β-Migrating very low density lipoproteins induce foam cell formation in mouse mesangial cells

ATHEROSCLEROSIS

EmfER

Atherosclerosis 114 (1995) 123-132

P-Migrating

very low density lipoproteins induce foam cell formation in mouse mesangial cells

Takeshi Nishikawa*, Shozo Kobori, Haruo Takeda, Takayuki Higashi, Yoshihiro Sato, Takayuki Sasahara, Toshihiro Yano, Masaya Kasho, Yoshichika Anami, Motoaki Shichiri Depurtment

of’ Metabolic

Medicine,

Kumamoto

University

School

of Medicine,

I- I- I Honjo,

Kumamoto

860, Japan

Received 12 August 1994; revision received 8 November 1994; accepted 17 November 1994

Abstract To elucidate whether p-migrating very low density lipoproteins (P-VLDL) induce foam cell formation in mesangial cellsor not, surfacebinding and foam cell formation with /?-VLDL were studied in mousemesangialcells. Specific binding kineticsfor p-VLDL and low density lipoproteins(LDL) on the mesangialcellswere observedwith Kd = 3.8 and 13.7 ,ug/ml, and B,,,= 65.9 and 71.9 ng/mg cell protein at 4°C respectively. The binding of fi-VLDL was inhibited by excessamountsof LDL or P-VLDL, but not by acetyl-low density lipoproteins. Ligand blotting using j’-VLDL or LDL and immunoblotting using anti-human LDL receptor monoclonal antibody detected the same apparentsingleprotein (approx. 130kDa). Incorporation of [i4C]oleateinto cholesterylesterin mousemesangialcells was enhancedby P-VLDL to 3-fold higher than that by LDL, and it was inhibited by chloroquine or anti-human LDL receptor monoclonal antibody. The light microscopic findings also demonstrated that cholesteryl ester deposition increasedin these cells incubated with j?-VLDL, but not with LDL. In conclusion, p-VLDL was specificallytaken up by receptor-mediatedendocytosisin mousemesangialcellsthrough LDL receptors,resultingin foam cell formation. Keywords: Mesangialcells;D-Migrating very low density lipoproteins; Foam cell formation; Low density lipoprotein receptors

1. Introduction

Mesangial cell lipid accumulation is a recognized feature of glomerular disease and has been implicated as a factor in the pathogenesis of renal * Corresponding author, Tel.: 81-96-373-5169, Ext. 5883; Fax: 81-96-366-8397.

injury [l-5]. Recently, Coritsidis et al., Fernando et al. and Gupta et al. demonstrated a scavenger function for mesangial cells with respect to oxidized-low density lipoproteins (oxidized-LDL) and acetyl-low density lipoproteins (acetyl-LDL) [6-91. In a previous study, we indicated biochemically and morphologically that acetyl-LDL receptors were present in rat mesangial cells, indicating

0021-9150/95/$09.50 0 1995 El sevier Science Ireland Ltd. All rights reserved SSDI 0021-9150(94)05476-Y

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the scavenger function of rat mesangial cell-like macrophages [lo]. These findings indicate that mesangial cells take up modified lipoproteins such as oxidized-LDL and acetyl-LDL, resulting in mesangial cell lipid accumulation. However, it remains to be clarified whether p-migrating very low density lipoproteins (p-VLDL) induce foam cell formation in mesangial cells or not. To our knowledge, only Griine et al. reported that rabbit P-VLDL was bound, internalized, and degraded by human mesangial cells [l 11. Therefore, in the present experiments, surface binding, ligand blotting and immunoblotting, cholesteryl ester formation, and oil red 0 staining were carried out using the 5th passages of cultured mouse mesangial cells, in which no macrophage-like mesangial cells were contaminated.

