625 @ 1992 The Japanese Society of Pathology
Review
Accumulation of Cho lesteryI Ester in Atherosclerotic Lesions
Tatsuya Takano
This article reviews aspects of the molecular pathology of cholesteryl ester accumulation in atherosclerotic lesions. 1. Transcytosis of lipoproteins through a cultured endothelial monolayer. 2. Effects of platelets and PGI, on intercellular transport of endothelial cells. 3. Transformation of macrophages to foam cells. 4. Cholesteryl ester deposition in the extracellular space of atherosclerotic lesions. The development and use of novel monoclonal antlbodies recognizing atherosclerotic lesions and peroxidized lipoproteins prepared from then are also discussed. Acta Pathol Jpn 42: 625-631,1992. Key words : Atherosclerosis, Endothelial cell, Foam cell, Extracellular space, Transcytosis, Cholesteryl ester, Lipoprotein, Peroxidized lipoprotein, Monoclonal antibody, Vitronectin
A characteristic of atherosclerosis is the accumulation of large amounts of lipids, mainly cholesteryl ester, in the arterial wall. Histochemical and ultrastructural observations have shown that these lipids accumulate in both the cytoplasm of foam cells and the extracellular space. It has been suggested that the foam cells originate from macrophages (1) and/or modified smooth muscle cells (2). It has also been hypothesized that the extracellular lipids originate from circulating lipoproteins penetrating endothelial cells and/or denatured lipoproteins derived from ruptured foam cells (3). The lipids deposited in the extracellular space may be endocytosed by macrophages and/or modified smooth muscle cells, resulting in the Received April 3, 1992. Accepted for publication June 1, 1992. Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa. Mailing address : Tatsuya Takano, Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagarniko, Kanagawa 199-01, Japan. This work was supported by Grants for Scientific Research from the Ministry of Education, Science and Culture of Japan
formation of foam cells. This article reviews aspects of the molecular pathology of cholesteryl ester accumulation in atherosclerotic lesions.
1. Transcellular Transport of Lipoprotein The vascular endothelium is believed to act as a selective barrier to the passage of macromolecules between the blood plasma in the vascular lumen and the interstitial fluid in the perivascular spaces. Studies of the transport of low-density lipoprotein (LDL) are important f o r determining the mechanism of cholesteryl ester accumulation in the arterial wall. Extensive studies of the internalization of LDL through receptors have been made using cultured endothelial cells. Recent cytochemical studies have shown that LDL passes through the endothelium in transcytotic vesicles in situ (4). However, very
4
3
2
1
0 0
1
2
3
Incubation Time (h) Figure 1. Time course of RB-LDL transport. Rhodamine Blabeled LDL (RB-LDL) (0.2mg protein/ml) was introduced into the upper compartment, and the transport of RB-LDL through the membrane to the lower compartment was monitored : Dacron sheet alone (m); Dacron sheet with gelated collagen (A); endothelial monolayer on the Dacron sheet with gelated collagen at 37'C (o), O'C (o),and 37'C for 2 h then a t O'C (A).
62 6
Cholesteryl Ester in Atheroma (Takano)
little is known about the mechanism, kinetics and requirements of the transcellular transport of LDL across the endothelium. Rhodamine 6-labeled LDL (RB-LDL) can be used as a fluorescent probe to investigate transport (5). A considerable amount of the RB-LDL was found to be transported at 37"C,but not a t O X , and on reducing the temperature from 37°Cto O X , transport decreased dramatically (Fig. 1). LDL transport was also shown to be energydependent, since it was inhibited by a combination of 2deoxyglucose and NaN,, inhibitors of ATP generation. After transport at 37"C,no degradation products of apoprotein B were detected by SDS-polyacrylamide gel electrophoresis, suggesting that LDL was not metabolized during tra,nsport. These results suggest that LDL is transported in transcytotic vesicles by a temperatureand energy-dependent process, but not through cellular junctions nor by endocytosis and exocytosis via a lysosomal system.
2.
