(Received for publication, August 16, 1995)
From the
Cholesterol was poorly available to free apolipoprotein
(apo)A-I-mediated cellular lipid efflux from cholesterol-loaded rat
vascular smooth muscle cells generating cholesterol-poorer
pre--HDL particles than those generated from macrophages by the
same reaction (Li, Q., Komaba, A., and Yokoyama, S. (1993) Biochemistry 32, 4597-4603). The factors known to induce
transformation of the smooth muscle cells into a macrophage-like stage
were used in order to modulate this reaction, such as human
platelet-derived growth factor, macrophage colony-stimulating factor,
and phorbol 12-myristate-13acetate (PMA). When the cells were
stimulated by PMA following the pretreatment with platelet-derived
growth factor plus macrophage colony-stimulating factor, cholesterol
efflux mediated by free apoA-I increased 3-fold without changing
phospholipid efflux, resulting in generation of pre-
-HDL particles
more rich in cholesterol. This treatment had only a little or no effect
on apparent cellular cholesterol efflux to HDL or lipid microemulsion,
respectively. Overall cellular free cholesterol pool size was
unaffected by the treatment, and probing by extracellular cholesterol
oxidase did not detect gross change in the cellular surface
cholesterol. This specific enrichment of cholesterol in the
apoA-I-mediated cellular lipid efflux was reversed by protein kinase C
inhibitors. Measurement of intracellular cholesterol esterification
suggested that PMA induced translocation of intracellular cholesterol
to a specific pool for apoA-I-mediated efflux, and a protein kinase C
inhibitor reversed this effect.
When cholesterol is transported from the peripheral cells to the
liver for its biological degradation, the first step of the pathway is
efflux of cellular cholesterol to plasma lipoproteins. High density
lipoprotein (HDL) is believed to play a primary role in
this step by ``accepting'' the cellular cholesterol. Cellular
free cholesterol is exchangeable with extracellular lipoprotein surface
by a nonspecific physicochemical mechanism such as its diffusion
through aqueous phase or direct collision between the membranes
(Rothblat and Phillips, 1982; Johnson et al., 1986; Karlin et al., 1987; Johnson et al., 1988). The net efflux
could therefore be induced by a gradient of cholesterol content between
cell membrane and lipoprotein surface, so that cholesterol
esterification on HDL may play an important role in such a mechanism
(Murphy, 1962; Glomset, 1968; Ray et al., 1980; Fielding and
Fielding, 1981; Czarnecka and Yokoyama, 1995). Specific function of HDL
to accommodate cholesterol may also be attributed to physicochemical
properties of its surface (Stein et al., 1986; Phillips et
al., 1987). On the other hand, a HDL-binding protein(s) on the
cell surface is proposed to play a specific role being linked to
intracellular signal transduction that results in mobilization of
intracellular cholesterol to cellular surface (Graham and Oram, 1987;
Slotte et al., 1987; Mendez et al., 1991; McKnight et al., 1992).
Recent development of the investigation in
this field shed light for further understanding of the mechanism for
cellular cholesterol efflux. Fielding and co-workers discovered that
cellular cholesterol appeared in a specific HDL fraction,
pre--HDL, in the very early stage of its efflux and then was
transferred to other lipoproteins (Castro and Fielding, 1988; Miida et al., 1990, 1992) and proposed that this lipoprotein
fraction is the most efficient ``acceptor'' of cellular
cholesterol. Later, we demonstrated that lipid-free apolipoproteins
having multisegments of amphiphilic helix mediate the net efflux of
cholesterol and phospholipid from various types of cells, generating
new HDL-like particles with these cellular lipids (Hara and Yokoyama,
1991, 1992; Hara et al., 1992; Komaba et al., 1992).
The particles were consistent with pre-
-HDL with respect to the
chemical and physical properties. Other groups have also reported
similar data using lipid-free apolipoproteins (Bielicki et
al., 1992; Forte et al., 1993). These results may explain
why cellular cholesterol appears in pre-
-HDL fraction in the very
early stage of the efflux (Castro and Fielding, 1988; Miida et
al., 1990, 1992); pre-
-HDL particles are newly generated with
cellular lipid by apolipoproteins dissociated from HDL, rather than the
particles in the medium preferably ``accepting'' cholesterol
from cellular surface.
