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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1066-1074.)
© 1999 American Heart Association, Inc.

Original Contributions

Depletion of Preß₁LpA1 and LpA4 Particles by Mast Cell Chymase Reduces Cholesterol Efflux From Macrophage Foam Cells Induced by Plasma

Miriam Lee; Arnold von Eckardstein; Leena Lindstedt; Gerd Assmann; Petri T. Kovanen

From the Wihuri Research Institute, Helsinki, Finland (M.L., L.L., P.T.K.); and the Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium. Westfälische Wilhelms-Universität Münster and Institut für Arterioskleroseforschung an der Universität Münster, Germany (A.v.E., G.A.).

Correspondence to Dr Petri T. Kovanen, Wihuri Research Institute, Kalliolinnantie 4, 00140 Helsinki, Finland. E-mail petri.kovanen{at}wri.fi

	Abstract

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Abstract—Exposure of the LpA1-containing particles present in HDL₃ and plasma to a minimal degree of proteolysis by the neutral protease chymase from exocytosed rat mast cell granules (granule remnants) leads to a reduction in the high-affinity component of cholesterol efflux from macrophage foam cells. In this study, we demonstrate for the first time, a role for mast cell chymase in the depletion of the lipid-poor minor components of HDL that are specifically involved in reverse cholesterol transport as initial acceptors of cellular cholesterol. Thus, addition of proteolytically active granule remnants or human skin chymase to cholesterol-loaded macrophages of mouse or human origin incubated with human apoA1, ie, a system in which preß₁LpA1 is generated, resulted in a sharp reduction in the high-affinity cholesterol efflux promoted by apoA1. As determined by nondenaturing 2-dimensional polyacrylamide gradient gel electrophoresis, the granule remnants effectively depleted the preß₁LpA1, but not the LpA1, in HDL₃ and in plasma during incubation at 37°C for <1 hour. Incubation of plasma with granule remnants for 1 hour also led to near disappearance of the LpA4–1 and LpA4–2 particles, but did not affect the distribution of the apoA2-containing lipoproteins present in the plasma. We conclude that the reduced ability of granule remnant-treated HDL₃ and granule remnant-treated plasma to induce cholesterol efflux from macrophage foam cells is caused by selective depletion by mast cell chymase of quantitatively minor A1- and A4-containing subpopulations of HDL. Because these particles, ie, preß₁LpA1 and LpA4, are efficient acceptors of cholesterol from cell surfaces, their depletion by mast cells may block the initiation of reverse cholesterol transport in vivo and thereby favor foam cell formation in the arterial intima, the site of atherogenesis.

Key Words: mast cells • reverse cholesterol transport • preß₁LpA1 • LpA4 • chymase • proteolysis

	Introduction

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Removal of cholesterol from the plasma membranes of cells is essential for maintenance of the physiological intracellular cholesterol balance.^{1 2} This process is especially critical for cells such as macrophages, which lack the ability to regulate the influx of cholesterol, and therefore are easily transformed into cholesterol-loaded foam cells.³ Indeed, accumulation of cholesteryl esters in macrophages and smooth muscle cells is typical for early atherosclerotic lesions.⁴ The primary event that triggers the uptake of cholesterol by macrophages is modification of LDLs in the arterial intima, by proteolytic,⁵ oxidative,⁶ or lipolytic⁷ mechanisms.

Several different lipoproteins and natural proteins, as well as some synthetic peptides or artificial substrates, can function as cholesterol acceptors when added to cell cultures.⁸ HDLs appear to play the most important role in vivo; their concentration in plasma is inversely correlated with the risk of coronary artery disease.⁹ A prerequisite for the action of HDL particles as efficient acceptors of cellular cholesterol is their integrity. Thus, chemical modifications of HDLs in vitro, such as copper-mediated oxidation,¹⁰ treatment with trypsin or pronase,¹¹ or glycation,¹² all reduce the efficiency with which HDLs induce cholesterol efflux from cells. In a more physiological system, we have observed that mast cells reduce a high-affinity component of the cholesterol efflux promoted by HDL₃ from cholesterol-loaded macrophages in culture.¹³ This mast cell-mediated mechanism was caused by proteolytic degradation of HDL₃ apoproteins by the neutral protease chymase, which is present in exocytosed mast cell granules (ie, granule remnants). Most important, only a minimal degree of proteolysis was sufficient to cause loss of HDL₃ function, suggesting that the high-affinity process involves a minor subfraction of HDL that is particularly susceptible to proteolytic cleavage.

HDLs are a highly heterogeneous family of particles that have been classified into several subgroups according to their density (HDL₂, HDL₃), apolipoprotein composition (LpA1 without apoA2, LpA1 with apoA2), or electrophoretic mobility (-, preß-migrating HDL).¹⁴ In recent years, much attention has been focussed on a small fraction of lipid-poor HDL particles that exhibit electrophoretic preß-mobility in contrast to the major component of HDL, which exhibits electrophoretic -mobility. PreßLpA1 particles can be generated by lipolysis of HDL through the action of hepatic lipase,¹⁵ by interconversion of HDL through the action of the cholesteryl ester transfer protein,^{16 17 18 19} or by the phospholipid transfer protein.^{20 21} They can also be formed when apoA1 is incubated with macrophages,²² fibroblasts, or vascular smooth muscle cells.²³ These preßLpA1 particles are discoidal and remove cholesterol from cells very rapidly by a process that involves interaction with protease-sensitive domains on the cell surface.^{24 25} From preß₁LpA1, cell-derived cholesterol is rapidly transferred to the bulk of -LpA1, presumably by conversion of particles of the former type into those of the latter.^{26 27} Incubation of plasma at 37°C has been shown to induce redistribution of apoA1 within plasma, with a variable decrease in its content of preß₁LpA1. However, when plasma was incubated with cells of various types, such as fibroblasts or HL-60 macrophages, this decrease was inhibited.²³ Compared with other HDL species, the apoA1 in preß₁LpA1 has been shown to expose different epitopes to monoclonal antibodies,^{28 29 30} indicating a different conformation and, hence, suggesting differences in its accessibility to modification by proteolysis, oxidation, or glycation.

