From the Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
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ABSTRACT |
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Both oxidized low density lipoprotein (ox-LDL)
and platelet-derived growth factor (PDGF) have been implicated in the
genesis of various inflammatory responses, including atherosclerosis. We demonstrate here a novel interaction between specific oxidized lipids derived from ox-LDL and PDGF. The lipid moieties of ox-LDL caused concentration-dependent inactivation of PDGF as
measured by loss of its mitogenic activity and its binding to high
affinity receptors. Reverse-phase and normal-phase HPLC were used to
purify the inactivating component in the lipid mixture. By fast atom bombardment mass spectrometry and infrared spectroscopy, we identified the inactivating lipids as the 9- and 13-hydroperoxy derivatives of
cholesteryl linoleate, cholesteryl hydroperoxyoctadecadienoate. When a
series of cholesteryl esters were subjected to oxidizing conditions,
only those containing two or more double bonds caused inactivation of
PDGF; the extent of inactivation increased with increased levels of
oxidation. Exposing PDGF to cumene hydroperoxide, t-butyl
hydroperoxide, or hydrogen peroxide did not affect the activity of the
mitogen. The oxidized lipid had no effect on the mitogenic activity of
epidermal growth factor but did abolish the mitogenic activity of basic
fibroblast growth factor and the antiproliferative activity of
transforming growth factor 1. The inactivation of PDGF and other
cytokines by lipid hydroperoxides may occur in such processes as
vascular disease, inflammation, and wound healing.
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INTRODUCTION |
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Platelet-derived growth factor (PDGF)1 is a potent mitogen for smooth muscle cells, fibroblasts, and other cells of mesenchymal origin in culture (1). PDGF is secreted by cells that play important roles in vascular biology, such as endothelial cells (EC) (2), macrophages (3, 4) and, under certain conditions, arterial smooth muscle cells (5, 6). Inappropriate production of PDGF by EC following injury or activation in vivo may play a significant role in the development of the atherosclerotic lesion by increasing the proliferation and migration of neighboring smooth muscle cells, a key event in lesion formation (7).
PDGF production by cultured EC is constitutive but is stimulated by injury as well as exposure to a number of agents, such as lipopolysaccharide (8), phorbol esters (8), and thrombin (9). EC production of active PDGF is also specifically inhibited under certain culture conditions. Initial observations from our laboratory showed that chemically modified LDL, such as acetylated LDL or oxidized LDL (ox-LDL), completely inhibited the production of active PDGF from bovine aortic EC without affecting total protein synthesis (10). Native LDL or its lipid extract had no effect on production of active PDGF by bovine aortic EC.
In the present report, we demonstrate that the lipid moiety of ox-LDL is capable of inactivating PDGF. We have also purified and characterized the major PDGF inactivating component from the lipid extract of ox-LDL. This lipid component, cholesteryl hydroperoxyoctadecadienoate (cholesteryl HPODE) has been identified as the major oxidation product in LDL that has been oxidized by activated human monocytes (11). More recently, cholesteryl HPODE was detected, using the same detection system described in this report, in all human atherosclerotic lesions analyzed (n = 80), regardless of the type of lesion or stage of progression (12). The inactivation of PDGF by this component of ox-LDL, as well as other oxidized lipids, may occur in lesion development and/or the process of inflammation and wound healing.
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MATERIALS AND METHODS |
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LDL and Cholesteryl Ester Oxidation-- LDL was isolated from human plasma by differential ultracentrifugation at 1.019 < d < 1.063 (13). LDL was oxidized in vitro by dialysis against 0.9% NaCl, 5 µM CuSO4 for 6-8 h at 37 °C; ox-LDL was then lyophilized, and lipids were extracted with diethyl ether. Recovery of total cholesterol in the extract was consistently greater than 90%. Cholesteryls stearate, oleate, linoleate, linolenate, arachidate, and arachidonate (Sigma) were subjected to oxidizing conditions by exposure to air in a 37 °C water bath for 48-72 h. Lipid peroxidation was measured as thiobarbituric acid-reacting substances (TBARS) by a modification (14) of the method of Schuh et al. (15) and expressed as malondialdehyde equivalents per milligram of cholesteryl ester. Cholesterol and cholesteryl ester masses were determined by the ferric chloride method of Zak et al. (16) after saponification (17).
Separation of Lipids by HPLC and Confirmation of Chemical Structures-- HPLC analyses were performed with a SpectraPhysics liquid chromatograph system (pump, SP8800; detector, SP8490) equipped with either a µBondaPak reverse-phase C18 preparative (19 mm × 15 cm; Millipore, Bedford, MA) or a µBondaPak normal phase CN (39 mm × 30 cm; Millipore) column. The C18 column was eluted at room temperature with 100% acetonitrile for 10 min, followed by a linear gradient from 100% acetonitrile to 100% 2-propanol in 45 min at a flow rate of 10 ml/min. The CN column was eluted at room temperature in an isocratic system of 100% hexane at a flow rate of 1 ml/min. Both columns were monitored at 206 nm. Data were recorded, and chromatograms were integrated by Ramona software (Raytest; Pittsburgh, PA).
