EXPERIMENTAL AND MOLECULAR PATHOLOQY 13, 118-129 (1970)

Poorly Differentiated Subendothelial Ceils in Swine Aortas’

K. T. LEE, K. J. LEE, S. K. LEE, H. IMAI, AND R. M. O’NEAL

Department of Pathology, Albany Medical College, Albany, New York

Received February 20, 1970

Hyperlipemic diets induce grossly visible masses of smooth-muscle cells in the intima of swine aorta in l-2 months. Because of the location of these lesions in the

intima, the possibility must be considered that a special subpopulation of subendo- thelial cells might be the origin of the cell masses. An electron microscopic study

of 385 poorly differentiated cells lying in the intima of the aortic trifurcation of swine revealed the following: (1) most were in close apposition to the endothelium;

(2) their incidence was approximately 7/100 endothelial cells; (3) by using subjec- tive morphologic criteria, the cells could be divided into three groups: smooth-

muscle cell-like, 30%; monocyte-like, 4Oyc; and unclassified, consisting of cells without specialized features, 30%; (4) no consistent effect on frequency and fine

structure of these cells was induced by 3 days’ feeding of a hyperlipemic diet.

Swine fed hyperlipemic (HL) diets containing large amounts of cholesterol and triglycerides develop grossly visible masses of smooth muscle cells (SMC) in the intima of large arteries within l-2 months (Florentin and Nam, 1968). We are carrying out studies directed at determining the genesis of these proliferative lesions that develop by 30-60 days after initiation of HL diets (Thomas et al., 1968). Mitotic counts and 3H-thymidine-labeling indices have been determined for these SMC masses (proliferative lesions) and both are many-fold higher than corresponding values for grossly normal areas of the aortic wall. We have also found that even within 7 days of initiation of the HL diet, and before the develop- ment of a grossly visible lesion, mitotic counts and 3H-thymidine-labeling indices are increased in both the media and intima of the trifurcation region of the abdomi- nal aorta (Thomas et al., 1968; Florentin et al., 1969).

If we consider that the cells which are multiplying at 7 days may be the popula- tion of cells responsible for the later development of gross intimal lesions, it be- comes important to identify the multiplying cells in the aortic intima and define them as precisely as we can. Of special interest is the possibility that a subpopula- tion of poorly differentiated cells existing in the subendothelial area serves as the principal precursors for the cells in the developing intimal lesions.

We have carried out two electron microscopy studies directed to these questions. One of these studies is the subject of this report. The aims were (1) to make a systematic study of poorly differentiated subendothelial (intimal) cells in the abdominal aorta of swine with regard to their overall frequency and possible identity and (2) to compare the frequency and fine structure of these cells in cho- lesterol-fed and control swine. The results of the other study, in which we have

1 Supported by NIH Grant HE-07155 from the National Heart Institute, Bethesda, Mary- land.

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examined by electron microscopy large numbers of cells in mitosis in swine aortas and have attempted to establish their identity, is being reported separately (Imai el cd., 1970).

MATERIAL AND METHODS

Eighteen male Yorkshire swine, approximately 8 weeks old and weighing from 8-10 kg were used in this study. All animals had been on commercial mash diet (Agway Hog Feed) ad &turn prior to the experiment. At the beginning of the experiment, animals were divided into three groups of six swine each: milk-cho- lesterol, milk-control, and mash-control groups.

Animals were housed and fed individually. Swine in the milk-cholesterol group were offered daily 530 gm of dry whole milk powder (Borden’s Starlac, 2400 cal- ories) suspended in water with 8 gm of crystalline cholesterol. The milk-control group was offered the same amount of milk powder without added cholesterol, and the mash-control group was offered 680 gm of commercial hog mash (Agway Hog Feed, 2400 calories). All swine on the milk and milk-cholesterol diets received vitamins and 120 mg of iron per day. According to the manufacturer’s specifica- tions the milk powder consisted of 22 % protein, 40 % fat, and 29 % carbohydrate by calories. With infrequent exceptions all consumed the entire amount of food that was offered. Water was given 2-3 times a day in an amount suf&ient to assure proper hydration.

Three days after beginning the experimental diets, the swine were sacrificed by a blow to the head. At sacrifice, the abdominal aorta with its trifurcation was re- moved, after which a routine autopsy was performed. The entire aortic trifurcation was fixed immediately in cold 4% glutaraldehyde buffered at pH 7.4. Within 10 minutes the aorta was opened with care under a dissecting microscope, and blocks approximately 4-5 cu mm were cut from cushion regions in the trifurcation. These blocks were washed in Verona1 acetate buffer for 5 minutes, fixed again in 5 % glutaraldehyde in cacodylate buffer at pH 7.4 for 30-60 minutes, and postfixed in 1% osnsic acid buffered at pH 7.4 for 60 minutes. After repeated washing in graded ethanol, blocks were embedded in Epon, sectioned with Porter-Blum microtomes, and stained with uranyl acetate and lead citrate. Electron micrographs were taken with a Philips 200 or RCA EMU-4.

