Degradation of Phospholipid Transfer Protein (PLTP) and PLTP-generated Pre-beta -high Density Lipoprotein by Mast Cell Chymase Impairs High Affinity Efflux of Cholesterol from Macrophage Foam Cells*

Miriam LeeDagger §, Jari Metso, Matti Jauhiainen, and Petri T. KovanenDagger ||

From the Dagger  Wihuri Research Institute, Kalliolinnantie 4, FIN-00140 Helsinki, Finland, § Faculty of Biology, University of Havana, 10400 Havana, Cuba, and  National Public Health Institute, Department of Molecular Medicine, Biomedicum, Haartmaninkatu 8, FIN-00290, Helsinki, Finland

Received for publication, October 23, 2002, and in revised form, December 30, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human atherosclerotic lesions contain mast cells filled with the neutral protease chymase. Here we studied the effect of human chymase on (i) phospholipid transfer protein (PLTP)-mediated phospholipid (PL) transfer activity, and (ii) the ability of PLTP to generate pre-beta -high density lipoprotein (HDL). Immunoblot analysis of PLTP after incubation with chymase for 6 h revealed, in addition to the original 80-kDa band, four specific proteolytic fragments of PLTP with approximate molecular masses of 70, 52, 48, and 31 kDa. This specific pattern of PLTP degradation remained stable for at least 24 h of incubation with chymase. Such proteolyzed PLTP had reduced ability (i) to transfer PL from liposome donor particles to acceptor HDL3 particles, and (ii) to facilitate the formation of pre-beta -HDL. However, when PLTP was incubated with chymase in the presence of HDL3, only one major cleavage product of PLTP (48 kDa) was generated, and PL transfer activity was almost fully preserved. Moreover, chymase effectively depleted the pre-beta -HDL particles generated from HDL3 by PLTP and significantly inhibited the high affinity component of cholesterol efflux from macrophage foam cells. These results suggest that the mast cells in human atherosclerotic lesions, by secreting chymase, may prevent PLTP-dependent formation of pre-beta -HDL particles from HDL3 and so impair the anti-atherogenic function of PLTP.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reverse cholesterol transport is a physiologically important route for removal of excess cholesterol from the membranes of peripheral cells and its transport to the liver for secretion into the bile. This process is of special importance for macrophages, which, having no ability to regulate incoming cholesterol, are readily transformed into cholesterol-loaded foam cells. Several factors, such as apolipoproteins, lipolytic enzymes, lipid transfer proteins, and lipoprotein receptors, are involved in reverse cholesterol transport (1). Initially, efflux of cellular cholesterol is promoted by extracellular cholesterol acceptors. The primary and most efficient acceptors of cholesterol seem to be small discoidal lipid-poor pre-beta -migrating high density lipoproteins (pre-beta -HDL)1 (2). These particles interact with the cell membrane through a microsolubilization process that leads to uptake first of phospholipids and then of cholesterol from the membrane (3). This process appears to be controlled by the ATP-binding cassette transporter protein 1 on the macrophage cell membrane (4), its transcription being regulated in part by the orphan nuclear receptor LXR. Activation of LXR in macrophages increases not only ATP-binding cassette transporter A1 but also the expression of genes encoding ABCG1 and apoE (5, 6), which may be also involved in efflux of cholesterol toward HDL. Recently, it was shown (7) that LXR activation in macrophages also up-regulates phospholipid transfer protein (PLTP) expression, but this up-regulation does not have any direct effect on cholesterol efflux from mouse peritoneal macrophages.

PLTP contributes to the remodeling of HDL by promoting net transfer and exchange of phospholipids among HDL subclasses and other lipoproteins (8). PLTP-mediated remodeling of HDL can occur via two major pathways. (i) PLTP facilitates the transfer of excess surface phospholipids from post-lipolytic chylomicrons and very low density lipoproteins to the HDL fraction, demonstrating the importance of this process for the maintenance of HDL levels (9). (ii) Human plasma PLTP in vitro converts small HDL3 particles into larger particles, with concomitant release of poorly lipidated apoA-I, which displays pre-beta -mobility in agarose electrophoresis (10, 11). Moreover, in vivo, in both transiently expressed and transgenic mouse models of PLTP, an increased capacity of the plasma of mice overexpressing human PLTP to produce pre-beta -HDL has been demonstrated (11-13). Thus, by influencing HDL size and composition, PLTP plays an important role in HDL metabolism and modulates its anti-atherogenic potential.

