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The topography of cholesterol-induced fatty streaks
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
The Topography
of Pathology,
Virginia
(1974)
of Cholesterol-Induced w.
Department
374-386
20,
Medical Commonwealth
J.
s.
Streaks1
STILL
College University,
Received
Fatty
November
of Virginia, Richmond,
Health Science Division, Virginia 23298
2, 1973
The appearance of ‘the arterial endothelium of rabbits which had been fed cholesterol is examined by scanning (SEM) and transmission electron microscopy (TEM). Attention is dsrawn to the large number of circulating cells attached to the endothelial surface and the number of these as seen by TEM which appear to be penetrating into the intima. It is proposed that these penetrating cells form an important component of cholesterol-induced fatty streaks in the rabbit.
The following communication describes certain features, as seen by scanning electron microscopy ( SEM ), of fatty intimal thickenings produced in the rabbit by cholesterol feeding, with special emphasis on the changes in the endothelial surface and the relation of these changes to the pathogenesis of the thickenings. Intimal lesions produced in this manner have been exhaustively studied by various histological methods over the last 60 years and recently scanning electron microscopy has been applied (Sunaga et al., 1970; Shimamato et al., 1971; Weber and Tosi, 1971 [a-b]; Christensen and Garbarsch, 1972). Although the results of the latter provide valuable information about the arterial endothelial surface, both normal and abnormal, the investigations are not generally directed to investigating the relationship of the endothelial changes to the formation and growth of the plaque. MATERIALS
AND METHODS
Nine New Zealand white male rabbits initially weighing 2.0 kg were divided into two groups. The animals in the first group, consisting of six rabbits, were fed rabbit chow coated with cholesterol (Cholesterol U.S.P.) which had been dissolved in petroleum ether. The second group of three rabbits were fed untreated chow. The test animals were killed at weekly intervals beginning two weeks after the commencement of the diet. Serum cholesterol levels varied from 300 mg to 1050 mg%. Control animals were killed at intervals during the test period. The animals were given 100 units of heparin (Sodium Heparin, Upjohn) to prevent or lessen clotting and then killed with an overdose of barbiturate. The aorta and carotid arteries were removed entire, opened, washed vigorously in saline and cut into segments approximately f/2 cm in length. Most of these were 1 This
work
was supported
by USPH
Grant
HL 374
Copyright All rights
0 1974 by Academic Press, of reproduction in any form
Inc. reserved.
09622,
National
Heart
and
Lung
Institute.
CHOLESTEROL-INDUCED
FATTY
375
STREAKS
prepared for scam-ring electron microscopy ( SEM ) but one segment from each aortic zone, arch, thoracic and abdominal, was prepared for transmission electron microscopy (TEM). Material for TEM was fixed in 1% buffered osmium tetroxide in the cold, treated with 0.5% n-phenyl-p-phenylenediamine (Eastman Kodak) in 80% alcohol as an osmium tetroxide enhancing agent, and as a stain for 2 /~.m sections which were cut in all blocks (Dennison et al., unpublished observations), and eventually embedded in epoxy resin (Maraglas DER). The SEM specimens, after washing in saline, were notched to mark the direction of blood flow and fixed in Karnovsky’s paraformaldehyde-gluteraldehyde fixative for 2 hr in the cold (Karnovsky, 1965). Thereafter the material was washed in buffer for 2 hr then post fixed in 1% buffered osmium tetroxide for 1 hr in the cold. The specimens were then dehydrated in alcohol and treated with amyl acetate prior to critical point drying (Anderson, 1951). After drying, the material was coated with gold palladium and viewed in an AMR 1000 scanning electron microscope at 20 KV. When the material had been scanned, it was embedded in epoxy resin for TEM and light microscopic studies. Although inferior technically to material prepared specifically for TEM, the reprocessed material had certain obvious advantages when trying to correlate TEM and SEM appearances. RESULTS The surface characteristics of the normal arterial endothelium have been well described by others (Shimamoto et al., 1971; Sunaga et al., 1971; Christensen and Garbarsch, 1972). Endothelial cells and their intervening gullies form
FIG. I. Normal arterial
endothelium
of rabbit.
Blood
flow is from
right
to left.
X1,500.