2. Materials

and methods

2.1. Preparation

and radiolabeling

of lipoproteins

Preparation and radiolabeling of lipoproteins was performed as described elsewhere [10,12]. Briefly, human LDL (d = 1.019-1.063 g/ml) was isolated from the plasma of healthy subjects by ultracentrifugation. ,8-VLDL (d < 1.006 g/ml) was isolated by ultracentrifugation from the plasma of New Zealand white rabbits fed 0.5% cholesterol diet. An agarose electrophoretogram of isolated lipoproteins demonstrated broad ,8band. Acetyl-LDL was prepared by the method of Basu et al. [13]. These lipoproteins were radiolabeled with [1251]Na (Amersham, Buckinghamshire, UK) by the iodine monochloride method and separated from unreacted iodine using a GF-5 column (Pierce Chemical Co., Rockford, IL) [14,15]. More than 95% of the labeled ligand was precipitable by trichloroacetic acid. Specific radioactivities for the lipoproteins were 200-600 counts/min per ng protein. These lipoproteins were stored at 4°C and used in the experiments within 1 week. Storage of labeled lipoproteins within 1 week did not damage lipoproteins. Protein assay was performed by the protein assay method (Pierce Chemical Co., Rockford, IL).

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2.2. Cells Isolation of glomeruli was performed by the method of Mori et al., with slight modification [ 10,161. Female DDY mice weighing approximately 30 g were anesthetized with diethyl ether. The kidneys were removed and the cortices were pressed through a series of decreasing pore-size (150, 106 and 63 pm) sieves. The tissue remaining on the finest sieve was rinsed twice in Hank’s balanced salt solution (HBSS: Nissui Pharmaceutical Co., Tokyo, Japan) and centrifuged. The pellet was incubated in 5 ml of HBSS containing 750 units/ml collagenase (Sigma Chemical Co., St. Louis, MO) for 30 min at 37°C. After incubation, digested glomeruli were washed three times and passed over a 38-pm pore-size sieve. These glomerular cores were cultured at 37°C in RPM1 1640 (Life Technologies, Inc., Grand Island, NY) with 20% fetal calf serum (FCS), 100 units/ml penicillin, 10 pg/ml streptomycin and ITS premix (Becton Dickinson Lab., Bedford, MA) in 75 cm2 plastic tissue culture &asks in a 5% CO, incubator. In these cultured mesangial cells after five serial passages, no contamination of circulating macrophages was confirmed by immunohistochemistry using the specific monoclonal antibody of macrophages as previously described [lo]. Mesangial cells were subcultured at 2-week intervals and used between the fifth and tenth passages for experiments. Mesangial cells were plated onto 24 well plates at 20 000 cells/well and grown in RPM1 1640 with 20% FCS, 100 units/ml penicillin, 10 pg/ml streptomycin and ITS premix to confluence. Cells were incubated for 36 h in lipoprotein-deficient medium which consisted of DMEM (Nissui Pharmaceutical Co., Tokyo, Japan) containing 3% bovine serum albumin (BSA). Then, each experiment was performed. 2.3. Assay of surfuce binding

Binding assay was performed by the method of Goldstein et al. with slight modification [10,17]. The reaction mixture, in a total volume of 1 ml, contained confluent cells and O-100 pug of ‘251-labeled lipoproteins with or without unlabeled lipoproteins (50-fold excess of labeled lipoproteins) in DMEM containing 3% BSA. Binding was achieved by a 2-h incubation at 4°C on a mi-

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mixer. After incubation, cultured croplate medium was discarded and cells were washed three times with 1 ml of ice-cold PBS (pH 7.4) containing 0.5% BSA, followed by three washes with 1 ml of ice-cold PBS (pH 7.4). The cells were dissolved twice in 0.5 ml of 0.1 N NaOH by incubation at 37°C for 30 min. The cell suspension was removed quantitatively from the well by trituration with a cell scraper. One aliquot (OSml) of the cell suspension was used for counting radioactivity by a gamma counter and another aliquot (50-~1) was used for the assay of cellular protein. For lipoprotein competition experiments, unlabeled lipoproteins were added at increasing concentrations along with a constant amount of [‘2s1]/?-VLDL or [12’I]LDL. In addition, to determine whether the binding site on mouse mesangial cells is down-regulated or not, preloading studies with fi-VLDL or LDL were performed. Mesangial cells were incubated for 24 h at 37°C with DMEM containing 3% BSA alone and with the addition of 500 pg cholesterol/ml of lipoproteins for 24 h at 37°C prior to the experiment. The cells were then washed three times with PBS and incubated for 1 h at 37°C to internalize bound lipoproteins. Surface binding assay was performed with 50 pg/ml of [“‘I]lipoproteins (saturation dosage) for 2 h at 4°C. 2.4. Ligand blotting and Immunoblotting Cells were homogenized in Buffer A (50 mM Tris-HCl, pH 8.0, containing 160 mM NaCl, 2 mM EDTA, 3 mM phenylmethanesulfonyl fluoride, and 100 units/ml Aprotinin) at 4°C. After centrifugation at 1500 x g for 10 min, the extract was centrifuged at 100 000 x g for 1 h [18]. Membrane pellets were mixed with sample buffer and heated for 10 min at 60°C. Aliquots of these samples (100 pg) were applied to 4-12% sodium dodecyl sulfate-polyacrylamide gradient gels (TEF Co., Nagano, Japan) in the Laemmli buffer system [ 191. After electrophoresis, samples were transferred to the nitrocellulose membrane according to the method of Towbin et al. [20]. Then the nitrocellulose membrane was incubated in blocking buffer (50 mM Tris-HCl, pH 8.0, containing 2 mM CaCl,, 50 mg/ml BSA, and 90 mM NaCl) at 4°C for 14 h. Immunoblotting was