Effect of Platelets a n d PGI, o n lntercellular Transport t h r o u g h a Denuded Arterial Endothelial Monolayer Any damage to the endothelial layer results in accumulation of lipoproteins. Platelet adhesion and aggregation may also occur at the site of injury which may, in turn, lead to thrombosis (6). Malfunction of endothelial cells appears to be an initial event in atherogenesis. Histamine, serotonin and PAF secreted from platelets are all potent mediators of vascular permeability (7,8). In previous studies, we developed an in vitro model to invest igate int ercelIula r j unct io na I t ra nsport of fluorescein dextran (FD) (9). FDs of various MW were studied in order to determine a suitable size of FD for use as a marker of intercellular transport through the endothelial monolayer. FD (150 kDa) was found to be a suitable marker, since it passed through the denuded area and transport was restricted to that found through an intact endothelial monolayer. We also showed that addition of fibrinogen and fibronectin to the collagen gel led to a synergistic increase of platelet binding to a partially denuded endothelial monolayer (10). Under these conditions, marked aggregation of platelets was observed in the denuded area by scanning electron microscopy. In addition, transport was also studied in the presence of mediators released f r o m activated platelets (11). Supernatant of platelets activated by denudation of the endothelial monolayer did not affect the transport, nor did supernatants of platelets activated with ADP or thrombin. In our current model, mediator(s) secreted from platelets did not enhance endothelial permeability,
T
0
1
2
3
4
T i m e (h)
Figure 2. Effect of platelets and isocarbacyclin on transport of fluorescein dextran (FD) through a partially denuded endothelial monolayer. FD transport was measured through an intact endothelial monolayer (o), through a partially denuded endothelial monolayer in the absence (a)or presence of platelets with (A) or without (m) 3 ~ 1 0 M -~ isocarbacyclin, and through a gel layer (u). The values are the means +-SD of three experiments.
and platelet binding to a denuded endothelial monolayer resulted in a decrease in transport (Fig. 2). This finding may be explained by covering of the denuded area by adherent and aggregated platelets, thus inhibiting transport. Isocarbacyclin (stable derivative of prostacyclin) inhibited platelet binding by more than 80% but had little effect o n transport through the endothelial layer when platelets were present. We demonstrated that although prostacyclin strongly inhibited platelet aggregation, a small number of platelets forming a monolayer were adherent to the denuded area. We suggest that formation of a platelet monolayer at the denudation site is sufficient to suppress the transport of FD (Fig. 3). It is known that prostacyclin production by blood vessels is increased in endothelial injury (12), and adherent platelets can often be seen in sections of injured vessel wall. These results suggest that, in vivo, the barrier function may be maintained by adherent platelets after prostacyclin secretion.
3. Accumulation of Cholesteryl Ester in Foam Cells In early lesions, lipids accumulate mainly in the cytoplasm of cells such as macrophages. Thin-section electron microscopy has shown that intracellular lipids are stored in two forms in the cytoplasm of foam cells: in
627
Acta Pathologica Japonica 42 (9): 1992 FD
FD
Q
/
A : Control
B : PGI,
Figure 3. Adherent and aggregated platelets inhibit FD transport by covering the denuded area (A :
control). FD transport was still inhibited, though platelet binding was inhibited by more than 80% by prostacyclin (B: PGI,).
membrane- bound vacuoles, which probably correspond to lysosomes, and in vacuoles without a peripheral membrane. These lipid droplets, both with and without membranes, were also observed in quick-freeze replicas of foam cells possibly derived from macrophages and/or smooth muscle cells (Fig. 4) (13). The membrane-free droplets were more numerous than membrane-bound droplets, and appeared to consist of onion-like concentric lamellae. Onion-like lamellae were also observed in anisotropic cholesteryl ester droplets prepared in vitro. These studies suggest that the onion-like droplets consist mainly of cholesteryl ester and correspond to the cholesteryl ester-rich lipid inclusions. The other type of lipid droplet in foam cells is membrane-bound, as found in lysosomes. These lipid-laden organelles may corre spond to low-density lysosomes, which can be prepared by flotation sucrose density gradient centrifugation. The lipid droplets in lysosomes vary in size. Cholesteryl esters may accumulate as cytoplasmic lipid droplets after microsomal re-esterification of free cholesterol liberated from lysosomal particles. On the other hand, in highly lipid-laden cells, elements of the endoplasmic reticulum, which is involved in reesterification of cholesterol, are rarely found. An alternative explanation for the accumulation of membranefree droplets in these highly lipid-laden foam cells is that lipid inclusion bodies accumulate, not via microsomes, but are transferred to the cytoplasm from phagolysosomes with concomitant partial hydrolysis of cholesteryl esters via the “vesicular pathway” (Fig. 5) (14).
