Lipoprotein-like Phospholipid Particles Inhibit the Smooth Muscle Cell Cytotoxicity of Lysophosphatidycholine and Platelet-Activating Factor
Abstract—Oxidation of LDL is associated with degradation of phosphatidylcholine into platelet-activating factor (PAF)–like phospholipids and lysophosphatidylcholine (LPC). Exposure of cultured human smooth muscle cells to PAF and LPC in a concentration of 25 μmol/L was found to result in complete cell death, as assessed by the MTT cytotoxicity assay and cell counting. Addition of 50 μg/mL apolipoprotein A-I– and apolipoprotein A-IMilano–containing phospholipid particles completely inhibited this cytotoxicity. Phospholipid complexes alone were almost as effective, whereas free apolipoprotein A-IMilano and albumin were without effect, suggesting that the effect was phospholipid dependent. Experiments using [14C]LPC demonstrated that apolipoprotein A-I– and apolipoprotein A-IMilano–containing phospholipid particles effectively bind LPC. The results show that HDL-like phospholipid particles effectively inhibit the toxic effect of phospholipids and other lipid-soluble factors. The ability of HDL to inhibit the proinflammatory and toxic effects of phospholipids generated during oxidation of LDL may be responsible for part of the antiatherogenic properties of HDL.
- Received February 11, 1997.
- Accepted August 27, 1997.
Oxidation of LDL lipids in the arterial wall is believed to play a key role in the formation of atherosclerotic lesions.1 2 3 Oxidized LDL particles are removed by macrophages via scavenger receptors, resulting in the formation of foam cells. Oxidation of LDL is associated with major changes in phospholipid composition. Phosphatidylcholine is degraded to short side-chain phospholipids, including PAF-like molecules.4 5 6 These extremely reactive and proinflammatory phospholipids are hydrolyzed by an LDL-associated phospholipase A2, recently shown to be identical to PAF-acetylhydrolase, into LPC.6 7 In LDL oxidized in vitro by exposure to cupric ions, LPC constitutes approximately 30% of all LDL phospholipids.5 Although less reactive than PAF, LPC markedly affects the function of inflammatory cells.8 It also inhibits endothelial-dependent arterial relaxation9 ; activates endothelial expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1,10 as well as endothelial release of platelet-derived growth factor and heparin-binding epidermal growth factor–like protein11 ; stimulates the macrophage production of tumor necrosis factor-α12; and promotes SMC proliferation.13 These findings suggest that PAF and LPC accumulation during intimal oxidation of LDL may be involved in activation of the inflammatory process and SMC proliferation associated with the formation of fibrous and complicated atherosclerotic plaque.
On the basis of epidemiological data, HDL particles have been assigned a protective role in coronary heart disease in humans.14 15 16 Experimental studies in hypercholesterolemic rabbits injected with homologous HDL17 and in cholesterol-fed apoA-I transgenic mice18 have provided direct evidence for the antiatherogenic properties of HDL. The antiatherogenic effect of HDL has been attributed to its ability to extract cholesterol from peripheral tissues and to mediate reverse cholesterol transport.19 However, alternative mechanisms, including inhibition of complement polymerization,20 LDL oxidation,21 and protection against LDL cytotoxicity,22 have also been suggested. HDL carries enzymes that hydrolyze oxidized derivatives of phosphatidylcholine into LPC.23 The present study was designed to investigate whether apoA-IMilano reconstituted in phospholipid particles has the capacity to protect against the harmful effects of generation of PAF-like molecules and accumulation of LPC. The effect of purified human apoA-I reconstituted in phospholipid liposomes was compared with that of liposomes containing recombinant apoA-IMilano, a mutant form of apoA-I believed to have increased antiatherogenic capacity.24
Isolation and Culture of SMCs
SMCs were isolated from the media of human saphenous vein grafts. Tissue specimens were carefully dissected, cut into small pieces, and allowed to attach to the surface of six-well multiplates by drying for 15 minutes. The explants were then cultivated in F-12/DMEM medium (GIBCO) containing 10% NCS (GIBCO) and 50 μg/mL gentamycin at 37°C in an atmosphere of 5% CO2 in air. Cells began migrating out from the explants within 1 to 2 weeks and reached confluence within another 2 weeks. Secondary cultures were established by trypsinization and seeding of the cells in 75-cm2 culture flasks. The purity of the SMC populations was determined by the presence of muscle-specific α-actin immunoreactivity, using the HHF 35 antibody (DAKO). Cells up to the 10th passage were used in the experiments.