W. J. S. STILL
376
FIG. 2. Normal
Varioussmall
hollows
endothelial are seen.
surface X250.
just
proximal
to
a branch
opening
(bottom
right).
longitudinal lines which show some localized convergence and curving as mouths of branches are approached (Figs. l-2). The corrugation of the endothelial surface may be partly or largely due to the contraction and wrinkling of the underlying internal elastic lamina ( Christensen and Garbarsch, 1972). Of the large number of lesions available for study, attention was concentrated on small lesions, edges of larger ones and areas surrounding bifurcations and branches. The smallest alteration which could be termed a lesion consisted of a smooth surfaced bleb covering the area of two or three endothelial cells (Fig. 3). Cross sections showed that these lesions were comprised of two or three foam cells situated between the endothelium and internal elastic lamina (Fig. 4). Althmoughthese lesions were quite evident and discrete, the surrounding endothelium frequently appeared swollen, smoother and more protuberant than normal. Invariably the endothelial cells showed multiple small cytoplasmic processes,These tend to be more conspicuous on cells not covering lesions (see Fig. 3). Such processeswere much finer than those displayed by cells foreign to the endothelium (Fig. 3 and see below). Many of these small discrete lesions had circulating blood cells, or cells foreign to the endothelium, attached to their surface as Fig. 3 shows. Further, as can be seen in the same illustration, some of the foreign cells appeared to be penetrating the endothelium. As we shall see, these were common occurrences in lesions of all sizes and were confirmed by TEM (see Figs. 5-S). Lesions of a larger size invariably had their long axis in the direction of the blood ilow and the majority were roughly torpedo-shaped hillocks arising more or less abruptly from the plane of the endothelium (Fig. 7). Frequently, at the
CHOLESTEROL-INDUCED
FATTY
FIG. 3. An early (small) lesion, center. The covering endothelium but bulbous. The surrounding endothelium shows multiple surface foreign to the endothelium are present ( C ). The one on the left penetration, Cell on right has at least two RBC’s attached. Blood x2,250.
FIG. 4. Transverse present.
TEM
specimen.
(non-longitudinal) X5,000.
section
of
an
377
STREAKS
early
lesion.
is generally smooth processes. Two cells appears in the act of flow, bottom to top.
A
single
foam
cell
is
378
W.
J. S. STILL
FIG. 5. Transverse multiple at right.
section showing a monocyte perched on an endothelial cytoplasmic processes. Another circulating cell (C) is firmly attached Fat filled cells are present at bottom. X13,000.
FIG. 6. endothelium
Transverse at arrows.
section. X10,000.
Edge
of
lesion.
A
cell
containing
lipid
is
cell. Both show to endothelium
penetrating
the
CHOLESTEROL-INDUCED
FATTY
FIG. 7. A plaque in the abdominal aorta main area with more bulbous endothelium (arrows). Smooth object on surface at right X360.
FIG. 8. Transverse The
endothelial
section. The smooth cell contains two rounded
379
STREAKS
showing the smooth endorhelium covering the at edge. A few circulating cells are adherent is fat (see Fig. 8). Blood flow is right to left.
surface body lipid inclusions
is lipid (see previous (see Fig. 10). X18,000.
illustration).
380
W.
J. S. STILL
rims of the plaque, the endothelium tended to be more bulbous than normal, but that on the main surface of the plaque tended to be flattened and apparently stretched over the underlying tissue. In addition to this, many of the endothelial cells of plaques this size showed multiple small pale blebs in their cytoplasm. These appeared to correlate with lipid inclusions in the endothelium (see Figs. 8 and 10). Another frequent finding on the endothelial surface was small smooth globules (see Fig. 7). These on cross section were shown to consist of lipid (Fig. 8). Like the rest of the endothelium in the test animals, endothelium overlying these plaques, particularly cells at the rims, showed multiple cytoplasmic processes (See Figs. 3 and 5). These cytoplasmic processes have been described as “hooklets” in previous studies and have been thought to play some role in cellular adhesion under these and other experimental conditions (Still and O’Neal, 1962; Still and Dennison, 1970).
FIG. 9. Thoracic aorta. Two larger plaques are present either side with relatively normal endothelium between. Numerous circulating cells are attached to the plaque surfaces some with coarse surface processes (arrows). Blood flow bottom to top. x350.
All larger plaques had numerous cells sticking to the surface (Fig. 9). This was in contrast to the other areas of endothelial surface which showed very few adherent cells. On the plaque surface adherent cells were more prevalent at the edges where the whole endothelial surface appeared more bulbous, or at least, less flattened, than on the upper surface of the lesion. The adherent cells could be clearly distinguished from the endothelium and also from other circulating elements such as red cells and platelets (Fig. 10). The latter were quite rarely found attached to the endothelium. The obvious deduction that these adherent cells were monocytes, lymphocytes or foam cells was confirmed by cross sectional studies (see Figs. 5, 6, 9 and 11).