Proteolytic enzymes, such as the mast cell-derived neutral protease chymase, have been shown to modify the composition and function of HDL particles profoundly. Mast cell chymase efficiently degrades apoA-I in isolated HDL3 fractions by specifically depleting the minor pre-beta -migrating HDL particles, thus impairing the first step of reverse cholesterol transport in vitro (14). Chymase is a chymotrypsin-like protease with broad cleavage specificity; thus, not only apoA-I (15) but also other apolipoproteins (16, 17) and plasma proteins (18) constitute substrates for this enzyme. In this paper, we investigated whether chymase can degrade PLTP and whether such proteolysis would impede its key function in reverse cholesterol transport, i.e. generation of pre-beta -HDL particles and their participation in the process of cholesterol efflux from cholesterol-loaded macrophages.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of Plasma Lipoproteins-- LDL (1.019-1.050 g/ml) and HDL3 (1.125-1.210 g/ml) were isolated from fresh normolipidemic human plasma by sequential ultracentrifugation, using KBr for density adjustments, and their quantities are expressed by their total protein content. LDL was first acetylated (acetyl-LDL) (19) and then radiolabeled by treatment with [3H]cholesteryl linoleate ([1,2-3H]cholesteryl linoleate, Amersham Biosciences) dissolved in 10% dimethyl sulfoxide (20), yielding preparations of [3H]cholesteryl linoleate incorporated into acetyl-LDL ([3H]CL-acetyl-LDL) with specific activities ranging from 30 to 100 dpm/ng protein. Isolated HDL3 preparations containing variable amounts of both alpha - and pre-beta -migrating HDL were used.

Purification and Phospholipid Transfer Assay of PLTP-- PLTP was purified from fresh human plasma by a combination of chromatographic techniques, as described (10, 21). The purified PLTP preparation displayed a single 80-kDa band in SDS-PAGE analysis and did not express cholesteryl ester transfer protein or lecithin:cholesterol acyltransferase activity. PLTP activity was measured by a radiometric assay, following PL transfer from radiolabeled donor PL liposomes to acceptor HDL3 particles (10, 22).

Human Chymase-- Recombinant human chymase (specific activity 80 BTEE units/µg) was provided by Teijin Ltd., Hino, Tokyo, Japan. The preparation was diluted in 5 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA (TNE buffer) before use. The enzyme preparation was fully inhibited by adding soybean trypsin inhibitor (SBTI, Sigma) at a final enzyme:inhibitor mass ratio of 1:100.

Proteolysis of PLTP by Chymase-- PLTP (2.5 µg, corresponding to PL transfer activity of 1000 nmol/h) and chymase (0.5 µg, equal to 40 BTEE units) were incubated in TNE buffer (final volume 170 or 230 µl) at 37 °C for up to 24 h in the absence or presence of HDL3 (1 mg of protein/170 or 230 µl). In an additional experiment PLTP and chymase were incubated with either HDL3 (0.5 mg/ml) or LDL (1.3 mg/ml). Incubations were stopped by immersing the tubes in ice and adding 50 µg of SBTI to each tube.

Aliquots of the incubation mixtures were used immediately for the HDL conversion assay (see below) and for Western blot analysis of PLTP. The residual samples were stored at -20 °C until PLTP phospholipid transfer activity was measured. Sample storage at -20 °C followed by one thawing does not affect PLTP activity. In one set of experiments, recombinant human PLTP expressed in the baculovirus-insect cell system (23) was incubated with chymase.

Proteolysis of HDL3 by Chymase-- HDL3 (1 mg of protein) was incubated with chymase (0.5 µg, equal to 40 BTEE units) in TNE buffer (final volume 170 µl) in the absence of PLTP. Purified PLTP was added to aliquots of this chymase-treated HDL3 to study PLTP-mediated HDL size conversion and pre-beta -HDL generation. For the cellular cholesterol efflux experiments, HDL3 (350 µg of protein) was incubated for 24 h at 37 °C with PLTP (PL transfer activity of 1000 nmol/h) or in TNE buffer only. These samples were divided into two identical aliquots, and the PLTP-treated and control HDL3 were incubated for 6 h at 37 °C in the presence of chymase (7 BTEE units). After full inhibition of the chymase activity by addition of SBTI, HDL3 aliquots were added to the macrophage foam cell medium in the concentrations shown in Fig. 6. "Blank" samples of PLTP incubated for 6 h at 37 °C in the absence or presence of chymase and then incubated in TNE buffer for 24 h were also added to the macrophage cultures in order to test their ability to promote efflux of cellular cholesterol in the absence of HDL3.

