119
Atherosclerosis, 90 (1991) 119-126 0 1991 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved 0021.9150/91/$03.50 ADONIS 002191509100171Q
ATHERO 04699
Biologically modified LDL increases the adhesive properties of endothelial cells Johan Frostegkd
‘, Anders Haegerstrand
2, Magnus Gidlund 3 and Jan Nilsson
’
’Department of Medicine, 2 Department of Anatomy, ‘Department of Immunology, Karolinska Institutet, Stockholm (Sweden) (Received 29 January, 1991) (Revised, received 18 June, 1991) (Accepted 21 June, 1991)
Summary
Adhesion of monocytes to the arterial endothelium is an important early event in atherosclerosis. Several lines of evidence have suggested that oxidation of low density lipoprotein (LDL) in the arterial wall may initiate the inflammatory-like process that generally is present in atherosclerotic lesions. In vitro, oxidition of LDL can be obtained both by exposure to divalent ions, such as Cu2+, or by incubation with different cell types, including monocytes and endothelial cells. The present study was designed to investigate the possible influence of oxidized LDL on the adhesive properties of endothelial cells. We report here that Cu2+ -oxidized LDL is as effective as interleukin l/I in stimulating the ability of cultured human endothelial cells to bind U937 monocytic cells. The stimulation was inhibited by cycloheximide, indicating that de novo protein synthesis is required. Biologically modified LDL, obtained by incubation with human peripheral blood monocytes, also enhanced the adhesiveness of endothelial cells. This effect was not due to an increased secretion of interleukin l/3 from the monocytes exposed to LDL. Treatment of endothelial cells for 24 h with native LDL was also found to increase the adhesion of U937 cells. Exposure of endothelial cells to LDL for 24 h resulted in an oxidative modification of LDL. Furthermore, the antioxidant butylated hydroxytoluene inhibited both the endothelial-dependent oxidation of LDL as well as the increased adhesion of U937 cells, suggesting a coupling between these two processes. The results indicate that LDL, modified by exposure to monocytes or endothelial cells in the arterial wall, may increase the adhesive properties of the endothelium.
Key words: Atherosclerosis;
Endothelial
cells; Adhesion
Introduction Correspondence
to: Johan Frostegird,
Dept. of Medicine, Karolinska Hospital, Box 60500, S-104 01 Stockholm, Sweden. Fax: 46 8 30 78 04.
In cholesterol-fed animals, monocytes adhere to the arterial endothelium and migrate into the
120 intima, where they differentiate into macrophages [l-3]. This process is believed to be an important initiating mechanism in atherogenesis. Accumulating evidence suggests that oxidation of low density lipoprotein (LDL) may play a role in the development of atherosclerosis [4]. Oxidized lipoproteins have been identified in atherosclerotic lesions both in humans and in experimental animals [5]. In contrast to native LDL, oxidized LDL is taken up by macrophages, by a specific family of receptors, referred to as scavenger receptors [6,7]. Scavenger receptors are also present on the surface of endothelial cells [8]. Oxidized LDL has been identified as a chemoattractant for monocytes [9], and to induce differentiation and increased adhesive properties of monocytes [lo]. Gerrity et al. [ll] have reported that the increased adhesion of monocytes observed in hypercholesterolemic swine is explained both by a release of a chemotactic factor from the arterial wall and by an increased senisitivity of monocytes for this factor. Endothelial cells, exposed to minimally oxidized LDL, secrete macrophage colony stimulating factor [12]. LDL, minimally modified by storage or mild iron oxidation, enhance the adhesive properties of endothelial cells [13]. /3VLDL [14,15] and LDL [16] from non-fasting subjects have been shown to increase the adhesiveness of endothelial cells. Furthermore, oxidized LDL has been shown to impair endothehum-dependent arterial relaxation [171 and to be cytotoxic for endothelial cells [18]. Recently interest has focused on the interaction between endothelial cells and monocytes in immune and inflammatory reactions. Interleukinl/3 (IL-l@), a cytokine produced by activated monocytes/ macrophages increase endothelial adhesiveness. Endothelial cells also produce factors such as IL-l and IL-6 that activate immune competent cells [193. Endothelial cells and monocytes have been found to induce oxidation of LDL in vitro [20]. To further analyse the interactions between lipoproteins, monocytes and endothelial cells, we investigated the effect of Cu2+-oxidized and biologically oxidized LDL on the adhesive properties of human umbilical vein endothelial cells. We report that both these forms of oxidized LDL
increase the adhesive properties of endothelial cells. The possible implications of these findings in atherogenesis are discussed. Materials
and methods
Cell culture U937 cells [21] were grown in medium RPM1
1640 (GIBCO BRL, Glasgow, Scotland) supplemented with 10% heat-inactivated fetal calf serum (FCS; GIBCO BRL) and 50 pg/ml of gentamycin sulphate and kept in an atmosphere of 5% CO, in air. Fresh medium was added twice weekly, and the cell density was kept at 2-8 x 10’ cells/ml. Cell number was determined in an electronic cell counter (VDA 140, Analys Instrument AB, Stockholm, Sweden). Peripheral blood monocytes (PBM) were isolated from human buffy coats. The buffy coat was layered onto Ficoll-Hypaque and centrifuged for 20 min at 800 X g. Cells at the interface were removed and washed twice in phosphate-buffered saline (PBS). They were then resuspended in RPM1 1640 medium supplemented with 50 pg/ml gentamycin and 10% FCS. After incubating the cells for 1 h in 37 ‘C in 60-mm petri dishes the nonadherent cells were removed by washing the plates twice in PBS. The remaining adherent cells were cultured in RPM1 1640 containing 10% FCS, 50 lg/ml gentamycin at a concentration of 1 X lo6 cells/ml. Cell viability was determined by trypan blue dye exclusion and exceeded 90% in all experiments. The endothelial cells were prepared from human umbilical veins essentially as described by Jaffe et al. [22]. Briefly, veins of fresh umbilical cords were rinsed with 50-100 100 ml PBS and subsequently filled with a collagenase solution (O.l%, Cooper, U.S.A.) and incubated at 37°C for 20 min. Cells were harvested, pooled, centrifuged at 800 X g and resuspended in Medium 199 (GIBCO) with addition of 20% FCS, 50 pg/ml penicillin and 50 U/ml streptomycin. Primary cultures were grown in gelatin-coated (0.2% gelatin in PBS for 30 min) 25-cm3 tissue culture flasks until confluence was reached (2-3 days). Cells were then detached using 0.1% trypsin/0.02% EDTA in calcium and magnesium-free PBS, seeded on gelatin-coated glass
121 cover slips (diameter 13 mm>, placed in culture wells with a diameter of 16 mm/well (24-well plates, Costar, Cambridge, MA, U.S.A.) and allowed to grow to confluence.