CHOLESTEROL-INDUCED
FATTY
STREAKS
381
Apart from adhering, a number of these cells gave the distinct impression, as they did in smaller lesions, that they were breaching the endothelium; and cells were seen in apparently every stage of this process (see Figs. 3, 10, 12, and 13). Such penetration was also confirmed just as in the smaller lesions by cross sectional views of these plaques and has, of course, been well illustrated in numerous previous studies on dietary-induced arterial lesions in animals (see Discussion). At the edges of large plaques, particularly those surrounding a bifurcation or branch, large groups of foreign or circulating cells were seen lying in hollows of the endothelial surface (Fig. 14). Closer inspection showed that most or many of the surface cells in these groups were somewhat flattened, although still retaining their distinctive appearance (Fig. 15), i.e., distinct from endothelium, and that most collections contained a few red cells and an
FIG. 10. View of the edge of a plaque. The surface cells are clearly distinguished and at least one seems to be penetrating (large arrow). Several endothelial cells show pale blebs under their luminal surface (small arrows) (see Figure 8). X1,100.
odd platelet. We were uncertain about the significance of these findings although endothelial hollows are seen in the same areas in the normal vessel (see Fig. 2) and inspection of human fatty streaks showed foreign or circulating cells fixed and flattened in similar hollows ( Fig. 16). None of the surface changes described were fundamentally altered by the duration of the diet or the height of the serum cholsterol. We were impressed, however, in individual animals by the signs of activity and growth, as measured by surface ‘adherent cells in lesions in the aortic arch and thoracic portion of the aorta as compared with the abdominal areas (see Figs. 7 and 9).
382
W.
FIG. 11. Foam
cell attached
FIG. 12. Distal edge of large middle seems to be penetrating endothelium. X3.200.
to intact
J. S. STILL
endothelium.
SEM
plaque. One circulating cell and one on right (arrow)
preparation.
(bottom) is farther
X9,500.
is attached. in (or out)
One in of the
CHOLESTEROL-INDUCED
FIG.
13. A high
power
view
of two
FIG. 14. Part of a large sessile cells are seen ( arrows) together
cells
FATTY
(arrows)
lesion distal with single
penetrating
to an aortic cells adherent
383
STREAKS
the endothelium
branch (right). to endothelium.
(E).
X8,000.
Numerous X150.
nests
of
W.
FIG. 15. A higher still look
unlike
power endothelium.
FIG. 16. Human firmly
embedded
view of a single X1,000.
J. S. STILL
cell
nest.
Cells
fatty streak. Two cells (arrows) differing in hollows on endothelial surface. X1,000.
on left
from
(arrow)
the
are flattened
endothelium
but
appear
CHOLESTEROL-INDUCED
FATTY
STREAKS
385
DISCUSSION It has been proposed by a number of investigators that circulating white cells, in particular monocytes, with or without lipid, play a role in the formation of the cholest.erol-induced arterial lesions in experimental animals (Leary, 1941; Rannie & Duguid, 1954; Poole & Florey, 1958; Still & O’Neal, 1962; Still, 1964). It has even been proposed that this might happen during the formation of fatty streaks in man (Still & Marriott, 1964), a suggestion which is given some support in Fig. 16. The actual mechanisms whereby circulating cells reach the subendothelial space has caused some disagreement even among protagonists for the process as a whole. Rannie and Duguid (1954) believed that groups of circulating cells were deposited on the endothelium and then incorporated into the intima by endothelial overgrowth. Some appearances, particularly those seen in Figs. 14 and 15 seem to support this contention, with the additional indication that in some instances hollows occur in the endothelial surface which appear conducive to the aggregation of circulating cells. There is a possibility that some of the hollows described are artifactual cracks revealing cells already lying in the intima. However, #actual cracks in other areas due to preparatory techniques were easily recognizable as such (Christensen and Garbarsch, 1972) and moreover the presence of groups of cells sticking on the surface without the benefit of hollows, the presence of hollows in similar areas of normal endothelium and the presence of red cells and platelets among the cells within hollows all lessen this possibility and its significance. Despite this, however, the significance of these changes remains obscure if not dubious. Other investigators (Poole & Florey, 1958), including the present authors (Still & O’Neal, 1962; Still, 1964), have considered and shown that circulating cells with and without lipid actively penetrate the endothelial layer to reach the subendotheilal space, both in dietary induced .&ease and under the influence of hypertension (Sill, 1968). Certain appearances in this study reinforce that contention. It is, of course, always possible that cells are leaving rather than entering the intima. The balance of opinion is against this as the direction of flow, since the lesions steadily become larger. The comparatively large areas surveyed by SEM display very clearly the great frequency with which circulating cells stick to the plaque surface and how relatively seldom they stick to areas of endothelium not covering the plaque. In the past the use of a few or even many transverse histological or transmission EM sections has given the impression to many observers that both adherent and penetrating cells are an unusual occurrence. The SEM shows that this view may be erroneous for purely technical reasons and that adherence, and perhaps proportionally, penetration, may be relatively common even in human lesions. *Adherence occurs despite the fact that the endothelium covering the plaque is flattened in a physical sense and appears to have less filamentous processes than the surrounding endothelium. Previous TEM studies have either not brought this point out or have believed the opposite to be true (Still and O’Neal, 1962). There is, however, no doubt that hyperlipemic conditions induce surface filaments to become more conspicuous in the arterial endothelium as a whole. Although it was only an impression, survey of many lesions showed that cell adherence was particularly prevalent at the rims of plaques, where you would
386
W.