It has also been observed that apoA1-free HDL particles containing apoA4 contribute to the cholesterol uptake from cells (see Reference 8⁸ for review), namely LpA4–1 and LpA4–2. Like preß₁LpA1, they are quantitatively minor components of HDL, exhibiting electrophoretic "slow -mobility" and contributing to reverse cholesterol transport. Interestingly, studies on cellular cholesterol efflux have shown that, during 1-minute pulse-incubation of plasma with ³H-cholesterol-loaded fibroblasts, cholesterol has been taken from cells more efficiently by LpA4–1 than by preß₁LpA1.³¹

The present study was directed toward understanding the mechanisms underlying the blocking effect of chymase on the cholesterol efflux promoted by HDL₃ as previously described.^{13 32} For this purpose, we investigated the effect of mast cell granule remnants on the small subpopulations of HDL containing apoAs (preß₁LpA1, LpA4–1, and LpA4–2), which play a role as early cholesterol acceptors from cells. Specifically, we determined whether proteolysis of preß₁LpA1 would be responsible for the previously observed inhibitory effect of mast cell chymase on the high affinity component of cholesterol efflux from macrophage foam cells.³²

	Methods

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Animals
Adult male Wistar rats and female NMRI mice were purchased from the Viikki Laboratory Animal Center of the University of Helsinki.

Rat Mast Cell Granule Remnants and Human Chymase
Serosal mast cells were isolated from peritoneal and pleural cavities of rats. Degranulation was induced with compound 48/80 (Sigma) and the exocytosed granules (ie, granule remnants) were isolated from the released material by centrifugation as described.³³ The quantity of granule remnants is expressed in terms of their total protein content³⁴ or proteolytic activity with BTEE as a substrate.³⁵ The amounts of granule remnants used in the different experiments reported here ranged from 6 to 60 µg total protein/assay, corresponding to 8 to 85 BTEE units of chymase (granule remnants from rat peritoneal mast cells contain, on average, 1.41±0.8 BTEE units/µg total protein, n=11). Chymase from human skin was prepared as previously described.³⁶

Inactivation of Chymase Activity in Granule Remnants
Granule remnants were incubated in 5 mg/mL BSA, 1 mmol/L EDTA, 5 mmol/L Tris-HCl, pH 7.4 containing 250 µg/mL PMSF, at 37°C for 15 minutes. The granule remnants were washed with the above buffer without PMSF, and their proteolytic activity was measured as described.³⁷ The degree of chymase inactivation in PMSF-treated granule remnants was >99%.

Human Fresh Plasma
Normolipidemic blood was collected from healthy, fasting volunteers into precooled plastic tubes with streptokinase (Kabikinase, Pharmacia Upjohn) (final concentration 150 IU/mL) as a fibrinolytic agent. The blood was centrifuged at 4°C for 30 minutes to obtain plasma. The plasma was placed on ice and used immediately for experiments, or preserved at -70°C until use.

Isolation of Plasma Lipoproteins and LPDS
Human LDL (d=1.019 to 1.063 g/mL) and HDL₃ (d=1.125 to 1.210 g/mL) were isolated from fresh normolipidemic plasma by sequential ultracentrifugation using KBr.³⁸ LDL was acetylated by the addition of acetic anhydride, dialyzed extensively, and labeled with ³H-cholesteryl linoleate (Amersham International) as described previously.¹³ The preparations had specific activities from 50 to 100 dpm of ³H-cholesteryl linoleate/ng protein. The quantities of the lipoproteins are expressed in terms of their protein content. The LPDS fraction (d>1.215 g/mL) was obtained by sequential ultracentrifugation of plasma. Thrombin was then added to convert fibrinogen into insoluble fibrin. The fibrin clot was removed by centrifugation, and the supernatant was designated as fibrinogen-free LPDS. The concentration of the fibrinogen-free LPDS is expressed in terms of its total protein content.

Isolation of ApoA1 From Human Plasma
HDL (d=1.063 to 1.210 g/mL) was delipidated by ethanol:ether extraction,³⁹ followed by separation of apoA1 from apoA2 by anion exchange chromatography on a HiTrap Q column (Pharamcia LKB Biotechnology).⁴⁰ The purity of the apoprotein was checked by electrophoresis in a 15% SDS-PAGE gel under nonreducing conditions.

Cell Cultures and Loading of Macrophages With Cholesteryl Esters
Peritoneal cells were harvested from unstimulated mice in PBS (Gibco) containing 1 mg/mL BSA. The cells were recovered after centrifugation and resuspended in DMEM (Gibco) with 100 U/mL penicillin and 100 µg/mL streptomycin (medium A) supplemented with 20% FCS, and plated into 24-well plates (Becton Dickinson Labware). After incubation at 37°C for 2 hours in humidified CO₂, nonadherent cells were removed. After washing with PBS, the adherent cells (macrophages) were loaded with ³H-cholesterol by incubation for 18 hours in the presence of 20 µg/mL of ³H-cholesteryl linoleate acetyl LDL in medium A containing 20% FCS. In some experiments, such as those in which the net efflux of cholesterol from macrophage foam cells was studied, the cells were loaded with 20 µg/mL of unlabeled acetyl LDL. Human monocytes were separated from buffy coat cells, seeded into 24-well plates, and transformed into macrophage foam cells, as described previously.³² In short, the cells were loaded for 2 days with cholesterol by incubating them in medium A containing 5 mg/mL of fibrinogen-free LPDS in the presence of 25 µg/mL of ³H-labeled acetyl-LDL.