The reaction can be carried out by a number
of amphiphilic helical apolipoproteins (Hara and Yokoyama, 1991; Hara et al., 1992; Bielicki et al., 1992) or their model
peptides (Mendez et al., 1994), and it may induce specific
translocation of intracellular cholesterol that would render
cholesterol readily available for the efflux (Mendez et al.,
1994). The involvement of a protein binding site(s) for such
apolipoproteins has not clearly been identified on cellular surface.
However, many circumstantial evidences may support a potential role of
such a site(s) in this reaction. Apparent K values for apolipoprotein-mediated lipid efflux were as low
(Hara and Yokoyama, 1991; Komaba et al., 1992) as that of the
LDL-receptor interaction (
10
M) (Brown
and Goldstein, 1986), and lower than that of the lipid-apolipoprotein
interaction (
10
M) (Tajima et
al., 1983; Yokoyama et al., 1985), indicating some type
of specific interaction. Proteolytic treatment of cellular surface
resulted in complete inhibition of this pathway (Li et al.,
1995). Furthermore, specific lack of interaction with lipid-free apoA-I
in fibroblasts from the patients with Tangier disease strongly
suggested that there is a genetically defined cellular interaction site
for lipid-free apolipoproteins, and that this reaction is a major
source of plasma HDL (Francis et al., 1995).
Thus, free
apolipoprotein-mediated cellular cholesterol efflux seems to be a
distinct pathway from nonspecific physicochemical cholesterol exchange
reaction between cellular surface and lipoproteins. This may lead us to
the hypothesis that HDL-mediated lipid efflux includes two distinct
mechanisms. One is physicochemical lipid exchange between the cell and
HDL surface in which gradient of cholesterol between the two surfaces
causes the net efflux and therefore cholesterol esterification on HDL
may act as its driving force. The other is generation of new
pre--HDL with cellular lipids, as apolipoproteins locally
dissociate from HDL surface to interact with the cellular surface (Hara
and Yokoyama, 1991, 1992). The maximum contribution of the free
apolipoprotein-mediated portion could be as much as 40% of apparent
HDL-mediated efflux (Komaba et al., 1992; Francis et
al., 1995).
Interestingly, vascular smooth muscle cells are
very resistant to the free apolipoprotein-mediated cholesterol efflux
(Komaba et al., 1992) due to generation of cholesterol-poor
pre--HDL (Li et al., 1993). This was specific to
lipid-free apolipoprotein-mediated cholesterol efflux, whereas little
significant difference was found among cell lines in the nonspecific
cholesterol efflux mediated by low density lipoprotein (LDL) or lipid
microemulsions (Li et al., 1993). The low
cholesterol/phospholipid ratio in the HDL-mediated lipid efflux from
smooth muscle cells implied that free apoA-I-mediated mechanism is in
fact involved in cellular lipid efflux to HDL (Li et al.,
1993). These findings indicated that cell-specific intracellular
factors are involved in regulation of cholesterol incorporation into
the cellular lipid efflux mediated by apolipoprotein-cell interaction.
Vascular smooth muscle cells are known to be stimulated by certain growth factors and transformed to different stages, and such transformation may be related to its roles in development of vascular atherosclerotic lesions. The smooth muscle cells isolated from arteriosclerotic lesion were found to express the c-fms gene encoding the receptor for macrophage colony-stimulating factor (MCSF) (Inaba et al., 1992a). In vitro, platelet-derived growth factor (PDGF) BB-chain homodimer stimulates the smooth muscle cells to induce c-fms gene as well as the scavenger receptor gene, as transforming the cells to a macrophage-like stage (Inaba et al., 1992b). On the other hand, in the undifferentiated macrophage cell line cell THP-1, phorbol esters are known to induce differentiation of the cells with respect to many macrophage activities, including expression of the scavenger receptor (Hara et al., 1987; Takata et al., 1989) and apolipoprotein E and lipoprotein lipase (Tajima et al., 1985; Menju et al., 1989). Hence, the in vitro transformation of the cells by these factors may modulate the cell specific factors involved in apolipoprotein-mediated lipid efflux and would provide an important model to study the intracellular mechanism for cholesterol efflux by this particular pathway.
