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An electron microscope study of cholesterol atherosclerosis in the rabbit
EXPERIMENTAL ANDMOLECULAR PATHOLOGY 2, 491-502(1963)
An
Electron
Study of Cholesterol
Microscope
Atherosclerosis w. Department
of
Pathology,
Washington
in the J.
s.
STILL’
University,
Received
Rabbit
School
November
of
Medicine,
St. Louis,
Missouri
-7, 1962
INTRODUCTION Arterial lesions produced in rabbits by cholesterol have been the subject of a large number of investigations, among them two using the electron microscope (Buck, 1958a; Parker, 1960). Despite this, the morphology and pathogenesis of these lesions are still the subject of debate (Felch and Van Itallie, 1960). The electron microscopic study reported here presents additional evidence on the nature of the early lesion in the rabbit with particular regard to the origin of the various intimal cells. MATERIALS AND METHODS The aortas from eight mature male albino rabbits were used. These animals were fed Ralston Purina chow pellets coated with cholesterol dissolved in cottonseed oil (Wesson). The amount of cholesterol fed to each was not calculated, but at weekly intervals the total and free serum cholesterol levels were estimated in each animal by the method of Zak et al. (1954). Two similar rabbits fed Purina chow without cholesterol were used as controls. After 2 weeks on the cholesterol and cottonseed oil diet the animal with the highest cholesterol level was killed, and thereafter at 2 week intervals one animal was killed on the same basis. The aortas were opened longitudinally along the anterior border, the ascending aorta and arch discarded, and the remainder cut into three segments which were pinned on cardboard strips. These were immersed in osmium dichromate fixative (Dalton, 1955) for 30 minutes at 4°C. After fixation, ten transverse strips were dehydrated in graded ethanols, which contained 0.570 phosphotungs’tic acid, and infiltrated by methacrylate. The tissue was then embedded in partially prepolymerized methacrylate to which benzyl peroxide had been added as a catalyst. Phase sections from all blocks were examined and any lesion over which the endothelium was intact was viewed in the microscope. Lesions which were thought to be representative were examined by multiple sections. An RCA EMU 3B electron microscope was used. RESULTS NORMAL
AORTA
The ultrastructure of elastic arteries has been described by others (Buck, 1958b; 1960; Pease and Paule, 1960). One feature not emphasized in previous
Keech,
1 Present
address:
Royal
Free
Hospital,
Gray’s 191
Inn
Road,
London,
W. C. 1, England
492
W. J.
S. STILL
descriptions but of some importance is the presence in the intima of cells apparently derived from smooth muscle, These cells are found in crescentic spaces formed by a focal division of the internal elastic lamina (Fig. 1). Some are typical if compared with the smooth muscle cells of the media; the majority, however, are smaller, rounded, or oval, rather than fusiform, and contain few identifiable myofibrils.
FIG. 1. Kormal rabbit aorta. A smooth lying in a crescentic division of the internal
muscle elastic
cell (M) is seen under lamina (L). X 5000.
the
endothelium
(E)
On many occasions these cells were seen with cytoplasmic processes passing through gaps in the overlying elastic tissue. Similar structures are seen in the aorta of the rat (Still and O’Neal, 1962). MAIN
INTIMAL
CELLS
Examination of a large number of lesions of all sizes, and therefore presumably of all ages from the eight animals fed the cholesterol-cottonseed o’il diet, showed that two main cell types were present. The first was the “foam cell” of light microscopy, that is, a large vacuolated cell measuring up to 40 p with a round nucleus (Figs, 2 and 3). The cytoplasmic vacuoles were spherical, variable in size with a limiting membrane, and for the most part empty of contents. Some contained clusters of dense black granules. In addition the cytoplasm occasionally contained one or more densely osmiophilic myelin figure inclusions and more frequently inclusions of a finer whorled nature of varying size and electron density (Fig. 4). It seemed likely that these inclusions and vacuoles represented various forms of ingested lipid. All cells showed mitochondria, elements of endoplasmic reticulum, and Golgi apparatus. The second type of cell found in large numbers in all lesions was much smaller
CHOLESTEROL
FIG. 2. An early intimal muscle cell and aL smooth
ATHEROSCLEROSIS
IN
THE
lesion showing a single foam cell (F) (S) within a division of the internal
RABBIT
493
just deep to the endothelium, elastic lamina (L). x 4500.
