465
Atherosclerosis, 31 (1978) 465-471 @ Elsevier/North-Holland Scientific Publishers,
Ltd.
THE ACTION OF HUMAN HIGH DENSITY CHOLESTEROL CRYSTALS Part 1. Light-microscopic
LIPOPROTEIN
ON
Observations
C.W.M. ADAMS and ‘Y.H. ABDULLA Department (Received (Accepted
of Pathology, Guy’s Hospital Medical School, London SE1 9RT (Great Britain) 5 August, 1978) 18 August, 1978)
Summary High density lipoprotein (HDL) was found in vitro to form myelin buds (liposomes) from washed crystals of free cholesterol (commercial or atheroma sources). This activity led to the progressive destruction and solubilization of the crystals. Low density and very low density lipoproteins did not have any effect. Liposome formation and solubilization were accelerated by calcium ions, phospholipase A and polyunsaturated lecithin (Lipostabil). Cholesterol crystals were nearly completely destroyed after 18 h incubation with HDLLipostabil. Key words:
- Cholesterol Atherosclerosis Micelle - Phospholipid
-
High density
lipoprotein
-
Liposome
-
Introduction Resorption of atheroma lipids and regression of atherosclerosis is at best a somewhat slow process [l-3], in comparison with removal rates for lipids from other tissues [4]. Likewise, the turnover rate of cholesterol in atheroma is relatively slow [ 5-71. This metabolic inertia of cholesterol in atherosclerosis has been attributed in part to the crystalline nature of some of the deposited cholesterol, and in part to the absence of effective phagocytic cells and degradative enzymes for cholesterol in the atherosclerotic arterial wall [El]. However, in recent years it has become apparent that high density lipoprotein (HDL) will Partly
supported
by a grant
from
the
British
Heart
Foundation.
466
remove cholesterol from incubated atherosclerotic tissue [ 91, cultured arterial smooth muscle cells [ lO,ll] and fibroblasts [ 121. It, therefore, seemed relevant to see whether HDL is active against cholesterol in the supposedly inert crystalline form, in addition to its apparent action against cholesterol taken up by cells. It will be shown that HDL is, in fact, active against cholesterol crystals, in that it promotes the formation of myelin buds [ 131 or liposomes [ 141 over the crystal surface. This budding results in the progressive erosion and destruction of the cholesterol crystal. Methods Lipoproteins were prepared from pooled human blood-bank serum. High density (HDL), low density (LDL) and very low density (VLDL) lipoproteins were respectively precipitated with sodium phosphotungstate, heparinmanganese and heparin-magnesium, following Burstein and Scholnick [ 151. These fractions were dialysed exhaustively against 10 mM Tris (pH 7.4) in 0.15 M sodium chloride, and were then concentrated by vacuum dialysis until semisolid. These stock solutions were stored at 4°C. The relevant fractions were further purified for use by flotation over buffered salt with respective densities of 1.210, 1.063 and 1.009 [15]. The purity of each fraction was then confirmed by crossed immunoelectrophoresis, using antibody against whole human serum (Behringwerke). Each purified fraction was stored at 4°C and was used over a maximum period of 7 days. Crystalline free cholesterol was obtained from BDH and further purified by formation of its dibromide [ 161. Material from the core of advanced human lesions in subjects aged over 65 years was repeatedly washed with 0.15 M sodium chloride and centrifuged at 500 X g for 15 min. The precipitate contained a mixture of rhomboidal crystals and flat plate-like rectangular crystals. Approximately 1 mg saline-washed samples of cholesterol crystals were suspended in increasing dilutions of HDL (15, 3, 0.6, 0.12 mg protein/ml 0.15 M saline-5 mM barbital buffer, pH 7.3). Budding activity was assessed by direct transmission and polarizing microscopy after incubation of the mixtures (with or without shaking) for 15 min to 18 h at 37°C. Buffered 0.15 M saline, whole serum, LDL (- 5 mg protein/ml), VLDL (- 5 mg protein/ml) and human serum albumin (- 5 mg protein/ml; Miles) were also tested for budding activity against cholesterol crystals. The following other crystalline substances were tested to see whether they formed buds with HDL: cholesteryl stearate (Hopkin and Williams), glyceryl tristearate (Hopkin and Williams), stearic acid (Hopkin and Williams), stearyl alcohol (BDH), cholestan-3a-ol (Sigma), cholestan-3&ol (Sigma), cholesteryl digitonide and octadecane (L. Light). Activation or inhibition of bud formation with HDL was tested with l-20 mM calcium chloride [lo] and with a sonicated polyunsaturated lecithin (Lipostabil, Nattermann, Cologne; about 70% of the phospholipid was dilinoleyl phosphatidyl choline). HDL was also tested for budding activity after preliminary digestion for 2 h at 37°C with the following enzymes: trypsin (Sigma grade l), and phospholipase A (Crotulus adumanteus, Sigma) C and D (Sigma). All enzymes were used at 1 mg/ml 0.15 M saline-5 mM barbital buffer at pH 7.3.