Thus, we undertook the study of lipid-free apoA-I-mediated lipid efflux from rat vascular smooth muscle cells in comparison to the apparent cholesterol efflux to lipoproteins and lipid microemulsions under the influence of stimulation of the cells by the growth factors and phorbol esters.
Cellular cholesterol was oxidized by extracellular cholesterol oxidase (Sigma) in order to assess accessibility from the cellular surface, and the oxidized product, cholestone, was analyzed by thin layer chromatography after extracting the cellular lipid, as described in detail previously (Lange and Ramos, 1983; Li et al., 1993).
Intracellular cholesterol esterification activity
was measured for the cholesterol-loaded cells after treatment with
growth factors and PMA, according to the method previously described
(Francis et al., 1993). The cells were treated in the same
manner as the efflux experiment for cholesterol loading with cationized
LDL, the growth factor treatment, and PMA stimulation in the presence
and absence of H-7. After washing the medium containing
[1-C]oleic acid (57 mCi/mmol, Amersham), 1.5
µCi (26 nmol) was added to each culture dish. After a 1-h
incubation at 37 °C, the cells were washed with ice-chilled medium
and lipid was extracted for the analysis by thin layer chromatography.
The radioactivity in cholesteryl ester fraction was counted.
Lipid efflux profile was compared between rat aortic smooth
muscle cells and mouse peritoneal macrophages. The efflux to lipid
microemulsion represents nonspecific physicochemical pathway and
apoA-I-mediated efflux represents the efflux pathway by generating new
pre--HDL particles with cellular lipid. The condition was chosen
for the V
of the efflux rate at the 24-h
incubation (Hara and Yokoyama, 1991, 1992), and the results shown in Table 2were consistent with our previous observation (Li et
al., 1993). In nonspecific pathway (to lipid microemulsion), the
rate of cholesterol efflux was much higher than that of phospholipid
and the ratio of cholesterol to total phospholipid was very similar for
the two types of cell, giving a weight ratio around 4. In contrast,
phospholipid efflux was substantially high in the apoA-I-mediated
efflux from both cells. Specific difference was observed in cholesterol
efflux between smooth muscle cells and macrophages. Cholesterol was
very poorly incorporated from smooth muscle cells into this pathway,
making its weight ratio to phospholipid substantially lower than the
efflux from macrophages, 0.24 versus 1.57. In lipid efflux to
HDL, the value of this parameter was intermediate between the
emulsion-mediated and apoA-I-mediated pathways.
Smooth muscle cells were exposed to the potential stimuli to induce macrophage-like functions. Such factors as PDGF, MCSF, and PMA were used in an attempt to modulate lipid efflux profile for the reasons mentioned earlier, and the results are shown in Table 3. When each of the three factors was individually used, there was no effect on the cholesterol efflux from the cells either by HDL or apoA-I. Since PDGF is known to induce MCSF receptor (Inaba et al., 1992b), the two growth factors were used together, but no additional effect was found by this combination. When the cells were stimulated by PMA following the treatment with growth factors, cholesterol efflux was somewhat enhanced from the PDGF-pretreated cells but not from the MCSF-treated cells. No increase was observed in the apparent cholesterol efflux to HDL in this condition. Finally, when both growth factors were used together in the pretreatment for the PMA-stimulation, the efflux of cholesterol mediated by lipid-free apoA-I was enhanced 3-fold. The increase was only 30% with the efflux to HDL by the same treatment. In contrast to the results for the cholesterol efflux, phospholipid efflux mediated by apoA-I was not significantly affected by the treatment with PDGF/MCSF and PMA. Total cellular protein has not reached significant difference by any of these treatments (Table 3). To examine potential contribution to the efflux of intracellular cholesteryl ester hydrolysis as the cellular reaction to the growth factors, free/total cholesterol ratio was determined in the cells during the pretreatment period (24 h). There was no significant change in this ratio (Table 3).
