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Macrophages modify β-VLDL by proteolysis and enhance subsequent lipid accumulation in arterial smooth muscle cells
Atherosclerosis, II (1989) 203-208 Elsevier Scientific Publishers Ireland,
203 Ltd.
ATH 04320
Macrophages modify P-VLDL by proteolysis and enhance subsequent lipid accumulation in arterial smooth muscle cells John B. Davis and David E. Bowyer Pathology Department, University of Cambridge, Cambridge CB2 1 QP (U.K.) (Received 11 November, 1988) (Accepted 30 January, 1989)
Summary Murine resident peritoneal macrophages (MRPM), incubated with beta-very low density lipoprotein (P-VLDL), modify the /3-VLDL, producing an increase in the mobility of the lipoprotein. The modification does not result in an increase of thiobarbituric acid-reactive substances (TBARS) in the lipoprotein, and is not inhibited by butylated hydroxyanisole (BHA), EDTA, removal of copper and iron from the medium, or by diphenyliodonium (DPI), suggesting that the mechanism of modification is independent of oxidation. Macrophage conditioned medium performed the modification in the absence of cells, and phenylmethylsulphonyl fluoride (PMSF) inhibited P-VLDL modification, whereas other protease inhibitors did not, suggesting that a secreted neutral serine protease may possibly be involved in the mechanism. The modified P-VLDL enhanced the accumulation of cholesterol esters by smooth muscle cells (SMC).
Key words:
Macrophages;
/3-VLDL;
Modification;
Smooth
Introduction During atherogenesis, there is accumulation of lipids within smooth muscle cells (SMC) and monocyte-derived macrophages in the arterial intima. Although this lipid is ultimately derived largely from circulating plasma lipoproteins, there is increasing evidence to suggest that modification of native plasma lipoproteins may enhance or may
Correspondence to: Dr. John B. Davis, Ludwig Institute for Cancer Research, 91 Riding House Street, London WlP 8BT, U.K. 0021-9150/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland,
muscle
cell; Atherosclerosis
even be necessary to produce this lipid accumulation both in SMC [1,2] and in macrophages [3]. It is well recognised that low-density-lipoprotein (LDL) may be modified in vitro by SMC [4,5], endothelial cells [3], and macrophages [6]. The process is thought to be mediated via a free radical mechanism [6], which is initiated by superoxide [7,8], requires the presence of iron and copper in the medium [5] and, in the case of macrophages, a competent respiratory burst [8]. It may be inhibited by superoxide dismutase [8], free radical scavengers, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) [4,6] and vitamin E [7], and by diphenyliodonium (DPI) compounds (unpublished observations), Ltd.
204 which inhibit the macrophage respiratory burst. Modified LDL causes lipid accumulation in macrophages largely because it is recognised by a scavenger receptor [3,6]. It is not known whether scavenger receptors, or only non-specific low affinity pathways, are responsible for the accumulation of lipid from modified lipoprotein in SMC
19Jl. Another lipoprotein that appears to be important in causing lipid accumulation in arterial cells is beta-migrating very low density lipoprotein (/3-VLDL). It occurs in patients with type III dyslipoproteinaemia and appears in the serum of experimental animals fed cholesterol-enriched diets [lO,ll]. Native P-VLDL stimulates lipid accumulation in macrophages [12], apparently consequent upon its uptake by a high affinity receptor [12-141, and in SMC [15]. In the studies reported here we have now investigated whether biological modification of P-VLDL by macrophages results in enhanced lipid accumulation in SMC. The results show that modification of P-VLDL by macrophages does occur with a resultant enhancement of uptake by SMC. This modification may be caused by proteolytic activity, inhibitable by PMSF, but not leupeptin and pepstatin, which is released or secreted by the macrophages. Materials and methods Preparation of LDL and /3-VLDL LDL and /?