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Macrophages, oxidised lipids and atherosclerosis
Medical Hypotheses
12 : 171 - 178, 1983
MACROPHAGES, M.J. Mitchinson,
OXIDISED LIPIDS AND ATHEROSCLEROSIS.
Dept. of Pathology,
Tennis Court Road, Cambridge.
ABSTRACT The suggestion has frequently been made that lipid-laden blood monocytes might contribute to atherosclerosis by emigrating into the arterial intima. In spite of much evidence that this occurs, the mechanism has never attracted widespread support as being of major importance, mainly because of the apparently small numbers of monocytes involved in this traffic, compared to the larger numbers of smooth muscle cells in the lesion. Recent observations suggest that some at least of the macrophages within the early lesion may be oxidising their lipid contents. Because some oxidised lipids are known to be toxic to cells, it is proposed that the production of oxidised lipids by macrophages may cause the death of these and other cells in the intima; and that cell death begins the vicious circle of injury and further lipid accumulation which characterise the enlarging plaque. Key words Macrophages
- Atherosclerosis
- Oxidised
lipids - Ceroid.
INTRODUCTION The first proponent of the role of blood-borne mononuclear cells in the origin of atherosclerosis was Leary, who suggested that Kupffer cells took up lipid droplets and travelled in the blood to invade the arterial intima (1, 2). Later workers made observations supporting the idea of the emigration of monocytes into early plaques in experimental animals (3-10) and in man (11). The macrophage-like cells in human atherosclerotic lesions are however heavily outnumbered by smooth muscle cells and probably for this reason smooth muscle cells There is have recently attracted much more attention than macrophages. however some evidence which can be interpreted as providing a much more important role for macrophages, despite their small numbers.
M.H
F
171
MONOCYTES/MACROPHAGES
AND EARLY ATHEROSCLEROSIS
Leary's observations were followed by other evidence of lipidladen mononuclear cells adhering to rabbit endothelium and penetrating it (3, 10). Electron microscopic studies confirmed that this occurs in the rat (4, 5), rabbit (6, 7), swine (8, 9) and in human atherosclerosis (11). There is also a report that mononuclear cells, labelled in a donor rabbit, and introduced into a recipient cholesterol fed rabbit, were found within the recipient's atheromatous lesions within 24-28 hours (12). Several methods have been used to confirm that some cells in early lesions are indeed macrophages, including their content of cytochrome oxidase (13), adherence to glass, carriage of Fc and C3b receptors, content of lysozyme (14) and acid esterase activity (7). All these reports conclude that the source of intimal macrophagelike cells is blood mononuclear cells, presumably monocytes. Only Leary considered that they took up lipid as Kupffer cells originally; later workers have not identified the source of the lipid with certainty, although there is some interesting data on the subject. LIPIDS AND MONOCYTES The presence of lipid droplets in human neutrophils and monocytes has been well-known for many years (15, 16). O'Neal and his coworkers demonstrated that the numbers of lipid-laden blood monocytes are greatly increased in hypercholesterolaemic rats on atherogenic diets (17-20). A study in progress shows wide variation between individual human subjects in the amounts of stainable lipid in blood neutrophils and monocytes, and suggests that this variation is not wholly explained by the differences in plasma cholesterol levels (21). One experiment demonstrated that human monocytes cultured in soft agar increased in size due to the accumulation of neutral fat in the cytoplasm (22). Some of the lipid is therefore present in the cells before emigration into the vessel wall, but more may accumulate after the cell enters the intima. None of these findings answer the question of the source of the lipid. Some may be taken up by pinocytosis and some synthesised by In vitro the cell. Which system predominates in vivo in unknown. it is found that high levels of cholesterol in the medium increase its uptake by macrophages, and decrease the synthesis of cholesterol by the cells (23). In man, dietary cholesterol supplements cause an increase in monocyte content of cholesterol (241, but differing lipid diets are reported not to affect sterol synthesis by blood mononuclear cells studied in vitro (25). The composition of human leucocyte lipids differs from that in the plasma (26) but this is hardly surprising in view of the metabolic activities of the cells. Much information is accumulating on handling of lipids and lipoproteins by monocytes and, although more work is required to clarify the mechanisms involved, it already seems that the relationship of leucocyte lipid content to plasma lipids is consistent with the idea of participation of the monocyte in early atherogenesis.