PLTP Immunoblots-- Western blot analysis was carried out essentially as described (24). Briefly, aliquots from incubations of PLTP with chymase performed in the absence or presence of HDL3 were applied to a 12.5% SDS-PAGE. After electrophoresis, the proteins were electrotransfered to nitrocellulose filters and immunoblotted with rabbit anti-human PLTP polyclonal IgG. The antibody, produced against the full-length human PLTP protein, was diluted 1:1000, and the filters were treated with the antibody overnight. After washing, peroxidase-labeled goat anti-rabbit IgG (1:2000 dilution) was added, and incubation was continued for 2 h. After washing, the proteins were visualized by ECL (Amersham Biosciences).

HDL Conversion Assay, Analysis of HDL Particle Size, and Quantitation of Pre-beta -HDL-- HDL particle size was determined by nondenaturing polyacrylamide gradient gel electrophoresis (GGE), as described previously (25). To study the effect of pretreatment with chymase on PLTP and on HDL3, respectively, assays were carried out after further 24 h incubation of (a) chymase-treated PLTP with fresh HDL3 and (b) chymase-treated HDL3 with fresh PLTP. The pre-beta -HDL band was further quantified by running the samples on two-dimensional crossed immunoelectrophoresis, as reported recently (11, 13). Another experiment was carried out with simultaneous incubation of PLTP, HDL3, and chymase, and the pre-beta -HDL band was quantified after various periods up to 24 h.

Cell Cultures and Loading of Macrophages with Cholesteryl Esters-- Peritoneal cells from unstimulated mice were harvested into PBS containing 1 mg/ml BSA. The cells were recovered after centrifugation, resuspended in DMEM (Invitrogen) containing 100 units/ml penicillin, 100 µg/ml streptomycin, and 20% fetal calf serum, and plated onto 24-well plates (BD Biosciences). After incubation at 37 °C for 2 h in a humidified CO2 incubator, nonadherent cells were removed by washing with PBS. The adherent cells (i.e. the macrophages) were loaded and radiolabeled by incubation for 18 h in the presence of 20 µg of protein/ml of [3H]cholesteryl linoleate-acetyl-LDL in DMEM supplemented with 20% fetal calf serum.

Cholesterol Efflux Assay-- [3H]Cholesterol-loaded macrophages were washed with PBS and incubated with DMEM supplemented with SBTI (final concentration in the medium 100 µg/ml) and the indicated concentrations of HDL3. After 4 h, the media were collected and centrifuged at 200 × g for 5 min, and the radioactivity of each supernatant was determined by liquid scintillation counting and normalized for the cellular protein mass. Under the conditions used, the [3H]cholesterol efflux from the macrophage foam cells is linear for up to 4 h of incubation and reflects the net flux of cholesterol from the macrophages into the medium (14, 15). The data presented are means ± S.D. of triplicate incubations.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of HDL3 on Degradation of PLTP by Chymase-- PLTP isolated from human plasma was incubated with human chymase at 37 °C in the absence or presence of 5 mg/ml of HDL3 for up to 24 h, and degradation products were analyzed by Western blotting, using a monospecific polyclonal anti-PLTP antibody (Fig. 1, panels A and B). In the absence of HDL3, chymase treatment of PLTP (80 kDa) for only 5 min led to the appearance of a degradation band of 48 kDa (panel A, arrow). At 6 h, three other distinct fragments with apparent molecular masses of 70, 52, and 31 kDa were also present. Densitometric analysis used for quantitation showed that at 6 and 24 h the decrease in the 80-kDa band PLTP band was 16 and 58%, respectively. Next, we incubated PLTP with chymase in the presence of HDL3 (panel B). Notably, all the HDL3 preparations used in this study contained both PLTP (80 kDa) and the 48-kDa band (panel B, 0 h lane), and the intensity of the latter band was enhanced after as little as 5 min of incubation with chymase. Importantly, prolongation of incubation from 6 to 24 h did not change the specific degradation patterns of PLTP produced by chymase (panels A and B). Similar results were obtained when a preparation of recombinant human PLTP was treated with chymase (not shown). In additional experiments, the formation of the PLTP 48-kDa band was observed when PLTP and chymase were incubated for 6 h in the presence of a low concentration of HDL3 (0.4 mg/ml, instead of 5 mg/ml, as above) (Fig. 2). Interestingly, incubation of PLTP with chymase in the presence of LDL (1.3 mg/ml) produced a PLTP fragmentation pattern displaying four major fragments (Fig. 2). The ultracentrifugally isolated LDL did not contain any immunodetectable PLTP (not shown).


