Biochimica et Biophysica Acta, 1091 (1991) 63-67 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 016%4889/91/$03.50 ADONIS 016748899100057E
63
BBAMCR 12837
Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein N o r i a k i K u m e 1,,, H i d e n o r i A r a i 1, C h u i c h i K a w a i 1 a n d T o r u K i t a 2 Third Division, 1 Department of Internal Medicine and 2 Department of Geriatric Medicine, Faculty of Medicine, Kyoto University, Kyoto (Japan)
(Received 5 February 1990) (Revised manuscript received 21 August 1990)
Key words: Oxidized low-density lipoprotein; Acetylated Pow-density lipoprotein receptor; Atherosclerosis; (Human endothelial cell)
We examined the upt.ake pathway of acetylated low-density lipoprotein and oxidafively modified LDL (oxidized LDL) in human umbilical veto endothelial cells in culture. Proteolytic degradation of 12Sl-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent arid showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 12sI-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of t2Sl-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized L D L On the contrary, they may have additional receptors, as we previously indicated on moust~ macrophages, which recognize acetylated LDL, but not oxidized LDL.
Introduction
Several lines of evidence suggest that oxidative modification of low-density lipoprotein (LDL) can occur in vivo [1-5]. Oxidized LDL (Ox-LDL) is taken up by macrophages and changes them into foam cells which are characteristic in the early lesion of atheroselerosis. We have recently shown that mouse peritoneal macrophages have at least three different receptors for Ox-
Abbreviations: HEC, human endothelial cells; LDL, low-density lipoprotein; Ac-LDL, acetylated low-density lipoprotein; Ox-LDL, oxidized low-density lipoprotein; HDL, high-density iipoprotein; MDA, malondialdehyde; ECGS, endothelial cell growth supplement; LPDS, lipoprotein deficient serum; PBS, phosphate-buffered saline; TCA, trichloroacetic acid; TBARS, thiobarbituric acid-reactive substances. Correspondence (* present address): N. Kume, Vascular Research Division, Department of Pathology, Brigham and Women's Hospital, Harvard Medical Scool, 75 Francis Street, Boston, MA 02115, U.S.A.
LDL and acetylated LDL (Ac-LDL); one is common for both Ac-LDL and Ox-LDL and the others are specific only for either Ac-LDL or Ox-LDL [6]. Sparrow et al. also demonstrated that mouse peritoneal macrophages have another receptor which recognizes Ox-LDL but not Ac-LDL in addition to a receptor common for both of the two forms of modified LDL [7]. On the other hand, vascular endothelial ceils were reported to have a receptor for modified LDL such as Ac-LDL and malondialdehyde (MDA)-conjugated LDL [8-111. In the present study, we investigated the uptake pathway of Ox-LDL, which is thought to be a form of modified LDL existing in vivo, by cultured human endothelial cells (HEC).
Materials and Methods Materiais, Medium 199 (Cat. No. M0393), heparin (Cat. No. H3125) and endothelial cell growth supplement (ECGS) (Cat. No. E2759) were purchased from Sigma. Fetal bovine serum was obtained from Hyclone
64
(Lot No. 1111797). Penicillin G and streptomycin were from M.A. Bioproducts. Sodium [12~I]iodide was purchased from New England Nuclear (Code No. NEZ033A). All other chemicals were of reagent grade. Cells. HEC were harvested from human umbilical veins by collagenase digestion as previously described [12,13]. Harvested cells were suspended in medium 199 with 15~ fetal bovine serum, 90/~g/ml of heparin, 30 /~g/ml of ECGS, 100 units/ml of penicillin (3 and 100 /~g/rnl of streptomycin, seeded in a gelatin-coated flask and cultured in humidified air containing 5% CO2 at 37°C. We identified these cells as endothelial cells by their typical cobblestone morphology at confluence and their production of Von Willebrand factor. The cells were passaged onto gelatin-coated plastic petri dishes and used as first-passaged confluent monolayers in the following experiments. Lipoproteins. Lipoproteins and lipoprotein deficient serum (LPDS) were isolated from human plasma by sequential ultracentrifugation. Oxidative modification was performed by incubating LDL with phosphatebuffered saline (PBS) supplemented with 5/~M CuSO4 at 370C without cells for 24 h and then dialyzing it against excess volume of PBS without cupric ion at 4°C for at least 24 h. Acetylation of LDL was carried out by acetic anhydride. Radioiodination of lipoproteins were performed by iodine monochloride method [14]. The specific radioacti~,ity of 12SI-labeled lipoproteins were around 200 cpm/ng protein.