-VLDL were prepared from human serum and hyperlipidic rabbit serum respectively, by density ultracentrifugation, using fractions of LDL 1.065 > d > 1.019 g/ml and /3-VLDL d < 1.006 g/ml [16,17]. Protein was measured using a modified Lowry assay [18]. Isolation of murine resident peritoneal macrophages MRPM were isolated from 8-lo-week-old Balb/c mice by intraperitoneal injection and witbdrawal of 3 ml sterile Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO Laboratories). The cells were pelleted, red blood cells lysed by hypotonic shock in 0.2% saline for 30 set, returned to 0.9% saline, pelleted, and the white blood cells resuspended in DMEM, 3.7 g/l NaHCO,, 10% fetal calf serum (FCS), 100 III/ml penicillin, 100 pg/ml streptomycin, 100 pg/ml kanamycin, and
seeded at 1 X lo6 cells/ml in 35-mm dishes (Falcon). After overnight incubation, non-adherent cells were removed by vigorous washing. Incubation of /3-VLDL with macrophages MRPM were incubated for 48 h at 37“ C, 5% CO, in a medium containing 125 pg /3-VLDL protein/ml in DMEM containing 3.7 g/l NaHCO,, 0.2% lactalbumin hydrolysate, 3.0 pmol/l FeSO,, 0.01 hmol/l CuSO,, plus antibiotics. The inhibitors, 1 mm01 EDTA, 50 pmol/l BHA, 5 pmol/l DPI, were added singly at the commencement of cultures, at the above final concentrations, to inhibit superoxide or free radical-mediated processes. Experiments were also conducted by incubation in medium lacking iron and copper. One mmol/l PMSF, 5 mmol/l leupeptin and 35 mmol/l pepstatin (in combination) and 1 mmol/l EDTA were used to inhibit protease action in macrophage cultures. The medium was then collected and modified /3-VLDL isolated by density ultracentrifugation at d < 1.065 g/ml, and modified LDL at d < 1.21 g/ml. Control media were incubated and treated in parallel but without macrophages. Agarose gel electrophoresis of lipoproteins The lipoproteins incubated and isolated as above were run in duplicate on agarose universal gels (Corning) in 1 mmol/l EDTA, 0.05 mol/l sodium barbitone buffer, pH 8.6, and stained for lipid using Fat red 7B, and protein using Amido black 10B (G.T. Gurr Ltd.) according to the Coming-Eel instruction manual. Thiobarbituric say
acid-reactive
substance (TBARS)
as-
The amount of TBARS in macrophage modified LDL and P-VLDL was assayed according to the method of Schuh et al. [19]. 400 ~1 H,O, 500 ~120% tricholoroacetic acid in 0.6 mol/l HCI, and 200 ~1 1% thiobarbituric acid in 120 mmol/l Tris was added to 100 1.11of sample and heated at 100°C for 15 min. The reaction mixtures were then cooled, centrifuged to remove precipitates and the pink reaction product measured at 532 nm. A standard was prepared from malondialdehyde bis(dimethylaceta1) as described by Schuh et al. [19] and run in parallel.
205 b
nl
IL b
c
Lipid accumulation in SMC
-
d
e
f
Fig. 1. (a) SMC were incubated for 48 h at 37 o C, 5% CO,, in medium alone (a), or in medium containing 125 pg b-VLDL protein/ml that had previously been conditioned for 48 h with (c), or without (b) MRPM. (B) SMC were incubated, as above, in medium (d), or in fresh medium containing 125 pg re-isolated &VLDL protein/ml that had been prepared from medium conditioned with (f), or without (e) MRPM. The SMC were washed and harvested in PBS, pelleted, stored frozen, and assayed for cholesterol ester content. (n = 4; error bars = SEM; P i 0.025, r-test for means of b&c, and e&f).