Preparation of LDL Venous blood from healthy donors was drawn after overnight fasting into precooled vacutainer tubes containing disodium EDTA (1 mg/ml). Plasma was recovered by means of low-speed centrifugation (1400 x g, 20 min) at 1 o C and kept at this temperature throughout the separation procedures. LDL was isolated from plasma in the density interval 1.025-1.050 kg/l by sequential preparative ultracentrifugation [23] in a 50.3Ti Beckman fixed angle rotor (Beckman LS-80 ultracentrifuge) for 20 h. The total protein content of the LDL preparation was determined by the method of Lowry et al. [24].
Oxidation of LDL Copper mediated oxidation of LDL was performed by adding 0.2 mg/ml LDL to F-10 medium (Dulbecco) containing 10 PM CuSO, and kept overnight at 37 o C. The lipid peroxide content of oxidized and native LDL was determined by analyzing thiobarbituric acid-reactive substances expressed as malondialdehyde (MDA) equivalents [25]. 500 ~1 of the LDL preparation (200 pg/ml) was mixed with 1 ml of MDA reagent (10 ml 30% trichloroacetic acid, 1.25 ml 4 M hydrochloric acid, 112 ~1 2% butylated hydroxytoluene and 0.755% thiobarbituric acid). The samples were heated in a boiling water bath for 20 min, cooled and then centrifuged at 12000 x g for 2 min. Optical density was read at 532 nm. Fresh tetramethoxypropane which produces malondialdehyde was used as a standard. The presence of endotoxins in the lipoprotein preparations was analyzed using the Limulus assay (Kabi, Stockholm, Sweden). All endotoxin levels were below 0.5 ng/ml in the stock solutions and below 1 pg/ml in the test samples. There was no difference in endotoxin levels between native and oxidized LDL.
Biological modification of LDL Biological modification of LDL was performed by adding 100 pg/ml LDL to PBM, at a concentration of lo6 cells/ml, or to EC which had been grown to confluence in 12-well plates (Costar). The cell-lipoprotein suspensions were kept for 24 h at 37 o C in serum-free F-10 medium with addition of 0.1% bovine serum albumin (BSA). The lipid peroxide content of the supernatants was determined by measuring the TBAR content as described above and the content of IL-l/3 as described below.
Determination of adhesion The adhesive properties of endothelial cells was studied by determining the adhesion of U937 cells to a confluent layer of cultured human umbilical vein EC [lo]. EC were prepared as described previously and incubated with the different LDL-preparations. U937 cells were then added to each well containing an endothelial cell monolayer on glass cover slips. The final concentration of U937 cells was 5 X 10” cells/ml. After 30 min of cocultivation at 37 o C, each glass cover slip was washed 6 times in PBS to remove unspecifically attached U937 cells, and placed in a well containing trypsin/EDTA solution to detache the cells. Cells were suspended into single cells and counted in an electronic cell counter. The number of cells, after subtraction of a mean of EC from cover slips without the addition of U937 cells, was regarded as adherent U937 cells. Cell viability was determined by trypan blue dye exclusion and exceeded 90% in all experiments. The endothelial cell monolayers were intact in all experiments.
Determination of IL-ID PBM were prepared as described above and suspended in RPM1 1640 medium at a concentration of lo6 cells/ml. The cells were then seeded out in 24-well plates and incubated with native LDL for 24 h. The concentration IL-1p was then determined using an enzyme linked immunosorbent assay (ELISA, Eurogenetics, Belgium). Briefly, the procedure is a 4-stage test carried out in a microtitration plate which has been coated with a monoclonal antibody specific for IL-lfi.
122 Expression of ICAM-
The monoclonal antibody against ICAM- was a kind gift from Dr M. Patarroyo, Karolinska Institute. EC in 24 well plates (Costar) were washed in PBS and incubated with 0.5 ml of 0.25% trypsin until the cells had detached. 0.5 ml of FCS was then added in order to further inhibit enzyme activity. The cells were then gently suspended and washed twice in PBS. The monoclonal antibodies were added to the cells and the suspension placed on ice for 30 min. Thereafter, the cells were washed twice in PBS and a FITC (fluorescein-isothiocyanate)-conjugated rabbit anti-mouse antibody (Becton Dickinson) was added. The cell suspension was then placed on ice for 30 min, rinsed twice in PBS, and the surface expression of ICAM- determined in a FACS-Scan from Becton Dickinson. Mean fluorescence was determined and the surface expression of ICAM- was expressed as percent increase in mean fluorescence as compared to control cells.