J. S. STILL
expect peripheral growth to occur and a further impression was that plaques in the aortic arch and thoracic aorta seemed more active in this sense than plaques in the abdominal aorta. Both impressions fit fairly well with the natural history of dietary-induced arterial disease in animals. The present study was concerned solely with the changes occurring on the endothelial surfaces. Clearly, if circulating cells stick and penetrate more frequently under hyperlipemic conditions, the effect of hyperlipemia on these cells must be considered. However, preliminary studies in this laboratory on the buffy coats of animals used in this experiment, have not so far showed any consistent differences between the surfaces of lymphocytes and monocytes from hyperlipemic and normolipemic animals. REFERENCES ANDERSON, T. F. 1951. Techniques for the preservation of three dimensional structures in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci. Ser. 11, 13, 130. CHRISTENSEN, B. C., and GARBARSCH, C. 1972. A scanning electron microscopic (SEM) study on the endothelium of the normal rabbit aorta. Angliologica. 9, 15-26. DENNISON, S. M., FREEMAN, R., and Still, W. J. S. “N-phenyl-p-phenylenediamine as a stain for electron microscopy” unpublished observations. KARNOVSKY, J. (1965). A formaldehyde-gluteraldehyde fixative of high osmolarity for use in electron microscopy. .I. Cell Biol. 27, 137 (A). LEARY, T. 1941. The genesis of atherosclerosis. Arch. Pathok 32, 507-585. POOLE, J. C. F., and FLOREY, H. W. 1958. Changes in the endothelium of the aorta and the behavior of macrophages in experimental atheroma in the rabbit. .I. Puthol. Bact. 75,
245-251. RANNIJZ, I., and DUGUID, J. B. 1954. The pathogenesis of cholesterol arteriosclerosis in the rabbit. 1. Pathol. Bud. 66, 395-398. SHIMAMMO, T., YAMASHJTA, Y., NUMANO, F., and SUNAGA, T. 1971. Scanning and transmission electron microscopic observations of endothelial cells in the normal condition and in initial stages of atherosclerosis. Acta Path. Jup. 21(l), 93-119. STILL, W. J. S. 1964. Electron microscope studies in experimental atherosclerosis. Symp. zooz. sot. London 11, 181-188. STILL, W. J. S., and O’NEAL, R. M. 1962. An electron microscopic study of experimental atherosclerosis in the rat. Amer. J. Path. 40, 21-30. STILL, W. J. S., and MARRIOTT, P. R. 1964. Comparative morphology of the early atherosclerotic lesion in man and cholesterol-atherosclerosis in the rabbit. J. Atheroccler. Res.
4, 373-386. STILL, W. J. S. 1968. The pathogenesis of intimal thickenings produced by hypertension in large arteries in the rat. Lab. Inuest. 19, 84-94. SUNAGA, T., YAMASHITA, Y., NUMANO, F., and SnrhUh~oro, T. 1970. Luminal surface of normal and atherosclerotic arteries observed by scanning electron microscope. Proc. 3rd. Scan. E. M. Symposium, Chicago, Ill. 243-244. WEBER, G., and TOSI, P. ( 1971). Some observations with the scanning electron microcope on the rabbit. Puthol. Europ. 6, 407-410. WEBER, G., and TOSI, P. ( 1971). Observations with the scanning electron microscope on the development of cholesterol atherosclerosis in the guinea pig. Virchow’s Arch. Path. Angt.