Preparation of ApoA-I– and ApoA-IMilano–Containing Phospholipid Particles
Human apoA-I was obtained from Organon Teknika Corporation and recombinant apoA-IMilano was a gift from Pharmacia and Upjohn, Stockholm, Sweden. Complexes of apoA-I (or apoA-IMilano) and EYL-PC were made as described by Matz and Jonas.25 In short, 36 mg of EYL-PC in ethanol was dried in a glass tube under nitrogen, and 2 mL of sodium cholate (10 mg/mL) was added. The tube was vigorously vortexed and left under agitation at 4°C for at least 2 hours. A solution of 1.5 mg/mL apoA-I (or apoA-IMilano) in PBS was prepared and the EYL/cholate solution was added until a final ratio of EYL to apoA-I of 2.5 (wt/vol) was obtained, and the tube was left under agitation overnight at 4°C. Finally, the solution was dialyzed for 3 days at 4°C against 10×1 L of PBS. The concentration of apoA-I in the final preparation was determined as described by Lowry et al26 and phospholipids by an enzymatic kit (Wako). The size of the complexes was determined by electrophoresis under nondenaturing conditions as described by Cheung,27 using commercially available 4% to 20% linear polyacrylamide gradient gels. The gel was stained by incubation in 0.1% (wt/vol) Coomassie brilliant blue R 250 in methanol/acetic acid/water (5:1:4, vol/vol/vol) for 16 hours at 20°C and destained in methanol/acetic acid/water (5:1:4, vol/vol/vol) until background staining had completely disappeared. The gels were scanned in an LKB Ultroscan XL scanner, and particle sizes were determined from a standard curve. The hydrated Stokes diameter of free apoA-IMilano was approximately 7.0 nm, whereas that of apoA-I– and apoA-IMilano–containing particles was approximately 8.0 to 8.5 nm. In the complexes, approximately 99% by weight of apoA-I was in the monomeric form, whereas 90% by weight of apoA-IMilano was in dimeric form. The difference in monomeric molecular weight between the two types of apoA-I is negligible, which means that the molar ratio of protein monomer to phospholipid is the same in the two types of particles. The molecular ratio of phosphatidylcholine/apoA-I monomer is approximately 103.
Experiments were also performed with apoA-I/soybean phosphatidylcholine particles (kindly provided by Dr J. Doran, Swiss Red Cross) and apoA-IMilano/dimethylphosphatidylcholine particles (kindly provided by Dr H. Ageland, Pharmacia and Upjohn). The effect of these particles was the same as for those prepared as described above.
MTT Assay of Cell Viability
SMCs were seeded out in 24-well plates and grown to subconfluent density in 10% NCS/DMEM. The cultures were than rinsed in DMEM/F-12 (1:1) without phenol red and incubated with respective test substance in DMEM/F-12 without phenol red/0.1% BSA (Sigma) for 20 hours. Cytotoxicity was then analyzed by using the MTT assay (Sigma). MTT was dissolved in DMEM/F-12 without phenol red at a concentration of 5 mg/mL. An amount of this solution equal to 10% of the culture medium volume was added to cell cultures. After 1 hour, cultures were removed from the incubator and the formazan crystals solubilized by adding solubilization solution (10% [vol/vol]) Triton X-100 and 0.1N HCl in isopropanol) equal to the original culture medium volume. Metabolic activity was quantified by measuring light absorbance at 570 nm.
SMCs were seeded out in 12-well plates and grown to subconfluent density in 10% NCS/DMEM. They were then serum starved by transfer to 0.1% BSA/DMEM for 48 hours to arrest cell growth and incubated with the respective test substance in 0.1% BSA/DMEM for another 48 hours. The cultures were subsequently trypsinized and cell numbers determined in an electronic cell counter (Analys Instrument).
