PREVENTIVE
303-309
(1979)
MEDICINE
8,
Carbon
Monoxide, Tobacco Smoking, Pathogenesis of Atherosclerosis’
and the
D. M. TURNER Immunochemistry Section, Glaxo-Allenbury Research (Greenford) Limited, Greenford, Middlesex, UB6 OHE, England Carbon monoxide (CO) exposure, which results in mean carboxyhemoglobin levels of 1096, enhances the incidence of coronary artery atherosclerosis in female White Cameau pigeons which have been made hypercholesterolemic by addition of cholesterol to their diet. CO is, however, without effect on normocholesterolemic birds. Aortic cholesterol levels are increased by CO exposure but phospholipid and triglyceride levels are reduced in hypercholesterolemic 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 cholesterol are critically interdependent factors which can influence the extent of CO involvement in the pathogenesis of the disease. CO enhancement of atherosclerosis seems to be significant 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 evidence 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 pathogenesis of coronary artery atherosclerosis. Of the many thousand components in tobacco smoke, nicotine has been ascribed a role in aggravating the progression of coronary artery atherosclerosis and in precipitating sequelae such as arrhythmias and myocardial 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 mechanisms whereby risk factors such as CO or hyperlipidemia exert their effects and have been concentrated on biochemical or morphological characterization 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 (1, 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 a Workshop American Health Foundation October lo- 12, 1978.
on Carbon Monoxide and Cardiovascular Disease, sponsored by the and the Federal Health Offtce, Federal Republic of Germany, Berlin,
303 0091-7435/79/030303-07$02.00/O Copyright Q 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.
304
D.
M.
TURNER
arterial disease in this species is significant and can be accelerated by the addition of small amounts of cholesterol to the diet (16). We also believed that the biological fate of CO 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, resulting in mean daily blood levels of 10% carboxyhemoglobin (10% COHb), increased the incidence of coronary artery atherosclerosis in birds made hypercholesterolemic by feeding a diet containing 1% cholesterol. CO had no effect, however, on the incidence of coronary artery atherosclerosis in normocholesterolemit birds. Similar findings have been reported for normocholesterolemic rabbits (28), squirrel monkeys (13), and cynomolgus monkeys (9) after CO exposure. This effect in hyperlipidemic birds manifested itself, however, only after a significant 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 2% cholesterol enhanced 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 exposure 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 1 illustrates the hypothetical 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 significant 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. Endogenous processes governing lesion development may then tend to predominate and, because some substrates including oxygen are limiting, the process will slow down. CO exposure 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, because 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. 1). When 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). When 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 Cameau pigeon, a 1% dietary addition of cholesterol, 10% COHb, and an exposure of 52-week duration seem to
WORKSHOP: CARBON MONOXIDE
AND CVD
305
FIG. 1. Hypothetical relationship between the observed incidence of coronary artery atherosclerosis and duration of hypercholesterolemia in the White Cameau pigeon. (I) Indicates the presumed rate of progression in birds fed 1% cholesterol which results in a two- to three fold hypercholesterolemia. (I + CO) Indicates the presumed rate of progression in birds fed 1% cholesterol and exposed to 150 ppm CO which results in the same degree of hypercholesterolaemia but raises the mean carboxyhemoglobin concentration to 10%.
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 IO-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 al. (34) observed a CO enhancement of coronary artery atherosclerosis after 7 months in moderately hyperlipidemic squirrel monkeys, with COHb levels of 20%. This was similar in extent to that which we, by exposure to 10% COHb, observed in the pigeon after 12 months. The incidence of disease in control hypercholesterolemic monkeys was 17%. In studies with cynomolgus monkeys by Malinow et al. (18), 1Cmonth 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 pre-existing disease at the start of the study may also have been relatively great. Exposure of experimental animals and birds to levels of CO which are similar
306
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M.
