Proc. Nati. Acad. Sci. USA Vol. 75, No. 1, pp. 356-60, January 1978
Cell Biology
Role of contact inhibition in thexregulation of receptor-mediated uptake of low density lipoprotein in cultured vascular endothelial cells (membrane fluidity/vascular endothelium/wounding) I. VLODAVSKY*, P. E. FIELDINGt, C. J. FIELDINGtt, AND D. GOSPODAROWICZ*§ * Cancer Research Institute, tCardiovascular Research Institute, and § Departments of Medicine and t Physiology, University of California, San Francisco,
California 94143
Communicated by Abraham White, November 4, 1977
ABSTRACT Bovine vascular endothelial cells during logarithmic growth bind, internalize, and degrade low density lipoprotein (LDL) via a receptor-mediated pathway. However, contact-inhibited (confluent) monolayers bind but do not internalize LDL. This is in contrast to aortic smooth muscle cells or endothelial cells that have lost the property of contact inhibition. These cells internalize and degrade LDL at both high and low cell densities. The LDL receptors of smooth muscle and sparse endothelial cells down-regulate in response to LDL. In contrast, normal endothelial cells at confluency show little response. When contact inhibition in endothelial monolayers was locally released by wounding, and LDL was present, only cells released from contact inhibition accumulated LDL cholesterol. In smooth muscle cells under the same conditions, the entire culture interiorized lipid. It thus appears that in endothelial cells, unlike smooth muscle cells, contact inhibition is the major factor regulating cellular uptake of LDL cholesteryl ester. Reversal of contact inhibition by wounding provides a mechanism by which the endothelium could be the primary initiator of the atherosclerotic plaque. A number of recent'studies have identified a pathway in several cultured cell lines by which low density lipoprotein (LDL) is taken up into the cells via a specific receptor (1-3y. After internalization, the lipoprotein is catabolized in the lysosomes. The lipoprotein apoprotein is degraded to low molecular weight material and the cholesteryl ester content is hydrolyzed by lysosomal cholesterol esterase and re-esterified by the acylCoA:cholesterol acyl-transferase pathway (1). Most of the studies reported so far have been carried out with cultured fibroblasts and smooth muscle cells. These cells are exposed under physiological conditions to only low concentrations of plasma lipoproteins such as are present in the intercellular lymph (4). However, because the vascular endothelium meets the full circulating concentrations of lipoproteins, it is of interest to determine to what extent endothelial cells regulate cholesterol uptake according to the fibroblast model. The vascular endothelium in vivo reveals a characteristic appearance as a single layer of highly flattened, contact-inhibited cells. The use of the peptide fibroblast growth factor (FGF) (5) has now permitted the long-term maintenance-of pure endothelial clones from a variety of sources, including the coronary bed and aortic arch of several~species (6). These cells retain the in vivo properties of endothelium and have been used in the present research to define the significance of the LDL receptor-mediated pathway in vascular endothelium.
MATERIALS AND METHODS Preparation of LDL and Lipoprotein-Deficient Serum. Human and bovine LDL (1.019 < density < 1.063 g/cm3), human very low density lipoprotein (VLDL) (density < 1.006 g/cm3), human high density lipoprotein (HDL) (1.063 < density < 1.21 g/cm3), and the homologous lipoprotein-deficient sera (LPDS) (density > 1.21 g/cm3) were obtained from plasma by differential ultracentrifugal flotation (7). LDL was iodinated with iodine monochloride (8). After labeling, unbound 125I was removed by column chromatography on Sephadex G-50 followed by extensive dialysis. Specific activity of'the labeled LDL was 165-508 cpm-/ng of protein. Lipidbdund radioactivity was 5-7%. Culture of Endothelial and Smooth Muscle Cells. Bovine eridothelial and smooth muscle cells were obtained from the fetal heart or the aortic arches of adult animals and were maintained and cloned as previously described (5). Cells were routinely cultured at 370 (10% CO2 atmosphere) on Falcon dishes in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (Gibco, Grand Island, NY). Endothelial cells were passaged weekly at 1:64 split ratio and FGF (100 ng/ihil) was added every other day until the cells were nearly confluent. The presence of antihemophilic factor antigen and a strict contact-inhibited morphology at confluency have been a constant feature of all subcultures of the vascular endothelial cells (9). Cultures that were no longer maintained in the presence of FGF lost, within two passages, their contact inhibition property and are referred to as "non-contact-inhibited" endothelial cells. For the binding experiments, monolayers of cells from stock plates were dissociated with 0.05% trypsin/0.02% EDTA solution and 2 X 104 cells were seeded into each 35-mm dish. Cells from subconfluent cultures or 6-8 days after reaching confluency were washed and 2.5 ml of fresh medium containing LPDS (4 mg/ml) was added to each plate. After an additional 32-48 hr the monolayers were washed and 1 ml of the same medium containing the indicated concentration of 125I-labeled LDL (125I-LDL) was added to initiate the experiment. Determination of LDL Binding, Interiorization, and Degradation. After the appropriate incubation period at 370, the medium was removed and treated with an equal volume of cold 25% wt/vol trichloroacetic acid. After centrifugation, the supernatant was extracted with hydrogen peroxide and chloroform to correct for radioiodide (10), and the radioactivity
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Abbreviations: LDL, low density lipoprotein; FGF, fibroblast growth factor; LPDS, lipoprotein-deficient serum.