4. Cholesteryl Ester Deposits in the Extracellular Space In advanced lesions, lipids are found in the extracellular matrix. However, due to the limitations of the thinsection method, the fine structure of the extracellular lipids cannot be shown, since the neutral lipids are extracted during the dehydration procedure. However, using the quick-freeze and etching technique, we have
Figure4. Quick-freeze replica of the perinuclear area of a foam cell (13). An organelle containing various sizes of granules is seen among droplets with onion-like layers. Part of the
membrane of this organelle shows an E-fracture face (arrowhead). The fractured face of some granular contents are smooth and that of others contains membrane particles (arrows). (N), nucleus (~20,000).
clearly demonstrated the ultrastructural characteristics of these extracellular lipids. They appeared as irregularly shaped vesicles, some of which had a surface membrane. The vesicles and electron-lucent structures among collagen fibers in thin sections corresponded to these extracellular lipids (Fig. 6)(13). Some extracellular vesicles were aggregated, and the clustered vesicles were similar in size t o the cells destined to collapse in the atherosclerotic aorta. Some extracellular vesicles containing lipids might be derived from these disrupted foam cells. A t the same time, lipoprotein or denatured lipoprotein transported through the endothelial barrier might be deposited in the extracellular space.
628
Cholesteryl Ester in Atheroma (Takano)
LIPOPROTEINS
/L-7
FOAM C E L L S
RUPTURED C E L L S Figure 5. lntracellular lipids in foam cells are stored in two forms: 1) membrane-bound vacuoles which probably correspond t o lysosomes, and 2) vacuoles without a peripheral membrane. Cholesteryl esters may accumulate after microsomal re-esterification of free cholesterol liberated from lysosomal particles (Path I),and/or after transfer t o the cytoplasm from phago-lysosomes via the "vesicular pathway" (Path 11).
5. Monoclonal Antibodies Recognizing Extracellular Regions w i t h Lipid Deposits in t h e Atherosclerotic Aorta In order to study the mechanisms of cholesteryl ester accumulation in the extracellular space of the atherosclerotic aorta, we prepared novel monoclonal antibodies against atherosclerotic lesions using a delipidated crude homogenate of atherosclerotic aorta from Watanabeheritable hyperlipidemic (WHHL) rabbits as a complex mixture of immunogens (15). By screening the antibodies histochemically, we were able to obtain for the first time monoclonal antibodies which specifically recognized the extracellular matrix with lipid-laden deposits (EMRla/212D) in atherosclerotic lesions (Figs. 7a, b). The purified antigenic material is a glycoprotein with a molecular mass of 66 kDa, and the epitope of the antigenic material contains sialic acid as a major element (16). We have isolated from rabbit liver cDNA library clones coding for the 66-kDa glycoprotein (GP66). The clone spans the sequence coding for the entire GP66 (456 amino acids) and 19 amino acids of a signal peptide. GP66 was deduced to be rabbit vitronectin (17) from its nucleotide sequence, containing an Arg-Gly-Asp cell attachment sequence and showing 76% amino acid sequence homology with human vitronectin. Furthermore, EMR 1a/2 12D recognized rabbit vit ronect in purified by heparin affinity chromatography. RNA blot
Figure 6. Appearance o f the extracellular connective tissue space of the atherosclerotic aorta by the quick-freeze. etching technique (13). Large amounts of lipids with a vesicular structure are observed among collagen fibers, which show a characteristic repeating pattern (arrowheads). Some of these vesicular structures appear t o be covered by a membrane. Arrows indicate fractured faces of vesiculated lipids corre sponding t o the P-fracture face of a biomembrane; for details see text. These vesicular structures correspond to vesicles rimmed with a unit membrane structure (inset, arrow) among collagen fibers (inset, arrowhead) in thin sections ( ~ 4 4 , 0 0 0 ; inset, x 40,000).