-
A
B
C
origin
D
Fig. 1. Agarose gel electrophoresis of native LDL (lane B), oxidized LDL (lane C), acetylated LDL (lane D) and HDL (lane A). Lipoproreins (50-100 ~g protein) were applied on 0.5~ agarose gel electrophoresis.
Lowry et al. using bovine serum albumin as standard [16]. Agarose gel electrophoresis was performed by the previously described method [15]. tO '-. D
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Assays of proteolytic degradation and ceU-association of lipoproteins. Proteolytic degradation and cell-associa-
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tion of nSI-labeled lipoproteins were performed as previously described [14]. Briefly, confluent monolayers were washed with PBS, fed with fresh medium 199 with 10~ LPDS and the indicated amount of ~25I-labeled lipoproteins, and incubated at 37°C in a CO2 incubator. At the end of the incubation period, culture medium was collected and the radioactivity in the trichloroacetic acid (TCA)-soluble and non-iodine fraction was counted as proteolytic degradation. On the other hand, the cells were immediately placed at 4°C, washed with buffer A (150 mM NaC1, 50 mM Tris-HCl (pH 7.4) and 2 mg/rnl bovine serum albumin) twice and buffer B (150 mM NaCI, 50 mM Tris-HCl (pH 7.4)) once, dissolved in 0.2 M N aOH and then counted as cell-association (binding plus internalization). Specific degradation or cell-association was calculated by subtracting the amount of ~25I-labeled lipoprotein degraded or cell-associated in the presence of more than 20-fold excess amount of the unlabeled lipoprotein from that degraded in the absence of the unlabeled lipoprotein. Other assays. Thiobarbituric acid-reactive substances (TBARS) were measured as previously described [15]. Protein concentration was determined by the method of
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Fig. 2. Time-course of the specific degradation of t25I-labeled acetylated LDL (upper panel) or oxidized LDL (lower panel). 125[. labeled ace~ylated LDL (25/~g protein/ml) or oxidized LDL (5 pg protein/ml) were added to medium 199 with 10~ LPDS and incubated with the confluent monolayers of endothelial cells at 37°C in a CO 2 incubator for the indicated periods. Then the culture medium were collected and the TCA-soluble fraction were counted as the amount of the degraded ]25I-labeled iipoproteins as described in Materials and Methods. Each point indicates the mean of duplicate determinations.
65 C 0
Results and Discussion
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Oxidized LDL we prepared contained lipid peroxides as much as around 10 nmol MDA equivalent/mg protein in TBARS assay after dialysis against PBS. As we showed previously, Ox-LDL we used was negatively charged almost at the same degree as Ac~LDL, as shown by agarose gel electrophoresis (Fig. 1). Ox-LDL is reported to be cytotoxic to proliferating endothelial cells or fibroblasts in culture [17]. We used confluent monolayers and did not detect any morphological changes or loss of the cellular protein content under our experimental conditions. When HEC were incubated with ]25I-labeled Ac-LDL or Ox-LDL, degraded materials of ~25I-labeled lipoproteins accumulated in the culture medium linearly for up to 24 h (Fig. 2). Dose-response relationship of the specific degradation of ~251labeled Ac-LDL or Ox-LDL showed saturation kinetics and it suggested their uptake is receptor-mediated (Fig. 3). The amount of the degraded Ac-LDL or Ox-LDL by
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~Jg/ml ~=Sl-Upoproteln Fig. 4. Dose-response relationship of the specific cell-association of 12~l-labeled acetylated LDL (upper panel) or oxidized LDL (lower panel). Endothelial monolayers were incubated with the indicated amount of 12~l-labeled acetylated or oxidized LDL in medium 199 with 10% LPDS for 6 h. The amount of radioligands specifically associated to the cells were measured as described in Materials and Methods. Each point indicates the mean of duplicate determinations.
