Introduction Materials and methods

Atherosclerosis 150 2000 357 – 363 Calcium and lipoprotein lipase synergistically enhance the binding and uptake of native and oxidized LDL in mouse peritoneal macrophages Xiaosong Wang, Joachim Greilberger, Gu¨nther Ju¨rgens Institute of Medical Biochemistry, Karl-Franzens Uni6ersita¨t Graz, A- 8010 Graz, Austria Received 7 June 1999; received in revised form 9 September 1999; accepted 29 September 1999 Abstract The influence of Ca 2 + and Mg 2 + , together with lipoprotein lipase LPL, on the binding and uptake of Eu 3 + -labeled native and oxidized low density lipoprotein LDL to mouse peritoneal macrophages MPM, and on the deposition of esterified cholesterol in these macrophages, were studied. We found that both LPL and Ca 2 + but not Mg 2 + increased the binding and uptake of native and mildly or moderately oxidized LDL, and the subsequent deposition of cholesterol esters in MPM. When added together, LPL and Ca 2 + synergistically increased the binding and uptake of native and oxidized LDL, and the deposition of esterified cholesterol derived from native and mildly or moderately oxidized LDL, in MPM. Since both calcium and LPL are found in the atherosclerotic lesions, our results suggest that Ca 2 + and LPL may synergistically promote foam cell formation and atherogenesis. Furthermore, future research in the metabolism of lipoproteins should take into account the calcium levels in the experimental conditions. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Low density lipoprotein; Lipoprotein lipase; Calcium; Macrophages; Oxidation www.elsevier.comlocateatherosclerosis

1. Introduction

Lipoprotein lipase LPL is the major enzyme re- sponsible for the hydrolysis of triglycerides in circulat- ing chylomicrons and very low density lipoproteins VLDL. Aside from this enzymatic function, LPL was found to act as a bridge linking apoB of low density lipoprotein LDL with cell surface heparan sulfate proteoglycans HSPG and the LDL receptor related protein LRP [1 – 3]. In this way, LPL could enhance the binding and uptake of LDL by macrophages and smooth muscle cells. It was shown that oxidation of LDL modified the structural and functional properties of this lipoprotein [4,5]. Binding of oxidized LDL followed by the uptake in macrophages in an unregulated fashion would lead to the formation of foam cells, the hallmark of the formation of atherosclerotic lesions [6]. Recently, it was shown that exogenously added bovine LPL [7] and LPL produced by macrophages [8] also stimulated the bind- ing and uptake of moderately oxidized LDL by macrophages. The deposition of calcium was found in human atherosclerotic lesions, and at the end stage of atherosclerosis some areas of the intima are calcified. Calcium was found to increase the binding of native and oxidized LDL to extracellular matrix [9] and the binding of VLDL to subendothelial cell matrix [10]. Therefore, it was of pathophysiological importance to investigate whether Ca 2 + could influence the foam cell formation, and what is its effect on LPL-mediated binding and uptake of native and oxidized LDL in macrophages. Corresponding author. Tel.: + 43-316-3804195, fax: + 43-316- 3809615. E-mail address : guenther.juergenskfunigraz.ac.at G. Ju¨rgens 0021-915000 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 9 9 0 0 4 1 3 - X