467
Four advanced human examined for activity with sections of formol-fixed relevant solution, enclosed 10 min-2 h.
atherosclerotic plaques (WHO grade III) were also HDL with and without added Lipostabil. Cryostat tissue were mounted on slides, covered with the with a cover-slip and incubated at 22” or 37°C for
Results Morphological aspects Myelin buds (liposomes) were seen within 20 min over the surface of cholesterol crystals incubated in the undiluted HDL solution (15 mg/ml). HDL at physiological dilution (3 mg/ml) showed buds within 2 h, while the weaker dilutions (0.6 and 0.1 mg/ml) produced some effect by 18 h. Stages in the destruction of cholesterol crystals by bud formation with HDL alone are shown in Figs. 1 and 2. Buds were initially isotropic, but often developed Maltese Cross anisotropism with the passage of time. The thick rhomboidal crystals from the core of atherosclerotic lesions formed buds with HDL, but the thin flat plate-like crystals did not. However, with both LDL and VLDL fine crystals were often precipitated over the surface of the original cholesterol crystals from 2 h onwards. Freshly prepared HDL was inactive after heat denaturation at 60°C for 20 min and mainly so after treatment with 8 M urea. HDL was not active against cholesteryl stearate and the other test crystals mentioned under Methods. In 4 replicate studies, undiluted LDL (5 mg/ml), VLDL (5 mg/ml) and albumin (5 mg/ml) produced no buds with cholesterol crystals over 18 h.
Fig. 1. Myelin buds over partly dissolved cholesterol crystals after 3 hours’ incubation in HDL. Partly polarized light, X 300.
Fig. 2. X 300.
Mydin
buds
around
a cholesterol
crystal,
3 hours’
incubation
in HDL.
Partly
polarized
light,
Myelin buds are known to form with phospholipids, and with phospholipids-cholesterol and other lipid mixtures [ 13,14,17]. In this connection, preincubation of HDL with phospholipases C and D largely inhibited liposome formation, presumably by removing phospholipid from the HDL molecule. By contrast, preincubation of HDL with phospholipase A caused some activation of budding, which could be consistent with the formation of a surface-active
Fig. 3. Acceleration and increased density of myelin bud formation after addition of Lipostabil Note erosion of cholesterol crystal after 1 hour’s incubation. Partly polarized light, X 500.
to HDL.
Fig. 4. Effect of incubation with Lipostabil and HDL. Left zero time; middle complete disappearance of crystalline cholesterol. Fully polarized light, X 190.