FIG. 3. An intimal lesion in which foam cells (F) predominate. Two of the smaller intimal cells, each with a single dense inclusion are seen at bottom corners. An endothelial cell (E) appears in the process of being overgrown by the spread of the lesion. x 3000.
494
W. J.
S. STILL
than the foam cell and displayed only a few inclusions or frequently only one (Fig. 5). The majority of these had a uniformly dense osmiophilic core with a clear area between this and the limiting membrane of the inclusion, while others had strands of osmiophilic material radiating from the core to the periphery (Fig. 6). Besides the osmiophilic inclusions the cytoplasm also contained complex arrays
FIG. 4. A higher inclusion of moderate
An intimal FIG. 5. The foam cells present
power view of the complex lipid inclusions of the foam density has a whorled laminated structure. x 10,000.
lesion in which the smaller cell (S) with the dense inclusions are typically placed just under the endothelium. X 3500.
cell.
The
largest
predominates.
CHOLESTEROL
ATHEROSCLEROSIS
IN
THE
495
RABBIT
of endoplasmic reticulum and Golgi apparatus; a few cells showed rudimentary fibrils just under the cytoplasmic membrane. The relative number of these two main cell types varied in any one lesion, but a scattering of foam cells was present just under the endothelium even in lesions largely composed of the smaller cell.
FIG. 6. A higher power view of a small intimal cell. The osmiophilic radiating type and the cytoplasm contains a number of vesicles. Some just visible under the cell membrane. X 8000.
FIG. 7. A vacuolated cell (V) at center is lying in the endothelium surface. The endothelium appears normal. A nonvacuolated cell (N), seen at left also deposited on surface. X 4600.
inclusions rudimentary
(E) and probably
are of fibrils
the are
is molded to its a monocyte, is
496
W. J. SURFACE
S. STILL
CHANGES
In this investigation particular attention was paid to the endothelial surface of all lesions and the changes occurring there. It was common to find vacuolated cells lying on and molded to an area of endothelium which appeared morphologically normal (Fig. 7). At times nonvacuolated cells with the appearance of monocytes were found in the same relationship to the endothelium. Less often, but still of frequent occurrence, vacuolated cells were seen penetrating the normal surface (Fig. 8). These vacuolated cells, whether lying oa the surface or penetrating it, were identical in all respects with the foam cells within the intima. The endothelium overlying these lesions showed several variations from the normal. A number showed densely osmiophilic inclusions similar to those present in the smaller type of intimal cell (Fig. 9) and all showed large numbers of vesicles interpreted as part of the endoplasmic reticulum filled with granular material. A number, apparently increasing with the age of the lesion, showed spherical or oval bodies composed of a compact array of complex membranes of high electron density (Fig. 10). On numerous occasions degeneration of individual endothelial cells was apparent. The change was distinguished by the increased density and granularity of the cell and the disruption of its cytoplasmic structures. The change from the degenerating cell to the contiguous healthy one was abrupt and striking (Fig. 11). At times these degenerating cells were seen apparently in the process of desquamation, leaving the cells of the underlying lesion exposed. Since the endothelium is a delicate structure and easily damaged during processing, desquamation such as this may have been an artifact. However, the smaller partial ruptures of individual endothelial cells which were seen on occasion, often with an underlying intimal cell extruding into the aortic lumen, appeared to be an actual process (Fig. 12). OTHER
INTIMAL
STRUCTURES
Beside the two main cell types, other cells and structures were seen in the majority of lesions. Prominent among these were monocytes and lymphocytes usually found immediately beneath the end&helium (Fig. 13). Occasio’nally, eosinophils and plasma cells were present, usually as single cells. In a number of lesions elongated cells interpreted as fibroblasts were seen, and these occasionally contained osmiophilic inclusions. In all lesions a number of cells were present which could not be identified; some, as those illustrated in Fig. 14, were cells with numerous cytoplasmic vesicles containing granular material, well-developed Golgi apparatus, and an occasional spherical osmiophilic inclusion. In all lesions studied the extracellular material was of a granular nature and low electron density. In many areas of the aorta this granular material alone was found deposited in, and thereby widening, the subendothelial zone, without the presence of cells. Osmiophilic lipid was not present in this granular material, but in some places small aggregates of striated fibrils, considered to be fibrin, were seen (Fig. 15). This, however, was an uncommon finding, and for the most part the extracellular substance had no identifiable characters. In the larger, older lesions collagenous fibers and fragmentary elastic tissue were frequently found.