For counting the frequency of poorly differentiated subendothelial cells five blocks were taken from the cushion regions in eight swine (four milk-cholesterol, three milk-control, one mash-control), and three grids were made from each block. Thus, 15 grids from each swine were examined. For this purpose lOO- or 150-mesh grids were used. From one grid an average of 25-90 endothelial cells could be counted, making a total of up to 1300 endothelial cells per aorta. Only cells with nuclei in the plane of section were included in the count. Excluded from the count were polymorphonuclear leukocytes, which were rare, and well-differentiated SMC with closely packed arrays of filaments, fusiform densities, and basement membrane, which were common. The ratio of poorly differentiated subendothelial cells was determined.

By subjective morphologic criteria these poorly differentiated cells (Figs. 1 and 2) were subdivided into three groups: smooth muscle cell-lie; monocyte-like, and

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FIG. 1. Diagrammatic representation of a portion of the trifurcation region of swine aorta.

Most of the poorly differentiated cells (PDC) are located immediately subjacent to the endo- thelial cells. Smooth muscle cells (SMC) make up the majority of the cell population of the

intimal cushion in this region, and are usually separated by bundles of collagen (C) and elastica

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unclassified. If features suggesting the presence of the usual identifying char- acteristics of SMC, such as arrays of filaments, fusiform densities, or basement membrane, were recognizable in the poorly differentiated cells, these were classified as “smooth muscle cell-like” (Fig. 3). When the subendothelial cell contained homogeneous dense bodies consistent with primary lysosomes, a moderate number of mitochondria, abundant ribosomes in clusters, and a moderate number of dis- persed profiles of granular endoplasmic reticulum it was classified as “monocyte- like” (Fig. 4). This category was designed to include lymphocytes, monocytes, and macrophages, which we consider as migratory and not indigenous to the aorta. Cells with no recognizable specialized features were designated “unclassified” (Figs. 5 and 6). Regardless of location, cells that were readily recognizable as to type (Figs. 7 and 8) were not included in the current study.

RESULTS

All animals appeared healthy and showed no ill effects attributable to the diets during the short periods of the experiment. At autopsy no significant changes were observed in the thorax or abdomen and no proliferative lesions were present in the aorta of any swine.

In the cushion areas the intima is normally thick and a large number of cells are present in the subendothelial region. Most of the subendothelial cells in these

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FIG. 2. Low-power electron micrograph of two poorly differentiated subendothelial cells lying close to the endothelium. Portions of cytoplasm of mature SMC and bundles of elastica lie below. X 5500.

cushion regions are SMC, and only a small percentage of subendothelial cells are those we are referring to as poorly differentiated subendothelial cells. Only a few cells are present in the intima of noncushion areas.

The poorly differentiated subendothelial cells were almost always located im- mediately beneath and in close apposition to the endothelium (Figs. 1 and 2); mature SMC were almost always deeper. In general, poorly differentiated sub- endothelial cells were round or oval and had nuclei with marginal condensation of chromatin, large numbers of free ribosomes, sparse endoplasmic reticulum, and few mitochondria.

The overall frequency of the poorly differentiated subendothelial cells for the eight swine in milk-cholesterol, milk-control, and mash-control groups combined was 6.9/100 endothelial cells (385 poorly differentiated cells to 5566 endothelial cells counted). The frequency was 3.8/100 endothelial cells for milk and mash controls and 9.3 for milk-cholesterol swine, but variation among individual swine in both groups from 0.7-15 % precluded meaningful statistical analysis. The relative frequencies of the three categories in the 385 poorly differentiated cells were SMC- like, 30%, monocyte-like, 40 %, and unclassified, 30%. Again, no consistent dif- ferences in frequencies and fine structure were apparent between milk-cholesterol and control groups.

DISCUSSION

Our observations confirm that a subpopulation of cells exists in the subendothelial region of the aortic wall of swine and this subpopulation differs distinctly from the well-differentiated SMC that constitute the predominant cell type in this location. This minority population is heterogeneous and can be further divided into (1) a group of cells with a few filaments that can be classified as poorly differentiated SMC, (2) a group that resemble monocytes, and (3) an “unclassified” group. In

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FIG. 3. Example of a poorly differentiated subendothelial cell classified as SMC-like (SL).