1 h; right 18 h. Note near-
lysophosphatide from phospholipid within the HDL molecule. Preincubation of HDL with trypsin largely inhibited budding and this indicates that the apoprotein moiety is required as well as the phospholipid. Although Leathes [13] reported that calcium completely abolished bud formation with isolated phospholipids, calcium, in fact, activated bud formation from cholesterol crystals with HDL over the range l--20 mM Ca++, with a maximum around 8 mM. Lipostabil (polyunsaturated lecithin) had no direct action against cholesterol in concentrations up to 8 mg/ml but, when added (0.25 mg) to a low concentration of HDL (0.1 mg in 1 ml buffered saline) or to undiluted whole serum, it caused a remarkable increase in the destruction of cholesterol crystals (Figs. 3 and 4). Marked activity was noted by 15 min, while 18 hours’ incubation led to near complete destruction of the suspension of cholesterol crystals (Fig. 4). Stronger concentrations of HDL and Lipostabil produced even more active budding. Discussion In a preliminary study we noted that crystals of tritium-labelled cholesterol readily reacted with non-radioactive HDL, with a resulting marked increase of radioactivity in the supernatant (HDL phase). This finding was unexpected in view of previous indications that cholesterol crystals, with their low surface/ volume ratio, might be in a metabolically sequestered or inaccessible state [ 81. This unexpected result led us to examine the cholesterol--HDL reaction under the microscope. The present in vitro findings show that macromicellar bodies (known as myelin buds, myelin figures or liposomes) form at the surface of the cholesterol crystal in contact with HDL, and that this process goes on under experimental in vitro conditions to the destruction of the crystal. This effect is not seen with other lipoproteins or albumin. Liposome formation does not depend on a micellar reaction with phospholipid released from degraded HDL, as denatura-
470
tion of the lipoprotein with heat at 60°C prevented the process. Nevertheless, HDL phospholipid is required for liposome formation and, indeed, addition of a polyunsaturated lecithin (Lipostabil) remarkably accelerates the process. This finding is consistent with the known enhancing action of phospholipids on HDL as regards cholesterol uptake [ 191, the marked clearing action of Lipostabil on hyperlipidaemic serum [ 201 and the antiatherogenic action of intravenous Lipostabil in the rabbit [ 20,211. As will be shown in the next paper [ 181, the liposome is a large body of high molecular weight and shows a characteristic appearance under the electron microscope. As will be discussed, these features distinguish it from the smaller HDL molecule. The liposomes which are considered in this present paper and which are visible with the light microscope, probably correspond to the large and aggregated medium-sized ultrastructural bodies described in the following paper [17]. Glomsett [ 221 outlined a physiological role for HDL in cholesterol metabolism, whereby the lipoprotein carried cholesterol from cell membrane back to the liver. The liposomes described in this present paper seem to differ in that their cholesterol - as studied with tritium-cholesterol - is carried to the reticuloendothelial system as well as the liver (Adams, Abdulla, Bayliss and Morgan, to be published). Whether or not liposome formation is of importance in actual or potential regression of atherosclerotic lesions remains to be determined. HDL is active in vitro against cholesterol crystals in incubated sections of human atherosclerotic lesions but, in vivo, a limitation might be imposed by the relative avascularity and, thus, inaccessibility of the arterial wall. However, in comparison with other lipoproteins, HDL apoproteins are known to enter the arterial wall with some ease [23]. Thus, liposome formation may prove to play a pathophysiological role in atherosclerosis. A further possible target for HDL would be the embolus that consists largely of cholesterol crystals derived from atheromatous gruel.