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performed using 10 lug/ml of anti-human LDL receptor monoclonal antibody, followed by 1251labeled protein A (Amersham, Buckinghamshire, UK). Ligand blots were obtained by incubating the nitrocellulose membrane with fresh blocking buffer containing 10 fig/ml ‘251-labeled lipoprotein for 2 h at room temperature. Each membrane was washed five times at room temperature with washing buffer (50 mM Tris-HCl, pH 8.0, containing 2 mM CaCl,, and 90 mM NaCl) [21]. Autoradiograms were obtained by exposing the dried membrane to Fuji X-Ray film for 8-20 h at - 80°C with a Cronex Lightning Plus enhancing screen (DuPont, Boston, MA). 2.5. Assay of cholesteryl ester formation The assay procedure was performed by using the method of Goldstein et al. with slight modification [10,22]. Cells were incubated at 37°C for 24 h in a 5% CO2 incubator in 24 well plates containing 1 ml of DMEM with 3% BSA and various amounts of lipoproteins (O-100 pg/ml) plus 10 ml of sodium [14C]oleate-BSA conjugate (84 &i/pl). After incubation, each well was washed three times with PBS containing 0.5% BSA (pH 7.4), followed by three washes with PBS (pH 7.4). An aliquot (0.5-ml) of hexane/isopropanol (3:2) was added to each well and incubated twice for 30 min at room temperature. The organic solvent extract was transferred into a glass tube. The solvent was evaporated to dryness under N, gas, and the lipids in each tube were resuspended in 60 ~1 of isopropanol and spotted on a silica gel G plate for thin-layer chromatography. The chromatogram was developed in hexane/ethyl ether/methanol/ acetic acid (85:20:1:1 by vol.), and the cholesteryl ester spot was identified with iodine vapor (R, = 0.9), cut from the chromatogram, and counted in 3 ml of Sintisol EX-HTM (Dojindo Laboratories, Kumamoto, Japan). The cells were dissolved in 1 ml of 0.1 N NaOH and aliquots (50 ~1) were used for protein assay. In addition, to determine whether P-VLDL was taken up through a pathway similar to the LDL receptor-mediated pathway, mesangial cells were preincubated with various concentrations of anti-human LDL receptor monoclonal antibody (Oncogene science, Inc., Unionda, NY) and incorporation of [‘4C]oleate

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0

3 F

0

50 Bound

et al. I Atherosclerosis

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1

loo

20406080100

[’ 251]-p-VLDL (&ml)

123-132

'=,

0

20

0

50 Bound

40

60

loo

60

100

[1251]-LDL (&ml)

Fig. 1. Surface binding of p-VLDL (left) and LDL (right) to mesangial cells. Confluent mesangiual cells were incubated for 2 h at 4°C with 0- 100 pg of [‘*‘1],9-VLDL or [‘2SI]LDL. The non-specific binding (A) determined in the presence of 50-fold excess unlabeled B-VLDL or LDL was subtraced from the total binding (O), resulting in the specific binding (0). Each point represents mean value of three duplicate determinations.

