High-density lipoprotein inhibits the synthesis of platelet-activating factor in human vascular endothelial cells

EISEVIER

J. Lipid Mediators Cell SignaIling 13 (1996) 73-88

High-density lipoprotein inhibits the synthesis of platelet-activating factor in human vascular endothelial cells Junko Sugatani a,*, Masao Miwa b, Yutaka Komiyama a Department

‘, Seiji Ito a

ofMedical Chemistry, Kansai Medical University, IO-IS Fumizonocho, Moriguchi, Osaka 570,

Japan b Department of Biochemistry. School of Pharmaceutical Science. University of Shizuoka. Shizuoka 422, Japan ’Department of Clinical Science and Laboratory Medicine, Kansai Medical Unioersity, IO-IS Fumizonocho, Moriguchi, Osaka 570, Japan Received 9 January

1995; revised 18 May 1995; accepted

30 May 1995

Abstract The regulation of platelet-activating factor (PAFI synthesis by serum lipoproteins was investigated in human umbilical vein endothelial cells. High-density lipoprotein (HDL) inhibited PAF synthesis in agonist (thrombin, histamine, and A23187)-stimulated endothelial cells, that was determined by incorporation of [ 3H]acetate into PAF and by bioassay. The inhibition by HDL was increased in a concentration-dependent manner, but was reversed as the concentration of thrombin increased. HDL did not affect the time course of PAF production. HDL lipids suppressed the PAF production to a lesser extent than HDL. The reduction of PAF accumulation by HDL did not result from degradation of PAF but inhibition of PAF synthesis, which was mainly mediated via the blockade of acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activation. HDL did not prevent the release of [ 3H]arachidonic acid in thrombin-stimulated endothelial cells. The binding of “‘1-HDL to endothelial cells and its uptake were not enhanced by thrombin stimulation. These results demonstrate that HDL may inhibit the activation of acetyhransferase by thrombin at the cell surface. This observation may explain a part of mechanism of HDL action. Keywords:

Platelet-activating

factor; Human; High-density

lipoprotein;

Endothelial

cells; Acetyltransferase

1. Introduction Platelet-activating unique phospholipid,

* Corresponding

factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine), has a wide spectrum of biological activities (Hanahan,

author. Tel. 81-6-992-1001 ext. 2454; Fax 81-6-992-1781.

0929.7855/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0929-7855(95)00047-X

a 1986;

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Lipid Mediators Cell Signnlling 13 (1996) 73-88

Braquet et al., 1987; Venable et al., 1993). Research over the last 15 years has provided evidence that PAF functions as a potent chemical mediator, which is generated in a variety of cells as a result of chemical or immune stimulation associated with inflammatory reactions and anaphylaxis. Although newly synthesized PAF in activated basophils, eosinophils, peritoneal and alveolar macrophages, and polymorphonuclear leukocytes (PMNs) has been reported to be released from cells of origin in vivo and act on their neighbouring cells as an autacoid (Pinckard et al., 1979; Cromwell et al., 1990; Mencia-Huerta and Benveniste, 1981; Albert and Snyder, 1983; Camussi et al., 1987; Cluzel et al., 1989; Miwa et al., 1992), the PAF synthesized in endothelial cells remains in the surface membrane, plays a role in cell-cell interactions such as PMN and monocytic-cell adhesion to endothelial cells, and appears to function as a potent chemical mediator in pathophysiological processes of inflammation and atherogenesis (Zimmerman et al., 1985; DiCorleto and Dela Motte, 1989). Endothelial cells produce PAF in early response to inflammatory and vasoactive agents such as thrombin, and outer-membrane porins from histamine, bradykinin, angiotensin II, ATP, H,O,, Gram-negative bacteria within minutes of exposure (Zimmerman et al., 1985; Whatley et al., 1988; Lewis et al., 1988; Tufano et al., 1993). Endothelial cells stimulated by such inflammatory and vasoactive agents synthesize PAF primarily by a remodelling pathway involving two sequential enzymatic steps: (1) deacylation of 1-alkyl-2-acyl-sn-glycero3-phosphocholine by phospholipase A 2 yielding the precursor 1-alkyl-2-lyso-snglycero-3-phosphocholine (lyso-PAF) and (2) the subsequent acetylation by acetylCoA:lyso-PAF acetyltransferase (EC 2.3.1.67). On the other hand, intracellular PAF acetylhydrolase, a PAF-degrading enzyme, is also considered to control the level of PAF in endothelial cells, since the pretreatment of cells with phenylmethylsulfonyl fluoride to inactivate PAF acetylhydrolase increases the level of PAF in thrombin-stimulated endothelial cells (Hirafuji et al., 1987). To date, there have been many reports on intracellular factors regulating PAF synthesis by influencing these enzyme reactions, e.g., calcium, G-proteins, protein kinase C and cyclic AMP, and Naf/Hf transport system, and availability in levels of substrates such as acetyl-CoA and lyso-PAF (Whatley et al., 1989, Whatley et al., 1990; Heller et al., 1991; Ghigo et al., 1988; is known Sugiura et al., 1989; Hayashi et al., 1992). However, little information concerning an extracellular facto&) regulating PAF synthesis in endothelial cells in vivo. In the present study, we demonstrate (1) that high-density lipoprotein (HDL), in vivo extracellular factor, suppresses thrombin-stimulated PAF synthesis in human umbilical vein endothelial cells and (2) that HDL inhibits the activation of acetyltransferase in stimulated endothelial cells through its binding to the cell surface.