Cells cultured on glass coverslips were fixed in 100% methanol at −20°C for 30 minutes. The coverslips were air dried, rinsed twice with water, and transferred to cell-culture dishes covered with wet tissue paper. Seventy-five microliters of the following solution was added to each coverslip: 20 μmol/L biotin-16-dUTP, 200 U/mL terminal deoxytransferase, 300 mmol/L Tris-HCl (pH 7.2), 10 mmol/L CoCl2, and 300 mg/mL freshly added cacodylate. The samples were incubated at 37°C for 60 minutes. The coverslips were transferred to TB buffer (300 mmol/L NaCl, 30 mmol/L sodium citrate) for 15 minutes, rinsed twice with PBS, incubated in 2% BSA for 10 minutes, and rinsed twice for 5 minutes with PBS. Extravidin-FITC (100 μL) diluted to 15 μg/mL in PBS was added to each coverslip. After 30 minutes at 37°C, the samples were washed three times with PBS and once with PBS containing 0.1% Triton X-100. The coverslips were mounted on slides with antifading solution (1 mg/mL p-phenylenediamine, 10% [vol/vol] PBS, 90% [vol/vol] glycerol). At least 200 cells were counted for quantitative analyses. TUNEL-positive cells had brightly stained nuclei, showing either homogeneous staining of intact nuclei or condensation of chromatin into distinct, brightly stained fragments.
Binding of LPC to ApoA-I–Containing Phospholipid Particles
l-Lyso-3-phosphatidylcholine (1 μCi), 1-[1-14C]palmitoyl (Amersham) was evaporated, together with 100 μL of 2 mmol/L unlabeled LPC to serve as carrier. The LPC was then dissolved in 2 mL of PBS containing various concentrations of apoA-I–containing phospholipid particles, apoA-IMilano–containing phospholipid particles, or phospholipid complexes alone and incubated for 2 hours at 20°C. A 0.5-mL portion of this solution was put on a NAP 5 column (Pharmacia) and eluted in water to separate [14C]LPC bound to apoA-I–containing phospholipid particles from free [14C]LPC. Aliquots of 1.0 mL were collected and analyzed for protein and radioactivity. The column was finally eluted with ethanol to remove remaining labeled LPC.
Data are expressed as mean±SD and were evaluated by the nonparametric Wilcoxon test. Statistical analyses were made on groups including at least six independent samples. A value of P<.05 was taken as the level of statistical significance.
Both PAF (Fig 1A⇓) and LPC (Fig 1B⇓) were found to be highly cytotoxic for cultured human SMCs in a dose-dependent manner. Exposure of SMCs to PAF for 24 hours in concentrations up 5 μmol/L had only minor effects on cell viability, whereas exposure of the cells to 25 μmol/L PAF decreased viability by more than 90%, as assessed by the MTT test. A similar dose-response relationship was observed for LPC. Addition of 50 μg/mL human apoA-I–containing phospholipid particles (which equals 185 μg/mL of EYL-PC) completely inhibited PAF-induced cytotoxicity to a PAF concentration of up to 50 μmol/L (Fig 1A⇓). ApoA-IMilano–containing phospholipid particles and protein-free phospholipid particles were as effective as apoA-I–containing phospholipid particles in inhibiting PAF cytotoxicity, whereas free apoA-IMilano without phospholipid was without effect. ApoA-I– and apoA-IMilano–containing phospholipid particles also inhibited the cytotoxicity of LPC (Fig 1B⇓). Again, addition of phospholipid liposomes without apoA-I was almost as effective, whereas free apoA-IMilano was without effect. Incubation of SMCs with medium containing 10 μmol/L PAF for 3 days resulted in a 93.5±3.0% reduction in cell number compared with cells grown in medium alone (P<.005). Addition of 50 μg/mL apoA-I and apoA-IMilano–containing phospholipid particles completely inhibited this cytotoxicity (Fig 2⇓).
Dose-response experiments adding various concentrations of apoA-I–containing phospholipid particles to medium containing 50 μmol/L PAF and LPC showed that a complete inhibition of cytotoxicity was obtained at an apoA-I concentration of 25 μg/mL (Fig 3A⇓). Also, apoA-IMilano–containing phospholipid particles inhibited PAF- and LPC-induced cytotoxicity in a dose-dependent manner but could not completely restore cell viability in this assay (Fig 3B⇓). Cell counting of SMCs cultured for 3 days in the presence of 50 μmol/L LPC demonstrated a complete or almost complete inhibition of cytotoxicity in cultures given 10 μg/mL apoA-I–containing phospholipid particles or 25 μg/mL apoA-IMilano–containing phospholipid particles (Fig 4A⇓). ApoA-I–containing phospholipid particles (25 μg/mL) and apoA-IMilano–containing phospholipid particles (50 μg/mL) were required to achieve a complete inhibition of cytotoxicity induced by addition of 50 μmol/L PAF in this system (Fig 4B⇓). Addition of free apoA-IMilano did not affect cell survival (Fig 4B⇓). For both PAF and LPC, protein-free phospholipid liposomes were almost as effective as apoA-I–containing phospholipid liposomes. An almost complete inhibition of cytotoxicity was observed at an EYL-PC concentration of 185 μg/mL, which equals the phospholipid concentration in samples with 50 μg/mL apoA-I (Figs 4A⇓ and 4B⇓). HDL isolated from human plasma protected against the cytotoxicity of 50 μmol/L LPC with similar dose-response relation to that of apoA-I– and apoA-IMilano–containing phospholipid particles (data not shown). Addition of human LDL also inhibited PAF and LPC cytotoxicity. For example, addition of 25 μg/mL LDL completely inhibited the cytotoxicity of 50 μmol/L LPC. However, the interpretation of these findings was complicated by the fact that addition of LDL at this concentration in itself increased MTT activity by 41.1±20.1% and at a concentration of 100 μg/mL, by 172.3±65.0%.
