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M. TURNER - arterial disease in this species is significant and can be accelerated by the addition of small amounts of cholesterol to the diet (I6). fj We also believed that the iologica fate o O would not be too dissimilar to that which occurred in mammals, including man. ' - As a result of our studies (1, 6), it has been observed that CO exposure. re- sulting in mean daily blood levels of 109'o carboxyhemoglobin ( I0'lc COHbI. increased the incidence or coronary artery atherosclerosis in birds made hyper- cholesterolemic by feeding a diet containing 1% cholesterol. CO had no effect, however, on the incidence of coronary artery atherosclerosis in normocholesterol- emic birds. Similar findings have been reported for normocholesterolemic rabbits (28), squirrel monkeys (13), and cynomolgus monkeys (9) after CO exposure. This effect in hyperlipfdemic birds manifested-itself, however, only after a sig- nificant period of CO exposure (in our hands, 52 weeks), but after continued exposure to CO (84 weeks) we observed a similar incidence of disease in both CO- and sham-exposed birds. Feeding birds a diet containing 2S'c cholesterol en- hanced the rate of disease-progression,though not the plasma cholesterol levels. relative to 1% cholesterol feeding. After 52 weeks, the incidence of coronary artery atherosclerosis was similar, in both CO- and-sham-exposed birds, to that observed after 84 weeks in the earlier study. - The variable effects of_CO on arterial disease, which are dependent on expo- sure duration, degree of hypercholesterolemia, and the level of CO exposure, may be explained if one assumes that the process of arterial lipid infiltration and lesion development is multiphasic. Figure I illustrates the h}pothetical scheme. Feeding 1% cholesterol enhances the disease development after-a lag phase and this proceeds relatively rapidly due to increased arterial uptake of plasma lipids. Eventually the process of lipid uptake is reduced due to the presence of a signifi- cant number of lesions that may affect the local intimal morphology and hence membrane transport processes. Enhanced lipid uptake may also affect membrane transport of lipid in adjacent apparently uninvolved regions. Endoeenous pro- cesses governing lesioh development may then-tend to predominate and, because some substrates including oxygen are limiting-,-the process will slow down. CO ex- posure appears to enhance-, by methods which are not yet clear, the initial uptake of plasma lipid and so this phase of lesion development accelerates. Eventually, however, the "endogenous" lesion development starts to predominate and. be- cause it is less affected by COHb, the rate of progression eventually becomes similar to that in the controls. - - Because of the subjective nature of our method for assessment of coronary artery disease, with its resultant effects on group variability, it was not possible to detect CO-mediated effects in birds where the disease incidence in controls was less than 10% (at the point A-A of Fig. YbWhen the incidence of disease reached 17--20% in control birds, however, it became more feasible to detect enhancement produced by CO (B-B in Fig. 1/)OWhen the incidence reached approximately 30-35% (C-C), the rate of disease progression appeared to have slowed so that similar levels of coronary artery atherosclerosis were observed in both CO- and sham-exposed birds. For the White Carneau pigeon, a 1% dietary addition of cholesterol, 10% COHb, and an exposure of 52-week duration seem to L ~ ICePyri9hr ~ --- I I I

WORKSHOP: CARBON MONOXIDE AND CVD 29. Thomsen. H. K. Carbon-monozide induced atherosclerosis in primates: An clcctron microscopic study on the coronary aneries o( 3(rrrnra iru: monkeys_ Ar6rrnsrlrrr.mfr 20. -'33-_'40 (1974). 30. Topping. D. L. Acute effects of carbon monoaidc on the metabolism of perfused rat liver. Binrhrnr J. 152, 425,927 (197A. . 31. Topping. D. L.. and Tumer, D. \f. Plasma triglyceride secretion in squurel monkeys' Effects of nicotine. Nurr. ,ifrrnhol. ig, 89-98 (1975). - 32. Turner. D. h1., and Topping. D. L. The effect of tobacco smoke and some of its constituents on Iriglyceride secretion in the squirrel monkey. Rrr. Corrrmnn. Clrern. Patlml. Plrnrnmrul. 12, g5-100 (1975). 33. Wa(d, N.. Howard. S.. Smith. P. G.. and Kjcldscn. K. Associaiion b<twzcn atherosclerotic diseases and carho.yhcmoglobin levels in tobacco-smokcs. Brir. Med. J. 1. 761-765 (1973). 34. Webster. W. S.. Clarkson. T. B.. and LoOand. H. B. "Carbon Nlonocidc Exposure on Atherosclerosis in the White Camaau Pigeon," Proc. 4th Intemat. Atherosclcrosis A4eting. Tokya, 1976. 35. Zflvcrsmil. D. B. A proposal linking alhcrogencsis to the interaction of endothelial lipoprotein lipase with triglyceride rich iipoproleins. Cbralation Rra. 33, 633-63,8 f 19731. 4Zy

