198
Biochimica et Biophysics Acta, 388 (1975) 198-202 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56593
LIPOPROTEIN UPTAKE MUSCLE CELLS
BY CULTURED
HUMAN ARTERIAL
SMOOTH
EDWIN L. BIERMAN and JOHN J. ALBERS Department of Medicine, Division of Metabolism and Gerontology, Northwest Lipid Research Clinic, and Department of Biochemistry, University of Washington School of Medicine, Seattle, Wash. 98195
(U.S.A.)
(Received December 9th, 1974)
Summary Human arterial smooth muscle cells growing in tissue culture, in contrast to rat cells, preferentially bind and take up large, lipid-rich lipoproteins ( ’ * ‘Ilabeled low density and very low density lipoproteins) in comparison to smaller, high density lipoproteins. These results may be relevant to the known difference in the propensity of these two species to develop atherosclerosis. Introduction The multipotential smooth muscle cell is the predominant cell type in the intima and media of large arteries. It is also the cell that proliferates early in the development of atheroma [l-4], perhaps as a monoclonal response [ 51, to eventually become the lipid-filled foam cell. The recent development by Ross of a technique for the growth in homogeneous culture of smooth muscle cells isolated from aorta of guinea pigs [6] and subhuman primates [7] has allowed a direct examination of the ability of that cell to take up and metabolize different lipoproteins. Results in recent studies, using smooth muscle cells successfully cultured from rat aorta, suggested that some specificity for uptake and degradation of different lipoprotein fractions might exist [8,9]. However, since man appears to be particularly prone to accumulate lipid in arterial smooth muscle cells in vivo with age [lo] and to develop atherosclerosis, it is essential to test these questions with cells derived from human arteries. We have now successfully grown homogeneous cultures of human smooth muscle cells from explants of intimal-medial segments of normal appearing pieces of large arteries (aortic, iliac, femoral or renal), obtained during vascular surgery for coarctation, atherosclerosis, or renal transplantation, using the method of Ross [ 61 as modified previously [9]. These cells share the morphological and growth characteristics of smooth muscle cells cultured from
199
other species and the cultures are free of fibroblast contamination. They differ from human fibroblasts (both skin and adventitial) by their longer lag period before outgrowth from explants, their slower growth rate, and their morphology (smooth muscle cells characteristically contain numerous myofilaments with dense bodies). Methods For the present study, the cells were grown in Falcon plastic tissue-culture flasks in modified Dulbecco-Vogt medium containing 10% fetal calf serum and used after 2-5 passages. Experiments were performed on cells plated in plastic Falcon petri dishes (60 mm diameter) after the cells had grown to confluency, with typical multilayer formation, usually containing about lo6 cells per dish after three weeks. Lipoproteins were separated from plasma of normal donors by standard methods of sequential ultracentrifugation as follows. The plasma was centrifuged at 40 000 rev./mm for 18 h in a Beckman L5-50 ultracentrifuge using a Spinco 60 Ti rotor. The top very low density lipoprotein fraction was removed and recentrifuged in 0.85% NaCl/0.05% EDTA solution (pH 7.4) under identical conditions. For low density lipoprotein isolation, the nonprotein solvent density was adjusted to 1.019 g/ml with solid KBr, then centrifuged at 40 000 rev./min for 22 h. The top fraction was discarded and the bottom fraction adjusted to d = 1.063 g/ml and centrifuged for 22 h at 42 000 rev./min. The top low density lipoprotein fraction was washed by recentrifugation under identical conditions. Following labeling by iodination with ’ * 51 by a modified method of MacFarlane [ll],the lipoproteins were filtered through a molecular sieve (Biogel A5m) to remove denatured products and free iodine. After dialysis and sterile filtration (Millipore Swinnex 0.45 pm), they were added to fresh medium bathing the cells. Cells were incubated with labeled lipoproteins for periods up to 48 h at 37” C. Analysis of radioactivity in medium and cells was performed as described previously [9] (legend, Fig. 1). Analysis of precision of low density lipoprotein uptake in replicate human smooth muscle cell cultures incubated with low density lipoprotein for 8, 24, and 48 h yielded a coefficient of variation of 21%. Results and Discussion The uptake of very low density and low density lipoproteins by cultured human arterial smooth muscle cells considerably exceeded that previously observed in the rat (Fig. 1). In four experiments with both cells and lipoproteins isolated from different donors, low density lipoprotein uptake in cells ranged from 500 to 3000 ng/lOO I.rg lipoprotein protein added/lo6 cells in 24 hours. Very low density lipoprotein uptake approximated these levels (320-1100 ng). In contrast, as reported in previous studies [9], homologous very low density lipoprotein uptake by rat aortic smooth muscle cells averaged only 5 ng/lOO pg lipoprotein added/lo6 cells in 24 h. (For conversion of uptake of radioactivity to ng protein uptake, it has been assumed that all labeled lipoprotein apoproteins are taken up at the same rate.) The affinity of arterial smooth muscle cells for lipoproteins of different
HUMAN
RAT
.A...