a
b
d
C
Fig. 2. 125 pg j?-VLDL protein/ml was incubated with MRPM and no iron or copper supplements, EDTA, (g) 5 mmol/l leupeptin and 35 nunol/l
Smooth muscle cells were prepared from porcine aortic primary explants and maintained in DMEM, 3.7 g/l sodium bicarbonate, 10% FCS, plus antibiotics [20,21]. Medium conditioned with or without MRPM was centrifuged at 500 X g, to remove detatched cells, and plated onto secondary SMC for a second 48 h incubation. Alternatively, lipoprotein re-isolated from conditioned medium was buffer exchanged on a PDlO column (Pharmacia) and incubated in fresh medium, at 125 pg P-VLDL protein/ml, with the secondary SMC. Lipid accumulation in the SMC was assayed according to the methods of Bowyer and Ring [22]. Results Macrophage conditioned medium, containing j?-VLDL, raised cholesterol ester accumulation in SMC compared to medium conditioned in the absence of macrophages (Fig. la). @-VLDL re-iso-
e
in medium for 48 h at 37 o C, 5% CO,, in the presence of: (a & h) no MRPM. (b) (c) plus 50 pmol/l BHA, (d) 5 gmol/l DPI, (e) 1 mmol/l PMSF, (f) 1 mmol/l pepstatin. The conditioned /I-VLDL was isolated by density centrifugation at
d = 1.065 g/ml
and run on agarose
gels.
206 lated from the conditioned medium and incubated with SMC in fresh basal medium also raised SMC lipid accumulation compared to controls (Fig. lb). The P-VLDL isolated from medium incubated with macrophages was found to have increased mobility on agarose gel when compared to pVLDL incubated in the absence of cells (Fig. 2). The increased mobility after incubation with macrophages was not inhibited by the removal of iron and copper from the medium, nor by the addition of 1 mmol/l EDTA, or 50 pmol/l BHA, or by 5 pmol/l DPI to the medium at the commencement of conditioning (Fig. 2). Conditioning of P-VLDL did not lead to an increase in the TBARS content of the lipoprotein, unlike LDL (data not shown). Addition of protease inhibitors leupeptin and pepstatin, or EDTA did not inhibit the modifi-
4
b
C
d
Fig. 3. lk pg fi-VLDL protein/ml was incubated for 48 h at 37 o C, 5% CO,, with (a) or without (b) MRPM, and similarly in niedium that had been previously conditioned for 48 h with (c) or without (d) MRPM and had been centrifuged at 500 X g for 10 min to remove any detached cells. The incubated &VLDL was re-isolated and run on agarose gel.
cation of P-VLDL, whereas PMSF completely inhibited the increase in P-VLDL mobility (Fig. 2). Native /3-VLDL was found to be modified equally well by lipoprotein-free medium that had been previously conditioned by macrophages, suggesting a role for a secreted mediator (Fig. 3). Discussion
The results presented demonstrate that treatment of fi-VLDL by incubation with macrophages enhances its ability to induce lipid accumulation in SMC. Such enhancement might be an indirect effect of the lipoprotein, such as secretion of a macrophage monokine into the conditioned medium. However, the effect is still observed after re-isolation of the conditioned P-VLDL by ultracentrifugation, and, unless a putative monokine remains bound to the /3-VLDL fraction following re-isolation, this mechanism would seem to be an unlikely explanation of the phenomenon. A more likely explanation is that a modified form of pVLDL is responsible, as occurs with LDL [23,6,4]. This is supported by our observation that, following incubation with macrophages, the b-VLDL has altered electrophoretic mobility on agarose gel. In the case of LDL, in order that modification of the lipoprotein can occur it is necessary to incubate LDL with macrophages or endothelial cells; conditioned medium alone having no effect [23,8]. It thus suggests that either intracellular modification occurs, or that the modification is brought about by a mediator with short half-life, e.g., superoxide anion. With /%VLDL, however, we have found that macrophage-conditioned medium is able to cause modification, suggesting that a more stable mediator is involved. Furthermore, removal of iron and copper ions, which are necessary for cellular modification of LDL [5], did not affect modification of fi-VLDL, and no evidence of lipid peroxides was found in the modified /?-VLDL. Macrophages secrete considerable amounts of hydrolytic enzymes [24], either constitutively or when stimulated, and these might be involved in lipoprotein modification. This is supported by our data that the serine protease inhibitor, PMSF (1 mmol/l), completely inhibited modification of /3-
