Effects of normolipidemic high-density lipoproteins on proliferation of monkey aortic smooth muscle cells induced by hyperlipidemic low-density lipoproteins

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

41, 258-266 (1984)

Effects of Normolipidemic High-Density Lipoproteins on Proliferation of Monkey Aortic Smooth Muscle Cells Induced by Hyperlipidemic Low-Density Lipoproteins’ YOJI YOSHIDA,~ KATTI FISCHER-DZOGA~ AND ROBERT

W. WISSLER~*~

2Department of Pathology, Yamanashi Medical School, 110 Shimogato, Tamaho Mura, Nakakoma Gun, Yamanashi 409-38, Japan, and 3Department of Pathology and Specialized Center of Research in Atherosclerosis, University of Chicago, Chicago, Illinois 60637 Received

March

24, 1984

The primary outgrowth of medial explants of thoracic aorta from rhesus monkeys was used to study the influence of normolipidemic (N) high-density lipoproteins (HDL) on cell proliferation induced by hyperlipidemic (H) low-density lipoprotein (LDL). The experiments were initiated about 6 weeks after explantation when the celiular outgrowth had almost reached the stationary phase of growth. After being added to the culture media, 5% HLDL induced another proliferative phase in the cultures, as measured by increase in culture area and [3H]thymidine incorporation. The cell proliferation stimulation by 5% H-LDL was prevented by adding 15% N-HDL along with the 5% H-LDL, so that the increment of colony size and incorporation rate of [3H]thymidine into nuclei were similar to those of a group maintained in a medium containing 5% N-HDL. In a second experiment the addition of 5% to 20% of N-HDL to the culture medium containing 5% H-LDL reduced the percentage of nuclei labeled by [‘HJthymidine to control levels at all concentrations of N-HDL. In both experiments, the addition of N-HDL by itself at any concentration, from 5 to 20%, did not stimulate cell proliferation.

INTRODUCTION There is a well-documented negative correlation between plasma concentrations of high-density lipoproteins (HDL) and the incidence of coronary heart disease in the human (Carlson 1960; Rhoads et al., 1976; Castelli et al., 1977; Miller et al., 1977; Pearson et al., 1979). These reports suggest a beneficial effect of a high HDL level in the serum on atherogenesis. Experimental studies investigating the cellular mechanism of these beneficial effects of HDL have shown that HDL is effective not only in reducing LDL entry into the aortic smooth muscle cells in culture (Carew et al., 1976), but also in the removal of intracellular free cholesterol and cholesterol esters (Stein et al., 1975; St. Clair et al., 1977; Bates, 1979). In this study we have investigated the direct effects of normolipidemic high-density lipoprotein (N-HDL) on the proliferation of monkey aortic smooth muscle cells induced by hyperlipidemic low-density lipoproteins (H-LDL) in tissue culture, an effect that has been studied extensively in this laboratory (Kao et al., 1968; Dzoga et al., 1971; Fischer-Dzoga et al., 1974, 1976; FischerDzoga and Wissler 1976; Wissler et al., 1981). MATERIALS

AND METHODS

Aortas from young healthy adult male rhesus monkeys were obtained and the adventitia and intima were stripped off under sterile conditions. Round explants ’ Supported in part by funds from Grant HL-150624 To whom reprint requests should be addressed. 258 0014-4800/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

10 from the National Institutes of Health.