172
OXIDISED
LIPIDS AND ATHEROSCLEROSIS
A completely different avenue of thought ascribes an important role in atherogenesis to lipids that have been oxidised. It has often been proposed that lipid peroxidation may cause tissue damage in a wide variety of conditions (27). In both man and experimental animals, oxidised lipids (including lipoperoxides and 26-OH cholesterol) are present in atherosclerotic plaques, in amounts proportional to the severity of the lesions (28-33). Their source has been suggested to be dietary (34). The anti-oxidant tocopherol has also been reported to be deficient in human atherosclerotic aorta (35). Oxidised lipids such as 26-OH cholesterol and other oxidised sterols are cytotoxic (36, 37) angiotoxic and atherogenic (38, 39). Rats on a diet deficient in the anti-oxidant, vitamin E, developed aortic intimal lesions whichwere prevented by e-tocopherol (40). It has also been suggested that lipoperoxides inhibit oxidative enzymes in the plaques (30) and inhibit prostacyclin production (41). Oxidised lipids seem to be more difficult to extract from the vessel wall than other lipids (42). This "bound fraction" is greater in atherosclerotic areas (43). Such observations recall the frequent descriptions of the finding of ceroid or lipofuscin in atherosclerotic plaques (44-50). The nature of this group of pigments is complex and variable, but is generally accepted to consist largely of polymerised oxidised lipids (51). THE ROLE OF MACROPHAGES
IN THE EARLY INTIMAL LESION
Returning to the macrophage, it is obviously important to consider whether, in addition to their role as simple carriers of lipid, macrophages in the early lesion might have any specific effects which would accelerate the development of the lesion. Some fragments of information are beginning to accumulate on this question. First, it has been noted that the lipid-laden macrophages in early lesions soon begin to degenerate and die, spilling their contents into the intima (4, 6). In view of the known 'harmful effects of released macrophage enzymes in other inflammatory conditions, this mechanism may be of importance. But other effects have also been proposed. Macrophage-dependent factors have been discovered which stimulate the proliferation of fibroblasts (52) and smooth muscle cells (53). Macrophages have been shown to release hydrogen peroxide, which is toxic to other cells (54). Human monocytes in culture release products which increase DNA synthesis, low density lipoprotein binding and degradation, in smooth muscle cells and fibroblasts (55). In all these ways macrophages might accelerate the development of the early plaque.
173
One final possibility, which has not attracted much attention, may be more cogent than the rest. It has recently been pointed out that some of the subendothelial macrophage-like cells in the earliest human atherosclerotic plaques, but not in the nearby normal intima, contain oil-red 0 positive material which is insoluble in alcohol, xylene and chloroform-methanol (50). To a lesser extent similar insoluble lipid was present in endothelial and smooth muscle cells. If histochemical dogma is correct, this represent oxidised lipids. Since the lipid in circulating monocytes is soluble in methyl alcohol (21), ethyl alcohol and xylene (561, this suggests that when the lipid-laden monocyte has entered the injured intima it begins to oxidise the lipids it contains. No cell is better equipped than the macrophage to oxidise lipids, and this capability was known many years ago (57, 58). In recent years it has become clear that an important part of the bactericidal capability of neutrophils and macrophages is their production of toxic oxygen radicals and other oxidation products (59-62). These same mechanisms also seem to cause intracellular lipid peroxidation, by macrophages more than by neutrophils (62). In neutrophils, at least,these oxidative mechanisms are enhanced in hyperlipoproteinaemia (63). It is therefore probable that the intimal macrophages in early atherosclerotic plaques are capable of oxidising lipids. In doing so, they may produce soluble oxidised lipids that are exocytosed, or released when the macrophage dies, and prove toxic to neighbouring cells. The insoluble lipid products visible in sections may also be toxic, but it is perhaps more likely that they represent no more than an indication of the site at which other, soluble oxidised lipids were first produced. The soluble oxidised lipids would presumably be more toxic than the polymerised insoluble products. Oxidised lipids produced by macrophages might therefore be responsible for initiating the intimal necrosis which is widely considered the hallmark of irreversibility of the plaque (64). CONCLUSIONS The appearance of smooth muscle cells in the intima is not the Smooth muscle cells enter the intima first event of atherosclerosis. as a diffuse reaction of the vessel wall to many forms of injury such as immune complex deposition, irradiation and hypertension; most importantly, they are responsible for the diffuse intimal thickening of advancing age. Thus the earliest focal lesions of atherosclerosis in the human aorta and its branches are found as local accentuations of this diffuse thickening, associated with lipid accumulation. If smooth muscle cells are present in the intima before the plaque is initiated, some focal superimposed event is necessary to explain the focal lesion. Although it is generally agreed that this event is caused by haemodynamic injury, the nature of the event resulting from the injury is disputed. The evidence is now sufficient to propose a specific role in this A crucial focal event to the ingress of monocytes from the blood.