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in a portion of the aqueous phase was measured to determine the amount of '25I-labeled degradation products released to the medium. Plates without cells were incubated under the same conditions to control for spontaneous breakdown of the labeled LDL. To determine the amount of LDL bound to the cell surface and that which had entered the cells, each monolayer was chilled to 40 and washed 12 times at 40 with phosphate-buffered saline containing 0.2% bovine serum albumin. One milliliter of solution containing 50 mM NaCl, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) and sodium heparin (Sigma) at 10 mg/ml was then added, and Ihe-plates were incubated at 40 for 1 hr to release the 125I-L9;L bound to the cell surface (11). Each monolayer was then washed six times with phosphate-buffered saline containing albumin, dissolved in 0.1 M NaOH, and the radioactivity was measured to determine the amount of 125I-LDL. that had entered the cell and hence was not accessible for heparin release. A sample was also taken for determination of cell protein (12). The protein content of endothelial cells was 360 Ag/106 cells, and that of smooth muscle cells was 540( ,g/106 cells. Each value for LDL binding and catabolism represents the average of duplicate determinations, and the data are expressed as specific 125I-LDL binding, interiorization, and .degradation of the lipoprotein. Duplicate determinations varied by less than 10% of the mean values. The nonspecific value at each concentration of LDL, which has been subtracted, was determined by incubating the monolayer with 1251-LDL in the presence of unlabeled LDL protein at 350-700 ,ug/ml. Values for nonspecific binding ranged from 10% at nonsaturating concentration of LDL (2.5-25 ,ug/ml) up to 40% at saturation (100 ,ug/ml). Nonspecific uptake of LDL at these concentrations ranged from 3.to 20% of the cell-associated radioactivity. RESULTS in Subconfluent Cultures. Sparse, LDL Catabolism of subconfluent cultures of fetal bovine heart endothelial cells show binding, interiorization, and degradation of LDL in a manner similar to that described for fibroblasts and smooth muscle cells (1-3). The presence of specific, high-affinity surface receptor sites was indicated by the observations that binding was saturable (half-maximal at 15 ,ug of LDL protein/ml) (Fig. 1A), and showed the expected competition with unlabeled LDL. The amount of LDL bound reached a plateau within 1 hr (Fig. 1B). There was no significant displacement of the bound 125I-LDL by high density lipoprotein; however, a 2-fold excess of very low density lipoprotein competed with the labeled LDL as efficiently as native LDL. Subsequent to
binding, the bound material was internalized and degraded to acid-soluble products that were released into the culture medium (Fig. 1). The degradation of 125I-LDL was inhibited by excess of unlabeled LDL and reached a maximal rate at the same LDL concentration as that required to saturate the high-affinity receptor binding sites (Fig. 1A). It seems, therefore, that the high-affinity uptake and degradation of LDL by endothelial cells is, as in other cell systems, dependent-upon the prior binding of LDL to its specific cell surface receptor site. Similar results have been obtained in the present research with adult bovine aortic endothelial and smooth muscle cells. Under equilibrium conditions, sparse and subconfluent endothelial cells showed, per mg of cell protein, a 2- to 3-fold higher LDL binding and uptake than did smooth muscle cells, but values per cell were comparable. When binding was carried out at 40, 70-80% of the cell-associated 125I-LDL radioactivity was released into the medium by heparin. At 370 the total cellular uptake of 125I-LDL was 3- to 5-fold greater than at 40, but only 10-20% of the total radioactivity was released by heparin. These results indicate that at 40 there was little or no interiorization of LDL and that only LDL particles bound to the cell surface were accessible to heparin release. Similar results were obtained with smooth muscle cells. To demonstrate that the results obtained with human LDL were quantitatively similar for the homologous bovine LDL, experiments were carried out with LDL from each species and 2 1250
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358
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FIG. 3. Binding and uptake of 125I-LDL by sparse and confluent cultures of endothelial and smooth muscle cells. (A) Adult bovine aortic endothelial cells. (B) Adult bovine aortic endothelial cells that have lost contact inhibition of growth. (This clone was derived from cells that were maintained in the absence of FGF for four passages). (C) Adult bovine aortic smooth muscle cells. (D, E, and F) Confluent cultures at the time of the experiments. (Phase contrast, X96.) The endothelial and smooth muscle cells were plated at an initial density of 2 X 104 cells per 35-mm dish and tested before (A, ,) and 8 days after (@, 0) reaching confluency. Similar results were obtained with cells that were seeded at high and low density to obtain confluent and sparse cultures on the day of the experiment. To obtain a highly confluent and contact-inhibited monolayer of endothelial cells, cells were seeded at low density and FGF was added every other day until the cells were nearly confluent (5-7 days). The cultures were then maintained for an additional 8-10 days to assume the distinct appearance of contact-inhibited cells (D). The growth medium was replaced with fresh medium containing LPDS (4 mg/ml) 32-48 hr before the experiments. Monolayers were then washed and incubated (3 hr, 370) with various concentrations of 125I-LDL (412 cpm/ng) in 1 ml of the same medium. Specific binding (A and 0, accessible for heparin release) and internalization (A and 0, heparin-resistant) of 125I-LDL were determined. (D) Adult bovine endothelial cells, highly flattened and contact inhibited. (E) Adult bovine aortic endothelial cells, a clone that no longer shows contact inhibition of growth. These are elongated cells that can grow on top of each other; even at confluency, spaces are available between the cells. (F) Bovine aortic smooth muscle cells. Cells grow on top of each other as a multiple layer and in various directions.
its respective LPDS. These lipoproteins showed similar binding properties (Fig. 2), and competed with each other.
