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Fibrin and atherogenesis — A hypothesis
409
Atherosclerosis, 33 (1979) 409-413 @ Elsevier/North-Holland Scientific
Publishers,
FIBRIN AND ATHEROGENESIS
JULIAN
Ltd.
- A HYPOTHESIS
L. KADISH
Department
ofsurgery,
Children’s Hospital Medical Center, Boston, MA 02115
(U.S.A.)
(Received 11 January, 1979) (Revised, received 9 March, 1979) (Accepted 24 March, 1979)
summary It has recently been found that endothelial cells exhibit an unusual change in cellular behavior in response to contact with fibrin. The possible implications of this finding with regard to the mechanism of atherogenesis are discussed. It is proposed that mural fibrin in vivo may produce a disorganized endothelium which can act as a nidus for further fibrin deposition and platelet aggregation. In the presence of inadequate fibrinolysis, a prolonged endothelial lesion could occur which may eventually result in atheromatous plaque formation. This view of atherogenesis requires reduced fibrinolytic activity as a prerequisite for plaque formation, a requirement which is in agreement with currently known data associating atherogenic risk factors with inhibited fibrinolysis.
Key words:
Atherogenesis ysis
-Disorganized
endothelium
-Fibrin
-Inhibition
of fibrinol-
At present, no single theory of atherogenesis can explain in detail all of the known risk factors which predispose to arteriosclerotic vascular disease. We present here the view, based on recent experimental observations, that atherogenesis represents an abnormal form of repair of intimal injuries fostered by reduced levels of plasma fibrinolytic activity. When considered from this perspective, the data currently known of risk factors which predispose to atherosclerosis may be viewed as acting through a common central initiating mechanism. It has recently been found that endothelial cells in culture exhibit an unusual change in cellular behavior in response to contact with fibrin [l]. Ordinarily endothelial cells at confluence display a uniform monolayer morphology (Fig. 1A) much like that of endothelium in vivo. If such a culture is overlaid with a fibrin clot, the cells in contact with the fibrin undergo a rapid morpho-
410
logic change within 2-4 h (Fig. 1B) in which they separate from each other, send out numerous pseudopods, and when recorded by time-lapse photography are seen to become migratory. The effect is reversible in that removal of the fibrin either enzymatically or mechanically results in the resumption of typical endothelial morphology within 2-4 h. This response is apparently specific for endothelium since fibroblasts, epithelial cells, and a variety of other normal and neoplastic cell lines failed to. show any change in behavior in response to contact with fibrin. The mechanism of this response to fibrin is unclear. It is intriguing to speculate that a similar phenomenon may occur in vivo and may possibly contribute to the pathogenesis of atherosclerosis. Endothelial injury has been taken to be a necessary component in the generation of atherosclerosis [2,3]. Presumably it is accompanied by a small amount of platelet aggregation and fibrin deposition. In the presence of adequate levels of plasminogen activator, such deposits would be rapidly cleared and normal endothelial organization would resume as the lesion heals. However, if fibrinolysis were inadequate, fibrin deposits might affect the endothelium in a manner similar to that observed in our in vitro culture system. This would lead to the exposure of more and more subendothelium and increased platelet aggregation over an extended period of time resulting in an ever expanding area of disorganized endothelium. This disorganized endothelium would no longer be an effective barrier to plasma proteins which are normally excluded from the vessel wall. Such an abnormally permeable endothelium is in fact frequently observed in atherogenesis [4]. Moreover, platelet adhesion and local release of the contents of the platelet granules have been implicated as a stimulus for local proliferation of smooth muscle cells [2,5] resulting in the eventual development of an apparently monoclonal fibrous plaque [6]. The prolonged disorganizing effect of fibrin in contact with endothelium could thus generate a lesion which could eventually result in myointimal thickening, From this point of view, the key defect that would foster atherogcnesis would be ineffective fibrinolysis. This concept was initially presented by Astrup in 1956 [ 71. His suggestion that ineffective fibrinolysis may be the key defect in atherosclerosis spawned numerous studies of plasminogen activator
Fig. 1A. Normal
confluent
Fig. 1B. The came culture
monolayer
morphology
12 h after being covered
of bovine aortic with a fibrin clot.
endothelium X600.
in culture.
X600.
