©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Oxidized Low Density Lipoprotein Reduces Thrombomodulin Transcription in Cultured Human Endothelial Cells through Degradation of the Lipoprotein in Lysosomes (*)

(Received for publication, November 6, 1995)

Hidemi Ishii (§) Keiichiro Kizaki Shuichi Horie Mutsuyoshi Kazama

From the Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui, Kanagawa 199-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Oxidized low density lipoprotein (LDL), a potent atherogenic lipoprotein, has been shown to cause the alteration of various endothelial functions. We have examined the effect of oxidized LDL on the cofactor activity for thrombin-dependent protein C activation and expression of thrombomodulin (TM), a cell surface antithrombotic glycoprotein, on cultured human umbilical vein endothelial cells. Oxidized LDL prepared by irradiation of LDL with 254-nm ultraviolet light did not directly affect the cofactor activity of isolated TM. Exposure of the cells to oxidized LDL (25-200 µg/ml), but not native LDL and acetylated LDL, reduced TM cofactor activity in parallel with its antigen levels on the cell surface in an oxidation-, concentration- and time-dependent manner. TM mRNA levels were reduced prior to decrease in TM antigen levels and were 50% of the control levels at 3.0 h after treatment of the cells with oxidized LDL. The apparent half-life time (t = 2.8 h) of TM mRNA in the oxidized LDL-treated cells, however, did not significantly differ from that (t = 2.6 h) in the control cells when the cells were coincubated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a transcriptional inhibitor. Treatment of the cells with bafilomycin A1, an inhibitor for the proton pump of the lysosomes, inhibited intracellular degradation of the LDL and prevented down-regulations of the mRNA and the cell surface TM antigen levels caused by oxidized LDL. The inhibitor molecule in oxidized LDL was shown to be a lipid; organic solvent extracts (300 mg/ml cholesterol, an equivalent concentration with lipids in 200 µg/ml oxidized LDL) of oxidized LDL inhibited expression of TM antigen to nearly the same extent as the oxidized LDL, although water extracts did not affect TM expression on the cells. These results suggested that down-regulation of TM on endothelial cells exposed to oxidized LDL resulted from inhibition of its transcription mediated by lysosomal degradation of oxidized LDL and that a lipid component in the LDL could be an active species. A decrease in TM expression on the surface of endothelial cells may contribute to promote thrombosis in atherosclerotic lesions.


INTRODUCTION

Vascular endothelial cells play an active role in the regulation of blood coagulation and fibrinolysis(1) . The thrombin-thrombomodulin (TM)(^1)-protein C pathway is of major physiologic significance for the antithrombotic properties of endothelial cells(2, 3, 4) . A key cofactor in this pathway is TM, which is a high affinity receptor for thrombin on the endothelial cell surface(2, 3, 4) . The TM-thrombin complex acts as a potent activator for circulating protein C. Activated protein C functions as an anticoagulant by proteolytically degrading the coagulation factors Va and VIIIa, which are essential for blood coagulation(2, 3, 4) . The physiologic relevance of this pathway for the antithrombotic properties of endothelial cells is emphasized by the thrombotic disorders frequently observed in patients with protein C-resistant factor V (5, 6) or protein C deficiency(7) .

The prothrombotic properties of endothelial cells are enhanced in response to bacterial endotoxin (8) and inflammatory cytokines such as interleukin 1 (9, 10, 11) or tumor necrosis factor-alpha(10, 11, 12, 13, 14, 15, 16, 17) . The major components of the increase in prothrombotic properties are a decrease in the expression of TM(8, 9, 10, 11, 12, 13, 14, 15, 16, 17) and induction of the expression of tissue factor(8, 9, 10, 12, 13, 17) , which functions as the cellular trigger for the coagulation cascade(18) . The down-regulation of TM activity is due to increased internalization of surface TM(11, 14) and/or inhibition of TM transcription (15, 16) by the inflammatory cytokines. The combination of reduced TM activity and increased tissue factor expression on the cell surface would be expected to promote coagulation and contribute to thrombosis in inflammatory disorders, such as septic shock, disseminated intravascular coagulation, and malignancy(2, 19) . Homocysteine also down-regulates TM activity on endothelial cells(20, 21) . Patients with hereditary homocystinuria have high levels of plasma homocysteine and suffer from frequent arterial and venous thrombosis(22) . The down-regulation of TM in response to homocysteine is due to direct inhibition of TM activity on the cell surface (20, 21) as well as inhibition of TM transport between the endoplasmic reticulum and Golgi apparatus after transcription(20) . Down-regulation of TM has been also reported in endothelial cells exposed to hypoxic conditions (23) or cyclosporine A(24) , which is associated with the development of post-transplantation glomerular thrombosis and thrombotic microangiopathy. Thus, down-regulation of TM on endothelial cells potentiates prothrombotic properties of the vascular wall.

Thrombosis is also frequently observed in atherosclerotic lesions. Low density lipoprotein (LDL) in plasma is a major risk factor for atherosclerosis, and the LDL can be oxidized by lipid peroxidation (25, 26) and accumulate in atherosclerotic lesions(27) . Oxidized LDL induces the expression of tissue factor (28) and reduces endothelial fibrinolytic activity through increased secretion of plasminogen activator inhibitor-1 and decreased secretion of tissue-type plasminogen activator(29) . The induction of tissue factor and increased secretion of plasminogen activator inhibitor-1 were mediated by increases in transcription levels(28, 29) . It was recently suggested that the cofactor activity of TM on the surface of endothelial cells also decreases in response to stimulation with oxidized LDL(30) . The decreased mechanism is, however, unknown with respect to whether oxidized LDL directly inhibits TM cofactor activity or decreases TM antigen levels on the cell surface. The potential mechanism for reduced TM activity on the surface of cultured human endothelial cells exposed to oxidized LDL is investigated in the present work.