174
step in atherosclerosis may be that these macrophages, and perhaps to a lesser extent other cells, release lipids which have'been oxidised in the cytoplasm; these oxidised lipids are capable of rendering the plaque irreversible by killing other cells, including smooth muscle cells. The diet of the individual when these events are proceeding, (mainly during the 2nd and 3rd decades), may have important effects. Monocytes may carry more lipid in subjects on high fat diets; oxidised lipids in the diet would presumably worsen the outcome; and inadequate dietary anti-oxidants such as vitamin E would also be detrimental. Thus this view of atherosclerosis suggests that the smooth muscle cells are innocent, the macrophages guilty. Of course, no one who has followed the history of ephemeral opinions on the origin of atherosclerosis would suggest that this is the whole truth. But since this is a view that is easily susceptible to testing, and even provides the possibility of simple dietary intervention, it deserves consideration. It may emerge that the macrophage, thought only to have a walk-on part, is in fact the villain of the piece. REFERENCES 1.
Leary, T. The genesis of atherosclerosis. Arch. Path. 32: 507, 1941. 2. Leary, T. Crystalline ester cholesterol and atherosclerosis. Arch. Path. 47: 1, 1949. 3. Rannie, L., Duguid, J.B. The pathogenesis of cholesterol arteriosclerosis in the rabbit. J. Path. Bact. 66: 395, 1953. 4. Still, W.J.S., O'Neal, R.M. Electron microscopic study of experimental atherosclerosis in the rat. Am. J. Path. 40: 21, 1962 5. Gerrity, R.G., Cliff, W.J. The aortic tunica intima in young and ageing rats. Exp. mol. Path. 16: 382, 1972. 6. Cookson, F.B. The origin of foam cells in atherosclerosis. Brit. J exp. Path. 52: 62, 1971. 7. Gaton, E., Wolman, M. The role of smooth muscle cells and hematogenous macrophages in atheroma. J. Path. 123: 123, 1977. 8. Gerrity, R-G., Naito, H.K., Richardson, M., Schwartz, C.J. Dietary-induced atherosclerosis in swine: morphology of the intima in prelesion stages. Am. J. Path. 95: 775, 1979. 9. Gerrity, R.G. The role of the monocyte in atherogenesis. I. Transition of blood-borne monocytes into foam cells in fatty lesions. Am. J. Path. 103: 181, 1981. 10. Poole, J.C.F., Florey, H.W. Changes in the endothelium of the aorta and the behaviour of macrophages in experimental atheroma of rabbits. J. Path. Bact. 75: 245, 1958. 11. Geer, J.C. Fine structure of human aortic intimal thickening and fatty streaks. Lab. Invest. 14: 1764, 1965. 12. Spraragen, S-C., Giordano, A.R., Poon, T.P., Hamel, H. Participation of circulating mononuclear cells in the genesis of atheromata. Circ. 40 (supple. 3): 24, 1965.