2. Materials

and methods

2. I. Materials

1 -Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine and 1-hexadecyl-2-lyso-snglycero-3-phosphocholine were purchased from Bachem Feinchemikalien (Bubendorf, Switzerland). 1-Hexadecyl-2-[ 3H]acetyl-sn-glycero-3-phosphocholine (370 GBq/mmol),

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75

[5,6,8,9,11,12,14,15-3H]arachidonic acid (3.7 TBq/mmol) and [3H]acetyl-CoA (141 GBq/mmol) were obtained from DuPont-New England Nuclear Japan (Tokyo). [ 3H]Acetate (123 GBq/mmol) and Na’251 (carrier free in 0.1 N NaOH) were purchased from Amersham Japan (Tokyo). l-Hexadecanoyl-2-acetyl-sn-glycero-3-phosphocholine was synthesized by acetylating l-hexadecanoyl-2-lyso-sn-glycero-3-phosphocholine with acetic anhydride in the presence of perchloric acid as a catalyst (Kumar et al., 1983). Human cr-thrombin was highly purified by minor modification of method reported by Kawabata et al. (1985). Histamine dichloride, calcium-ionophore A23187, trypsin (EC 3.4.21.4) (bovine pancreas, type I), TLCK-treated cY-chymotrypsin (EC 3.4.21.1) (bovine pancreas, type VII) and bovine serum albumin (BSA, fraction V, essentially fatty acid-free) were products of Sigma Chemical Co. (St. Louis, MO, USA). Bacillus cereus phospholipase C (EC 3.1.4.3) was from Asahi Chemical Industry (Tokyo, Japan); palmitic acid was from Wako Pure Chemical Industries (Osaka, Japan); 6-ketoprostaglandin F,, was from Funakoshi (Tokyo, Japan). 2.2. Cells Endothelial cells from human umbilical vein segments were isolated by treatment with 0.1% collagenase from Clostridium histolyticum for 20 min at 37°C and serially cultured in Medium 199 (Gibco, Grand Island, NY, USA) containing 10 mM Hepes, penicillin (100 U/ml>, streptomycin (100 Fg/ml), porcine heparin (90 pg/ml) (Sigma, St. Louis, MO, USA), and endothelial cell growth factor (60 pg/ml) (Collaborative Research, Lexington, MA, USA) supplemented with 20% fetal bovine serum (Gibco, Grand Island, NY, USA). Cells were grown in plastic tissue culture flasks (Coming Glass Works, Coming, NY, USA) that were precoated with gelatin (Difco Laboratories, Inc., Detroit, MI, USA). The final passage was into C-12 plates with 22 mm diameter wells except otherwise indicated, in which cells were seeded at an initial cell density 1 X lo5 cells/well and reached confluence after 3-4 days. Cells were routinely used for experiments at passage levels 2-3 and confluent densities. These cells were characterized by morphologic criteria and positive immunofluorescence for factor VIII antigen. The release of cytosolic lactate dehydrogenase activity was determined by a calorimetric assay using a kit (LDH-UV Test, Wako Pure Chem. Industries, Osaka, Japan). Total enzyme values were measured after lysis by freeze-thawing and sonication. Rabbit platelets were prepared from platelet-rich plasma obtained with Ficoll-Paque as described previously (Sugatani et al., 1987). 2.3. Lipoproteins Lipoproteins were isolated from human serum by sequential ultracentrifugation according to standard techniques using solid KBr for density adjustment (Hatch and Lees, 1968). The lipoprotein fractions were separated according to densities as follows: low density (LDL, 1.019- 1.063 g/cm3 density) and high density (HDL, 1.063-1.210 g/cm3 density). To further remove contaminating plasma proteins, the HDL preparation was washed by recentrifugation at 1.210 g/cm3 density. Isolated fractions were extensively dialyzed at 4°C against a buffer containing 0.15 M NaCl and 1 mM EDTA