‘..
:
. . . ‘.., . . . .
.‘...
LDL:” ___---
24 HOURS
36
__--
0
48 HOURS
G Fig. 1. Uptake of lipoprotein protein by cultured arterial smooth muscle cells from rat (left panel) and human (right panel). Note lo-fold difference in ordinate units. Data from the rat was adapted from a previous report [9] and depicts an experiment in which each dish contained 4 X 106 cells grown for 15 days after passage to which 140 pg lipoprotein protein was added to 4 ml medium. Human cells were obtained from explants of a grossly normal segment of the femoral artery from a 50-year-old man. Each dish contained 0.96 X lo6 cells grown for 19 days after passage. Homologous lipoproteins (52 Pg protein/dish) were added to 4 ml medium in each dish. Total lipoprotein protein uptake by cells (“cell protein uptake”) represents radioactivity in the cell pellet (obtained after washing and release of surface-bound counts by trypsinization of the cell layer followed by centrifugation and resuspension in buffer, repeated twice), from which trichloroacetic acid-soluble counts (after addition of 5% trichloroacetic acid to cell pellet and centrifugation) and lipid-soluble counts (after extraction of trichloroacetic acid precipitate of cells with chloroform/methanol, 2 : 1) have been subtracted. Each point represents the mean of results from replicate culture dishes. HDL, LDL and VLDL. high, low and very low density lipoproteins.
classes differed markedly between these species. In the rat the smaller high density lipoprotein particles (approx. 600 000 mol. wt) [12] were taken up to a greater extent than the much larger (3-128 million mol. wt) [13] very low density lipoprotein particles. In striking contrast, cholesterol-rich low density lipoprotein and triacylglycerol-rich very low density lipoprotein were taken up by human cells to a much greater extent (approx. lo-fold) than the smaller high density lipoprotein (28-40 ng/lOO pg lipoprotein protein added lo6 cells). Control 1 ’ ‘I-labeled human serum albumin was taken up to an even lesser extent than high density lipoprotein (16 ng). Thus, while homologous high density lipoprotein uptake by human and rat arterial smooth muscle cells was similar, the larger, lipid-rich, low and very low density lipoproteins, appeared to be preferentially taken up by human cells. This suggests that there might be receptors on arterial smooth muscle cells, specific for different lipoproteins. This possibility is consistent with the studies of Brown and Goldstein [14-161 who have documented the presence of receptors on cultured human skin fibroblasts which specifically bind low density lipoproteins. Specific binding of these lipoproteins by human arterial smooth muscle cells was studied by analysis of radioactive protein released from cells by trypsin after incubation of cell layers with ’ * ‘I-labeled lipoprotein for varying times and extensive washing with buffer. These trypsin-released counts (presumably surface-bound [17] ) were corrected for non-specific binding by sub-
201
3
6
9 HOURS
24
3
6
9
24
HOURS
Fig. 2. Biding, uptake and degradation of ’ 2s I-labeled low density lipoprotein protein by human arterial smooth muscle cells. Cells were obtained from explants of aorta distal to a coarctation in a 12-year-old male. Low density lipoprotein (1.6 X lo5 cpm/wg protein) was added (9.2 c(g protein/dish) to 4 ml fresh medium containing 1% fetal calf serum and incubated for 24 h. Cell protein uptake (right panel) as in Fig. 1. Surface-bound protein (right panel) represents radioactivity released after trypsinization of cell layer subsequent to removal of medium containing labeled low density lipoprotein and 5 washes with buffer 191, corrected for non-specific binding assessed by measurement of trypsin-released counts after addition of a lOO-fold excess unlabeled low density lipoprotein to other dishes. Protein degradation represents water-soluble radioactivity appearing in medium during incubation with cells, corrected for free iodide by chloroform extraction of the trichloroacetic acid-soluble fraction of the medium after mixture with KI and Hz02 191. Negligible degradation occurred at 37’C over 24 h in the absence of cells. Each point represents the mean of measurements from replicate culture dishes. Each dish contained 1.04 X lo6 cells, grown for 28 days after passage.