Effect of Cell Density on Binding and Uptake of LDL. Contact-inhibited endothelium forms a monolayer of closely apposed, flattened cells as shown in Fig. 3D. In contrast, endothelial cells that have been cultured in the absence of FGF and have lost the property of contact inhibition, or vascular smooth muscle cells, form multilayers at high cell densities (Fig. SE and F). By comparison with subconfluent vascular endothelial cells, a contact-inhibited monolayer bound 2- to 3-fold less LDL and interiorized hardly at all (Fig. 3A); a 4- to 5-fold decrease in the ability of the cells to internalize LDL was already seen 24-48 hr after confluency had been reached. As with the subconfluent cultures, the LDL that bound to confluent cells was released by heparin and only the nonspecific binding was detected in the presence of an excess of unlabeled LDL. In subconfluent and confluent smooth muscle cells and in the non-contact-inhibited endothelium there was little difference in the degree of binding and interiorization of LDL at a low or high cell density (Fig. 3B and C). Effects of Release from Contact Inhibition. The cells of confluent endothelium in a medium containing LPDS were disrupted by EDTA and plated in the same medium at one-fifth
the density. An 8- to 10-fold increase in the extent of 125I-LDL internalization and degradation was obtained at 24 hr after seeding although the cells did not proliferate in LPDS-containing medium (Fig. 4). The efficiency of plating was similar to the 90% regularly obtained. The recovery of the ability of the cells to internalize bound LDL after dissociation of the confluent monolayer indicates that this function is regulated by cell-cell contacts rather than by inhibition of cell division. To avoid possible artifacts due to EDTA dissociation, the confluent monolayer was wounded locally by removing about 10% of the cells with a rubber policeman. Upon incubation in a medium containing LPDS for 24 hr at 370, cells migrated into the wound, and there was a 2- to 2.5-fold increase in the total uptake of 125I-LDL, although the rest of the cells remained highly contact inhibited. To visualize the region of LDL uptake, wounded monolayers were incubated with LDL (300 ,g/ml, added every day) and then stained with Oil Red 0. Under these conditions there was a massive uptake of lipid in the periphery of the wound area (Fig. 5B), but there was no significant staining in the rest of the monolayer (Fig. 5A). In similar experiments with cultured aortic smooth muscle cells, there was a uniform uptake of-lipoprotein lipid over the entire culture (Fig. 5C and D).
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Muscle Cells. In cultured fibroblasts and smooth muscle cells the number of LDL receptors is diminished when the cells are incubated with LDL-containing medium over a period of days (13). It has been proposed that this feedback regulation is in response to increased levels of cellular cholesterol; a similar type of regulation has been reported for a variety of surface receptors (14). Confluent and sparse cultures of endothelial and smooth muscle cells were incubated for 24-72 hr with 50 ,ug of unlabeled LDL protein per ml. The cells were washed and the LDL binding capacity was determined by incubation with various concentrations of 125I-LDL. In both confluent and nonconfluent smooth muscle cells, a 24-hr preincubation with LDL produced a 70-80% decrease in 125I-LDL binding and uptake (Table 1). On the other hand, while subconfluent endothelium showed a similar extent of down-regulation, confluent endothelium showed only a small (less than 30%) inhibition even after preincubation with LDL for 72 hr. In all cases the binding caTable 1. Feedback regulation of the LDL receptor sites in sparse and confluent cultures of endothelial and smooth muscle cells 125I-LDL 125I-LDL binding internalization +LDL -LDL -LDL +LDL Cells Smooth muscle 100.6 334.9 55.4 6.0 Sparse 80.6 7.8 268.3 43.2 Confluent Endothelium 142.0 11.1 682.2 66.4 Sparse 64.7 42.8 21.7 27.1 Confluent Confluent and sparse cultures of adult bovine aortic endothelial and smooth muscle cells were obtained as described in the legend to Fig. 3. The medium was replaced with fresh medium containing LPDS, and the cells were incubated for 24 hr and then with or without unlabeled human LDL (50 ,ug of protein per ml) for a further 24 hr. Each plate was washed free of unbound LDL, incubated (1 hr, 370) in LPDS-containing medium, and washed again to allow complete interiorization of the receptor-bound LDL. Cells were then incubated (3 hr, 370) with 1251-LDL (15 gg/ml) to determine the amount of 125I-LDL specifically bound to the cell surface and hence accessible for heparin release, and the amount of 125I-LDL internalized by the cells (heparin-resistant). Data are expressed as ng of LDL per mg of cell protein.
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FIG. 5. Confluent monolayers of vascular endothelial and smooth muscle cells exposed to high levels of LDL. Confluent monolayers of adult bovine aortic endothelial or smooth muscle cells were wounded and exposed for 5 days to LDL (300 gg of protein per ml, added each day). The plates were then fixed and stained with Oil Red 0 and hematoxylin. (X 210.) (A) Endothelial cells, an area away from the wound. Contact-inhibited cells do not accumulate stained lipid droplets (lack of black dots). (B) Endothelial cells in the wound area. Cells migrating into the wound show large numbers of Oil Red Opositive inclusions. (C) Smooth muscle cells, an area away from the wound. Cells are heavily labeled with stained particles reflecting the accumulation of lipid. (D) Smooth muscle cells in the wound arealarge numbers of stained particles in cells migrating into the wound.
pacity was restored by a 24-hr incubation of the cells in LPDS. DISCUSSION Two mechanisms have been proposed for the regulation of the intracellular level of cholesterol. These involve (a) regulation via a receptor-mediated inhibition of the enzymes of cellular cholesterol synthesis and (b) down-regulation of the number of receptor sites through which the uptake of plasma lipoprotein is mediated. It is of obvious interest to determine which of these pathways is significant in vascular endothelium, particularly because these cells provide the primary barrier against high circulating levels of plasma LDL. The data obtained in this study indicate that the primary regulation of LDL uptake in confluent aortic endothelium is via neither of these pathways, but is through inhibition of uptake brought about by the formation of cell-cell contacts, without major changes in the number of receptor sites. It has been-previously shown that confluent 3T3 cells transformed by simian virus 40 bind and internalize Ricinus communes agglutinin II molecules, whereas
360
Cell Biology: Vlodavsky et al.