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(pH 7.4): Enzyme activities of trypsin and thrombin were assayed by platelet aggregation and that of cY-chymotrypsin by hydrolysis of benzoyl+tyrosine ethyl ester (Hummel, 1959). The compositions of LDL and HDL by percent weight were as follows: 27.5 and 57.6% protein, 24.7 and 22.5% phospholipid, and 39.6 and 15.2% total cholesterol, respectively. LDL and HDL incubated at pH 2.8 for 30 min at room temperature to inactivate PAF acetylhydrolase and then neutralized with 1 .O N NaOH were used unless otherwise stated, since the lipoproteins include PAF acetylhydrolase by which PAF in endothelial cells was degraded (Zimmerman et al., 1990). HDL was labelled by a modification of the Iodogen method as described by Virgolini et al. (1991). After labelling, unbound ‘25I was removed by gel filtration on a Sephadex G-25 column. Specific activity of the labeled HDL was 2830 cpm/ng protein. Radiochemical purity analysis determined by sodium dodecylsulfate polyacrylamide gel electrophoresis indicated that over 95% of radioactivity was bound to HDL. Protein content was determined using BCA protein assay reagent (Pierce, Rockford, IL, USA). The concentrations of LDL and HDL were expressed based on their protein contents. 2.4. Quantitation

of PAF

The synthesis of PAF in response to agonist stimulation was investigated using both the incorporation of radioactive precursors and bioassay (rabbit platelet aggregation). Endothelial cells were washed twice with 1 ml of Hank’s buffered salt solution (HBSS) containing 4 mM NaHCO,, 10 mM Hepes (pH 7.4) and 0.25% BSA and preincubated for 5 min at 24°C with appropriate concentration of HDL or BSA as a control in 1 ml of the same buffer containing 25 &i of carrier free [ 3H]acetate unless otherwise indicated. Then the reaction was initiated by the addition of stimulant (thrombin, histamine or A23187) on a rotary shaker (60 oscillations per min). After the appropriate incubation period, the reaction was stopped by adding methanol containing 50 mM acetic acid and unlabeled PAF carrier (50 lug), the cells were scraped from the plate and it was washed twice with 1 ml of methanol. Total lipids were extracted from the pooled samples using a modification of the method of Bligh and Dyer (1959). 3 ml of chloroform and 1.7 ml of 50 mM sodium acetate were added to bring the mixture ratio to 1:1:0.9 (CHCl,:CH,OH:H,O). The chloroform phase was removed and another volume of chloroform was added to the aqueous phase. After vigorous mixing, the chloroform phase was removed and combined with the first fraction. The lipids in the chloroform extract were separated by thin-layer chromatography (TLC) on silica gel G plates (250 pm, 20 X 20 cm, Uniplate, Analtech, Newark, NJ, USA) with a solvent system of CHC13:CH30H:CH3COOH:H,0 (50:25:8:4, by volume). After development, the plate was exposed to 6-p-toluidine-2-naphthalenesulfonic acid and the area containing PAF was scraped from the plate. The radioactivity was determined in a liquid scintillation counter (Packard TriCarb 460). The recovery of PAF through extraction and purification procedures was 81.6 f 3.8% (3 experiments) as determined by radiotracer studies using 1-hexadecyl-2-[ 3H]acetyl-sn-glycero-3-phosphocholine. In the platelet aggregation assay, changes in light transmittance were monitored by an aggregometer (Nikko Hematracer PAT-4A). The sample, which was extracted by the method of Bligh and Dyer and purified by Amprep octadecyl mini-column chromatog-

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Cell Signalling 13 (1996) 73-88

77

raphy and TLC as reported previously (Sugatani et al., 19931, was dispersed in 0.1% BSA/saline solution. The amount of PAF in the original sample was quantified by comparison of the increase in the maximal percentage of light transmission through platelet suspensions stimulated by experimental samples with that through a suspension stimulated by a known quantity of 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine. 2.5. Assay of [3H]arachidonic

acid release

Subconfluent endothelial cells (3.2 X 10’ cells) were incubated with 0.25 ,&i of [3H]arachidonic acid in 1 ml of Ml99 medium containing 20% fetal bovine serum for 24 h at 37°C. Cells were then washed two times with 1 ml of HBSS containing 10 mM Hepes and 0.25% BSA and preincubated in the same medium with HDL (3 mg/ml) or BSA (3 mg/ml) as a control for 30 s at 24°C. The cells were stimulated with thrombin (0.1 U/ml) or saline as vehicle for the indicated periods of time at 24°C on a rotary shaker (60 oscillations per min). Cellular lipid separation was carried out by TLC on silica gel G in hexane: diethyl ether: acetic acid: methanol (85:20:2:4, by volume). The radioactivities of [ 3H]arachidonic acid released in the medium and incorporated cellular phospholipids were determined by liquid scintillation counting. For analyzing [3H]arachidonic acid and its radiolabeled metabolites released from cells into the medium, the medium was acidified to pH 3 with 1 N HCl, extracted with a 6-fold volume of ethyl acetate washed against water, and evaporated with nitrogen gas. (40 pg) were added to the samples. The Palmitic acid (40 pg) and 6-keto-PGF,, samples were developed on silica gel G plates in the organic phase of ethyl acetate:2,2,4_trimethylpentane: acetic acid:water (11:5:2: 10, by volume). Lipids that comigrated with authentic fatty acid and 6-keto-PGF,, were detected by UV fluorescence after spraying the plate with 1 mM 6-ptoluidine-2-naphthalenesulfonic acid and the radioactivities of the samples were determined by scintillation spectroscopy. 5 min after thrombin stimulation over 90% of radioactive substances in the medium was free fatty acid. 2.6. Measurement drolase activity