Results Effect of Cu ‘+-oxidized LDL adhesiveness
on endothelial cell
Exposure of native LDL to 10 PM of Cu2+ for 16 h at 37 ’C resulted in a marked increase in the amount of TBAR material present in LDL (data not shown) and in an increased mobility during agarose gel electrophoresis (data not shown). Incubation of native LDL in F-10 medium without Cu*+ did not alter the TBAR content of the lipoprotein preparation. To analyse the effect of Cu*+-oxidized LDL on the adhesive properties of EC, confluent cultures of human umbilical vein EC were exposed to medium containing different concentrations of native or Cu2+-oxidized LDL for 6 h. The lipoprotein containing medium was then removed and the ability of the cells to induce adhesion of U937 cells determined. Cu*+oxidized LDL was found to stimulate the adhesiveness of EC in a dose-dependent manner, whereas native LDL was without effect (Fig. 1). The adhesive-stimulatory effect of 50 pg/ml of Cu*+-oxidized LDL was comparable to that of 1 U/ml of interleukin-l/3. Exposure of U937 cells
-101. 0
I. 20
I. 40
Llpoprotoin
8. 60
I 60
*, 100
(pa/ml)
Fig. 1. Adhesion of U937 cells to a cultured monolayer of endothelial cells previously exposed to native ( ??) or oxidized (0) LDL for 6 h. Each value represents the mean and SD of triplicate determinations. * * * P < 0.005.
to oxidized LDL for 30 min had no effect on their adhesive properties (data not shown). Effect of biologically modified LDL on endothelial cell adhesiveness
Incubation of native LDL with human peripheral, blood monocytes (PBM) or human umbilical vein endothelial cells (EC) for 24 h at 37 “C resulted in a marked increase in LDL TBAR material (Fig. 2). The PBM-modified LDL also ***
4-
3-
2-
-14 0
20
40
Lipoprotein
60
60
100
@O/ml)
Fig. 2. Modification of LDL by PBM and EC. The lipid peroxide content of native ( ??), EC-modified (0) and PBMmodified (0) LDL was determined as thiobarbituric acid-reactive (TBAR) substances and expressed as malondialdehyde (MDA) equivalents. Each value represents the mean and SD of triplicate determinations. * * * P < 0.005
123
60
0 0
x n
60
t
s C .P
40
0 $
20,. 0
I. 20
I 40
'
Llpoproteln
I. 60
I. 60
1 100
20
01 0
20
40
60
Llpoproteln
(pg/ml)
60
100
(pglml)
Fig. 3. Adhesion of U937 cells to a cultured monolayer of endothelial cells previously exposed to 50 wg/ml of PBMmodified LDL for 6 h. Each value represents the mean and SD of triplicate determinations. * * * P < 0.005.
Fig. 4. Adhesion of U937 cells to a cultured monolayer of endothelial cells previously exposed to native LDL at the indicated concentrations for 24 h. Each value represents the mean and SD of triplicate determinations. * * P < 0.01.
stimulated EC adhesiveness in a dose-dependent manner (Fig. 3). The adhesive-stimulatory effect of 50 pgg/ml PBM-modified LDL was comparable to that of 50 pg/ml of Cu’+-modified or 1 U/ml IL-lp. In order to study whether also EC themselves have the capacity to modify LDL in a similar way, cultured EC were exposed to medium containing different amounts of native LDL for 24 h at 37°C. Exposure of native LDL to EC was found to result in a significant increase in LDL TBAR material (Fig. 2). Incubation of EC with native LDL for 24 h was also found to enhance the adhesive properties of EC with a maximal effext obtained at a concentration of 50 pg/ml of LDL (Fig. 4).
of Cu*+-exposed LDL was completely inhibited by addition of 20 Kg/ml BHT (data not shown). Pretreatment of the EC with protein synthesis inhibitor cykloheximide at a concentration of 1 Kg/ml for 1 h effectively blocked the ability of Cu’+-oxidized LDL to stimulate EC adhesiveness (Fig. 5). LDL at the concentrations used in the adhesion experiments, did not influence the release of IL-l/3 (data not shown) from EC or
Mechanisms involued in LDL mediated increase in endothelial cell adhesion
The antioxidant butylated hydroxytoluene (BHT) was found to inhibit the Cu2+-mediated increase in LDL TBAR material as well as the adhesive-stimulatory effect of Cu2+ exposed LDL. LDL at a concentration of 50 pg/ml was incubated in F-10 medium containing 10 PM Cu2+ and increasing concentrations of the antioxidant butylated hydroxytoluene (BHT) for 24 h. BHT at a concentration of 20 WM completely inhibited CuZC-mediated increase in LDL TBAR material (data not shown). The adhesive-stimulatory effect
COntrOl
OXLDL
ch+oxLDL
Fig. 5. Effect of cycloheximide on Cu2+-oxidized LDL-mediated increase in EC adhesiveness. EC were pretreated with 1 pg/ml cycloheximide (ch) for 1 h, then exposed to 50 pg/ml of Cu2+-oxidized LDL (ox LDL) for another 6 h and the adhesion of U937 was then determined. Each value represents the mean and SD of triplicate determinations. *P< 0.05.