2. Materials and methods

2 . 1 . Lipoprotein preparation LDL and lipoprotein-deficient serum LPDS were isolated from the plasma of normolipidemic human donors with serum lipoproteina levels lower than 1 mg100 ml, by differential ultracentrifugation at density ranges between 1.020 and 1.050 gml and \ 1.235, respectively, as previously described [9]. Protein concen- tration of LDL was measured by the method of Lowry et al. [11] using bovine serum albumin BSA as stan- dard. LDL concentration in this study is referred to as its protein content. 2 . 2 . Labeling of LDL with Europium 3 + Eu 3 + Eu 3 + -labeling of LDL was performed as described [9]. Briefly, 2 mg LDL in 50 mM NaHCO 3 pH 8.5, containing 20 m M Trolox 2-carboxy-2,5,7,8-te- tramethyl-6-chromanol, a water-soluble vitamin E derivative from Hofmann LaRoche, was incubated with 0.2 mg Eu 3 + -chelate of N 1 -p-isothiocyanatoben- zyl-diethylentriamine-N 1 , N 2 , N 3 , N 3 -tetraacetic acid DELFIA Eu-labeling kit; Wallac Oy at 25°C in the dark for 12 h. Sephadex G-25 chromatography Phar- macia Biotech was used for the separation of the labeled protein from free europium in 50 mM Tris – HCl pH 7.8, containing 0.05 NaN 3 and 20 mM Trolox. The labeling yield of Eu 3 + -LDL was between 4 and 22 Eu 3 + protein molmol. 2 . 3 . Cu 2 + -mediated oxidation of LDL and Eu 3 + -LDL Prior to oxidation, LDL and Eu 3 + -LDL were dia- lyzed against 10 mM PBS pH 7.4. Cu 2 + -mediated oxidation of LDL 500 mgml was performed at 37°C with 30 mM CuCl 2 . At intervals between 0 and 24 h the reaction was terminated by adding a stop solution to achieve a final EDTA concentration of 0.27 mM. The samples were saturated with nitrogen and stored at 4°C. The degree of the oxidative modification of LDL was estimated as the relative electrophoretic mobility REM to the respective labeled and non-labeled native LDL on 1 agarose gels at pH 8.05 using the Lipi- dophor-system Immuno AG. In our experiments, LDL and Eu 3 + -LDL which were oxidized for 1 and 3 – 4 h revealed REM values around 1.3 and 2.4, respec- tively. They were designated mildly 1 h and moder- ately 3 or 4 h oxidized LDL, respectively. LDL which was oxidized for 8 h REM value 3.0 – 3.3 and 24 h REM value 3.5 – 3.8 were designated strongly and extensively oxidized LDL, respectively. In some sam- ples lipidperoxides LPO were estimated by a spec- trophotometric assay with CHOD-iodide color reagent Merck at 365 nm, as developed in this laboratory [12]. 2 . 4 . Cell cultures Resident peritoneal macrophages from Balbc mice MPM were elicited by intraperitoneal injection of 2 ml of 3 thioglycollate medium Gibco BRL 3 days before harvesting. Primary cultures were prepared at a density of 1.5 × 10 5 well in 96-well plates Costar, Aus- tria, in RPMI-1640 medium Gibco BRL containing 10 fetal calf serum FCS Gibco BRL, 100 unitsml of penicillin and 100 mgml of streptomycin. Cells were maintained in a humidified incubator with 5 CO 2 at 37°C. Three hours after the plating, non-adherent cells were washed away with 10 mM PBS pH 7.4. The cells were cultured in the above medium overnight before use. Cell viability was greater than 98 as assessed by trypan blue exclusion. 2 . 5 . Purification and biotinylation of LPL LPL was purified from fresh unpasteurized milk us- ing Heparin-Sepharose Pharmacia chromatography as described by Sexana et al. [13], followed by affinity chromatography on a HiTrap Heparin column Phar- macia eluted with 10 mM phosphate buffer pH 6.8, containing 0.75 – 2 M NaCl. Purified LPL showed a major protein band of 55 kDa, and a minor band of 40 kDa in some fractions, when analyzed by SDS- PAGE and stained with Coomassie Blue. Using an anti-LPL monoclonal antibody the 55 kDa band was visible in Western blot analysis, while the smaller 40 kDa band was not shown. This 40 kDa band corresponds to the digested LPL fragment suggested by Hendriks et al. [14]. Purified LPL was biotinylated using D -biotin-N-hydroxysuccinimide ester Boehringer Manheim, Germany, as described [15]. The biotin-la- beled LPL was 55 kDa in size as checked with SDS-PAGE, and enzymatically active. When biotin-la- beled LPL was transblotted to nitrocellulose after SDS- PAGE, and then detected with peroxidase-labeled streptavidin, a major 55 kDa band was found. 2 . 