EXPERIMENTAL PROCEDURES

Materials

Reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan), unless otherwise indicated. Bovine serum albumin (BSA), human thrombin (4000 NIH units/mg), and o-phenylenediamine were obtained from Sigma. 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was purchased from Calbiochem. Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were obtained from Flow Laboratories (Irvine, United Kingdom). t-Butoxycarbonyl-Leu-Ser-Thr-Arg-4-methylcoumarin-7-amide was purchased from Peptide Institute Inc. (Osaka, Japan). Human antithrombin III was donated by Green Cross Co. (Osaka, Japan). [alpha-P]dCTP and NaI were purchased from DuPont NEN. Protein C was isolated from bovine plasma according to the method of Hashimoto et al.(31) . Human placental TM was isolated by the method previously described(32) . Mouse monoclonal anti-human TM IgG and rabbit polyclonal anti-human TM IgG were prepared as reported previously(33) .

Cell Culture

Endothelial cells were harvested from human umbilical cord veins and were grown in DMEM supplemented with 20% heat-inactivated FCS, 72 units/ml penicillin, and 50 µg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO(2) as described previously(34) . Experiments were performed with confluent monolayers (within three passages) on gelatin-coated 96-well plates (Falcon; Becton Dickinson, Lincoln Park, NJ) or collagen-coated 60-mm diameter dishes (Corning; Iwaki Glass, Tokyo, Japan). For each experiment, cells were washed in serum-free medium and then incubated in DMEM supplemented with 10% FCS containing LDLs (native LDL, acetylated LDL, or oxidized LDL) and/or various compounds. After incubation for various periods, cell viability was determined by trypan blue dye exclusion. In all cases, trypan blue dye exclusion indicated that more than 90% of the cells remained viable after treatment with these inducers. It has been reported that oxidized LDL is cytotoxic to proliferating endothelial cells and fibroblasts in culture(35) . Therefore, we used the confluent monolayer of endothelial cells in the present experiments.

Preparation of LDL

LDL (density range 1.019-1.063 g/ml) was separated from fresh human plasma of healthy subjects by a sodium bromide stepwise density gradient centrifugation and dialyzed extensively against 10 mM phosphate-buffered saline (PBS; pH 7.4) containing 0.25 mM EDTA according to the method of Redgrave et al.(36) . Before oxidation or acetylation of LDL, EDTA was removed by chromatography on a Sephadex G-25 column (PD-10; Pharmacia Fine Chemicals, Uppsala, Sweden). The protein content was determined by the method of Lowry et al.(37) using BSA as a standard. The indicated amounts of LDL used in the experiments were based upon the LDL protein concentration. Cholesterol content was measured by using Monotest® cholesterol kit (Boehringer Mannheim). Oxidized LDL was prepared by short UV irradiation according to the method of Dousset et al.(38) . LDL (1 mg of protein/ml) was irradiated by UV light (254 nm, 0.4 mW/cm^2) for variable times (0-12 h). Oxidative modification of LDL was estimated as formation of thiobarbituric acid reactive substances (TBARS) and expressed as malondialdehyde equivalents per mg of protein using 1,1,3,3-tetraethoxypropane, a precursor of malondialdehyde, as a standard(39) . Acetylated LDL was prepared by the method of Basu et al.(40) . LDL (3 mg of protein/ml) was mixed with an equal volume of saturated sodium acetate, and then 1 µl of acetic anhydride/mg of LDL was added to the solution with continuous mixing for 90 min at 4 °C. The reaction solution was then dialyzed against PBS. The extent of acetylation was confirmed by agarose gel electrophoresis and oil red O staining, and the mobility of acetylated LDL was 2.9 ± 0.7 times faster than that of native LDL (n = 3). In addition, modification of lysine residues in acetylated LDL were measured by using trinitrobenzenesulfonic acid according to the method of Steinbrecher(41) , and more than 50% of lysine residues were modified. The various LDL preparations were not contaminated by endotoxin as assayed by the limulus lysate test (Wako Pure Chemical Industries).

Measurement of TM Cofactor Activity

TM cofactor activity for thrombin-dependent protein C activation on the cell surface was determined by measuring the amidolytic activity of activated protein C generated by the cell surface TM as described previously(17, 42) . LDLs and/or various compounds incubated with cultured human umbilical vein endothelial cells (HUVECs, 2 times 10^4 cells/well in 96-well plates) were removed, and the cells were washed 3 times with Hanks' balanced salt solution containing 0.2% BSA (HBS-BSA, pH 7.4). The washed cells were incubated with 100 µg/ml protein C, 1 NIH unit/ml thrombin, 1 mM CaCl(2) and 5 mg/ml BSA in 20 mM Hepes-NaOH (pH 7.4) containing 0.15 M NaCl for 30 min at 37 °C under an atmosphere consisting of 95% air and 5% CO(2). Activation of protein C was terminated by the addition of a mixture of antithrombin III (final concentration 2 units/ml) and heparin (final concentration 8 units/ml) in 20 mM Tris-HCl (pH 8.5) containing 0.1 M NaCl and 1 mM CaCl(2). A mixture of 0.2 ml of the conditioned medium and 0.2 ml of 400 µMt-butoxycarbonyl-Leu-Thr-Arg-4-methylcoumarin-7-amide, a synthetic substrate for activated protein C, was incubated in a test tube for 10 min at 37 °C, and the reaction was terminated by adding acetic acid to a final 10% (v/v) concentration. The liberated 7-amino-4-methylcoumarin was then measured using a spectrofluorophotometer (Hitachi 650-10S, Hitachi Co., Tokyo, Japan) with excitation at 380 nm and emission at 440 nm. The amounts of activated protein C in the reaction mixture were calculated using a standard curve established with known amounts of activated protein C.