H003
222
6PL
630
Cholesteryl Ester in Atherorna (Takano)
Q
Figure 9.
Working hypothesis; How cholesteryl ester accumulates in atherosclerotic lesions.
ger cells accumulate in the extracellular spaces of the arterial wall. Vitronectin, possibly associated with scavenger cells, may be involved in the elimination of lipid deposits in the extracellular space of atherosclerotic lesions. Acknowledgements : We would like to thank Drs. K.M. Arnanurna, G.G. Ecsedi, M. Enornoto, R. Hashida, N. Hayashi, T. Irnanaka, C.A. Kawagoe, J. Kirnura, Y. Komine, M. Kurosaka, C. Mineo, K.C. Miyasato, M. Mori, H. Mowri, K. Nakagami, S. Ohkurna, R. Sato, 0. Shirnasaki, H. Takahashi and Y.M. Yagyu for their collaboration in this research work. We are grateful to Drs. Y. Watanabe and K. Hirohata (Kobe University) for providing us with WHHL rabbits, and to S. lkegarni (Teikyo University), T. Kanaseki (Tokyo Metropolitan Institute for Neurosciences) and Y. Yoshida (Yamanashi Medical College) for their helpful comments and discussions. Finally we thank Miss H. Kobayashi for her kind assistance in the various stages of preparation of the manuscript.
REFERENCES 1. Shaffner T, Taylor K, Bartucci EJ, et a/. Arterial foam cells with distinctive immunornorphologic and histochemical features of rnacrophages. Am J Pathol 100 : 57-73, 1980. 2. Ross R and Glornset JA. The pathogenesis of atherosclerosis. N Engl J Med 295: 369-377, 1976. 3. Takano T, Kanaseki T, Amanurna K, et a/. Macrophages and accumulation of cholesterol ester in atherornatous aorta. In Richard S and Alan R, eds. The Reticuloendothelial System, Liss, New York, 1985 : 323-337. 4. Sirnionescu N. Cellular aspects of transcapillary exchange. Physiol Rev 63: 1536-1579, 1983. 5. Hashida R, Anamizu C, Kirnura J, et a/. Transcellular transport of lipoprotein through arterial endothelial cells in monolayer culture. Cell Struct Funct 11: 31-
6. 7. 8. 9.
10. 11.
12. 13.
14.
15.
16.
42, 1986. Ross R. Platelet: Cell proliferation and atheroscle rosis. Metabolism 28: 410-414, 1979. Majno G, Shea SM, and Leventhal M. Endothelial contraction induced by histamine-type mediators. J Cell Biol 42 : 647-672, 1969. Carnussi G, Arese P, Tetta C, et a/. Platelet activating factor. In Bertani T and Rernuzzi G, eds. Glornerular Injury, Wichtig Editore, Milan, 1983 : 89-1 18. Hashida R, Anamizu C, Yagyu YM, et a/. Transcellular transport of fluorescein dextran through an arterial endothelial cell monolayer. Cell Struct Funct 11: 343-349, 1986. Yagyu Y, Hashida R, lwasaki K, et a/. Effect of PGI, on platelet binding to partially denuded endothelial rnonolayer in vitro. Thrornb Res 64: 733-744, 1991. Yagyu Y, Mineo C, lrnanaka T, et a/. Intercetlular transport through a partially denuded arterial endothe lial monolayer: Effect of platelets and PGI,. Thrornb Res 66: 215-222, 1992. Eldor A, Falcone DJ, Hajjar DP, et a/. Recovery of prostacyclin production by deendothelialized rabbit aorta. J Clin Invest 6 7 : 735-741, 1981. Arnanurna K, Kanaseki T, lkeuchi Y, et a/. Studies on fine structure and location of lipids in quick-freeze replicas of atherosclerotic aorta of WHHL rabbits. Virchows Arch [A] 410: 231-238, 1986. Mineo C, Kanaseki T, Enornoto M, et a/. lntracellular transport of cholesteryl esters from lysosornes to cytoplasm in macrophages. Cell Struct Funct 1 3 : 435443, 1988. Kirnura J, Nakagarni K, Arnanurna K, et a/. Monoclonal antibodies recognizing lipid-laden cells and extracellular regions with lipid-deposits in atheroscle rotic aorta. Virchows Arch [A] 410: 159-164, 1986. Nakagami K, Shirnazaki 0, Sato R, et a/. Monoclonal antibody EMRla/212D recognizing site of deposition of extracellular lipid in atherosclerosis : Purification and characterization of the antigen. Am J Pathol