into cellular cholesteryl ester in mesangial cells was measured after the incubation with lipoproteins. 2.6. Oil red 0 staining Cells were plated in chamber slides for tissue culture (Nunc, Inc., Naperville, IL) at approximately 20 000 cells/ml and incubated in DMEM containing 3% BSA and 100 pg/ml of lipoproteins with or without 10 PM probucol, changing the medium with lipoproteins every 3 days as described elsewhere [lo]. After 1, 2 or 4 weeks of incubation, chamber slides were washed three times by PBS and the chamber was removed from the slide. Samples fixed with paraformaldehyde vapor for 10 min were stained with oil red 0 for 10 min and counterstained with hematoxylin for 5 min. Then the samples were examined by light microscopy. 3. Results

3.1. Binding of lipoproteins to mesangial cells Dose-dependencies of the lipoprotein binding to mesangial cells were measured at 4°C. As shown in Fig. 1, the existence of a specific binding

site for j?-VLDL or LDL was demonstrated. The specific binding kinetics for p-VLDL and LDL were obtained by Scatchard’s analysis (& = 3.8 and 13.7 pg/ml, B,,,= 65.9 and 71.9 ng/mg cell protein), respectively. To determine whether /3-VLDL and LDL bind to the same binding site or not, competitive binding experiments were performed using the mesangial cells. As shown in Fig. 2, the bindings of [‘251]LDL and [1251]jZ-VLDL were strongly inhibited by excess unlabeled LDL or p-VLDL, but not by excess acetyl-LDL. The binding of [12’I]LDL on mesangial cells was suppressed by preloading with LDL, but that of [‘251]p-VLDL was poorly suppressed by preloading with P-VLDL (Table 1). 3.2. Identljication of receptors for p-VLDL on mesangial cells by ligand blotting and immunoblotting Receptors on the mesangial cells which bound B-VLDL were identified by ligand blotting. Ligand blotting using a-VLDL or LDL detected the same apparent single protein (approx. 130 kDa) in membrane pellets of the mesangial cells. In the immunoblotting experiment, a protein of the same

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II4 (1995)

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123- 132

kDa

c

D D ;

2w i d

40

g + C A c

20

0

z

w

a Q

40

iI = 3 =

ligand (pgIml)

0

0 100200300400soa

0100200300400500

Unlabeled

20

Unlabeled

ligand (pg/ml)

Fig. 2. Effects of unlabeled ligdnds on binding of [“‘I]~VLDL (left) and [lZ51]LDL (right) to mesangial cells. Confluent mesangial cells were incubated for 2 h at 4°C with 10 pg of [‘251]b-VLDL or [“sI]LDL in the presence of increasing amounts of unlabeled P-VLDL ( l ), LDL ( n ) or acetyl-LDL (A). Binding is expressed as the percentage of the value obtained in the absence of unlabeled ligands. Each point represents mean value of three duplicate determinations.

apparent molecular weight reacted with anti-human LDL receptor monoclonal antibody (Fig. 3). 3.3. Cholesteryl ester formation in mesangial cells To evaluate the ability of /3-VLDL to stimulate cholesteryl ester formation in mesangial cells, incorporation of [‘4C]oleate into cholesteryl ester was studied. As shown in Fig. 4, /J-VLDL markedly stimulated incorporation of [‘4C]oleate Table 1 Surface bindings of [‘251]P-migrating very low density lipoprotein (p-VLDL) and [‘2sI]low density lipoprotein (LDL) to mouse mesangial cells after preincubation with /?-VLDL or LDL Preincubated lipoprotein

[‘251]a-VLDL bound (ng/mg cell protein)

[12’I]LDL bound (ng/mg cell protein)

None p-VLDL LDL

70.5 + 4.7 (0%) 45.5 * 3.1 (35.4%) 43.8 * 3.5 (37.9%)

71.2 k 5.0 (0%) 37.0 * 3.3 (48.0%) 33.1 +- 2.6 (53.5%)

Mesangial ceils were incubated for 24 h at 37°C with DMEM containing 3% BSA alone and with the addition of 500 fig cholesterol/ml of lipoproteins for 24 h at 37°C prior to the experiment. Surface binding assay was performed with 50 pg/ml of [‘251]lipoproteins for 2 h at 4°C. Values are mean k SE. of five experiments with percentage of inhibition given in parentheses.