The general contour of nucleus and cytoplasm, the short segment of basement membrane (BM), and the suggestion of fusiform densities (D) near the cell membrane were the features which determined the classification. X 7000.

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FIG. 4. Example of a poorly differentiated subendothelial cell classified as monocyte-like. Dense bodies consistent with primary lysosomes are seen. Mitochondria, ribosomes in clusters. and granular endoplasmic reticulum were present in moderate amounts. X 26,000.

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FIG. 5. Example of a poorly differentiated subendothelial cell considered as unclassified.

The cell has no recognizable specialized features. Clusters of ribosomes, the most prominent cytoplasmic component, are abundant. X 28,000.

FIG. 6. An “unclassified” cell from the subpopulation of poorly differentiated subendothe- lial cells in the cushion region of a swine aorta. This cell has prominent Golgi, many lysosomes,

and arrays of rough endoplasmic reticulum, which are features found in monocytes. However, the arrays of filaments (F) and the elongation of the cell body are features found in endothelial

or SMC. This illustration is included to demonstrate the difficulties encountered in classifying individual cells. X 42,000.

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FIG. 7. A subendothelial cell clearly recognizable as a SMC. The cytoplasm of this cell is less electron dense than that of the underlying SMC at the bottom of the micrograph. However,

the cytoplasm is largely occupied by arrays of filaments and fusiform densities, characteristic of SMC. Such cells were not included in the counts. X 8000.

FIG. 8. A “subendothelial” cell classified as an endothelial cell and, therefore, not included in the counts of cells. Large numbers of tonofi!aments in an irregular whorling pattern, without fusiform densities, characterized these cells. The apparent subendothelial location of such a cell is considered to be a “three-dimensional” artifact of sectioning and was encountered rarely. X 34,000.

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some instances, cells that are considered unclassified in one profile prove to have SMC features in another profile; even those that remain unclassified in the profiles available might prove to be SMC, monocytes, or endothelial cells if studied in a large number of serial sections. This difficulty in the ultrastructural classification of subendothelial cells has been well illustrated by Hoff and Gottlob (1968). If these cells are early forms of SMC it is not clear why they appear to be limited largely to the innermost layer of the aortic wall. Perhaps they are analogous to the primitive angioblast which forms the anlage of blood vessels.

The first description of a distinct subpopulation of cells in the subendothelial intima is attributed to Langhans (1866) and since then these cells have been the subject of considerable study and speculation (Seifert, 1962). Prior to the wide- spread use of electron microscopy in the study of atherosclerosis, Duff, McMillan, and Ritchie (1957) used light microscopy and a surface (en face) technique to demonstrate the presence of intimal cells lying immediately beneath intact endo- thelium of rabbit aorta. These cells were classified as fibrocytes, histiocytes, and monocytoid cells. Since the electron microscopic studies of Geer et al. (1961) and their summary of supporting work it has become widely accepted that the intimal cells previously designated as “fibrocytes” are principally smooth muscle cells; the cells we have designated monocyte-like and unclassified probably would have been designated as histiocytes or monocytoid cells by Duff et al. (1957) by light micros- copy. Still and O’Neal (1962) and Still and Dennison (1967) have described similar mononuclear cells lying in apposition to endothelial cells in the rat and rabbit.

The possibility exists that the poorly differentiated subendothelial cells divide more rapidly than do other cells in the aorta. For this reason, we made a search by electron microscopy for mitotic figures but found only 3 such cells in mitosis, whereas more than 75 recognizable smooth muscle cells have been found in mitosis in the same general area and deeper in the aortic wall (Imai et al., 1970). In other studies, by light microscopy, we have examined mitotic figures and cells labeled with 3H-thymidine and found these to be distributed at random throughout the aortic wall (Florentin et al., 1969). Thus, the innermost layer of the aorta is not the major site of rapid cell division when the animals are subjected to HL diets for a short period.

Cells similar to the poorly differentiated subendothelial cells described in this study are relatively frequent in atherosclerotic lesions, where they appear to be scattered at random (Daoud et al., 1968). This similarity invites speculation re- garding the role of the poorly differentiated subendothelial cells in the pathogenesis of intimal proliferative lesions of atherosclerosis. On the other hand, SMC are dividing more rapidly in atherosclerotic lesions (Thomas et at., 1968) than in the grossly normal portions of aortas, and dedifferentiation to the point of loss of recognizable specialized features could occur. We know that the rate of cell division is considerably greater in atherosclerotic lesions than in adjacent tissue, but it is still far less than the rate in tissue culture where dedifferentiation is known to occur.

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