of acetyl-CoA:lyso-PAF

acetyltransferase

actiuity and PAF acetylhy-

Acetyl-CoA:lyso-PAF acetyltransferase activity was determined essentially as described previously (Yamazaki et al., 1994). Endothelial cell lysates (1.25-5.00 pg protein) were incubated for 15-30 min at 37°C in a final volume of 100 ~1 of 0.1 M Tris/HCl buffer (pH 6.9) containing 1 mM dithiothreitol (DTT), 25 mM NaF and 0.1% BSA with 4 nmol lyso-PAF and 30 nmol of [‘Hlacetyl-PAF (3.08 kBq/nmol). The reaction was stopped by addition of 20 ~1 of 5% BSA/saline solution. After addition of 80 ~1 of 30% trichloroacetic acid, the reaction mixture was centrifuged at 750 X g for 2 min to obtain [3H]acetyl-PAF bound to the denatured BSA. The pellet was washed with 400 ~1 of 7% trichloroacetic acid and centrifuged twice again at 750 X R for 2 min. The resulting pellet was dissolved in 200 ~1 of 0.1 M potassium phosphate buffer containing 1% SDS (pH 8.0) and the radioactivity was determined by liquid scintillation counting. PAF acetylhydrolase activity was determined by measurement of [3H]acetate pro-

78

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Lipid Mediators Cell Signalling 13 (1996) 73-88

duced from 1-alkyl-2-[ 3Hlacetyl-sn-glycero-3-phosphocholine upon precipitation of the complex of radioactive substance and albumin with trichloroacetic acid as described by Miwa et al. (1988). 2.7. Binding of “‘I-HDL

to cells

endothelial cells at 4 or 24”C, To determine ‘*‘I-HDL binding to thrombin-stimulated cells in 35-mm dishes were washed twice with 1 ml of HBSS containing 0.25% BSA and 10 mM Hepes (pH 7.4), preincubated with various concentrations of lz51-HDL ranging from 0.5 to 1000 pg/ml in 1 ml of the same medium for 30 s and stimulated with thrombin (1 U/ml) or saline as a control on a rotary shaker (60 oscillations per min). After 5 min of incubation at 4°C or 24”C, the incubation medium was removed and discarded. The cells were washed rapidly three times with 1 ml of ice-cold HBSS containing 0.25% BSA and 10 mM Hepes (pH 7.4) followed by three rapid washes with ice-cold HBSS containing 10 mM Hepes (pH 7.41, and were dissolved by incubation at room temperature for at least 15 min in 1.0 ml of 0.1 N NaOH. An aliquot (50 ~1) was used to determine the content of cellular proteins and an aliquot (0.5 ml> was counted to determine the residual ‘25I radioactivity. The nonspecific binding was determined by incubating the cells with ‘251-HDL in the presence of a 20-fold excess of unlabelled HDL. Radioactivity was determined in a Packard gamma counter (MINAXIy 5550). 2.8. Statistical

analysis

The data are expressed as means + SD of three to four experiments. The results concerning PAF production are representative of at least three separate experiments, in which each experiment was duplicated by using two different dishes. The significance of differences was determined using the unpaired Student’s r-test. Differences at p < 0.05 were considered to be significant.

3. Results 3.1. Effect of HDL on thrombin-stimulated

PAF synthesis

in endothelial

cells

PAF accumulation started 30 s after the addition of thrombin, reached a peak at 5 min, and decreased thereafter at 24°C (Fig. I). While the time course of PAF synthesis was not affected, the level of newly synthesized PAF was decreased by HDL. The inhibitory effect of HDL (3 mg/ml) was prominent when the incubations were carried out at 24°C (49.1 + 0.6% inhibition) rather than at 37°C (32.8 + 2.5% inhibition). From this reason all experiments were done at 24°C. HDL includes PAF acetylhydrolase (1.15 pmol/min per mg protein), and after incubation at pH 2.8 for 30 min at room temperature the enzyme activity was negligible (0.38 fmol/min per mg protein). Acid-treated HDL inhibited the PAF production to the same extent as native HDL; the extent of inhibition by acid-treated and native HDL at 5 min was 41.8 _t 0.9 and 43.9 + 1.3%, respectively. Since PAF acetylhydrolase in LDL has been reported to

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Lipid Mediators Cell Signalling 13 11996) 73-88