³H-Cholesterol Efflux From Macrophage Foam Cells
³H-Cholesterol-loaded macrophages were washed with PBS and incubated with fresh medium A. Free apoA1, HDL₃, fresh plasma, or plasma preincubated at 37°C was added to the cell cultures, and the mixtures were incubated in the presence or absence of mast cell granule remnants or human chymase as described in the figure and table legends. In some cases the cholesterol efflux-inducing ability of samples preincubated with granule remnants was determined. After efflux times ranging between 30 minutes and 6 hours, the media were collected and centrifuged at 15 000 rpm for 5 minutes. The radioactivity in each supernatant was determined by liquid scintillation counting. Under these conditions, ³H-cholesterol efflux was linear through the initial 6 hours of incubation with foam cells.³² The experimental data points are means of triplicate measurements unless otherwise specified.

Measurement of Cholesterol Net Efflux From Macrophage Foam Cells
Cholesterol-loaded macrophages were incubated with acceptors as described above. After 6 hours the macrophages were washed, cellular lipids were extracted with hexane:isopropanol (3:2, vol/vol), and cholesteryl esters (including palmitate, oleate, and linoleate) were determined by HPLC as described before.⁴¹

Regulation of the Cholesterol Efflux-Inducing Capacity of Plasma by Preincubation at 37°C in the Presence of Macrophages
Fresh plasma (5%, vol/vol in medium A) was preincubated with unlabeled macrophage cultures at 37°C for up to 6 hours, and aliquots of the plasma-containing medium were then added to other culture dishes with ³H-cholesterol-loaded macrophages. After incubation for 30 minutes, the medium was removed and ³H-radioactivity was measured. In parallel dishes, aliquots of plasma (5%) were preincubated in the absence of cells, as described above, and the cholesterol efflux-inducing capacity was then determined by incubation with ³H-cholesterol-loaded macrophages for 30 minutes. A repeated measures model was fitted, incorporating time, macrophages, and their interactions as fixed effects. The model-based contrasts were estimated at the following preincubation times: 15 minutes, 30 minutes, 90 minutes, and 6 hours. As shown in Figure 1, preincubation of streptokinase-plasma at 37°C with macrophage cultures did not reduce its subsequent ability to promote cholesterol efflux from ³H-cholesterol-loaded macrophages. In contrast, preincubation for 6 hours in the absence of macrophages produced a significant decrease in the ability of plasma to induce cholesterol efflux compared with the parallel control incubations. The decrease was more rapid during the first 90 minutes, then slowed so that, after 6 hours of preincubation, it was 70% of the level in nonincubated samples (Figure 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of preincubation at 37oC in the presence or absence of macrophages on the subsequent ability of plasma to promote cholesterol efflux from mouse macrophage foam cells. To obtain 3H-cholesterol labeled macrophage foam cells, mouse macrophages were incubated at 37°C for 18 hours in 300 µL of medium A supplemented with 20% FCS and 20 µg/mL of 3H-cholesterol labeled acetyl-LDL. The cells were then washed, and received 300 µL of medium A containing 5% plasma that had been preincubated at 37°C for different times up to 6 hours in either the presence () or absence () of fresh unloaded macrophages in 300 µL of medium A. After incubation for 30 minutes, the 3H-cholesterol radioactivity of the medium was then determined and plotted as a function of the preincubation time. Values are means±SEM (n=5); 100% corresponds to the absolute mean value of 2434±105 dpm/dish per 30 minutes, n=5. *P<0.05, **P<0.01, ***P<0.001.

Proteolysis of HDL₃ and Plasma by Granule Remnants, and Subsequent Quantification of PreßLpA1 Particles
To measure ³H-cholesterol efflux, samples of fresh plasma (5%, vol/vol in medium A) were incubated at 37°C for 90 minutes with ³H-cholesterol-loaded macrophages in the presence of increasing quantities of granule remnants. The foam cells were then washed with PBS, and a second 90-minute efflux period was started by adding fresh plasma (final concentration 5%) to the cultures. In a separate experiment, samples of native HDL₃ (1 mg/mL) or fresh plasma (17.5 mg/mL) were incubated at 37°C for different times up to 24 hours in the presence of granule remnants, keeping the proportions of HDL₃ and plasma to granule remnants the same as in the incubation media of cholesterol efflux experiments. Control incubations were performed either in the presence of granule remnants at 4°C, or in the absence of granule remnants at 37°C. The incubations were stopped by centrifugation at 4°C, 15 000 rpm for 5 minutes, and the samples were frozen immediately and kept at -70°C until analyzed by 2-dimensional electrophoresis. Finally, 5% fresh plasma was preincubated for 6 hours at 4°C (native) or at 37°C (preß depleted). Both samples of plasma were then incubated at 37°C with ³H-cholesterol-loaded macrophages in the presence or absence of granule remnants, and the levels of radioactivity of the media were measured after 90 minutes.