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259

PROLIFERATION

of the media, 2 mm in diameter, were individually placed in 30-ml plastic tissue culture flasks and maintained in basal Eagle’s medium (BME), supplemented with 10% calf serum. Experiments were initiated about 6 weeks after explantation, when the cultures had almost reached the stationary phase of growth (FischerDzoga et al., 1977). In the first experiment, groups of seven or eight cultures of approximately the same size were treated for 10 days with medium containing 5% calf serum to which 5-15% of the various lipoproteins were added. Five percent represents a quantity equivalent to the lipoprotein present in 5% (v/v) whole serum. Two milliliters of culture medium were added to each flask, containing 0.02 mg (5%) or 0.06 mg (15%) N-HDL cholesterol and/or 0.18 mg H-LDL cholesterol, in addition to the 0.2 mg total cholesterol provided by the calf serum present in the medium. In addition total cholesterol levels of the different media used in the first experiment were determined on an Auto Analyzer II (Technicon Corp.). They ranged from 11 mg/lOO ml for the medium with 5% N-HDL to 22 mg/lOO ml for the medium with 5% H-LDL and 15% N-HDL. In all groups the respective media was changed at Days 0, 1, 3, and 7. Increase in culture area was used to evaluate cell growth: The diameter of the circular cellular outgrowth was measured on two perpendicular axes on Days 3, 5, and 10 (Fig. l), the surface area calculated and expressed as percentage of the culture surface area similarly determined before treatment (Day O), thus using a “withingroup repeated-measurements” experimental design. To obtain another measurement of growth, four or five cultures in each group were labeled for the first 3 days of the experimental period by adding [3H]thymidine (sp ac. 6.7 Ci/mmole) to the culture medium (Fischer-Dzoga and Wissler, 1976). [3H]thymidine (1 &i/ml) was added to the media at Day 0. Twenty-four hours later the [3H]thymidine content was decreased to 0.2 t.&i/ml and the cultures incubated for another 48 hr (Fig. 1). After that (72 hr) the culture medium contained no [3H]thymidine. On Day 10 the cultures were fixed in situ in 10% neutral buffered Formalin. The bottoms of the flasks were then removed with a hot knife and the cultures processed for autoradiography and subsequently Experimental

plan Days

0

[3H] thymidine Measurement Change

Measurements

I

I

2I

3I

4L

5I

6I

7

8I

91

L

of s!ze

t

of medium

t

t t

and calculation

of surface

c

t

t

t

,IO

t

area

IA+Bl-d I

2

1

Idl’,=S I 2 1

*Autoradaography

of

selected

cultures

at day

IO

1. Primary cultures of approximately the same size were treated for 10 days with the experimental media. [3H]Thymidine was added to some cultures for the first 3 days and autoradiographs prepared after 10 days. The surface area of all cultures was determined at Days 0, 3, 5, and 10. FIG.

260

YOSHLDA,

FISCHER-DZOGA,

AND WISSLER

stained with oil red 0 and Ehrlich’s acid hematoxylin (Fischer-Dzoga er al., 1977). In each culture five selected fields totaling no less than 1000 cells were examined. Four fields were chosen from four edges of the colony and the fifth field was from near the center of the outgrowth. In a second experiment the effects of several concentrations of N-HDL on cell proliferation were investigated. Eight comparable groups of six to eight cultures each were exposed to BME containing 5% calf serum supplemented with one of the following lipoprotein fractions: (a) 5% N-LDL, (b) 5% N-HDL, (c) 5% HLDL, (d) 5% H-LDL + 5% N-HDL, (e) 5% H-LDL + 10% N-HDL, (f) 5% HLDL + 20% N-HDL, (g) 5% N-LDL + 20% N-HDL, or (h) 20% N-HDL. Monkey normolipidemic and hyperlipidemic sera were obtained from healthy young male rhesus monkeys fed either a monkey chow diet5 or the chow diet supplemented with both 25% coconut oil and 2% cholesterol for at least 2 months. They were bled from the femoral vein following a 12- to 14-hr fast. LDL and HDL fractions were separated by fractional ultracentrifugation with increasing density of the solvent. LDL was isolated at a density of 1.019-1.050 g/ml and HDL at a density of 1.067-1.210 g/ml (Have1 et al., 1955). Analyses of the NHDL apoproteins indicated the presence of two major polypeptides, Apo A-I and Apo A-II (Edelstein et al., 1973; Scanu ez al., 1973). Acrylamide electrophoresis of the H-LDL fraction revealed one strong band in the P-region. RESULTS The strong stimulatory effect of hyperlipidemic LDL on these stationary cells has been confirmed and resulted in an increase in culture size and [3H]thymidine incorporation. The presence of N-HDL in the culture medium diminished or abolished this effect. This is shown in Fig. 2, which documents the growth of each group in the first experiment. During the lo-day experimental period the cultures exposed to the medium containing 5% H-LDL showed a significantly greater increase in average culture size than the group receiving 5% N-HDL. The growth of colony size in the group exposed to 5% H-LDL together with 15% N-HDL was similar to that of the cultures in 5% N-HDL. Cultures exposed to 5% H-LDL and 5% N-HDL did show some increase in colony size, however less than that produced by 5% H-LDL alone. Thus there was some negative effect on the H-LDL-induced proliferation, but not nearly as much as the suppression by 15% of HDL (Fig. 2). The rate of colony area increase per day was calculated by dividing the relative increase of colony size for the different time points of the experimental period by the number of days between measurements. It was consistently greater during the first 5 days and appeared to slow down for the second half of the IO-day experimental period in every group. Thus H-LDL appears to sustain an increased rate of growth of these relatively stationary colonies only for a relatively short time (Fig. 3). Half of the cultures, randomly selected, from each group of the first experiment were exposed to r3H]thymidine for autoradiography. There were no significant differences in the rates of colony size increase between flasks with and without thymidine in any group. Therefore it appears that the concentration of J This primate ration was purchased in pulverized form from Ralston Purina and was prepared to our specifications from a constant formulation of ingredients with no animal fat added.