353, 325-329.
AND
MOLECULAR
PATHOLOGY
The Topography
of Pathology,
Virginia
(1974)
of Cholesterol-Induced w.
Department
374-386
20,
Medical Commonwealth
J.
s.
Streaks1
STILL
College University,
Received
Fatty
November
of Virginia, Richmond,
Health Science Division, Virginia 23298
2, 1973
The appearance of ‘the arterial endothelium of rabbits which had been fed cholesterol is examined by scanning (SEM) and transmission electron microscopy (TEM). Attention is dsrawn to the large number of circulating cells attached to the endothelial surface and the number of these as seen by TEM which appear to be penetrating into the intima. It is proposed that these penetrating cells form an important component of cholesterol-induced fatty streaks in the rabbit.
The following communication describes certain features, as seen by scanning electron microscopy ( SEM ), of fatty intimal thickenings produced in the rabbit by cholesterol feeding, with special emphasis on the changes in the endothelial surface and the relation of these changes to the pathogenesis of the thickenings. Intimal lesions produced in this manner have been exhaustively studied by various histological methods over the last 60 years and recently scanning electron microscopy has been applied (Sunaga et al., 1970; Shimamato et al., 1971; Weber and Tosi, 1971 [a-b]; Christensen and Garbarsch, 1972). Although the results of the latter provide valuable information about the arterial endothelial surface, both normal and abnormal, the investigations are not generally directed to investigating the relationship of the endothelial changes to the formation and growth of the plaque. MATERIALS
AND METHODS
Nine New Zealand white male rabbits initially weighing 2.0 kg were divided into two groups. The animals in the first group, consisting of six rabbits, were fed rabbit chow coated with cholesterol (Cholesterol U.S.P.) which had been dissolved in petroleum ether. The second group of three rabbits were fed untreated chow. The test animals were killed at weekly intervals beginning two weeks after the commencement of the diet. Serum cholesterol levels varied from 300 mg to 1050 mg%. Control animals were killed at intervals during the test period. The animals were given 100 units of heparin (Sodium Heparin, Upjohn) to prevent or lessen clotting and then killed with an overdose of barbiturate. The aorta and carotid arteries were removed entire, opened, washed vigorously in saline and cut into segments approximately f/2 cm in length. Most of these were 1 This
work
was supported
by USPH
Grant
HL 374
Copyright All rights
0 1974 by Academic Press, of reproduction in any form
Inc. reserved.
09622,
National
Heart
and
Lung
Institute.
CHOLESTEROL-INDUCED
FATTY
375
STREAKS
prepared for scam-ring electron microscopy ( SEM ) but one segment from each aortic zone, arch, thoracic and abdominal, was prepared for transmission electron microscopy (TEM). Material for TEM was fixed in 1% buffered osmium tetroxide in the cold, treated with 0.5% n-phenyl-p-phenylenediamine (Eastman Kodak) in 80% alcohol as an osmium tetroxide enhancing agent, and as a stain for 2 /~.m sections which were cut in all blocks (Dennison et al., unpublished observations), and eventually embedded in epoxy resin (Maraglas DER). The SEM specimens, after washing in saline, were notched to mark the direction of blood flow and fixed in Karnovsky’s paraformaldehyde-gluteraldehyde fixative for 2 hr in the cold (Karnovsky, 1965). Thereafter the material was washed in buffer for 2 hr then post fixed in 1% buffered osmium tetroxide for 1 hr in the cold. The specimens were then dehydrated in alcohol and treated with amyl acetate prior to critical point drying (Anderson, 1951). After drying, the material was coated with gold palladium and viewed in an AMR 1000 scanning electron microscope at 20 KV. When the material had been scanned, it was embedded in epoxy resin for TEM and light microscopic studies. Although inferior technically to material prepared specifically for TEM, the reprocessed material had certain obvious advantages when trying to correlate TEM and SEM appearances. RESULTS The surface characteristics of the normal arterial endothelium have been well described by others (Shimamoto et al., 1971; Sunaga et al., 1971; Christensen and Garbarsch, 1972). Endothelial cells and their intervening gullies form
FIG. I. Normal arterial
endothelium
of rabbit.
Blood
flow is from
right
to left.
X1,500.