traction of counts released in other dishes which had been incubated with more than a 100.fold excess of unlabeled low density lipoprotein (an average of 11% of low density lipoprotein remained bound; n = 4). Specific protein binding was decreased 55% by incubation of cells at 4°C. Trypsin-releasable low density lipoprotein accounted for half the protein radioactivity associated with the cell layer during the first 4 h of incubation (Fig. 2). Subsequently, cell uptake progressed more rapidly. Lipoprotein degradation, assessed by appearance of trichloroacetic acidsoluble, non-iodide radioactivity in the incubation medium, increased in parallel with uptake of lipoprotein after an initial delay of 2 h (Fig. 2). This phenomenon has also been observed with cultured skin fibroblasts [16] and suggests that specific binding of lipoprotein to cell surface receptors, presumably via specific apolipoprotein polypeptides, is a critical first step prior to subsequent cellular uptake and degradation of lipoprotein. These results are of particular interest since rat and man differ markedly in their propensity to develop atherosclerosis with aging. The apparent specificity of cultured human arterial smooth muscle cells for binding and uptake of large, lipid-rich lipoproteins is consistent with the possibility that these cells, which play a central role in atherogenesis, accumu-
202
late lipid as a result of specific lipoprotein binding and subsequent they are exposed and in contact with lipoproteins in vivo.
uptake
when
Acknowledgements This study was supported by grants from the National Institutes of Health and the American Heart Association. We thank M. Stewart and V. Cabana for their expert help. References 1 Parker, F. (1960) Am. J. Pathol. 36, 19-53 2 Geer, J.C., McGill, Jr. H.C. and Strong, J.P. (1961) Am. J. Pathol. 38, 263-287 3 Haust, M.D. and More. R.H. (1963) in Evolution of the Atherosclerotic Plaque (Jones, R.J., ed.), pp. 51-63, University of Chicago Press. Chicago 4 Geer, J.C. and Haust, M.D. (1972) Smooth Muscle Cells in Atherosclerosis, S. Karger. Basel. 5 Benditt, E.P. and Benditt, J.M. (1973) Proc. Natl. Acad. Sci. U.S. 70. 1753-1756 6 Ross, R. (1971) J. Cell Biol. 50. 172-186 7 Ross, R. and Glomset, J. (1973) Science 180, 1332-1338 8 Bierman, E.L., Eisenberg, S.. Stein, 0. and Stein, Y. (1973) Biochim. Biophys. Acta 329, 163-169 9 Bierman, E.L., Stein, 0. and Stein, Y. (1974) Circ. Res. 35. 136-150 10 Smith, E.B. (1970) in The Artery and the Process of Arteriosclerosis: Patbogenesis. (Wolf, S.. ed.). pp. 81-120, Plenum Press, New York 11 Bilheimer. D.W.. Eisenberg, S. and Levy, R.I. (1972) B&him. Biophys. Acta 260, 212-221 12 Koga, S., Honvitz, A.L. and Scanu. A.M. (1969) J. Lipid Res. 10, 577-588 13 Scanu, A.M. (1972) Biochim. Biophys. Acta 265, 471-508 14 Brown. M.S. and Goldstein, J.L. (1974) Proc. Natl. Acad. Sci. U.S. 71, 788-792 15 Brown, M.S. and Goldstein, J.L. (1974) Science 185, 6143 16 Goldstein, J.L. and Brown, M.S. (1974) J. Biol. Chem. 249, 5153-5162 17 Werb, 2. and Cohn, Z.A. (1971) J. Exp. Med. 134. 1570-1590
Biochimica et Biophysics Acta, 388 (1975) 198-202 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56593
LIPOPROTEIN UPTAKE MUSCLE CELLS
BY CULTURED
HUMAN ARTERIAL
SMOOTH
EDWIN L. BIERMAN and JOHN J. ALBERS Department of Medicine, Division of Metabolism and Gerontology, Northwest Lipid Research Clinic, and Department of Biochemistry, University of Washington School of Medicine, Seattle, Wash. 98195
(U.S.A.)