LIPOPROTEIN A B

0,

I

EFFECT

--o--

261

5%N-HDL 5%H-LOL

C D --o--

i

ON CELL PROLIFERATION

5%H-LDL+l5%N-HDL 5% H-LDL+lS%

i

9

N-HDL

i

j

6

_.o

i

i

b

8

IO Days

Time

FIG. 2. The diameter of each culture was measured on two perpendicular axes before treatment and on Days 3, 5, and 10. The relative increase in culture size over the value at Day 0 (arbitrarily set at 100) was determined for each culture and averaged for each group. At Day 10, group B (5% H-LDL) is substantially different (137.6 k 11.7) from the control group A (120.6 f 5.9) and from the group receiving 15% N-HDL along with the H-LDL (D). The addition of only 5% N-HDL had less effect on the H-LDL-induced proliferation.

[3H]thymidine used had no effect on colony growth under these experimental conditions. The percentage of isotope-labeled nuclei in the cultures exposed to [3H]thymidine is summarized in Table I. The percentage of labeled nuclei was significantly higher (45.5 -+ 6.47%) in the cultures treated with the hyperlipidemic LDL fraction than in the group receiving 5% N-HDL (27.4 k 4.45%) (P < 0.01). The addition of 15% N-HDL to the cultures which contained the hyperlipidemic LDL fraction depressed the percentage of labeled nuclei to a value (22.5 +_ 6.16%) which was similar to that of the group maintained in 5% N-HDL. In contrast the m

BME+5%

calf

serumC5X

0

BME+S%

calf

serum+5%

N-HDL H-LDL

m

EME+S%

calf

serum+5%

H-LDL+S%

N-HDL

Intervals FIG. 3. Cell colonies were measured at Days 0, 3, 5, and 10. The relative increase in diameter over the value of the preceding measuring date was determined for each culture and averaged for each group.

262

YOSHIDA,

Effect of Normolipidemic

5% 5% 5% 5%

FISCHER-DZOGA,

AND WISSLER

TABLE I HDL on [3H]Thymidine Incorporation Muscle Cells in Culture

Addition to the culture medium I N-HDL H-LDL H-LDL + 5% N-HDL H-LDL + 15% N-HDL

Number of cultures counted

Percentage of cells labeled with [3H]thymidine (mean f SE) 27.4 45.4 37.8 22.5

5 6 6 7

Note. All groups were different from 5% H-LDL 0.001 (***).