if0L!e=_. - D. 61. TURNER to levels attained during tobacco smoking, therefore, appears to aggravate cholesterol-induced coronary artery atherosclerosis at a certain phase of disease development. If similar effects occur in man, such CO-mediated-effects on atherogenesis may be greatest only when the degiee of arterial disease is small. Where significant disease is present, CO may have little effect. The observation, based on epidemiological studies, that the association of coronary artery disease with tobacco smoking tends to be greatest in young smokers may find explanation in the above-mentioned hypothesis. - The mechanism(s) by which CO exposure effects the pathogenesis of athero- sclerosis is not entirely clear and recent evidence (28) has cast doubt on the once widely quoted (2, 15) edematous changes.in the arterial wall resulting from CO exposure. Studies with arterial smooth muscle_cells in culture (23) and with isolated perfused arteries (26) have, however, shown that hypoxia or CO ex- posure does enhance lipid uptake but other indirect effects of CO cannot be ex- cluded. It is well documented that in hypercholesterolemic rabbits (3, 7) and birds (I), CO tends to increase plasma cholesterol levels. The variation between individuals, however, tends to be so great as to eliminate significance of group data: Small changes in plasma cholesterol may produce profound alterations in arterial cholesterol levels and Ho et aL (12) have observed an exponential relationship between plasma and tissue cholesterol concentrations in the rabbit. It is probable that plasma cholesterol is too insensitive a measure for detection of CO-mediated effects and it may well be that more significant changes in lipoprotein compositions occur. In recent years, evidence has been produced (20, 24, 27) suggesting that an increased risk of ischemic heart disease is associ- ated with raised levels of plasma low-density lipoprotein (LDL) and lowered levels of plasma high-density lipoprotein (HDL). Indeed, very low-density lipo- protein levels also may mediate the arterial lipoprotein uptake (35). We have shown in the perfused rat liver (20) and in the aresthetized squirrel monkey (29) that CO exposure can alter hepatic lipid secretion. If such CO- mediated effects can alter plasma-lipoprotein composition in favor of a relative increase in LDL at the expeserf of HDL, enhanced arterial uptake of lipids might result. - - We have also observed, in the White Carneau pigeon, that CO exposure is associated with raised aortic cholesterol levels but decreased triglyceride and phospholipid levels (6). Bowyer (personal communication) has observed in moderately hypercholesterolemic cynomolgus monkeys and rabbits that CO ex- posure enhances the arterial content of both cholesterol and cholesterol esters. Patelski et nl. (22) have reported alterations in arterial lipid enzyme activities consequent upon feeding an atherogenic diet to rabbits. They observed a decrease in cholesterol esterase and increase of lipolytic enzyme activities. The effects of such changes would be-to-favor accumulation of-cholesterol esters but to act against retention-of iriglyceride and phospholipid. Changes in lys4mal esterase activities have been reported by Dc Duve-(8) as a basis for the transformation of arterial smooth muscle cells to foam cells. Our observations on the tissue lipid changes which occur with CO exposure in the pigeon (6) are consistent with theabove findings and suggest that CO may produce rl, - ---------- ---- --- --- ~ o ---_ _ . C I opja3nr I I