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 degree 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 affects the pathogenesis of atherosclerosis 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 exposure does enhance lipid uptake but other indirect effects of CO cannot be excluded. 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 associated with raised levels of plasma low-density lipoprotein (LDL) and lowered levels of plasma high-density lipoprotein (HDL). Indeed, very low-density lipoprotein levels may also mediate arterial lipoprotein uptake (35). We have shown in the perfused rat liver (20) and in the anesthetized squirrel monkey (29) that CO exposure can alter hepatic lipid secretion. If such COmediated effects can alter plasma lipoprotein composition in favor of a relative increase in LDL at the expense 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 exposure enhances the arterial content of both cholesterol and cholesterol esters. Patelski et al. (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 triglyceride and phospholipid. Changes in lysosomai esterase activities have been reported by De 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 the above findings and suggest that CO may produce
WORKSHOP: CARBON MONOXIDE
AND CVD
307
its atherogenic effects by inhibition of lysosomal lipase activity. An increase in arterial cholesterol ester content would inhibit intracellular transport and subsequent removal via the HDL-mediated mechanism. Thus CO-mediated lipid changes may effect both the influx and efflux of cholesterol 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 as 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 demonstrated in model studies are relevant to those effects which might occur in the tobacco smoker. Few people would question the association with tobacco smoking 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 nicotine exposure conditions. Though the effects on plasma-free fatty acids, for example, 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 extrapolate 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 studies have, 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 et al. (19) seems to offer a useful possibility of addressing this crucial question. The role of CO in tobacco smoke as an important factor 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 be vigorously pursued. REFERENCES 1. Armitage, A. K., Davies, R. F., and Turner, D. M. The effects of carbon monoxide on the development of atherosclerosis in the white cameau pigeon. Atherosclerosis 23, 333-344 (1976). 2. Astrup, P. Some physiological and pathological effects of moderate carbon monoxide exposure. Brit. Med. J. 2, 447-452 (1972). 3. Astrup, P., Kjeldsen, K., and Wanstrup, J. Enhancing influence of carbon monoxide on the development of atheromatosis in cholesterol fed rabbits. J. Atheroscler. Res. 7,343-354 (1%7).
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4. Auerbach, O., Hammond, E. C., and Gartinkel, L. Smoking in relation to atheromatosis of the coronary arteries. New Engl. J. Med. 273, 775-779 (1965). 5. Bimstingl, M., Hawkins, L., and McEwen, T. Experimental atherosclerosis during chronic exposure to carbon monoxide. Eur. J. Surg. Res. 2, 92-93 (1976). 6. Davies, R. F., and Turner, D. M. “The Effects of Carbon Monoxide Exposure on Atherosclerosis in the White Cameau Pigeon,” Proc. 4th Internat. Atherosclerosis Meeting, Tokyo, 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. Afherosclerosis 24, 527536 (1976). 8. De Duve, C. The participation of lysosomes in the transformation of smooth muscle cells to foamy cells in the aorta of cholesterol fed rabbits. Acta Cardiol., suppl. 20, 9-25 (1974). 9. Eckhardt, R. E., MacFarland, H. N., Alaine, Y. C. E., and Busey, W. M. The biologic effects from long term exposure of primates to carbon monoxide. Arch. Environ. Health 25,381-387 (1972). 10. Heliovaara, M., Karvonen, M. J., Vilhurien, R., and Purisar, S. Smoking, carbon monoxide, and atherosclerotic diseases. Brit. Med. J. 1, 268-270 (1978). 11. Hill, P., and Wynder, E. Smoking and cardiovascular disease. Effect of nicotine on the serum epinephrine and corticoids. Amer. Heart J. 87, 491-496 (1974). 12. Ho, K. J., Eiland, S. H., and Taylor, C. B. Mode of cholesterol accumulation in various tissues of rabbits with various serum cholesterol levels. Proc. Sot. Exp. Biol. Med. 141, 277-281 (1972). 13. Jones, R. A., Strickland, J. A., Stunkard, J. A., and Siegel, J. Effects on experimental animals of long term inhalation exposure to carbon monoxide. TOX.Appl. Pharmacol. 19,46-53 (1971). 14. Kershbaum, A., Pappajohn, D. J., Bellet, S., Hirabayashi, M., and Shafiita, H. Effect of smoking and nicotine on adrenocortical secretion. JAMA 203, 275-278 (1%8). 15. Kjeldsen, K.,Astrup, P., and Wanstrup, J. Ultrastructural changes in the rabbit aorta after moderate carbon monoxide exposure. Atherosclerosis 16, 67-82 (1972). 16. Kritchevsky, D. Laboratory models for atherosclerosis. Adv. Drug. Res. 9, 41-53 (1974). 17. Lefkowitz, R. J. Smoking, catecholamines and the heart. New Engl. 1. Med. 295,615-616 (1976). 