contact-inhibited, normal 3T3 cells do not internalize the bound toxin (15). In rapidly growing, sparse cultures of endothelial cells we have demonstrated an active LDL-receptor pathway whose capacity and properties are similar to those found in this study for smooth muscle cells. Such results have been reported previously for smooth muscle cells and fibroblasts (1-3). A similar pathway has also been reported for subconfluent cultures of umbilical vein endothelial cells (16). However, in vivo the endothelium neither grows rapidly nor has the morphology of subconfluent endothelial cells. Studies in situ indicate that the endothelial cells of the major vessels are highly contact inhibited and that cell division takes place slowly if at all, except in response to endothelial injury (17). It is therefore apparent that the natural regulatory mechanisms of endothelial cells are only expressed in confluent monolayers. A possible exception to this could be the capillary endothelium from liver, kidney, and endocrine organs, which may not show strict contact inhibition, because this type of endothelium is fenestrated (18). We have now shown that the LDL receptor-mediated pathway has little or no physiological significance in contact-inhibited, normal endothelium but is fully retained in altered endothelial cells, which no longer show contact inhibition even at high cell density. In fact, the impossibility of obtaining a perfect cell monolayer in tissue culture, due to the surface heterogeneity of the plastic petri dishes (19), may account for the very low residual level of LDL uptake in the contact-inhibited cells. In contrast, when cells were released from contact inhibition, as in endothelial injury, there was a greatly increased uptake of LDL into the cells at the wound periphery but not in the cells that remained contact inhibited. As a result of such an injury, the subendothelial smooth muscle cells would be presented with the full plasma concentration of LDL, which could lead to an uncontrolled uptake of lipid. Thus, the wound area would accumulate cholesteryl ester-laden endothelial and smooth muscle cells, which could give rise to foam cells and lipid deposits such as occur in the atherosclerotic lesion. Another example that demonstrates how the morphology of a cell layer can dictate a metabolic function is the inhibition of phagocytosis in cultured epithelial sheets due to the formation of cell-cell contacts (20). The barrier property of endothelium of the large vessels, which at confluency is reflected by the lack of uptake of LDL, may result from changes in the cell surface membrane that impede the process of adsorptive endocytosis. This would be the case if LDL receptors are randomly distributed over the surface of the cell, but, on binding the lipoprotein particle, they are directed to a specific region for internalization. This would require a long-range lateral movement of the receptor-LDL complex in the membrane plane. Such a phenomenon has been described for the migration of concanavalin A binding sites into phagocytic vesicles (21), and for the redistribution of various cell surface receptors to form caps prior to their endocytosis (22). This type of mechanism has been demonstrated with concanavalin A and sparse endothelial cultures (23). Recent research has demonstrated that fibroblasts internalize by direct endocytosis or via an induced receptor clustering rather than by surface capping (24, 15). This is in agreement with experiments in human fibroblasts, showing that ferritin-conjugated LDL binds to receptors on coated regions of the cell membrane (25), which are then invaginated to form coated vesicles. Such research-has indicated that the lipoprotein-specific receptors are located in the coated regions and do not move there under
Proc. Nati. Acad. Sci. USA 75 (1978)
the stimulus of LDL binding. Therefore, in endothelial cells, unlike fibroblasts, cell-cell contacts and the massive accumulation of fibronectin at confluency (23) could restrict receptor mobility and thus prevent the entire internalization process. Consequently, any disruption of the endothelial monolayer would release the cells from contact inhibition, increase the fluidity of the membrane, and initiate a chain reaction resulting in the eventual accumulation of LDL lipid in the endothelial
layer. Although the above conclusions are the result of observations made in vitro, they provide a basis for an in ioo study and for a reevaluation of the role of the endothelial surface in the early development of the atherosclerotic plaque. This work was supported by Grants HL 20197 and HL 14237 from the National Institutes of Health. I.V. is a recipient of the Chaim Weizmann Research Training Fellowship. 1.
Goldstein, J. L. & Brown, M. S. (1977) Annu. Rev. Biochem. 46, Weinstein, D. B., Carew, T. E. & Steinberg, D. (1976) Biochim. Biophys. Acta 424,404-421. Stein, 0. & Stein, Y. (1975) Biochim. Biophys. Acta 398, 377384. Reichl, D., Simons, L. A., Myant, N. B., Pflug, J. J. & Mills, G. L. (1973) Clin. Sci. Mol. Med. 45,313-329. Gospodarowicz, D., Moran, J., Braun, D. & Birdwell, C. (1976) Proc. Natl. Acad. Scd. USA 73,4120-4124. Gospodarowicz, D., Greenburg, G., Bialecki, H. & Zetter, B., (1977) in Cellular Control of Proliferation, ed. Freed, C. In Vitro 13, in press. Havel, R. J., Eder, H. S. & Bragdon, J. A. (1955) J. Clin. Invest. 34, 1345-1353. MacFarlane, A. S. (1958) Nature 182, 5S. Gospodarowicz, D., Moran, J. & Braun, D. (1977) J. Cell Physiol. 91,377-386. Bierman, E. L., Stein, 0. & Stein, Y. (1974) Circ. Res. 35, 136150. Goldstein, J. L., Basu, S. K., Brunschede, G. Y. & Brown, M. S. (1976) Cell 7,85-95. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. Goldstein, J. L., Anderson, R. G., Buja, L. M., Basu, S. K. & Brown, M. S. (1977) J. Clin. Invest. 59, 1196-1202. Kahn, C. R. (1976) J. Cell Biol. 70, 261-286. Nicolson, G. L., Lacorbiere, M. & Hunter, T. R. (1975) Cancer Res. 35, 144-155. Stein, 0. & Stein, Y. (1976) Biochim. Biophys. Acta 431, 363368. Wright, H. P. (1972) Atherosclerosis 15,93-100. Greep, R. 0. (1966) Histology (McGraw Hill, New York), pp. 285-289. Schubert, D., Harris, A. J., Heinemann, S., Kidokoro, Y. & Patrick, J. (1973) in Tissue Culture of the Nervous System, ed. Sato, G. (Plenum Press, New York), p. 58. Vasiliev, J. M., Gelfand, I. M., Domnina, L. V., Zacharova, 0. S. & Ljubimov, A. V. (1975) Proc. Natl. Acad. Scd. USA 72,719722. Oliver, J. M., Ukena, T. E. & Berlin, R. D. (1974) Proc. Natl. Acad. Sci. USA 71, 394-398. Raff, M. C. & De Petris, S. (1973) Fed. Proc. Fed. Am. Soc. Exp. Biol. 32, 48-54. Gospodarowicz, D., Vlodavsky, I. & Fielding, P. E. (1977) in International Congress of Birth Defects 5th, ed. Peereboom, T. (Excerpta Medica, Amsterdam), in press. Storrie, B. & Edelson, P. J. (1977) Cell 11, 707-717. Anderson, R. G. W., Brown, M. S. & Goldstein, J. L. (1977) Cell
897-930. 2. 3. 4.