CHOLESTEROL
FIG.
arrows.
8. A vacuolated cell (F) Note the miniature clefts
FIG. 9. An endothelial the smaller intimal cells.
ATHEROSCLEROSIS
IN
is seen penetrating apparently in the foam cell. X 4000.
cell conlaining X 6500.
a dense
osmiophilic
THE
497
RABBIT
normal
endothelium
inclusion
similar
(E)
to
those
between
seen in
498
FIG. 10. An endothelial cell left of nucleus with two similar
W. J. S. STILL
that shows but smaller
a large rounded masses elsewhere
mass of compact in the cytoplasm.
membranes x 10,000.
to the
FIG. 11. A degenerating endothelial cell (E) is seen together with a healthy endothelial cell at left. The abrupt change is marked by arrows. A foam cell (F! is situated beneath the degenerating surface cell. X 3750.
CHOLESTEROL
ATHEROSCLEROSIS
FIG. 12. A degenerating foam cell (C) is protruding
endothelial at arrows.
FIG. 13. it appears x 6000.
(L) situated lymphocyte
A lymphocyte likely that this
cell (E) X 7000.
with
IN
dense
just deep to has penetrated
THE
499
RABBIT
granular
the endothelium the endothelium
cytoplasm
(E). from
through
In the
which
this situation aortic lumen.
a
500
W.
J.
S. STILL
cells in the intima; they contain FIG. 14. Unidentified and a large number of cytoplasmic vesicles with granular
FIG. 15. The granular appearance of the extracellular (E) and the aortic lumen are at top left. Dense material are seen at center (arrows). X 10,000.
osmiophilic inclusions contents. X 5000.
material fragments
(EX) is shown. of indistinctly
of various
types
The endothelium striated fihrillar
CHOLESTEROL
ATHEROSCLEROSIS
IN
THE
RABBIT
501
DISCUSSION Buck (1958a), in his description of cholesterol-induced aortic atherosclerosisin the rabbit, indicated there were two types of cells present in the intimal lesions,one resembling the surface lining cells and the other similar to the foam cell described here. Parker (1960), in a description of the coronary artery lesions, believed that the majority of the lipid-containing cells were smooth muscle in origin. In this investigation the lesions were found to be much more complex. Cells were present which corresponded to the foam cell of light microscopy, and with them a variety of others both with and without lipid inclusions. The most numerous of the other cells was a small cell with denseosmiophilic inclusions and at times with rudimentary cytoplasmic myofibrils. The cells without lipid were monocytes and lymphocytes, both present in significant numbers, and an occasional polymorphonuclear leucocyte and plasma cell. With regard to the origin of these cells this study has shown that foam cells, identical with those in the intima, are deposited on the surface of the endothelium and apparently are often in the processof penetrating it. This is in agreement with the findings in some light microscopic studies of rabbit cholesterol atherosclerosis (Anitschow, 1933; Leary, 1941; Rannie and Duguid, 1953; Poole and Florey, 1958) and with the results of an electron microscopic study of aortic atherosclerosisin the rat (Still and O’Neal, 1962). It can be further assumedthat both monocytes and lymphocytes can penetrate the aortic endothelium since they were found regularly in a subendothelial position. On the other hand the smaller intimal cell, which morphologically appears quite different from the foam cell, seemedsimilar in some respects to the smSoothmuscle cells normally found in the intima and to those in the upper layer of the media; for example, some of the smaller intimal cells showed myofibrils in the cytoplasm. Very close similarities exist between this small intimal cell and the LLmyo-intimal” cell described by Buck (1961) in the intima of ligated arteries and regarded by him as being primarily smooth muscle in origin. The variation in the degree of osmiophilia and physical appearance of the lipid in these two types of cells, that is, the foam cell