of Monkey Aortic Medial Smooth

k t f f

4.5* 6.5 4.9** 6.2***

group at P < 0.01 (*), P < 0.1 (**), and P <

addition of 5% N-HDL resulted in a lesser degree of enhanced thymidine incorporation rate produced by the H-LDL (37.8 t 4.9%). These results were confirmed in the second experiment: the percentage of labeled nuclei indicates a similar incorporation rate of [3H]thymidine for the cultures exposed to either 20% N-HDL alone or to 20% N-HDL and 5% N-LDL (P > 0.05) (Table II). It is equally low in the group receiving 5% N-LDL or 5% NHDL. In contrast the labeling index of the cultures treated with 5% H-LDL was significantly higher than that of most of the other groups. However 5, 10, or 20% N-HDL added to the media containing 5% H-LDL lowered the incorporation of [3H]thymidine into the nuclei to near control levels. An increase in rate of growth by H-LDL is in general agreement with results previously reported from this laboratory (Fischer-Dzoga et al., 1976; FischerDzoga and Wissler, 1976) and although in this experiment the 5% N-HDL group yielded a slightly higher percentage of labeled nuclei than that of the 5% N-LDL group, the difference was not significant (P > 0.01). DISCUSSION Arterial smooth muscle cell proliferation has been assumed to be one of the important parts of the pathogenesis of both human (Haust et al., 1960; Geer et TABLE II Effect of Hyperlipidemic and/or Normolipidemic Lipoprotein Fractions on the Proliferation of Monkey Aortic Medial Smooth Muscle Cells in Culture as Measured by [‘H]Thymidine Incorporation Addition to the culture medium 5% N-LDL 5% N-HDL 5% H-LDL 5% H-LDL 5% H-LDL 5% H-LDL 5% N-LDL 20% N-HDL

+ + + +

5% N-HDL 10% N-HDL 20% N-HDL 20% N-HDL

Number of cultures counted 6 8 7 6 7 8 7 7

Percentage of cells labeled (mean f SE) 21.2 26.4 33.4 22.9 23.3 20.3 21.8 20.0

k f t 2 k k ” 2

5.3* 6.2** 7.8 4.3* 5.9*** 7.3* 6.0* 4.37

Note. All groups were different from the group receiving 5% H-LDL at P < 0.025 (*). P < 0.1 (**) P < 0 05 (***), and P < 0.01 (t). There was significant difference between the control group (S%‘N-LDL) and the groups receiving 5% H-LDL with 5, 10, or 20% N-HDL added.

LIPOPROTEIN

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263

al., 1961; Wissler, 1968, 1974b) and experimental (Parker and Odland, 1966; Scott er al., 1967; Wissler et al., 1981) atherosclerosis. Furthermore, previous studies have documented the increased in viva incorporation by arterial smooth muscle cells of [3H]thymidine when diets that raise the serum cholesterol level are fed to experiment animals (Stary and McMillan, 1970; Thomas et al., 1971). Recently a number of possible mechanisms have been proposed to link endothelial cell damage (Sternerman and Ross, 1972) and/or elevated blood lipids (Wissler, 1974a) to accelerated atherogenesis, and evidence from in vitro studies indicated that potent cell proliferation stimulating factors may be responsible for at least some of the atherogenic effects (Wissler, 1979). Two factors which have received sustained attention are the one derived from the platelet (Ross et al., 1974) and one that seems to be closely associated with the elevation of LDL in the serum (Fischer-Dzoga et al., 1976; Fischer-Dzoga and Wissler, 1976) and which appears to act even when the platelet factor in serum has already exerted its maximum effect (Fischer-Dzoga et al., 1983). When interest in the protective effects of HDL against atherogenesis was recently reawakened (Miller and Miller, 1975; Carew et al., 1976) it was only natural that investigations should be directed toward the ways in which increasing concentrations of this lipoprotein might counteract the atherogenic effects of LDL from hyperlipidemic serum. Several studies have already indicated that HDL may play an important part in preventing the accumulation of cholesterol or in removing excess cholesterol from arterial smooth muscle cells or from the artery wall (Bondjers and Bjorkerud, 1974; Stein et al., 1976; Bondjers et al., 1977; Tall and Small, 1978; Bates, 1979). These data represent the first study of which we are aware that specifically addresses the question of whether the HDL fraction will counteract the atherogenie smooth muscle cell proliferation which results from elevated cholesterolrich LDL levels in the serum. The results seem to indicate that normal HDL over a range of concentrations will diminish and/or largely prevent the cell proliferation induced by H-LDL as measured by [3H]thymidine incorporation. However, concentrations as high as 15 or 20% of normolipidemic HDL in the culture medium have no appreciable effect on cell proliferation, as measured either by colony size or percentage of [3H]thymidine incorporation when compared to the control groups with 5% normolipidemic HDL or LDL. The cellular and/or molecular mechanisms by which HDL inhibits the cell proliferation induced by the LDL fraction from hypercholesterolemic serum are obscure. Evidence gained from the study of *251-labeled LDL from hyperlipemic serum suggests that the excess HDL may interfere with specific LDL binding to the cells (Bates, 1980). Whether this means that some part of rhesus HDL apoprotein binds more avidly to the SMC surface than does the LDL apo B, remains to be demonstrated. Evidently HDL does bind to the membranes of vascular smooth muscle cells, but appears to be much more slowly internalized than LDL (Biermann and Albers, 1975; Stein and Stein, 1975; Carew et al., 1976). Moreover it has been reported that the presence in the culture medium of HDL inhibits the binding and internalization of LDL by arterial smooth muscle cells (Biermann and Albers, 1976; Carew et al., 1976) and human fibroblasts, presumably due to interaction between the two lipoproteins during binding to the cell surface (Bondjers et al., 1977). Mahley has recently reported a minor subfraction, rich in Apo