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Fig. 1.   Effect of HDL3 on the degradation of PLTP by chymase. Incubation of PLTP (2.5 µg, specific activity = 400 nmol/h/µg) with chymase (0.5 µg, specific activity = 80 BTEE units/µg) in TNE buffer (final volume 170 µl) was carried out at 37 °C for different times up to 24 h. Two sets of incubations were performed, in the absence or presence of 1 mg of HDL3 protein/tube (final concentrations in the assay are 5 mg/ml protein; 1.5 mg/ml cholesterol). Incubations were stopped by placing the tubes on ice and by addition of soybean trypsin inhibitor (50 µg/tube). Aliquots of the incubation mixtures were applied to Western blot analysis of PLTP. Panel A, incubations of PLTP with chymase; panel B, incubations of PLTP with chymase in the presence of HDL3. Intact PLTP (80 kDa) and PLTP degradation fragments (apparent molecular mass of about 48 kDa) are indicated by arrows.


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Fig. 2.   Degradation of PLTP by chymase in the presence of physiological (human arterial intima) concentrations of HDL or LDL. Incubations of PLTP with chymase were carried out at 37 °C for 6 h under the conditions described in Fig. 1 but in the presence of HDL3 (final concentration 0.4 mg/ml protein; 0.1 mg/ml cholesterol) or LDL (final concentration 1.3 mg/ml protein; 2 mg/ml cholesterol). Aliquots of the incubation mixtures were used for Western blot analysis of PLTP. Intact PLTP (80 kDa) and a PLTP degradation fragment (apparent molecular mass of about 48 kDa) are indicated by arrows.

Phospholipid Transfer Activity of Chymase-treated PLTP-- To evaluate whether the phospholipid transfer activity of PLTP was impaired after degradation with chymase, incubation was terminated after 6 and 24 h by adding SBTI, and the mixtures were then incubated, using radiolabeled PL liposomes as donors and HDL3 as acceptors (Table I). When PLTP was treated with chymase in the absence of HDL3, the activity of PLTP to transfer PL from liposomes to HDL3 was reduced by 20% after 6 h and by 40% after incubation for 24 h (p < 0.05 for both reductions). However, the presence of HDL3 during the preincubation of PLTP with chymase for at least 24 h fully maintained PLTP phospholipid transfer activity.


                              
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Table I
Effect of chymase treatment on the phospholipid transfer activity of PLTP
PLTP (2.5 µg, specific activity = 400 µmol/µg/h) was incubated with chymase (0.5 µg, specific activity = 80 BTEE units/µg) in TNE buffer (final volume 170 µl) at 37 °C for 6 and 24 h. Incubations were performed in the absence or presence of HDL3 as described in Fig. 1. Four µl of the preincubation mixtures were taken, after stopping the reaction, to measure PLTP phospholipid transfer activity, as described under "Experimental Procedures." Percentages relative to incubations of PLTP alone are given in parentheses for each incubation time. Each measurement was made in triplicate and each value is a mean ± S.D.

Effect of Chymase Treatment of PLTP or HDL3 on PLTP-mediated HDL3 Size Conversion-- To study whether chymase would modify the ability of PLTP to promote HDL3 conversion (formation of large fused alpha - and small pre-beta -migrating HDL particles), a two-step incubation protocol was used. PLTP was first incubated with chymase to yield "chymase-treated PLTP" or with buffer alone (control PLTP) for 6 h. HDL3 was then added to the incubation mixture, and the incubation was continued for 24 h (size conversion assay). The distribution of HDL subclasses was analyzed by nondenaturing polyacrylamide gradient gel (GGE) electrophoresis (4-26%). Two subpopulations, of large and small sized HDL, were clearly separated (Fig. 3). On agarose gel electrophoresis, the large particles displayed alpha - and the small ones pre-beta -mobility (not shown). As shown in Fig. 3, panel A, when incubations were carried out in the presence of PLTP (lanes 2 and 3), the alpha -HDL particles were larger than in the control HDL3 (lane 1), whether or not PLTP had been pretreated with chymase (average sizes of 10.9 and 10.7 nm, respectively, as compared with the control 10.1 nm). However, when the conversion assay was performed with the control PLTP (lane 2), but not with the chymase-treated PLTP (lane 3), an intense protein band corresponding to poorly lipidated apoA-I was observed in the size range of about 6.5-7.0 nm. Even a short period of treatment of PLTP with chymase (10 min) reduced the PLTP-dependent generation of pre-beta -HDL (not shown). We also preincubated HDL3 with chymase (chymase-treated HDL3) or with buffer alone (control HDL3) for 6 h and performed the size conversion assay after adding PLTP (panel B). As previously observed, such treatment of HDL3 with chymase leads to (i) efficient degradation of apoA-I with formation of a limited number of large and medium size polypeptides, (ii) low efficiency degradation of apoA-II (15), and (iii) depletion of the pre-beta -migrating HDL present in the HDL3 preparation (14) as also seen here (lane 3). Interestingly, however, chymase did not abolish the ability of untreated (non-incubated) PLTP to generate pre-beta -HDL from the chymase-treated HDL3 (lane 2 compared with lane 4). A similar distribution of HDL particles was observed even after incubation of HDL3 in the presence of chymase for 24 h (not shown).