(Received December 9th, 1974)
Summary Human arterial smooth muscle cells growing in tissue culture, in contrast to rat cells, preferentially bind and take up large, lipid-rich lipoproteins ( ’ * ‘Ilabeled low density and very low density lipoproteins) in comparison to smaller, high density lipoproteins. These results may be relevant to the known difference in the propensity of these two species to develop atherosclerosis. Introduction The multipotential smooth muscle cell is the predominant cell type in the intima and media of large arteries. It is also the cell that proliferates early in the development of atheroma [l-4], perhaps as a monoclonal response [ 51, to eventually become the lipid-filled foam cell. The recent development by Ross of a technique for the growth in homogeneous culture of smooth muscle cells isolated from aorta of guinea pigs [6] and subhuman primates [7] has allowed a direct examination of the ability of that cell to take up and metabolize different lipoproteins. Results in recent studies, using smooth muscle cells successfully cultured from rat aorta, suggested that some specificity for uptake and degradation of different lipoprotein fractions might exist [8,9]. However, since man appears to be particularly prone to accumulate lipid in arterial smooth muscle cells in vivo with age [lo] and to develop atherosclerosis, it is essential to test these questions with cells derived from human arteries. We have now successfully grown homogeneous cultures of human smooth muscle cells from explants of intimal-medial segments of normal appearing pieces of large arteries (aortic, iliac, femoral or renal), obtained during vascular surgery for coarctation, atherosclerosis, or renal transplantation, using the method of Ross [ 61 as modified previously [9]. These cells share the morphological and growth characteristics of smooth muscle cells cultured from
199
other species and the cultures are free of fibroblast contamination. They differ from human fibroblasts (both skin and adventitial) by their longer lag period before outgrowth from explants, their slower growth rate, and their morphology (smooth muscle cells characteristically contain numerous myofilaments with dense bodies). Methods For the present study, the cells were grown in Falcon plastic tissue-culture flasks in modified Dulbecco-Vogt medium containing 10% fetal calf serum and used after 2-5 passages. Experiments were performed on cells plated in plastic Falcon petri dishes (60 mm diameter) after the cells had grown to confluency, with typical multilayer formation, usually containing about lo6 cells per dish after three weeks. Lipoproteins were separated from plasma of normal donors by standard methods of sequential ultracentrifugation as follows. The plasma was centrifuged at 40 000 rev./mm for 18 h in a Beckman L5-50 ultracentrifuge using a Spinco 60 Ti rotor. The top very low density lipoprotein fraction was removed and recentrifuged in 0.85% NaCl/0.05% EDTA solution (pH 7.4) under identical conditions. For low density lipoprotein isolation, the nonprotein solvent density was adjusted to 1.019 g/ml with solid KBr, then centrifuged at 40 000 rev./min for 22 h. The top fraction was discarded and the bottom fraction adjusted to d = 1.063 g/ml and centrifuged for 22 h at 42 000 rev./min. The top low density lipoprotein fraction was washed by recentrifugation under identical conditions. Following labeling by iodination with ’ * 51 by a modified method of MacFarlane [ll],the lipoproteins were filtered through a molecular sieve (Biogel A5m) to remove denatured products and free iodine. After dialysis and sterile filtration (Millipore Swinnex 0.45 pm), they were added to fresh medium bathing the cells. Cells were incubated with labeled lipoproteins for periods up to 48 h at 37” C. Analysis of radioactivity in medium and cells was performed as described previously [9] (legend, Fig. 1). Analysis of precision of low density lipoprotein uptake in replicate human smooth muscle cell cultures incubated with low density lipoprotein for 8, 24, and 48 h yielded a coefficient of variation of 21%. Results and Discussion The uptake of very low density and low density lipoproteins by cultured human arterial smooth muscle cells considerably exceeded that previously observed in the rat (Fig. 1). In four experiments with both cells and lipoproteins isolated from different donors, low density lipoprotein uptake in cells ranged from 500 to 3000 ng/lOO I.rg lipoprotein protein added/lo6 cells in 24 hours. Very low density lipoprotein uptake approximated these levels (320-1100 ng). In contrast, as reported in previous studies [9], homologous very low density lipoprotein uptake by rat aortic smooth muscle cells averaged only 5 ng/lOO pg lipoprotein added/lo6 cells in 24 h. (For conversion of uptake of radioactivity to ng protein uptake, it has been assumed that all labeled lipoprotein apoproteins are taken up at the same rate.) The affinity of arterial smooth muscle cells for lipoproteins of different
HUMAN
RAT
.A...