264

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WISSLER

E and induced by cholesterol feeding, to be responsible for this interaction (Mahley et al., 1978). However, under our experimental conditions the HDL was from normal monkeys on a low fat, cholesterol-free chow diet. Analysis of this HDL revealed the presence of two major apoproteins, Apo AI and Apo A-II (Edelstein et al., 1973, Scanu et al., 1973), and a minor apoprotein occasionally found in normal serum of this species is evidently not identical with APO-E (Rude1 et al., 1977). Whatever the mechanism(s), HDL in the culture medium effectively inhibits the proliferation of arterial smooth muscle cells induced by LDL from hyperlipidemic serum. ACKNOWLEDGMENTS The authors are grateful to Mr. Randolph Hughes, Mr. Lance Lusk, and Miss Yea-Fhey Kuo for their excellent technical assistance. They also appreciate the assistance of Mrs. Gertrud Friedman and Mrs. Gwen Matthews in preparing the manuscript for submission.

REFERENCES BATES, S. R. (1979). Accumulation and loss of cholesterol esters in monkey arterial smooth muscle cells exposed to normal and hyperlipemic serum lipoproteins. Atherosclerosis 32, 16% 176. BATES, S. R. (1980). Effect of HDL on the interaction of hyperlipemic LDL with monkey smooth muscle cells. Artery 7, 303-315. BIERMANN, E. L., and ALBERS, J. J. (1975). Lipoprotein uptake by cultured human arterial smooth muscle cells. Biochim. Eiophys. Acta 388, 198-202. BIERMANN, E. L., and ALBERS, J. J. (1976). Lipoprotein uptake and degradation by cultured human arterial smooth cells. In “Atherosclerosis Drug Discovery” (C. E. Day, ed.), pp. 437-452. Plenum, New York. BONDJERS,G., and BJORKERUD,S. (1974). Cholesterol transfer between arterial smooth muscle tissue and serum lipoproteins in vitro. Artery 1, 3-9. BONDJERS,G., OLSSON, G., NYMAN, L.-L., and BJORKERUD,S. (1977). High density lipoprotein (HDL) dependent elimination of cholesterol from normal arterial tissue in man. In “Atherosclerosis IV.” (G. Schettler, Y. Goto, Y. Hata, and G. Klose, eds.), pp. 70-71. Springer-Verlag, New York/Berlin. CAREW, T. E., KOSCHINSKY, T., HAYES, S. B., and STEINBERG, D. (1976). A mechanism by which high-density lipoproteins may slow the atherogenic process. Lance? 1, 1315-1317. CARLSON, L. A. (1960). Serum lipids in men with myocardial infarction. Acta Med. Scud. 167, 399413. CASTELLI, W. P., DOYLE, J. T., GORDON, T., HAMES, C. G., HJORTLAND, M. C., HULLEY, S. B., KAGAN, A., and ZUKEL, W. J. (1977). HDL cholesterol and other lipids in coronary heart disease: The cooperative lipoprotein phenotyping study. Circulation 55, 767-770. DZOGA, K., VESSELINOVITCH, D., FRASER, R., and WISSLER, R. W. (1971). The effect of lipoproteins on the growth of aortic smooth muscle cells in vitro, Amer. J. Pathol. 62, 32a. EDELSTEIN, C., LIM, C. T., and SCANU, A. M. (1973). The serum high density lipoproteins of Macaws rhesus. II. Isolation, purification and characterization of their two major polypeptides. J. Biol. Chem.