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Fig. 3.   Effect of chymase treatment of PLTP or HDL3 on PLTP-mediated HDL3 conversion. Incubation of PLTP (2.5 µg, specific activity = 400 nmol/h/µg) or HDL3 (1 mg of protein/tube) with chymase (0.5 µg, specific activity = 80 BTEE units/µg) was performed in TNE buffer (final volume 170 µl). After 6 h, the incubations were stopped by transferring the tubes to ice and by addition of soybean trypsin inhibitor (50 µg/tube). Fresh HDL3 or fresh PLTP was added to the chymase-treated PLTP (panel A) or chymase-treated HDL3 (panel B), respectively, and the incubation mixtures were further incubated for 24 h at 37 °C. The size of the HDL particles was measured in aliquots of the incubation mixtures by nondenaturing GGE, as described under "Experimental Procedures."

Effect of Chymase Treatment of PLTP or HDL3 on Pre-beta -HDL Content-- As shown above, the pre-beta -HDL band was also detected in the samples corresponding to control incubations performed in the absence of PLTP. This may reflect spontaneous generation of pre-beta -HDL from HDL3 during the incubation period at 37 °C (see Fig. 3, panels A and B, lanes 1). Consequently, the qualitative GGE data did not allow us to determine quantitatively the specific PLTP-dependent increase in pre-beta -HDL particles during the HDL3 conversion assay. To obtain this information, the above experimental protocol was used; the samples were then analyzed by two-dimensional crossed immunoelectrophoresis, using anti-apoA-I for quantitation, and the rocket areas corresponding to both alpha - and pre-beta -HDL were quantified. The coefficient of variation of the crossed immunoelectrophoresis was 2-3% among the assays performed. The amount of pre-beta -HDL was calculated as a percentage of the sum of alpha - and pre-beta -HDL (Fig. 4). This analysis indicated that, after 24 h of incubation in the absence of PLTP, the proportion of pre-beta -HDL particles present in the HDL3 sample that was preincubated for 6 h at 37 °C in buffer was 6% (panel A) versus 3-4% in the non-incubated HDL3 (not shown). However, the ability of HDL3, when pretreated with chymase for 6 h, to spontaneously generate pre-beta -HDL during a subsequent 24-h incubation at 37 °C was strongly reduced (panel B). When the control PLTP (preincubated in buffer only) was incubated with untreated HDL3, the amount of pre-beta -HDL increased to 37% (panel C), whereas the ability of chymase-treated PLTP to generate pre-beta -HDL particles was less (25%; panel D). Next, we tested whether the inhibitory effect of chymase pretreatment on the ability of HDL3 to generate pre-beta -HDL spontaneously, as observed above (panels B versus A), also applied to the PLTP-dependent generation of pre-beta -particles. For this purpose, HDL3 was incubated for 6 h in the absence or presence of chymase, and after inhibition of chymase, a second incubation for 24 h with untreated PLTP was performed. Interestingly, PLTP was able to generate smaller amounts of pre-beta -HDL from chymase-treated HDL3 as compared with that from the control HDL3 (increases from 6 to 36%; panels A and E, and from 2 to 27%; panels B and F).


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Fig. 4.   Effect of pretreatment of PLTP or HDL3 with chymase on pre-beta -HDL levels. PLTP (3 µg, specific activity = 331 nmol/h/µg) or HDL3 (1 mg protein/tube) were incubated with chymase (0.5 µg, specific activity = 80 BTEE units/µg) in TNE buffer (final volume 170 µl) for 6 h. The reaction was stopped by adding soybean trypsin inhibitor (50 µg/tube). HDL size conversion assays were performed after adding fresh PLTP to HDL3 samples that had been preincubated in the absence or presence of chymase (panels E and F), and adding fresh HDL3 to samples of PLTP that had been preincubated in the absence or presence of chymase (panels C and D). A second incubation of these samples was carried out for 24 h, and the amounts of pre-beta -HDL and alpha -HDL were measured by two-dimensional crossed immunoelectrophoresis, as stated under "Experimental Procedures." The amounts of pre-beta -HDL are expressed as percentages of the total alpha -HDL + pre-beta -HDL. The values of the control and chymase-treated HDL3 incubated in buffer only (HDL blanks) are shown in panels A and B.