5. 6. 7. 8. 9.
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13. 14. 15. 16.
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21. 22. 23. 24. 25.
10,351-364.
Cell Biology
Role of contact inhibition in thexregulation of receptor-mediated uptake of low density lipoprotein in cultured vascular endothelial cells (membrane fluidity/vascular endothelium/wounding) I. VLODAVSKY*, P. E. FIELDINGt, C. J. FIELDINGtt, AND D. GOSPODAROWICZ*§ * Cancer Research Institute, tCardiovascular Research Institute, and § Departments of Medicine and t Physiology, University of California, San Francisco,
California 94143
Communicated by Abraham White, November 4, 1977
ABSTRACT Bovine vascular endothelial cells during logarithmic growth bind, internalize, and degrade low density lipoprotein (LDL) via a receptor-mediated pathway. However, contact-inhibited (confluent) monolayers bind but do not internalize LDL. This is in contrast to aortic smooth muscle cells or endothelial cells that have lost the property of contact inhibition. These cells internalize and degrade LDL at both high and low cell densities. The LDL receptors of smooth muscle and sparse endothelial cells down-regulate in response to LDL. In contrast, normal endothelial cells at confluency show little response. When contact inhibition in endothelial monolayers was locally released by wounding, and LDL was present, only cells released from contact inhibition accumulated LDL cholesterol. In smooth muscle cells under the same conditions, the entire culture interiorized lipid. It thus appears that in endothelial cells, unlike smooth muscle cells, contact inhibition is the major factor regulating cellular uptake of LDL cholesteryl ester. Reversal of contact inhibition by wounding provides a mechanism by which the endothelium could be the primary initiator of the atherosclerotic plaque. A number of recent'studies have identified a pathway in several cultured cell lines by which low density lipoprotein (LDL) is taken up into the cells via a specific receptor (1-3y. After internalization, the lipoprotein is catabolized in the lysosomes. The lipoprotein apoprotein is degraded to low molecular weight material and the cholesteryl ester content is hydrolyzed by lysosomal cholesterol esterase and re-esterified by the acylCoA:cholesterol acyl-transferase pathway (1). Most of the studies reported so far have been carried out with cultured fibroblasts and smooth muscle cells. These cells are exposed under physiological conditions to only low concentrations of plasma lipoproteins such as are present in the intercellular lymph (4). However, because the vascular endothelium meets the full circulating concentrations of lipoproteins, it is of interest to determine to what extent endothelial cells regulate cholesterol uptake according to the fibroblast model. The vascular endothelium in vivo reveals a characteristic appearance as a single layer of highly flattened, contact-inhibited cells. The use of the peptide fibroblast growth factor (FGF) (5) has now permitted the long-term maintenance-of pure endothelial clones from a variety of sources, including the coronary bed and aortic arch of several~species (6). These cells retain the in vivo properties of endothelium and have been used in the present research to define the significance of the LDL receptor-mediated pathway in vascular endothelium.
MATERIALS AND METHODS Preparation of LDL and Lipoprotein-Deficient Serum. Human and bovine LDL (1.019 < density < 1.063 g/cm3), human very low density lipoprotein (VLDL) (density < 1.006 g/cm3), human high density lipoprotein (HDL) (1.063 < density < 1.21 g/cm3), and the homologous lipoprotein-deficient sera (LPDS) (density > 1.21 g/cm3) were obtained from plasma by differential ultracentrifugal flotation (7). LDL was iodinated with iodine monochloride (8). After labeling, unbound 125I was removed by column chromatography on Sephadex G-50 followed by extensive dialysis. Specific activity of'the labeled LDL was 165-508 cpm-/ng of protein. Lipidbdund radioactivity was 5-7%. Culture of Endothelial and Smooth Muscle Cells. Bovine eridothelial and smooth muscle cells were obtained from the fetal heart or the aortic arches of adult animals and were maintained and cloned as previously described (5). Cells were routinely cultured at 370 (10% CO2 atmosphere) on Falcon dishes in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (Gibco, Grand Island, NY). Endothelial cells were passaged weekly at 1:64 split ratio and FGF (100 ng/ihil) was added every other day until the cells were nearly confluent. The presence of antihemophilic factor antigen and a strict contact-inhibited morphology at confluency have been a constant feature of all subcultures of the vascular endothelial cells (9). Cultures that were no longer maintained in the presence of FGF lost, within two passages, their contact inhibition property and are referred to as "non-contact-inhibited" endothelial cells. For the binding experiments, monolayers of cells from stock plates were dissociated with 0.05% trypsin/0.02% EDTA solution and 2 X 104 cells were seeded into each 35-mm dish. Cells from subconfluent cultures or 6-8 days after reaching confluency were washed and 2.5 ml of fresh medium containing LPDS (4 mg/ml) was added to each plate. After an additional 32-48 hr the monolayers were washed and 1 ml of the same medium containing the indicated concentration of 125I-labeled LDL (125I-LDL) was added to initiate the experiment. Determination of LDL Binding, Interiorization, and Degradation. After the appropriate incubation period at 370, the medium was removed and treated with an equal volume of cold 25% wt/vol trichloroacetic acid. After centrifugation, the supernatant was extracted with hydrogen peroxide and chloroform to correct for radioiodide (10), and the radioactivity
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Abbreviations: LDL, low density lipoprotein; FGF, fibroblast growth factor; LPDS, lipoprotein-deficient serum.