50
100
150
pg/ml taSI-Lipoprotein Fig. 3. Dose-response relationship of the specific degrad~,~ior, of 125,l-labeled acetylated LDL (upper panel) or oxidized LDL (lower panel). Endothelial monolayers were incubated with the indicated amount of ]251-labeled acetylated or oxidized LDL in medium 199 with 10~ LPDS for 6 h. Degraded materials of 1251-labeled lipoproreins were measured and the specific degradation was calculated as described in Materials and Methods. Each point shows the mean of duplicate determinations.
HEC was much less than that degraded by mouse peritoneal macrophages which we observed previously [6], and was comparable with the previous data which measured the degradation of Ac-LDL in cultured bovine aortic or human umbilical vein endothelial cells [8,10,11 ]. The dose-response relationship of the cell-association of lzsI-labeled Ac-LDL or Ox-LDL also showed similar saturation kinetics (Fig. 4). To investigate the uptake pathway of Ac-LDL and Ox-LDL in HEC, we performed the cross-competition study of the degradation, and also the cell-association, between Ac-LDL and Ox-LDL. The degradation of lzsI-labeled Ox-LDL was almost equally inhibited by 100-fold excess amount of unlabeled Ac-LDL or OxLDL. On the other hand, the degradation of lesI-labeled Ac-LDL was only partially inhibited by the excess amount of unlabeled Ox-LDL, while the excess amount of Ac-LDL inhibited it completely (Fig. 5). Cell-association of these two forms of modified LDL in HE('
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heterogeneity of Ox-LDL particles that the excess amount of Ox-LDL could not completely inhibit the uptake of Ac-LDL. At present, we do not know the pathophysiological significance of endothelial receptors for Ac-LDL and Ox-LDL in vivo. They may play a protective role against atherogenesis locally by removing atherogenic lipoproreins, which would be taken up by ,nacrophages and change them into foam cells. However, the transient increase in the cellular cholesterol content caused by the uptake of modified lipoproteins may impair the endothelial function regulating thrombus formation, transendothelial permeability or vascular tone [18]. Further studies would be needed to elucidate the interaction between endothelial cells and modified LDL, especially Ox-LDL which is thought to be a form of modified LDL existing in vivo. The present study demonstrates the difference in the recognition for Ac-LDL and Ox-LDL in HEC, and also suggests the difference in the receptors for modified LDL between vascular endothelial cells and macrophages. Recently, two types of macrophage scavenger
I
250 500 Unlabeled Upoprotelna (pglml)
Fig. 5. Cross-competition of the degradation of acetylated LDL and oxidized LDL in endothelial cells. Each dish received 5/~g protein/ml of 1251-labeledacetylated LDL (upper panel) or oxidized LDL (lower panel) and the indicated amount of unlabeled acetylated LDL (e), oxidized LDL (0) or native LDL (z~). After the incubation for 4 h, the culture medium was collected and the radioactivities in the degraded materials were measured as described in Materials and Methods. Each point indicates the mean of duplicate dishes. This figure is the representative of three separate experiments.
(%) 100 L
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showed almost the same re.suits as those shown in the degradation assay (Fig. 6). The data presented above indicate that HEC take up Ox-LDL via the same receptors as those for Ac-LDL. We have already shown the results of a similar crosscompetition study in mouse peritoneal macrophages, which suggest the existence of at least three different types of receptor; one is common for both Ac-LDL ~nd Ox-LDL, and the others are specific only for either Ac-LDL or Ox-LDL. Unlike mouse macrophages, HEC do not have any receptors which recognize Ox-LDL but not Ac-LDL. On the contrary, the present data, which show that 100-fold excess amount of Ox-LDL could only partially inhibit the degradation or cell-association of Ac-LDL, suggest the existence of additional receptors on endothelial cells, as we indicated on mouse macrophages, which recognize Ac-LDL but not OxLDL. Ox-LDL may not consist of homogeneous particles, because some enlarged particles were found by electron microscopy [15]. However, it cannot be explained by the
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Fig. 6. Cross-competition of the cell-association of acetylated LDL and oxidized LDL. Each dish contained 5 /~g protein/ml of 1251labeled acetylated LDL (upper panel) or oxidized LDL (lower panel) and the indicated amount of unlabeled acetylated LDL (®), oxidized LDL (o) or native LDL (z~). After the incubation for 4 h, the cell-associated radioactivities were measured as described in Materials and Methods. Each point shows the mean value of duplicate dishes.