79

Time, min Fig. 1. Effect of HDL on PAF production by thrombin in human vascular endothelial cells. Cultured endothelial cells (4 X IO5 cells) were preincubated for 5 min with HDL (0, ??) or BSA as a control (3 mg/ml) (0.0) and then stimulated with thrombin (0.5 U/ml) (0.W ) or saline as vehicle (0,~) for the indicated periods of time at 24°C. The incorporation of f3H]acetate into PAF was determined. Results are presented as mean f half-range of duplicate determinations.

hydrolyze PAF in endothelial cells (Zimmerman et al., 1990) acid-treated HDL was used to avoid the influence of PAF acetylhydrolase in HDL in the present study. Camussi et al. (1988) have reported that synthesis and release of PAF in human PMNs, macrophages and vascular endothelial cells stimulated by elastase are inhibited by plasma cr , -proteinase inhibitor and cr , antichymotrypsin. Therefore, we confirmed that the HDL preparation was not contaminated by these antiproteinases: The extent of platelet aggregation by trypsin (10 to 200 pg/ml) and thrombin (0.5 to 2 U/ml) in the presence of HDL (3 mg/ml) and that of the hydrolysis of benzoyl-L-tyrosine ethyl ester by cY-chymotrypsin (0.05 to 0.8 U/ml) in the presence of HDL (0.2 mg/ml) were almost the same as those in the absence of HDL. On the other hand, when the amount of PAF in thrombin-stimulated cells and the medium was determined by bioassay based on washed rabbit platelet aggregation after purification from the lipid extract, the level after thrombin (0.5 U/ml) stimulation for 5 min in the presence of HDL (3 mg/ml) (164 + 18 pg/105 cells) was 42.3% of the control in the absence of HDL (282 + 31 pg/105 cells), indicating that the decrease in the radioactivity of the [ 3H]acetylated lipids was consistent with the result obtained by bioassay. Accordingly, in this study, the amount of newly synthesized PAF was determined by the radioactivity of [3H]acetate incorporated into 1-radyl-2-acetyl-sn-glycero-3-phosphocholine. The dose-dependent effect of HDL on PAF synthesis is recorded in Fig. 2A. The inhibition of thrombin (0.5 U/ml)-stimulated PAF synthesis by HDL was increased in a concentration-dependent manner, and reached the maximum at the concentration of 3 mg/ml. The HDL preparation was not toxic to the culture because the release of lactate dehydrogenase from endothelial cells was less than 1% after treatment with HDL. On the other hand, PAF synthesis reached a plateau at the thrombin concentration of 0.1 U/ml (Fig. 2B). The inhibition of thrombin-stimulated PAF synthesis by HDL (3 mg/ml) was reversed as the concentration of thrombin increased, indicating that

80

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Lipid Mediators

-0

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1 .o

2.0

13 (1996)

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3.0

HDL, mg/ml

Thrombin,

U/ml

Fig. 2. (A) Inhibitory effect of HDL on thrombin-stimulated PAF production. Cultured endothelial cells were preincubated for 5 min with the indicated concentrations of HDL or BSA as a control and then stimulated with thrombin (0.5 U/ml) or saline as vehicle at 24°C for 5 min. (B) Dose-response curve for inhibitory effect of HDL on thrombin-stimulated PAF production. Cultured endothelial cells were preincubeted for 5 min with HDL or BSA as a control (3mg/ml) and then stimulated with the indicated concentrations of thrombin or saline as vehicle at 24°C for 5 min. In both experiments, the incorporation of [‘HIacetate into PAF was determined. Results in all instances are presented as mean f half-range of duplicate determinations of an increase in the radioactivity of PAF after subtraction of each of the basal values of unstimulated controls.

enhanced thrombin stimulation could overcome the inhibitory action of HDL. Moreover, in order to determine whether HDL was effective or not even after the addition of thrombin, we studied the influence of addition time of HDL on PAF synthesis. The addition of HDL 5 s after addition of thrombin caused 64.5% inhibition toward PAF synthesis. However, when HDL was added to the cells 30 s, 1 min, and 2 min after addition of thrombin, the magnitude of inhibition decreased to 42.9, 27.5 and 8.7%, respectively. These observations show that HDL was not effective if added after the cell activation. On the other hand, when the cells were preincubated with HDL or BSA as a control (3 mg/ml) for 60 min and then washed with buffer, the PAF production in the absence of HDL was 85.9 f 1.8% of the control (10575 f 862 dpm). The inhibition of PAF synthesis (14.1 + 1.8% inhibition) was not significant, compared with that in the presence of HDL (3 mg/ml) (63.3 + 0.1% inhibition), suggesting that HDL may affect PAF production upon stimulation by thrombin. Furthermore, in order to investigate whether the inhibition of PAF synthesis by HDL was only toward thrombin, we examined the effects of HDL on histamine- and A23 187stimulated PAF syntheses at 10 min and 15 min, at which PAF synthesis reached a peak, respectively. As shown in Table 1, histamine (10 PM)- and A23 187 (2.5 PM)-stimu-