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FIG. 1. Cell surface binding, internalization, and degradation of 125I-LDL as a function of (A) 1251-LDL concentration and (B) time of incubation with LDL. Subconfluent cultures of fetal bovine heart endothelial cells (FBHE) were incubated for 48 hr in growth medium containing LPDS (4 mg/ml), washed, and incubated for 3 hr (at 370) with various concentrations of human 1251-LDL (165 cpm/ng) (experiment A), or for 10 of 125I-LDL per ml (experiment B). After the appropriate interval, the amounts of radioactive trichloroacetic acid-soluble various times with Jg material released to the medium (A) and heparin-releasable (0) and heparin-resistant (0) 125I-LDL were determined.
in a portion of the aqueous phase was measured to determine the amount of '25I-labeled degradation products released to the medium. Plates without cells were incubated under the same conditions to control for spontaneous breakdown of the labeled LDL. To determine the amount of LDL bound to the cell surface and that which had entered the cells, each monolayer was chilled to 40 and washed 12 times at 40 with phosphate-buffered saline containing 0.2% bovine serum albumin. One milliliter of solution containing 50 mM NaCl, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) and sodium heparin (Sigma) at 10 mg/ml was then added, and Ihe-plates were incubated at 40 for 1 hr to release the 125I-L9;L bound to the cell surface (11). Each monolayer was then washed six times with phosphate-buffered saline containing albumin, dissolved in 0.1 M NaOH, and the radioactivity was measured to determine the amount of 125I-LDL. that had entered the cell and hence was not accessible for heparin release. A sample was also taken for determination of cell protein (12). The protein content of endothelial cells was 360 Ag/106 cells, and that of smooth muscle cells was 540( ,g/106 cells. Each value for LDL binding and catabolism represents the average of duplicate determinations, and the data are expressed as specific 125I-LDL binding, interiorization, and .degradation of the lipoprotein. Duplicate determinations varied by less than 10% of the mean values. The nonspecific value at each concentration of LDL, which has been subtracted, was determined by incubating the monolayer with 1251-LDL in the presence of unlabeled LDL protein at 350-700 ,ug/ml. Values for nonspecific binding ranged from 10% at nonsaturating concentration of LDL (2.5-25 ,ug/ml) up to 40% at saturation (100 ,ug/ml). Nonspecific uptake of LDL at these concentrations ranged from 3.to 20% of the cell-associated radioactivity. RESULTS in Subconfluent Cultures. Sparse, LDL Catabolism of subconfluent cultures of fetal bovine heart endothelial cells show binding, interiorization, and degradation of LDL in a manner similar to that described for fibroblasts and smooth muscle cells (1-3). The presence of specific, high-affinity surface receptor sites was indicated by the observations that binding was saturable (half-maximal at 15 ,ug of LDL protein/ml) (Fig. 1A), and showed the expected competition with unlabeled LDL. The amount of LDL bound reached a plateau within 1 hr (Fig. 1B). There was no significant displacement of the bound 125I-LDL by high density lipoprotein; however, a 2-fold excess of very low density lipoprotein competed with the labeled LDL as efficiently as native LDL. Subsequent to
binding, the bound material was internalized and degraded to acid-soluble products that were released into the culture medium (Fig. 1). The degradation of 125I-LDL was inhibited by excess of unlabeled LDL and reached a maximal rate at the same LDL concentration as that required to saturate the high-affinity receptor binding sites (Fig. 1A). It seems, therefore, that the high-affinity uptake and degradation of LDL by endothelial cells is, as in other cell systems, dependent-upon the prior binding of LDL to its specific cell surface receptor site. Similar results have been obtained in the present research with adult bovine aortic endothelial and smooth muscle cells. Under equilibrium conditions, sparse and subconfluent endothelial cells showed, per mg of cell protein, a 2- to 3-fold higher LDL binding and uptake than did smooth muscle cells, but values per cell were comparable. When binding was carried out at 40, 70-80% of the cell-associated 125I-LDL radioactivity was released into the medium by heparin. At 370 the total cellular uptake of 125I-LDL was 3- to 5-fold greater than at 40, but only 10-20% of the total radioactivity was released by heparin. These results indicate that at 40 there was little or no interiorization of LDL and that only LDL particles bound to the cell surface were accessible to heparin release. Similar results were obtained with smooth muscle cells. To demonstrate that the results obtained with human LDL were quantitatively similar for the homologous bovine LDL, experiments were carried out with LDL from each species and 2 1250
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its respective LPDS. These lipoproteins showed similar binding properties (Fig. 2), and competed with each other.
Effect of Cell Density on Binding and Uptake of LDL. Contact-inhibited endothelium forms a monolayer of closely apposed, flattened cells as shown in Fig. 3D. In contrast, endothelial cells that have been cultured in the absence of FGF and have lost the property of contact inhibition, or vascular smooth muscle cells, form multilayers at high cell densities (Fig. SE and F). By comparison with subconfluent vascular endothelial cells, a contact-inhibited monolayer bound 2- to 3-fold less LDL and interiorized hardly at all (Fig. 3A); a 4- to 5-fold decrease in the ability of the cells to internalize LDL was already seen 24-48 hr after confluency had been reached. As with the subconfluent cultures, the LDL that bound to confluent cells was released by heparin and only the nonspecific binding was detected in the presence of an excess of unlabeled LDL. In subconfluent and confluent smooth muscle cells and in the non-contact-inhibited endothelium there was little difference in the degree of binding and interiorization of LDL at a low or high cell density (Fig. 3B and C). Effects of Release from Contact Inhibition. The cells of confluent endothelium in a medium containing LPDS were disrupted by EDTA and plated in the same medium at one-fifth
the density. An 8- to 10-fold increase in the extent of 125I-LDL internalization and degradation was obtained at 24 hr after seeding although the cells did not proliferate in LPDS-containing medium (Fig. 4). The efficiency of plating was similar to the 90% regularly obtained. The recovery of the ability of the cells to internalize bound LDL after dissociation of the confluent monolayer indicates that this function is regulated by cell-cell contacts rather than by inhibition of cell division. To avoid possible artifacts due to EDTA dissociation, the confluent monolayer was wounded locally by removing about 10% of the cells with a rubber policeman. Upon incubation in a medium containing LPDS for 24 hr at 370, cells migrated into the wound, and there was a 2- to 2.5-fold increase in the total uptake of 125I-LDL, although the rest of the cells remained highly contact inhibited. To visualize the region of LDL uptake, wounded monolayers were incubated with LDL (300 ,g/ml, added every day) and then stained with Oil Red 0. Under these conditions there was a massive uptake of lipid in the periphery of the wound area (Fig. 5B), but there was no significant staining in the rest of the monolayer (Fig. 5A). In similar experiments with cultured aortic smooth muscle cells, there was a uniform uptake of-lipoprotein lipid over the entire culture (Fig. 5C and D).