Acta Pathologica Japonica 42 (9): 1992
135: 93-100, 1989. 17. Sato R, Komine Y, lmanaka T, et a/. Monoclonal antibody EMRla/212D recognizing site of deposition of extracellular lipid in atherosclerosis : Isolation and characterization of a cDNA clone for the antigen. J
631
Biol Chem 265: 21232-21236, 1990. 18. Takano T and Mineo C. Atherosclerosis and molecular pathology : Mechanisms of cholesteryl ester accurnulation in foam cells and extracellular space of atheroscle rotic lesion. J Pharmacobiodyn 13: 385-413, 1990.
Review
Accumulation of Cho lesteryI Ester in Atherosclerotic Lesions
Tatsuya Takano
This article reviews aspects of the molecular pathology of cholesteryl ester accumulation in atherosclerotic lesions. 1. Transcytosis of lipoproteins through a cultured endothelial monolayer. 2. Effects of platelets and PGI, on intercellular transport of endothelial cells. 3. Transformation of macrophages to foam cells. 4. Cholesteryl ester deposition in the extracellular space of atherosclerotic lesions. The development and use of novel monoclonal antlbodies recognizing atherosclerotic lesions and peroxidized lipoproteins prepared from then are also discussed. Acta Pathol Jpn 42: 625-631,1992. Key words : Atherosclerosis, Endothelial cell, Foam cell, Extracellular space, Transcytosis, Cholesteryl ester, Lipoprotein, Peroxidized lipoprotein, Monoclonal antibody, Vitronectin
A characteristic of atherosclerosis is the accumulation of large amounts of lipids, mainly cholesteryl ester, in the arterial wall. Histochemical and ultrastructural observations have shown that these lipids accumulate in both the cytoplasm of foam cells and the extracellular space. It has been suggested that the foam cells originate from macrophages (1) and/or modified smooth muscle cells (2). It has also been hypothesized that the extracellular lipids originate from circulating lipoproteins penetrating endothelial cells and/or denatured lipoproteins derived from ruptured foam cells (3). The lipids deposited in the extracellular space may be endocytosed by macrophages and/or modified smooth muscle cells, resulting in the Received April 3, 1992. Accepted for publication June 1, 1992. Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa. Mailing address : Tatsuya Takano, Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagarniko, Kanagawa 199-01, Japan. This work was supported by Grants for Scientific Research from the Ministry of Education, Science and Culture of Japan
formation of foam cells. This article reviews aspects of the molecular pathology of cholesteryl ester accumulation in atherosclerotic lesions.
1. Transcellular Transport of Lipoprotein The vascular endothelium is believed to act as a selective barrier to the passage of macromolecules between the blood plasma in the vascular lumen and the interstitial fluid in the perivascular spaces. Studies of the transport of low-density lipoprotein (LDL) are important f o r determining the mechanism of cholesteryl ester accumulation in the arterial wall. Extensive studies of the internalization of LDL through receptors have been made using cultured endothelial cells. Recent cytochemical studies have shown that LDL passes through the endothelium in transcytotic vesicles in situ (4). However, very
4
3
2
1
0 0
1
2
3
Incubation Time (h) Figure 1. Time course of RB-LDL transport. Rhodamine Blabeled LDL (RB-LDL) (0.2mg protein/ml) was introduced into the upper compartment, and the transport of RB-LDL through the membrane to the lower compartment was monitored : Dacron sheet alone (m); Dacron sheet with gelated collagen (A); endothelial monolayer on the Dacron sheet with gelated collagen at 37'C (o), O'C (o),and 37'C for 2 h then a t O'C (A).