and the smaller intimal cells, also indicates that they may be of differing types. It is known that unsaturated lipid is more osmiophilic than the saturated forms (Thomas and O’Neal, 1960), and in the present lesionsosmiophilic lipid was found predominantly in the smaller intimal cells, in endothelium, and in smooth muscle. In the foam cell the visible lipid took two forms, namely coarse myelin figures and whorled structures of moderate density, the latter regarded as representing lipo-protein complexes, while the empty vacuoles were presumed to have contained saturated lipid before processing. It is interesting to note that in human atherosclerotic lesionsthe cells identified as smooth muscle contain the sametype of osmiophilic lipid inclusions as those in the smaller intimal cell of the rabbit lesions (Geer et al., 1961). Thus the foam cell and the smaller intimal cell not only differ morphologically but in the type of lipid they contain. This, taken with the presenceof foam cells on the surface of the lesions and the similarities between the smaller intimal cell and smooth muscle, suggeststhat the foam cell may be derived from cells in the circulating blood, while the smaller intimal cell is probably derived from the smooth muscle of the aorta.
502
W.
J.
S. STILL
One unexpected finding in this investigation was endothelial cells, and, further, the apparent ability the aortic lumen because of this. It is not possible in do more than infer in which direction cells seen going; however, the presence of both penetration of sion through degenerating endothelium may indicate directions across the surface.
the degeneration of individual of intimal cells to extrude into a study of the present nature to breaching the endothelium are normal endothelium and extruthat foam cells can pass in both
SUMMARY Lesions produced in the rabbit aorta by cholesterol and cottonseed oil were studied with the electron microscope. These lesions showed two main cell types: vacuolated cells containing complex lipid inclusions (foam cells) and smaller cells containing inclusions of mainly unsaturated lipid. Foam cells and monocytoid cells were seen deposited on the endothelial surface and in some instances apparently penetrating it. Degeneration of individual endothelial cells was also observed with the occasional extrusion of underlying intimal cells. It is postulated that the foam cell is derived from the circulating blood as distinct from the other main cell type, which is derived from the smooth muscle of the aorta. ACKNOWLEDGMENTS I am grateful for this study, and Education
to Mrs. Shirley Carroll and Miss Virginia Dunman who cut the thin sections which was supported by a grant from the Wesson Fund for Medical Research and Research Grant No. 209 from the Nutrition Foundation. REFERENCES
N. (1933). In “Arteriosclerosis” (Cowdry, E. V. ed.), p. 288. MacMillan, New York. Buts, R. C. (1958a). The fine structure of the aortic endothelial lesions in experimental atherosclerosis of rabbits. Am. J. Pathol. 34, 897-909. BUCK, R. C. (1958b). The fme structure of endothelium of large arteries. 1. Biophys. Biockem. Cytol. 4, 187-190. BUCK, R. C. (1961). Intimal thickening after ligature of arteries. An electron microscopic study. Circulation Res. 9, 418-426. DALTON, A. V. (1955). A chrome-osmium fixative for electron microscopy. Anat. Rec. 121, 281. FELCH, W. C., and VAN ITALLIE, T. B. (1960). Reflections on the pathologic physiology of atherosclerosis. New Engl. J. Med. 2, 1125-1128. GEER, J. C., MCGILL, H. C., and STRONG, J. P. (1961). The fine structure of human atherosclerotic lesions. Am. 1. Pathol. 36, 263-275. KEECH, M. K. (1960). Electron microscope study of the normal rat aorta. J. Biophys. Biochem. Cytol. 7, 533-538. LEARY, T. (1941). The genesis of atherosclerosis. Arch. Pathol. 