Simultaneous Incubation of PLTP and HDL3 in the Presence of Chymase: Effect on Pre-beta -HDL Content-- Because treatment of PLTP with chymase only partially reduced its ability to generate pre-beta -HDL (see Fig. 4, panels C and D), and chymase treatment of HDL3 only slightly reduced the PLTP-dependent formation of pre-beta -HDL from these particles (Fig. 4, panels E and F), we next determined the effect of chymase on pre-beta -HDL levels when PLTP, HDL3, and chymase were incubated simultaneously for up to 24 h (Fig. 5). As expected, in the absence of chymase, the proportions of pre-beta -HDL increased in a time-dependent manner (Fig. 5, left panels, from 3 to 14 to 30%). In sharp contrast, when chymase was present in the mixture, accumulation of pre-beta -HDL was almost totally abolished. Accordingly, in all incubations in which chymase was present, the amounts of pre-beta -HDL remained close to zero, irrespective of the incubation period (Fig. 5, right panels). These results were further confirmed by apoA-I immunoblot analysis of a parallel set of samples fractionated in agarose gels. When both HDL3 and PLTP were incubated with chymase for a short time (up to 60 min), depletion of the endogenously formed pre-beta -HDL was observed after 10 min (results not shown). This indicates that chymase was highly efficient in degrading pre-beta -HDL even in the presence of partially active PLTP.


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Fig. 5.   Simultaneous incubation of PLTP and HDL3 in the presence of chymase: effect on pre-beta -HDL levels. PLTP (3 µg, specific activity = 331 nmol/h/µg) and HDL3 (1 mg protein/tube) were simultaneously incubated in the absence or presence of chymase (0.5 µg, specific activity = 80 BTEE units/µg) in TNE buffer (final volume 230 µl) for 10 min, 6 h, and 24 h. The incubations were stopped by adding SBTI (50 µg/tube). The amounts of the pre-beta - and alpha -HDL subpopulations were analyzed and expressed as described in Fig. 4.

Effect of Chymase on PLTP-treated HDL3-induced Cholesterol Efflux-- Because incubation of HDL3 with PLTP promoted strong formation of pre-beta -HDL (Fig. 5, incubation times up to 24 h), we next studied the ability of the PLTP-pretreated HDL3 to induce efflux of cellular cholesterol from macrophage foam cells and the effect of chymase on this process. We observed that both the control and the PLTP-treated HDL3 induced a dose-dependent and saturable increase in [3H]cholesterol efflux (Fig. 6, panel A). It was observed that, at 3 µg/ml, the rate of efflux induced by the PLTP-treated HDL3 (pre-beta -HDL content 33 ± 10%) was twice that observed with the control HDL3 (pre-beta -HDL content 12 ± 5%). Thus, the efficiency of the process, especially at low concentrations of HDL3 (up to 12.5 µg/ml), appeared to depend on the pre-beta -HDL content of HDL3. Indeed, the efficiency of the process increased by 2-fold after PLTP treatment, as demonstrated by a decrease in Km from 18 µg/ml in the control HDL3 to 8 µg/ml (panel B). After chymase treatment, the kinetics of the efflux promoted by PLTP-treated and by the control-HDL3 were identical (31 and 32 µg/ml, respectively), and reflected loss of the high affinity component of the efflux, i.e. a decrease in the rate of efflux within the low range of cholesterol acceptor concentrations (below 12.5 µg/ml). Chymase treatment also caused full depletion of pre-beta -HDL from the HDL3 preparations. The efflux of cholesterol promoted by PLTP alone was insignificant.