.I. Sugntani et al./J. Table 1 Inhibitory A23187

Lipid Mediators Cell Signalling 13 (19961 73-88

effect of HDL on PAF production

cells stimulated

with thrombin,

PAF (dpm)

Assay

Thrombin

+ _

Histamine

+ _ + _

A23187

in endothelial

81

histamine,

and

Inhibition (%I

Control

Plus HDL

11420+78 260+ 15 10396+475 242 + 25 3 264 + 608 221+5

4999 + 495 329*9 3 705 + 548 249f4 1204+3 38756

56.7 66.0 73.2

Cultured endothelial cells were preincubated for 5 min in the presence or absence of HDL (3 mg/ml) and then stimulated with thrombin in saline (0.5 U/ml), histamine in saline (10 PM), A23187 dissolved in 0.2% dimethylsulfoxide (2.5 PM) or vehicle for 5, 10 or I5 min, at which PAF synthesis reached a peak, respectively, at 24°C. Results are presented as mean f half-range of duplicate determinations

lated PAF syntheses were induced to similar extents as thrombin (0.5 U/ml and 0.1 U/ml, respectively), and were similarly reduced by HDL at the concentration of 3 mg/ml, indicating that HDL inhibited not only thrombin stimulation but also other receptor-mediated stimulation. In addition, since unsaturated fatty acids inhibit the PAF synthesis in neutrophils and eosinophilia leukemia cells (Remy et al., 1989; Shikano et al., 1993), the effect of HDL lipids on thrombin-stimulated PAF synthesis was determined (Table 2). Althogh HDL lipids inhibited PAF synthesis by 16.5%, such inhibition was less than that of HDL. This observation suggested that HDL component except HDL lipids is required for the inhibition of PAF synthesis in endothelial cells. Next, the effect of HDL on thrombin-stimulated PAF synthesis was compared with that of LDL. Since the protein content of LDL particles is less than 50% of that of HDL particles, the effects of lipoproteins at an equimolar concentration were compared. HDL at the concentration of 1.5 mg/ml suppressed thrombin (0.5 U/ml)-induced PAF

Table 2 Effect of HDL lipids on thrombin-stimulated

PAF production

in endothelial

Assay

Thrombin

PAF (dpm)

Control

+ _ + _

8299 f 627 202+ 16 5444k69 576*6 7050+ 165 289 f 29

Plus HDL Plus HDL lipids

+ _

cells Inhibition (%I

39.9 16.5

Cultured endothelial cells were preincubated for 5 min in the presence or absence of HDL (1 mg/ml) or HDL lipids and then stimulated with thrombin in saline (0.5 U/ml) or saline as vehicle for 5 min at 24°C. HDL lipids: total lipids in HDL (I mg) were extracted according to the method of Bligh and Dyer (1959) and dispersed in 1 ml of 0.25% BSA/saline. Results are presented as meanf half-range of duplicate determinations

82

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.I. Sugatani et cd/J. Lipid Mediators Cell Signailing 13 (1996) 73-88

more strongly than LDL (62.0 + 1.2% inhibition by LDL, respectively).

by HDL and 23.1 + 1.6%

acid, 3.2. Effects of HDL on PAF acetylhydrolase actiuity, release of 13H]arachidonic and acetyl-CoA:lyso-PAF acetyltransferase activity in thrombin-stimulated endothelial cells

The majority of newly synthesized PAF was retained in the cells in the presence and absence of HDL (94.9 + 3.3 and 98.6 & 5.7%, respectively). Since HDL might augment the degradation of newly synthesized PAF in thrombin-stimulated endothelial cells, leading to the decrease in the level of accumulated PAF, we studied the effect of HDL on PAF acetylhydrolase activity in endothelial cells and the medium. In non-stimulated endothelial cells, intracellular and extracellular PAF acetylhydrolase activities were 405 + 21 fmol/min per mg protein and 23 f 2 fmol/min per ml, respectively. In the presence and absence of HDL at the concentration of 1 mg/ml, the enzyme activity in intact cells and extracellular medium 2 and 5 min after thrombin (0, 0.1, and 1 .O U/ml> stimulation was not significantly changed (data not shown). The PAF synthesis in agonist-stimulated endothelial cells occurs via two steps; activation of phospholipase A 2, which hydrolyzes a long chain fatty acid from a precursor phospholipid of PAF (1-alkyl-2-acyl-sn-glycero-3-phosphocholine) to yield lyso-PAF, and acetylation of the lyso-PAF. First, in order to investigate the possibility that phospholipase A, activity was suppressed by HDL, we determined the amount of [ 3H]arachidonic acid released from thrombin-stimulated endothelial cells. 5 min after thrombin stimulation, the amount of [ 3Hlarachidonic acid released from endothelial cells into the medium was not affected by HDL, whereas it was rather enhanced by HDL at

0

10

20

30 Time,

40

50

60

min

Fig. 3. Effect of HDL on thrombin-induced [‘Hlarachidonic acid release. [‘Hkachidonic acid release induced by thrombin (0.1 U/ml) (0, W ) or saline as a control (0.0 ) in the presence ( 0, W) or absence (0,O) of HDL (3 mg/ml) was determined as described in section 2. Ratio of [‘Hlarachidonic acid release to the total [‘Hjarachidonic acid incorporated in cells was calculated. Results are presented as mean f SD of three determinations. ’ ’ p < 0.01 vs the corresponding control group (Student’s t-test for unpaired data).