22, 507-585. PARKER, F. (1960). An electron microscopic study of experimental atherosclerosis. Am. J. Pathot. 36, 19-33. PEASE, D. C., and PAULE, W. J. (1960). Electron microscopy of e!astic arteries: the thoracic aorta of the rat. J. Ultrastruct. Res. 3, 469-483. (1958). Changes in the endothelium of the aorta and the POOLE, J. C. F., and FLOREY, H. W. behaviour of macrophages in experimental atheroma of rabbits. J. Pathol. Bacterial. 76, 245-251. RANNIE, I., and Ducum, J. B. (1953). Pathogenesis of cholesterol arteriosclerosis in the rabbit. J. Pathol. Bacterial. 66, 395-398. STILL, W. J. S., and O’NEAL, R. M. (1962). Electron microscopic study of experimental atherosclerosis in the rat. Am. J. Pathol. 40, 21-30. THOMAS, W. A., and O’NEAL, R. M. (1960). Electron microscopy studies of butter and corn oil in jejunal mucosa. Arch. Pathol. 69, 121-129. ZAK, B., DICKENMAN, R. C., WRITE, P. C., BURNETT, H., and CHERNEY, P. J. (1954). A rapid method for the estimation of total and free cholesterol. Am. J. Clin. Pathol. 24, 1307-1315. ANITSCHOW,
An
Electron
Study of Cholesterol
Microscope
Atherosclerosis w. Department
of
Pathology,
Washington
in the J.
s.
STILL’
University,
Received
Rabbit
School
November
of
Medicine,
St. Louis,
Missouri
-7, 1962
INTRODUCTION Arterial lesions produced in rabbits by cholesterol have been the subject of a large number of investigations, among them two using the electron microscope (Buck, 1958a; Parker, 1960). Despite this, the morphology and pathogenesis of these lesions are still the subject of debate (Felch and Van Itallie, 1960). The electron microscopic study reported here presents additional evidence on the nature of the early lesion in the rabbit with particular regard to the origin of the various intimal cells. MATERIALS AND METHODS The aortas from eight mature male albino rabbits were used. These animals were fed Ralston Purina chow pellets coated with cholesterol dissolved in cottonseed oil (Wesson). The amount of cholesterol fed to each was not calculated, but at weekly intervals the total and free serum cholesterol levels were estimated in each animal by the method of Zak et al. (1954). Two similar rabbits fed Purina chow without cholesterol were used as controls. After 2 weeks on the cholesterol and cottonseed oil diet the animal with the highest cholesterol level was killed, and thereafter at 2 week intervals one animal was killed on the same basis. The aortas were opened longitudinally along the anterior border, the ascending aorta and arch discarded, and the remainder cut into three segments which were pinned on cardboard strips. These were immersed in osmium dichromate fixative (Dalton, 1955) for 30 minutes at 4°C. After fixation, ten transverse strips were dehydrated in graded ethanols, which contained 0.570 phosphotungs’tic acid, and infiltrated by methacrylate. The tissue was then embedded in partially prepolymerized methacrylate to which benzyl peroxide had been added as a catalyst. Phase sections from all blocks were examined and any lesion over which the endothelium was intact was viewed in the microscope. Lesions which were thought to be representative were examined by multiple sections. An RCA EMU 3B electron microscope was used. RESULTS NORMAL
AORTA
The ultrastructure of elastic arteries has been described by others (Buck, 1958b; 1960; Pease and Paule, 1960). One feature not emphasized in previous
Keech,
1 Present
address:
Royal
Free
Hospital,
Gray’s 191
Inn
Road,
London,
W. C. 1, England
492
W. J.
S. STILL
descriptions but of some importance is the presence in the intima of cells apparently derived from smooth muscle, These cells are found in crescentic spaces formed by a focal division of the internal elastic lamina (Fig. 1). Some are typical if compared with the smooth muscle cells of the media; the majority, however, are smaller, rounded, or oval, rather than fusiform, and contain few identifiable myofibrils.