W. J. S. STILL
376
FIG. 2. Normal
Varioussmall
hollows
endothelial are seen.
surface X250.
just
proximal
to
a branch
opening
(bottom
right).
longitudinal lines which show some localized convergence and curving as mouths of branches are approached (Figs. l-2). The corrugation of the endothelial surface may be partly or largely due to the contraction and wrinkling of the underlying internal elastic lamina ( Christensen and Garbarsch, 1972). Of the large number of lesions available for study, attention was concentrated on small lesions, edges of larger ones and areas surrounding bifurcations and branches. The smallest alteration which could be termed a lesion consisted of a smooth surfaced bleb covering the area of two or three endothelial cells (Fig. 3). Cross sections showed that these lesions were comprised of two or three foam cells situated between the endothelium and internal elastic lamina (Fig. 4). Althmoughthese lesions were quite evident and discrete, the surrounding endothelium frequently appeared swollen, smoother and more protuberant than normal. Invariably the endothelial cells showed multiple small cytoplasmic processes,These tend to be more conspicuous on cells not covering lesions (see Fig. 3). Such processeswere much finer than those displayed by cells foreign to the endothelium (Fig. 3 and see below). Many of these small discrete lesions had circulating blood cells, or cells foreign to the endothelium, attached to their surface as Fig. 3 shows. Further, as can be seen in the same illustration, some of the foreign cells appeared to be penetrating the endothelium. As we shall see, these were common occurrences in lesions of all sizes and were confirmed by TEM (see Figs. 5-S). Lesions of a larger size invariably had their long axis in the direction of the blood ilow and the majority were roughly torpedo-shaped hillocks arising more or less abruptly from the plane of the endothelium (Fig. 7). Frequently, at the
CHOLESTEROL-INDUCED
FATTY
FIG. 3. An early (small) lesion, center. The covering endothelium but bulbous. The surrounding endothelium shows multiple surface foreign to the endothelium are present ( C ). The one on the left penetration, Cell on right has at least two RBC’s attached. Blood x2,250.
FIG. 4. Transverse present.
TEM
specimen.
(non-longitudinal) X5,000.
section
of
an
377
STREAKS
early
lesion.
is generally smooth processes. Two cells appears in the act of flow, bottom to top.
A
single
foam
cell
is
378
W.
J. S. STILL
FIG. 5. Transverse multiple at right.
section showing a monocyte perched on an endothelial cytoplasmic processes. Another circulating cell (C) is firmly attached Fat filled cells are present at bottom. X13,000.
FIG. 6. endothelium
Transverse at arrows.
section. X10,000.
Edge
of
lesion.
A
cell
containing
lipid
is
cell. Both show to endothelium
penetrating
the
CHOLESTEROL-INDUCED
FATTY
FIG. 7. A plaque in the abdominal aorta main area with more bulbous endothelium (arrows). Smooth object on surface at right X360.
FIG. 8. Transverse The
endothelial
section. The smooth cell contains two rounded
379
STREAKS
showing the smooth endorhelium covering the at edge. A few circulating cells are adherent is fat (see Fig. 8). Blood flow is right to left.
surface body lipid inclusions
is lipid (see previous (see Fig. 10). X18,000.
illustration).
380
W.
J. S. STILL
rims of the plaque, the endothelium tended to be more bulbous than normal, but that on the main surface of the plaque tended to be flattened and apparently stretched over the underlying tissue. In addition to this, many of the endothelial cells of plaques this size showed multiple small pale blebs in their cytoplasm. These appeared to correlate with lipid inclusions in the endothelium (see Figs. 8 and 10). Another frequent finding on the endothelial surface was small smooth globules (see Fig. 7). These on cross section were shown to consist of lipid (Fig. 8). Like the rest of the endothelium in the test animals, endothelium overlying these plaques, particularly cells at the rims, showed multiple cytoplasmic processes (See Figs. 3 and 5). These cytoplasmic processes have been described as “hooklets” in previous studies and have been thought to play some role in cellular adhesion under these and other experimental conditions (Still and O’Neal, 1962; Still and Dennison, 1970).
FIG. 9. Thoracic aorta. Two larger plaques are present either side with relatively normal endothelium between. Numerous circulating cells are attached to the plaque surfaces some with coarse surface processes (arrows). Blood flow bottom to top. x350.
All larger plaques had numerous cells sticking to the surface (Fig. 9). This was in contrast to the other areas of endothelial surface which showed very few adherent cells. On the plaque surface adherent cells were more prevalent at the edges where the whole endothelial surface appeared more bulbous, or at least, less flattened, than on the upper surface of the lesion. The adherent cells could be clearly distinguished from the endothelium and also from other circulating elements such as red cells and platelets (Fig. 10). The latter were quite rarely found attached to the endothelium. The obvious deduction that these adherent cells were monocytes, lymphocytes or foam cells was confirmed by cross sectional studies (see Figs. 5, 6, 9 and 11).
