Prostacyclin and atherosclerosis

TIPS - February, 1980

164 TABLE

II. done results of progressive ratio test.scondlrcted by intravenous self-administration in rhesus

monkeys Unir close

Drug

(mg:kg in each mjeclion) --0.5 0.25 0.21 0.11** 800

Morphine Codeine Pcnlazocine Cocarne Alcohol

Final ratios in each monkey Ivatreated-Prclreat~.) xw-64OO 16aLl2.800. wlO-12.800, 16OO-l&XI 4530-IO.-60. 6400-12.800. 2690-3200 3200-320). 6400-3200. 6400-3200 1600-Ha). 6400-3200 16@3-6401. 32006400. 3200-3200. 6400-6400

-. Pretreated for development of physical dependence. **The ratio of 12,800 achieved al. a unit dose of 0.48.

ment has been observed with morphine .md codeine, but not with cocaine or sntazocine (Table 11). The result with alcohol falls between those of the opiates and pentazoclne because pretreatment was found to intensify the behavior less markediy thas that of opiates. These results have proved that although the ‘craving or ‘appetancy’ spoken of by Kruger et al. and Mardones have been clearly demonstrated without the necessity for prior development of physical dependence, still it is clear that such strong and active desire can be intensified by physical dependence when such dependence is developed on the opiates in particular, and to a lesser extent on alcohol.

Reading list I.

2.

3.

4.

5. 6.

Deneau. G. A . Yanagita, T. and Sewers. M. H. (1964) Prmmiings of the 26th Annual Scienrific Meeting, Commit&e on Problems of N.R.C.. N.A.S.. Dependence, DW? Washington, D.C., pp. 3812-3821. Kruger. H.. Eddy. N. B. and Sumwalt. M. (1941) ne phormacologv of the Opium Alkaloids, Supplement No. 165. Public Health Reports. U.S. Governmem Printing Office. for references see Schuster, C. R. and Thompson, T. (I%91 Annu. SPY. Pharmocol. 9.483-502. Wikler. A., Green. P. C.. Smith, H. D. and Pescor. F. T. (1960) Proceedings oJ rhe Zlsr rlnnwl &ienrijic Meeting. Committee on Problems of Drug Dependence, N.A.S.. N.R.C.. Washington, D.C.. pp.2244-2259. Seevers. M. H. (1968)JAMA 206.1263-1266. Mardones, J. (1963) In: W. S. Root, and F. G. Hofmann, @is), Physiologicul Phormucology.

Prostacyclinand atherosclerosis R. J, Gryglewski fiPnrtmClt Poland.

ofPharmacology, fli’. Copernicus .Ued~cal Academy,

Concepts of the dewlopmeat of atberoszluosis Varic~s hypotheses and conceptions of the pathogenesi s of atherosclerosis continually transform our clinical approach to the problems of prevenrion and treatment of this disease. The hundred years’ dispute between follower! of &?!I Rokitansky’s ‘incrustation hypothesis” and Vircliow’s ‘infiltrat.ion hypothesis” is still not closed. Do intravascular thrombi initiate atherosclerotic dam&e of arterial wall or do thq crown arterial iesions already ushered in by lipid infiltration? This last point of view has PrevaiIed among scientists for many years owing ~XZSIYto &ichkor’s experimental demonaration that lipid streaks crmr in arteries of rabbit:; which have been fed a ‘F!.mm

Ym+

Hd!aod

Rmewr,~d

Pm,,

,9*0

31-531 Cmcow. Gmegdmecko 16,

Vol I. Academic Press. New York and London. pp. 99-183. 7. Deneau. G. A.. Yanagrta. f. and Seevers, Al. tl (I %‘?I P~.~ho~~Jhormocol~Jgiaf i9erl.I 16. 10-48. 8. Johanson. C. E. and Balster. R. I.. (IY78) Bull. .Varc. 30,43-54. 9. Uello. N. K. (1973) Phormclr. Bwhrm. Hehav. I, RP-lOI. IO. Yanagita, T. and Takahashi. S. (1973) .1. Pharmacol. Exp. Ther. IIJS. 307-3 16. II. Yanagits, f. el al. (1970) Jap. 1. Clime. Pharmarol. I, 13-16. I?. Wood, IR. W., Grubman, J. and Weiss, B. (1977) J. Pharmacol. Esp. The,. 202.491-499. 13. Jarvik. M. E. (1967) Ann. N. Y. Arad. Sci. 142.280-294. 14. Ando, K. and Yanaaita. T. Submitted IO Psychopharmocologia. 15. Jones, 8. E. and Prada, J. A. (1973) Psychopharmarologra 30, I -I 2. 16. Yanagitr. T. (1976) P’tormacol. Rev. 27, 503-509. If. Griffitha, R. R., Brady. J. V. and Snell. J. D. (1978) Psychophermaco’ogy 56. S-13. 111. Johanson. C. E. and Sc’tuster, C. R. (1975) J. Pharmocol. E&p. Ther. !93.676-688.