VLDL by macrophages. This contrasts with the modification of LDL by oxygen, which is inhibited by EDTA and radical scavengers, but not by PMSF [19]. Other protease inhibitors tested in these experiments, leupeptin and pepstatin or EDTA, did not block modification, suggesting that a secreted serine protease may be involved in macrophage modification of P-VLDL. Proteolytic modification of lipoproteins and consequent alteration of their binding determinants is an intriguing possibility that may play a role in /3-VLDL metabolism. Subspecies of /3VLDL, from hyperlipidic dogs and patients with type III dyslipoproteinaemia, contain different apo B-100 and apo B-48 content depending upon their hepatic or intestinal origin [25], and cause different levels of cholesteryl ester accumulation in macrophages. The properties of the subspecies may be due in part to their different apo B content controlling their binding affinities. It is possible that proteolytic degradation of apo B or apo E may control the maturation of /3-VLDL particles and their subsequent metabolism [26], and that extracellular proteolysis in the pathological, atherosclerotic lesion may be responsible for enhanced accumulation of lipid from /3-VLDL within SMC. A recent paper [27] reports that coculture of endothelial cells with SMC can enhance the binding of /3-VLDL to, and lipid accumulation by, SMC of different phenotypes. It is not yet known if a common mechanism for the modification of P-VLDL by endothelial cells, SMC and macrophages exists. Acknowledgements The authors wish to acknowledge the expert technical assistance of Janice Ring and Cheryl Godliman. This work was funded by the SERC and May & Baker Ltd. References 1 Stein, O., Halperin, G. and Stem, Y., Interaction between macrophages and aortic smooth muscle cells. Enhancement of cholesterol esterification in smooth muscle cells by media of macrophages incubated with acetylated LDL, B&him. Biophys. Acta, 665 (1981) 477.
2 Stein, O., Stein, Y., Chajek-Shaul, T., Halperin, G., Olivercrona, T. and Friedman, G., Interaction between macrophages and mesenchymal cells. Effect of LDL- or HDL-containing media, added to cholesteryl ester loaded macrophages, on cholesteryl esterification in mesenchymal cells, B&him. Biophys. Acta, 712 (1982) 597. 3 Henricksen, T., Mahoney, E.M. and Steinberg, D., Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells: recognition by receptors for acetylated low density lipoprotein, Proc. Natl. Acad. Sci. USA, 78 (1981) 6499. 4 Morel, D.W., DiCorletto, P.E. and Chisholm, G.M., Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation, Arteriosclerosis, 4 (1984) 357. 5 Heinecke, J.W., Rosen, H. and Chait, A., Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells, J. Clin. Invest., 74 (1984) 1890. 6 Parthasarathy, S., Printz, D.J., Boyd, D., Joy, L. and Steinberg, D., Macrophage oxidation of low density lipoprotein generates a modified form recognised by the scavenger receptor, Arteriosclerosis, 7 (1986) 505. 7 Heinecke, J.W., Baker, L., Rosen, H. and Chait, A., Superoxide-mediated modification of low density lipoprotein by arterial smooth muscle cells, J. Clin. Invest., 77 (1986) 757. 8 Hiramatsu, K., Rosen, H., Heinecke, J.W., Wolfbauer, G. and Chait, A., Superoxide initiates oxidation of low density lipoprotein by human monocytes, Arteriosclerosis, 7 (1987) 55. 9 Rothblat, G.H., Arbogast, L.Y. and Ray, E.K., Stimulation of esterified cholesterol accumulation in tissue culture cells exposed to high density lipoproteins enriched in free cholesterol, J. Lipid Res., 19 (1978) 350. 10 Mahley, R.W., Dietary fat, cholesterol, and accelerated atherosclerosis, Atheroscler. Rev., 5 (1979) 1. 11 Mistry, P., Nicoll, A., Niehaus, C., Christie, I., Janus, E. and Lewis, B., Cholesterol feeding revisited, Circulation, 54 (1976) H-178. 12 Mahley, R.W., Innerarity, T.L., Brown, MS., Ho, Y.K. and Goldstein, J.L., Cholesterol ester synthesis in macrophages. Stimulation by P-very low density lipoprotein from cholesterol-fed animals of several species, J. Lipid Res., 21 (1980) 970. 13 Goldstein, J.L., Ho, Y.K., Brown, M.S., Innerarity, T.L. and Mahley, R.W., Cholesteryl ester accumulation in macrophages resulting from receptor-mediated uptake and degradation of hypercholesterolemic canine P-very low density lipoprotein, J. Biol. Chem., 255 (1980) 1839. 14 Van-Lenten, B.J., Fogelman, A.M., Hokom, M.M., Benson, L., Haberlund, M.E. and Edwards, P.A., Regulation of the uptake and degradation of P-very low density lipoprotein in human monocyte macrophages, J. Biol. Chem., 258 (1983) 5151. 15 Chen, R.M. and Fischer-Dzoga, K., Effect of hyperlipemic lipoproteins on the lipid accumulation of rabbit aortic medial cells, Atherosclerosis, 28 (1977) 339. 16 Gherardi, E., The hepatic low density lipoprotein receptor. PhD thesis, Cambridge University, 1987.