References Armstrong, M.L. and Megan. M.B.. Lipid depletion in atheromatous coronary arteries in rhesus monkeys after regression diets, Circulat. Res.. 30 (1972) 675. Vesselinovitch, D.. Wissler. R.W., Hughes, R. and Borensztajn. J.. Reversal of advanced atherosclerosis in rhesus monkeys, Part 1 (Light-microscopic studies), Atherosclerosis, 23 (1976) 165. Adams, C.W.M., Morgan, R.S. and Bayliss, O.B.. No regression of atheroma over one year in rabbits previously fed a cholesterol-enriched diet, Atherosclerosis, 18 (1973) 429. Adams, C.W.M.. Bay&s, O.B. and Turner, D.R., Phagocytes. lipid-removal and regression of atheroma. J. Path., 116 (1975) 225. Gould, R.J., Jones, R.J. and Wissler. R.W., Lability of cholesterol in human atherosclerotic plaques, Circulation, 20 (1959) 967. Field, H.. Swell, L.. Schools, P.E. and Treadwell. C.R., Dynamic aspects of cholesterol metabolism in different areas of aorta and other tissues in man and their relationships to atherosclerosis, Circulation, 22 (1960) 547. 1 Goodman. De W.S. and Noble.,R.P.. Turnover of plasma cholesterol in man, J. Clin. Invest., 47 (1968) 231. 8 Adams, C.W.M., Tissues changes and lipid entry in developing atheroma. In: Atherogenesis: Initiating Factors, CIBA Foundation Symposium NS 12.1973, pp. 5-37. S., Cholesterol transfer between arterial smooth muscle tissue and serum 9 Bondjers. G. and BjSrkerud, lipoproteins in vitro, Artery, 1 (1974) 3-9. 10 Stein, Y.. Glangeaud. MC.. Fatnaru. M. and Stein, 0.. The removal of cholesterol ester from aortic
smooth Biochim. 11 12
13 14 15 16 17 18 19 20
21 22 23
muscle
cells in culture
Biophys.
Acta,
and Landschutz
380 (1975)
ascites
cells by fractions
of high density
lipoprotein,
106.
Carew, T.E.. Hayes, S.B., Koschinsky, T. and Steinberg, D., A mechanism by which high density lipoproteins may slow the atherogenic process, Lance& 1 (1976) 1315. Stein, 0.. Vanderhoek, J. and Stein, Y., Cholesterol content and sterol synthesis in human skin fibroblasts and rat aortic smooth muscle cells exposed to lipoprotein depleted serum and high density lipoproteinlphospholipid mixtures, Biochim. Biophys. Acta, 431 (1976) 347. Leathes, J.B., Role of fats in vital phenomena, Lecture III. Myelin forms of lecithine. Lancet, 1 (1925) 957. Bangham, A.D., Lipid bilayers and biomembranes, Ann. Rev. Biochem., 41 (1972) 753. Burstein, M. and Scholnick, J.R.. Lipoprotein-polyanion-metal interactions, Adv. Lipid Res., 11 (1973) 67. Fieser, L.F., Cholesterol and comparisons, Part 7 (Steroid dibromides), J. Amer. Chem. Sot., 75 (1953) 5421. Adams, C.W.M., Histochemical mechanism of the Marchi reaction for degenerating myelin, J. Neurothem.. 2 (1958) 178. Abdulla, Y.H. and Adams, C.W.M.. The action of human high density lipoprotein on cholesterol crystals, Part 2 (Biochemical observations), Atherosclerosis, 31 (1978) 473. Rothblat. G.H., Buchko. M.K. and Kritchevsky. D., Cholesterol uptake by L 5178 Y tissue culture cells - Studies with delipidized serum, Biochim. Biophys. Acta. 164 (1968) 327. Adams, C.W.M.. Abdulla. Y.H., Bayliss. O.B. and Morgan, R.S., Modification of aortic atheroma and fatty liver in cholesterol-fed rabbits by intravenous injection of saturated and polyunsaturated lecithins, J. Path. Bact., 94 (1967) 77. 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 (1970) 41. Stein, Y. and Stein, 0.. Lipid synthesis and degradation and lipoprotein transport in mammalian aorta. In: Atherogenesis: Initiating Factors, CIBA Foundation Symposium NS 12. 1973, p. 165. Glomsett, J.A., The plasma lecithin : cholesterol acyltransferase reaction. J. Lipid Res., 9 (1968) 155.