Tomoji Yanogrra grodiaored in meddrcinefroa rhe Jikei Universi?v, Tok 1’0 b 1955. .After dorng research there he worked af rho University of Michigan from 1960 to 1’965. He fhen rerurned ro rhe Jikei University and was oppoinred Direcror of rhe Freriinical Research Luhorarories in 196.9. His mon.v public offices include membership of rhe WHO Advisory Panel on Drug Dependence.

We may also reverse this question: why are platelets not activated inside arteries of healthy subjects? Our answer is that in healthy subjects platelets are prevented from activation by prosta.cyclin (PGI,)4*B-the hormone generated continuously by blood vessels, kidneys and hmgsb. By raising CAMP levels in platelets3 prostacyclin keeps them in a non-agressive state, i.e. it suppresses their natural tendency to adhesion, aggregation and release reaction. By raising CAMP levels in arterial walls’ prostacyclin decreases the permeability of the intimo. and prevents hyperplasia of thr media. We suggest, consequently, that atherosclerosis develops when secretion of prostacyclin is impaired. In other words, we propose that atherosclerosis is a disease due to a deficiency of prostacyclin’.

diet rich in lipids. On the other hand, in the early stages of human atherosclerosis blood platelets and fibrin indeed appear to ‘incrust’ the arterial wall while myointimal oells of arteries grow over them and thus form an atheromatic plaque. Ross and Glom& believe that intravascular activation of platelets is the key event for development of an atheromatic plaque. Activated platelets release a growth fi4ctor that stimulates prolifera. tion of arterial myocytes and promoteFS Prostacydin and tbromboxane A2 their migration towards the arising atheromatic plaque. This attractive The products of cycle-oxygenation of concept provokes the next question: what arachidonic acid, cyclic endoperoxides makes platelets release their growth (PGG, and PGH,). give rise to prostafactor as well as other factors inside (e.g. in noids, i.e. lo prostacyclin arteries of a subject in whom the atheroarteries), to thromboxane A, (e.g. in sclerotic process is just about to begin? platelets) and to a number of prosta-

TIPS - February,

1980

gldndins in various tissues. Prostacyclin (half-life 5 min at 37°C pH 7.4) and thromboxane A, (half-life 30 set at 37°C. pH 7.4) although unstable, are potent circulatory hormones. Between prostacyclin and thromboxane A, there exists a direct physiological antagonism. Prostacyclin is a vasodilator which inhibits activation of platelets while thromboxane A, is a vasoconstrictor with pro-aggregating properties. lsomerization of PGH, to prostacyclin or to thromboxane A, requires the presence of specific microsomal enzymes, so called

165

Lipid peroxides and ntherosclerosis

cyclin it is kept within the range of ‘physiological stimulation’ while for thromboxane the nfotion of a ‘patho-

In one of the early papers on the discovery of prostacyclin‘ we described how IS-hydroperonyarachidonic acid is a potent inhibitor (IC., = I .5 PM) of prostacyclin synthetase in porcine aortic microsomes. Otherwise, the enzyme is resistant to intoxication by many other chemicals. l5-hydroperoxyarachidonic acid also inhibits generation of prostacyclin by arterial strips and by cultured endothelial cells. A powerful inhibition of prostacyclin synthetase by a number of other linear peroxides of polyunsaturated fatty acids has been also reported. It might be possible that inactivation of the enzyme is brought about by oxygen-centered free radicals which are generated during decomposition of lipid peroxides. These free radicals may also arise from a number of hydroperoxyeicosatetraenoic acids (HPETE) and from prostaglandin G, which are formed from arachidonic acid in mammalian tissues. At the same time thromboxane synthetase is insensitive to the inhibitory action of lipid peroxides. An atherogenic diet consisting of olive oil and cholesterol causes an early and dramatic suppression of prostacyclin generation by arteries in rabbits as well as a diversion of arachidonate metabolism to PGE, in arteries and kidneys: and to thromboxane A2 in platelets!. These findings prompted us to speculare that hyperlipidemia may induce an increased non-enzymatic lipid peroxidation, and then lipid peroxides or corresponding oxygen-centered free radicals inactivate prostacyclin synthetase in arteries. The subsequent deficiency of prostacyclin would be responsible for development of atherosclerosis. There is no direct evidence that in experimental or human atherosclerosis lipid peroxidation is the earliest sign of development of the disease. hlethodological difficulties in evaluation of intensity of lipid peroxidation in vivo do not help in solving this important problem. Nonetheless, lipid peroxides have been found in arteries from atherosclerotic patients and in ceroid atheromatic plaques. At the same time, in human atheromatic plaques prostacyclin is hardly generated, although experimental venous thrombosis is not associated with a decrease in production of prostacyclin by damaged rat veins. On the other hand Sinzinger and his colleagues have recently shown that in diabetic and atherosclerotic patients the samples of venous tissue generate less prostacyclin than veins of

logical range of intensity’ comes to mind.