WORKSHOP: CARROY MONOXIDE AND CVD I , .«. Fto. 1. Hypothetical relationship between the observed incidence of coronary anery atherosclero- sis and duration of hypercholesterolemia in the 8'hite Carneau pigeon_ (t) Indicates (he presumed rate of progression in birds fed Lr cholesterol which results in a two- to three told hyperchodes terolemia. p+ CO) Indicates Ihe presumed rate of pm¢ression in birds fed 1r cholesterol and ezposed to I W ppm CO which results in the same degree of hppercholesterolaemia but raises the mean rarboxyhemoglobin concentration to IOS. - be critical if CO enhancement is to be observed (1). For the New Zealand White rabbit using a relatively high fat diet (7) containing 2% cholesterol with 20% COHb. a significant diet-induced incidence of coronary artery atherosclerosis and a marked enhancement of the incidence in CO-exposed rabbits was seen after only 10-week exposure. The points at which these critical factors interact thus vary with the species and may explain the apparently conflicting results reported by other investigators regarding the effects of CO on cholesterol-induced-arterial disease in various animal models employing different experimental protocols. Webster et at. (34) observed a CO enhancement of coronary artery athero- sclerosis after 7 months in moderately hyperlipidemic squirrel monkeys, with COHb levels of 20 c. This was similar in extent to that which we, by exposure to tOry COHb, obserti'ed in the pigeon after 12 months. The incidence of disease in control hypercholesterolemic monke}3was 17%. In studies with cynomolgus monkeys by Malinow er al. (18), 14-month exposure to 20% COHb did not produce any evidence of a CO effect on atherosclerosis. The incidence of the disease in control animals was, however, some 50% and_ it.may have been that the duration of hypercholesterolemia was too long, thus masking an earlier CO-mediated enhancement. The extent of pr~ xisting disease at the start of the study also may have been relatively great. , Exposure of experimental animals and birds to levels of CO which are similar (. '/ ~~ ~c~ (

enevcnrrve r.iEOtciWE 8, 000-000 c19:91 Carbon Monoxide. Tobacco Smoking, and the Pathogenesis of Atherosclerosis' D. M. TURNER fmnrunorhnnisrq Section. G)nro-.i!lrnhnnr Rrsearrh (Greenford) Ln:irrd. Grrrnf~rJ, ,Sfiddfrsrz, U86 OHE, Engfund Received November 14. 1978 - - Carbon monoxide (CO) exposure. which results in mean carbox}hemo¢lobin levels of 1070, enhances the incidencc of-coronary anery atherosclerosis in female Nhite Carncau pigeons which have been made hyperchelesterolcmic by addition of cholesterol to Iheir diet. CO is, however, without effect on normocholcsterolemic birds. Aortic cholesterol levels are increased-by CO exposure but phospholipid and triglyceridr levels are reduced in hyperchdeslerofemic birds. These effects might be due to inhibition of lysosomal enzyme activity. The level of CO exposure, the duration of that exposure. and the level of dietary choles- terol are critically interdependent factors which can in0uence the extent of CO in•okement in the pathogenesis of the disease. CO enhancement of atherosclerosis seems to be sig- nificant in the pigeon. during early lesion development when arterial lipid accumulation may be greatest. - For some time. the tobacco smoking habit has been known to be a major risk factor in the development of coronary heart disease (4, 25). The majority of evi- dence implicating tobacco smoking exposure, however, has been epidemiological and the last-decade has seen much effort devoted to isolating a component, or components, from smoke that could be shown to be important in the patho- genesis of coronary artery atherosclerosis. Of the many thousand components in tobacco smoke, nicotine has been ascribed in a role in aggravating the progression of coronary artery atherosclerosis and in precipitating sequelae such as arrhyth- mias and myocardiai infarction (11, 14, 17, 21) while carbon monoxide (CO) has been implicated in the pathogenesis of atherosclerosis (2, 10, 33). Much of the basic research on atherogenesis has been concerned with mech- anisms whereby such risk factors -such as CO or hyperlipidemia exert their effects and have been concentrated on biochemical or morphological character- ization of lesions obtained postmortem. Studies of the possible mechanism(s) for the role of (CO) have been reported by many groups of investigators and wide use has been made of a number of models such as the rabbit (3, 5, 7, 15, 28), various species of monkey (9, 13, 18, 29, 34), and the pigeon (l, 6). In studies over a number of years at the former Tobacco Research Council Laboratories, the role of CO has been investigated using the White Carneau pigeon as a model, essentially because the spontaneous incidence of progressive ~ ' Presented at„Workshop on-Carbon Monoside and Cardio<ascular Disease, sponsored by the / American Health Foundation and the Federal Health Office. Federal Republic of Germany. Berlin, October 10-12. 