411
levels as affected by atherogenic risk factors. All of the results obtained are consistent with his original view. High lipid diets resulting in increased plasma low density lipoprotein levels have been noted to cause inhibition of fibrinolysis [S-11]. The low density lipoprotein fraction transports most of the cholesterol in the blood and elevated levels have been correlated with an increased incidence of arteriosclerotic heart disease. Thus, the primary effect of LDL may be through inhibition of fibrinolysis. Diabetes mellitus, smoking, obesity, and hypertension are also all associated with reduced fibrinolysis [ 12191. Those people with clinical ischemic heart disease and/or infarction have a significantly lower blood level of plasminogen activator [ 141. The converse appears to be true as well. High levels of plasminogen activator have been associated with decreased susceptibility to myocardial infarction. Plasminogen activator levels are highest in the young and progressively decline during aging [ 201. Essien [21] has noted that the Nigerian population has an unusually high level of plasminogen activator and he hypothesizes that this feature is related to the low incidence of atherosclerotic heart disease in the Nigerian. As well, vigorous exercise causes a marked increase in plasminogen activator levels [22] and has been found to offer at least some protection against ischemic heart disease [ 231. The view presented here is that atherogenesis represents an abnormal reaction to intimal injury. That is, in conditions of adequate fibrinolysis (or inhibited fibrin deposition), mural fibrin deposits would be rapidly lysed and normal endothelial organization would resume as an endothelial injury heals. However, in conditions of inhibited fibrinolysis, mural fibrin would remain for prolonged periods, causing continued endothelial disorganization and prolonged platelet adhesion, with the eventual development of smooth muscle proliferation in the effected region. This view rests on two major assumptions. First, it is assumed that endothelium in vivo responds to fibrin in a manner similar to that seen in vitro. Second, it is implied that atherogenesis can be considered as analogous to the intimal thickening seen in animal models following endothelial injury and therefore fibrin should be involved in that response as well. The literature provides data on both of these points. Vassalli et al. [24], in an electron-microscopic study of experimentally induced disseminated intravascular coagulation, found striking morphologic changes in the endothelium of glomerular capillaries in the areas of fibrin deposits. The endothelial cells were noted to be “swollen”, rounded up, and numerous spaces were present between the cells, thus exposing the subendothelium. It is likely that this change in endothelial organization in response to the presence of intravascular fibrin represents the in vivo analog of the response seen in endothelial cell cultures. As well, data has been obtained which implicates fibrin as a key component in producing intimal thickening following endothelial injury. In the rat, an endothelial injury caused by infusing air into the carotid artery results in a predictable course of myointimal thickening followed by slow regression of the lesion over a period of months [25,26]. However, the intimal thickening stage of this response can be totally suppressed by heparinization of the experimental animal after the injury procedure [ 271. In such animals, regeneration of
412
the endothelium rapidly occurred such that by two weeks after injury, normal arterial architecture had returned. This data, interpreted with the effect of fibrin on endothelial organization kept in mind, indicates that relative suppression of fibrin deposition in the area of intimal injury, permitted the rapid return of normal endothelial integrity, thereby preventing smooth muscle proliferation secondary to continued platelet adhesion and degranulation. The predicted experimental result of the foregoing hypothesis of the mechanism of atherogenesis is that maintenance of adequate fibrinolysis may be antiatherogenic. Data showing such an effect have recently been reported [ 281. Note added in proof A view similar to that given here has recently Kwaan in Artery, 5 (1979) 285.
been presented
by Dr. Han C.