Atherosclerosis, 31 (1978) 465-471 @ Elsevier/North-Holland Scientific Publishers,
Ltd.
THE ACTION OF HUMAN HIGH DENSITY CHOLESTEROL CRYSTALS Part 1. Light-microscopic
LIPOPROTEIN
ON
Observations
C.W.M. ADAMS and ‘Y.H. ABDULLA Department (Received (Accepted
of Pathology, Guy’s Hospital Medical School, London SE1 9RT (Great Britain) 5 August, 1978) 18 August, 1978)
Summary High density lipoprotein (HDL) was found in vitro to form myelin buds (liposomes) from washed crystals of free cholesterol (commercial or atheroma sources). This activity led to the progressive destruction and solubilization of the crystals. Low density and very low density lipoproteins did not have any effect. Liposome formation and solubilization were accelerated by calcium ions, phospholipase A and polyunsaturated lecithin (Lipostabil). Cholesterol crystals were nearly completely destroyed after 18 h incubation with HDLLipostabil. Key words:
- Cholesterol Atherosclerosis Micelle - Phospholipid
-
High density
lipoprotein
-
Liposome
-
Introduction Resorption of atheroma lipids and regression of atherosclerosis is at best a somewhat slow process [l-3], in comparison with removal rates for lipids from other tissues [4]. Likewise, the turnover rate of cholesterol in atheroma is relatively slow [ 5-71. This metabolic inertia of cholesterol in atherosclerosis has been attributed in part to the crystalline nature of some of the deposited cholesterol, and in part to the absence of effective phagocytic cells and degradative enzymes for cholesterol in the atherosclerotic arterial wall [El]. However, in recent years it has become apparent that high density lipoprotein (HDL) will Partly
supported
by a grant
from
the
British
Heart
Foundation.
466
remove cholesterol from incubated atherosclerotic tissue [ 91, cultured arterial smooth muscle cells [ lO,ll] and fibroblasts [ 121. It, therefore, seemed relevant to see whether HDL is active against cholesterol in the supposedly inert crystalline form, in addition to its apparent action against cholesterol taken up by cells. It will be shown that HDL is, in fact, active against cholesterol crystals, in that it promotes the formation of myelin buds [ 131 or liposomes [ 141 over the crystal surface. This budding results in the progressive erosion and destruction of the cholesterol crystal. Methods Lipoproteins were prepared from pooled human blood-bank serum. High density (HDL), low density (LDL) and very low density (VLDL) lipoproteins were respectively precipitated with sodium phosphotungstate, heparinmanganese and heparin-magnesium, following Burstein and Scholnick [ 151. These fractions were dialysed exhaustively against 10 mM Tris (pH 7.4) in 0.15 M sodium chloride, and were then concentrated by vacuum dialysis until semisolid. These stock solutions were stored at 4°C. The relevant fractions were further purified for use by flotation over buffered salt with respective densities of 1.210, 1.063 and 1.009 [15]. The purity of each fraction was then confirmed by crossed immunoelectrophoresis, using antibody against whole human serum (Behringwerke). Each purified fraction was stored at 4°C and was used over a maximum period of 7 days. Crystalline free cholesterol was obtained from BDH and further purified by formation of its dibromide [ 161. Material from the core of advanced human lesions in subjects aged over 65 years was repeatedly washed with 0.15 M sodium chloride and centrifuged at 500 X g for 15 min. The precipitate contained a mixture of rhomboidal crystals and flat plate-like rectangular crystals. Approximately 1 mg saline-washed samples of cholesterol crystals were suspended in increasing dilutions of HDL (15, 3, 0.6, 0.12 mg protein/ml 0.15 M saline-5 mM barbital buffer, pH 7.3). Budding activity was assessed by direct transmission and polarizing microscopy after incubation of the mixtures (with or without shaking) for 15 min to 18 h at 37°C. Buffered 0.15 M saline, whole serum, LDL (- 5 mg protein/ml), VLDL (- 5 mg protein/ml) and human serum albumin (- 5 mg protein/ml; Miles) were also tested for budding activity against cholesterol crystals. The following other crystalline substances were tested to see whether they formed buds with HDL: cholesteryl stearate (Hopkin and Williams), glyceryl tristearate (Hopkin and Williams), stearic acid (Hopkin and Williams), stearyl alcohol (BDH), cholestan-3a-ol (Sigma), cholestan-3&ol (Sigma), cholesteryl digitonide and octadecane (L. Light). Activation or inhibition of bud formation with HDL was tested with l-20 mM calcium chloride [lo] and with a sonicated polyunsaturated lecithin (Lipostabil, Nattermann, Cologne; about 70% of the phospholipid was dilinoleyl phosphatidyl choline). HDL was also tested for budding activity after preliminary digestion for 2 h at 37°C with the following enzymes: trypsin (Sigma grade l), and phospholipase A (Crotulus adumanteus, Sigma) C and D (Sigma). All enzymes were used at 1 mg/ml 0.15 M saline-5 mM barbital buffer at pH 7.3.