The cofactor activity of TM isolated from human placenta was measured by the method described previously(33) . TM (200 ng/ml) was incubated with or without 200 µg/ml oxidized or native LDL in PBS for 24 h at 37 °C. After incubation, the solution was diluted 10-fold in assay buffer, which consisted of 100 µg/ml protein C, 2 NIH units/ml thrombin, 3 mM CaCl(2), 0.15 M NaCl, and 5 mg/ml BSA in 20 mM Tris-HCl (pH 7.4), and then the reaction mixture was further incubated for 30 min at 37 °C. Termination of protein C activation and quantification of activated protein C were performed as described above.

Measurement of TM Antigen on Cell Surface and in Cell Lysate

TM antigen levels on the cell surface were determined by the described previously(17, 42) . Briefly, the cell monolayer (96-well plates, 2 times 10^4 cells/well) was washed with HBS-BSA, and 200 µl of horseradish peroxidase-labeled monoclonal antibodies (TM mAb 2 and TM mAb 11) was placed in each well. After incubation for 10 min at room temperature, the cells were washed 3 times with HBS-BSA. The horseradish peroxidase activity of the antibodies bound to the cell surface was measured in the presence of 0.02% hydrogen peroxide and 0.5 mg/ml o-phenylenediamine in 0.1 M citrate buffer (pH 5.0) at room temperature for 10 min, and color development was terminated with 100 µl of 2 N H(2)SO(4). Results are shown as the relative expression (percentage) of cell surface TM on treated cells compared with control cells. TM antigen in cell lysates was measured as described previously(17, 42) . The cells were washed quickly with HBS-BSA and extracted with 200 µl of 50 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl, 0.5% Triton X-100, and 1 mM benzamidine hydrochloride for 1 h at 4 °C. The TM antigen levels in cell extracts were measured by enzyme immunoassay using monoclonal antibodies (TM mAb 2, TM mAb 11, and TM mAb 20) as reported previously(33) . Purified human placental TM was used as a standard.

Immunocytochemistry

Immunocytochemical detection of TM antigen was performed by the indirect immunofluorescence technique as described previously(43) . The cells were washed 3 times in PBS containing 0.02% NaN(3) and then fixed with 2% paraformaldehyde in PBS for 20 min at 4 °C. The cells were treated with blocking solution (PBS containing 5% normal goat serum) for 20 min and then incubated with blocking solution containing 10 µg/ml rabbit anti-human TM IgG or preimmune IgG at 4 °C. One hour later, cells were washed with PBS, and detection was performed with goat anti-rabbit IgG fluorescein isothiocyanate conjugate (Sigma). Parallel experiments using preimmune IgG instead of rabbit anti-human TM IgG were performed to evaluate nonspecific immunofluorescence.

Northern Blot Analysis

Northern blot hybridization was performed as described previously(42) . Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform method (44) from untreated or oxidized LDL-treated cells (60-mm diameter dishes, 10^6 cells/dish). After determination of RNA purity and concentration by a spectrophotometer, 20 µg of total RNA was fractionated on a 1% agarose/formaldehyde gel. RNA was then transferred overnight by capillary action in 10 times standard saline citrate (10 times SSC: 1.5 M NaCl, 0.15 M sodium citrate, pH 7.4) onto Hybond-N+ nylon filter (Amersham Japan, Tokyo, Japan) and immobilized by ultraviolet irradiation. The filter was prehybridized for 1 h in prehybridization buffer consisting of 50% formamide, 5 times SSC, 5 times Denhardt's solution (1% polyvinylpyrolidone, 1% Ficoll 400, and 1% BSA), 50 mM Tris-HCl (pH 7.4), 0.25 mg/ml denatured salmon sperm DNA, and 0.1% SDS. Hybridization was performed with a random primed P-labeled human TM cDNA (42) and human beta-actin cDNA (Nippon gene, Tokyo, Japan) in prehybridization buffer containing 10% dextran sulfate at 42 °C for overnight. After hybridization, the filter was washed twice with 2 times SSC and 0.1% SDS for 15 min at room temperature followed by two washes of 30 min in 0.1 times SSC and 0.1% SDS at 60 °C, and exposed to Kodak XAR-5 film using intensifying screen for 1 day. The autoradiograph was analyzed densitometrically with a Shimazu flying-spot scanner CS-9000 (Kyoto, Japan).

DRB Experiments

The half-life of mRNA was determined by the method of Xie et al.(45) . Cells were incubated with 65 µM DRB, a transcriptional inhibitor, and/or oxidized LDL (200 µg/ml) for 0, 2, 4, and 6 h at 37 °C. Total RNA was isolated, and Northern blot hybridization was performed as described above. After densitometric scanning of the autoradiogram, the t was calculated by linear regression analysis of data from semilog plots of the percentage of TM mRNA remaining versus incubation time.