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Muscle Cells. In cultured fibroblasts and smooth muscle cells the number of LDL receptors is diminished when the cells are incubated with LDL-containing medium over a period of days (13). It has been proposed that this feedback regulation is in response to increased levels of cellular cholesterol; a similar type of regulation has been reported for a variety of surface receptors (14). Confluent and sparse cultures of endothelial and smooth muscle cells were incubated for 24-72 hr with 50 ,ug of unlabeled LDL protein per ml. The cells were washed and the LDL binding capacity was determined by incubation with various concentrations of 125I-LDL. In both confluent and nonconfluent smooth muscle cells, a 24-hr preincubation with LDL produced a 70-80% decrease in 125I-LDL binding and uptake (Table 1). On the other hand, while subconfluent endothelium showed a similar extent of down-regulation, confluent endothelium showed only a small (less than 30%) inhibition even after preincubation with LDL for 72 hr. In all cases the binding caTable 1. Feedback regulation of the LDL receptor sites in sparse and confluent cultures of endothelial and smooth muscle cells 125I-LDL 125I-LDL binding internalization +LDL -LDL -LDL +LDL Cells Smooth muscle 100.6 334.9 55.4 6.0 Sparse 80.6 7.8 268.3 43.2 Confluent Endothelium 142.0 11.1 682.2 66.4 Sparse 64.7 42.8 21.7 27.1 Confluent Confluent and sparse cultures of adult bovine aortic endothelial and smooth muscle cells were obtained as described in the legend to Fig. 3. The medium was replaced with fresh medium containing LPDS, and the cells were incubated for 24 hr and then with or without unlabeled human LDL (50 ,ug of protein per ml) for a further 24 hr. Each plate was washed free of unbound LDL, incubated (1 hr, 370) in LPDS-containing medium, and washed again to allow complete interiorization of the receptor-bound LDL. Cells were then incubated (3 hr, 370) with 1251-LDL (15 gg/ml) to determine the amount of 125I-LDL specifically bound to the cell surface and hence accessible for heparin release, and the amount of 125I-LDL internalized by the cells (heparin-resistant). Data are expressed as ng of LDL per mg of cell protein.
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FIG. 5. Confluent monolayers of vascular endothelial and smooth muscle cells exposed to high levels of LDL. Confluent monolayers of adult bovine aortic endothelial or smooth muscle cells were wounded and exposed for 5 days to LDL (300 gg of protein per ml, added each day). The plates were then fixed and stained with Oil Red 0 and hematoxylin. (X 210.) (A) Endothelial cells, an area away from the wound. Contact-inhibited cells do not accumulate stained lipid droplets (lack of black dots). (B) Endothelial cells in the wound area. Cells migrating into the wound show large numbers of Oil Red Opositive inclusions. (C) Smooth muscle cells, an area away from the wound. Cells are heavily labeled with stained particles reflecting the accumulation of lipid. (D) Smooth muscle cells in the wound arealarge numbers of stained particles in cells migrating into the wound.
pacity was restored by a 24-hr incubation of the cells in LPDS. DISCUSSION Two mechanisms have been proposed for the regulation of the intracellular level of cholesterol. These involve (a) regulation via a receptor-mediated inhibition of the enzymes of cellular cholesterol synthesis and (b) down-regulation of the number of receptor sites through which the uptake of plasma lipoprotein is mediated. It is of obvious interest to determine which of these pathways is significant in vascular endothelium, particularly because these cells provide the primary barrier against high circulating levels of plasma LDL. The data obtained in this study indicate that the primary regulation of LDL uptake in confluent aortic endothelium is via neither of these pathways, but is through inhibition of uptake brought about by the formation of cell-cell contacts, without major changes in the number of receptor sites. It has been-previously shown that confluent 3T3 cells transformed by simian virus 40 bind and internalize Ricinus communes agglutinin II molecules, whereas
360
Cell Biology: Vlodavsky et al.