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Lipid Mediarors Cell Signalling 13 (1996) 73-88

00’ 2

4

6

a

83

10

Time, min Fig. 4. Effect of HDL on acetyl-CoA:lyso-PAF acetyltransferase activity in thrombin-stimulated endothelial cells. Cultured endothelial cells in HDL (0, ??) or BSA (0 ,O) as a control (1 mg/ml) were stimulated with thrombin (0.1 U/ml) (O,M ) or saline as vehicle (O,O) for the indicated periods of time at 24°C. After stimulation of the cells, the medium was removed and discarded. The cells were immediately chilled on ice, washed with 1 ml of ice-cold 10 mM Tris/HCl buffer (pH 6.9) containing 0.25 M sucrose, 1 mM D’IT and 25 mM NaF twice and then freezed in liquid nitrogen. The cells in 1 ml of the same buffer were scraped using a rubber policeman and sonicated for 30 s in an ice bath. The acetyltransferase activity in the cell lysates was determined as described in section 2. Results are presented as mean&SD of three to four determinations. * p < 0.05, * * p < 0.01 vs the corresponding control group (Student’s r-test for unpaired data).

60 min (Fig. 3). It was confirmed by counting the radioactivity from the [ 3H]arachidonic acid-labelled cellular phospholipids. The 3H radioactivities of cellular phospholipids 5 min after stimulation with thrombin (0.1 U/ml> were 92.8 f 5.1 and 93.2 + 0.8% of the control in the presence and absence of HDL (3 mg/ml), respectively. These findings demonstrated that phospholipase A, activity was not inhibited by HDL. Furthermore, the effect of HDL on acetyl-CoA:lyso-PAF acetyltransferase activity was studied in parallel with PAF synthesis. In non-stimulated endothelial cells, the basal activity was 5.02 + 1.72 nmol/min per mg protein. After thrombin stimulation, the enzyme activity transiently increased about 4-fold during PAF synthesis and gradually returned to the basal level. The time course of the acetyltransferase activity after thrombin stimulation in the presence of HDL was the same as that in the absence of HDL, whereas the level of the enzyme activity significantly decreased and the maximal increase 2 min after thrombin stimulation was 60% of the control (Fig. 4). The extent of inhibition of the enzyme activity was near that of the PAF production (53.8% of the control). On the other hand, when the cell lysates were incubated with acetyl-CoA without exogenous lyso-PAF and with 2.5fold concentration (100 PM) of lyso-PAF, the enzyme activity 2 min after thrombin stimulation in the presence of HDL was decreased to 57.3 and 56.7% of the control, respectively. These observations show that the decrease in the PAF production in the presence of HDL resulted from the blockade of the acetyltransferase activation rather than the enhancement of acyltransferase activity, i.e., the decrease in supply of substrate lyso-PAF.

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0

0.2

0.4

0.6

0.0

1 .o

HDL, mg protein/ml Fig. 5. Effect of thrombin on binding of ‘251-HDL and its uptake to cultured endothelial cells. The cells were preincubated for 30 s with varying concentrations of ‘lSI-HDL at 4°C (0.0) or 24°C (a,= ) and then stimulated for 5 min with thrombin (1 .O U/ml) (0, ?? ) or saline as a control (0,O). The amount of protein in each dish was measured and the amount of total cellular “‘I-HDL bound/mg cell protein was calculated. Results are presented as mean + SD of three determinations. * p < 0.05, * * p < 0.01 vs the corresponding control group (Student’s t-test for unpaired data).

3.3. Effect of thrombin on binding of “‘I-HDL

and its uptake to endothelial

cells

We studied whether thrombin stimulation enhanced incorporation of HDL into endothelial cells, leading to the subsequent inhibition of intracellular acetyltransferase activity. Fig. 5 shows the influence of thrombin stimulation on binding of ‘251-HDL and its uptake to endothelial cells. In order to minimize internalization of HDL, endothelial cells were incubated with 12’I-HDL at 4°C. The amount of HDL bound to normal endothelial cells was almost the same as that to thrombin-stimulated ones at 4°C. The specific binding to normal and thrombin-stimulated endothelial cells at 4°C was near 20.4 f 5.4 and 21.2 + 9.9 ng HDL/mg cell protein, respectively (data not shown). The amount of HDL incorporated into endothelial cells for 5 min incubation, which is obtained by subtraction of the amount of total binding at 4”C, from that at 24”C, was slight (less than 0.05% of addd HDL) and was not able to prevent the acetyltransferase activity in the cell lysates (IC,,, 70 pg HDL protein toward 2.5 pg protein of cell lysates). Thrombin stimulation rather reduced the amount of HDL uptake. These results demonstrate that the inhibition of PAF synthesis might be mediated via cell surface receptors but not due to the increase in the binding of HDL and its uptake.