Two-Dimensional Electrophoresis of HDL₃ and Plasma
Native HDL₃ (d=1.125 to 1.210 g/mL) and plasma samples were analyzed for their contents of apoA1-, apoA2-, and apoA4-containing lipoproteins by 2-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) in the sequence: agarose gel electrophoresis, polyacrylamide gradient gel electrophoresis, and immunoblotting, as described previously.^{26 27 31 42} Briefly, in the first dimension, aliquots of pretreated plasma samples with 650 µg protein or pretreated HDL₃ with 1 µg protein were separated by electrophoresis at 4°C in 0.75% agarose gel, using a 50 mmol/L Merbital buffer (pH 8.7) that contained, per liter, 44.3 g Tris, 19.23 g Merbital (Serva), 0.53 g calcium lactate, and 1 g sodium azide. Bromophenol blue was added to a standard sample to visualize the albumin in the native gel. Electrophoresis was stopped when the albumin/bromophenol blue marker had migrated 6 cm. The agarose gel strips containing the preseparated lipoproteins were then transferred to a 3% to 20% polyacrylamide gradient gel. Separation in the second dimension was performed at 40 mA for 4 to 5 hours at 10°C. During this time, the endogenous plasma albumin had migrated 10 cm and was visible in the native gel as a faint blue band because bromophenol blue was present in the cathodic buffer (300 µL/L of buffer). The proteins separated in the PAGGE gel were electroblotted onto a nitrocellulose membrane. The apoA1- or apoA2-containing lipoproteins were immunocomplexed with goat antibodies against human apoA1 (Boehringer Mannheim); the apoA4-containing lipoproteins were immunocomplexed with rabbit antibodies against human apoA4. Antigen-antibody complexes were visualized with peroxidase-conjugated anti-goat IgG antibodies from rabbit or anti-rabbit IgG antibodies from donkey (DAKO), and chloronaphthol and hydrogen superoxide as substrates. To quantify preßLpA1, plasma was separated only by agarose gel electrophoresis before its proteins were blotted onto nitrocellulose. ApoA1-containing particles were immunodetected as described above. The intensity of immunostaining was quantified by photoimaging (BAS 1500, Fuji).

Statistical Analysis
Statistical analysis was carried out using Student's test for paired samples. ANOVA (Friedman's 2-way, or parametric 2-way) and a repeated measures ANOVA were also used for specific experiments as described in each case.

	Results

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Effect of Incubation With Granule Remnants on ³H-Cholesterol Efflux From Macrophages Promoted by ApoA1
To investigate if the ability of granule remnants to block the high affinity component of the cholesterol efflux reported previously^{13 32} resides in its capacity to proteolyze preßLpA1, we selected an experimental system in which preß₁LpA1 is generated by incubating lipid-free apoA1 with mouse peritoneal macrophages.^{22 27 43} For this purpose, mouse peritoneal macrophages were loaded with ³H-cholesterol by incubating them with ³H-cholesteryl linoleate-labeled acetyl LDL for 18 hours. The ³H-cholesterol-macrophage foam cells were then incubated for 6 hours in media containing various concentrations of lipid-free human apoA1, and the efflux of ³H-cholesterol from the cells into the medium was measured. Addition of apoA1 (up to 100 µg/mL) to the ³H-cholesterol-loaded macrophages resulted in a rapid increase in the radioactivity of the medium until a steady level was reached at a protein concentration of 25 µg/mL. Addition of granule remnants to the incubation system greatly reduced the ability of apoA1 to remove cholesterol from the cells. When incubation was performed with lower concentrations of apoA1 (1.5 to 25 µg/mL), the blocking effect of the remnants on the high-affinity component of the efflux process was clearly visible (Figure 2). Thus, in the presence of granule remnants, the cholesterol efflux promoted by apoA1 was almost linear and reached only 1/3 of the total efflux observed at the highest concentration of apoA1 used (1570±83 versus 481±96; P<0.05). The proteolytic action of chymase can be irreversibly inhibited when granule remnants are treated with PMSF.⁴⁴ When such PMSF-granule remnants were added to macrophage cultures containing apoA1, no significant reduction in cholesterol efflux was observed, showing that the inhibitory effect was caused by the proteolytic activity of the granule remnants (not shown). These results strongly suggested that the preß-species formed during incubation of apoA1 with foam cells and responsible for the high affinity uptake of cellular cholesterol, were efficiently degraded by mast cell granule remnants. Essentially the same results were observed when apoA1 was preincubated with human chymase and then added to ³H-cholesterol-loaded human monocyte-macrophages. The results shown in Table 1 reveal a significant reduction of the cholesterol efflux promoted by human chymase-treated apoA1 compared with the native apolipoprotein. This demonstrates that the observed inhibition of cholesterol transfer from mouse macrophage foam cells to apoA1 caused by the proteolytic action of rat chymase is also exerted by its human counterpart in a system containing human macrophage foam cells.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of mast cell granule remnants on cholesterol efflux from mouse macrophage foam cells mediated by lipid-free apoA1. 3H-cholesterol-loaded foam cells were incubated at 37°C in 300 µL of medium A containing the indicated concentrations of apoA1 in the presence () or absence () of granule remnants (6 µg total protein equal to 8 BTEE units of chymase/300 µL). After incubation for 6 hours, the 3H-cholesterol-associated radioactivity of the medium was determined and plotted as a function of the protein concentration of the acceptor.

View this table: [in this window] [in a new window]   Table 1. 3H-Cholesterol Efflux From Human Monocyte-Derived Macrophages by ApoA1 or Human Skin Chymase-Treated ApoA1

Effect of Granule Remnants on Net Cholesterol Efflux From Macrophage Foam Cells on Incubation With Cholesterol Acceptors
Table 2 shows that the ability of granule remnant-treated cholesterol acceptors (serum, HDL₃, and apoA1) to induce mass transfer of cellular cholesterol to the acceptors is strongly reduced. Thus, the reduced cellular efflux of ³H-cholesterol by the granule remnant-treated acceptors (see above) was caused by net transfer of cholesterol from the foam cells to the medium.