18. Malinow, M. R., McLaughlin, P., Dhindsa, D. S., Metcalf, J., Ochsner, A. J., III, Hill, J., and McNutty, W. P. Failure of carbon monoxide to induce myocardial infarction in cholesterol fed cynomolgus monkeys (Mucaca fascicularis). Cardiovascular Res. 10, lOl- 108 (1976). 19. McGill, H. C., Jr., Rogers, W. R., Wilbur, R. L., and Johnson, D. E. Cigarette smoking baboon model: Demonstration of feasibility. Proc. Sot. Exp. Biol. Med. 157,672-676 (1978). 20. Miller, G. J., and Miller, N. E. Plasma high density lipoprotein concentration and development of ischaemic heart disease. Lancer 4, 16-19 (1975). 21. Mjos, 0. D., and Ilebekk, A. Effects of nicotine on myocardial metabolism and performance in dogs. Stand. J. C/in. Lab. Invest. 32, 75-80 (1978). 22. Patelski, J., Bowyer, D. E., Howard, A. N., Jennings, I. W., Thorne, C. J. R., and Gresham, G. A. Modification of enzyme activities in experimental atherosclerosis in the rabbit. Atherosclerosis 12, 41-53 (1970). 23. Paula, W. J., Zemplenyi, T. K., Rounds, D. E., and Blankenhom, D. H. Light and electron microscopic characteristics of arterial smooth muscle cells cultures subjected to hypoxia or carbon monoxide. Atherosclerosis 25, 111- 123 (1976). 24. Rossner, S., Mettinger, K. L., Kjellin, K. G., Siden, A., and Soderstrom, C. E. Normal serum cholesterol but low HDL cholesterol concentration in young patients with ischaemic cerebrovascular disease. Lancer, 18, 577-579 (1978). 25. Sackett, D. L., Gibson, R. W., Bross, I. D. J., and Pickren, J. W. Relation between aortic atherosclerosis and the use of cigarettes and alcohol: An autopsy study. New Engl. J. Med. 279, 1413-1420 (1968). 26. Sarma, J. S. M., Tillmanns, H., Ikeda, S., and Bing, R. J. The effect of carbon monoxide on lipid metabolism of human coronary arteries. Atherosclerosis 22, 193- 198 (1975). 27. Scan& A. M. Plasma lipoproteins and coronary heart disease. Ann. C/in. Lab. Sci. 8, 79-83 (1978). 28. Stender, S., Astrup, P., and Kjeldsen, K. The effect of carbon monoxide in the aortic wall of rabbits. Atherosclerosis 28, 357-367 (1977).
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29. Thomsen, H. K. Carbon monoxide induced atherosclerosis in primates. An electron microscopic study on the coronary arteries of Macaca irus monkeys. Atherosclerosis 20, 233-240 (1974). 30. Topping, D. L. Acute effects of carbon monoxide on the metabolism of perfused rat liver. Biochem. .l. 152,425-427 (1975). 31. Topping, D. L., and Turner, D. M. Plasma triglyceride secretion in squirrel monkeys: Effects of nicotine. Nutr. Metabol. 18, 89-98 (1975). 32. Turner, D. M., and Topping, D. L. The effect of tobacco smoke and some of its constituents on triglyceride secretion in the squirrel monkey. Res. Commun. Chem. Pathol. Pharmacol. 12, 85-100 (1975). 33. Wald, N., Howard, S., Smith, P. G., and Kjeldsen, K. Association between atherosclerotic diseases and carboxyhemoglobin levels in tobacco smoke. Brit. Med. J. 1, 761-765 (1973). 34. Webster, W. S., Clarkson, T. B., and Lofland, H. B. Carbon Monoxide-Aggravated Atherosclerosis in the Squirrel Monkey. Exp. MO/. Path. 13, 36-50 (1968). 35. Zilversmit, D. B. A proposal linking atherogenesis to the interaction of endothelial lipoprotein lipase with triglyceride rich lipoproteins. Circulation Res. 33, 633-638 (1973).
303-309
(1979)
MEDICINE
8,
Carbon
Monoxide, Tobacco Smoking, Pathogenesis of Atherosclerosis’
and the
D. M. TURNER Immunochemistry Section, Glaxo-Allenbury Research (Greenford) Limited, Greenford, Middlesex, UB6 OHE, England Carbon monoxide (CO) exposure, which results in mean carboxyhemoglobin levels of 1096, enhances the incidence of coronary artery atherosclerosis in female White Cameau pigeons which have been made hypercholesterolemic by addition of cholesterol to their diet. CO is, however, without effect on normocholesterolemic birds. Aortic cholesterol levels are increased by CO exposure but phospholipid and triglyceride levels are reduced in hypercholesterolemic 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 cholesterol are critically interdependent factors which can influence the extent of CO involvement in the pathogenesis of the disease. CO enhancement of atherosclerosis seems to be significant 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 evidence 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 pathogenesis of coronary artery atherosclerosis. Of the many thousand components in tobacco smoke, nicotine has been ascribed a role in aggravating the progression of coronary artery atherosclerosis and in precipitating sequelae such as arrhythmias and myocardial 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 mechanisms whereby risk factors such as CO or hyperlipidemia exert their effects and have been concentrated on biochemical or morphological characterization 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 (1, 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 a Workshop American Health Foundation October lo- 12, 1978.
on Carbon Monoxide and Cardiovascular Disease, sponsored by the and the Federal Health Offtce, Federal Republic of Germany, Berlin,
303 0091-7435/79/030303-07$02.00/O Copyright Q 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.