4. Discussion In this report, we investigated the influence of lipoprotein on PAF synthesis in human vascular endothelial cells. First, we confirmed that stimulation of endothelial cells by thrombin elicited PAF synthesis in a concentration-dependent way, which peaks within 5

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min, being consistent with previous results reported by Prescott et al. (1984), Hirafuji et al. (1987) and Ghigo et al. (1988). The results of the present study demonstrate that agonist (thrombin, histamine and A23 187)-stimulated PAF production in endothelial cells was inhibited by HDL, based on the facts that the incorporation of [ 3HIacetate into [3H]acetyl-PAF was reduced and PAF accumulation determined by bioassay (platelet aggregation) was decreased. The majority of the newly synthesized PAF remained associated with the endothelial cells in the presence and absence of HDL. HDL therefore appears not to remove the newly synthesized PAF from cells to HDL particles, which are subsequently released back into the extracellular fluid and deacylated by ecto-type PAF acetylhydrolase on endothelial cells. We therefore attempted to determine which pathway in thrombin-mediated PAF synthesis was influenced by HDL. Whereas HDL did enhance [3H]arachidonic acid release for prolonged incubation with thrombin (60 min), it did not affect [ 3H]arachidonic acid release for the short-time incubation (5 min). Our results further demonstrate that HDL did not enhance PAF acetylhydrolase activity in the stimulated cells and did not induce the enzyme secretion, i.e., that the reduction of PAF accumulation by HDL was not due to the degradation of newly synthesized PAF. These results show that early events in the PAF synthesis pathway, i.e., the binding of thrombin to endothelial cells and the phospholipase A, activation, were not affected by HDL. On the other hand, acetyl-CoA:lyso-PAF acetyltransferase activity in thrombin-stimulated cells was reduced by HDL, which was not restored to normal by adding exogenous lyso-PAF. Accordingly, it is possible that the activation of the acetyltransferase is blocked by HDL, rather than that the acetyl-CoA:lyso-PAF acyltransferase activity is enhanced by HDL leading to the decrease in the supply of lyso-PAF for PAF synthesis. Endothelial cells cause the shape changes in response to stimulants such as thrombin to enhance permeability to macromolecules, e.g., albumin (Del Vecchio et al., 1987) and to facillitate the transport of LDL into the cells which activates intracellular lysophosphatidylcholine acyltransferase (Ezaki et al., 1994). However, since the incorporation of HDL into the cells was the acetyltransferase might be not directly not enhanced by thrombin stimulation, attacked by incorporated HDL. Concerning the mechanism of activation of the acetyltransferase, it has been reported that the enzyme is activated by phosphorylation via the involvement of protein kinase C, protein kinase A, and/or calmodulin-dependent kinase in cell-free experiments (Lenihan and Lee, 1984, Domenech et al., 1987; Nieto et al., 1988) and the involvement of protein kinase C in whole-cell experiments (Whatley et al., 1989; Heller et al., 1991). In addition, Ca2+ at micromolar concentrations has been shown to regulate the acetyltransferase activity in rat splenic microsomes by decreasing the K, for acetyl-CoA (Gomez-Cambronero et al., 1985). HDL may modulate such phosphorylation or Ca2+-dependent activation of the acetyltransferase in endothelial cells, resulting in the reduced enzyme activity. Whereas there are many reports on agents and factors influencing PAF synthesis, few inhibitors existing in vivo like HDL have been found. HDL has a mitogenic effect and serves as a scavenger of tissue cholesterol in a reverse cholesterol transport pathway. In addition to these events, our present study demonstrates the new action of HDL as inhibiting the PAF synthesis in agonist-stimulated endothelial cells. PAF synthesized in endothelial cells and PMNs participates in initial recruitment of PMNs, monocytes and

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platelets to blood vessels by increasing cell adhesion to the endothelium (Zimmerman et al., 1985; DiCorleto and Dela Motte, 1989; Ding et al., 1992; Hirafuji and Shinoda, 1993). Bourgain et al. (1985) have reported that PAF infusion to the mesenteric artery induces pathological alterations similar to that found in early atherogenesis (Koltai and Braquet, 1992). Thus, PAF is considered to play a potential role in the initiation and progression of atherosclerosis. The inhibitory effect on PAF synthesis of HDL might be associated with its anti-atherosclerotic action by suppressing cell interaction such as PMN, monocytes and platelet adhesion to endothelial cells.

Acknowledgements This work was supported in part by a Research Grant for the study of Intractable Diseases from the Ministry of Health and Welfare of Japan and by a grant from Ono Medical Research Foundation.

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