124 *** X
-5o.b.4b.$0.o Lipoprotein
(pglml)
Fig. 6. Effect of oxidized LDL or IL-l/3 on expression of ICAMon EC. The cells were exposed to 50 pg/ml of Cu’+-oxidized LDL (0) or 1 U/ml IL-lp (x) for 6 h and the surface expression of ICAMdetermined using a FACS scan. The surface expression of ICAMis expressed as percent increase in mean fluorescence. * * * P < 0.005
PBM. Furthermore, the surface expression of the endothelial cell adhesion molecule ICAM- (Fig. 6) was not enhanced after exposure to oxidized LDL for 6 h. In contrast, a marked increase in the endothelial surface expression of ICAMwas noted after treatment with 1 U/ml of IL-lp. Discussion
Adhesion of monocytes to the artery wall endothelium is one of the earliest events in the development of atherosclerotic plaques [l-3]. The mechanisms that initiate this process are not fully understood. In this study, we have focused on the adhesive properties of endothelial cells, and how they are influenced by LDL. Experimental studies of monocyte adherence to cultured endothelial cells are complicated by the fact that isolated peripheral blood monocytes suspensions usually are contaminated with lymphocytes, which may lead to unwanted immunological reactions. To avoid this problem we chose to use the monocytic cell line U937, which has been well characterized as a model for monocytes [26,27], to study adhesion. The present results demonstrate that Cu2+oxidized LDL as well as LDL modified by human monocytes and endothelial cells increase en-
dothelial adhesiveness. The cellular modification of LDL is coupled to lipid peroxidation. The antioxidant BHT inhibits both lipid peroxidation and the adhesive stimulatory effect of LDL suggesting a coupling between these two processes. The mechanism by which Cu2+-oxidized and biologically modified LDL stimulate EC adhesiveness remains to be determined. It requires de novo protein synthesis in the cells but is not mediated by an increased secretion of IL-l@ or by an increased surface expression of ICAM-1. It is possible that the effect of LDL and oxidized LDL is due to an endotoxin contamination in the lipoprotein preparations, since endotoxin can increase the adhesive properties of endothelial cells. Using the limulus assay we found trace amounts of endotoxins in our lipoprotein preparations. However, since there were similar levels of endotoxins in native and oxidized LDL it does not seem likely that the increased effect of oxidized LDL on endothelial adhesiveness is due to endotoxins. The finding that the antioxidant BHT inhibited adhesion of U937 cells to endotelial cells exposed to LDL for 24 h also argues against any major influence of endotoxins. Furthermore, presence of endotoxins in the LDL preparation would be expected to result in an increased release of IL-l from monocytes exposed to LDL. The finding that no such increase was observed further argue against the possibilty that the adhesive-stimulatory effect of lipoproteins is explained by contamination of endotoxins. Several lines of evidence have suggested that oxidation of LDL play an important role in atherogenesis (for review, see [4]). Oxidized LDL has been found to act as a chemoattractant for monocytes [9] and to stimulate differentiation of monocytes into macrophages [lo]. Oxidized LDL also stimulate release of IL-lp from human monocytes and activates T cells (Frostegsrd et al., unpublished data) and promotes growth of smooth muscle cells (Stiko-Rahm et al., unpublished data). Oxidized LDL has been shown to be cytotoxic to endothelial cells under certain circumstances [183. We found no signs of detachment of endothelial monolayers or decreased cell viability as determined by trypan blue dye exclusion after treatment with oxidized LDL. However, the pos-
125 siblity of slight damage to the endothelial cells, cannot be ruled out. At lower concentrations oxidized LDL has been found to inhibit secretion of PDGF from cultured EC [28]. Alderson et al. [16] have reported that native LDL enhances monocyte adhesion to endothelial cells. However, this increase was mainly found using postprandial LDL, while LDL from fasting subjects had no significant effect. Endemann et al. [15] reported that P-VLDL and triglycerideand cholesterolrich LDL but not normal LDL and VLDL enhance the monocyte adhesion to endothelial cells. It is possible that postprandial LDL may enhance adhesion due to an inreased proneness to oxidation, as compared to LDL from fasting subjects. LDL, minimally modified by storage or slight Fe’+-induced oxidation, was recently demonstrated to induce a monocyte chemotactic protein [29] and increase EC adhesiveness [13]. The present results suggest that also LDL modified by cells present in both normal arteries and in atherosclerotic lesions may enhance the adhesive properties of EC. Acknowledgements This work was supported by Swedish Medical Research Council (8311,7126), the Knut and Alice Wallenberg Foundation, the Swedish Heart and Lung Foundation, Marcus and Amalia Wallenberg Memorial Foundation, the Swedish Cancer Society, King Gustaf V 80th Birthday Fund, the Swedish Society of Medicine, and the Nanna Swartz Fund.
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References Gerrity, R.G.. The role of the monocyte in atherogenesis: II. Migration of foam cells from atherosclerotic lesions, Am. J. Pathol., 103 (1981) 181. Joris. I., Nunnari, J.J., Krolikowski, F.J. and Ajno, G.. Studies on the pathogenesis of atherosclerosis. I. Adhesion and emigration of mononuclear cells in the aorta of hypercholestorolemic rats, Am. J. Pathol., 113 (1983) 341. Faggiotto, A.. Ross. R. and Harker, L., Studies of hypercholesterolemia in the nonhuman primate. I. Changes that lead to fatty streak formation, Arteriosclerosis, 4 (1983) 323. Steinberg, D.. Parthasarathy, S., Carew, T.E., Khoo, J.C. and Witztum. J.L.. Beyond cholesterol. Modifications of
IS
I6
17
low-density lipoprotein that increase its atherogenicity, N. Engl. J. Med., 320 (1989) 915. Palinski, W., Rosenfeld, M.E., Yla-Herttuala, S., Gurtner. G. C., Socher, S.S., Butler, SW., Parthasarathy, S., Carew, T.E., Steinberg, D. and Witztum J.L., Low density lipoprotein undergoes modification in vivo, Proc. Natl. Acad. Sci. USA, 86 (1989) 1372. Kodama, T.. Freeman, M., Rohrer, L.. Zabrecky. J, Matsudaira, P. and Krieger, M., Type 1 macrophage scavenger receptor contains o-helical and collagen-like coiled coils, Nature, 343 (1990) 531. Rohrer. L, Freeman, M, Kodama T., Penman, M. and Krieger, M., Coiled fibrous domains mediate ligand bidning by macrophage scavenger receptor type II, Nature, 343 (1990) 570. Nagelkerke, J.F., Barto, K.P. and van Berkel. T.J.. In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer and parenchymal cells. J. Biol. Chem., 258 (1983) 12221. Quinn, M.T.. Parthasarathy, S., Fong. G.L. and Steinberg, D., Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/ macrophages during atherogenesis. Proc. Natl. Acad. Sci. USA. X4 (1987) 2995. Frostegird, J., Nilsson, J., Haegerstrand, A.. Hamsten, A., Wigzell, H. and Gidlund, M.. Oxidized low-density lipoprotein induces differentiation and adhesion of human monocytes and the monocytic cell line U937. Proc. Natl. Acad. Sci. USA, X7 (1990) 904. Gerrity, R.G., Goss, J.A. and Soby. L., Control of monocyte recruitment by chemotactic factor(s) in lesion-prone areas of swine aorta, Arteriosclerosis, 5 (19x5) 55. Rajavashisth, T.B., Andalibi. A.. Territo. M.C., Berliner. J.A.. Navab. M., Fogelman. A.M. and Lusis. A.J., Induction of endothelial cell expression of granulocyte and macrophage colony stimulating factor by modified low density lipoproteins, Nature, 344 (1990) 254. Berliner. J.A., Territo, M.C., Sevanian, A., Ramin, S., Kim, J.A., Bamshad, B.. Esterson. M. and Fogelman, A.M., Minimally modified low density lipoprotein stimulates monocyte endothelial interactions, J. Clin. Invest., 85 (1990) 1260. Territo, M.C.. Berliner. J.A., Almada, L.O., Ramirez. R. and Fogelman, A.M.. B-Very low density lipoprotein pretreatment of endothelial monolayers increases monocyte adhesion, Arteriosclerosis, 9 (1989) X24. Alderson, L.M., Endemann, G., Lindsey, S., Pronczuk, A., Hoover. R.I. and Hayes. K.C., LDL enhances monocyte adhesion to endothelial cells in vitro. Am. J. Pathol., 123 (1986) 334. Endemann. G.. Pronzcuk, A., Friedman, G., Lindsey, S., Alderson, L. and Hayes, K.C., Monocyte adherence to endothelial cells in vitro is increased by P-VLDL, Am. J. Pathol., 126 (1987) 1. Kugiyama. K., Kerns, S.A.. Morrisett. J.D.. Roberts, R. and Henry, P.D., Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins, Nature. 344 (1990) 160.