Fig. 1shows the time course of the efflux of these lipids mediated by lipid-free apoA-I and HDL from control smooth muscle cells and those treated with PDGF/MCSF plus PMA. While free apoA-I-mediated cholesterol efflux was high from the stimulated smooth muscle cells, phospholipid efflux was essentially the same between the control and stimulated cells. The stimulation of the cell did not have a prominent effect on the lipid efflux to HDL. The enhancement of cholesterol efflux by the treatment was also demonstrated in an apoA-I dose-dependent manner (Fig. 2). Cellular cholesterol efflux was more than doubled by the PDGF/MCSF plus PMA treatment of smooth muscle cells in this particular experiment, while phospholipid efflux was almost identical between the control and the treated cells. Thus, the relative enhancement effect of the combined treatment was specific to cholesterol availability to the apoA-I-mediated cellular lipid efflux that had otherwise been poor in smooth muscle cells (Li et al., 1993).
Figure 1: Time course of lipid efflux from smooth muscle cells induced by apoA-I (top) and by HDL (bottom). Circles, control; triangles, treatment with PDGF and MCSF plus PMA. Left panels, cholesterol efflux. Right panels, phosphatidylcholine efflux. The cells were labeled for cholesterol and choline phospholipid as described in the text, treated with growth factors and PMA, and the lipid efflux was monitored up to 24 h in the presence of 10 µg of apoA-I or 100 µg of HDL as protein in the 1-ml medium. Cellular protein per dish was 310.5 ± 19.4 µg. Radioactivity of cellular free cholesterol per dish was 20842 ± 2840 dpm, and cellular free cholesterol was 48.5 ± 2.2% of total cellular cholesterol in radioactivity. Radioactivity of cellular phosphatidylcholine was 6516 ± 668 cpm/dish.
Figure 2: Dose dependence of the apoA-I-mediated lipid efflux from smooth muscle cells. Circles, control; triangles, treatment with PDGF and MCSF, plus PMA. Panel A, cholesterol efflux; panel B, phosphatidylcholine efflux. The cells were labeled for cholesterol and choline phospholipid as described in the text, treated with growth factors and PMA, and the lipid efflux was observed in the presence of various amounts of apoA-I for 24 h. Cellular protein per dish was 346.8 ± 15.3 µg. Radioactivity of cellular free cholesterol per blank dish was 22526 ± 2697 cpm, and that of cellular phosphatidylcholine was 19539 ± 2021 cpm. Cellular free cholesterol was 42.3 ± 2.6% of total cellular cholesterol in radioactivity.
To confirm
generation of new pre--HDL by this reaction and incorporation of
more cholesterol into this particle by the activation of smooth muscle
cells, the culture medium was analyzed by density gradient
ultracentrifugation. Fig. 3demonstrates that the newly formed
HDL in the medium was more cholesterol-rich when the cells were
stimulated. Thus, more cholesterol molecules (relatively to
phospholipid) were incorporated into the pre-
-HDL particles
generated by lipid-free apolipoproteins when the smooth muscle cells
were treated with PDGF/MCSF plus PMA.
Figure 3: Density gradient analysis of the product in the culture medium after incubation of apoA-I with the smooth muscle cells, control (left panel) and activated (right panel). Smooth muscle cells were activated by PDGF, MCSF, and PMA as described in the text. ApoA-I, 10 µg/medium, induced cellular lipid efflux for 24 h. The medium was then analyzed in sucrose density gradient ultracentrifugation as described in the text. Specific radioactivity of cholesterol and phosphatidylcholine was obtained for respective cellular lipid pool and used for calculation of weight amount of cholesterol and phosphatidylcholine in each density fraction of the medium. Cellular protein was 375.2 ± 31.9 µg/dish, and total cellular cholesterol was 5.45 ± 0.55 µg. Free cholesterol was 72.3 ± 0.5% of total cellular cholesterol. The data are expressed as weight percentage of each lipid in each fraction to total phosphatidylcholine in the medium. Circles, cholesterol; triangles, phosphatidylcholine; solid line without symbol, density.
In order to observe whether this specific increase of cholesterol incorporation into apoA-I-mediated cellular lipid efflux pathway is related to gross change in cellular cholesterol distribution, surface cholesterol of smooth muscle cells was probed by extracellular cholesterol oxidase. Fig. 4illustrates the percentage of oxidized cellular cholesterol in the control and activated conditions. There was no difference in cellular surface cholesterol observed by this procedure either in the initial rate of the oxidation or the end point of the reaction between the control cells and the cells after the combined treatment.