View this table: [in this window] [in a new window]   Table 2. Effect of Granule Remnants on Net Efflux of Cholesterol From Macrophage Foam Cells to Various Cholesterol Acceptors

Effect of Incubation With Granule Remnants on PreßLpA1 Subspecies of HDL₃
The original experiments on the cholesterol efflux-reducing effect of granule remnants were done with HDL₃.¹³ In those experiments, it appeared that significant inhibition of cholesterol efflux was observable when only 5% of the apolipoprotein had been degraded, suggesting that a minor subpopulation of HDL₃ was involved. Furthermore, apoA1 was a critical component of those HDL particles that were demonstrated to be highly sensitive to proteolysis.³² To study whether this component was preß₁LpA1, aliquots of HDL₃ were incubated in the absence or presence of granule remnants at 37°C for different periods of time up to 90 minutes. Parallel incubations with granule remnants were carried out at 4°C. After removal of the granule remnants by centrifugation, the samples were analyzed for their content of preß₁LpA1 by 2D-PAGGE. As shown in Figure 3, preß₁LpA1 was present in the HDL₃ fraction, but rapidly disappeared during incubation with granule remnants at 37°C. Indeed, it was clear from examination of the electrophoretogram that, when incubation was performed at 37°C in the presence of granule remnants, practically no preß₁LpA1 was visible after 30 minutes of incubation. This loss of preß₁LpA1 was paralleled by a reduced ability to induce efflux of cholesterol from the macrophage cultures (80% of reduction in cholesterol efflux when 25 µg/mL of HDL₃ was incubated with 6 µg/well of granule remnants in the cell culture system described in Figure 2). When incubation of HDL₃ with granule remnants was carried out at 4°C, no loss of preß₁LpA1 was observed, reflecting inhibition of the enzymatic activity of granule chymase (not shown). Nor did any loss of preß₁LpA1 occur in control samples incubated at 37°C without granule remnants (Figure 3).

View larger version (46K): [in this window] [in a new window]   Figure 3. Effect of mast cell granule remnants on the content of preß1LpA1 in HDL3. HDL3 (1 mg/mL) was incubated in 50 µL of 150 mmol/L NaCl, 1 mmol/L EDTA, 5 mmol/L Tris-HCl, pH 7.4 in the presence of granule remnants (40 µg total protein equal to 56 BTEE units of chymase/50 µL) at 37°C or at 4°C for different time periods. Parallel incubations were carried out at 37°C in the absence of granule remnants. The samples were centrifuged at 4°C, 15 000 rpm for 5 minutes, and analyzed for their LpA1 subpopulations by 2D-PAGGE.

Effect of Granule Remnants on ³H-Cholesterol Efflux Promoted by Fresh Plasma
Preincubation of plasma at 37°C for 90 minutes has been found to be associated with a reduced (40%) rate of short-term cholesterol efflux (0.5 to 5 minutes) from cultured fibroblasts. However, this effect, which was associated with conversion of preßHDL to other HDL particles, is not observed if incubation is performed in the presence of fibroblasts, vascular smooth muscle cells, or macrophages.²³ In Figure 1, it was demonstrated that incubation of plasma with macrophages for various lengths of time (up to 6 hours) does not change its ability to promote subsequent cholesterol efflux from the foam cells during a 30 minutes incubation. This result allowed us to design experiments in which the effect of mast cell granule remnants on the cholesterol efflux-inducing capacity of plasma could be studied for prolonged periods in a system comprising plasma, granule remnants, and macrophages.

To determine the ability of proteolytically active granule remnants to block the function of the cholesterol acceptors contained in plasma, ie, in the presence of the physiological inhibitors of chymase, the cholesterol efflux-promoting ability of plasma was measured after addition to the incubation system of different amounts of granule remnants (6 to 60 µg total protein/well). When fresh plasma (5%) was incubated with ³H-cholesterol-loaded macrophages in the presence of increasing concentrations of granule remnants, a progressive reduction in cholesterol efflux capacity was observed (Figure 4). Although as little as 6 µg was enough to reduce the cholesterol efflux activity of plasma significantly, the inclusion of 15 µg of granule remnants in the incubation medium reduced cholesterol efflux by 50%. Further additions of granule remnants to the cell culture caused relatively smaller reductions in the efflux. Accordingly, in the following experiments we selected 15 µg as the quantity of granule remnants to be added to the plasma. As shown by a previous study, preßLpA1-dependent cholesterol efflux can be blocked by pretreatment of the cells with protease.²⁵ However, incubation of foam cells with granule remnants did not affect the rate of cholesterol efflux because removal of the granule remnants from the medium and addition of fresh plasma to the cells did not influence the rate of efflux (not shown). This finding, also made previously,¹³ may be caused by the inability of the chymase embedded in the proteoglycan meshwork of the granule remnants to attack the cell membrane.

View larger version (17K): [in this window] [in a new window]   Figure 4. Dose-dependent effects of granule remnants on the cholesterol efflux capacity of plasma. Fifteen microliters of plasma was incubated in 300 µL of medium A (to give a final concentration of 5% of plasma) with 3H-cholesterol-labeled macrophage foam cells at 37°C for 6 hours in the presence of increasing concentrations of granule remnants (6 to 60 µg total protein equal to 8 to 84 BTEE units of chymase/300 µL). The 3H-cholesterol-associated radioactivity of the medium was determined and plotted as a function of µg of granule remnants (total protein) in the incubation medium.

Effect of Incubation With Granule Remnants on Minor ApoA-Containing Subspecies of HDL Found in Plasma
To study whether the observed inhibitory effect of granule remnants on the ability of whole plasma to induce cholesterol efflux from macrophages was related to degradation of preßLpA1, as demonstrated in the HDL₃ fraction, samples of fresh plasma were incubated at 37°C in the presence or absence of granule remnants (15 µg), after which the preß₁LpA1 subpopulation of particles was analyzed by 2D-PAGGE. This analysis revealed that plasma treated with granule remnants suffered a time-dependent and progressive loss of preß₁LpA1. Thus, as shown in Figure 5, a decrease or even a disappearance of preß₁LpA1 was clearly observed after incubation of plasma with granule remnants at 37°C (Figure 5C and 5F) but was not observed after incubation with granule remnants at 4°C (Figure 5B and 5E) or without granule remnants at 37°C (Figure 5A and 5D). The incomplete disappearance of preß₁LpA1 after prolonged control incubation at 37°C is in contrast with another report,¹⁵ but may be caused by regeneration by HDL interconversions through cholesteryl ester transfer protein and phospholipid transfer protein.^{16 17 18 19 20 21} Densitometric analysis of anti-apoA1 immunoblots of agarose gels revealed that, even after incubation for 5 and 15 minutes at 37°C with granule remnants (not shown), 60% of the preß₁LpA1 had disappeared, and 90% had disappeared after incubation for 24 hours. In contrast, during incubation at 4°C for up to 24 hours, the presence of granule remnants did not lead to loss of preß₁LpA1. In the control experiment without granule remnants, the amount of preß₁LpA1 had not changed after incubation for 5 or 15 minutes (not shown), and had decreased by only 40% after incubation for 24 hours at 37°C.