‘..
:
. . . ‘.., . . . .
.‘...
LDL:” ___---
24 HOURS
36
__--
0
48 HOURS
G Fig. 1. Uptake of lipoprotein protein by cultured arterial smooth muscle cells from rat (left panel) and human (right panel). Note lo-fold difference in ordinate units. Data from the rat was adapted from a previous report [9] and depicts an experiment in which each dish contained 4 X 106 cells grown for 15 days after passage to which 140 pg lipoprotein protein was added to 4 ml medium. Human cells were obtained from explants of a grossly normal segment of the femoral artery from a 50-year-old man. Each dish contained 0.96 X lo6 cells grown for 19 days after passage. Homologous lipoproteins (52 Pg protein/dish) were added to 4 ml medium in each dish. Total lipoprotein protein uptake by cells (“cell protein uptake”) represents radioactivity in the cell pellet (obtained after washing and release of surface-bound counts by trypsinization of the cell layer followed by centrifugation and resuspension in buffer, repeated twice), from which trichloroacetic acid-soluble counts (after addition of 5% trichloroacetic acid to cell pellet and centrifugation) and lipid-soluble counts (after extraction of trichloroacetic acid precipitate of cells with chloroform/methanol, 2 : 1) have been subtracted. Each point represents the mean of results from replicate culture dishes. HDL, LDL and VLDL. high, low and very low density lipoproteins.
classes differed markedly between these species. In the rat the smaller high density lipoprotein particles (approx. 600 000 mol. wt) [12] were taken up to a greater extent than the much larger (3-128 million mol. wt) [13] very low density lipoprotein particles. In striking contrast, cholesterol-rich low density lipoprotein and triacylglycerol-rich very low density lipoprotein were taken up by human cells to a much greater extent (approx. lo-fold) than the smaller high density lipoprotein (28-40 ng/lOO pg lipoprotein protein added lo6 cells). Control 1 ’ ‘I-labeled human serum albumin was taken up to an even lesser extent than high density lipoprotein (16 ng). Thus, while homologous high density lipoprotein uptake by human and rat arterial smooth muscle cells was similar, the larger, lipid-rich, low and very low density lipoproteins, appeared to be preferentially taken up by human cells. This suggests that there might be receptors on arterial smooth muscle cells, specific for different lipoproteins. This possibility is consistent with the studies of Brown and Goldstein [14-161 who have documented the presence of receptors on cultured human skin fibroblasts which specifically bind low density lipoproteins. Specific binding of these lipoproteins by human arterial smooth muscle cells was studied by analysis of radioactive protein released from cells by trypsin after incubation of cell layers with ’ * ‘I-labeled lipoprotein for varying times and extensive washing with buffer. These trypsin-released counts (presumably surface-bound [17] ) were corrected for non-specific binding by sub-
201
3
6
9 HOURS
24
3
6
9
24
HOURS
Fig. 2. Biding, uptake and degradation of ’ 2s I-labeled low density lipoprotein protein by human arterial smooth muscle cells. Cells were obtained from explants of aorta distal to a coarctation in a 12-year-old male. Low density lipoprotein (1.6 X lo5 cpm/wg protein) was added (9.2 c(g protein/dish) to 4 ml fresh medium containing 1% fetal calf serum and incubated for 24 h. Cell protein uptake (right panel) as in Fig. 1. Surface-bound protein (right panel) represents radioactivity released after trypsinization of cell layer subsequent to removal of medium containing labeled low density lipoprotein and 5 washes with buffer 191, corrected for non-specific binding assessed by measurement of trypsin-released counts after addition of a lOO-fold excess unlabeled low density lipoprotein to other dishes. Protein degradation represents water-soluble radioactivity appearing in medium during incubation with cells, corrected for free iodide by chloroform extraction of the trichloroacetic acid-soluble fraction of the medium after mixture with KI and Hz02 191. Negligible degradation occurred at 37’C over 24 h in the absence of cells. Each point represents the mean of measurements from replicate culture dishes. Each dish contained 1.04 X lo6 cells, grown for 28 days after passage.