1978. _ - . -- 0091-7435179.0083-OOOOS01.000 CapYrilM : 1919 Ef AaaGvi< Ar++. Im. NI rirkn M rtCrulucinn m.et rrnn rrrnrJ. J-I - _1--

WORKSHOP: CARBON MONOXIDE AND CVD its atherogenic effects by inhibition of lysosomal lipase activity. An increase in arterial cholesterol ester content would inhibit intracellular transport and subse- quent removal via the HDL-mediated-mecha . Thus CO-mediated lipid changes may effect both the influx and efflux of c olesterol from the artery wall. Though the epidemiological evidence suggests a significant role for tobacco smoking in the pathogenesis of a number of types of cardiovascular disease, the evidence linking CO/5s the principal factor in tobacco smoke/is less convincing. In studies on man it is difficult to separate the effects of CO from those of tobacco smoke and it may be that the statistical association-between CO exposure in tobacco smoke and atherogenesis (14) is a reflection of the fact that CO is a marker of smoke intake. It tends to be assumed that the biological effects of CO which can be demon- strated in model-studies are-relevant to those effects which might occur in the tobacco smoker. Few people would question_the association with tobacco smok- ing and the increased risk of cardiovascular disease but the interpretation and extrapolation of data from model studies with an isolated smoke component can be hazardous. We have investigated the effects of CO and nicotine on hepatic lipid secretion (31, 32) in the anesthetized squirrel monkey and compared those effects with tobacco smoke while maintaining quantitatively similar CO and nico- tine exposure conditions. Though the effects on plasma-free fatty acids, for ex- ample, can be explained in terms of the CO and nicotine content of the smoke. the effects on hepatic lipid secretion were quite different from those seen with CO or nicotine alone. Tobacco smoke appears to contain factors which inhibit the response of liver lipids to CO alone and, if the effects on hepatic secretion relate ultimately to the development of atherosclerosis, it may be most unwise to extrap- olate from a CO effect to that of CO in tobacco smoke. It is clearly important to establish in a suitable animal model whether tobacco smoke influences the pathogenesis of atherosclerosis but such studiesjhave- for technical reasons, not yet been reported in detail as far as I am aware. Our pigeon model does not lend itself to studies with tobacco smoke because the respiratory system is so-different from that in mammals as is the metabolism of xenobiotics. Therefore, the recent development of a smoking baboon model by McGill el a!. (19) seems to offer a useful possibility of addressing this crucial question. - - - - The role of CO in tobacco smoke as an important fact in the development of atherosclerosis remains unclear and the search, therefore, for other agents in ' tobacco smoke which may be involved in the pathogenesis of this disease should I be vigorously pursued. - - - - -t REFERENCES - 1. Armiiage, A. K., Davies- R. F., and Turner. D. \I. The effects of carbon monoxide on the development of atherosclerosis in the white carneau pigeon. Atherosderosis 23, 333-314 (t976). 2. Astrup. P. Some physioloeical and pathological effects of moderale carbon monoxide exposure. Brir. .tled J. 2. L17-452 (1972). 3. Asnup- P., Kjatdsen- K.. and Wanstrup- 7. Enhancing influence of carbon monoxide on ihe development of atheromatosis in chotesterol fed rabbits.J. Arherusder. Res.7, 343-354 ( 1967). W