View larger version (67K): [in this window] [in a new window]   Figure 5. Effect of mast cell granule remnants on the distribution of apoA1-containing lipoproteins in plasma. Fifteen microliters of plasma was incubated in 60 µL of 150 mmol/L NaCl, 1 mmol/L EDTA, 5 mmol/L Tris-HCl, pH 7.4, for either 1 hour (A to C) or 24 hours (D to F). In control experiments A and D, the buffer did not contain granule remnants; in B, C, E, and F, the buffer contained granule remnants (15 µg total protein equal to 21 BTEE units of chymase/60 µL) in a final plasma concentration of 17.5 mg total protein/mL. Incubations were performed either at 37°C (A, C, D, F), at which chymase was active, or at 4°C, at which it had low activity (B, E). The incubations were stopped after the indicated time intervals by centrifuging the vials at 4°C to sediment the granule remnants. Forty microliters of the supernatants were analyzed for their LpA1 subpopulations by 2D-PAGGE. Note the decrease or disappearance of preß1LpA1 on incubation of plasma with granule remnants at 37°C (C, F), which was not observed after incubation without granule remnants at 37°C (A, D) or with granule remnants at 4°C (B, E).

Incubation of plasma without cells at 37°C makes it possible to define 2 fractions of cholesterol acceptors containing apoA1, one labile at this temperature (preß₁LpA1; the fraction responsible for the high-affinity efflux), and the other resistant to incubation at 37°C (-HDL; the fraction promoting slow cholesterol efflux; see Figure 1). Using prolonged preincubation times, we were able to study the influence of granule remnants on the ability of these 2 fractions to promote efflux of cholesterol. In this experimental approach, samples of undiluted fresh plasma were preincubated for 6 hours at 4°C or 37°C to preserve or reduce the concentrations of temperature-labile acceptors, and added to ³H-cholesterol-loaded macrophage cell cultures in the absence or presence of granule remnants. Preincubation of plasma for 6 hours at 37°C in the absence of granule remnants resulted in a significant reduction in its cholesterol efflux-promoting activity compared with plasma preincubated for 6 hours at 4°C (from 1589 to 1197 dpm) (Table 3; see also Figure 1). When the 2 preincubated plasmas were incubated with ³H-cholesterol-loaded macrophage cultures in the presence of granule remnants, further reductions in their cholesterol efflux capacity were observed (Table 3). As expected, the granule remnant-dependent reduction was more pronounced and significant in those plasma samples that had been preincubated at 4°C (from 1589 to 826 dpm), ie, those without previous loss of their content of preß₁LpA1. It is possible that the further reduction in residual cholesterol efflux (from 1197 to 820 dpm) observed when granule remnants were added to plasma that had been preincubated at 37°C was attributable to granule remnant-dependent degradation of residual amounts of preß₁LpA1 and other cholesterol acceptors such as LpA4–1, which had not been depleted from plasma by preincubation at 37°C (see later Figure 6).

View this table: [in this window] [in a new window]   Table 3. Effect of Mast Cell Granule Remnants on the Ability of Plasma Preincubated for 6 Hours at 4°C or at 37°C to Promote Cholesterol Efflux From 3H-Cholesterol-Loaded Macrophages

View larger version (74K): [in this window] [in a new window]   Figure 6. Effect of mast cell granule remnants on the distribution of apoA4-containing lipoproteins in plasma. Incubations of plasma were performed as described in Figure 5. After sedimentation of granule remnants, 40 µL of the supernatants were analyzed for their LpA4 subpopulations by 2D-PAGGE. Note the granule remnant-dependent decrease of the smaller LpA4–1 particles (C, F), which was not observed after incubation without granule remnants at 37°C (A, D) or with granule remnants at 4°C (B, E).

To evaluate the overall impact of chymase proteolysis on other cholesterol acceptor particles, we studied the potential effect of mast cell granule remnants on the other apoAs of lipoprotein particles present in plasma. Thus, we incubated plasma with granule remnants after the experimental procedure shown in Figure 5, and the LpA4 subpopulations were analyzed by 2D-PAGGE in the chymase-treated plasma samples (Figure 6). As described previously,³¹ apoA4 is present in 2 subpopulations of particles that have preß-mobility to -mobility. Incubation of plasma with granule remnants at 37°C for 1 hour (Figure 6C) or 24 hours (Figure 6F) led to the almost complete disappearance of LpA4–1 (smaller particles), which was not observed after incubation in the presence of granule remnants at 4°C (Figure 6B and 6E), or in the absence of granule remnants at 37°C (Figure 6A and 6D). Incubation of plasma at 37°C with granule remnants for 1 hour (Figure 6C) led to disappearance of LpA4–2 (larger particles). However, after incubation for a longer time, this change was independent of the presence of chymase: the larger particles disappeared in the absence of granule remnants at 37°C (Figure 6D) and in the presence of granule remnants at 4°C (Figure 6E) if incubation was continued for 24 hours, ie, even when chymase was absent or its activity was low (at 4°C). By contrast, treatment of plasma with granule remnants under the conditions described above did not change the immunoelectrophoretic profile of apoA2-containing particles. As shown previously,⁴² apoA2 is found in a single type of particle, which has electrophoretic -mobility. Figure 7 shows that the intensity of immunostaining of this particle population was unaffected by the experimental conditions used, even by incubation of plasma in the presence of granule remnants at 37°C for 24 hours (Figure 7D).