CHOLESTEROL-INDUCED
FATTY
STREAKS
381
Apart from adhering, a number of these cells gave the distinct impression, as they did in smaller lesions, that they were breaching the endothelium; and cells were seen in apparently every stage of this process (see Figs. 3, 10, 12, and 13). Such penetration was also confirmed just as in the smaller lesions by cross sectional views of these plaques and has, of course, been well illustrated in numerous previous studies on dietary-induced arterial lesions in animals (see Discussion). At the edges of large plaques, particularly those surrounding a bifurcation or branch, large groups of foreign or circulating cells were seen lying in hollows of the endothelial surface (Fig. 14). Closer inspection showed that most or many of the surface cells in these groups were somewhat flattened, although still retaining their distinctive appearance (Fig. 15), i.e., distinct from endothelium, and that most collections contained a few red cells and an
FIG. 10. View of the edge of a plaque. The surface cells are clearly distinguished and at least one seems to be penetrating (large arrow). Several endothelial cells show pale blebs under their luminal surface (small arrows) (see Figure 8). X1,100.
odd platelet. We were uncertain about the significance of these findings although endothelial hollows are seen in the same areas in the normal vessel (see Fig. 2) and inspection of human fatty streaks showed foreign or circulating cells fixed and flattened in similar hollows ( Fig. 16). None of the surface changes described were fundamentally altered by the duration of the diet or the height of the serum cholsterol. We were impressed, however, in individual animals by the signs of activity and growth, as measured by surface ‘adherent cells in lesions in the aortic arch and thoracic portion of the aorta as compared with the abdominal areas (see Figs. 7 and 9).
382
W.
FIG. 11. Foam
cell attached
FIG. 12. Distal edge of large middle seems to be penetrating endothelium. X3.200.
to intact
J. S. STILL
endothelium.
SEM
plaque. One circulating cell and one on right (arrow)
preparation.
(bottom) is farther
X9,500.
is attached. in (or out)
One in of the
CHOLESTEROL-INDUCED
FIG.
13. A high
power
view
of two
FIG. 14. Part of a large sessile cells are seen ( arrows) together
cells
FATTY
(arrows)
lesion distal with single
penetrating
to an aortic cells adherent
383
STREAKS
the endothelium
branch (right). to endothelium.
(E).
X8,000.
Numerous X150.
nests
of
W.
FIG. 15. A higher still look
unlike
power endothelium.
FIG. 16. Human firmly
embedded
view of a single X1,000.
J. S. STILL
cell
nest.
Cells
fatty streak. Two cells (arrows) differing in hollows on endothelial surface. X1,000.
on left
from
(arrow)
the
are flattened
endothelium
but
appear
CHOLESTEROL-INDUCED
FATTY
STREAKS
385
DISCUSSION It has been proposed by a number of investigators that circulating white cells, in particular monocytes, with or without lipid, play a role in the formation of the cholest.erol-induced arterial lesions in experimental animals (Leary, 1941; Rannie & Duguid, 1954; Poole & Florey, 1958; Still & O’Neal, 1962; Still, 1964). It has even been proposed that this might happen during the formation of fatty streaks in man (Still & Marriott, 1964), a suggestion which is given some support in Fig. 16. The actual mechanisms whereby circulating cells reach the subendothelial space has caused some disagreement even among protagonists for the process as a whole. Rannie and Duguid (1954) believed that groups of circulating cells were deposited on the endothelium and then incorporated into the intima by endothelial overgrowth. Some appearances, particularly those seen in Figs. 14 and 15 seem to support this contention, with the additional indication that in some instances hollows occur in the endothelial surface which appear conducive to the aggregation of circulating cells. There is a possibility that some of the hollows described are artifactual cracks revealing cells already lying in the intima. However, #actual cracks in other areas due to preparatory techniques were easily recognizable as such (Christensen and Garbarsch, 1972) and moreover the presence of groups of cells sticking on the surface without the benefit of hollows, the presence of hollows in similar areas of normal endothelium and the presence of red cells and platelets among the cells within hollows all lessen this possibility and its significance. Despite this, however, the significance of these changes remains obscure if not dubious. Other investigators (Poole & Florey, 1958), including the present authors (Still & O’Neal, 1962; Still, 1964), have considered and shown that circulating cells with and without lipid actively penetrate the endothelial layer to reach the subendotheilal space, both in dietary induced .