Degradation of I-Oxidized LDL

Radioiodination of oxidized LDL was performed by the iodine monochloride method(46) . The specific radioactivity of I-oxidized LDL was approximately 200 cpm/ng of protein. Intracellular degradation of I-oxidized LDL was measured by the method of Naganuma et al.(47) . Cells grown in collagen-coated 35-mm diameter dishes (Corning; Iwaki Glass, Tokyo, Japan) were washed once with DMEM containing 0.1% BSA (DMEM-BSA) and then incubated in DMEM-BSA containing 10 µg/ml I-oxidized LDL for 2 h at 4 °C. After three washings with DMEM-BSA, cells were incubated with or without 1 µM bafilomycin A1 in DMEM-BSA for 1 h at 4 °C. Culture medium was changed to prewarmed (37 °C) DMEM containing 10% FCS and 10 µg/ml nonlabeled oxidized LDL with or without 1 µM bafilomycin A1, and cells were incubated for 0, 0.5, 1, 2, and 4 h at 37 °C. At the end of incubation, culture medium was collected, and an aliquot of the medium was mixed with a final 10% (w/v) trichloroacetic acid. The radioactivity recovered in the trichloroacetic acid-insoluble fraction was counted as oxidized LDL in the medium. Free [I]iodide (I) in the trichloroacetic acid-soluble fraction of the conditioned medium was removed by the method of Goldstein et al.(48) . An aliquot (1 ml) of the trichloroacetic acid-soluble fraction was mixed with 10 µl of 40% (w/v) KI and 40 µl of 30% (v/v) H(2)O(2). After 5 min at room temperature, 2 ml of chloroform was added to the solution, and then free iodine (I(2)) was extracted into the chloroform layer with agitation. The radioactivity of the upper aqueous layer was counted as [I]monoiodotyrosine degraded from oxidized LDL.

In a similar fashion, the cells were washed 3 times with 50 mM Tris-HCl (pH 7.4) containing 0.15 M NaCl and 0.2% BSA and followed by two washes with 50 mM Tris-HCl (pH 7.4) containing 0.15 M NaCl. The washed cells were solubilized with 1% Triton X-100 for 1 h at 4 °C and then centrifuged to remove insoluble material. Proteins in the solution were precipitated with final 10% (w/v) trichloroacetic acid, and free [I]iodide in trichloroacetic acid-soluble fraction was removed by the method described above. The radioactivities of the trichloroacetic acid-insoluble or -soluble fraction were counted as undegraded or degraded oxidized LDL, respectively, in the cells. Specific association and degradation of the LDL in the cells was calculated by subtracting the radioactive count of each fraction measured in the presence of a 20-fold excess amount of the unlabeled oxidized LDL from the radioactivity of each fraction measured in the absence of the unlabeled oxidized LDL.

Fractionation of LDL

Organic solvent-soluble and -insoluble components in native or oxidized LDL were separated with chloroform/methanol (1:2) by the method of Bligh and Dyer(49) . An aliquot (1 ml) of native or oxidized LDL was mixed with 1.25 ml of chloroform and 2.5 ml of methanol. After 10 min at room temperature, 1.25 ml of chloroform and 1.25 ml of deionized water were added to the solution, and the phases were separated by centrifugation. The organic solvent phase was evaporated under nitrogen gas, and the residual extracts were dispersed in DMEM containing 10% FCS by sonication just before use for the experiments. The water-soluble samples were concentrated by evaporation in vacuo and dissolved in DMEM containing 10% FCS.


RESULTS

Effect of Oxidized LDL on TM Cofactor Activity

LDL was oxidized by UV irradiation at 254 nm for various periods, and values of TBARS were measured for the irradiated LDL as an indicator of the extent of oxidation (Fig. 1A). The TBARS values increased with increasing time of UV irradiation, while no TBARS was observed in unirradiated native LDL. Endothelial cells were cultured for 24 h with 100 µg/ml LDL oxidized for 0-12 h and the thrombin-dependent protein C activation on the cell surface was measured (Fig. 1B). Protein C activation decreased with the increasing oxidation level of LDL, while activation was not influenced by incubation of the cells with unirradiated native LDL. Cell viability as measured by trypan blue dye exclusion was over 90% in all of these experiments, and no increase in TM antigen levels in the culture medium was detected (data not shown). Since measurable thrombin-dependent protein C activation on HUVEC surfaces requires TM cofactor activity, these results indicated that oxidized LDL reduced the cofactor activity of TM on the cell surface without induction of cell injury and release of the surface TM into the culture medium.


Figure 1: Oxidation of LDL by UV irradiation and effects on endothelial cell surface TM cofactor activity. A, human LDL was oxidized by UV irradiation (254 nm, 0.4 mW/cm^2) for various times. The degree of LDL oxidation was evaluated by formation of TBARS and is expressed as nmol of malondialdehyde/mg of protein. B, confluent endothelial cells were incubated with LDL (100 µg/ml) irradiated for various periods for 24 h. After incubation, TM-dependent activation of protein C on the cell surface was measured by the method described under ``Experimental Procedures.'' The data shown are the mean ± S.D. from four independent experiments.