FIG. 1. Kormal rabbit aorta. A smooth lying in a crescentic division of the internal
muscle elastic
cell (M) is seen under lamina (L). X 5000.
the
endothelium
(E)
On many occasions these cells were seen with cytoplasmic processes passing through gaps in the overlying elastic tissue. Similar structures are seen in the aorta of the rat (Still and O’Neal, 1962). MAIN
INTIMAL
CELLS
Examination of a large number of lesions of all sizes, and therefore presumably of all ages from the eight animals fed the cholesterol-cottonseed o’il diet, showed that two main cell types were present. The first was the “foam cell” of light microscopy, that is, a large vacuolated cell measuring up to 40 p with a round nucleus (Figs, 2 and 3). The cytoplasmic vacuoles were spherical, variable in size with a limiting membrane, and for the most part empty of contents. Some contained clusters of dense black granules. In addition the cytoplasm occasionally contained one or more densely osmiophilic myelin figure inclusions and more frequently inclusions of a finer whorled nature of varying size and electron density (Fig. 4). It seemed likely that these inclusions and vacuoles represented various forms of ingested lipid. All cells showed mitochondria, elements of endoplasmic reticulum, and Golgi apparatus. The second type of cell found in large numbers in all lesions was much smaller
CHOLESTEROL
FIG. 2. An early intimal muscle cell and aL smooth
ATHEROSCLEROSIS
IN
THE
lesion showing a single foam cell (F) (S) within a division of the internal
RABBIT
493
just deep to the endothelium, elastic lamina (L). x 4500.
FIG. 3. An intimal lesion in which foam cells (F) predominate. Two of the smaller intimal cells, each with a single dense inclusion are seen at bottom corners. An endothelial cell (E) appears in the process of being overgrown by the spread of the lesion. x 3000.
494
W. J.
S. STILL
than the foam cell and displayed only a few inclusions or frequently only one (Fig. 5). The majority of these had a uniformly dense osmiophilic core with a clear area between this and the limiting membrane of the inclusion, while others had strands of osmiophilic material radiating from the core to the periphery (Fig. 6). Besides the osmiophilic inclusions the cytoplasm also contained complex arrays
FIG. 4. A higher inclusion of moderate
An intimal FIG. 5. The foam cells present
power view of the complex lipid inclusions of the foam density has a whorled laminated structure. x 10,000.
lesion in which the smaller cell (S) with the dense inclusions are typically placed just under the endothelium. X 3500.
cell.
The
largest
predominates.
CHOLESTEROL
ATHEROSCLEROSIS
IN
THE
495
RABBIT
of endoplasmic reticulum and Golgi apparatus; a few cells showed rudimentary fibrils just under the cytoplasmic membrane. The relative number of these two main cell types varied in any one lesion, but a scattering of foam cells was present just under the endothelium even in lesions largely composed of the smaller cell.
FIG. 6. A higher power view of a small intimal cell. The osmiophilic radiating type and the cytoplasm contains a number of vesicles. Some just visible under the cell membrane. X 8000.
FIG. 7. A vacuolated cell (V) at center is lying in the endothelium surface. The endothelium appears normal. A nonvacuolated cell (N), seen at left also deposited on surface. X 4600.