126 18 Hessler, J.R., Robertson, A.L. and Chishohn, G.M., LDLinduced cytotoxicity and its inhibition by HDL in human vascular smooth muscle cells and endothelial cells in culture, Atherosclerosis, 32 (1979) 213. 19 Mantovani, A. and Dejana, E., Cytokines as communication signals between leukocytes and endothelial cells, Immunol. Today, 10 (1989) 370. 20 Henriksen, T., Mahoney, E.M. and Steinberg, D., Enhanced macrophage degradation of biological modified low density lipoprotein. Arteriosclerosis, 3 (1983) 149. 21 Sundstrom, C. and Nilsson, K., Establishment and characterization of a human histiocytic lymphoma cell line (U937), Int. J. Cancer, 17 (1976) 565. 22 Jaffe, E.A., Nachman, R.L., Becker, C.G. and Minick, C.G., Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria, J. Clin. Invest., 52 (1973) 2745. 23 Havel, R.J., Eder, H.A. and Bragdon, J.H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 24 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265.
25 Regnstrom, J., Walldius, G, Carlson, L. and Nilsson, J., Effect of probucol treatment on the susceptibility of low density lipoprotein isolated from hypercholesterolemic patients to become oxidatively modified in vitro, Atherosclerosis, 82 (1990) 43. 26 Harris, P. and Ralph, P., Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines, J. Leukocyte Biol., 37 (1985) 407. 27 Gidlund, M., Rossi, P., Cotran, P., Ramstedt, U. and Wigzell, H., In humans a strong correlation exists between expression of the M3 antigen, Fc mediated phagocytic activity and failure to participate in extracellular antibody dependent cytotoxicity, Eur. J. Immunol., 18 (1988) 477. 28 Fox, P.L and DiCorleto, P.E., Modified low density lipoproteins suppress production of a platelet derived growth factor-like protein by cultured endothelial cells, Proc. Natl. Acad. Sci. USA, 83 (1986) 4774. 29 Cushing, S.D., Berliner, J.A., Valentine, A.J., Territo, M.C., Navab, M., Parhami, F., Gerrity, R., Schwartz, C.J. and Fogelman, A.L., Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells, Proc. Natl. Acad. Sci. USA, 87 (1990) 5134.
Atherosclerosis, 90 (1991) 119-126 0 1991 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved 0021.9150/91/$03.50 ADONIS 002191509100171Q
ATHERO 04699
Biologically modified LDL increases the adhesive properties of endothelial cells Johan Frostegkd
‘, Anders Haegerstrand
2, Magnus Gidlund 3 and Jan Nilsson
’
’Department of Medicine, 2 Department of Anatomy, ‘Department of Immunology, Karolinska Institutet, Stockholm (Sweden) (Received 29 January, 1991) (Revised, received 18 June, 1991) (Accepted 21 June, 1991)
Summary
Adhesion of monocytes to the arterial endothelium is an important early event in atherosclerosis. Several lines of evidence have suggested that oxidation of low density lipoprotein (LDL) in the arterial wall may initiate the inflammatory-like process that generally is present in atherosclerotic lesions. In vitro, oxidition of LDL can be obtained both by exposure to divalent ions, such as Cu2+, or by incubation with different cell types, including monocytes and endothelial cells. The present study was designed to investigate the possible influence of oxidized LDL on the adhesive properties of endothelial cells. We report here that Cu2+ -oxidized LDL is as effective as interleukin l/I in stimulating the ability of cultured human endothelial cells to bind U937 monocytic cells. The stimulation was inhibited by cycloheximide, indicating that de novo protein synthesis is required. Biologically modified LDL, obtained by incubation with human peripheral blood monocytes, also enhanced the adhesiveness of endothelial cells. This effect was not due to an increased secretion of interleukin l/3 from the monocytes exposed to LDL. Treatment of endothelial cells for 24 h with native LDL was also found to increase the adhesion of U937 cells. Exposure of endothelial cells to LDL for 24 h resulted in an oxidative modification of LDL. Furthermore, the antioxidant butylated hydroxytoluene inhibited both the endothelial-dependent oxidation of LDL as well as the increased adhesion of U937 cells, suggesting a coupling between these two processes. The results indicate that LDL, modified by exposure to monocytes or endothelial cells in the arterial wall, may increase the adhesive properties of the endothelium.
Key words: Atherosclerosis;
Endothelial
cells; Adhesion
Introduction Correspondence
to: Johan Frostegird,
Dept. of Medicine, Karolinska Hospital, Box 60500, S-104 01 Stockholm, Sweden. Fax: 46 8 30 78 04.