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Adams, C.W.M., Bayliss, O.B. Detection of macrophages in atherosclerotic lesions with cytochrome oxidase. Brit. J. exp. Path. 57: 30, 1976. Schaffner, T., Taylor, K., Bartucci, E.J., Fischer-Dzoga, K., Beeson, J.H., Glagov, S., Wissler, R.W. Arterial foam cells with distinctive immunomorphologic and histochemical features of macrophages. Am. J. Path. 100: 57, 1980. Hayhoe, F.G.J. The cytochemical demonstration of lipids in blood and bone-marrow cells. J. Path. Bact. 65: 413, 1953. Hayhoe, F.G.J., Quaglino, D. Lipids and sudanophilia. In: Haematological Cytochemistry. Churchill Livingstone, Edinburgh: 68, 1980. Simon, R.C., Still, W.J.S., O'Neal, R.M. The circulating lipophage and experimental atherosclerosis. J. Ath. Res. 1: 395, 1961. Suzuki, M., O'Neal, R.M. Accumulation of lipids in the leukocytes of rats fed atherogenic diets. J. Lipid Res. 5: 624, 1964. Marshall, J.R., O'Neal, R.M. The lipophage in hyperlipemic rats - an electron microscopic study. Exp. Mol. Path. 5: 1, 1966. Suzuki, M., O'Neal, R.M. Circulating lipophages, serum lipids and atherosclerosis in rats. Arch. Path. 83: 169, 1967. Mitchinson, M.J. Human leucocyte lipids and serum cholesterol. To be published. Zucker-Franklin, D., Grusky, G., Marcus, A. Transformation of monocytes into "fat" cells. Lab. Invest. 38: 620, 1978. Kar, S., Day, A.J. Composition and metabolism of lipid in macrophages from normally fed and cholesterol fed rabbits. Exp. mol. Path. 28: 65, 1978. Mistry, P., Miller, N.E., Laker, M., Hazzard, W-R., Lewis, B. Individual variation in the effects of dietary cholesterol on plasma lipoproteins and cellular cholesterol homeostasis in man. J. Clin. Invest. 67: 493, 1981. Illingworth, D.R., Sundberg, E.E. The influence of dietary fats on lipid synthesis by human mononuclear cells. Biochem. Sot. Trans 9: 49, 1981. Kim, H.-S., Suzuki, M., O'Neal, R.M. Leukocyte lipids of human blood. Am. J. Clin. Path. 48: 314, 1967. Its measurement, Barber, A.A., Bernheim, F. Lipid peroxidation: occurrence and significance in animal tissues. Adv. Gerontol. Res. 2: 355, 1967. Glavind, J., Hartmann, S., Clemmesen, J., Jessen, K-E., Dam, H. Studies on the role of lipoperoxides in human pathology. II. The presence of peroxidised lipids in the atherosclerotic aorta. Acta. Path. Microbial. Stand. 30: 1, 1952. Iwakami, M. Peroxides as a factor of atherosclerosis. Nagoya J. med. Sci. 28: 50, 1965. Aoyama, S., Iwakami, M. On the role of fatty acid peroxides in 1965. atherogenesis. Jap. Heart J. 6: 128, Part 9. 26Smith, L.L., van Lier, J.E. Sterol metabolism: hydroxy-cholesterol levels in the human aorta. Atherosclerosis. 12:
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32. Brooks, C.J.W., Steel, G., Gilbert, J.D., Harland, W.A. Polar sterol esters in human atherosclerotic plaques. Atherosclerosis. 13: 223. 1971.