inclusions rudimentary
(E) and probably
are of fibrils
the are
is molded to its a monocyte, is
496
W. J. SURFACE
S. STILL
CHANGES
In this investigation particular attention was paid to the endothelial surface of all lesions and the changes occurring there. It was common to find vacuolated cells lying on and molded to an area of endothelium which appeared morphologically normal (Fig. 7). At times nonvacuolated cells with the appearance of monocytes were found in the same relationship to the endothelium. Less often, but still of frequent occurrence, vacuolated cells were seen penetrating the normal surface (Fig. 8). These vacuolated cells, whether lying oa the surface or penetrating it, were identical in all respects with the foam cells within the intima. The endothelium overlying these lesions showed several variations from the normal. A number showed densely osmiophilic inclusions similar to those present in the smaller type of intimal cell (Fig. 9) and all showed large numbers of vesicles interpreted as part of the endoplasmic reticulum filled with granular material. A number, apparently increasing with the age of the lesion, showed spherical or oval bodies composed of a compact array of complex membranes of high electron density (Fig. 10). On numerous occasions degeneration of individual endothelial cells was apparent. The change was distinguished by the increased density and granularity of the cell and the disruption of its cytoplasmic structures. The change from the degenerating cell to the contiguous healthy one was abrupt and striking (Fig. 11). At times these degenerating cells were seen apparently in the process of desquamation, leaving the cells of the underlying lesion exposed. Since the endothelium is a delicate structure and easily damaged during processing, desquamation such as this may have been an artifact. However, the smaller partial ruptures of individual endothelial cells which were seen on occasion, often with an underlying intimal cell extruding into the aortic lumen, appeared to be an actual process (Fig. 12). OTHER
INTIMAL
STRUCTURES
Beside the two main cell types, other cells and structures were seen in the majority of lesions. Prominent among these were monocytes and lymphocytes usually found immediately beneath the end&helium (Fig. 13). Occasio’nally, eosinophils and plasma cells were present, usually as single cells. In a number of lesions elongated cells interpreted as fibroblasts were seen, and these occasionally contained osmiophilic inclusions. In all lesions a number of cells were present which could not be identified; some, as those illustrated in Fig. 14, were cells with numerous cytoplasmic vesicles containing granular material, well-developed Golgi apparatus, and an occasional spherical osmiophilic inclusion. In all lesions studied the extracellular material was of a granular nature and low electron density. In many areas of the aorta this granular material alone was found deposited in, and thereby widening, the subendothelial zone, without the presence of cells. Osmiophilic lipid was not present in this granular material, but in some places small aggregates of striated fibrils, considered to be fibrin, were seen (Fig. 15). This, however, was an uncommon finding, and for the most part the extracellular substance had no identifiable characters. In the larger, older lesions collagenous fibers and fragmentary elastic tissue were frequently found.
CHOLESTEROL
FIG.
arrows.
8. A vacuolated cell (F) Note the miniature clefts
FIG. 9. An endothelial the smaller intimal cells.
ATHEROSCLEROSIS
IN
is seen penetrating apparently in the foam cell. X 4000.
cell conlaining X 6500.
a dense
osmiophilic
THE
497
RABBIT
normal
endothelium
inclusion
similar
(E)
to
those
between
seen in
498
FIG. 10. An endothelial cell left of nucleus with two similar
W. J. S. STILL
that shows but smaller
a large rounded masses elsewhere
mass of compact in the cytoplasm.
membranes x 10,000.
to the
FIG. 11. A degenerating endothelial cell (E) is seen together with a healthy endothelial cell at left. The abrupt change is marked by arrows. A foam cell (F! is situated beneath the degenerating surface cell. X 3750.
CHOLESTEROL
ATHEROSCLEROSIS
FIG. 12. A degenerating foam cell (C) is protruding
endothelial at arrows.
FIG. 13. it appears x 6000.
(L) situated lymphocyte
A lymphocyte likely that this
cell (E) X 7000.
with
IN
dense
just deep to has penetrated
THE
499
RABBIT
granular
the endothelium the endothelium
cytoplasm
(E). from
through
In the
which
this situation aortic lumen.
a
500
W.
J.
S. STILL
cells in the intima; they contain FIG. 14. Unidentified and a large number of cytoplasmic vesicles with granular
FIG. 15. The granular appearance of the extracellular (E) and the aortic lumen are at top left. Dense material are seen at center (arrows). X 10,000.
osmiophilic inclusions contents. X 5000.
