,26

table 2: 'H-thymidine label uptake in the different wall layers of irradiat d abdominal and non-irradiated thoracic 0 aorta in x - SEM in animals exposed to 1000 rad.

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PROSTAGLANDINS ever, it never reaches in any of the groups the level of siIt is interesting to note, that the highest peak gnificance. can be seen using the highest irradiation doses (4000 and 5000 rad), consequently followed by the lowest levels after two weeks (table 1). Autoradiography revealed a very severe increase in uptake of 'H-thymidine (table 2) by the vascular cells of the different wall layers. In all the 3 layers the highest mitotic activity can be seen 1 week after single irradiation, decreasing moderately thereafter. Again, in the thoracic aortic segment there is a trend towards an enhanced mitotic activity being in none of the groups significant (table 2). DISCUSSION The findings of an increased PGI, -formation are paralleled by a very severe endothelial damage occuring in the early phase after irradiation. Thus the temporary increase in PGI*formation seems to be due to the cellular damage (Elder, 15). Previous studies after e lethal dose of whole body irradiation have been shown to cause increased levels of PGEz and PGF,alpha in Spraque-Dawley rats. Baluda (16) and co-workers reported on a decrease in PGI, -synthesis of abdominal aorta in rat and guinea pig after gamma-rays and fast neutrons. Hahn (17) found an increase in 6-keto-PGFlalpha release from pulmonary endothelial cells irradiated in vitro. In a very recent communication Schneidekraut (18) reported during the V. International Prostaglandin Conference an increase in urinary thromboxane B,-levels, whereas the 6-keto-PGF.,alpha-levels remained unchanged throughout the observation period. These findings obtained after whole body gamma-irradiation might contribute to the vascular pathology to a rather important degree. The changes observed 2 weeks after irradiation are much more pronounced as it could be explained by a detachment of endothelial cells alone, which contribute in the rabbit less than 15% of total (19) vascular wall PGI,. A decrease of more than 40% in total points to the fact, that cell death is occuring to an important degree in other layers too. The long-lasting effect of a decreased vascular prostacyclin formation fits well into the morphological and experimental data of extensive atherosclerotic lesions being found selectively at the site of irradiation. Assuming that prostacyclin is most probably acting as a local rather than a circulating hormone (19) it should be estimated whether there is in addition a decrease in stimulated PGI,-generation by the platelet derived growth factor (21) or the prostacyclin-synthesis stimulating plasma factor (22). Also the question of the behaviour of platelet sensitivity to antiaggregatory prostaglandins and the response of the vessel wall to repeated irradiation injuries is still unanswered. However, from these preliminary morphological and metabolical findings one could draw the conclusion that irradiation causes via cell damage a temporary increase in PGI,-formation followed by a depression after, which might initiate and perpetuate local platelet deposition and, consequently severe atherosclerotic lesions.

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PROSTAGLANDINS REFERENCES 1. WARREN,S.: Effects of irradiation on the normal tissues. IV. Effects on the cardiovascular system. Arch.Path. 34: 1070 (1942). 2. SHEEHAN,J.F.; Foam cell plaque in the intima of irradiated small arteries (one hundred to five hundred microns in external diameter). Arch. Path. -37: 297 (1944). 3. CASTER,W.O.,W.D.ARMSTRONG,E.SIMONSON: Changes in the cardiovascular system following total body x-irradiation. Amer.J.Physiol. _188: 169 (1957). 4. LAMBERTS,H.R.: Initial x-ray effect on the aortic wall and their late consequences. Symp. Cellular basis and etiology of late somatic effects of ionizing radiation. Academic Press 1963. 5. DeBOER,W.G.R.M.: Experimentele en therapeutische rontgenbestraling als oorzak van arteriele en cardiale beschadiging. Academisch proefschrift, Wolters, Groningen 1963. 6. RIGDON,R.H.,H.CLJRL: Effect of roentgen irradiation on capillary permeability and inflammation in the skin of the rabbit. Amer.J. Roentgenol. -49: 250 (1943).

7. BROWN,J.M.,L.F.FAJARDO,R.J.STEWART:

Mural thrombosis of the heart induced by radiation. Arch.Pathol. 96: 1 (1973).

8. SMITH,C.,A.LOEWENTHAL: A study of elastic arteries in irradiated mice of different ages. Proc.Soc.Exp.Biol.Med. 75: 859 (1950). 9. RLJBIN,P.,G.W.CASSARETT: In: Clinical Radiation Pathology. Saunders, Philadelphia 1, 1968. 10. ZOLLINGER,U.H.: Die Strahlenvaskulopathie. 11.

Path.Europ.l:45

SILBERBAUER,K.,H.SINZINGER,W.FEIGL,C.LEITmER: lenschadigungen. VASA 6: 183 (1977).

(1970).

Arterielle Strah-

12. SILBERBAUER,K.,R.WILLVONSEDER,W.FEIGL,K.KRISCH,H.SINZINGER: Morphologische und zellkinetische Untersuchungen an der Arterienwand nach Rontgenbestrahlung. Zellkinetik bei 1000-5000 rad Herdbestrahlungsdosis am Kaninchen. VASA 5: 215 (1976). 13. SINZINGER,H.,L.WICKE,R.WILLVONSEDER,W.FEIGL,P.HERNUSS,E.M~LLER-TYL,W.ERD: Morphologische und zellkinetische Untersuchungen an der Arterienwand nach Rontgenbestrahlung. I. Ergebnisse bei 3000 rad Herdbestrahlungsdosis am Kaninchen. Acta morph. neerl.scand. -14: 201 (1976). 14. LEITHNER,C .,M.WINTER,K.SILBERBAUER,H.SINZINGER;T.VUKOVICH,W. PINGGERA: Plasmatische und vaskulare Aspekte der haemorrhagische Diathese bei Uramie. 3.Donausymp. f.Nephrol. 270 (1978). 15. ELDOR,A.: Personal communication

(1982).

16. BALUDA,V.P., SUSHKEWICH,G.N.,PARSHKOV,E.M.,LUKOYANOVA,T.I.: Influence of gamma-rays 60-Co and fast neutrons on intravascular platelet aggregation and prostacyclin-like activity of the vascular wall (in Russian). Zkbl.Med. 62: 243 (1982). 17. HAHN,F.: In: Prostaglandins and Cancer. First International Conference, vol. 2: 381 (1982). 18. SCHNEIDKRAUT,M.J., P.A.KOT,P.W.RAMWELL: Urinary prostacyclin and thromboxane levels after whole body gamma-irradiation. Adv. in Prostaglandin, Thromboxane and Leukotriene Res. 10: Raven Press (1982) in press.

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19.

SINZINGER,H.,SILBERBAUER;K.,F.RING,M.WING,M.WINTER: Is the susceptibility to atherosclerosis due to species differences in vascular prostacyclin formation ? Experientia -81: 246 (1978).

20.

STEER,M.L.,D.E.McINTYRE,L.LEYINE,E.W.SALZMAN: Is prostacyclin a physiologically important circulating anti-platelet agent ? Nature 283: 194 (1980).

21. SINZINGER,H.,A.KEFALIDES,Ch.HOCHE: Is a diminished respinse in prostacyclin synthesis by human vascular lesions to the platelet derived growth factor at the local level the main determinant for atherosclerosis ? in press. 22. DADAK,C.,C.HOCHE,A.KEFALIDES,H.SINZINGER: Prostacyclin synthesis stimulating plasma factor in pregnancy and puerperium. Brit. J.Gynecol.Obstet. 1982 (in press).

Peter W. Ramwell Editor: Received: 6-9-82 Accepted: 7-3-82

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