67
receptors were cloned and sequenced [19,20]. Both of the above two types of the receptor equally recognize Ac-LDL; however, their recognition for Ox-LDL has not yet been elucidated. In addition, endothelial scavenger receptors have not been clarified as molecules. Further studies would be needed to elucidate the receptor multiplicity. However, the present results demonstrate that human endothelial cells do not have any receptors specific only for Ox-LDL and suggest the possibility that they also have some receptors specific only for Ac-LDL. Acknowledgements We thank doctors and nursing staff in the Delivery Unit, Obstetrics Ward of Kyoto University Hospital for their help in obtaining human umbilical cords. We also wish to thank Drs. K. Ishii, Y. Nagano, H. Otani and Y. Ueda for helpfull discussions. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (Nos. 01304063, 63480270) and grants from the Tokyo Biochemical Research Foundation. References 1 Kita, T., Nagano, Y., Yokode, M., lshii, K., Kume, N., Ooshima, A., Yoshida, H. and Kawai, C. (1987) Proc. Natl. Acad. Sci. USA 84, 5928-5931. 2 Kita, T., Nagano, Y., Yokode, M., Ishii, K., Kume, N., Narumiya, S. and Kawai, C. (1988) Am. J. Cardiol. 62 13B-19B. 3 Carew, T.E., Schwenke, D.C. and Steinberg, D. (1987) Proc. Natl. Acad. Sci. USA 84, 7725-7729.
4 Nagano, Y., Kita, T., Yokode, M., lshii, K., Kume, N., Otani, H., Arai, H. and Kawai, C. (1989) Arteriosclerosis 9, 453-461. 5 Palinski, W., Rosenfeld, M.E., Ylii-Herttuala, S., Gurtner, G.C., Socher S.S., Butler, S.W., Parthasarathy, S., Carew, T.E. and Steinberg, D. (1989) Proc. Natl. Acad. Sci. USA 86, 1372-1376. 6 Arai, H., Kita, T., Yokode, M., Narumiya, S. and Kawai, C. (1989) Biochem. Biophys. Res. Commun. 159, 1375-1382. 7 Sparrow, C.P., Parthasarathy, S. and Steinberg, D. (1989) J. Biol. Chem. 264, 2599-2604. 8 Stein, O. and Stein, Y. (1980) Biochim. Biophys. Acta 620, 631-635. 9 Baker, D.P., Van Lanten, B.J., Fogelman, A.M., Edwards, P.A., Kean, C. and Bediner, J.A. (1984) Arteriosclerosis 4, 248-255. 10 Striimpfer, A.E.M., Van der Westhuyzen, D.R. and Coetzee, G.A. (1985) Eur. J. Cell Biol. 36, 81-90. 11 Havekes, L., Mommaas-Kienhuis, A.M., Schouten, D., De Wit, E., Scheffer, M. and Van Hinsbergh, V.W.M. (1985) Atherosclerosis 56, 81-92. 12 Jaffe, E.A., Nachman, R.L., Becker, C.G. and Minick, C.R. (1973) J. Clin. Invest. 52, 2745-2756. 13 Gimbrone, M.A., Cotran, R.S. and Folkman, J. (1974~, J. Cell. Biol. 60, 673-684. 14 Goidstein, J.L., Ho, Y.K., Basu, S.K. and Brown, M.S. (1979) Proc. Natl. Acad. Sci. USA 76, 333-337. 15 Yokode, M., Kita, T., Kikawa, Y., Ogorochi, T., Narumiya, S. and Kawai, C. (1988) J. Clin. Invest. 81,720-729. 16 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 17 Kosugi, K., Morel, D.W., DiCorleto, P.E. and Chisolm, G.M. (1987) J. Cell. Physiol. 130, 311-320. 18 Navab, M.. Hough, G.P., Berliner, J.A., Frank, J.A., Fogelman, A.M., Haberland, M.E. and Edwards, P.A. (!986) J. Clin. Invest. 78, 389-397. 19 Kodama, T., Freeman, M., Rohrer L., Zabrecky, J., Matsudaira, P. and Krieger, M. (1990) Nature 343, 531-535. 20 Rohrer, L., Freeman, M., Kodama, T., Penman, M. and Krieger, M. {1990) Nature 343, 570-572.