References 1 Kadish, J.L., Butterfield, C.E. and Folkman, J., The effect of fibrin on cultured vascular endothelial cells, Tissue and Cell. 11 (1979) 99. 2 Ross, R., Glomset, J. and Harker, L., Response to Injury and atherogenesis, Amer. J. Path., 86 (1977) 675. 3 Spaet, T.H., Gaynor, E. and Sternerman, M.B., Thrombosis, atherosclerosis, and endothelium, Amer. Heart J., 87 (1974) 661. 4 Adams, C.W.M., Bay&s, O.B. and Morgan, R.S., Permeability in atherosclerosis - Fluorescence test in green light with trypan blue, Atherosclerosis. 27 (1977) 353. 5 Fuster, V., Bowie, E.J.W. and Brown. A.L., Spontaneous arterial lesions in normal pigs and pigs with van WiRebrand’s disease, Adv. Exp. Med. Biol., 82 (1977) 315. 6 Benditt. E.P. and Benditt. J.M., Evidence for a monoclonal origin of human atherosclerotic plaques, Proc. Nat. Acad. Sci. U.S.A., 70 (1973) 1753. 7 Astrup, T.. The biological significance of fibrinolysis, Lancet, 2 (1956) 565. 8 Bang, N.U. and Cliffton, E.E., The effect of alimentary hyperlipemia on thrombolysis in tivo, Thromb. Diath. Haemorrh., 4 (1960) 149. Inhibition of fibrinolysis by hyperlipemia. Thromb. Diath. 9 Cliffton. E.E. and BuscheII, A.R., Haemorrh., 5 (1961) 463. 10 Greig, H.B.W. and Runde. J.A., Studies on the inhibition of fibrinolysis by lipids, Lancet. 2 (1957) 461. 11 Sarkar, N., Reduced fibrinolytic activity of atherosclerotic sera caused by an increase in low-density lipoproteins in blood, Nature (Lond.), 189 (1961) 929. 12 Abner, L.O. and Nilsson. I.M., On fibrinolysis in diabetes mellitus, Acta Med. Stand.. 98 (1975) 101. 13 Bogie. W., George, J. and Crane. C.W., Fibrinolytic activity in treatment of diabetes, Lancet, 2 (1976) 312. 14 Meade, T.W., Chakrabarti, R. and North. W.R.S.. Associations between fibrinolytic activity and other variables in an industrial population, Adv. Exp. Med. Biol., 82 (1977) 219. 15 Shaw, D.A. and MacNaughton, D., Relationship between blood fibrinolytic activity and body fatness, Lancet, l(1963) 352. 16 Ogston. D. and McAndrew, G.M.. Fibrinolysis in obesity, Lancet, 2 (1964) 1205. 17 Prokopowicz, J., Worowski, K., PopIawski, A.. Gabryelewicz, A. and Niewiarowski. S.. Fibrinolytic system in patients with hypertensive cardiovascular disease, Thromb. Diath. Haemorrh., 17 (1967) 1. enzyme system in 18 Bennett, B.. Ogston, D.. Cramford. G.P.M. and Douglas. A.S., The fibrinolytic hypertension, J. CIin. Path., 26 (1973) 351. 19 Tsapogas, M.J., Peabody, R.A.. Wu. K., Dew&, K.T. and Eckert. C., Depressed endogenous fibrinolytic activity in essential hypertension, J. Cardiovasc. Sung., 15 (1974) 651. 20 Chakrabarti, R., Brozovic, M., North, W.R.S., Stirling. Y. and Meade. T.W.. Effects of age on fibrinolytic activity and factors V, VII, and VIII. Proc. Roy. Sot. Med., 68 (1975) 267. 21 E&en, E.M., Enhanced fibrinolysis In Nigerians -- Probable contributory factor to low PrevabmCe of atherosclerosis in the Nigerian, Afr. J. Med. Med. Sci.. 5 (1976) 287. 22 Ogston, D. and Fullerton, H.W., Changes in fibrinolytic activity produced by physical actitity. Lancet. 2 (1961) 730.
413
23 24 25
26
27 28
Shapiro, S.. Weinblatt, E., Frank, C.W. and Sager, R.V.. Incidence of coronary heart disease in a POPUlation insured for medical care (HIP), Amer. J. Public Health, Suppl. to Vol. 59 (1969) 16. Vassal& P.. Simon. G. and Rouiller, C., Electron microscopic study of glomerular lesions resulting from intravascular fibrin formation, Amer. J. Path., 43 (1963) 579. Fishman, J.A., Ryan, G.B. and Karnovsky, M.J., Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening, Lab. Invest., 32 (1975) 339. Clowes, A.W., Ryan, G.B., Breslow, J.L. and Karnovsky, M.J., Absence of enhanced intimalthickening in the response of the carotid arterial wall to endothelial injury in hypercholesterolemic rats, Lab. Invest., 35 (1976) 6. Clowes, A.W. and Kamovsky. M.J.. Suppression by heparin of smooth muscle cell proliferation in injured arteries, Nature (Land.), 265 (1977) 625. Bordia. A., Verma, S.K., Vyas, A.K., Khabya. B.L., Rathore, A.S., Bhu, N. and Bedi, H.K., Effect of essential oil of onion and garlic on experimental atherosclerosis in rabbits, Atherosclerosis, 26 (1977) 379.