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Part 18. On the Smith, L.L., Pandya, N.L. Sterol metabolism: uniqueness of the occurrence of 26-hydroxycholesterol in the human aorta. Atherosclerosis. 17: 21, 1973. Leading article, Atherosclerosis and auto-oxidation of cholesterol. Lancet. 1: 964, 1980. Ito, T. Tocopherol, non-protein SH and metals in the human aorta, Jap. Circ. J. 33: 25, 1969. Harland, W.A., Smith, A.G., Gilbert, J.D. Tissue reaction to atheroma lipids. J. Path. 111: 247, 1973. Baranowski, A., Adams, C.W.M., Bayliss High, O.B., Bowyer, D.E. Connective tissue responses to oxysterols. Atherosclerosis. 41: 225, 1982. Imai, H., Werthessen, N-T., Taylor, C.B., Lee, K.-T. Angiotoxicity and arteriosclerosis due to contaminants of USP-grade cholesterol. Arch. Path. 100: 565, 1976. Imai, H., Werthessen, N.T., Subramanyam, P.W., Le Quesne, P.W., Soloway, A.H. Angiotoxicity of oxygenated derivatives of cholesterol and their presumptive precursors. Science. 207: 651, 1980. Sulkin, N-M., Sulkin, D.F. Intimal lesions in arteries of Vit. E deficient rats. Proc. Sot. Exp. Biol. Med. 103: 111, 1960. Gryglewski, R.J. Prostacyclin and atherosclerosis. Trends Pharmacol. Sci. 1: 164, 1980. Joh, T.H., Fukazawa, K., Kummerow, F.A., Perkins, E.G. Studies on the distribution and chemical properties of the bound lipids of the human aorta. J. Ath. Res. 6: 164, 1966. Bradby, G.H.V., Walton, K.W., Watts, R. The binding of total lowdensity lipoproteins in human arterial intima affected and unaffected by atherosclerosis. Atherosclerosis. 32: 403, 1979. Pappenheimer, A.M., Victor, J. Ceroid pigment in human tissues. Am. J. Path. 22: 395, 1946. Burt, R.C. The incidence of acid-fast pigment (ceroid) in aortic atherosclerosis. Am. J. Clin. Path. 22: 135, 1952. Schornagel, H.E. The occurrence of iron and ceroid in coronary arteries. J. Path. Bact. 72: 267, 1956. Gyarkey, F., Shimamura, T., O'Neal, R.M. The fine structure of ceroid in human atheroma. J. Histochem. Cytochem. 15: 732, 1967. Gonzalez, E.L. Insoluble hard material found in human atherosclerotic aortas. Nature. 219: 274, 1968. Slater, R.S., Smith, E.B. The microdissection of large atherosclerotic plaques to give morphologically and topographically defined fractions for analysis: Part 2. Studies on "nile blue" cells. Atherosclerosis. 15: 57, 1972. Mitchinson, M.J. Insoluble lipids in human atherosclerotic plaques, Atherosclerosis. 45: 11, 1982. Pearse, A.G.E. The lipid pigments. In: Histochemistry (3rd Ed.), Churchill Livingstone, *Edinburgh: 1076, 1972. factor that Leibovich, S.J., Ross, R. A macrophage-dependent stimulates the proliferation of fibroblasts in vitro. Am. J. Path. 84: 501, 1976. Ziats, N.P., Robertson, A.L., Jr. Effects of peripheral blood monocytes on human vascular cell proliferation. Atherosclerosis. 38: 401, 1981.
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Nathan, C.F. The release of hydrogen peroxide from mononuclear phagocytes and its role in extracellular cytolysis. In: Mononuclear phagocytes. Functional aspects: Part II. Ed. R. van Furth, Nijhoff: 1165, 1980. Chait, A., Mazzone, T. A human macrophage secretory product increases low density lipoprotein uptake by arterial smooth muscle cells and fibroblasts. Clin. Res. 29: 493a, 1981. Mitchinson, M.J. Unpublished observations. Day, A.J. Oxidation of 14C-labelled chylomicron fat and 14C-labelled unesterified fatty acids by macrophages in vitro and the effect of clearing factor. Quart. J. exp. Physiol. 45: 220, 1960. Day. A.J. A comparison of the oxidation of cholesterol-26-14C, palmitate-l-14C, and tripalmitin-l-14C by macrophages in vitro. Quart. J. exp. Physiol. 46: 383, 1961. Babior, B.M. Oxygen-dependent microbial killing of phagocytes. New Engl. J. Med. 298: 721, 1978. Fantone, J.C., Ward, P.A. Role of oxygen-derived free radicals and metabolites in leucocyte-dependent inflammatory reactions. Am. J. Path. 107: 397, 1982. Weiss, S.J., Lobuglio, A.F. Phagocyte-generated oxygen-metabolites and cellular injury. Lab. Invest. 47: 5, 1982. Stossel, T.P., Mason, R.J., Smith, A.L. Lipid peroxidation by human blood phagocytes. J. Clin. Invest. 54: 638, 1974. Ludwig, P.W., Hunninghake, D.B. & Hoidal, J.R. Increased leucocyte oxidative metabolism in hyperlipoproteinaemia. Lancet ii: 348, 198 2. Velican, C., Velican, D. Coronary intimal necrosis occurring as an early stage of atherosclerotic involvement. Atherosclerosis. 39: 479, 1981.