A

B

C

Fig. 3. Ligand blotting of P-VLDL and LDL, and immunoblottting of anti-LDL receptor antibody on mesangial cells. Mesangial cells were prepared, electrophoresed, and transferred to nitrocellulose membrane as described under Materials and Methods. The scale gives migrations of molecular weight standards. In ligand blotting, the blot of cell proteins was incubated with 10 pg of [‘“‘I]B-VLDL (A) or [‘251]LDL (B). In immunoblotting (C), the blot was incubated with anti-human LDL receptor monoclonal antibody, followed by the incubation with [“51]protein A.

into cellular cholesteryl ester in mesangial cells as compared with LDL. At a ligand concentration of 100 pg/ml, a rate of cholesteryl ester formation induced by /J’-VLDL was approximately 3-fold higher than that by LDL. The cholesteryl [‘4C]oleate synthesis was completely inhibited by the addition of 100 PM chloroquine. In addition, to determine whether the LDL receptors on mesangial cell surface membranes were responsible for the receptor-mediated endocytosis of j?-VLDL, an experiment of inhibition was carried out using anti-human LDL receptor monoclonal antibody. As shown in Fig. 5, cholesteryl ester formation induced with /I-VLDL or LDL was dose-dependently inhibited with loading anti-human LDL receptor monoclonal antibody in mesangial cells.

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3.4. Light microscopic examination of lipid accumulation in mesangial cells

Fig. 6 demonstrates the light microscopic findings by oil red 0 staining of mesangial cells incubated with /?-VLDL or LDL for 1, 2 and 4 weeks. With time, almost all of the mesangial cells incubated with p-VLDL showed abundant deposits stained with oil red 0 in the cytoplasm, and there were striking alterations in the morphology of these cells. On the contrary, cells incubated with LDL showed little oil red 0 positive deposits. Thus, foam cell formation was microscopically recognized in mesangial cells incubated with p-VLDL. There was no significant change in foam cell formation with or without 10 PM of probucol (data not shown). 4. Discussion

We previously reported biochemically and morphologically that rat mesangial cells took up acetyl-LDL by receptor-mediated endocytosis, and that cholesterol in acetyl-LDL was converted to cholesterol ester, resulting in an increased cellu-

0 0

20

40

60

60

100

Ligand (&ml) Fig. 4. Cholesteryl ester formation in mesangial cells incubated with /?-VLDL and LDL. Confluent mesangial cells were incubated with 10 pg/ml of [“‘Cloleate-BSA conjugate and 0- 100 ,cg of /?-VLDL or LDL for 24 h at 37°C. Incorporation of [“Tloleate into cholesteryl ester by B-VLDL (0) or LDL (W) was examined. Open circle (0: /?-VLDL) and square (0: LDL) represent the data obtained by the addition of 100 PM chloroquine to the incubation medium. Each point represents mean value of three duplicate determinations.

0 0

I 50

I loo

I 160

206

Anti-LDL receptor antibody (&ml) Fig. 5. Inhibition of cholesteryl ester formation by anti-human LDL receptor monoclonal antibody in mesangial cells. Confluent mesangial cells were preincubated with antibody to the LDL receptor at 4°C for 2 h. Cells were incubated for 24 h at 37°C with 10 fig of /I-VLDL or LDL on 10 fig/ml of [‘4C]oleate-BSA conjugate. Incorporation of [“‘Cloleate into cholesteryl ester by /?-VLDL (0) or LDL (W) is represented by mean value of three duplicate determinations.

lar cholesterol content [lo]. This finding partially explained the mechanism of foam cell formation in cultured rat mesangial cells. However, it remains to be clarified whether p-VLDL induces foam cell formation in mesangial cells or not. In this experiment, it was clearly demonstrated that p-VLDL was specifically taken up by receptormediated endocytosis in mouse mesangial cells, resulting in foam cell formation. It is well known that p-VLDL is specifically bound and taken up by receptor-mediated endocytosis in mouse peritoneal macrophages and mouse macrophages-derived cells [23,24]. Koo et al. reported that in the binding experiment with mouse peritoneal macrophages, specific binding kinetics for /3-VLDL and LDL were Kd = 0.7 and 32.9 pg/ml, respectively [23]. In our binding experiment with mouse peritoneal macrophages, specific binding kinetics for p-VLDL and LDL were K,, = 5.3 and 18.1 pug/ml, and B,,,,, = 68.3 and 76.7 ng/mg cell protein, respectively (unpublished data). The difference of Kd values between their experiments and ours might be due to the