Figure 4: Oxidation of cholesterol in smooth muscle cells by extracellular cholesterol oxidase. Circles, control; triangles, treatment with PDGF and MCSF plus PMA. Rat vascular smooth muscle cells were loaded with radiolabeled cholesterol as described in the text. After pretreatment with the growth factors and PMA, cells were incubated with cholesterol oxidase (3 IU/ml) for various periods of time and the radioactivity of cellular free cholesterol and cholestone was determined by thin layer chromatography in order to measure percent oxidation of the cellular free cholesterol. Radioactivity of cellular free cholesterol was 2655 ± 550 cpm/dish, and the ratio of intracellular free cholesterol to total cellular cholesterol was 52.4 ± 3.4% % in radioactivity. Cellular protein was 342.4 ± 12.4 µg/dish.
Table 4shows the comparison of the effect on the apoA-I-mediated cholesterol efflux pathway and a nonspecific physicochemical efflux pathway to lipid microemulsion of the treatment of the smooth muscle cells with the growth factors and PMA. While the enhancement was demonstrated for the apoA-I-mediated efflux, there was no influence of the treatment on the nonspecific efflux. Another experiment shown in this table is the effect of sphingosine, a potent protein kinase C inhibitor, on the activated apoA-I-mediated cholesterol efflux by the growth factors/PMA treatment. The enhancement effect of PMA was completely reversed in the presence of 40 µM of sphingosine in the medium, while it did not have any effect on the nonspecific cholesterol efflux.
Fig. 5further demonstrates the effect of protein kinase C inhibitors on the enhancement of apoA-I-mediated cellular lipid efflux from smooth muscle cells. Both sphingosine and staurosporine suppressed the cholesterol efflux in a dose-dependent manner (left panel). The maximum inhibition by sphingosine and staurosporine was 81% and 47% of the increment of cholesterol efflux by PMA, respectively. In contrast, staurosporine showed no significant effect on phosphatidylcholine efflux mediated by apoA-I (right panel). A specific inhibitor of cyclic nucleotide-dependent protein kinase C, H-7, was used in a similar manner (Fig. 6). The increase of apoA-I-mediated cholesterol efflux by PMA from the growth factors-treated cells was almost completely reversed, while the effect on phospholipid efflux was negligible (left panel). The same compound demonstrated no effect on nonspecific lipid efflux from the similarly activated smooth muscle cells to lipid microemulsions (right panel).
Figure 5: Effect of protein kinase C inhibitors on the specific enhancement of apoA-I-mediated efflux of cholesterol (left panel) and phosphatidylcholine (right panel) induced by the growth factors and the phorbol ester. Smooth muscle cells were treated with PDGF and MCSF and then incubated with PMA in the presence or absence of sphingosine (µM) or staurosporine (nM) as described in the text. Efflux of cholesterol and phospholipid was induced by 10 µg of apoA-I in the medium. Efflux of each lipid was measured using specific radioactivity of respective cellular lipid. Cellular protein/dish was 483.0 ± 56.7 µg, and cellular total cholesterol was 6.64 ± 0.59 µg/dish. Cellular free cholesterol was 56.5 ± 2.5% of total cellular cholesterol. Triangles, sphingosine; circles, staurosporine. Broken horizontal lines represent the efflux level of each lipid from the cells without treatment. Error bars represent standard error of triplicated assay.
Figure 6: Effect of H-7, a cyclic nucleotide-dependent protein kinase C inhibitor, on the apoA-I-mediated efflux of cholesterol from the smooth muscle cells pretreated with the growth factors and stimulated by PMA. The cells were pretreated with PDGF and MCSF for 24 h. PMA was added to the medium in the presence of various amount of H-7 for the last 45 min of the growth factor treatment. The cells were washed and the lipid efflux by 10 µg of apoA-I (left panel) and by lipid microemulsion (as 156 µg of phospholipid) was observed for 24 h. Details of the method are described in the text. Circles represent cholesterol efflux, and squares represent phosphatidylcholine efflux. Each data point represents mean ± S.E. of triplicated assay. Broken horizontal line in the left panel represent the level of cholesterol efflux by apoA-I from the non-activated cells. Cellular protein was 168 ± 18 µg/dish, and cellular cholesterol was 2.1 ± 0.3 µg/dish.