healthy subjects.

synthetases. In vivo a selective inhibition of one of these synthetases may divert

metabolism of PGH, from prostacyclin to thromboxane A, and the contrary also happens. A realistic possibility exists also of influencing the prostacyclin/ thromboxane ratio by change in the composition of dietary polyunsaturated fatty acids. In vitro generation of prostacyclin by arteries and lungs is increased by mild mechanical Ltimuli, thrombin, calcium ionophore and low concentrations of arachidonic acid. In vivo release of prostacyclin into circulation is stimulated by angiotensin II, bradykinin and hyperventilation. Drastic stimuli are required for the stimulation of generation of thromboxane A, such as aggregation of platelets, anaphylactic shock in lungs or high concentrations of arachidonic acid. We are dealing with an unusual biochemical and physiological system. Two antagonistic circulatory hormones are generated from the same substrate and through the same intermediates. Both hormones are unstable and neither is stored in tissues. Apparently their biological activity depends exclusively on the rate of their generation. Only the last enzymes of this biochemical chain, i.e. prostacyclin synthetase and thromboxane synthetase are located in different types of cells. But do endothelial cells and platelets constitute impenetrable condominia? We believe that one elf the biochemical expressions of platelet-endothelial interaction is a modest flow of PGH, from platelets to endothelium and the resulting increase in generation of prostacyclin. In

respect to this interaction one should keep in mind that the intensity of any stimulus triggering generation of prostacyclin is much lower than that required for the energizing of the thromboxane synthetase system. One would say that for prosta-

The group of Mustard

has shown that feeding rabbits a diet enriched with safflower 011 cau,ec a marked suppression of prortacyilin generation by their vascular endothelium. Safflower oil is the richest plant source of polyunsaturated fatty acids, which tan be the substrates for formation of lipid peroxides. A potential hazard of o\erfeeding with unsaturated lipids has also been demonstrated in swine which were kept on a corn soybean diet free of cholesterol and saturated fatty acids. The animals developed atherosclerosis identical to that observed in humans. On the other hand antioxidants protect rats against vascular damage of acute choline deficiency-the damage resulting from increased lipid peroxidation m the body. In summary, a quantity of circumstantial evidence shows that atherosclerosis may be initiated by increased lipid peroxidation, but we lack direct evidence Experimental atherosclerosis and prostrcyclin

The early stage of experimental athrr+ sclerosis in rabbits is associated with i severe depression (by 60-80~0 I in generation of prostacyclin by heart, aorta. mesenteric arteries’, lun!gs and kidneys’. Suppression of prostacyclin generation is detected before any anaromtcal change in arteries is visible and before metabolism of arachidonic acid :n plareiets will be activated. This generalized suppression of the prostacyclin synthesizing system in rabbit atherosclerosis is associated with susceptibility of platelet increased and cyclase endothelial adenylate adenylare cyclase to the stimulator) action of exogenous prosracyclin. This hypersensitivity of the ‘second messenger system’ towards the primar! mediator rnAy be a consequence of prostacyiiin detisienc?, as has been observed in other b:ological systems deprived of a primary p!tysiological mediator. The fact that finctioning of prosra~vslin-stimulared $lenylate cyclase is activated in atheros’clerosis gives a sound basis for administration oi prostxkclin

in treatment of the

disease.

Clinical evidence

After we had established the rfleiti\e and safe doses of prostasyilin in health! hormone the synthetic volunteers’. (Wellcome Research and Upjohn Co.) was administered to five patients with advanced arteriosclerosis obliterans of the lower extremities (IV. category of atherosclerosib according to De Bakey). In an open clinical study prostacyclin was

TIPS - February, 1980

FIR. lo. Physiological balance between pluteiet and G,

infused upstream into the femoral artery at a dose of S-10 ng kg’ min-1 during a period of 72 hi”. All patients had chronic ischemic ulcers and in three, f&al necrosis was present. The lesions were painful and had resisted healing for from 3 months tG 3 years. In these patients various types of conservative treatment had been tried in the past and no further treatment could be offered except for amputation. In all patients pain was extinguished two days after prostacyclin therapy, and 4-8 weeks later in three patients rwession of necrosis and the healing of ischemic ulcers occurred. In two other patients we observed a remarkable improvement. The striking clinncal improvement after prostacyclin therapy was accompanied by persistent increase in capillary blood flow via the calf muscle. as measured by the clearance of “‘?Xe. The anatomical obstruction to m,ajor arteries remained unchanged as evidenced by angiogras. Since the pharmacological treat.nent of advanced obstructive vascular disease with various vasodilal:ors (except for prostaglandin E,) had been totally unsuccessful, we believe ex iuvuntibus that the prostacyclin thcaapy was in fact an hormonal substituoion therapy. Althouyh up to the present beneficial effects of prostacyclin have been observed in a further 20 patients, nonetheless, extensive double blind aadies are necessary to prove mr to disprove the validity of our conception. Hypothesis We postulate that athersclerosis cs a disease due to the deficiency of prostacyclm. This hormonal defect is caused by intoxication of prostacyclin synthetase with lipid peroxides or with corrcsponding free radicals which are likely to k generated during hyperlipidemia (Fig. 1).