Atherosclerosis, 33 (1979) 409-413 @ Elsevier/North-Holland Scientific
Publishers,
FIBRIN AND ATHEROGENESIS
JULIAN
Ltd.
- A HYPOTHESIS
L. KADISH
Department
ofsurgery,
Children’s Hospital Medical Center, Boston, MA 02115
(U.S.A.)
(Received 11 January, 1979) (Revised, received 9 March, 1979) (Accepted 24 March, 1979)
summary It has recently been found that endothelial cells exhibit an unusual change in cellular behavior in response to contact with fibrin. The possible implications of this finding with regard to the mechanism of atherogenesis are discussed. It is proposed that mural fibrin in vivo may produce a disorganized endothelium which can act as a nidus for further fibrin deposition and platelet aggregation. In the presence of inadequate fibrinolysis, a prolonged endothelial lesion could occur which may eventually result in atheromatous plaque formation. This view of atherogenesis requires reduced fibrinolytic activity as a prerequisite for plaque formation, a requirement which is in agreement with currently known data associating atherogenic risk factors with inhibited fibrinolysis.
Key words:
Atherogenesis ysis
-Disorganized
endothelium
-Fibrin
-Inhibition
of fibrinol-
At present, no single theory of atherogenesis can explain in detail all of the known risk factors which predispose to arteriosclerotic vascular disease. We present here the view, based on recent experimental observations, that atherogenesis represents an abnormal form of repair of intimal injuries fostered by reduced levels of plasma fibrinolytic activity. When considered from this perspective, the data currently known of risk factors which predispose to atherosclerosis may be viewed as acting through a common central initiating mechanism. It has recently been found that endothelial cells in culture exhibit an unusual change in cellular behavior in response to contact with fibrin [l]. Ordinarily endothelial cells at confluence display a uniform monolayer morphology (Fig. 1A) much like that of endothelium in vivo. If such a culture is overlaid with a fibrin clot, the cells in contact with the fibrin undergo a rapid morpho-
410
logic change within 2-4 h (Fig. 1B) in which they separate from each other, send out numerous pseudopods, and when recorded by time-lapse photography are seen to become migratory. The effect is reversible in that removal of the fibrin either enzymatically or mechanically results in the resumption of typical endothelial morphology within 2-4 h. This response is apparently specific for endothelium since fibroblasts, epithelial cells, and a variety of other normal and neoplastic cell lines failed to. show any change in behavior in response to contact with fibrin. The mechanism of this response to fibrin is unclear. It is intriguing to speculate that a similar phenomenon may occur in vivo and may possibly contribute to the pathogenesis of atherosclerosis. Endothelial injury has been taken to be a necessary component in the generation of atherosclerosis [2,3]. Presumably it is accompanied by a small amount of platelet aggregation and fibrin deposition. In the presence of adequate levels of plasminogen activator, such deposits would be rapidly cleared and normal endothelial organization would resume as the lesion heals. However, if fibrinolysis were inadequate, fibrin deposits might affect the endothelium in a manner similar to that observed in our in vitro culture system. This would lead to the exposure of more and more subendothelium and increased platelet aggregation over an extended period of time resulting in an ever expanding area of disorganized endothelium. This disorganized endothelium would no longer be an effective barrier to plasma proteins which are normally excluded from the vessel wall. Such an abnormally permeable endothelium is in fact frequently observed in atherogenesis [4]. Moreover, platelet adhesion and local release of the contents of the platelet granules have been implicated as a stimulus for local proliferation of smooth muscle cells [2,5] resulting in the eventual development of an apparently monoclonal fibrous plaque [6]. The prolonged disorganizing effect of fibrin in contact with endothelium could thus generate a lesion which could eventually result in myointimal thickening, From this point of view, the key defect that would foster atherogcnesis would be ineffective fibrinolysis. This concept was initially presented by Astrup in 1956 [ 71. His suggestion that ineffective fibrinolysis may be the key defect in atherosclerosis spawned numerous studies of plasminogen activator
Fig. 1A. Normal
confluent
Fig. 1B. The came culture
monolayer
morphology
12 h after being covered
of bovine aortic with a fibrin clot.
endothelium X600.
in culture.
X600.