Intracellular cholesterol esterification activity was determined in the same stages of activation of cholesterol-loaded smooth muscle cells by the growth factors and PMA as used for lipid efflux experiments (Fig. 7). Incorporation of radiolabeled oleate into cholesteryl ester was not significantly changed by the cellular treatment with both growth factors. Adding PMA for short periods (45 min) significantly decreased the reaction, suggesting the decrease of cholesterol pool available for this reaction. The presence of H-7 completely reversed the effect of PMA. Thus, the effect of PMA and H-7 on intracellular cholesterol esterification was reciprocal to the effect of these compounds on apoA-I-mediated cholesterol efflux from the growth factor-treated vascular smooth muscle cells.
Figure 7:
Effect of PMA and H-7 on intracellular
cholesterol esterification in the smooth muscle cells pretreated with
growth factors. After the cholesterol-loaded cells were treated with
PDGF and MCSF for 23.25 h, PMA or PMA + H-7 (25 µM)
was added to the medium for another 45-min incubation. The cells were
washed, and the incorporation of [C]oleic acid
into cholesterol ester was measured by 1-h incubation as described in
the text. Control, without treatment with growth factors; GFs, PDGF and MCSF only; GFs + PMA, PMA
stimulation after the growth factors treatment; GFs + PMA
+ H7, PMA stimulation in the presence of H-7. The measurement
was quadruplicated. Asterisk indicates significant difference
form all others by p < 0.001. Cellular protein was 341
± 9 µg/dish, cellular cholesterol was 6.2 ± 1.0
µg/dish, and free cholesterol was 53.8 ± 2.8% of total
cellular cholesterol.
Finally, the experiment was performed for the treatment of mouse peritoneal macrophages with PDGF and MCSF, individually or in combination. Cholesterol efflux from the control cells was already high, and no significant effect of any of these treatments was observed on cholesterol and phospholipid efflux either mediated by HDL or free apoA-I (data not shown). Further incubation with phorbol ester (PMA) of the pretreated cells with the growth factors, which led to a specific effect on smooth muscle cells as described above, did not show any further differential effect on the lipid efflux, either among the different pretreatment groups with the growth factors or between the efflux mediated by apoA-I and HDL (Table 5).
After short periods of incubation, PMA stimulated the
cholesterol efflux by free apoA-I from the rat vascular smooth muscle
cells pretreated with growth factors. The effect was achieved, but
marginally by the pretreatment with PDGF alone and more prominently
when the cells were pretreated with PDGF and MCSF. Without the
stimulation, cholesterol was poorly incorporated into the
apoA-I-mediated lipid efflux, generating cholesterol-poor pre--HDL
in the medium. Stimulation of the cells by PDGF and MCSF plus PMA
enhanced the apoA-I-mediated cholesterol efflux without changing
phospholipid efflux rate, generating pre-
-HDL more rich in
cholesterol. Interestingly, no effect was found on a nonspecific
physicochemical lipid efflux pathway to lipid microemulsions by the
same treatment of the cells. The effect of the treatment on cholesterol
efflux to HDL was only partial. The observation was consistent with the
view that the stimulation is specific to cholesterol incorporation to
the apoA-I-mediated cellular lipid efflux, while the interaction of
apoA-I with the cell itself is unaffected, since phosphatidylcholine
efflux was not influenced by these treatment and the same amount of
pre-
-HDL seemed to be produced regardless of the treatment. The
partial effect on the HDL-mediated cholesterol efflux may reflect
contribution of the apoA-I-mediated mechanism to the overall cellular
lipid efflux to HDL.
At least three protein kinase C inhibitors reversed the stimulating effect of PMA on the apoA-I-mediated cholesterol efflux from the vascular smooth muscle cells pretreated with PDGF and MCSF, again without changing the cellular phospholipid efflux by free apoA-I. The inhibitors had no effect on the nonspecific cellular lipid efflux to lipid microemulsions. In contrast, intracellular cholesterol esterification was suppressed by the same PMA treatment in the pretreated vascular smooth muscle cells with PDGF and MCSF, and this effect was also reversed by the protein kinase C inhibitor. However, overall cellular free cholesterol pool size was unaffected by these cellular treatment and probing of cellular surface cholesterol by extracellular cholesterol oxidase did not demonstrate any significant difference between the control cells and the stimulated cells. The data thus indicated that intracellular cholesterol is translocated to the specific pool readily available to apoA-I-mediated lipid efflux by the short term incubation of the pretreated cells with PMA, and this was inhibited by the protein kinase C inhibitors. This effect was, however, not to an extent that influences cellular surface lipid composition reflecting in nonspecific physicochemical cellular lipid efflux and probing surface cholesterol by cholesterol oxidase.