467
Four advanced human examined for activity with sections of formol-fixed relevant solution, enclosed 10 min-2 h.
atherosclerotic plaques (WHO grade III) were also HDL with and without added Lipostabil. Cryostat tissue were mounted on slides, covered with the with a cover-slip and incubated at 22” or 37°C for
Results Morphological aspects Myelin buds (liposomes) were seen within 20 min over the surface of cholesterol crystals incubated in the undiluted HDL solution (15 mg/ml). HDL at physiological dilution (3 mg/ml) showed buds within 2 h, while the weaker dilutions (0.6 and 0.1 mg/ml) produced some effect by 18 h. Stages in the destruction of cholesterol crystals by bud formation with HDL alone are shown in Figs. 1 and 2. Buds were initially isotropic, but often developed Maltese Cross anisotropism with the passage of time. The thick rhomboidal crystals from the core of atherosclerotic lesions formed buds with HDL, but the thin flat plate-like crystals did not. However, with both LDL and VLDL fine crystals were often precipitated over the surface of the original cholesterol crystals from 2 h onwards. Freshly prepared HDL was inactive after heat denaturation at 60°C for 20 min and mainly so after treatment with 8 M urea. HDL was not active against cholesteryl stearate and the other test crystals mentioned under Methods. In 4 replicate studies, undiluted LDL (5 mg/ml), VLDL (5 mg/ml) and albumin (5 mg/ml) produced no buds with cholesterol crystals over 18 h.
Fig. 1. Myelin buds over partly dissolved cholesterol crystals after 3 hours’ incubation in HDL. Partly polarized light, X 300.
Fig. 2. X 300.
Mydin
buds
around
a cholesterol
crystal,
3 hours’
incubation
in HDL.
Partly
polarized
light,
Myelin buds are known to form with phospholipids, and with phospholipids-cholesterol and other lipid mixtures [ 13,14,17]. In this connection, preincubation of HDL with phospholipases C and D largely inhibited liposome formation, presumably by removing phospholipid from the HDL molecule. By contrast, preincubation of HDL with phospholipase A caused some activation of budding, which could be consistent with the formation of a surface-active
Fig. 3. Acceleration and increased density of myelin bud formation after addition of Lipostabil Note erosion of cholesterol crystal after 1 hour’s incubation. Partly polarized light, X 500.
to HDL.
Fig. 4. Effect of incubation with Lipostabil and HDL. Left zero time; middle complete disappearance of crystalline cholesterol. Fully polarized light, X 190.