62 6
Cholesteryl Ester in Atheroma (Takano)
little is known about the mechanism, kinetics and requirements of the transcellular transport of LDL across the endothelium. Rhodamine 6-labeled LDL (RB-LDL) can be used as a fluorescent probe to investigate transport (5). A considerable amount of the RB-LDL was found to be transported at 37"C,but not a t O X , and on reducing the temperature from 37°Cto O X , transport decreased dramatically (Fig. 1). LDL transport was also shown to be energydependent, since it was inhibited by a combination of 2deoxyglucose and NaN,, inhibitors of ATP generation. After transport at 37"C,no degradation products of apoprotein B were detected by SDS-polyacrylamide gel electrophoresis, suggesting that LDL was not metabolized during tra,nsport. These results suggest that LDL is transported in transcytotic vesicles by a temperatureand energy-dependent process, but not through cellular junctions nor by endocytosis and exocytosis via a lysosomal system.
2.
Effect of Platelets a n d PGI, o n lntercellular Transport t h r o u g h a Denuded Arterial Endothelial Monolayer Any damage to the endothelial layer results in accumulation of lipoproteins. Platelet adhesion and aggregation may also occur at the site of injury which may, in turn, lead to thrombosis (6). Malfunction of endothelial cells appears to be an initial event in atherogenesis. Histamine, serotonin and PAF secreted from platelets are all potent mediators of vascular permeability (7,8). In previous studies, we developed an in vitro model to invest igate int ercelIula r j unct io na I t ra nsport of fluorescein dextran (FD) (9). FDs of various MW were studied in order to determine a suitable size of FD for use as a marker of intercellular transport through the endothelial monolayer. FD (150 kDa) was found to be a suitable marker, since it passed through the denuded area and transport was restricted to that found through an intact endothelial monolayer. We also showed that addition of fibrinogen and fibronectin to the collagen gel led to a synergistic increase of platelet binding to a partially denuded endothelial monolayer (10). Under these conditions, marked aggregation of platelets was observed in the denuded area by scanning electron microscopy. In addition, transport was also studied in the presence of mediators released f r o m activated platelets (11). Supernatant of platelets activated by denudation of the endothelial monolayer did not affect the transport, nor did supernatants of platelets activated with ADP or thrombin. In our current model, mediator(s) secreted from platelets did not enhance endothelial permeability,
T
0
1
2
3
4
T i m e (h)
Figure 2. Effect of platelets and isocarbacyclin on transport of fluorescein dextran (FD) through a partially denuded endothelial monolayer. FD transport was measured through an intact endothelial monolayer (o), through a partially denuded endothelial monolayer in the absence (a)or presence of platelets with (A) or without (m) 3 ~ 1 0 M -~ isocarbacyclin, and through a gel layer (u). The values are the means +-SD of three experiments.
and platelet binding to a denuded endothelial monolayer resulted in a decrease in transport (Fig. 2). This finding may be explained by covering of the denuded area by adherent and aggregated platelets, thus inhibiting transport. Isocarbacyclin (stable derivative of prostacyclin) inhibited platelet binding by more than 80% but had little effect o n transport through the endothelial layer when platelets were present. We demonstrated that although prostacyclin strongly inhibited platelet aggregation, a small number of platelets forming a monolayer were adherent to the denuded area. We suggest that formation of a platelet monolayer at the denudation site is sufficient to suppress the transport of FD (Fig. 3). It is known that prostacyclin production by blood vessels is increased in endothelial injury (12), and adherent platelets can often be seen in sections of injured vessel wall. These results suggest that, in vivo, the barrier function may be maintained by adherent platelets after prostacyclin secretion.