When HUVECs were incubated with 200 µg/ml of oxidized LDL produced by 12-h UV irradiation, the cell surface TM cofactor activity did not significantly change with a 1-h incubation, but a time-dependent decrease in TM cofactor activity was observed by 3 h, and the level declined to 30% of that of the control by 24 h (Fig. 2A). When the cells were incubated for 24 h with various concentrations of oxidized LDL, the cofactor activity decreased with increasing concentration of oxidized LDL, and the level was about 30% of that of the control at 200 µg/ml of the oxidized LDL (Fig. 2B). Native or acetylated LDL had no effect on surface TM activity within the 24-h incubation (Fig. 2A). When 200 ng/ml of isolated human placental TM was incubated with 200 µg/ml oxidized LDL for 24 h at 37 °C, no change in the cofactor activity (TM alone, 37.8 ± 7.2 pmol of protein C activated/min/nmol of TM; incubation with oxidized LDL, 41.8 ± 2.5 pmol of protein C activated/min/nmol of TM) was observed. These results indicated that oxidized LDL reduced surface TM activity on endothelial cells in a time- and concentration-dependent manner independent from the direct affect of the LDL on the TM molecule.


Figure 2: Changes in TM cofactor activity on the surface of endothelial cells exposed to oxidized LDL. A, confluent endothelial cells were incubated for various times with 200 µg/ml native LDL (circle), acetylated LDL (up triangle), or oxidized LDL produced by UV irradiation of LDL for 12 h (bullet). B, cells were incubated for 24 h with various concentrations of oxidized LDL, which was produced by UV irradiation of LDL for 12 h. The cofactor activity of TM on the cell surface was measured by the method described under ``Experimental Procedures.'' The data shown is the mean ± S.D. from four independent experiments.



Effect of Oxidized LDL on TM Antigen Levels on the Cell Surface and in Cell Lysate

Cell surface TM antigen and total TM antigen in cell lysates were measured after incubation of the cells with oxidized LDL prepared by 12-h UV irradiation (Fig. 3). TM antigen levels on the surface and in cell lysates did not significantly change at 1 h after treatment of the cells with 200 µg/ml oxidized LDL but decreased after 3 h of treatment with oxidized LDL in a time-dependent manner (Fig. 3A). Cell surface and lysate TM antigen levels were reduced by treatment of the cells with 200 µg/ml oxidized LDL for 24 h to 35 and 33%, respectively, of those of the control. TM antigen levels in the culture medium did not significantly increase during the incubation periods. No changes in the TM antigen levels of the surface and cell lysate were observed in cells incubated with 200 µg/ml native LDL for up to 24 h. In treatment of the cells with various concentrations of oxidized LDL for 24 h, a dose-dependent decrease in cell surface and lysate TM antigen levels was observed (Fig. 3B). Oxidized LDL did not affect the enzyme immunoassay system for measurement of TM antigen levels (data not shown).


Figure 3: Effect of oxidized LDL on surface and total TM antigen levels in endothelial cells. Oxidized LDL was produced by UV irradiation of LDL for 12 h. A, confluent endothelial cells were incubated with 200 µg/ml native LDL (circle,up triangle) or oxidized LDL (bullet,) for various times. TM antigen levels on the surface (circle,bullet) and in the cell lysate (up triangle,) in the cells were measured by the methods described under ``Experimental Procedures.'' B, cells were incubated with various concentrations of oxidized LDL for 24 h, and then TM antigen levels on the cell surface (box) and in the cell lysate (&cjs2090;) were measured. The data represent the mean ± S.D. from four independent experiments.



A decrease in TM antigen on endothelial cells incubated with oxidized LDL was also observed by immunocytochemistry using polyclonal rabbit anti-human TM IgG (Fig. 4A and B). In this experiment, when preimmune rabbit IgG was used instead of rabbit anti-human TM IgG in the immunocytochemical detection, no fluorescence was observed in the preparations (Fig. 4C), indicating that the immunocytochemical detection of TM was not dependent on nonspecific binding of IgG.


Figure 4: Immunofluorescence detection of TM antigen on the surface of endothelial cells. Oxidized LDL was produced by UV irradiation of LDL for 12 h. Confluent endothelial cells were treated with or without 200 mg/ml oxidized LDL for 24 h. After incubation, untreated cells (A) and oxidized LDL-treated cells (B) were fixed and then incubated with rabbit anti-human TM IgG. Antibody localization was detected with goat fluorescein isothiocyanate-conjugated anti-rabbit IgG as described under ``Experimental Procedures.'' Parallel experiments using preimmune rabbit IgG were performed to assess nonspecific immunofluorescence (C).



TM mRNA in HUVECs Exposed to Oxidized LDL

TM mRNA levels in endothelial cells were measured by Northern blot hybridization at various times after treatment of the cells with 200 µg/ml oxidized LDL (Fig. 5). TM mRNA levels decreased by 1 h after treatment with oxidized LDL, and a time-dependent decrease in the mRNA level was observed for 12 h. The normalized TM mRNA signal decreased by about 80%, and this reduced level was maintained for 24 h (Fig. 5B).


Figure 5: Changes in TM mRNA levels in endothelial cells exposed to oxidized LDL. Oxidized LDL was produced by UV irradiation of LDL for 12 h. Confluent endothelial cells were treated with or without 200 mg/ml oxidized LDL for various times, and total RNA was extracted from the cells (A). TM mRNA levels were analyzed by Northern blot as described under ``Experimental Procedures.'' The migration of the ribosomal RNA is indicated on the right. B, the autoradiogram of panel A was analyzed by densitometry, and the data are expressed as the percentage of the control values after normalization to the actin mRNA signal.



