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INTRODUCTION |
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-
-migrating high density lipoproteins
(pre-
-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-
-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-
-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-
-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-
-HDL particles and
their participation in the process of cholesterol efflux from
cholesterol-loaded macrophages.
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EXPERIMENTAL PROCEDURES |
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
-
and pre-
-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-
-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-
-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-
-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-
-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.
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RESULTS |
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.
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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.
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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
- and small pre-
-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
- and the
small ones pre-
-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
-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-
-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-
-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-
-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."
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Effect of Chymase Treatment of PLTP or HDL3 on
Pre-
-HDL Content--
As shown above, the pre-
-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-
-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-
-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
- and
pre-
-HDL were quantified. The coefficient of variation of the
crossed immunoelectrophoresis was 2-3% among the assays performed.
The amount of pre-
-HDL was calculated as a percentage of the sum of
- and pre-
-HDL (Fig. 4). This
analysis indicated that, after 24 h of incubation in the absence
of PLTP, the proportion of pre-
-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-
-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-
-HDL increased to 37%
(panel C), whereas the ability of chymase-treated PLTP to
generate pre-
-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-
-HDL
spontaneously, as observed above (panels B versus
A), also applied to the PLTP-dependent
generation of pre-
-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-
-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- -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- -HDL and -HDL were measured by
two-dimensional crossed immunoelectrophoresis, as stated under
"Experimental Procedures." The amounts of pre- -HDL are expressed
as percentages of the total -HDL + pre- -HDL. The values of the
control and chymase-treated HDL3 incubated in buffer only
(HDL blanks) are shown in panels A and
B.
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Simultaneous Incubation of PLTP and HDL3 in the
Presence of Chymase: Effect on Pre-
-HDL Content--
Because
treatment of PLTP with chymase only partially reduced its ability to
generate pre-
-HDL (see Fig. 4, panels C and D), and chymase treatment of HDL3 only slightly
reduced the PLTP-dependent formation of pre-
-HDL from
these particles (Fig. 4, panels E and F), we next
determined the effect of chymase on pre-
-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-
-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-
-HDL was almost totally abolished.
Accordingly, in all incubations in which chymase was present, the
amounts of pre-
-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-
-HDL was observed after 10 min (results not shown). This indicates that chymase was highly
efficient in degrading pre-
-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- -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- - and -HDL subpopulations were analyzed and expressed
as described in Fig. 4.
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Effect of Chymase on PLTP-treated HDL3-induced
Cholesterol Efflux--
Because incubation of HDL3 with
PLTP promoted strong formation of pre-
-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-
-HDL content 33 ± 10%) was twice that
observed with the control HDL3 (pre-
-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-
-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-
-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).
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 |
DISCUSSION |
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-
-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-
-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-
-HDL
promoted by PLTP is compatible with the view that the bulk of the
apoA-I remaining on the surface of
-HDL particles was available for
active generation of pre-
-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-
-HDLs have a crucial function as the primary
acceptors of cellular cholesterol (2, 37). The present data demonstrate
for the first time that pre-
-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-
-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-
-HDLs are involved in the
cholesterol efflux process. Because plasma PLTP activity is
significantly correlated with the ability of plasma to generate
pre-
-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-
-HDL. Also in mice with adenovirus-mediated overexpression of
PLTP, increased levels of pre-
-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-
-HDL particles.
However, in intimal areas with chymase-secreting mast cells,
chymase-dependent depletion of the PLTP-generated pre-
-HDL
particles could occur, thus causing an impairment of cholesterol efflux
from macrophages via this local anti-atherogenic function of PLTP.