&ease and under the influence of hypertension (Sill, 1968). Certain appearances in this study reinforce that contention. It is, of course, always possible that cells are leaving rather than entering the intima. The balance of opinion is against this as the direction of flow, since the lesions steadily become larger. The comparatively large areas surveyed by SEM display very clearly the great frequency with which circulating cells stick to the plaque surface and how relatively seldom they stick to areas of endothelium not covering the plaque. In the past the use of a few or even many transverse histological or transmission EM sections has given the impression to many observers that both adherent and penetrating cells are an unusual occurrence. The SEM shows that this view may be erroneous for purely technical reasons and that adherence, and perhaps proportionally, penetration, may be relatively common even in human lesions. *Adherence occurs despite the fact that the endothelium covering the plaque is flattened in a physical sense and appears to have less filamentous processes than the surrounding endothelium. Previous TEM studies have either not brought this point out or have believed the opposite to be true (Still and O’Neal, 1962). There is, however, no doubt that hyperlipemic conditions induce surface filaments to become more conspicuous in the arterial endothelium as a whole. Although it was only an impression, survey of many lesions showed that cell adherence was particularly prevalent at the rims of plaques, where you would
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expect peripheral growth to occur and a further impression was that plaques in the aortic arch and thoracic aorta seemed more active in this sense than plaques in the abdominal aorta. Both impressions fit fairly well with the natural history of dietary-induced arterial disease in animals. The present study was concerned solely with the changes occurring on the endothelial surfaces. Clearly, if circulating cells stick and penetrate more frequently under hyperlipemic conditions, the effect of hyperlipemia on these cells must be considered. However, preliminary studies in this laboratory on the buffy coats of animals used in this experiment, have not so far showed any consistent differences between the surfaces of lymphocytes and monocytes from hyperlipemic and normolipemic animals. REFERENCES ANDERSON, T. F. 1951. Techniques for the preservation of three dimensional structures in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci. Ser. 11, 13, 130. CHRISTENSEN, B. C., and GARBARSCH, C. 1972. A scanning electron microscopic (SEM) study on the endothelium of the normal rabbit aorta. Angliologica. 9, 15-26. DENNISON, S. M., FREEMAN, R., and Still, W. J. S. “N-phenyl-p-phenylenediamine as a stain for electron microscopy” unpublished observations. KARNOVSKY, J. (1965). A formaldehyde-gluteraldehyde fixative of high osmolarity for use in electron microscopy. .I. Cell Biol. 27, 137 (A). LEARY, T. 1941. The genesis of atherosclerosis. Arch. Pathok 32, 507-585. POOLE, J. C. F., and FLOREY, H. W. 1958. Changes in the endothelium of the aorta and the behavior of macrophages in experimental atheroma in the rabbit. .I. Puthol. Bact. 75,
245-251. RANNIJZ, I., and DUGUID, J. B. 1954. The pathogenesis of cholesterol arteriosclerosis in the rabbit. 1. Pathol. Bud. 66, 395-398. SHIMAMMO, T., YAMASHJTA, Y., NUMANO, F., and SUNAGA, T. 1971. Scanning and transmission electron microscopic observations of endothelial cells in the normal condition and in initial stages of atherosclerosis. Acta Path. Jup. 21(l), 93-119. STILL, W. J. S. 1964. Electron microscope studies in experimental atherosclerosis. Symp. zooz. sot. London 11, 181-188. STILL, W. J. S., and O’NEAL, R. M. 1962. An electron microscopic study of experimental atherosclerosis in the rat. Amer. J. Path. 40, 21-30. STILL, W. J. S., and MARRIOTT, P. R. 1964. Comparative morphology of the early atherosclerotic lesion in man and cholesterol-atherosclerosis in the rabbit. J. Atheroccler. Res.
4, 373-386. STILL, W. J. S. 1968. The pathogenesis of intimal thickenings produced by hypertension in large arteries in the rat. Lab. Inuest. 19, 84-94. SUNAGA, T., YAMASHITA, Y., NUMANO, F., and SnrhUh~oro, T. 1970. Luminal surface of normal and atherosclerotic arteries observed by scanning electron microscope. Proc. 3rd. Scan. E. M. Symposium, Chicago, Ill. 243-244. WEBER, G., and TOSI, P. ( 1971). Some observations with the scanning electron microcope on the rabbit. Puthol. Europ. 6, 407-410. WEBER, G., and TOSI, P. ( 1971). Observations with the scanning electron microscope on the development of cholesterol atherosclerosis in the guinea pig. Virchow’s Arch. Path. Angt.
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