63
BBAMCR 12837
Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein N o r i a k i K u m e 1,,, H i d e n o r i A r a i 1, C h u i c h i K a w a i 1 a n d T o r u K i t a 2 Third Division, 1 Department of Internal Medicine and 2 Department of Geriatric Medicine, Faculty of Medicine, Kyoto University, Kyoto (Japan)
(Received 5 February 1990) (Revised manuscript received 21 August 1990)
Key words: Oxidized low-density lipoprotein; Acetylated Pow-density lipoprotein receptor; Atherosclerosis; (Human endothelial cell)
We examined the upt.ake pathway of acetylated low-density lipoprotein and oxidafively modified LDL (oxidized LDL) in human umbilical veto endothelial cells in culture. Proteolytic degradation of 12Sl-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent arid showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 12sI-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of t2Sl-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized L D L On the contrary, they may have additional receptors, as we previously indicated on moust~ macrophages, which recognize acetylated LDL, but not oxidized LDL.
Introduction
Several lines of evidence suggest that oxidative modification of low-density lipoprotein (LDL) can occur in vivo [1-5]. Oxidized LDL (Ox-LDL) is taken up by macrophages and changes them into foam cells which are characteristic in the early lesion of atheroselerosis. We have recently shown that mouse peritoneal macrophages have at least three different receptors for Ox-
Abbreviations: HEC, human endothelial cells; LDL, low-density lipoprotein; Ac-LDL, acetylated low-density lipoprotein; Ox-LDL, oxidized low-density lipoprotein; HDL, high-density iipoprotein; MDA, malondialdehyde; ECGS, endothelial cell growth supplement; LPDS, lipoprotein deficient serum; PBS, phosphate-buffered saline; TCA, trichloroacetic acid; TBARS, thiobarbituric acid-reactive substances. Correspondence (* present address): N. Kume, Vascular Research Division, Department of Pathology, Brigham and Women's Hospital, Harvard Medical Scool, 75 Francis Street, Boston, MA 02115, U.S.A.
LDL and acetylated LDL (Ac-LDL); one is common for both Ac-LDL and Ox-LDL and the others are specific only for either Ac-LDL or Ox-LDL [6]. Sparrow et al. also demonstrated that mouse peritoneal macrophages have another receptor which recognizes Ox-LDL but not Ac-LDL in addition to a receptor common for both of the two forms of modified LDL [7]. On the other hand, vascular endothelial ceils were reported to have a receptor for modified LDL such as Ac-LDL and malondialdehyde (MDA)-conjugated LDL [8-111. In the present study, we investigated the uptake pathway of Ox-LDL, which is thought to be a form of modified LDL existing in vivo, by cultured human endothelial cells (HEC).
Materials and Methods Materiais, Medium 199 (Cat. No. M0393), heparin (Cat. No. H3125) and endothelial cell growth supplement (ECGS) (Cat. No. E2759) were purchased from Sigma. Fetal bovine serum was obtained from Hyclone
64
(Lot No. 1111797). Penicillin G and streptomycin were from M.A. Bioproducts. Sodium [12~I]iodide was purchased from New England Nuclear (Code No. NEZ033A). All other chemicals were of reagent grade. Cells. HEC were harvested from human umbilical veins by collagenase digestion as previously described [12,13]. Harvested cells were suspended in medium 199 with 15~ fetal bovine serum, 90/~g/ml of heparin, 30 /~g/ml of ECGS, 100 units/ml of penicillin (3 and 100 /~g/rnl of streptomycin, seeded in a gelatin-coated flask and cultured in humidified air containing 5% CO2 at 37°C. We identified these cells as endothelial cells by their typical cobblestone morphology at confluence and their production of Von Willebrand factor. The cells were passaged onto gelatin-coated plastic petri dishes and used as first-passaged confluent monolayers in the following experiments. Lipoproteins. Lipoproteins and lipoprotein deficient serum (LPDS) were isolated from human plasma by sequential ultracentrifugation. Oxidative modification was performed by incubating LDL with phosphatebuffered saline (PBS) supplemented with 5/~M CuSO4 at 370C without cells for 24 h and then dialyzing it against excess volume of PBS without cupric ion at 4°C for at least 24 h. Acetylation of LDL was carried out by acetic anhydride. Radioiodination of lipoproteins were performed by iodine monochloride method [14]. The specific radioacti~,ity of 12SI-labeled lipoproteins were around 200 cpm/ng protein.