411
levels as affected by atherogenic risk factors. All of the results obtained are consistent with his original view. High lipid diets resulting in increased plasma low density lipoprotein levels have been noted to cause inhibition of fibrinolysis [S-11]. The low density lipoprotein fraction transports most of the cholesterol in the blood and elevated levels have been correlated with an increased incidence of arteriosclerotic heart disease. Thus, the primary effect of LDL may be through inhibition of fibrinolysis. Diabetes mellitus, smoking, obesity, and hypertension are also all associated with reduced fibrinolysis [ 12191. Those people with clinical ischemic heart disease and/or infarction have a significantly lower blood level of plasminogen activator [ 141. The converse appears to be true as well. High levels of plasminogen activator have been associated with decreased susceptibility to myocardial infarction. Plasminogen activator levels are highest in the young and progressively decline during aging [ 201. Essien [21] has noted that the Nigerian population has an unusually high level of plasminogen activator and he hypothesizes that this feature is related to the low incidence of atherosclerotic heart disease in the Nigerian. As well, vigorous exercise causes a marked increase in plasminogen activator levels [22] and has been found to offer at least some protection against ischemic heart disease [ 231. The view presented here is that atherogenesis represents an abnormal reaction to intimal injury. That is, in conditions of adequate fibrinolysis (or inhibited fibrin deposition), mural fibrin deposits would be rapidly lysed and normal endothelial organization would resume as an endothelial injury heals. However, in conditions of inhibited fibrinolysis, mural fibrin would remain for prolonged periods, causing continued endothelial disorganization and prolonged platelet adhesion, with the eventual development of smooth muscle proliferation in the effected region. This view rests on two major assumptions. First, it is assumed that endothelium in vivo responds to fibrin in a manner similar to that seen in vitro. Second, it is implied that atherogenesis can be considered as analogous to the intimal thickening seen in animal models following endothelial injury and therefore fibrin should be involved in that response as well. The literature provides data on both of these points. Vassalli et al. [24], in an electron-microscopic study of experimentally induced disseminated intravascular coagulation, found striking morphologic changes in the endothelium of glomerular capillaries in the areas of fibrin deposits. The endothelial cells were noted to be “swollen”, rounded up, and numerous spaces were present between the cells, thus exposing the subendothelium. It is likely that this change in endothelial organization in response to the presence of intravascular fibrin represents the in vivo analog of the response seen in endothelial cell cultures. As well, data has been obtained which implicates fibrin as a key component in producing intimal thickening following endothelial injury. In the rat, an endothelial injury caused by infusing air into the carotid artery results in a predictable course of myointimal thickening followed by slow regression of the lesion over a period of months [25,26]. However, the intimal thickening stage of this response can be totally suppressed by heparinization of the experimental animal after the injury procedure [ 271. In such animals, regeneration of
412
the endothelium rapidly occurred such that by two weeks after injury, normal arterial architecture had returned. This data, interpreted with the effect of fibrin on endothelial organization kept in mind, indicates that relative suppression of fibrin deposition in the area of intimal injury, permitted the rapid return of normal endothelial integrity, thereby preventing smooth muscle proliferation secondary to continued platelet adhesion and degranulation. The predicted experimental result of the foregoing hypothesis of the mechanism of atherogenesis is that maintenance of adequate fibrinolysis may be antiatherogenic. Data showing such an effect have recently been reported [ 281. Note added in proof A view similar to that given here has recently Kwaan in Artery, 5 (1979) 285.
been presented
by Dr. Han C.