traction of counts released in other dishes which had been incubated with more than a 100.fold excess of unlabeled low density lipoprotein (an average of 11% of low density lipoprotein remained bound; n = 4). Specific protein binding was decreased 55% by incubation of cells at 4°C. Trypsin-releasable low density lipoprotein accounted for half the protein radioactivity associated with the cell layer during the first 4 h of incubation (Fig. 2). Subsequently, cell uptake progressed more rapidly. Lipoprotein degradation, assessed by appearance of trichloroacetic acidsoluble, non-iodide radioactivity in the incubation medium, increased in parallel with uptake of lipoprotein after an initial delay of 2 h (Fig. 2). This phenomenon has also been observed with cultured skin fibroblasts [16] and suggests that specific binding of lipoprotein to cell surface receptors, presumably via specific apolipoprotein polypeptides, is a critical first step prior to subsequent cellular uptake and degradation of lipoprotein. These results are of particular interest since rat and man differ markedly in their propensity to develop atherosclerosis with aging. The apparent specificity of cultured human arterial smooth muscle cells for binding and uptake of large, lipid-rich lipoproteins is consistent with the possibility that these cells, which play a central role in atherogenesis, accumu-
202
late lipid as a result of specific lipoprotein binding and subsequent they are exposed and in contact with lipoproteins in vivo.
uptake
when
Acknowledgements This study was supported by grants from the National Institutes of Health and the American Heart Association. We thank M. Stewart and V. Cabana for their expert help. References 1 Parker, F. (1960) Am. J. Pathol. 36, 19-53 2 Geer, J.C., McGill, Jr. H.C. and Strong, J.P. (1961) Am. J. Pathol. 38, 263-287 3 Haust, M.D. and More. R.H. (1963) in Evolution of the Atherosclerotic Plaque (Jones, R.J., ed.), pp. 51-63, University of Chicago Press. Chicago 4 Geer, J.C. and Haust, M.D. (1972) Smooth Muscle Cells in Atherosclerosis, S. Karger. Basel. 5 Benditt, E.P. and Benditt, J.M. (1973) Proc. Natl. Acad. Sci. U.S. 70. 1753-1756 6 Ross, R. (1971) J. Cell Biol. 50. 172-186 7 Ross, R. and Glomset, J. (1973) Science 180, 1332-1338 8 Bierman, E.L., Eisenberg, S.. Stein, 0. and Stein, Y. (1973) Biochim. Biophys. Acta 329, 163-169 9 Bierman, E.L., Stein, 0. and Stein, Y. (1974) Circ. Res. 35. 136-150 10 Smith, E.B. (1970) in The Artery and the Process of Arteriosclerosis: Patbogenesis. (Wolf, S.. ed.). pp. 81-120, Plenum Press, New York 11 Bilheimer. D.W.. Eisenberg, S. and Levy, R.I. (1972) B&him. Biophys. Acta 260, 212-221 12 Koga, S., Honvitz, A.L. and Scanu. A.M. (1969) J. Lipid Res. 10, 577-588 13 Scanu, A.M. (1972) Biochim. Biophys. Acta 265, 471-508 14 Brown. M.S. and Goldstein, J.L. (1974) Proc. Natl. Acad. Sci. U.S. 71, 788-792 15 Brown, M.S. and Goldstein, J.L. (1974) Science 185, 6143 16 Goldstein, J.L. and Brown, M.S. (1974) J. Biol. Chem. 249, 5153-5162 17 Werb, 2. and Cohn, Z.A. (1971) J. Exp. Med. 134. 1570-1590