17 Havel, R.J., Eder, H.A. and Bragdon, J.H., The distribution and chemical composition of ultracentrifugationally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 18 Lowry, O.H., Rosebrough, N.J., Fat-r, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 19 Schuh, J., Fairclough, G.F. and Haschemeyer, R.H., Oxygen-mediated heterogeneity of apo-low density lipoprotein, Proc. Natl. Acad. Sci. USA, 75 (1978) 3173. 20 Kokubu, T. and Pollack, O.J., In vitro cultures of aortic cells of untreated and of cholesterol fed rabbits, J. Atheroscler. Res., 1 (1961) 229. 21 Ross, R., The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibres, J. Cell Biol., 50 (1971) 172. 22 Bowyer, D.E. and King, J.P., A rapid and accurate method for the separation and estimation of neutral lipids, J. Chromatogr., 143 (1977) 473.
23 Henricksen, T., Mahoney, E.M. and Steinberg, D., Enhanced macrophage degradation of biologically modified low density lipoprotein, Arteriosclerosis, 3 (1983) 149. 24 Davies, P. and Bonney, R.J., Secretory products of mononuclear phagocytes. A brief review, J. Leukocyte Biol., 26 (1979) 37. 25 Fainaru, M., Mabley, R.W., Hamilton, R.L. and Innerarity, T.L., Structural and metabolic heterogeneity of /I-very low density lipoproteins from cholesterol-fed dogs and from humans with type III hyperlipoproteinemia, J. Lipid Res., 23 (1982) 702. 26 Gianturco, S.H. and Bradley, W.A., The role of apolipoprotein processing in receptor recognition, Methods Enxymol., 129 (1986) 319. 27 Hot-&an, S., Campbell, J.H. and Campbell, G.R., Effect of endothelium on /3-VLDL metabolism by cultured smooth muscle cells of differing phenotype, Atherosclerosis, 71 (1988) 57.
203 Ltd.
ATH 04320
Macrophages modify P-VLDL by proteolysis and enhance subsequent lipid accumulation in arterial smooth muscle cells John B. Davis and David E. Bowyer Pathology Department, University of Cambridge, Cambridge CB2 1 QP (U.K.) (Received 11 November, 1988) (Accepted 30 January, 1989)
Summary Murine resident peritoneal macrophages (MRPM), incubated with beta-very low density lipoprotein (P-VLDL), modify the /3-VLDL, producing an increase in the mobility of the lipoprotein. The modification does not result in an increase of thiobarbituric acid-reactive substances (TBARS) in the lipoprotein, and is not inhibited by butylated hydroxyanisole (BHA), EDTA, removal of copper and iron from the medium, or by diphenyliodonium (DPI), suggesting that the mechanism of modification is independent of oxidation. Macrophage conditioned medium performed the modification in the absence of cells, and phenylmethylsulphonyl fluoride (PMSF) inhibited P-VLDL modification, whereas other protease inhibitors did not, suggesting that a secreted neutral serine protease may possibly be involved in the mechanism. The modified P-VLDL enhanced the accumulation of cholesterol esters by smooth muscle cells (SMC).