References 1 Kadish, J.L., Butterfield, C.E. and Folkman, J., The effect of fibrin on cultured vascular endothelial cells, Tissue and Cell. 11 (1979) 99. 2 Ross, R., Glomset, J. and Harker, L., Response to Injury and atherogenesis, Amer. J. Path., 86 (1977) 675. 3 Spaet, T.H., Gaynor, E. and Sternerman, M.B., Thrombosis, atherosclerosis, and endothelium, Amer. Heart J., 87 (1974) 661. 4 Adams, C.W.M., Bay&s, O.B. and Morgan, R.S., Permeability in atherosclerosis - Fluorescence test in green light with trypan blue, Atherosclerosis. 27 (1977) 353. 5 Fuster, V., Bowie, E.J.W. and Brown. A.L., Spontaneous arterial lesions in normal pigs and pigs with van WiRebrand’s disease, Adv. Exp. Med. Biol., 82 (1977) 315. 6 Benditt. E.P. and Benditt. J.M., Evidence for a monoclonal origin of human atherosclerotic plaques, Proc. Nat. Acad. Sci. U.S.A., 70 (1973) 1753. 7 Astrup, T.. The biological significance of fibrinolysis, Lancet, 2 (1956) 565. 8 Bang, N.U. and Cliffton, E.E., The effect of alimentary hyperlipemia on thrombolysis in tivo, Thromb. Diath. Haemorrh., 4 (1960) 149. Inhibition of fibrinolysis by hyperlipemia. Thromb. Diath. 9 Cliffton. E.E. and BuscheII, A.R., Haemorrh., 5 (1961) 463. 10 Greig, H.B.W. and Runde. J.A., Studies on the inhibition of fibrinolysis by lipids, Lancet. 2 (1957) 461. 11 Sarkar, N., Reduced fibrinolytic activity of atherosclerotic sera caused by an increase in low-density lipoproteins in blood, Nature (Lond.), 189 (1961) 929. 12 Abner, L.O. and Nilsson. I.M., On fibrinolysis in diabetes mellitus, Acta Med. Stand.. 98 (1975) 101. 13 Bogie. W., George, J. and Crane. C.W., Fibrinolytic activity in treatment of diabetes, Lancet, 2 (1976) 312. 14 Meade, T.W., Chakrabarti, R. and North. W.R.S.. Associations between fibrinolytic activity and other variables in an industrial population, Adv. Exp. Med. Biol., 82 (1977) 219. 15 Shaw, D.A. and MacNaughton, D., Relationship between blood fibrinolytic activity and body fatness, Lancet, l(1963) 352. 16 Ogston. D. and McAndrew, G.M.. Fibrinolysis in obesity, Lancet, 2 (1964) 1205. 17 Prokopowicz, J., Worowski, K., PopIawski, A.. Gabryelewicz, A. and Niewiarowski. S.. Fibrinolytic system in patients with hypertensive cardiovascular disease, Thromb. Diath. Haemorrh., 17 (1967) 1. enzyme system in 18 Bennett, B.. Ogston, D.. Cramford. G.P.M. and Douglas. A.S., The fibrinolytic hypertension, J. CIin. Path., 26 (1973) 351. 19 Tsapogas, M.J., Peabody, R.A.. Wu. K., Dew&, K.T. and Eckert. C., Depressed endogenous fibrinolytic activity in essential hypertension, J. Cardiovasc. Sung., 15 (1974) 651. 20 Chakrabarti, R., Brozovic, M., North, W.R.S., Stirling. Y. and Meade. T.W.. Effects of age on fibrinolytic activity and factors V, VII, and VIII. Proc. Roy. Sot. Med., 68 (1975) 267. 21 E&en, E.M., Enhanced fibrinolysis In Nigerians -- Probable contributory factor to low PrevabmCe of atherosclerosis in the Nigerian, Afr. J. Med. Med. Sci.. 5 (1976) 287. 22 Ogston, D. and Fullerton, H.W., Changes in fibrinolytic activity produced by physical actitity. Lancet. 2 (1961) 730.
413
23 24 25
26
27 28
Shapiro, S.. Weinblatt, E., Frank, C.W. and Sager, R.V.. Incidence of coronary heart disease in a POPUlation insured for medical care (HIP), Amer. J. Public Health, Suppl. to Vol. 59 (1969) 16. Vassal& P.. Simon. G. and Rouiller, C., Electron microscopic study of glomerular lesions resulting from intravascular fibrin formation, Amer. J. Path., 43 (1963) 579. Fishman, J.A., Ryan, G.B. and Karnovsky, M.J., Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening, Lab. Invest., 32 (1975) 339. Clowes, A.W., Ryan, G.B., Breslow, J.L. and Karnovsky, M.J., Absence of enhanced intimalthickening in the response of the carotid arterial wall to endothelial injury in hypercholesterolemic rats, Lab. Invest., 35 (1976) 6. Clowes, A.W. and Kamovsky. M.J.. Suppression by heparin of smooth muscle cell proliferation in injured arteries, Nature (Land.), 265 (1977) 625. Bordia. A., Verma, S.K., Vyas, A.K., Khabya. B.L., Rathore, A.S., Bhu, N. and Bedi, H.K., Effect of essential oil of onion and garlic on experimental atherosclerosis in rabbits, Atherosclerosis, 26 (1977) 379.