Previous studies have indicated that 7β-hydroxycholesterol is responsible for much of the cytotoxicity of oxidized LDL.28 Incubation of SMCs with 7β-hydroxycholesterol in concentrations up to 25 μg/mL decreased cell viability by 20.1±12.3% (P<.01), as assessed by the MTT assay. Addition of 50 μg/mL apoA-I–containing phospholipid particles and apoA-IMilano–containing phospholipid particles completely inhibited this cytotoxicity. Free apoA-IMilano protein was without effect (Fig 5⇓). We have recently shown that 25-hydroxycholesterol induces apoptosis of human SMCs.28A Dose-response experiments demonstrated that 25-hydroxycholesterol was more toxic to SMCs than 7β-hydroxycholesterol. At a concentration of 10 μmol/L, 25-hydroxycholesterol reduced SMCs viability by 91.6±1.2% (P<.005; Fig 6⇓). Addition of 50 μg/mL apoA-I and apoA-IMilano complexes completely inhibited this cytotoxicity, and no effect was observed with free apoA-IMilano. Exposure of cultures to 10 μmol/L 25-hydroxycholesterol resulted in complete cell death and necrosis. In SMC cultures exposed to 5 μmol/L 25-hydroxycholesterol, cell viability was decreased by 78.6±5.9%, and 7.7±1.3% of the cells were TUNEL positive compared with 0.9±0.4% of control cells (Fig 7⇓). Addition of 50 μg/mL apoA-IMilano–containing phospholipid particles inhibited both the decrease in cell viability and the initiation of apoptosis induced by 5 μmol/L 25-hydroxycholesterol.
Incubation of SMCs with 300 μmol/L hydrogen peroxide decreased cell viability by 88.2±3.6% (P<.01). However, addition of apoA-I– and apoA-IMilano–containing phospholipid particles in concentrations up to 100 μg/mL did not affect hydrogen peroxide–induced toxicity (data not shown), suggesting that the effect of these complexes is selective for lipophilic factors. A possible explanation for the discrepant effect of apoA-I–containing phospholipid particles is that they act by incorporating lipophilic substances into the phospholipid complex.
To test this hypothesis, 1 μCi [14C]LPC was incubated with various concentrations of apoA-I– and apoA-IMilano–containing phospholipid particles followed by separation on a PD-10 column. Both apoA-I– and apoA-IMilano–containing phospholipid particles were found to effectively bind [14C]LPC. Phospholipid complexes alone were somewhat less effective (Fig 8⇓).
Oxidation of LDL is associated with the formation of PAF-like oxidized phospholipids and LPC.4 5 6 These phospholipid subspecies are extremely bioactive and have physiological functions in inflammatory cell signal transduction and thrombin-receptor activation of endothelial cells.29 30 Cell-culture studies have shown that LPC increases endothelial leukocyte adhesion molecule expression10 and mitogen production by endothelial cells11 and macrophages12 and stimulates SMC growth.13 Uncontrolled formation of these phospholipid species as a result of intimal lipoprotein oxidation may thus have an important role in the development of atherosclerotic plaque. The present findings that HDL-like apoA-I– and apoA-IMilano–containing phospholipid particles effectively bind and inhibit the cytotoxicity of PAF and LPC suggest that a similar mechanism may be responsible for some of the antiatherogenicity of HDL.