Key words:
Macrophages;
/3-VLDL;
Modification;
Smooth
Introduction During atherogenesis, there is accumulation of lipids within smooth muscle cells (SMC) and monocyte-derived macrophages in the arterial intima. Although this lipid is ultimately derived largely from circulating plasma lipoproteins, there is increasing evidence to suggest that modification of native plasma lipoproteins may enhance or may
Correspondence to: Dr. John B. Davis, Ludwig Institute for Cancer Research, 91 Riding House Street, London WlP 8BT, U.K. 0021-9150/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland,
muscle
cell; Atherosclerosis
even be necessary to produce this lipid accumulation both in SMC [1,2] and in macrophages [3]. It is well recognised that low-density-lipoprotein (LDL) may be modified in vitro by SMC [4,5], endothelial cells [3], and macrophages [6]. The process is thought to be mediated via a free radical mechanism [6], which is initiated by superoxide [7,8], requires the presence of iron and copper in the medium [5] and, in the case of macrophages, a competent respiratory burst [8]. It may be inhibited by superoxide dismutase [8], free radical scavengers, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) [4,6] and vitamin E [7], and by diphenyliodonium (DPI) compounds (unpublished observations), Ltd.
204 which inhibit the macrophage respiratory burst. Modified LDL causes lipid accumulation in macrophages largely because it is recognised by a scavenger receptor [3,6]. It is not known whether scavenger receptors, or only non-specific low affinity pathways, are responsible for the accumulation of lipid from modified lipoprotein in SMC
19Jl. Another lipoprotein that appears to be important in causing lipid accumulation in arterial cells is beta-migrating very low density lipoprotein (/3-VLDL). It occurs in patients with type III dyslipoproteinaemia and appears in the serum of experimental animals fed cholesterol-enriched diets [lO,ll]. Native P-VLDL stimulates lipid accumulation in macrophages [12], apparently consequent upon its uptake by a high affinity receptor [12-141, and in SMC [15]. In the studies reported here we have now investigated whether biological modification of P-VLDL by macrophages results in enhanced lipid accumulation in SMC. The results show that modification of P-VLDL by macrophages does occur with a resultant enhancement of uptake by SMC. This modification may be caused by proteolytic activity, inhibitable by PMSF, but not leupeptin and pepstatin, which is released or secreted by the macrophages. Materials and methods Preparation of LDL and /3-VLDL LDL and /?-VLDL were prepared from human serum and hyperlipidic rabbit serum respectively, by density ultracentrifugation, using fractions of LDL 1.065 > d > 1.019 g/ml and /3-VLDL d < 1.006 g/ml [16,17]. Protein was measured using a modified Lowry assay [18]. Isolation of murine resident peritoneal macrophages MRPM were isolated from 8-lo-week-old Balb/c mice by intraperitoneal injection and witbdrawal of 3 ml sterile Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO Laboratories). The cells were pelleted, red blood cells lysed by hypotonic shock in 0.2% saline for 30 set, returned to 0.9% saline, pelleted, and the white blood cells resuspended in DMEM, 3.7 g/l NaHCO,, 10% fetal calf serum (FCS), 100 III/ml penicillin, 100 pg/ml streptomycin, 100 pg/ml kanamycin, and
seeded at 1 X lo6 cells/ml in 35-mm dishes (Falcon). After overnight incubation, non-adherent cells were removed by vigorous washing. Incubation of /3-VLDL with macrophages MRPM were incubated for 48 h at 37“ C, 5% CO, in a medium containing 125 pg /3-VLDL protein/ml in DMEM containing 3.7 g/l NaHCO,, 0.2% lactalbumin hydrolysate, 3.0 pmol/l FeSO,, 0.01 hmol/l CuSO,, plus antibiotics. The inhibitors, 1 mm01 EDTA, 50 pmol/l BHA, 5 pmol/l DPI, were added singly at the commencement of cultures, at the above final concentrations, to inhibit superoxide or free radical-mediated processes. Experiments were also conducted by incubation in medium lacking iron and copper. One mmol/l PMSF, 5 mmol/l leupeptin and 35 mmol/l pepstatin (in combination) and 1 mmol/l EDTA were used to inhibit protease action in macrophage cultures. The medium was then collected and modified /3-VLDL isolated by density ultracentrifugation at d < 1.065 g/ml, and modified LDL at d < 1.21 g/ml. Control media were incubated and treated in parallel but without macrophages. Agarose gel electrophoresis of lipoproteins The lipoproteins incubated and isolated as above were run in duplicate on agarose universal gels (Corning) in 1 mmol/l EDTA, 0.05 mol/l sodium barbitone buffer, pH 8.6, and stained for lipid using Fat red 7B, and protein using Amido black 10B (G.T. Gurr Ltd.) according to the Coming-Eel instruction manual. Thiobarbituric say