3. Accumulation of Cholesteryl Ester in Foam Cells In early lesions, lipids accumulate mainly in the cytoplasm of cells such as macrophages. Thin-section electron microscopy has shown that intracellular lipids are stored in two forms in the cytoplasm of foam cells: in
627
Acta Pathologica Japonica 42 (9): 1992 FD
FD
Q
/
A : Control
B : PGI,
Figure 3. Adherent and aggregated platelets inhibit FD transport by covering the denuded area (A :
control). FD transport was still inhibited, though platelet binding was inhibited by more than 80% by prostacyclin (B: PGI,).
membrane- bound vacuoles, which probably correspond to lysosomes, and in vacuoles without a peripheral membrane. These lipid droplets, both with and without membranes, were also observed in quick-freeze replicas of foam cells possibly derived from macrophages and/or smooth muscle cells (Fig. 4) (13). The membrane-free droplets were more numerous than membrane-bound droplets, and appeared to consist of onion-like concentric lamellae. Onion-like lamellae were also observed in anisotropic cholesteryl ester droplets prepared in vitro. These studies suggest that the onion-like droplets consist mainly of cholesteryl ester and correspond to the cholesteryl ester-rich lipid inclusions. The other type of lipid droplet in foam cells is membrane-bound, as found in lysosomes. These lipid-laden organelles may corre spond to low-density lysosomes, which can be prepared by flotation sucrose density gradient centrifugation. The lipid droplets in lysosomes vary in size. Cholesteryl esters may accumulate as cytoplasmic lipid droplets after microsomal re-esterification of free cholesterol liberated from lysosomal particles. On the other hand, in highly lipid-laden cells, elements of the endoplasmic reticulum, which is involved in reesterification of cholesterol, are rarely found. An alternative explanation for the accumulation of membranefree droplets in these highly lipid-laden foam cells is that lipid inclusion bodies accumulate, not via microsomes, but are transferred to the cytoplasm from phagolysosomes with concomitant partial hydrolysis of cholesteryl esters via the “vesicular pathway” (Fig. 5) (14).
4. Cholesteryl Ester Deposits in the Extracellular Space In advanced lesions, lipids are found in the extracellular matrix. However, due to the limitations of the thinsection method, the fine structure of the extracellular lipids cannot be shown, since the neutral lipids are extracted during the dehydration procedure. However, using the quick-freeze and etching technique, we have
Figure4. Quick-freeze replica of the perinuclear area of a foam cell (13). An organelle containing various sizes of granules is seen among droplets with onion-like layers. Part of the
membrane of this organelle shows an E-fracture face (arrowhead). The fractured face of some granular contents are smooth and that of others contains membrane particles (arrows). (N), nucleus (~20,000).
clearly demonstrated the ultrastructural characteristics of these extracellular lipids. They appeared as irregularly shaped vesicles, some of which had a surface membrane. The vesicles and electron-lucent structures among collagen fibers in thin sections corresponded to these extracellular lipids (Fig. 6)(13). Some extracellular vesicles were aggregated, and the clustered vesicles were similar in size t o the cells destined to collapse in the atherosclerotic aorta. Some extracellular vesicles containing lipids might be derived from these disrupted foam cells. A t the same time, lipoprotein or denatured lipoprotein transported through the endothelial barrier might be deposited in the extracellular space.
628
Cholesteryl Ester in Atheroma (Takano)
LIPOPROTEINS
/L-7
FOAM C E L L S
RUPTURED C E L L S Figure 5. lntracellular lipids in foam cells are stored in two forms: 1) membrane-bound vacuoles which probably correspond t o lysosomes, and 2) vacuoles without a peripheral membrane. Cholesteryl esters may accumulate after microsomal re-esterification of free cholesterol liberated from lysosomal particles (Path I),and/or after transfer t o the cytoplasm from phago-lysosomes via the "vesicular pathway" (Path 11).