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Fig. 6.   Effect of chymase on the efflux of cholesterol from macrophage foam cells mediated by PLTP-treated HDL3. HDL3 was preincubated at 37 °C for 24 h in the absence or presence of PLTP, and the incubation was continued for 6 h after adding chymase or an equal volume of TNE buffer. Degradation by chymase was stopped by adding SBTI. Aliquots of the incubation mixtures were added in the indicated final concentrations of HDL3 to [3H]cholesterol-loaded macrophage foam cells cultured in an SBTI-containing medium. Panel A, the 3H radioactivity in the medium was determined after incubation for 6 h at 37 °C and normalized for the cellular protein. Values are means ± S.D. of triplicate wells. From the values for each plate, blank values (efflux measured in the absence of HDL3) were subtracted. PLTP blanks promoted non-significant levels of efflux. The percentage of cholesterol efflux from the macrophages in the presence of control HDL3 ranged from 7 to 9%. Panel B, data in panel A were transformed to their reciprocal values, and the kinetics of the cholesterol efflux (Km values) promoted by the various cholesterol acceptors were analyzed by the program Prism. The statistical significance of the data (*, p < 0.05) was determined by Student's t test for paired samples (control HDL3 versus PLTP-treated HDL3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we describe how a human neutral protease, mast cell chymase, degrades and partially inactivates PLTP and how HDL3 modulates this process. Moreover, high affinity efflux of cholesterol from the cholesterol-loaded macrophages, as mediated by PLTP-derived pre-beta -HDL, was abolished by chymase treatment.

Degradation of PLTP by chymase was a rapid process, and the first cleavage was demonstrated within 10 min of incubation. Interestingly, upon prolonged incubation, a pattern was observed reflecting that, despite the broad cleavage specificity of chymase, only four main, relatively stable PLTP fragments were produced. Such limited protease susceptibility suggests the presence of a restricted number of exposed amino acid sequences leading to specific chymase-accessible domains in PLTP.

Two forms of PLTP have been fractionated from native human plasma by size exclusion chromatography (26). One of these fractions corresponds to an inactive form of PLTP that elutes between HDL and LDL, and most important, the other fraction containing active PLTP elutes in the position of HDL (27, 28). Recent data based on PLTP molecular modeling and mutations at the N- and C-terminal regions of PLTP suggest that PLTP may have more than one HDL-binding site (29-31). Interestingly, all the suggested docking sites of PLTP on HDL3 involve peptide bonds that are potential cleavage sites for chymase, i.e. they contain aromatic or branched-chain aliphatic amino acids (32). When HDL3 was added to the incubation mixture containing PLTP and chymase, only one major degradation product of PLTP (of about 48 kDa) was observed, a finding compatible with the view that docking of PLTP onto HDL3 did alter the chymase-accessible sequences on PLTP. Interestingly, digestion of PLTP by chymase in the presence of HDL3 did not impair the phospholipid transfer function of PLTP. Notably, each of the HDL3 preparations used in this study also contained, in addition to intact PLTP (80 kDa), a PLTP degradation fragment of 48 kDa, i.e. similar in size to the major PLTP fragment produced by chymase in the presence of HDL3. Interestingly, the active form of PLTP purified from fresh human plasma has been resolved into two bands by PAGE, one corresponding to the 80-kDa band of intact PLTP and the other to a PLTP proteolytic fragment of 51 kDa with the N-terminal region located between amino acids 163 and 184 of the PLTP molecule (33). Provided the Phe161 in the PLTP molecule is susceptible to cleavage by chymase, chymase-induced formation of a PLTP degradation fragment of this size could take place. Indeed, we cannot exclude the possibility that such a fragment would be included in the broad 48-kDa band generated by chymase. Identification of the hydrolytic sites on PLTP that are susceptible to chymase cleavage is currently under investigation in our laboratory.

The mechanism underlying functional protection by a high concentration of HDL3 (5 mg/ml) of PLTP against chymase is of interest. This effect was also observed when HDL3 was present at a lower concentration (0.4 mg/ml; see Fig. 2), i.e. close to the physiological concentration range of HDL in the intimal fluid (34). Interestingly, LDL, when added at a concentration similar to that present in the arterial intima (34), did not protect PLTP against chymase. The fact that the active fraction of PLTP, when isolated from plasma by size exclusion chromatography, eluted in a position corresponding to that of large sized HDL particles (27) has suggested that PLTP is in a fully active conformation when associated with HDL. The present overall results, in addition, allow the speculation that binding of PLTP to HDL3 prevents inactivation of PLTP by proteolytic cleavage, at least with chymase. It will be important to study whether such protection applies to other naturally occurring proteases as well.

The finding that by treating HDL3 with chymase, the spontaneous, but not the PLTP-dependent, generation of pre-beta -HDL from spherical HDL3 particles was abolished suggests that chymase depleted the most readily dissociating apoA-I molecules. The fact that pretreatment of HDL3 with chymase was, nevertheless, unable to abolish the generation of pre-beta -HDL promoted by PLTP is compatible with the view that the bulk of the apoA-I remaining on the surface of alpha -HDL particles was available for active generation of pre-beta -HDL during HDL remodeling. Our results indicate that proteolysis of HDL3 by chymase, which produces limited degradation of apoA-I, does not impair its interaction with plasma proteins, such as lecithin:cholesterol acyltransferase (35) and PLTP (this study) which are involved in HDL remodeling.