rFDJ2--. D. M. TURNER 4. Auerbach. 0.. Hammond, E. C., and Gar6nkel, L. Smoking in relation to alheromatosis o(Ihe j ~ coronary arteries. New EngY. J. Sfed. 273, 775-779 (1965). - i . 5. Birnstingl, M., Hawkin5, L., and McEwen, T. Experimental atherosclerosis during chronic ex- , posure to carbon monoxide. Eur. J. Surg. Rer. 2, 92-93 (1976). 6. Davies. R. F.. and Turner. D. M. "The Effects of Carbon Monoxide Exposure on Atherosclerosis in the White Carnaau Pigcon," Proc. 4th Internat. Atherosclerosis Meet7ng, To:yo. 1976. ~ 7. Davies, R. F.. Topping, D. L., and Turner, D. M. The effect of intermittent carbon monoxide exposure on experimental atherosclerosis in the rabbit. Arherosderosis 24, 5'_7- 516 /1976). - 8. De Duve. C. The participation of lysosomes in thc transformation of smooth muscle cells ' I _ to foamy cells in the aorta of cholesterol fed rabbits. Acra CardioL, suppl. 20, 9-25 (1974). - 9. Eckhardt, R. E., MacFarland, H. N.. Alaine, Y. C. E., and Busey, W. M. The biologic cfTects from long term exposure of primates to carbon monoxide. Arch. Enrirnn. Health 25, 381-387 - I (1972). - . I 10. Helidvaara, bf., Karvonen, M. 1., Vilhurien, R., and Purisar, S. Smoking, carbon monoxide, - and atherosclerotic diseases. Brir. .lfed. l. 1, 268-270 (1978) ~ 11. Hill, P-, and Wynder, E Smoking and cardiovascular disease. Effect of nicotine on the scrum epinephrine and corticoids. Anrer. Heart J. 87, 491-496 (1974). 12. Ho, K. J.. Eiland, S. H., and Taylor, C. B. Mode of cholesterol accumulation in various tissucs of rabbits with various serum cholesterol levels- Proc. Soc. Erp. Biof. .tled. 141, 277-281 /19721, 13. Jones, R. A_, Strickland, J. A., Stunkard, J. A., and Siegel. J. Effects on experimental animals of long lerm inhalation exposure to carbon monoxide. Tar. Appl. Pharmacol. 19, 46-53 (1971)- 14. Kershbaum, A.. Pappajohn. D. J., Bellel. S., Hirabayashi. ht., and "Dhaftila, H- Effect of smoking - -- and nicotine on adrenocortical sccrelion_ JA.NA 203, 275-278 (1968). 15. Kjeldsen. K.,Aslrup, P., and Wanstrup, 1. Ultrastructural changes in the rabbit aorta after moderate carbon monoxide exposure. Atherosrlemsis 76, 67-82 (f972). - 16. Kritchcvsky. D. Laboratory models for atherosclerosis. Adr. Drng. Res. 9, 41-53 (1974). 17. Lelkowitz. R. 1. Smoking, catecholamines and the heart.,Vew Enqf. J..1/ed. 295, 615-616 (1976). 18. Malinow, M. R., McLaughlin, P., Dhindsa, D. S.. Metcalf. J.. Ochsner, A. J.. 111, Hill. J., and McNu11y..W. P. Failure of carbon monoxide to induce myocardial infarclion in cholesterol fed ' cynomolgus monkey'sl3locara fnecicuJaris). CardiorascrJor Res. 10, 101-108 (19i6). , 19- McGill, H. C., Jr., Rogers, W. R., Wilbur, R. L., and Johnson. D. E. Cigarette smoking baboon model: Demonstration of feasibility. Proc. Soc Ezp. Binl. ,tfed. 157, 672-676 (1978). 20, Miller. G. L, and Miller, N. E. Plasma high density lipoprotcin concentration and development of ischacmic heart disease. L nret 4, 16-19 (1975). _'1. Mjos. O. D.. and llebekk, A. Effects of nicotine on myocardial metabolism and performance in dogs. Srmid. J. Clin. Lnb. Jmesr. 32, 75-80 (1978). - - . 22. Patelski, J., Bowyer. D. E., Howard, A. N.. Jennings, 1. W., Thorne, C. 1. R_, and Gresham, O. A. Modification of enzyme activities in experimental atherosclerosis in the rabbit. Atheroscleroele 12. 41-53 (1970). ~ . 23. Paula, W. J., Zetnplenyi, T. K.. Rounds. D. E., and Blankenhorn, D. H. Light and electron . , microscopic characteristics of arterial smooth muscle cells cultures subjected to hypoxia or earbon mono,ude. Atheroscleeosis 25, 11 t-123 (1976). 24. Rosxner. S.. Mettinger, K. L., Kjellin, K. G.. Siden, A_,-and Soderstrom, C. E. Normal serum • J`SCSSn~Y cholesterol but low HDL cholesterol concentration in young patients with ischaemic cerobro- ~ vascular disease. Lanrel. 18, 577-579 (1978). ~i - 25. Sackett, D. L., Gibson^R. W., Bross, 1. D. J., and Pickren. 1. W. Relation bctwcen aortic a,r4herosclerosis and Ihe use of cigarettes and alcohol: An autopsy study. A'n, Engl. J. Sfedl 279, 1413-1420 (1968). ' 26. Sarma, 1. S M., Tillmanns. H.; Ikeda, S., and Ging, R. 1. The effect of carbon monoxide on lipid metabolism of human coronary arteries. Adrerosclrrusis 22, 193-198 (1975), 27. Scanu, A. 61. Plasma lipoproteins and coronary heart discase. Anrr. Clin. Lnb. Sri. 8, 79-83 (1978). 28. Slender, S.. Astrup, P., and Kjeldsen, K. The effect of carbon monoxide in the aortic wall of _.- rabbits. Atherosrloosis 28, 357-367 (1977). - IfaN ri3VsT -J.f7