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A
B
C
origin
D
Fig. 1. Agarose gel electrophoresis of native LDL (lane B), oxidized LDL (lane C), acetylated LDL (lane D) and HDL (lane A). Lipoproreins (50-100 ~g protein) were applied on 0.5~ agarose gel electrophoresis.
Lowry et al. using bovine serum albumin as standard [16]. Agarose gel electrophoresis was performed by the previously described method [15]. tO '-. D
5
L--
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Assays of proteolytic degradation and ceU-association of lipoproteins. Proteolytic degradation and cell-associa-
"0
tion of nSI-labeled lipoproteins were performed as previously described [14]. Briefly, confluent monolayers were washed with PBS, fed with fresh medium 199 with 10~ LPDS and the indicated amount of ~25I-labeled lipoproteins, and incubated at 37°C in a CO2 incubator. At the end of the incubation period, culture medium was collected and the radioactivity in the trichloroacetic acid (TCA)-soluble and non-iodine fraction was counted as proteolytic degradation. On the other hand, the cells were immediately placed at 4°C, washed with buffer A (150 mM NaC1, 50 mM Tris-HCl (pH 7.4) and 2 mg/rnl bovine serum albumin) twice and buffer B (150 mM NaCI, 50 mM Tris-HCl (pH 7.4)) once, dissolved in 0.2 M N aOH and then counted as cell-association (binding plus internalization). Specific degradation or cell-association was calculated by subtracting the amount of ~25I-labeled lipoprotein degraded or cell-associated in the presence of more than 20-fold excess amount of the unlabeled lipoprotein from that degraded in the absence of the unlabeled lipoprotein. Other assays. Thiobarbituric acid-reactive substances (TBARS) were measured as previously described [15]. Protein concentration was determined by the method of
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Fig. 2. Time-course of the specific degradation of t25I-labeled acetylated LDL (upper panel) or oxidized LDL (lower panel). 125[. labeled ace~ylated LDL (25/~g protein/ml) or oxidized LDL (5 pg protein/ml) were added to medium 199 with 10~ LPDS and incubated with the confluent monolayers of endothelial cells at 37°C in a CO 2 incubator for the indicated periods. Then the culture medium were collected and the TCA-soluble fraction were counted as the amount of the degraded ]25I-labeled iipoproteins as described in Materials and Methods. Each point indicates the mean of duplicate determinations.
65 C 0
Results and Discussion
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Oxidized LDL we prepared contained lipid peroxides as much as around 10 nmol MDA equivalent/mg protein in TBARS assay after dialysis against PBS. As we showed previously, Ox-LDL we used was negatively charged almost at the same degree as Ac~LDL, as shown by agarose gel electrophoresis (Fig. 1). Ox-LDL is reported to be cytotoxic to proliferating endothelial cells or fibroblasts in culture [17]. We used confluent monolayers and did not detect any morphological changes or loss of the cellular protein content under our experimental conditions. When HEC were incubated with ]25I-labeled Ac-LDL or Ox-LDL, degraded materials of ~25I-labeled lipoproteins accumulated in the culture medium linearly for up to 24 h (Fig. 2). Dose-response relationship of the specific degradation of ~251labeled Ac-LDL or Ox-LDL showed saturation kinetics and it suggested their uptake is receptor-mediated (Fig. 3). The amount of the degraded Ac-LDL or Ox-LDL by
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~Jg/ml ~=Sl-Upoproteln Fig. 4. Dose-response relationship of the specific cell-association of 12~l-labeled acetylated LDL (upper panel) or oxidized LDL (lower panel). Endothelial monolayers were incubated with the indicated amount of 12~l-labeled acetylated or oxidized LDL in medium 199 with 10% LPDS for 6 h. The amount of radioligands specifically associated to the cells were measured as described in Materials and Methods. Each point indicates the mean of duplicate determinations.
50
100
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pg/ml taSI-Lipoprotein Fig. 3. Dose-response relationship of the specific degrad~,~ior, of 125,l-labeled acetylated LDL (upper panel) or oxidized LDL (lower panel). Endothelial monolayers were incubated with the indicated amount of ]251-labeled acetylated or oxidized LDL in medium 199 with 10~ LPDS for 6 h. Degraded materials of 1251-labeled lipoproreins were measured and the specific degradation was calculated as described in Materials and Methods. Each point shows the mean of duplicate determinations.