1 h; right 18 h. Note near-
lysophosphatide from phospholipid within the HDL molecule. Preincubation of HDL with trypsin largely inhibited budding and this indicates that the apoprotein moiety is required as well as the phospholipid. Although Leathes [13] reported that calcium completely abolished bud formation with isolated phospholipids, calcium, in fact, activated bud formation from cholesterol crystals with HDL over the range l--20 mM Ca++, with a maximum around 8 mM. Lipostabil (polyunsaturated lecithin) had no direct action against cholesterol in concentrations up to 8 mg/ml but, when added (0.25 mg) to a low concentration of HDL (0.1 mg in 1 ml buffered saline) or to undiluted whole serum, it caused a remarkable increase in the destruction of cholesterol crystals (Figs. 3 and 4). Marked activity was noted by 15 min, while 18 hours’ incubation led to near complete destruction of the suspension of cholesterol crystals (Fig. 4). Stronger concentrations of HDL and Lipostabil produced even more active budding. Discussion In a preliminary study we noted that crystals of tritium-labelled cholesterol readily reacted with non-radioactive HDL, with a resulting marked increase of radioactivity in the supernatant (HDL phase). This finding was unexpected in view of previous indications that cholesterol crystals, with their low surface/ volume ratio, might be in a metabolically sequestered or inaccessible state [ 81. This unexpected result led us to examine the cholesterol--HDL reaction under the microscope. The present in vitro findings show that macromicellar bodies (known as myelin buds, myelin figures or liposomes) form at the surface of the cholesterol crystal in contact with HDL, and that this process goes on under experimental in vitro conditions to the destruction of the crystal. This effect is not seen with other lipoproteins or albumin. Liposome formation does not depend on a micellar reaction with phospholipid released from degraded HDL, as denatura-
470
tion of the lipoprotein with heat at 60°C prevented the process. Nevertheless, HDL phospholipid is required for liposome formation and, indeed, addition of a polyunsaturated lecithin (Lipostabil) remarkably accelerates the process. This finding is consistent with the known enhancing action of phospholipids on HDL as regards cholesterol uptake [ 191, the marked clearing action of Lipostabil on hyperlipidaemic serum [ 201 and the antiatherogenic action of intravenous Lipostabil in the rabbit [ 20,211. As will be shown in the next paper [ 181, the liposome is a large body of high molecular weight and shows a characteristic appearance under the electron microscope. As will be discussed, these features distinguish it from the smaller HDL molecule. The liposomes which are considered in this present paper and which are visible with the light microscope, probably correspond to the large and aggregated medium-sized ultrastructural bodies described in the following paper [17]. Glomsett [ 221 outlined a physiological role for HDL in cholesterol metabolism, whereby the lipoprotein carried cholesterol from cell membrane back to the liver. The liposomes described in this present paper seem to differ in that their cholesterol - as studied with tritium-cholesterol - is carried to the reticuloendothelial system as well as the liver (Adams, Abdulla, Bayliss and Morgan, to be published). Whether or not liposome formation is of importance in actual or potential regression of atherosclerotic lesions remains to be determined. HDL is active in vitro against cholesterol crystals in incubated sections of human atherosclerotic lesions but, in vivo, a limitation might be imposed by the relative avascularity and, thus, inaccessibility of the arterial wall. However, in comparison with other lipoproteins, HDL apoproteins are known to enter the arterial wall with some ease [23]. Thus, liposome formation may prove to play a pathophysiological role in atherosclerosis. A further possible target for HDL would be the embolus that consists largely of cholesterol crystals derived from atheromatous gruel.