High affinity efflux of cholesterol from macrophage foam cells has been defined as the component of the efflux of cellular cholesterol which operates in the low concentration range of a cholesterol acceptor (15). This component of the efflux process has been demonstrated to be highly susceptible to protease treatment that specifically depletes various lipid-free or lipid-poor apolipoproteins from different kinds of cholesterol acceptors (14, 15, 17). Thus, it likely reflects the apolipoprotein-mediated pathway of cholesterol efflux from macrophage foam cells (36). These observations provide further support for the notion that pre-beta -HDLs have a crucial function as the primary acceptors of cellular cholesterol (2, 37). The present data demonstrate for the first time that pre-beta -HDL particles generated by PLTP are responsible for the increased efflux of cholesterol from cultured macrophages and that this process is fully blocked by proteolysis of the formed pre-beta -HDL by chymase, therefore suggesting that PLTP functions as an anti-atherogenic factor and contributes to the removal of accumulated cholesterol from lesion macrophages. Previous studies (11, 38) carried out with cholesterol-loaded fibroblasts or using the human PLTP transgenic mouse model clearly support the concept that PLTP-generated pre-beta -HDLs are involved in the cholesterol efflux process. Because plasma PLTP activity is significantly correlated with the ability of plasma to generate pre-beta -HDL (11, 13, 39), the present observation, made with cholesterol-loaded macrophages, is potentially of physiological relevance. Interestingly, it has also been reported that the ability of mildly trypsinized HDL to remove cholesterol from cultured fibroblasts is restored by PLTP (40). The anti-atherogenicity of PLTP was also reported recently by van Haperen et al. (11) in mice overexpressing PLTP. Despite a lower HDL level in plasma of these mice, the elevated PLTP was more effective in preventing in vitro accumulation of cholesterol in macrophages via increased formation of pre-beta -HDL. Also in mice with adenovirus-mediated overexpression of PLTP, increased levels of pre-beta -HDL were observed (12, 13). In contrast, PLTP deficiency in hyperlipidemic mice models has resulted in decreased atherosclerosis that was explained via the effects on VLDL secretion (41), and strong overexpression of PLTP in mice heterozygous for the LDL receptor has been shown to increase the susceptibility to atherosclerosis (42). However, both the overexpression and gene knockout models are quite extreme conditions, and therefore, depending on the metabolic status, PLTP may display anti- or proatherogenic properties. Further studies are definitely needed to unravel the detailed mechanisms on the association of PLTP with atherosclerosis.

In summary, the present results enable us to frame the following hypothesis. A fraction of the active PLTP in association with a subclass of HDL from the plasma compartment enters the arterial intima, where degranulated chymase-containing mast cells are present (43). In the intimal fluid, the HDL-associated PLTP may maintain its activity despite the presence of chymase, so producing pre-beta -HDL particles. However, in intimal areas with chymase-secreting mast cells, chymase-dependent depletion of the PLTP-generated pre-beta -HDL particles could occur, thus causing an impairment of cholesterol efflux from macrophages via this local anti-atherogenic function of PLTP.

    ACKNOWLEDGEMENTS

We thank the Teijin Company Ltd., Japan, for providing the recombinant chymase and Päivi Ihamuotila and Laura Vatanen for excellent technical assistance.

    FOOTNOTES

* This work was supported by a grant from the Sigrid Juselius Foundation, Helsinki, Finland (to M. L.), by a grant from the Paavo Nurmi Foundation (to M. L. and M. J.), by the Finnish Foundation for Cardiovascular Research and by Pfizer International HDL Research Awards Program 2001-2003 (to M. J.). Part of this work was presented in abstract form at the European Lipoprotein 25th Anniversary, 9-11 September, 2002 Tutzing, Germany.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Wihuri Research Institute, Kalliolinnatie, 4, Helsinki 00140, Finland. Tel.: 358-9-681- 4131; Fax: 358-9-637-476; E-mail: petri.kovanen@wri.fi.

Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M210847200

    ABBREVIATIONS

The abbreviations used are: pre-beta -HDL, pre-beta -migrating high density lipoproteins; PLTP, phospholipid transfer protein; LDL, low density lipoproteins; SBTI, soybean trypsin inhibitor; BTEE, N-benzoyl-L-tyrosine ethyl ester; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; GGE, gradient gel electrophoresis; PL, phospholipid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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