178
12 : 171 - 178, 1983
MACROPHAGES, M.J. Mitchinson,
OXIDISED LIPIDS AND ATHEROSCLEROSIS.
Dept. of Pathology,
Tennis Court Road, Cambridge.
ABSTRACT The suggestion has frequently been made that lipid-laden blood monocytes might contribute to atherosclerosis by emigrating into the arterial intima. In spite of much evidence that this occurs, the mechanism has never attracted widespread support as being of major importance, mainly because of the apparently small numbers of monocytes involved in this traffic, compared to the larger numbers of smooth muscle cells in the lesion. Recent observations suggest that some at least of the macrophages within the early lesion may be oxidising their lipid contents. Because some oxidised lipids are known to be toxic to cells, it is proposed that the production of oxidised lipids by macrophages may cause the death of these and other cells in the intima; and that cell death begins the vicious circle of injury and further lipid accumulation which characterise the enlarging plaque. Key words Macrophages
- Atherosclerosis
- Oxidised
lipids - Ceroid.
INTRODUCTION The first proponent of the role of blood-borne mononuclear cells in the origin of atherosclerosis was Leary, who suggested that Kupffer cells took up lipid droplets and travelled in the blood to invade the arterial intima (1, 2). Later workers made observations supporting the idea of the emigration of monocytes into early plaques in experimental animals (3-10) and in man (11). The macrophage-like cells in human atherosclerotic lesions are however heavily outnumbered by smooth muscle cells and probably for this reason smooth muscle cells There is have recently attracted much more attention than macrophages. however some evidence which can be interpreted as providing a much more important role for macrophages, despite their small numbers.
M.H
F
171
MONOCYTES/MACROPHAGES
AND EARLY ATHEROSCLEROSIS
Leary's observations were followed by other evidence of lipidladen mononuclear cells adhering to rabbit endothelium and penetrating it (3, 10). Electron microscopic studies confirmed that this occurs in the rat (4, 5), rabbit (6, 7), swine (8, 9) and in human atherosclerosis (11). There is also a report that mononuclear cells, labelled in a donor rabbit, and introduced into a recipient cholesterol fed rabbit, were found within the recipient's atheromatous lesions within 24-28 hours (12). Several methods have been used to confirm that some cells in early lesions are indeed macrophages, including their content of cytochrome oxidase (13), adherence to glass, carriage of Fc and C3b receptors, content of lysozyme (14) and acid esterase activity (7). All these reports conclude that the source of intimal macrophagelike cells is blood mononuclear cells, presumably monocytes. Only Leary considered that they took up lipid as Kupffer cells originally; later workers have not identified the source of the lipid with certainty, although there is some interesting data on the subject. LIPIDS AND MONOCYTES The presence of lipid droplets in human neutrophils and monocytes has been well-known for many years (15, 16). O'Neal and his coworkers demonstrated that the numbers of lipid-laden blood monocytes are greatly increased in hypercholesterolaemic rats on atherogenic diets (17-20). A study in progress shows wide variation between individual human subjects in the amounts of stainable lipid in blood neutrophils and monocytes, and suggests that this variation is not wholly explained by the differences in plasma cholesterol levels (21). One experiment demonstrated that human monocytes cultured in soft agar increased in size due to the accumulation of neutral fat in the cytoplasm (22). Some of the lipid is therefore present in the cells before emigration into the vessel wall, but more may accumulate after the cell enters the intima. None of these findings answer the question of the source of the lipid. Some may be taken up by pinocytosis and some synthesised by In vitro the cell. Which system predominates in vivo in unknown. it is found that high levels of cholesterol in the medium increase its uptake by macrophages, and decrease the synthesis of cholesterol by the cells (23). In man, dietary cholesterol supplements cause an increase in monocyte content of cholesterol (241, but differing lipid diets are reported not to affect sterol synthesis by blood mononuclear cells studied in vitro (25). The composition of human leucocyte lipids differs from that in the plasma (26) but this is hardly surprising in view of the metabolic activities of the cells. Much information is accumulating on handling of lipids and lipoproteins by monocytes and, although more work is required to clarify the mechanisms involved, it already seems that the relationship of leucocyte lipid content to plasma lipids is consistent with the idea of participation of the monocyte in early atherogenesis.