The half-life of TM mRNA in cells treated with or without oxidized LDL was measured using DRB, a transcription inhibitor, to investigate the effect of oxidized LDL on TM mRNA stability (Fig. 6). The levels of TM mRNA detected by Northern blotting were analyzed by densitometry (Fig. 6, D, E, and F). TM mRNA levels in the cells treated with 65 µM DRB alone decreased in a time-dependent manner, and the apparent half-life of TM mRNA was 2.6 ± 0.2 h (Fig. 6D). The half-life of TM mRNA in cells treated with oxidized LDL alone was 3.0 ± 0.3 h (Fig. 6E). The half-life in cells treated with oxidized LDL and DRB also did not differ significantly and was calculated at 2.8 ± 0.2 h (Fig. 6F).


Figure 6: The half-life of TM mRNA in endothelial cells treated with DRB and/or oxidized LDL. Oxidized LDL was produced by UV irradiation of LDL for 12 h. Confluent endothelial cells were treated with 65 mM DRB alone (A and D), 200 mg/ml oxidized LDL (B and E), or DRB plus oxidized LDL (C and F) for various times. Total RNA was isolated from the cells, and Northern blot analysis was performed as described under ``Experimental Procedures.'' The autoradiogram was analyzed by densitometry, and the data are expressed as percentages of control after normalization to actin mRNA levels (D, E, and F). Each symbol (circle, up triangle, and box) represents three different experiments. The half-life (t) for each condition was calculated by linear regression analysis from semilog plots of the mRNA remaining versus incubation times and is reported as mean ± S.D.



TM Down-regulation and Lysosomal Function

The effect of bafilomycin A1, an inhibitor of the lysosomal proton pump, on regulation of TM mRNA and antigen levels in endothelial cells treated with oxidized LDL was investigated (Fig. 7). Cell viability was maintained over 90% within 7 h after treatment of the cells with 1 µM bafilomycin A1 but slightly decreased at 12 h after treatment. Therefore, this experiment was evaluated within 7 h after treatment of the cells with 1 µM bafilomycin A1. Treatment with bafilomycin A1 alone for 7 h did not affect TM mRNA levels. The marked decrease in TM mRNA levels in cells treated with oxidized LDL for 6 h was abolished by coincubation of the cells with bafilomycin A1 (Fig. 7A). In the cells treated with bafilomycin A1 alone, TM antigen levels on the cell surface were not affected, while total TM antigen levels in the cell lysate increased to 1.2 times the control levels (Fig. 7B). The 30 and 35% decrease in surface and total TM antigen levels, respectively, induced by oxidized LDL was not observed in cells coincubated with bafilomycin A1.


Figure 7: The effect of bafilomycin A1 on TM mRNA and antigen levels in endothelial cells exposed to oxidized LDL. Oxidized LDL was produced by UV irradiation of LDL for 12 h. Confluent endothelial cells were treated with or without 1 mM bafilomycin A1 and/or 200 µg/ml oxidized LDL. Bafilomycin A1 was added at 1 h before treatment of the cells with oxidized LDL and coincubated with oxidized LDL for 6 h. A, after incubation, total RNA in the cells was isolated as described under ``Experimental Procedures.'' RNA (10 mg) was blotted on a nylon membrane and hybridized with P-labeled TM or actin cDNA probes. B, after incubation, the surface TM antigen (box) and total TM antigen in cell lysate (&cjs2090;) of the cells were measured by the methods described under ``Experimental Procedures.'' The data represent the mean ± S.D. from four independent experiments and are analyzed by the Student t test, *, p < 0.01 when compared with control.



To investigate the degradation rate of oxidized LDL, cells were incubated with I-oxidized LDL for 2 h at 4 °C and then incubated with or without bafilomycin A1 for 1 h at 4 °C, and the LDL was chased with unlabeled oxidized LDL and/or bafilomycin A1 at 37 °C (Fig. 8). Culture medium and the cells were collected after the indicated incubation time, and radioactivity recovered in each trichloroacetic acid-soluble and -insoluble fraction was determined. At zero time, the trichloroacetic acid-insoluble radioactivity associated with the cells was the same for cells treated with oxidized LDL or oxidized LDL plus bafilomycin A1. The trichloroacetic acid-insoluble radioactivity (210 cpm; 11.7 ng of LDL protein/mg of cellular protein) associated with the cells decreased with time with a concomitant increase in trichloroacetic acid-soluble radioactivity in the culture medium. An increase of 120 cpm of trichloroacetic acid-soluble radioactivity (6.8 ng of LDL protein/mg of cellular protein) was observed in the medium at 4-h incubation of the cells at 37 °C. At 4 h, the increase in trichloroacetic acid-soluble radioactivity was inhibited by 70% in cells treated with bafilomycin A1. Trichloroacetic acid-soluble radioactivity associated with the cells and trichloroacetic acid-insoluble radioactivity in culture medium were negligible (data not shown). Thus, this result indicates that about 60% of the bound I-oxidized LDL was degraded and released to culture medium as trichloroacetic acid-soluble materials, and treatment of the cells with bafilomycin A1 inhibited the degradation.