In cholesterol-fed animals, monocytes adhere to the arterial endothelium and migrate into the
120 intima, where they differentiate into macrophages [l-3]. This process is believed to be an important initiating mechanism in atherogenesis. Accumulating evidence suggests that oxidation of low density lipoprotein (LDL) may play a role in the development of atherosclerosis [4]. Oxidized lipoproteins have been identified in atherosclerotic lesions both in humans and in experimental animals [5]. In contrast to native LDL, oxidized LDL is taken up by macrophages, by a specific family of receptors, referred to as scavenger receptors [6,7]. Scavenger receptors are also present on the surface of endothelial cells [8]. Oxidized LDL has been identified as a chemoattractant for monocytes [9], and to induce differentiation and increased adhesive properties of monocytes [lo]. Gerrity et al. [ll] have reported that the increased adhesion of monocytes observed in hypercholesterolemic swine is explained both by a release of a chemotactic factor from the arterial wall and by an increased senisitivity of monocytes for this factor. Endothelial cells, exposed to minimally oxidized LDL, secrete macrophage colony stimulating factor [12]. LDL, minimally modified by storage or mild iron oxidation, enhance the adhesive properties of endothelial cells [13]. /3VLDL [14,15] and LDL [16] from non-fasting subjects have been shown to increase the adhesiveness of endothelial cells. Furthermore, oxidized LDL has been shown to impair endothehum-dependent arterial relaxation [171 and to be cytotoxic for endothelial cells [18]. Recently interest has focused on the interaction between endothelial cells and monocytes in immune and inflammatory reactions. Interleukinl/3 (IL-l@), a cytokine produced by activated monocytes/ macrophages increase endothelial adhesiveness. Endothelial cells also produce factors such as IL-l and IL-6 that activate immune competent cells [193. Endothelial cells and monocytes have been found to induce oxidation of LDL in vitro [20]. To further analyse the interactions between lipoproteins, monocytes and endothelial cells, we investigated the effect of Cu2+-oxidized and biologically oxidized LDL on the adhesive properties of human umbilical vein endothelial cells. We report that both these forms of oxidized LDL
increase the adhesive properties of endothelial cells. The possible implications of these findings in atherogenesis are discussed. Materials
and methods
Cell culture U937 cells [21] were grown in medium RPM1
1640 (GIBCO BRL, Glasgow, Scotland) supplemented with 10% heat-inactivated fetal calf serum (FCS; GIBCO BRL) and 50 pg/ml of gentamycin sulphate and kept in an atmosphere of 5% CO, in air. Fresh medium was added twice weekly, and the cell density was kept at 2-8 x 10’ cells/ml. Cell number was determined in an electronic cell counter (VDA 140, Analys Instrument AB, Stockholm, Sweden). Peripheral blood monocytes (PBM) were isolated from human buffy coats. The buffy coat was layered onto Ficoll-Hypaque and centrifuged for 20 min at 800 X g. Cells at the interface were removed and washed twice in phosphate-buffered saline (PBS). They were then resuspended in RPM1 1640 medium supplemented with 50 pg/ml gentamycin and 10% FCS. After incubating the cells for 1 h in 37 ‘C in 60-mm petri dishes the nonadherent cells were removed by washing the plates twice in PBS. The remaining adherent cells were cultured in RPM1 1640 containing 10% FCS, 50 lg/ml gentamycin at a concentration of 1 X lo6 cells/ml. Cell viability was determined by trypan blue dye exclusion and exceeded 90% in all experiments. The endothelial cells were prepared from human umbilical veins essentially as described by Jaffe et al. [22]. Briefly, veins of fresh umbilical cords were rinsed with 50-100 100 ml PBS and subsequently filled with a collagenase solution (O.l%, Cooper, U.S.A.) and incubated at 37°C for 20 min. Cells were harvested, pooled, centrifuged at 800 X g and resuspended in Medium 199 (GIBCO) with addition of 20% FCS, 50 pg/ml penicillin and 50 U/ml streptomycin. Primary cultures were grown in gelatin-coated (0.2% gelatin in PBS for 30 min) 25-cm3 tissue culture flasks until confluence was reached (2-3 days). Cells were then detached using 0.1% trypsin/0.02% EDTA in calcium and magnesium-free PBS, seeded on gelatin-coated glass
121 cover slips (diameter 13 mm>, placed in culture wells with a diameter of 16 mm/well (24-well plates, Costar, Cambridge, MA, U.S.A.) and allowed to grow to confluence.
Preparation of LDL Venous blood from healthy donors was drawn after overnight fasting into precooled vacutainer tubes containing disodium EDTA (1 mg/ml). Plasma was recovered by means of low-speed centrifugation (1400 x g, 20 min) at 1 o C and kept at this temperature throughout the separation procedures. LDL was isolated from plasma in the density interval 1.025-1.050 kg/l by sequential preparative ultracentrifugation [23] in a 50.3Ti Beckman fixed angle rotor (Beckman LS-80 ultracentrifuge) for 20 h. The total protein content of the LDL preparation was determined by the method of Lowry et al. [24].
Oxidation of LDL Copper mediated oxidation of LDL was performed by adding 0.2 mg/ml LDL to F-10 medium (Dulbecco) containing 10 PM CuSO, and kept overnight at 37 o C. The lipid peroxide content of oxidized and native LDL was determined by analyzing thiobarbituric acid-reactive substances expressed as malondialdehyde (MDA) equivalents [25]. 500 ~1 of the LDL preparation (200 pg/ml) was mixed with 1 ml of MDA reagent (10 ml 30% trichloroacetic acid, 1.25 ml 4 M hydrochloric acid, 112 ~1 2% butylated hydroxytoluene and 0.755% thiobarbituric acid). The samples were heated in a boiling water bath for 20 min, cooled and then centrifuged at 12000 x g for 2 min. Optical density was read at 532 nm. Fresh tetramethoxypropane which produces malondialdehyde was used as a standard. The presence of endotoxins in the lipoprotein preparations was analyzed using the Limulus assay (Kabi, Stockholm, Sweden). All endotoxin levels were below 0.5 ng/ml in the stock solutions and below 1 pg/ml in the test samples. There was no difference in endotoxin levels between native and oxidized LDL.
Biological modification of LDL Biological modification of LDL was performed by adding 100 pg/ml LDL to PBM, at a concentration of lo6 cells/ml, or to EC which had been grown to confluence in 12-well plates (Costar). The cell-lipoprotein suspensions were kept for 24 h at 37 o C in serum-free F-10 medium with addition of 0.1% bovine serum albumin (BSA). The lipid peroxide content of the supernatants was determined by measuring the TBAR content as described above and the content of IL-l/3 as described below.