172
OXIDISED
LIPIDS AND ATHEROSCLEROSIS
A completely different avenue of thought ascribes an important role in atherogenesis to lipids that have been oxidised. It has often been proposed that lipid peroxidation may cause tissue damage in a wide variety of conditions (27). In both man and experimental animals, oxidised lipids (including lipoperoxides and 26-OH cholesterol) are present in atherosclerotic plaques, in amounts proportional to the severity of the lesions (28-33). Their source has been suggested to be dietary (34). The anti-oxidant tocopherol has also been reported to be deficient in human atherosclerotic aorta (35). Oxidised lipids such as 26-OH cholesterol and other oxidised sterols are cytotoxic (36, 37) angiotoxic and atherogenic (38, 39). Rats on a diet deficient in the anti-oxidant, vitamin E, developed aortic intimal lesions whichwere prevented by e-tocopherol (40). It has also been suggested that lipoperoxides inhibit oxidative enzymes in the plaques (30) and inhibit prostacyclin production (41). Oxidised lipids seem to be more difficult to extract from the vessel wall than other lipids (42). This "bound fraction" is greater in atherosclerotic areas (43). Such observations recall the frequent descriptions of the finding of ceroid or lipofuscin in atherosclerotic plaques (44-50). The nature of this group of pigments is complex and variable, but is generally accepted to consist largely of polymerised oxidised lipids (51). THE ROLE OF MACROPHAGES
IN THE EARLY INTIMAL LESION
Returning to the macrophage, it is obviously important to consider whether, in addition to their role as simple carriers of lipid, macrophages in the early lesion might have any specific effects which would accelerate the development of the lesion. Some fragments of information are beginning to accumulate on this question. First, it has been noted that the lipid-laden macrophages in early lesions soon begin to degenerate and die, spilling their contents into the intima (4, 6). In view of the known 'harmful effects of released macrophage enzymes in other inflammatory conditions, this mechanism may be of importance. But other effects have also been proposed. Macrophage-dependent factors have been discovered which stimulate the proliferation of fibroblasts (52) and smooth muscle cells (53). Macrophages have been shown to release hydrogen peroxide, which is toxic to other cells (54). Human monocytes in culture release products which increase DNA synthesis, low density lipoprotein binding and degradation, in smooth muscle cells and fibroblasts (55). In all these ways macrophages might accelerate the development of the early plaque.
173
One final possibility, which has not attracted much attention, may be more cogent than the rest. It has recently been pointed out that some of the subendothelial macrophage-like cells in the earliest human atherosclerotic plaques, but not in the nearby normal intima, contain oil-red 0 positive material which is insoluble in alcohol, xylene and chloroform-methanol (50). To a lesser extent similar insoluble lipid was present in endothelial and smooth muscle cells. If histochemical dogma is correct, this represent oxidised lipids. Since the lipid in circulating monocytes is soluble in methyl alcohol (21), ethyl alcohol and xylene (561, this suggests that when the lipid-laden monocyte has entered the injured intima it begins to oxidise the lipids it contains. No cell is better equipped than the macrophage to oxidise lipids, and this capability was known many years ago (57, 58). In recent years it has become clear that an important part of the bactericidal capability of neutrophils and macrophages is their production of toxic oxygen radicals and other oxidation products (59-62). These same mechanisms also seem to cause intracellular lipid peroxidation, by macrophages more than by neutrophils (62). In neutrophils, at least,these oxidative mechanisms are enhanced in hyperlipoproteinaemia (63). It is therefore probable that the intimal macrophages in early atherosclerotic plaques are capable of oxidising lipids. In doing so, they may produce soluble oxidised lipids that are exocytosed, or released when the macrophage dies, and prove toxic to neighbouring cells. The insoluble lipid products visible in sections may also be toxic, but it is perhaps more likely that they represent no more than an indication of the site at which other, soluble oxidised lipids were first produced. The soluble oxidised lipids would presumably be more toxic than the polymerised insoluble products. Oxidised lipids produced by macrophages might therefore be responsible for initiating the intimal necrosis which is widely considered the hallmark of irreversibility of the plaque (64). CONCLUSIONS The appearance of smooth muscle cells in the intima is not the Smooth muscle cells enter the intima first event of atherosclerosis. as a diffuse reaction of the vessel wall to many forms of injury such as immune complex deposition, irradiation and hypertension; most importantly, they are responsible for the diffuse intimal thickening of advancing age. Thus the earliest focal lesions of atherosclerosis in the human aorta and its branches are found as local accentuations of this diffuse thickening, associated with lipid accumulation. If smooth muscle cells are present in the intima before the plaque is initiated, some focal superimposed event is necessary to explain the focal lesion. Although it is generally agreed that this event is caused by haemodynamic injury, the nature of the event resulting from the injury is disputed. The evidence is now sufficient to propose a specific role in this A crucial focal event to the ingress of monocytes from the blood.