304
D.
M.
TURNER
arterial disease in this species is significant and can be accelerated by the addition of small amounts of cholesterol to the diet (16). We also believed that the biological fate of CO 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, resulting in mean daily blood levels of 10% carboxyhemoglobin (10% COHb), increased the incidence of coronary artery atherosclerosis in birds made hypercholesterolemic by feeding a diet containing 1% cholesterol. CO had no effect, however, on the incidence of coronary artery atherosclerosis in normocholesterolemit birds. Similar findings have been reported for normocholesterolemic rabbits (28), squirrel monkeys (13), and cynomolgus monkeys (9) after CO exposure. This effect in hyperlipidemic birds manifested itself, however, only after a significant 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 2% cholesterol enhanced 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 exposure 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 1 illustrates the hypothetical 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 significant 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. Endogenous processes governing lesion development may then tend to predominate and, because some substrates including oxygen are limiting, the process will slow down. CO exposure 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, because 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. 1). When 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). When 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 Cameau pigeon, a 1% dietary addition of cholesterol, 10% COHb, and an exposure of 52-week duration seem to
WORKSHOP: CARBON MONOXIDE
AND CVD
305
FIG. 1. Hypothetical relationship between the observed incidence of coronary artery atherosclerosis and duration of hypercholesterolemia in the White Cameau pigeon. (I) Indicates the presumed rate of progression in birds fed 1% cholesterol which results in a two- to three fold hypercholesterolemia. (I + CO) Indicates the presumed rate of progression in birds fed 1% cholesterol and exposed to 150 ppm CO which results in the same degree of hypercholesterolaemia but raises the mean carboxyhemoglobin concentration to 10%.
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 IO-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 al. (34) observed a CO enhancement of coronary artery atherosclerosis after 7 months in moderately hyperlipidemic squirrel monkeys, with COHb levels of 20%. This was similar in extent to that which we, by exposure to 10% COHb, observed in the pigeon after 12 months. The incidence of disease in control hypercholesterolemic monkeys was 17%. In studies with cynomolgus monkeys by Malinow et al. (18), 1Cmonth 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 pre-existing disease at the start of the study may also have been relatively great. Exposure of experimental animals and birds to levels of CO which are similar
306
D.
M.
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 degree 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 affects the pathogenesis of atherosclerosis 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 exposure does enhance lipid uptake but other indirect effects of CO cannot be excluded. 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 associated with raised levels of plasma low-density lipoprotein (LDL) and lowered levels of plasma high-density lipoprotein (HDL). Indeed, very low-density lipoprotein levels may also mediate arterial lipoprotein uptake (35). We have shown in the perfused rat liver (20) and in the anesthetized squirrel monkey (29) that CO exposure can alter hepatic lipid secretion. If such COmediated effects can alter plasma lipoprotein composition in favor of a relative increase in LDL at the expense 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 exposure enhances the arterial content of both cholesterol and cholesterol esters. Patelski et al. (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 triglyceride and phospholipid. Changes in lysosomai esterase activities have been reported by De 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 the above findings and suggest that CO may produce
WORKSHOP: CARBON MONOXIDE
AND CVD
307
its atherogenic effects by inhibition of lysosomal lipase activity. An increase in arterial cholesterol ester content would inhibit intracellular transport and subsequent removal via the HDL-mediated mechanism. Thus CO-mediated lipid changes may effect both the influx and efflux of cholesterol 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 as 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 demonstrated in model studies are relevant to those effects which might occur in the tobacco smoker. Few people would question the association with tobacco smoking 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 nicotine exposure conditions. Though the effects on plasma-free fatty acids, for example, 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 extrapolate 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 studies have, 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 et al. (19) seems to offer a useful possibility of addressing this crucial question. The role of CO in tobacco smoke as an important factor 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 be vigorously pursued. REFERENCES 1. Armitage, A. K., Davies, R. F., and Turner, D. M. The effects of carbon monoxide on the development of atherosclerosis in the white cameau pigeon. Atherosclerosis 23, 333-344 (1976). 2. Astrup, P. Some physiological and pathological effects of moderate carbon monoxide exposure. Brit. Med. J. 2, 447-452 (1972). 3. Astrup, P., Kjeldsen, K., and Wanstrup, J. Enhancing influence of carbon monoxide on the development of atheromatosis in cholesterol fed rabbits. J. Atheroscler. Res. 7,343-354 (1%7).
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