HEC was much less than that degraded by mouse peritoneal macrophages which we observed previously [6], and was comparable with the previous data which measured the degradation of Ac-LDL in cultured bovine aortic or human umbilical vein endothelial cells [8,10,11 ]. The dose-response relationship of the cell-association of lzsI-labeled Ac-LDL or Ox-LDL also showed similar saturation kinetics (Fig. 4). To investigate the uptake pathway of Ac-LDL and Ox-LDL in HEC, we performed the cross-competition study of the degradation, and also the cell-association, between Ac-LDL and Ox-LDL. The degradation of lzsI-labeled Ox-LDL was almost equally inhibited by 100-fold excess amount of unlabeled Ac-LDL or OxLDL. On the other hand, the degradation of lesI-labeled Ac-LDL was only partially inhibited by the excess amount of unlabeled Ox-LDL, while the excess amount of Ac-LDL inhibited it completely (Fig. 5). Cell-association of these two forms of modified LDL in HE('
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heterogeneity of Ox-LDL particles that the excess amount of Ox-LDL could not completely inhibit the uptake of Ac-LDL. At present, we do not know the pathophysiological significance of endothelial receptors for Ac-LDL and Ox-LDL in vivo. They may play a protective role against atherogenesis locally by removing atherogenic lipoproreins, which would be taken up by ,nacrophages and change them into foam cells. However, the transient increase in the cellular cholesterol content caused by the uptake of modified lipoproteins may impair the endothelial function regulating thrombus formation, transendothelial permeability or vascular tone [18]. Further studies would be needed to elucidate the interaction between endothelial cells and modified LDL, especially Ox-LDL which is thought to be a form of modified LDL existing in vivo. The present study demonstrates the difference in the recognition for Ac-LDL and Ox-LDL in HEC, and also suggests the difference in the receptors for modified LDL between vascular endothelial cells and macrophages. Recently, two types of macrophage scavenger
I
250 500 Unlabeled Upoprotelna (pglml)
Fig. 5. Cross-competition of the degradation of acetylated LDL and oxidized LDL in endothelial cells. Each dish received 5/~g protein/ml of 1251-labeledacetylated LDL (upper panel) or oxidized LDL (lower panel) and the indicated amount of unlabeled acetylated LDL (e), oxidized LDL (0) or native LDL (z~). After the incubation for 4 h, the culture medium was collected and the radioactivities in the degraded materials were measured as described in Materials and Methods. Each point indicates the mean of duplicate dishes. This figure is the representative of three separate experiments.
(%) 100 L
A ,
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showed almost the same re.suits as those shown in the degradation assay (Fig. 6). The data presented above indicate that HEC take up Ox-LDL via the same receptors as those for Ac-LDL. We have already shown the results of a similar crosscompetition study in mouse peritoneal macrophages, which suggest the existence of at least three different types of receptor; one is common for both Ac-LDL ~nd Ox-LDL, and the others are specific only for either Ac-LDL or Ox-LDL. Unlike mouse macrophages, HEC do not have any receptors which recognize Ox-LDL but not Ac-LDL. On the contrary, the present data, which show that 100-fold excess amount of Ox-LDL could only partially inhibit the degradation or cell-association of Ac-LDL, suggest the existence of additional receptors on endothelial cells, as we indicated on mouse macrophages, which recognize Ac-LDL but not OxLDL. Ox-LDL may not consist of homogeneous particles, because some enlarged particles were found by electron microscopy [15]. However, it cannot be explained by the
0
0
100
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t
250 500 Unlabeled Upoprotelns (pglml)
(%)
100
A
so A v
t
0
100
I
P
250 500 Unlabeled Upoprotalns ( • g / m l )
Fig. 6. Cross-competition of the cell-association of acetylated LDL and oxidized LDL. Each dish contained 5 /~g protein/ml of 1251labeled acetylated LDL (upper panel) or oxidized LDL (lower panel) and the indicated amount of unlabeled acetylated LDL (®), oxidized LDL (o) or native LDL (z~). After the incubation for 4 h, the cell-associated radioactivities were measured as described in Materials and Methods. Each point shows the mean value of duplicate dishes.