Determination of adhesion The adhesive properties of endothelial cells was studied by determining the adhesion of U937 cells to a confluent layer of cultured human umbilical vein EC [lo]. EC were prepared as described previously and incubated with the different LDL-preparations. U937 cells were then added to each well containing an endothelial cell monolayer on glass cover slips. The final concentration of U937 cells was 5 X 10” cells/ml. After 30 min of cocultivation at 37 o C, each glass cover slip was washed 6 times in PBS to remove unspecifically attached U937 cells, and placed in a well containing trypsin/EDTA solution to detache the cells. Cells were suspended into single cells and counted in an electronic cell counter. The number of cells, after subtraction of a mean of EC from cover slips without the addition of U937 cells, was regarded as adherent U937 cells. Cell viability was determined by trypan blue dye exclusion and exceeded 90% in all experiments. The endothelial cell monolayers were intact in all experiments.
Determination of IL-ID PBM were prepared as described above and suspended in RPM1 1640 medium at a concentration of lo6 cells/ml. The cells were then seeded out in 24-well plates and incubated with native LDL for 24 h. The concentration IL-1p was then determined using an enzyme linked immunosorbent assay (ELISA, Eurogenetics, Belgium). Briefly, the procedure is a 4-stage test carried out in a microtitration plate which has been coated with a monoclonal antibody specific for IL-lfi.
122 Expression of ICAM-
The monoclonal antibody against ICAM- was a kind gift from Dr M. Patarroyo, Karolinska Institute. EC in 24 well plates (Costar) were washed in PBS and incubated with 0.5 ml of 0.25% trypsin until the cells had detached. 0.5 ml of FCS was then added in order to further inhibit enzyme activity. The cells were then gently suspended and washed twice in PBS. The monoclonal antibodies were added to the cells and the suspension placed on ice for 30 min. Thereafter, the cells were washed twice in PBS and a FITC (fluorescein-isothiocyanate)-conjugated rabbit anti-mouse antibody (Becton Dickinson) was added. The cell suspension was then placed on ice for 30 min, rinsed twice in PBS, and the surface expression of ICAM- determined in a FACS-Scan from Becton Dickinson. Mean fluorescence was determined and the surface expression of ICAM- was expressed as percent increase in mean fluorescence as compared to control cells.
Results Effect of Cu ‘+-oxidized LDL adhesiveness
on endothelial cell
Exposure of native LDL to 10 PM of Cu2+ for 16 h at 37 ’C resulted in a marked increase in the amount of TBAR material present in LDL (data not shown) and in an increased mobility during agarose gel electrophoresis (data not shown). Incubation of native LDL in F-10 medium without Cu*+ did not alter the TBAR content of the lipoprotein preparation. To analyse the effect of Cu*+-oxidized LDL on the adhesive properties of EC, confluent cultures of human umbilical vein EC were exposed to medium containing different concentrations of native or Cu2+-oxidized LDL for 6 h. The lipoprotein containing medium was then removed and the ability of the cells to induce adhesion of U937 cells determined. Cu*+oxidized LDL was found to stimulate the adhesiveness of EC in a dose-dependent manner, whereas native LDL was without effect (Fig. 1). The adhesive-stimulatory effect of 50 pg/ml of Cu*+-oxidized LDL was comparable to that of 1 U/ml of interleukin-l/3. Exposure of U937 cells
-101. 0
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Llpoprotoin
8. 60
I 60
*, 100
(pa/ml)
Fig. 1. Adhesion of U937 cells to a cultured monolayer of endothelial cells previously exposed to native ( ??) or oxidized (0) LDL for 6 h. Each value represents the mean and SD of triplicate determinations. * * * P < 0.005.
to oxidized LDL for 30 min had no effect on their adhesive properties (data not shown). Effect of biologically modified LDL on endothelial cell adhesiveness
Incubation of native LDL with human peripheral, blood monocytes (PBM) or human umbilical vein endothelial cells (EC) for 24 h at 37 “C resulted in a marked increase in LDL TBAR material (Fig. 2). The PBM-modified LDL also ***
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20
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Lipoprotein
60
60
100
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Fig. 2. Modification of LDL by PBM and EC. The lipid peroxide content of native ( ??), EC-modified (0) and PBMmodified (0) LDL was determined as thiobarbituric acid-reactive (TBAR) substances and expressed as malondialdehyde (MDA) equivalents. Each value represents the mean and SD of triplicate determinations. * * * P < 0.005
123
60
0 0
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60
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40
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20,. 0
I. 20
I 40
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Llpoproteln
I. 60
I. 60
1 100
20
01 0
20
40
60
Llpoproteln
(pg/ml)
60
100
(pglml)
Fig. 3. Adhesion of U937 cells to a cultured monolayer of endothelial cells previously exposed to 50 wg/ml of PBMmodified LDL for 6 h. Each value represents the mean and SD of triplicate determinations. * * * P < 0.005.
Fig. 4. Adhesion of U937 cells to a cultured monolayer of endothelial cells previously exposed to native LDL at the indicated concentrations for 24 h. Each value represents the mean and SD of triplicate determinations. * * P < 0.01.
stimulated EC adhesiveness in a dose-dependent manner (Fig. 3). The adhesive-stimulatory effect of 50 pgg/ml PBM-modified LDL was comparable to that of 50 pg/ml of Cu’+-modified or 1 U/ml IL-lp. In order to study whether also EC themselves have the capacity to modify LDL in a similar way, cultured EC were exposed to medium containing different amounts of native LDL for 24 h at 37°C. Exposure of native LDL to EC was found to result in a significant increase in LDL TBAR material (Fig. 2). Incubation of EC with native LDL for 24 h was also found to enhance the adhesive properties of EC with a maximal effext obtained at a concentration of 50 pg/ml of LDL (Fig. 4).