Oxidized LDL has been shown to inhibit endothelial-derived relaxation through its increased LPC content.9 Matsuda and coworkers31 reported that this effect was reversed by HDL and that HDL also reversed inhibition of endothelial-derived relaxation caused by addition of LPC alone. Moreover, they demonstrated that HDL inhibited incorporation of radiolabelled LPC into cultured endothelial cells and stimulated the efflux of LPC from the cells into the culture medium. Incubation of oxidized LDL containing radiolabelled LPC with HDL was found to result in a transfer of LPC from LDL to HDL. These observations are in good agreement with our findings that apoA-I–containing phospholipid particles effectively incorporate LPC and inhibit LPC cytotoxicity.
The uptake of phospholipids by HDL is a less understood process than HDL cholesterol incorporation. By its secondary structure, consisting of eight folded amphipathic α helices, apoA-I has the ability to organize phospholipids into nascent HDL particles.32 In apoA-I–transfected CHO cells, apoA-I complexes with cellular phospholipid to be secreted as small HDL3-like particles.33 The transfer of phospholipids from cellular membranes to already existing HDL particles, as well as the transfer of phospholipids between lipoproteins, is mediated by specific phospholipid transfer proteins.34 35 The present findings that apoA-I–containing phospholipid particles may incorporate and inhibit the cytotoxicity of LPC in a serum-free environment suggest that HDL can complex free phospholipids also in the absence of phospholipid transfer protein. Our findings also demonstrate that phospholipid liposomes without apoA-I are almost as effective in this respect as complexes containing apoA-I, indicating that adsorption of PAF and LPC by the phospholipid layer is the principle mechanism involved in this effect. In contrast, addition of free apoA-I did not inhibit LPC cytotoxicity, demonstrating that the protein alone did not have the ability to complex LPC.
ApoA-I–containing phospholipid particles effectively inhibited the cytotoxicity of lipid-soluble substances such as LPC, PAF, and 7β-hydroxycholesterol but had little effect on the toxicity of H2O2, a substance with poor lipid solubility. These observations are well in agreement with the concept that the protective effect of apoA-I–containing phospholipid particles is due to incorporation of the toxic substance into the phospholipid layer rather than to a general cytoprotective effect.
The present study also compared the effect of wild-type apoA-I with that of the mutant apoA-IMilano. The apoA-IMilano is a naturally occurring variant of apoA-I, with a cysteine-for-arginine substitution at position 173 in the amino acid sequence that favors the formation of homodimers as well as heterodimers with apoA-II through disulfide linkage.36 The carriers of this mutation have markedly reduced HDL cholesterol levels and a high prevalence of hypertriglyceridemia without an increased incidence of vascular disease.24 The longer half-life of apoA-IMilano compared with the wild type may, at least in part, explain the former’s possible favorable effect.37 We have previously shown that apoA-IMilano inhibits the formation of intimal lesions after balloon injury of the aorta in hypercholesterolemic rabbits.38 This inhibition was associated with a reduced intimal inflammatory activity (assessed by a decreased number of intimal macrophages) but not with a decreased level of aortic tissue cholesterol. These findings suggest that apoA-IMilano may have functioned by scavenging proinflammatory phospholipid species rather than by the removal of cholesterol from the vessel wall of the hypercholesterolemic animals. The present study suggests that there is no difference between apoA-I and apoA-IMilano in inhibition of phospholipid cytotoxicity.
In conclusion, the present study demonstrates that apoA-I–containing phospholipid particles effectively bind and inhibit the cytotoxicity of proinflammatory phospholipids such as LPC and PAF and that this effect primarily is dependent on the phospholipid component of the particles. The ability of HDL to neutralize the effect of these phospholipids may have important protective functions in inhibiting the proinflammatory and toxic effects of lipid oxidation and restoring vasomotor function in atherosclerosis.
Selected Abbreviations and Acronyms
|DMEM||=||Dulbecco’s modified Eagle’s medium|
|EYL-PC||=||egg yolk phosphatidylcholine|
|NCS||=||newborn calf serum|
|SMC||=||smooth muscle cell|
|TUNEL||=||terminal deoxytransferase-mediated dUTP nick end labeling|
This study was supported by grants from Pharmacia and Upjohn, the Swedish Medical Research Council (8311), the Swedish Heart and Lung Foundation, the Grand Foundation, the Nanna Svartz fund, and King Gustav V 80th Birthday Fund. Recombinant apoA-IMilano was a gift from Dr H. Ageland, Pharmacia and Upjohn, Stockholm, Sweden. ApoA-I/soybean phosphatidylcholine particles were kindly provided by Dr J. Doran, Swiss Red Cross. Expert technical assistance was provided by Britt Elving.
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