he/u?/wll.

Fig. lb. How crrherosclerosr~may be mitiu:ed.

-t II~ inborn or acquired weakness of biochemical systems scavenging free radicals and/or a loss of balance between rates of lipoxygenation and cycle-oxygenation of arachidonic acids are other possible causes of disaster. The intoxication of prostacyclin synthetase by lipid peroxides may not be a diffuse phenomenon but rather a phenomenon restricted to the rheologically hazardous areas of circulation where shear force facilitates biochemical damage. Deficiency of prostacyclin leads to a decrease in CAMP levels both in platelets and in arterial walls, thus increasing platelet aggregability and endothelial permeability. Arterial endothelium when deprived of its capacity to synthesize prostacyclin becomes a surface prone to platelet adhesion and aggregation, as well as to invasion of macromolecules from blood to subendothelium. Aggregating platelets are activated not only by lack of ‘prostacyclin resistance’ at the endothelial surface but also by low concentrations of circulating prostacyclin and by diversion of arachidonic acid metabolism from prostacyclin to proaggregatory metabolites. Activated platelets adhere and aggregate on the endothelial cells and release harmful substances that cause endothelial damage, mural inflammatory raponre, migration of myocytes and formation of atheromatic plaque. The aggressive behaviour of platelets in atherosclerosis is facilitated by low levels of CAMP in arterial walls. Both phenomena grow from the same seed: damage done to the enzymatic system which is responsible for biosynthesis of prostacyclin. Therefore a long-lasting intravascular infusion of synthetic prostacyclin may prevent formation cf atheromatic plaques and protect against thrombo-emholic complications. Prostacyclin certainly cannot ‘dissolve’ athero-

matic deposits and disintegrate organized thrombi. Stimulation of biosynthesis of endogeneous prostacyclin is another approach to the same problem; however, protection against lipid peroxidation or scavenging of oxygen -centered free radicals may constitute the most important measure against the development of atherosclerosis. Reading list 1. Dembi6ska-Kie& 2.

3. 4.

5. 6. 7.

8.

9. IO.

A., Riicker, W., SchOnhdfer. P. S. (1979) Naunyn-Schmiedebergs Arch. Phurmocol. 308.2. 107-l IO. Dembibska-Kiti, A., Riicker. W. and Sch0nhOfer. P.S. (1979) Prostaglondins 17. 6, 831-837. Corman. R. R., Bunting. S. and Miller. 0. V. (1977) Prostaglondins I ?, 377-388. Gryglcwski, R. J.. Bunting. S.. Moncada, S., Flower. R. J. and Vane, J. R. (1976) Prostog/andins I2,685-7 13. Gtyglewski, R. J., Dembihska-Kick, A., Chytkowrki, A. and Gryglewska, T. (1978) Atherosclerosis 31.385-394. Gtyglewski. R. J.. Korbut. R. and Ocetkiewicz. A. (1978) Noture(London) 273,765767. Gryglewski, R. J.. Szczeklik. A. and Nizankowski, R. (1978) Thrombos. Res. 13, 153-163. Moncadal. S., Gtyglewski. R. J., Bunting, S. and Vane. J. R. (1976) Nature (London) 263. 663-665. Ross, R. and Glomset, J. A. (1976) New Engl. J. Med. 2953369-377; 420-425. Szczeklik, A., Nizankowski. R.. Skawihski. S., Szczeklik. J., Gluszko. P. and Gryglewski, R. 1. ~1979)Lcrncet1,1111-1114.

Ryszord J. Gryglewski is Pro/esor o/ Phormocolo,gy in the N. Copernicus Academy of Medicmne in Crcrcow. Poland, and u Member of The Polish Academy of Sciences. He has recently worked on prostoglondin-mediated mechanisms oJ ocrton oJ steroidal and non-steroidal anri-inf7ommotory drugs and in 1976, he and his British colleugues ducovered rhe e&fence of the ontithrombotic hormone, prostocyelirr. Presently, under his guidance, prostocyclin is being used in preliminary clinical trials /or rhe treotmettt of peripheral vascular disease.