67
receptors were cloned and sequenced [19,20]. Both of the above two types of the receptor equally recognize Ac-LDL; however, their recognition for Ox-LDL has not yet been elucidated. In addition, endothelial scavenger receptors have not been clarified as molecules. Further studies would be needed to elucidate the receptor multiplicity. However, the present results demonstrate that human endothelial cells do not have any receptors specific only for Ox-LDL and suggest the possibility that they also have some receptors specific only for Ac-LDL. Acknowledgements We thank doctors and nursing staff in the Delivery Unit, Obstetrics Ward of Kyoto University Hospital for their help in obtaining human umbilical cords. We also wish to thank Drs. K. Ishii, Y. Nagano, H. Otani and Y. Ueda for helpfull discussions. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (Nos. 01304063, 63480270) and grants from the Tokyo Biochemical Research Foundation. References 1 Kita, T., Nagano, Y., Yokode, M., lshii, K., Kume, N., Ooshima, A., Yoshida, H. and Kawai, C. (1987) Proc. Natl. Acad. Sci. USA 84, 5928-5931. 2 Kita, T., Nagano, Y., Yokode, M., Ishii, K., Kume, N., Narumiya, S. and Kawai, C. (1988) Am. J. Cardiol. 62 13B-19B. 3 Carew, T.E., Schwenke, D.C. and Steinberg, D. (1987) Proc. Natl. Acad. Sci. USA 84, 7725-7729.
4 Nagano, Y., Kita, T., Yokode, M., lshii, K., Kume, N., Otani, H., Arai, H. and Kawai, C. (1989) Arteriosclerosis 9, 453-461. 5 Palinski, W., Rosenfeld, M.E., Ylii-Herttuala, S., Gurtner, G.C., Socher S.S., Butler, S.W., Parthasarathy, S., Carew, T.E. and Steinberg, D. (1989) Proc. Natl. Acad. Sci. USA 86, 1372-1376. 6 Arai, H., Kita, T., Yokode, M., Narumiya, S. and Kawai, C. (1989) Biochem. Biophys. Res. Commun. 159, 1375-1382. 7 Sparrow, C.P., Parthasarathy, S. and Steinberg, D. (1989) J. Biol. Chem. 264, 2599-2604. 8 Stein, O. and Stein, Y. (1980) Biochim. Biophys. Acta 620, 631-635. 9 Baker, D.P., Van Lanten, B.J., Fogelman, A.M., Edwards, P.A., Kean, C. and Bediner, J.A. (1984) Arteriosclerosis 4, 248-255. 10 Striimpfer, A.E.M., Van der Westhuyzen, D.R. and Coetzee, G.A. (1985) Eur. J. Cell Biol. 36, 81-90. 11 Havekes, L., Mommaas-Kienhuis, A.M., Schouten, D., De Wit, E., Scheffer, M. and Van Hinsbergh, V.W.M. (1985) Atherosclerosis 56, 81-92. 12 Jaffe, E.A., Nachman, R.L., Becker, C.G. and Minick, C.R. (1973) J. Clin. Invest. 52, 2745-2756. 13 Gimbrone, M.A., Cotran, R.S. and Folkman, J. (1974~, J. Cell. Biol. 60, 673-684. 14 Goidstein, J.L., Ho, Y.K., Basu, S.K. and Brown, M.S. (1979) Proc. Natl. Acad. Sci. USA 76, 333-337. 15 Yokode, M., Kita, T., Kikawa, Y., Ogorochi, T., Narumiya, S. and Kawai, C. (1988) J. Clin. Invest. 81,720-729. 16 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 17 Kosugi, K., Morel, D.W., DiCorleto, P.E. and Chisolm, G.M. (1987) J. Cell. Physiol. 130, 311-320. 18 Navab, M.. Hough, G.P., Berliner, J.A., Frank, J.A., Fogelman, A.M., Haberland, M.E. and Edwards, P.A. (!986) J. Clin. Invest. 78, 389-397. 19 Kodama, T., Freeman, M., Rohrer L., Zabrecky, J., Matsudaira, P. and Krieger, M. (1990) Nature 343, 531-535. 20 Rohrer, L., Freeman, M., Kodama, T., Penman, M. and Krieger, M. {1990) Nature 343, 570-572.