View larger version (34K): [in this window] [in a new window]   Figure 7. Effect of mast cell granule remnants on the distribution of apoA2-containing lipoproteins in plasma. Incubations of plasma were performed as described in Figure 5. After sedimentation of granule remnants, 40 µL of the supernatants were analyzed for their LpA2 subpopulations by 2D-PAGGE. Note that the intensity of immunostaining of this particle population, which appears as a single band, was not affected by incubation of plasma with granule remnants at 37°C for 24 hours (D).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have previously described a cell-mediated mechanism that leads to proteolysis of HDL apoproteins and effectively reduces the ability of HDL₃ to remove cholesterol from macrophage foam cell cultures.¹³ Specifically, the high-affinity component of LpA1, HDL₃, and plasma that promotes cholesterol efflux from macrophage foam cells is sensitive to proteolysis by the neutral protease chymase contained in exocytosed mast cell granules, ie, the granule remnants.³² Because preß₁LpA1 promotes rapid and specific cholesterol efflux from cell cultures²⁵ and is sensitive to proteolysis,⁴⁵ it seemed very likely that the proteolysis-sensitive component of the cholesterol efflux capacity of HDL and plasma was, in fact, caused by the presence of this particle. To test this hypothesis, we used 2 experimental approaches. First, we added granule remnants to preß₁LpA1 that had been formed in vitro by incubation of lipid-free apoA1 with mouse peritoneal macrophage foam cells. Second, we used 2D-PAGGE and agarose gel electrophoresis to monitor changes in the levels of preß₁LpA1 in plasma and HDL₃, which were incubated in the presence or absence of mast cell granule remnants, and related these changes to the changes in cholesterol efflux capacity.

Mast cell granule remnants effectively prevented the high-affinity cholesterol efflux promoted by the preß₁LpA1 particles generated during the incubation of free apoA1 with cholesterol-loaded macrophages. Addition of granule remnants completely abolished the saturable high-affinity component involved in cholesterol efflux, which is found at rather low apoA1 levels (<25 µg/mL; Figure 2). The residual chymase-insensitive component responsible for cholesterol efflux may have been produced by phospholipids released into the medium when macrophages were incubated with apoA1 in the presence of granule remnants, which were unaffected by mast cell chymase. It was previously shown that cholesterol and phospholipids from various cells, including macrophages, are released into the incubation medium by amphipathic peptides.⁴⁶ Moreover, treatment of apoA1 with purified human skin chymase, derived from mast cells present in the skin, also inhibited the transfer of cholesterol from human monocyte macrophage foam cells induced by the apolipoprotein, suggesting that chymase-containing human mast cells, when activated, may prevent apoA1 from accepting cholesterol from human foam cells.

Incubation of plasma or HDL₃ at 37°C with granule remnants produced a progressive reduction in both the content of preß₁LpA1 and the cholesterol efflux ability (Figures 3 through 5). In agreement with our previous notion that mast cell granule remnants destroy the high-affinity cholesterol efflux component present in HDL and serum,³² we found that the cholesterol efflux-reducing effect of the granule remnants was greater in plasma rich in preß₁LpA1 than in plasma poor in preß₁LpA1 (Table 3).

When HDL₃ was incubated with granule remnants for as little as 30 minutes, no preß₁LpA1 was left, revealing the high efficiency of the proteolytic degradation (Figure 3). Incubation with granule remnants also decreased the content of preß₁LpA1 in plasma, but less rapidly than in HDL₃ (Figure 5). This difference can be explained by the partial inactivation of granule chymase by the protease inhibitors that are present in plasma but absent from the HDL₃ fraction. However, despite this partial inhibition, chymase effectively reduced the content of preß₁LpA1 during incubation for 1 hour; after incubation for 24 hours at 37°C, practically all the preß₁LpA1 had disappeared from the plasma samples. The inability of protease inhibitors to completely block the proteolysis of preß₁LpA1 may appear surprising. However, in contrast to the free enzyme, rat mast cell chymase bound to granule remnants has been found to be partially resistant to the action of the protease inhibitors present in human serum and intimal fluid.³² Thus, it seems that the binding of chymase to the heparin proteoglycans in granule remnants keeps the enzyme active against HDL₃ despite the presence of its natural inhibitors. Recently, the association between rat chymase and heparin proteoglycans has been shown also to protect the enzyme against protease inhibitors when acting on a small synthetic substrate.⁴⁷ The factors involved in the protection of chymase against its inhibitors seem to be related to the type of proteinase inhibitor and to the molecular features and sizes of the various substrates (L. Lindsfedt et al, unpublished data, 1994). The small extent of proteolysis necessary to disturb the function of HDL₃ as a cholesterol acceptor makes it appear that mast cell granule chymase could proteolytically block cholesterol efflux in the arterial intima even in the presence of its physiological inhibitors. Moreover, the specific depletion of preß₁LpA1 and LpA4 by chymase suggests that extracellular proteolysis can play a role in the control of the earliest step in removal of cholesterol from the arterial intima, where all the components for such an interaction are present. Indeed, increased numbers of degranulated mast cells have been found in human fatty streaks and in the shoulder regions of atheromas, ie, in locations where foam cells form.⁴⁸ The potential contribution of other intimal proteases to this process deserves further study.