6 . Cellular binding and association studies Cell binding and association studies were carried out using Eu 3 + -labeled native and oxidized LDL [16]. This non-radioactive time-resolved fluorometric assay can avoid the potential lipid peroxidation of LDL brought about by labeling lipoproteins with the widely used radioactive iodine, which is of special importance when one wants to compare the metabolism of native and mildly oxidized LDL [16], as in this study. Cell binding and association studies were carried out in RPMI-1640 medium Gibco containing 10 LPDS, 25 mM HEPES, pH 7.4, by incubating the cells with RPMI-1640 medium containing Eu 3 + -labeled native or oxidized LDL for 4 h at 4°C binding, in which the lipoproteins bound to the cell membrane or 37°C cell association, in which lipoproteins are associated with both cell membrane and intracellular compartments, in the absence or presence of exogenous LPL. Commer- cially purchased RPMI-1640 medium normal RPMI- 1640 medium contains 0.42 mM Ca 2 + and 0.4 mM Mg 2 + . CaCl 2 · 2H 2 O and MgCl 2 · 6H 2 O were added to this medium to achieve the desired final concentrations of Ca 2 + and Mg 2 + . At the concentrations of Ca 2 + and Mg 2 + used, there was no precipitation in the medium as checked by absorption at 450 nm. In some experi- ments, 1.34 mM EDTA was added to the medium to chelate Ca 2 + and Mg 2 + in the medium. After washing, the cells were dissolved with Triton X-100 0.05. Fluorescence in the cell lysate was measured in tripli- cates in enhancement solution with a VICTOR™ Mul- tilabel Fluorescence Counter Wallac Oy. Cell protein content was measured in duplicate with the method of Lowry et al. [11] using BSA as standard. Specific cellu- lar binding and association were calculated by subtract- ing the non-specific binding or association in the presence of an excess of unlabeled lipoproteins from those in the absence of unlabeled lipoproteins. Binding of biotin-labeled LPL to MPM was carried out in RPMI-1640 medium containing different concen- trations of Ca 2 + or Mg 2 + , for 2 h at 37°C. As control, biotin-labeled LPL was omitted from the medium. Af- ter washing with PBS, Eu 3 + -labeled streptavidin in RPMI-1640 medium was added to the cells and incu- bated for 30 min at 37°C. Cells were then washed and the fluorescence was measured as in cell binding and association studies. Specific binding was calculated by subtracting the fluorescence counts in the absence of biotin-labeled LPL from those in the presence of biotin- labeled LPL. 2 . 7 . Measurement of cellular cholesterol MPM were cultivated in 24-well plates Costar in RPMI-1640 medium containing 10 FCS. Twenty four hours before the experiment, RPMI-1640 medium sup- plemented with 10 human LPDS instead of FCS was added to the cells. At the start of the experiment, the cells were washed three times with RPMI-1640 medium. RPMI-1640 medium containing 1 BSA, 100 mgml of native and oxidized LDL, various concentrations of Ca 2 + or Mg 2 + , in the presence or absence of 10 mgml LPL, was then added to the cells and incubated for 24 h in a humidified incubator with 5 CO 2 at 37°C. Control incubations were performed in RPMI-1640 medium containing 1 BSA without other additions. At the end of the incubation, the cells were washed three times with PBS containing 0.1 BSA and two times with PBS. Cellular total and free cholesterol content were measured [17]. 2 . 8 . Binding of Eu 3 + -labeled nati6e and oxidized LDL to LPL An aliquot of 1.5 mg of LPL from bovine milk in 100 m l PBS was coated to each well of the microtitration plates Nunc at 4°C for 18 h. After three washes with PBS, each well was blocked with 200 ml PBS containing 3 BSA for 1 h at room temperature. The plates were washed three times with PBS, and then RPMI-1640 medium or Tris – HCl buffer containing 50 mM NaCl, 1 BSA, Eu 3 + -labeled native and oxidized LDL, and different concentrations of Ca 2 + or Mg 2 + was added to each well. After incubation for 1 h at 37°C, the wells were washed with PBS, and the fluorescence of bound Eu 3 + was measured in the presence of enhancement solution 200 mlwell. To measure the non-specific binding, bovine serum album instead of bovine milk LPL was coated to the plates, and the binding was measured the same way. The non-specific binding of Eu 3 + -labeled lipoproteins to BSA as compared to LPL was below 5, and was subtracted from the amount of binding to LPL.

3. Results