Figure 8: The effect of bafilomycin A1 on the degradation of I-oxidized LDL in endothelial cells. Confluent endothelial cells were incubated with 10 µg/ml I-oxidized LDL at 4 °C for 2 h. The unbound ligand was removed, and the cells were incubated in the absence (circle) or the presence of 1 µM bafilomycin A1 (bullet). After incubation for 1 h at 4 °C, cells were incubated at 37 °C for the indicated times in the absence (circle) or the presence of 1 µM bafilomycin A1 (bullet). A, at the end of incubation, conditioned medium was collected, and the radioactivity of the trichloroacetic acid-soluble fraction was counted as the amount of degraded I-oxidized LDL as described under ``Experimental Procedures.'' B, cells were solubilized, and the radioactivity in the trichloroacetic acid-insoluble fraction was counted as the amount of cell associated I-oxidized LDL as described under ``Experimental Procedures.'' The data shown are the mean of two different experiments.



Effect of Lipid or Aqueous Component of Oxidized LDL on TM Expression

Native and oxidized LDL were extracted with chloroform and methanol (1:2) by the method of Bligh and Dyer and separated into organic and aqueous phases. The effect of each extract on expression of cell surface TM antigen was investigated (Fig. 9). About a 40% decrease in TM antigen levels was observed by treatment with organic solvent extracts containing an equivalent concentration with lipid in 200 µg protein/ml oxidized LDL (300 µg cholesterol/ml) for 24 h. The extent of the decrease was comparable with that induced by treatment with 200 µg/ml oxidized LDL. Aqueous extracts of oxidized LDL and native LDL, as well as the organic or water-soluble extracts of native LDL, did not affect TM antigen levels on the surface of the cells.


Figure 9: The effect of organic or aqueous extraction of oxidized LDL on TM antigen levels on the surface of endothelial cells. The organic solvent-soluble and water-soluble components of native LDL or oxidized LDL were separated by the method described under ``Experimental Procedures,'' using chloroform, methanol, and water. Organic solvents in the chloroform fraction were evaporated, and the residual material was dispersed by sonication in DMEM containing 10% FCS. The aqueous extracts were concentrated by evaporation and dissolved in DMEM containing 10% FCS. All concentrations were adjusted to an equivalent concentration with lipid or protein in 200 µg protein/ml oxidized LDL. Confluent cultures of endothelial cells were incubated for 24 h in the absence or presence of native LDL (N-LDL), oxidized LDL (Ox-LDL), organic solvent extract of native (N-Lipid) or oxidized LDL (Ox-Lipid), or aqueous extract of native (N-Aq.) or oxidized LDL (Ox-Aq.). After incubation, TM antigen levels on the cell surface were measured by the method described under ``Experimental Procedures.'' Data represent the mean ± S.D. from three independent experiments.




DISCUSSION

The present study has characterized the effect of oxidized LDL on the cofactor activity and expression of TM and TM mRNA levels in endothelial cells. Weis et al.(30) suggested that TM cofactor activity for thrombin-dependent protein C activation on the surface of endothelial cells decreased in response to oxidized LDL prepared by incubation of LDL with CuSO(4). The present work used oxidized LDL prepared by UV irradiation of LDL, because the UV irradiation method can control the extent of oxidation. We also observed a decrease in TM cofactor activity on the surface of cells treated with oxidized LDL prepared by UV irradiation. The present work further demonstrates that the extent of reduction in the TM cofactor activity progressed with increasing oxidation levels of the added LDL. The decrease in TM cofactor activity could be a specific response in the cells exposed to oxidized LDL, since acetylated LDL and native LDL did not affect the activity. It was reported that Met in TM was oxidized by active neutrophil products and that the TM cofactor activity subsequently decreased(50) . In our experience, however, the cofactor activity of TM isolated from human placenta was not directly influenced by incubation with oxidized LDL, and the surface cofactor activity on cells treated with oxidized LDL was reduced in parallel with TM antigen levels (Fig. 2, 3, and 4). These results indicate that the decrease in TM cofactor activity on the surface of cells exposed to oxidized LDL is predominantly due to down-regulation of TM antigen levels on the cell surface irrespective of direct oxidation of the TM molecule.

Although significant changes in TM antigen levels on the cell surface and in the cell lysate were not observed at 1 h after treatment with oxidized LDL, a time-dependent decrease in each was observed from 3 h after the treatment. In contrast, TM mRNA levels had already decreased to about 80% of control values by 1 h after treatment with oxidized LDL, and a progressive decrease in TM mRNA levels was observed earlier than decreases in TM antigen levels. There was no increase in TM antigen levels in the culture medium in oxidized LDL-treated cells. These results suggested that the down-regulation of TM on oxidized LDL-treated cells could be predominantly dependent on reduced TM biosynthesis in the cells during decline of TM mRNA. The apparent half-life of TM mRNA in the cells treated with oxidized LDL plus DRB, a transcription inhibitor, was 2.8 h and did not significantly differ from the 2.6 h observed in the control cells treated with DRB alone or the 3.0 h in the cells treated with oxidized LDL alone. These results suggest that oxidized LDL did not affect the stability of TM mRNA and repressed TM biosynthesis through inhibition of TM transcription in the cells. The apparent half-life of TM mRNA in control cells observed in the present work was comparable with that reported previously(16) . The 5`-flanking promoter region of human TM contains two regions with a DNA binding sequence (CCGCCCC) for an inhibitory transcription factor from -842 to -836 base pair and from -1269 to -1262 base pair(51) . It is not known whether oxidized LDL repressed TM transcription through activation of this mechanism or through other unidentified elements.