References Armstrong, M.L. and Megan. M.B.. Lipid depletion in atheromatous coronary arteries in rhesus monkeys after regression diets, Circulat. Res.. 30 (1972) 675. Vesselinovitch, D.. Wissler. R.W., Hughes, R. and Borensztajn. J.. Reversal of advanced atherosclerosis in rhesus monkeys, Part 1 (Light-microscopic studies), Atherosclerosis, 23 (1976) 165. Adams, C.W.M., Morgan, R.S. and Bayliss, O.B.. No regression of atheroma over one year in rabbits previously fed a cholesterol-enriched diet, Atherosclerosis, 18 (1973) 429. Adams, C.W.M.. Bay&s, O.B. and Turner, D.R., Phagocytes. lipid-removal and regression of atheroma. J. Path., 116 (1975) 225. Gould, R.J., Jones, R.J. and Wissler. R.W., Lability of cholesterol in human atherosclerotic plaques, Circulation, 20 (1959) 967. Field, H.. Swell, L.. Schools, P.E. and Treadwell. C.R., Dynamic aspects of cholesterol metabolism in different areas of aorta and other tissues in man and their relationships to atherosclerosis, Circulation, 22 (1960) 547. 1 Goodman. De W.S. and Noble.,R.P.. Turnover of plasma cholesterol in man, J. Clin. Invest., 47 (1968) 231. 8 Adams, C.W.M., Tissues changes and lipid entry in developing atheroma. In: Atherogenesis: Initiating Factors, CIBA Foundation Symposium NS 12.1973, pp. 5-37. S., Cholesterol transfer between arterial smooth muscle tissue and serum 9 Bondjers. G. and BjSrkerud, lipoproteins in vitro, Artery, 1 (1974) 3-9. 10 Stein, Y.. Glangeaud. MC.. Fatnaru. M. and Stein, 0.. The removal of cholesterol ester from aortic
smooth Biochim. 11 12
13 14 15 16 17 18 19 20
21 22 23
muscle
cells in culture
Biophys.
Acta,
and Landschutz
380 (1975)
ascites
cells by fractions
of high density
lipoprotein,
106.
Carew, T.E.. Hayes, S.B., Koschinsky, T. and Steinberg, D., A mechanism by which high density lipoproteins may slow the atherogenic process, Lance& 1 (1976) 1315. Stein, 0.. Vanderhoek, J. and Stein, Y., Cholesterol content and sterol synthesis in human skin fibroblasts and rat aortic smooth muscle cells exposed to lipoprotein depleted serum and high density lipoproteinlphospholipid mixtures, Biochim. Biophys. Acta, 431 (1976) 347. Leathes, J.B., Role of fats in vital phenomena, Lecture III. Myelin forms of lecithine. Lancet, 1 (1925) 957. Bangham, A.D., Lipid bilayers and biomembranes, Ann. Rev. Biochem., 41 (1972) 753. Burstein, M. and Scholnick, J.R.. Lipoprotein-polyanion-metal interactions, Adv. Lipid Res., 11 (1973) 67. Fieser, L.F., Cholesterol and comparisons, Part 7 (Steroid dibromides), J. Amer. Chem. Sot., 75 (1953) 5421. Adams, C.W.M., Histochemical mechanism of the Marchi reaction for degenerating myelin, J. Neurothem.. 2 (1958) 178. Abdulla, Y.H. and Adams, C.W.M.. The action of human high density lipoprotein on cholesterol crystals, Part 2 (Biochemical observations), Atherosclerosis, 31 (1978) 473. Rothblat. G.H., Buchko. M.K. and Kritchevsky. D., Cholesterol uptake by L 5178 Y tissue culture cells - Studies with delipidized serum, Biochim. Biophys. Acta. 164 (1968) 327. Adams, C.W.M.. Abdulla. Y.H., Bayliss. O.B. and Morgan, R.S., Modification of aortic atheroma and fatty liver in cholesterol-fed rabbits by intravenous injection of saturated and polyunsaturated lecithins, J. Path. Bact., 94 (1967) 77. 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 (1970) 41. Stein, Y. and Stein, 0.. Lipid synthesis and degradation and lipoprotein transport in mammalian aorta. In: Atherogenesis: Initiating Factors, CIBA Foundation Symposium NS 12. 1973, p. 165. Glomsett, J.A., The plasma lecithin : cholesterol acyltransferase reaction. J. Lipid Res., 9 (1968) 155.