In the previous few years, a concept of a specific and biologically
regulated cellular cholesterol efflux has been gradually developed by
several research groups including ourselves. The efflux is mediated by
lipid-free helical apolipoproteins and the reaction results in
generation of pre--HDL with the apolipoprotein and cellular
phospholipid and cholesterol (Hara and Yokoyama, 1991, 1992; Hara et al., 1992; Bielicki et al., 1992; Forte et
al., 1993). This efflux reaction is distinguishable from
nonspecific physicochemical lipid exchange between cell and lipoprotein
in many aspects. The reaction has substantially lower K
(in the order of 10
M) (Hara and
Yokoyama, 1991; Komaba et al., 1992; Mendez et al.,
1994) than K
for apolipoprotein-lipid association
(Tajima et al., 1983; Yokoyama et al., 1985) and K
for cellular lipid efflux to HDL (Komaba et
al., 1992). A large amount of phospholipid accompanies cholesterol
efflux, while phospholipid efflux to lipoproteins is much lower than
that of cholesterol (Hara and Yokoyama, 1991; Forte et al.,
1993; Li et al., 1993). Extracellular cholesterol
esterification by lecithin:cholesterol acyltransferase takes place on
the pre-
-HDL generated by apoA-I (Forte et al., 1995;
Czarnecka and Yokoyama, 1995), but it does not have an impact on the
rate of lipid efflux by this pathway (Czarnecka and Yokoyama, 1995),
while it causes net efflux of cholesterol to HDL by reducing the influx
of cholesterol and by shifting equilibrium distribution of cholesterol
if the exchange rate is rapid (Czarnecka and Yokoyama, 1995).
Proteolytic treatment of cellular surface causes selective inhibition
of the apolipoprotein-mediated lipid efflux (Li et al., 1995).
Specific defect of this pathway has been identified in the fibroblasts
from the patients with Tangier disease (Francis et al., 1995)
as well as in normal human erythrocytes (Li et al., 1995). The
two distinct mechanisms were also differentiated by the lipid efflux
profiles from vascular smooth muscle cells. Although there is no
essential difference in nonspecific pathway between the smooth muscle
cells and mouse macrophages, the former cells are resistant to net
cholesterol efflux by free apolipoproteins, not because the cells are
less reactive to free apolipoproteins but because cholesterol is poorly
available to this reaction (Komaba et al., 1992; Li et
al., 1993).
In such a context, we undertook the attempt to modulate lipid-free apolipoprotein-mediated cellular lipid efflux by modification of cellular parameters. Vascular smooth muscle cells were chosen as a model because of their unique behavior in the apolipoprotein-mediated lipid efflux and because of their peculiar reaction to a numbers of factors leading to their various differential stages (Inaba et al., 1992a, 1992b; Shimada et al., 1992). They are known to be induced for macrophage-like functions, including expression of the genes encoding the scavenger receptor and the MCSF receptor in certain conditions such as stimulation by PDGF BB (Inaba et al., 1992a, 1992b). MCSF is known to enhance the activity of macrophage (Nakoinz and Ralph, 1988; Hume et al., 1988; Ishibashi et al., 1990; Shimano et al., 1990; Mori et al., 1991; Inaba et al., 1993; Ishii et al., 1994), and PMA is used for inducing differentiation of macrophage cell line cells such as THP-1 cells with respect to a number of macrophage-like functions, including expression of the scavenger receptor (Hara et al., 1987; Takata et al., 1989) and secretion of lipoprotein lipase and apolipoprotein E (Tajima et al., 1985; Menju et al., 1989).
Stimulation of the
smooth muscle cells by these factors brought us very interesting
results as summarized above. None of the individual factor alone
influenced lipid efflux profile either to HDL or by lipid-free apoA-I.