acid-reactive
substance (TBARS)
as-
The amount of TBARS in macrophage modified LDL and P-VLDL was assayed according to the method of Schuh et al. [19]. 400 ~1 H,O, 500 ~120% tricholoroacetic acid in 0.6 mol/l HCI, and 200 ~1 1% thiobarbituric acid in 120 mmol/l Tris was added to 100 1.11of sample and heated at 100°C for 15 min. The reaction mixtures were then cooled, centrifuged to remove precipitates and the pink reaction product measured at 532 nm. A standard was prepared from malondialdehyde bis(dimethylaceta1) as described by Schuh et al. [19] and run in parallel.
205 b
nl
IL b
c
Lipid accumulation in SMC
-
d
e
f
Fig. 1. (a) SMC were incubated for 48 h at 37 o C, 5% CO,, in medium alone (a), or in medium containing 125 pg b-VLDL protein/ml that had previously been conditioned for 48 h with (c), or without (b) MRPM. (B) SMC were incubated, as above, in medium (d), or in fresh medium containing 125 pg re-isolated &VLDL protein/ml that had been prepared from medium conditioned with (f), or without (e) MRPM. The SMC were washed and harvested in PBS, pelleted, stored frozen, and assayed for cholesterol ester content. (n = 4; error bars = SEM; P i 0.025, r-test for means of b&c, and e&f).
a
b
d
C
Fig. 2. 125 pg j?-VLDL protein/ml was incubated with MRPM and no iron or copper supplements, EDTA, (g) 5 mmol/l leupeptin and 35 nunol/l
Smooth muscle cells were prepared from porcine aortic primary explants and maintained in DMEM, 3.7 g/l sodium bicarbonate, 10% FCS, plus antibiotics [20,21]. Medium conditioned with or without MRPM was centrifuged at 500 X g, to remove detatched cells, and plated onto secondary SMC for a second 48 h incubation. Alternatively, lipoprotein re-isolated from conditioned medium was buffer exchanged on a PDlO column (Pharmacia) and incubated in fresh medium, at 125 pg P-VLDL protein/ml, with the secondary SMC. Lipid accumulation in the SMC was assayed according to the methods of Bowyer and Ring [22]. Results Macrophage conditioned medium, containing j?-VLDL, raised cholesterol ester accumulation in SMC compared to medium conditioned in the absence of macrophages (Fig. la). @-VLDL re-iso-
e
in medium for 48 h at 37 o C, 5% CO,, in the presence of: (a & h) no MRPM. (b) (c) plus 50 pmol/l BHA, (d) 5 gmol/l DPI, (e) 1 mmol/l PMSF, (f) 1 mmol/l pepstatin. The conditioned /I-VLDL was isolated by density centrifugation at
d = 1.065 g/ml
and run on agarose
gels.
206 lated from the conditioned medium and incubated with SMC in fresh basal medium also raised SMC lipid accumulation compared to controls (Fig. lb). The P-VLDL isolated from medium incubated with macrophages was found to have increased mobility on agarose gel when compared to pVLDL incubated in the absence of cells (Fig. 2). The increased mobility after incubation with macrophages was not inhibited by the removal of iron and copper from the medium, nor by the addition of 1 mmol/l EDTA, or 50 pmol/l BHA, or by 5 pmol/l DPI to the medium at the commencement of conditioning (Fig. 2). Conditioning of P-VLDL did not lead to an increase in the TBARS content of the lipoprotein, unlike LDL (data not shown). Addition of protease inhibitors leupeptin and pepstatin, or EDTA did not inhibit the modifi-
4
b
C
d
Fig. 3. lk pg fi-VLDL protein/ml was incubated for 48 h at 37 o C, 5% CO,, with (a) or without (b) MRPM, and similarly in niedium that had been previously conditioned for 48 h with (c) or without (d) MRPM and had been centrifuged at 500 X g for 10 min to remove any detached cells. The incubated &VLDL was re-isolated and run on agarose gel.