The importance of quantitatively minor HDL subfractions in reverse cholesterol transport has also been stressed by the demonstration that other HDL species that contain apoA4, but lack apoA1, possess the residual cholesterol efflux activity of plasma devoid of apoA1.³¹ As apoA4-containing particles are relatively abundant in interstitial fluid,⁴⁹ we studied the potential contribution of proteolysis of apoA4 particles to the overall effect of chymase on plasma cholesterol acceptors. Here, too, we found a role for chymase in the deletion of LpA4–1 and LpA4–2 from plasma. The susceptibility of apoA4 to proteolysis in these lipid-poor particles could be related to the hydrophilic properties of this apolipoprotein,⁵⁰ which limit its ability to penetrate lipid surfaces and cause a more expanded conformation that is more accessible to proteolytic modification.

Taken together, our observations strongly suggest that the mechanism responsible for the reduced cholesterol efflux capacity of HDL and plasma that have been preincubated with mast cell granule remnants is proteolysis of both preß₁LpA1 and LpA4. Our observations support the concept that lipid-poor preß-migrating HDL and lipid-free apoA1 remove cholesterol from the plasma membranes of many cell types more efficiently than native lipid-rich LpA1 or reconstituted phospholipid-containing particles.^{1 8 51} Interestingly, a small fraction of the most trypsin-labile apolipoproteins in HDL has been observed to mediate high-affinity binding of HDL to cholesterol-loaded fibroblasts, and treatment of HDL with trypsin has been found to cause a marked increase in the binding of the particles to low-affinity binding sites on the cell surface.⁵² Thus, it may be that proteolysis of preß₁LpA1 by mast cell granule remnants destroys structural domains in apoA1 that are required for this specific action.^{28 29 30 53 54} Moreover, because extravasal fluids are relatively enriched in preß₁LpA1⁵⁵ and LpA4 particles,⁴⁹ their proteolysis, and the consequent inhibition of cholesterol efflux by exocytosed mast cell granules, may be of special importance in the arterial intima, the site of atherogenesis. In the human aorta and coronary arteries this may specifically apply to fatty streak areas in which foam cells have formed and the number of degranulated mast cells has increased.⁵⁶

	Acknowledgments

 
This project was supported by grants from the Sigrid Juselius Foundation to Dr Lee, and from the Deutsche Forschungsgemeinschaft (Ec116,2-2 and Ec116,3-2) to Dr von Eckardstein. We thank Dr Armin Steinmetz, University of Marburg (Germany), for providing anti-apoA4 antisera, Bettina Bretz and Isabelle Schaukal for excellent technical assistance, and Petri Toivanen for careful statistical analysis.

Received September 8, 1998; accepted October 7, 1998.

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				M. Lee, J. Metso, M. Jauhiainen, and P. T. Kovanen 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 J. Biol. Chem., April 4, 2003; 278(15): 13539 - 13545. [Abstract] [Full Text] [PDF]

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				M. Lee, C. P. Sommerhoff, A. von Eckardstein, F. Zettl, H. Fritz, and P. T. Kovanen Mast Cell Tryptase Degrades HDL and Blocks Its Function as an Acceptor of Cellular Cholesterol Arterioscler Thromb Vasc Biol, December 1, 2002; 22(12): 2086 - 2091. [Abstract] [Full Text] [PDF]

				M. Lee, L. Calabresi, G. Chiesa, G. Franceschini, and P. T. Kovanen Mast Cell Chymase Degrades ApoE and ApoA-II in ApoA-I-Knockout Mouse Plasma and Reduces Its Ability to Promote Cellular Cholesterol Efflux Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1475 - 1481. [Abstract] [Full Text] [PDF]

				D. Sviridov, O. Miyazaki, K. Theodore, A. Hoang, I. Fukamachi, and P. Nestel Delineation of the Role of Pre-{beta}1-HDL in Cholesterol Efflux Using Isolated Pre-{beta}1-HDL Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1482 - 1488. [Abstract] [Full Text] [PDF]

				M. N. Nanjee, C. J. Cooke, W. L. Olszewski, and N. E. Miller Concentrations of Electrophoretic and Size Subclasses of Apolipoprotein A-I-Containing Particles in Human Peripheral Lymph Arterioscler Thromb Vasc Biol, September 1, 2000; 20(9): 2148 - 2155. [Abstract] [Full Text] [PDF]

				M. Lee, P. Uboldi, D. Giudice, A. L. Catapano, and P. T. Kovanen Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase: implications for HDL function J. Lipid Res., June 1, 2000; 41(6): 975 - 984. [Abstract] [Full Text]

				N. Fournier, V. Atger, J.-L. Paul, M. Sturm, N. Duverger, G. H. Rothblat, and N. Moatti Human ApoA-IV Overexpression in Transgenic Mice Induces cAMP-Stimulated Cholesterol Efflux From J774 Macrophages to Whole Serum Arterioscler Thromb Vasc Biol, May 1, 2000; 20(5): 1283 - 1292. [Abstract] [Full Text] [PDF]

				L. Lindstedt, J. Saarinen, N. Kalkkinen, H. Welgus, and P. T. Kovanen Matrix Metalloproteinases-3, -7, and -12, but Not -9, Reduce High Density Lipoprotein-induced Cholesterol Efflux from Human Macrophage Foam Cells by Truncation of the Carboxyl Terminus of Apolipoprotein A-I. PARALLEL LOSSES OF PRE-beta PARTICLES AND THE HIGH AFFINITY COMPONENT OF EFFLUX J. Biol. Chem., August 6, 1999; 274(32): 22627 - 22634. [Abstract] [Full Text] [PDF]

				J. G. Strauss, S. Frank, D. Kratky, G. Hammerle, A. Hrzenjak, G. Knipping, A. von Eckardstein, G. M. Kostner, and R. Zechner Adenovirus-mediated Rescue of Lipoprotein Lipase-deficient Mice. LIPOLYSIS OF TRIGLYCERIDE-RICH LIPOPROTEINS IS ESSENTIAL FOR HIGH DENSITY LIPOPROTEIN MATURATION IN MICE J. Biol. Chem., September 21, 2001; 276(39): 36083 - 36090. [Abstract] [Full Text] [PDF]

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