248, 7653-7660.

FISCHER-DZOGA, K., CHEN, R., and WISSLER, R. W. (1974). Effects of serum lipoproteins on the morphology, growth and metabolism of arterial smooth muscle cells. In “Arterial Mesenchyme and Arteriosclerosis” (W. D. Wagner and T. B. Clarkson, eds.), pp. 299-311. Plenum, New York. FISCHER-DZOGA,K., FRASER, R., and WISSLER, R. W. (1976). Stimulation of proliferation in stationary primary cultures of monkey and rabbit aortic smooth muscle cells. 1. Effects of lipoprotein fractions of hyperlipemic serum and lymph. Exp. Mol. Parho/. 24, 346-359. FISCHER-DZOGA, K., Kuo, Y-F., and WISSLER R. W. (1983). The proliferative effect of platelets and hyperlipidemic serum on stationary primary cultures. Atherosclerosis 47, 35-45. FISCHER-DZOGA, K., and WISSLER, R. W. (1976). Stimulation of proliferation in stationary primary cultures of monkey aortic smooth muscle cells. Part 2. Effect of varying concentrations of hyperlipemic serum and low density lipoproteins of varying dietary fat origins. Atherosclerosis 24, 515525. FISCHER-DZOGA,K., WISSLER, R. W., and SCANU, A. M. (1977). The lipoproteins and arterial smooth

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muscle cells: Cellular proliferation and morphology. In “Atherosclerosis” (G. W. Manning and M. D. Haust, eds.), pp. 915-920. Plenum, New York. GEER, J. C., MCGILL, H. C., and STRONG, J. P. (1961). The fine structure of human atherosclerotic lesions. Amer. J. Pathol. 38, 263-287. HAUST, M. D., MORE, R. H., and MOVAT, H. Z. (1960). The role of smooth muscle cells in the fibrogenesis of arteriosclerosis. Amer. J. Pathol. 37, 377-380. HAVEL, R. S., EDER, H. H., and BRAGDON, S. H. (1955). The distribution and chemical composition of ultracentrifugally-separated lipoproteins in human serum. J. C/in. Invest. 34, 1345- 1353. KAO, V., WISSLER, R. W., and DZOGA, K. (1968). The influence of hyperlipemic serum on the growth of medial smooth muscle cells of rhesus monkey aortas in vitro. Circulation 38, VI:12. MAHLEY, R. W., INNERARITY, T. L., BERSOT, T. P., LIPSON, A., and MARGOLIS, S. (1978). Alterations in human high-density lipoproteins, with or without increased plasma-cholesterol, induced by diets high in cholesterol. Lancer 2, 807-809. MILLER, G. J., and MILLER, N. E. (1975). Plasma high density lipoprotein concentration and development of ischaemic heart disease. Lance? 1, 16-19. MILLER, N. E., FORDE, 0. H., THELLE, D. S., and MJOS, 0. D. (1977). The Tromso heart study. High density lipoprotein and coronary heart disease: A prospective case-control study. Lancer 1, 965967.

PARKER, E, and ODLAND G. (1966). A correlative histochemical, biochemical and electron microscopic study of experimental atherosclerosis in the rabbit aorta with special reference to the myo-intimal cell. Amer. J. Parhol. 58, 197-239. PEARSON, T. A., BULKLEY, B. H., ACHUFF, S. C., KWITEROVICH, P. 0.. and GORDIS, L. (1979). The association of low levels of HDL cholesterol and arteriographically defined coronary artery disease. Amer. J. Epidemiol. 109, 285-298. RHOADS, G. G., GULBRANDSEN, C. L., and KAGAN, A. (1976). Serum lipoproteins and coronary heart disease in a population study of Hawaii Japanese men. N. Engl. J. Med. 293-298. Ross, R., GLOMSET, J., KARIYA, B., and HARKER, L. (1974). A platelet dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc. Nat/. Acad. Sci. USA 71, 1207-1210. RUDEL, L. L., GREENE, D. G., and SHAH, R. (1977). Separation and characterization of plasma lipoproteins of rhesus monkeys (Macaca mulatta). J. Lipid Res. 18, 734-744. SCANU, A. M., EDELSTEIN, C., VITELLO, L., JONES, R., and WISSLER, R. W. (1973). The serum high density lipoproteins of Macacus rhesus. I. Isolation, composition and properties, J. Biol. Chem. 243, 7648-7652.