cation of P-VLDL, whereas PMSF completely inhibited the increase in P-VLDL mobility (Fig. 2). Native /3-VLDL was found to be modified equally well by lipoprotein-free medium that had been previously conditioned by macrophages, suggesting a role for a secreted mediator (Fig. 3). Discussion
The results presented demonstrate that treatment of fi-VLDL by incubation with macrophages enhances its ability to induce lipid accumulation in SMC. Such enhancement might be an indirect effect of the lipoprotein, such as secretion of a macrophage monokine into the conditioned medium. However, the effect is still observed after re-isolation of the conditioned P-VLDL by ultracentrifugation, and, unless a putative monokine remains bound to the /3-VLDL fraction following re-isolation, this mechanism would seem to be an unlikely explanation of the phenomenon. A more likely explanation is that a modified form of pVLDL is responsible, as occurs with LDL [23,6,4]. This is supported by our observation that, following incubation with macrophages, the b-VLDL has altered electrophoretic mobility on agarose gel. In the case of LDL, in order that modification of the lipoprotein can occur it is necessary to incubate LDL with macrophages or endothelial cells; conditioned medium alone having no effect [23,8]. It thus suggests that either intracellular modification occurs, or that the modification is brought about by a mediator with short half-life, e.g., superoxide anion. With /%VLDL, however, we have found that macrophage-conditioned medium is able to cause modification, suggesting that a more stable mediator is involved. Furthermore, removal of iron and copper ions, which are necessary for cellular modification of LDL [5], did not affect modification of fi-VLDL, and no evidence of lipid peroxides was found in the modified /?-VLDL. Macrophages secrete considerable amounts of hydrolytic enzymes [24], either constitutively or when stimulated, and these might be involved in lipoprotein modification. This is supported by our data that the serine protease inhibitor, PMSF (1 mmol/l), completely inhibited modification of /3-
VLDL by macrophages. This contrasts with the modification of LDL by oxygen, which is inhibited by EDTA and radical scavengers, but not by PMSF [19]. Other protease inhibitors tested in these experiments, leupeptin and pepstatin or EDTA, did not block modification, suggesting that a secreted serine protease may be involved in macrophage modification of P-VLDL. Proteolytic modification of lipoproteins and consequent alteration of their binding determinants is an intriguing possibility that may play a role in /3-VLDL metabolism. Subspecies of /3VLDL, from hyperlipidic dogs and patients with type III dyslipoproteinaemia, contain different apo B-100 and apo B-48 content depending upon their hepatic or intestinal origin [25], and cause different levels of cholesteryl ester accumulation in macrophages. The properties of the subspecies may be due in part to their different apo B content controlling their binding affinities. It is possible that proteolytic degradation of apo B or apo E may control the maturation of /3-VLDL particles and their subsequent metabolism [26], and that extracellular proteolysis in the pathological, atherosclerotic lesion may be responsible for enhanced accumulation of lipid from /3-VLDL within SMC. A recent paper [27] reports that coculture of endothelial cells with SMC can enhance the binding of /3-VLDL to, and lipid accumulation by, SMC of different phenotypes. It is not yet known if a common mechanism for the modification of P-VLDL by endothelial cells, SMC and macrophages exists. Acknowledgements The authors wish to acknowledge the expert technical assistance of Janice Ring and Cheryl Godliman. This work was funded by the SERC and May & Baker Ltd. References 1 Stein, O., Halperin, G. and Stem, Y., Interaction between macrophages and aortic smooth muscle cells. Enhancement of cholesterol esterification in smooth muscle cells by media of macrophages incubated with acetylated LDL, B&him. Biophys. Acta, 665 (1981) 477.
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