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Fig. 6. The light microscopic examinations of mesangiai ceils incubated with j’-VLDL (D: 4 weeks), and stained with oil red 0. ( x 200)

different strains of mouse employed and different experimental conditions. In their experiment of ligand blotting and immunoblotting with these cells, ligand blot identified a single protein crossreacted with antibodies against bovine LDL receptors [23]. We also demonstrated that ligand blot identified a single protein cross-reacted with anti-human LDL receptor monoclonal antibody. These results revealed that p-VLDL might bind to LDL receptors in mouse peritoneal macrophages. Innerarity and Mahley demonstrated that lipoproteins contained multiple copies of apo E bound to LDL receptors with up to 20-fold higher affinity than LDL, which contained only one copy of apo B [25]. From these results, it has been considered that the binding affinity to the LDL receptors for pVLDL, apo E-rich lipoproteins, is higher than that for LDL in mouse peritoneal macrophages ~231.

In our cultured mesangial cells after 5th serial passages, no contamination of circulating macrophages and other cells, such as endothelial cells and epithelial cells, was evident on the basis

129

(A: I week, B: 2 weeks, C: 4 weeks) or LDL

of immunohistochemistry using specific monoclonal antibody to macrophages, immunohistochemical staining for desmin and vimentin, and negative staining for cytokeratin, as reported previously [lo]. The present study in mouse mesangial cells showed that the binding affinity (IQ for /3VLDL was 3.5-fold higher than that for LDL, while maximal capacity (B,,,) for binding of /?VLDL was similar to LDL. Ligand blotting using /?-VLDL or LDL and immunoblotting using anti-human LDL receptor monoclonal antibody detected a same apparent single protein (approx. 130 kDa). These experimental results of surface binding, ligand blotting and immunoblotting for fi-VLDL and LDL in mouse mesangial cells were quite similar to those in mouse peritoneal macrophages. This higher affinity for /3-VLDL than LDL may be explained by the difference of ligands (apo E and apo B) bound to LDL receptors. As observed in mouse peritoneal macrophages, it is considered that /?-VLDL might also specifically bind to the LDL receptors in mouse mesangial cells.

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It has been known that the heterologous lipoproteins bind with a lower affinity than the homologous lipoproteins [23,26], Wheeler et al. reported that the. binding affinity of human LDL to rat mesangial cells (K,, = 22.7 pg/ml) was lower than that of rat LDL (K,, = 1.3 pg/ml) [26]. Grijne et al. reported human LDL bound to human mesangial cells with a Kd value of 43.9 @g/ml [ll]. But, our data indicated that human LDL bound to mouse mesangial cells with a Kd value of 13.7 pg/ml. A question arises why the human mesangial cells have a low affinity for human LDL as compared with the binding affinity of human LDL to mouse mesangial cells and rat mesangial cells. In GrBne’s experiment, the assay of surface binding was studied at a temperature of 37°C. In our study, the assay was studied at 4°C. So, this difference might be due to the different experimental conditions. Recently, Takahashi et al. reported the presence of the specific receptors for apo E-containing lipoproteins (VLDL receptors) by isolating a cDNA from rabbit heart. This VLDL receptor mRNA was also abundant in tissues performing active fatty acid metabolism, including heart, muscle and adipose tissue, and a small content of this mRNA was detected in kidney [27]. In addition, Sakai et al. reported that VLDL receptor mRNA was present in human monocytic leukemia cells, THP- 1, and its expression was not down-regulated by sterols [28]. Further studies are required to clarify whether mesangial cells have specific VLDL receptors, and P-VLDL is partially taken up through VLDL receptors. In the experiment of cholesteryl ester formation, a-VLDL markedly stimulated incorporation of [14C]oleate into cellular cholesteryl ester in mouse mesangial cells as compared with LDL. At ligand concentration of 100 pgg/ml, a rate of cholesteryl ester formation, induced by p-VLDL, was approximately 3-fold higher than that by LDL. In addition, abundant foam cells were microscopically recognized in these cells incubated with /?-VLDL, but not with LDL. A question arises why foam cell formation is induced by p-VLDL, but not by LDL, despite the fact that both lipoproteins are taken up through the LDL receptor-mediated pathway in mesangial