material fragments
(EX) is shown. of indistinctly
of various
types
The endothelium striated fihrillar
CHOLESTEROL
ATHEROSCLEROSIS
IN
THE
RABBIT
501
DISCUSSION Buck (1958a), in his description of cholesterol-induced aortic atherosclerosisin the rabbit, indicated there were two types of cells present in the intimal lesions,one resembling the surface lining cells and the other similar to the foam cell described here. Parker (1960), in a description of the coronary artery lesions, believed that the majority of the lipid-containing cells were smooth muscle in origin. In this investigation the lesions were found to be much more complex. Cells were present which corresponded to the foam cell of light microscopy, and with them a variety of others both with and without lipid inclusions. The most numerous of the other cells was a small cell with denseosmiophilic inclusions and at times with rudimentary cytoplasmic myofibrils. The cells without lipid were monocytes and lymphocytes, both present in significant numbers, and an occasional polymorphonuclear leucocyte and plasma cell. With regard to the origin of these cells this study has shown that foam cells, identical with those in the intima, are deposited on the surface of the endothelium and apparently are often in the processof penetrating it. This is in agreement with the findings in some light microscopic studies of rabbit cholesterol atherosclerosis (Anitschow, 1933; Leary, 1941; Rannie and Duguid, 1953; Poole and Florey, 1958) and with the results of an electron microscopic study of aortic atherosclerosisin the rat (Still and O’Neal, 1962). It can be further assumedthat both monocytes and lymphocytes can penetrate the aortic endothelium since they were found regularly in a subendothelial position. On the other hand the smaller intimal cell, which morphologically appears quite different from the foam cell, seemedsimilar in some respects to the smSoothmuscle cells normally found in the intima and to those in the upper layer of the media; for example, some of the smaller intimal cells showed myofibrils in the cytoplasm. Very close similarities exist between this small intimal cell and the LLmyo-intimal” cell described by Buck (1961) in the intima of ligated arteries and regarded by him as being primarily smooth muscle in origin. The variation in the degree of osmiophilia and physical appearance of the lipid in these two types of cells, that is, the foam cell and the smaller intimal cells, also indicates that they may be of differing types. It is known that unsaturated lipid is more osmiophilic than the saturated forms (Thomas and O’Neal, 1960), and in the present lesionsosmiophilic lipid was found predominantly in the smaller intimal cells, in endothelium, and in smooth muscle. In the foam cell the visible lipid took two forms, namely coarse myelin figures and whorled structures of moderate density, the latter regarded as representing lipo-protein complexes, while the empty vacuoles were presumed to have contained saturated lipid before processing. It is interesting to note that in human atherosclerotic lesionsthe cells identified as smooth muscle contain the sametype of osmiophilic lipid inclusions as those in the smaller intimal cell of the rabbit lesions (Geer et al., 1961). Thus the foam cell and the smaller intimal cell not only differ morphologically but in the type of lipid they contain. This, taken with the presenceof foam cells on the surface of the lesions and the similarities between the smaller intimal cell and smooth muscle, suggeststhat the foam cell may be derived from cells in the circulating blood, while the smaller intimal cell is probably derived from the smooth muscle of the aorta.
502
W.
J.
S. STILL
One unexpected finding in this investigation was endothelial cells, and, further, the apparent ability the aortic lumen because of this. It is not possible in do more than infer in which direction cells seen going; however, the presence of both penetration of sion through degenerating endothelium may indicate directions across the surface.
the degeneration of individual of intimal cells to extrude into a study of the present nature to breaching the endothelium are normal endothelium and extruthat foam cells can pass in both
SUMMARY Lesions produced in the rabbit aorta by cholesterol and cottonseed oil were studied with the electron microscope. These lesions showed two main cell types: vacuolated cells containing complex lipid inclusions (foam cells) and smaller cells containing inclusions of mainly unsaturated lipid. Foam cells and monocytoid cells were seen deposited on the endothelial surface and in some instances apparently penetrating it. Degeneration of individual endothelial cells was also observed with the occasional extrusion of underlying intimal cells. It is postulated that the foam cell is derived from the circulating blood as distinct from the other main cell type, which is derived from the smooth muscle of the aorta. ACKNOWLEDGMENTS I am grateful for this study, and Education
to Mrs. Shirley Carroll and Miss Virginia Dunman who cut the thin sections which was supported by a grant from the Wesson Fund for Medical Research and Research Grant No. 209 from the Nutrition Foundation. REFERENCES
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