Scorr, R. F., JONES, R., DAOUD, A. S., ZUMBO, 0.. COULSTON, F., and THOMAS, W. A. (1967). Experimental atherosclerosis in rhesus monkeys. II. Cellular elements of proliferative lesions and possible role of cytoplasmic degeneration in pathogenesis as studied by electron microscopy. Exp. Mol.

Parhol.

7, 34-57.

STARY, H. C., and MCMILLAN, G. C. (1970). Kinetics of cellular proliferation in experimental atherosclerosis. Radioautography with grain counts in cholesterol-fed rabbits. Arch. Pathol. 89, 173183. ST. CLAIR, R. W., SMITH, B. P., and WOOD, L. L. (1977). Stimulation of cholesterol esterification in rhesus monkey arterial smooth muscle cells. Circ. Res. 40, 166-173. STEIN, Y., GLANGEAUD, M. C., FAINARU, M., and STEIN, 0. (1975). The removal of cholesterol from aortic smooth muscle cells in culture and Landschutz ascites cells by fractions of human highdensity apolipoprotein. Biochim. Biophys. Acta 380, 106- 118. STEIN, O., and STEIN, Y. (1975). Comparative uptake of rat and human serum low-density and highdensity lipoproteins by rat aortic smooth muscle cells in culture. Circ. Res. 36, 436-443. STEIN, O., VANDERHOEK, J., and STEIN, Y. (1976). Cholesterol content and sterol synthesis in human skin fibroblasts and rat aortic smooth muscle cells exposed to lipoprotein-depleted serum and high density apolipoprotein/phospholipid mixtures, Biochim. Biophys. Acta 431, 347-358. STEMERMAN, M. B., and Ross, R. (1972). Experimental atherosclerosis, I. Fibrous plaque formation in primates, an electron microscope study. J. Exp. Med. 136, 769-789. TALL, A. R., and SMALL, D. M. (1978). Plasma high density lipoproteins. N. Engl. J. Med. 299, 1232-1236. THOMAS, W. A., FLORENTIN, R. A., NAM, S. C., REINER, J. M., and LEE, K. T. (1971). Alteration in population dynamics of arterial smooth muscle cells during atherogenesis. I. Activation of inter-

266

YOSHIDA,

FISCHER-DZOGA,

AND WISSLER

phase cells in cholesterol-fed swine prior to gross atherosclerosis demonstrated by “postpulse salvage labeling.” Exp. Mol. Pathol. 15, 245-267. WISSLER, R. W. (1968). The arterial medial cell, smooth muscle or multifunctional mesenchyme? J. Atheroscler. Res. 8, 201-213. WISSLER, R. W. (1974a). Atherosclerosis-its pathogenesis in perspective. In “Comparative Pathology of the Heart” (E Homberger, ed.), pp. 10-31. Karger, Basel. WISSLER, R. W. (1974b). Development of the atherosclerotic plaque. In “The Myocardium: Failure and Infarction” (E. Braunwald, ed.), pp. 155-166. HP. Publishing, New York. WISSLER, R. W. (1979). Interaction of low-density lipoproteins from hypercholesterolemic serum with arterial wall cells and their extracellular products in atherogenesis and regression. In “The Biochemistry of Atherosclerosis” (A. M. Scanu, R. W. Wissler, and G. S. Getz, eds.), pp. 345-368. Dekker, New York. WISSLER, R. W. FISCHER-DZOGA, K., BATES, S. R., and CHEN, R. M. (1981). Arterial smooth muscle cells in tissue culture. In “Structure and Function of the Circulation” (C. J. Schwartz, N. T. Werthessen, and S. Wolf, eds.), pp. 427-474. Plenum, New York. WISSLER, R. W. (1984). Principles of the pathogenesis of atherosclerosis. In “Heart Disease. A Textbook of Cardiovascular Medicine” (E. Braunwald, ed.), 2nd ed., pp. 1183-1204. Saunders, Philadelphia.