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cells. The following reasons are considered as possible explanations for such a question. Firstly, the cholesterol content of p-VLDL from cholesterol-fed rabbits was 5.0-10.0 pg/pg protein and that of LDL was 2.0-3.0 ,ug/pg protein. Thus, cholesterol content of p-VLDL might be enough to induce cholesteryl ester formation, but that of LDL is too low. Secondly, the LDL receptors might not be effectively down-regulated by p-VLDL in mouse mesangial cells. Our present data indicated that the binding of /?-VLDL on mouse mesangial cells was poorly suppressed by preloading with BVLDL as compared with that of LDL (percentage of inhibition: 35.4 vs. 53.5%, respectively). Koo et al. demonstrated that LDL receptors on mouse peritoneal macrophages were significantly less sensitive to down-regulation than those on human fibroblasts [23]. Our recent data also indicated that the binding of p-VLDL on human fibroblasts was similarly suppressed by preloading with /3-VLDL as well as that of LDL (percentage of inhibition: 49.2 vs. 55.3%, respectively, unpublished data). Further studies would be required to clarify the mechanism where LDL receptors are not effectively down-regulated when mouse mesangial cells take up /I-VLDL. Thirdly, Tabas et al. reported that in mouse peritoneal macrophages, endocytosed /?-VLDL appeared in a distinct set of widely-distributed vesicles not acylseen with LDL and stimulated CoA:cholesterol acyltransferase (ACAT) activity, resulting in increased cholesteryl ester synthesis as compared with LDL despite the existence of down-regulation [29]. Finally, p-VLDL may be oxidized by mesangial cells and taken up partially from the scavenger receptors. Parthasarathy et al. suggested that /?-VLDL was highly sensitive to oxidative stress induced by endothelial cells and by copper ions, and that the oxidized-j?-VLDL stimulated cholesteryl esterification twice as much as unoxidized-P -VLDL in mouse peritoneal macrophages [30]. However, in our experiment with light microscopic examination, no significant change in foam cell formation induced by /3VLDL was observed in the presence or absence of an anti-oxidant, probucol.

T. Nishikawa et al. /Atherosclerosis I14 (1995) 123-132

In the previous study, we demonstrated the existence of acetyl-LDL receptors, through which acetyl-LDL induced foam cell formation in rat mesangial cells. In the present experiment with mouse mesangial cells, a rate of cholesteryl ester formation induced by acetyl-LDL was one-third of that by /?-VLDL. In the experiment with light microscopic findings, /? -VLDL also induced abundant foam cells, but they were scanty with acetyl-LDL (data not shown). In conclusion, the present study indicated biochemically and morphologically that p -VLDL was specifically taken up by receptor-mediated endocytosis through the LDL receptors, and that cholesterol in p-VLDL was converted to cholesteryl ester, resulting in foam cell formation in mouse mesangial cells. The foam cell formation by a-VLDL through LDL receptors was more marked in mouse mesangial cells than that by acetyl-LDL through scavenger receptors. Acknowledgements We gratefully appreciate the helpful advice and assistance of Professor Seikoh Horiuchi of the Department of Biochemistry and Mr. Kenshi Ichinose in our laboratory. This work was supported in part by research grants from the Scientific Research Fund of the Ministry of Education, Science and Culture, Japan (No. 06671041). References [I] Hiramatsu M, Karashima S, Hattori S, Matsuda I, Maeda H. A case of congenital nephrotic syndrome associated with partial deficiency of lecithin: cholesterol acyltransferase (LCAT) and hypothyroidism. lnt J Pediat Nephrol 1984;5:183. [2] McKenzie IFC, Kincaid-Smith P. Foam cells in the renal glomerulus. J Pathol 1969;97: 15 1. [3] Zollinger HU, Rohr H-P. Struktur und Bedeutung der renalen Schaumzellen. Virchows Arch (A) Pathol Anat Histol 1969;348:295. [4] Amatruda JM, Margolis S, Hutchins GM. Type III hyperlipoproteinemia. Arch Path01 1974;98:51. [5] Sheehan HL. Renal morphology in preeclampsia. Kidney lnt 1980;18:241. [6] Coritsidis G, Rifici V, Gupta S, Rie J, Shan Z, Neugarten J, Schlondorff D. Preferential binding of oxidized LDL to rat glomeruli in vivo and cultured mesangial cells in vitro. Kidney lnt 1991;39:858.

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