of Cu*+-exposed LDL was completely inhibited by addition of 20 Kg/ml BHT (data not shown). Pretreatment of the EC with protein synthesis inhibitor cykloheximide at a concentration of 1 Kg/ml for 1 h effectively blocked the ability of Cu’+-oxidized LDL to stimulate EC adhesiveness (Fig. 5). LDL at the concentrations used in the adhesion experiments, did not influence the release of IL-l/3 (data not shown) from EC or
Mechanisms involued in LDL mediated increase in endothelial cell adhesion
The antioxidant butylated hydroxytoluene (BHT) was found to inhibit the Cu2+-mediated increase in LDL TBAR material as well as the adhesive-stimulatory effect of Cu2+ exposed LDL. LDL at a concentration of 50 pg/ml was incubated in F-10 medium containing 10 PM Cu2+ and increasing concentrations of the antioxidant butylated hydroxytoluene (BHT) for 24 h. BHT at a concentration of 20 WM completely inhibited CuZC-mediated increase in LDL TBAR material (data not shown). The adhesive-stimulatory effect
COntrOl
OXLDL
ch+oxLDL
Fig. 5. Effect of cycloheximide on Cu2+-oxidized LDL-mediated increase in EC adhesiveness. EC were pretreated with 1 pg/ml cycloheximide (ch) for 1 h, then exposed to 50 pg/ml of Cu2+-oxidized LDL (ox LDL) for another 6 h and the adhesion of U937 was then determined. Each value represents the mean and SD of triplicate determinations. *P< 0.05.
124 *** X
-5o.b.4b.$0.o Lipoprotein
(pglml)
Fig. 6. Effect of oxidized LDL or IL-l/3 on expression of ICAMon EC. The cells were exposed to 50 pg/ml of Cu’+-oxidized LDL (0) or 1 U/ml IL-lp (x) for 6 h and the surface expression of ICAMdetermined using a FACS scan. The surface expression of ICAMis expressed as percent increase in mean fluorescence. * * * P < 0.005
PBM. Furthermore, the surface expression of the endothelial cell adhesion molecule ICAM- (Fig. 6) was not enhanced after exposure to oxidized LDL for 6 h. In contrast, a marked increase in the endothelial surface expression of ICAMwas noted after treatment with 1 U/ml of IL-lp. Discussion
Adhesion of monocytes to the artery wall endothelium is one of the earliest events in the development of atherosclerotic plaques [l-3]. The mechanisms that initiate this process are not fully understood. In this study, we have focused on the adhesive properties of endothelial cells, and how they are influenced by LDL. Experimental studies of monocyte adherence to cultured endothelial cells are complicated by the fact that isolated peripheral blood monocytes suspensions usually are contaminated with lymphocytes, which may lead to unwanted immunological reactions. To avoid this problem we chose to use the monocytic cell line U937, which has been well characterized as a model for monocytes [26,27], to study adhesion. The present results demonstrate that Cu2+oxidized LDL as well as LDL modified by human monocytes and endothelial cells increase en-
dothelial adhesiveness. The cellular modification of LDL is coupled to lipid peroxidation. The antioxidant BHT inhibits both lipid peroxidation and the adhesive stimulatory effect of LDL suggesting a coupling between these two processes. The mechanism by which Cu2+-oxidized and biologically modified LDL stimulate EC adhesiveness remains to be determined. It requires de novo protein synthesis in the cells but is not mediated by an increased secretion of IL-l@ or by an increased surface expression of ICAM-1. It is possible that the effect of LDL and oxidized LDL is due to an endotoxin contamination in the lipoprotein preparations, since endotoxin can increase the adhesive properties of endothelial cells. Using the limulus assay we found trace amounts of endotoxins in our lipoprotein preparations. However, since there were similar levels of endotoxins in native and oxidized LDL it does not seem likely that the increased effect of oxidized LDL on endothelial adhesiveness is due to endotoxins. The finding that the antioxidant BHT inhibited adhesion of U937 cells to endotelial cells exposed to LDL for 24 h also argues against any major influence of endotoxins. Furthermore, presence of endotoxins in the LDL preparation would be expected to result in an increased release of IL-l from monocytes exposed to LDL. The finding that no such increase was observed further argue against the possibilty that the adhesive-stimulatory effect of lipoproteins is explained by contamination of endotoxins. Several lines of evidence have suggested that oxidation of LDL play an important role in atherogenesis (for review, see [4]). Oxidized LDL has been found to act as a chemoattractant for monocytes [9] and to stimulate differentiation of monocytes into macrophages [lo]. Oxidized LDL also stimulate release of IL-lp from human monocytes and activates T cells (Frostegsrd et al., unpublished data) and promotes growth of smooth muscle cells (Stiko-Rahm et al., unpublished data). Oxidized LDL has been shown to be cytotoxic to endothelial cells under certain circumstances [183. We found no signs of detachment of endothelial monolayers or decreased cell viability as determined by trypan blue dye exclusion after treatment with oxidized LDL. However, the pos-
125 siblity of slight damage to the endothelial cells, cannot be ruled out. At lower concentrations oxidized LDL has been found to inhibit secretion of PDGF from cultured EC [28]. Alderson et al. [16] have reported that native LDL enhances monocyte adhesion to endothelial cells. However, this increase was mainly found using postprandial LDL, while LDL from fasting subjects had no significant effect. Endemann et al. [15] reported that P-VLDL and triglycerideand cholesterolrich LDL but not normal LDL and VLDL enhance the monocyte adhesion to endothelial cells. It is possible that postprandial LDL may enhance adhesion due to an inreased proneness to oxidation, as compared to LDL from fasting subjects. LDL, minimally modified by storage or slight Fe’+-induced oxidation, was recently demonstrated to induce a monocyte chemotactic protein [29] and increase EC adhesiveness [13]. The present results suggest that also LDL modified by cells present in both normal arteries and in atherosclerotic lesions may enhance the adhesive properties of EC. Acknowledgements This work was supported by Swedish Medical Research Council (8311,7126), the Knut and Alice Wallenberg Foundation, the Swedish Heart and Lung Foundation, Marcus and Amalia Wallenberg Memorial Foundation, the Swedish Cancer Society, King Gustaf V 80th Birthday Fund, the Swedish Society of Medicine, and the Nanna Swartz Fund.
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