174
step in atherosclerosis may be that these macrophages, and perhaps to a lesser extent other cells, release lipids which have'been oxidised in the cytoplasm; these oxidised lipids are capable of rendering the plaque irreversible by killing other cells, including smooth muscle cells. The diet of the individual when these events are proceeding, (mainly during the 2nd and 3rd decades), may have important effects. Monocytes may carry more lipid in subjects on high fat diets; oxidised lipids in the diet would presumably worsen the outcome; and inadequate dietary anti-oxidants such as vitamin E would also be detrimental. Thus this view of atherosclerosis suggests that the smooth muscle cells are innocent, the macrophages guilty. Of course, no one who has followed the history of ephemeral opinions on the origin of atherosclerosis would suggest that this is the whole truth. But since this is a view that is easily susceptible to testing, and even provides the possibility of simple dietary intervention, it deserves consideration. It may emerge that the macrophage, thought only to have a walk-on part, is in fact the villain of the piece. REFERENCES 1.
Leary, T. The genesis of atherosclerosis. Arch. Path. 32: 507, 1941. 2. Leary, T. Crystalline ester cholesterol and atherosclerosis. Arch. Path. 47: 1, 1949. 3. Rannie, L., Duguid, J.B. The pathogenesis of cholesterol arteriosclerosis in the rabbit. J. Path. Bact. 66: 395, 1953. 4. Still, W.J.S., O'Neal, R.M. Electron microscopic study of experimental atherosclerosis in the rat. Am. J. Path. 40: 21, 1962 5. Gerrity, R.G., Cliff, W.J. The aortic tunica intima in young and ageing rats. Exp. mol. Path. 16: 382, 1972. 6. Cookson, F.B. The origin of foam cells in atherosclerosis. Brit. J exp. Path. 52: 62, 1971. 7. Gaton, E., Wolman, M. The role of smooth muscle cells and hematogenous macrophages in atheroma. J. Path. 123: 123, 1977. 8. Gerrity, R-G., Naito, H.K., Richardson, M., Schwartz, C.J. Dietary-induced atherosclerosis in swine: morphology of the intima in prelesion stages. Am. J. Path. 95: 775, 1979. 9. Gerrity, R.G. The role of the monocyte in atherogenesis. I. Transition of blood-borne monocytes into foam cells in fatty lesions. Am. J. Path. 103: 181, 1981. 10. Poole, J.C.F., Florey, H.W. Changes in the endothelium of the aorta and the behaviour of macrophages in experimental atheroma of rabbits. J. Path. Bact. 75: 245, 1958. 11. Geer, J.C. Fine structure of human aortic intimal thickening and fatty streaks. Lab. Invest. 14: 1764, 1965. 12. Spraragen, S-C., Giordano, A.R., Poon, T.P., Hamel, H. Participation of circulating mononuclear cells in the genesis of atheromata. Circ. 40 (supple. 3): 24, 1965.
175
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15. 16. 17. 18. 19. 20. 21. 22. 23.
24.
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