Oxidized LDL is internalized after binding with the scavenger receptors on the surface of endothelial cells and degraded by the lysosomal pathway(46) . The decreased surface TM in endothelial cells exposed to inflammatory cytokines, such as tumor necrosis factor-alpha, were postulated to be due to increases in TM degradation through the lysosomal pathway and/or decreased TM transcription(11, 14, 15, 16) . Therefore, the present work was designed to address the potential contribution of lysosomal function to the down-regulation of TM in oxidized LDL-treated cells. It is known that bafilomycin A1, a specific inhibitor for vacuolar-type H-ATPase, inhibits the acidification of lysosomes(52) . Bafilomycin A1 inhibits the degradation of oxidized LDL but does not affect on the internalization of oxidized LDL and its transport into the lysosomes(47) . Treatment of endothelial cells with bafilomycin A1 alone did not affect TM mRNA levels or surface TM antigen levels but significantly increased total TM antigen levels. Thus bafilomycin A1 did not interfere with internalization of the surface TM, but it inhibited degradation of TM incorporated in the lysosomes. The present result supports previous studies showing that surface TM on endothelial cells is degraded through the lysosomal pathway under steady state conditions(11, 14) . Coincubation of the cells with oxidized LDL and bafilomycin A1 prevented the decrease in surface and total TM antigen levels. If the lysosomal degradation of surface TM was accelerated by oxidized LDL, then the decrease in surface TM levels could be not prevented even if the oxidized LDL-treated cells were coincubated with bafilomycin A1, because this compound did not inhibit internalization. This result implied that oxidized LDL did not accelerate TM degradation through the lysosomal pathway. The present work further indicates that bafilomycin A1 inhibited lysosomal degradation of oxidized LDL and the decrease in TM mRNA levels induced by oxidized LDL. Thus it was suggested that down-regulation of TM antigen in the cells exposed to oxidized LDL was predominantly due to inhibition of TM transcription and that the inhibition of TM transcription was mediated by degradation of oxidized LDL through the lysosomal function.

There is a variable distribution of scavenger receptors for modified LDL, including acetylated and oxidized LDL, on the surface of several cell types. Expression of the class A scavenger receptors (type I and II) was demonstrated on the surface of macrophages, and the receptors mediated internalization of the modified LDL(53) . We examined the effect of dextran sulfate or fucoidin, antagonists for the class A scavenger receptors, on down-regulation of TM caused by oxidized LDL and found no effect (data not shown). Therefore, internalization of oxidized LDL to induce the down-regulation of TM may be not mediated by the class A scavenger receptors. Naito et al.(54) recently demonstrated that internalization of the modified LDL in endothelial cells was not mediated by class A receptors and suggested that the cells may have other types of scavenger receptor. Acton et al.(55) have cloned the cDNA for a scavenger receptor, which is a new member of the CD36 family of membrane proteins (class B scavenger receptors), and the receptors are expressed in a variety of cells, including macrophages and endothelial cells(56) . In addition to class B scavenger receptors, De Rijke et al.(57) recently reported that a 95-kDa oxidized LDL-specific receptor was expressed in Kupffer and endothelial cells. Since antagonists for these scavenger receptors are not available, we could not examine whether the down-regulation of TM in oxidized LDL-treated cells was mediated by the class B scavenger receptors or the 95-kDa receptor.

Changes in the LDL particle associated with oxidative modification include lipid peroxidation, formation of conjugated dienes, carbonyl modification of apoB-100, and conversion of phosphatidylcholine to lysophosphatidylcholine by phospholipase A(2) in apoB-100 (25, 26) . It has been shown that organic extracts of oxidized LDL interfered with endothelial cell functions(29) . We found that TM expression on endothelial cells is negatively regulated by the degree of LDL oxidation and that organic extracts of oxidized LDL reduced TM antigen levels on the surface of cells to nearly the same extent as intact oxidized LDL, but aqueous extracts of oxidized LDL did not. Therefore, it is possible that the down-regulation of TM was mediated by lipid components of oxidized LDL degraded in the lysosomes. We have investigated the effect of native LDL or acetylated LDL treated with phospholipase A(2), lysophosphatidylcholine (palmitoyl- or stearoyl-lysophosphatidylcholine), or oxysterol (25-hydroxycholesterol or 7-ketocholesterol) on the TM antigen levels, although no affect was observed (data not shown). The active lipid component(s) of oxidized LDL that causes the down-regulation of TM remains to be determined.

Oxidized LDL induces reduction of tissue-type plasminogen activator release(29) , increase in plasminogen activator inhibitor-1 release (29) , and tissue factor expression (28) in endothelial cells. Therefore, oxidized LDL disturbs the balance between anti- and prothrombotic factors in endothelial cells. Decreases in TM expression on the surface of endothelial cells caused by oxidized LDL may further contribute to promotion of thrombosis in atherosclerotic lesions.


FOOTNOTES

*
This work was supported in part by a grant from the Ministry of Health and Welfare, Grants-in-aid 05837024 and 06836016 from the Ministry of Education, Science, and Culture of Japan, and by the Sasagawa Scientific Research Grant from the Japan Science Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Clinical Biochemistry, Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko, Tsukui, Kanagawa 199-01, Japan. Tel.: 81-426-85-3756; Fax: 81-426-85-2577.

(^1)
The abbreviations used are: TM, thrombomodulin; LDL, low density lipoprotein; BSA, bovine serum albumin; DRB, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HUVEC, human umbilical vein endothelial cell; TBARS, thiobarbituric acid reactive substance(s); HBS, Hanks' balanced salt solution; SSC, standard saline citrate.


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