5. Monoclonal Antibodies Recognizing Extracellular Regions w i t h Lipid Deposits in t h e Atherosclerotic Aorta In order to study the mechanisms of cholesteryl ester accumulation in the extracellular space of the atherosclerotic aorta, we prepared novel monoclonal antibodies against atherosclerotic lesions using a delipidated crude homogenate of atherosclerotic aorta from Watanabeheritable hyperlipidemic (WHHL) rabbits as a complex mixture of immunogens (15). By screening the antibodies histochemically, we were able to obtain for the first time monoclonal antibodies which specifically recognized the extracellular matrix with lipid-laden deposits (EMRla/212D) in atherosclerotic lesions (Figs. 7a, b). The purified antigenic material is a glycoprotein with a molecular mass of 66 kDa, and the epitope of the antigenic material contains sialic acid as a major element (16). We have isolated from rabbit liver cDNA library clones coding for the 66-kDa glycoprotein (GP66). The clone spans the sequence coding for the entire GP66 (456 amino acids) and 19 amino acids of a signal peptide. GP66 was deduced to be rabbit vitronectin (17) from its nucleotide sequence, containing an Arg-Gly-Asp cell attachment sequence and showing 76% amino acid sequence homology with human vitronectin. Furthermore, EMR 1a/2 12D recognized rabbit vit ronect in purified by heparin affinity chromatography. RNA blot
Figure 6. Appearance o f the extracellular connective tissue space of the atherosclerotic aorta by the quick-freeze. etching technique (13). Large amounts of lipids with a vesicular structure are observed among collagen fibers, which show a characteristic repeating pattern (arrowheads). Some of these vesicular structures appear t o be covered by a membrane. Arrows indicate fractured faces of vesiculated lipids corre sponding t o the P-fracture face of a biomembrane; for details see text. These vesicular structures correspond to vesicles rimmed with a unit membrane structure (inset, arrow) among collagen fibers (inset, arrowhead) in thin sections ( ~ 4 4 , 0 0 0 ; inset, x 40,000).
H003
222
6PL
630
Cholesteryl Ester in Atherorna (Takano)
Q
Figure 9.
Working hypothesis; How cholesteryl ester accumulates in atherosclerotic lesions.
ger cells accumulate in the extracellular spaces of the arterial wall. Vitronectin, possibly associated with scavenger cells, may be involved in the elimination of lipid deposits in the extracellular space of atherosclerotic lesions. Acknowledgements : We would like to thank Drs. K.M. Arnanurna, G.G. Ecsedi, M. Enornoto, R. Hashida, N. Hayashi, T. Irnanaka, C.A. Kawagoe, J. Kirnura, Y. Komine, M. Kurosaka, C. Mineo, K.C. Miyasato, M. Mori, H. Mowri, K. Nakagami, S. Ohkurna, R. Sato, 0. Shirnasaki, H. Takahashi and Y.M. Yagyu for their collaboration in this research work. We are grateful to Drs. Y. Watanabe and K. Hirohata (Kobe University) for providing us with WHHL rabbits, and to S. lkegarni (Teikyo University), T. Kanaseki (Tokyo Metropolitan Institute for Neurosciences) and Y. Yoshida (Yamanashi Medical College) for their helpful comments and discussions. Finally we thank Miss H. Kobayashi for her kind assistance in the various stages of preparation of the manuscript.
REFERENCES 1. Shaffner T, Taylor K, Bartucci EJ, et a/. Arterial foam cells with distinctive immunornorphologic and histochemical features of rnacrophages. Am J Pathol 100 : 57-73, 1980. 2. Ross R and Glornset JA. The pathogenesis of atherosclerosis. N Engl J Med 295: 369-377, 1976. 3. Takano T, Kanaseki T, Amanurna K, et a/. Macrophages and accumulation of cholesterol ester in atherornatous aorta. In Richard S and Alan R, eds. The Reticuloendothelial System, Liss, New York, 1985 : 323-337. 4. Sirnionescu N. Cellular aspects of transcapillary exchange. Physiol Rev 63: 1536-1579, 1983. 5. Hashida R, Anamizu C, Kirnura J, et a/. Transcellular transport of lipoprotein through arterial endothelial cells in monolayer culture. Cell Struct Funct 11: 31-
6. 7. 8. 9.
10. 11.
12. 13.
14.
15.
16.
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