When the two growth factors were used in combinations, additional PMA
caused prominent specific stimulation of cholesterol availability for
lipid-free apoA-I-mediated lipid efflux while phospholipid efflux in
this pathway was unchanged. Accordingly, this resulted in generating
pre--HDL particles with higher cholesterol/phospholipids ratio
than the particles generated with the non-stimulated cells. More
interestingly, the enhancement of cholesterol efflux by the
apoA-I-mediated by PMA was reversed by protein kinase C inhibitors.
These effects were not observed in a nonspecific lipid efflux pathway.
Thus, the apolipoprotein-mediated cellular lipid efflux pathway is well
distinguished from a physicochemical pathway.
The stimulation of
cholesterol efflux was independent of interaction of the
apolipoproteins with the cellular surface because phospholipid efflux
was intact. Thus, incorporation of cholesterol molecule into the free
apolipoprotein-mediated cellular lipid efflux pathway is regulated
independently of the cell-apolipoprotein interaction and of generation
of pre--HDL. From the fact that the stimulation was observed by
the short term incubation with PMA and reversed by the protein kinase C
inhibitors, it is fair to conclude that cholesterol enrichment in this
pathway is regulated by a mechanism that involves protein kinase C at
least in vascular smooth muscle cells. Thus, the mechanism of this
increase is more specific than overall activation of lipid metabolism
reported in the cells of dividing stage (Fielding et al.,
1982). In fact, the possibility of protein kinase C involvement in
cellular cholesterol efflux has been suggested in a few other previous
works although in a more general manner (Theret et al., 1990;
Mendez et al., 1991; Bernard et al., 1992;
Voyno-Yasenetskaya et al., 1993). Our data including
intracellular cholesterol esterification suggested that translocation
of cholesterol from the pool available for the esterification to the
specific pool readily available for the apolipoprotein-mediated efflux
is induced by PMA and prevented by the protein kinase C inhibitor. This
view may be consistent with the hypothesis proposed by Mendez et
al.(1991). A more specific approach is required for further
characterization of the reaction including phosphorylation of specific
proteins.
As discussed above, such specific translocation of cholesterol may result in general increase of cholesterol in the cell surface. However, the attempt of probing surface cholesterol by extracellular cholesterol oxidase failed to demonstrate any difference between the control and treated cells. Thus, overall surface cholesterol may not be a regulating factor for the cholesterol availability to apolipoprotein-mediated cellular lipid efflux unless this method may be inappropriate to probe the change of surface cholesterol correctly (Brasaemle and Attie, 1990; Liscum and Dahl, 1992). This may also be consistent with the unaffected nonspecific cellular lipid efflux from the stimulated smooth muscle cells. It should be noted that free/total cholesterol ratio in the cell was not changed by the growth factor treatment, suggesting no increase in overall intracellular free cholesterol pool by such a treatment. Thus, a concept of specific cholesterol pool available to the apolipoprotein-mediated efflux may have to be introduced to interpret these results, and the regulation would be strictly limited for such a specific cholesterol pool. This specific pool should be distinguishable from the cellular cholesterol pool that undergoes nonspecific exchange with lipoprotein surface.
Stimulation of mouse macrophages by human PDGF, MCSF, and PMA, or combinations of these factors, did not demonstrate specific effect on lipid efflux mediated either by lipid-free apoA-I or HDL. This may not be consistent with the report that MCSF stimulated HDL-mediated cholesterol efflux from human monocyte-derived macrophage (Ishibashi et al., 1990; Inoue et al., 1992). Although the general effect of human MCSF was demonstrated on mouse macrophage (Hume et al., 1988; Ishibashi et al., 1990), the effect may not be entirely similar between the species. Mouse macrophages are generally considered very active in comparison to other species so that they may already be in an activated stage and further activation may not be necessary to achieve the maximum cholesterol efflux level.
The results of this study implicate the importance of specific cellular factors in regulation of cholesterol efflux by the lipid-free apolipoprotein-mediated pathway. It seems to be particularly important to investigate its regulation by intracellular trafficking of cholesterol in relation to such a factor as manipulation of cellular protein phosphorylation in order to understand the entire process. Since apolipoprotein-cell interaction seems to be a major source of plasma HDL (Francis et al., 1995), it is increasingly important to study the cellular factors involved in this reaction system.