contact-inhibited, normal 3T3 cells do not internalize the bound toxin (15). In rapidly growing, sparse cultures of endothelial cells we have demonstrated an active LDL-receptor pathway whose capacity and properties are similar to those found in this study for smooth muscle cells. Such results have been reported previously for smooth muscle cells and fibroblasts (1-3). A similar pathway has also been reported for subconfluent cultures of umbilical vein endothelial cells (16). However, in vivo the endothelium neither grows rapidly nor has the morphology of subconfluent endothelial cells. Studies in situ indicate that the endothelial cells of the major vessels are highly contact inhibited and that cell division takes place slowly if at all, except in response to endothelial injury (17). It is therefore apparent that the natural regulatory mechanisms of endothelial cells are only expressed in confluent monolayers. A possible exception to this could be the capillary endothelium from liver, kidney, and endocrine organs, which may not show strict contact inhibition, because this type of endothelium is fenestrated (18). We have now shown that the LDL receptor-mediated pathway has little or no physiological significance in contact-inhibited, normal endothelium but is fully retained in altered endothelial cells, which no longer show contact inhibition even at high cell density. In fact, the impossibility of obtaining a perfect cell monolayer in tissue culture, due to the surface heterogeneity of the plastic petri dishes (19), may account for the very low residual level of LDL uptake in the contact-inhibited cells. In contrast, when cells were released from contact inhibition, as in endothelial injury, there was a greatly increased uptake of LDL into the cells at the wound periphery but not in the cells that remained contact inhibited. As a result of such an injury, the subendothelial smooth muscle cells would be presented with the full plasma concentration of LDL, which could lead to an uncontrolled uptake of lipid. Thus, the wound area would accumulate cholesteryl ester-laden endothelial and smooth muscle cells, which could give rise to foam cells and lipid deposits such as occur in the atherosclerotic lesion. Another example that demonstrates how the morphology of a cell layer can dictate a metabolic function is the inhibition of phagocytosis in cultured epithelial sheets due to the formation of cell-cell contacts (20). The barrier property of endothelium of the large vessels, which at confluency is reflected by the lack of uptake of LDL, may result from changes in the cell surface membrane that impede the process of adsorptive endocytosis. This would be the case if LDL receptors are randomly distributed over the surface of the cell, but, on binding the lipoprotein particle, they are directed to a specific region for internalization. This would require a long-range lateral movement of the receptor-LDL complex in the membrane plane. Such a phenomenon has been described for the migration of concanavalin A binding sites into phagocytic vesicles (21), and for the redistribution of various cell surface receptors to form caps prior to their endocytosis (22). This type of mechanism has been demonstrated with concanavalin A and sparse endothelial cultures (23). Recent research has demonstrated that fibroblasts internalize by direct endocytosis or via an induced receptor clustering rather than by surface capping (24, 15). This is in agreement with experiments in human fibroblasts, showing that ferritin-conjugated LDL binds to receptors on coated regions of the cell membrane (25), which are then invaginated to form coated vesicles. Such research-has indicated that the lipoprotein-specific receptors are located in the coated regions and do not move there under
Proc. Nati. Acad. Sci. USA 75 (1978)
the stimulus of LDL binding. Therefore, in endothelial cells, unlike fibroblasts, cell-cell contacts and the massive accumulation of fibronectin at confluency (23) could restrict receptor mobility and thus prevent the entire internalization process. Consequently, any disruption of the endothelial monolayer would release the cells from contact inhibition, increase the fluidity of the membrane, and initiate a chain reaction resulting in the eventual accumulation of LDL lipid in the endothelial
layer. Although the above conclusions are the result of observations made in vitro, they provide a basis for an in ioo study and for a reevaluation of the role of the endothelial surface in the early development of the atherosclerotic plaque. This work was supported by Grants HL 20197 and HL 14237 from the National Institutes of Health. I.V. is a recipient of the Chaim Weizmann Research Training Fellowship. 1.
Goldstein, J. L. & Brown, M. S. (1977) Annu. Rev. Biochem. 46, Weinstein, D. B., Carew, T. E. & Steinberg, D. (1976) Biochim. Biophys. Acta 424,404-421. Stein, 0. & Stein, Y. (1975) Biochim. Biophys. Acta 398, 377384. Reichl, D., Simons, L. A., Myant, N. B., Pflug, J. J. & Mills, G. L. (1973) Clin. Sci. Mol. Med. 45,313-329. Gospodarowicz, D., Moran, J., Braun, D. & Birdwell, C. (1976) Proc. Natl. Acad. Scd. USA 73,4120-4124. Gospodarowicz, D., Greenburg, G., Bialecki, H. & Zetter, B., (1977) in Cellular Control of Proliferation, ed. Freed, C. In Vitro 13, in press. Havel, R. J., Eder, H. S. & Bragdon, J. A. (1955) J. Clin. Invest. 34, 1345-1353. MacFarlane, A. S. (1958) Nature 182, 5S. Gospodarowicz, D., Moran, J. & Braun, D. (1977) J. Cell Physiol. 91,377-386. Bierman, E. L., Stein, 0. & Stein, Y. (1974) Circ. Res. 35, 136150. Goldstein, J. L., Basu, S. K., Brunschede, G. Y. & Brown, M. S. (1976) Cell 7,85-95. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. Goldstein, J. L., Anderson, R. G., Buja, L. M., Basu, S. K. & Brown, M. S. (1977) J. Clin. Invest. 59, 1196-1202. Kahn, C. R. (1976) J. Cell Biol. 70, 261-286. Nicolson, G. L., Lacorbiere, M. & Hunter, T. R. (1975) Cancer Res. 35, 144-155. Stein, 0. & Stein, Y. (1976) Biochim. Biophys. Acta 431, 363368. Wright, H. P. (1972) Atherosclerosis 15,93-100. Greep, R. 0. (1966) Histology (McGraw Hill, New York), pp. 285-289. Schubert, D., Harris, A. J., Heinemann, S., Kidokoro, Y. & Patrick, J. (1973) in Tissue Culture of the Nervous System, ed. Sato, G. (Plenum Press, New York), p. 58. Vasiliev, J. M., Gelfand, I. M., Domnina, L. V., Zacharova, 0. S. & Ljubimov, A. V. (1975) Proc. Natl. Acad. Scd. USA 72,719722. Oliver, J. M., Ukena, T. E. & Berlin, R. D. (1974) Proc. Natl. Acad. Sci. USA 71, 394-398. Raff, M. C. & De Petris, S. (1973) Fed. Proc. Fed. Am. Soc. Exp. Biol. 32, 48-54. Gospodarowicz, D., Vlodavsky, I. & Fielding, P. E. (1977) in International Congress of Birth Defects 5th, ed. Peereboom, T. (Excerpta Medica, Amsterdam), in press. Storrie, B. & Edelson, P. J. (1977) Cell 11, 707-717. Anderson, R. G. W., Brown, M. S. & Goldstein, J. L. (1977) Cell
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