For applications to the C18 column, standards and samples in CHCl3 containing 5 mg of total cholesterol were dried under N2, redissolved in 0.25 ml of 1:1 (v/v) 2-propanol/acetonitrile. For the CN column, 0.100 mg of sample were dissolved in 0.25 ml hexane. Fractions of interest were collected, dried under N2, redissolved in CHCl3, and stored under N2 atCell Culture--
Primary cultures of bovine aortic EC and human
foreskin fibroblasts were isolated and maintained in a 1:1 mixture of
Dulbecco-Vogt modified Eagle's medium and Ham's F-12 (DME/F-12) as
described previously (18). EC (passages 7-16) were plated in flasks at a split ratio of 1:3 to 1:5 in DME/F-12 containing 5% fetal bovine serum. For the collection of conditioned media containing PDGF, confluent cells were incubated for three days in DME/F-12 containing 2 mg/ml bovine serum albumin (Amresco, Solon, OH). Fibroblasts (passages
6-18) for the PDGF radioreceptor assay and mitogenic assay (see below)
were plated at subconfluent density (1.5-3 × 104
cells/cm2) in 24-well plates in 0.5 ml of medium containing
1% Zeta Sera-D (equivalent to CPSR-II, Sigma); cells were used within
2-10 days of plating. Mink lung epithelial cells (ATCC CCL64) for the
TGF-1 growth inhibition assay (see below) were cultured as described previously (19).
Preparation of Emulsions and Incubations-- Emulsions were prepared from the oxidized LDL extract, the unoxidized and oxidized cholesteryl esters, and the HPLC fractions always on the day of use. Up to 1 mg of sample, dimyristoylphosphatidylcholine (DMPC; Sigma) at 5% of the mass of the sample, and cardiolipin (CL; Sigma) at 1% of the mass of the sample were dried together under N2. Stable emulsions were formed by the addition of 0.020-0.200 ml (depending on the mass of lipid) of acetone/ethanol (1:1, v/v), which instantaneously generated a stable emulsion. DMPC/CL liposomes (5:1, w/w) were used as controls in all experiments involving emulsions. The water-soluble hydroperoxides, cumene hydroperoxide, t-butyl hydroperoxide, and hydrogen peroxide (Sigma), were added directly to the media containing PDGF.
Either conditioned media containing bovine PDGF, or human recombinant PDGF-AB (Boehringer Mannheim) in DME/F-12 containing 2 mg/ml bovine serum albumin (Amresco, Solon, OH), was used in the incubations. Lipid emulsions and/or water-soluble hydroperoxides were incubated with PDGF in siliconized polypropylene microfuge tubes for up to 72 h, rotating, at 37 °C. After 72 h, 25 µM HEPES (Life Technologies, Inc.) was added to preserve pH during the PDGF radioreceptor assay.PDGF Radioreceptor Assay-- PDGF was quantitated by a radioreceptor assay as described previously (2, 8). Briefly, unlabeled purified human recombinant PDGF-AB standard aliquots (0.05-0.40 ng/well) and aliquots of the incubations were applied in triplicate or quadruplicate to cultured fibroblasts for 1 h to allow binding of PDGF to PDGF receptors on the cells. The cells were washed, and media containing 125I-PDGF-AB (20) was placed on the cells for another hour to allow binding of 125I-PDGF-AB to any remaining receptor sites. Cells were washed again, solubilized, and radioactivity bound to cells was measured. Competition for binding of the PDGF in conditioned media versus 125I-PDGF-AB was compared with that of the purified standards; mass of active PDGF in conditioned media was expressed as ng/ml.
Mitogenic and Growth Inhibition Assays--
PDGF, EGF
(Collaborative Research, Inc.; Bedford, MA), bFGF (Austral Biological;
San Ramon, CA) and TGF-1 (Austral Biological) were incubated for
72 h in the absence and presence of oxidized lipid emulsions as
described above. Mitogenic activity of PDGF, EGF, and bFGF was
determined by the incorporation of [3H]thymidine into
trichloroacetic acid-precipitable material by quiescent fibroblasts,
18-22 h following treatment with the mitogen (3). Growth inhibition by
TGF-
1 was evaluated by the incorporation of
[3H]thymidine into trichloroacetic acid-precipitable
material in mink lung epithelial cells (CCL64) as described previously
(19).
SDS-PAGE of PDGF-- Human recombinant PDGF-AB and a trace of 125I-PDGF-AB (18) were incubated for 72 h in the absence and presence of oxidized lipid emulsions as described above. Samples and prestained molecular weight markers (Bio-Rad) were subjected under nonreducing and reducing conditions to 15% SDS-PAGE analysis (21); after autoradiography, gel lanes were sectioned into 0.5 cm pieces and analyzed for radioactivity.
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RESULTS |
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Previous studies from our laboratory demonstrated that intact ox-LDL inhibited the production of active PDGF from bovine aortic EC. We decided to test the effect of ox-LDL and the lipid extract of ox-LDL on PDGF activity in the absence and presence of bovine aortic EC (Fig. 1). The lipid extract of ox-LDL inactivated PDGF in a dose-dependent manner to the same degree as intact ox-LDL (Fig. 1A) and inactivated PDGF in both the presence and absence of cells (Fig. 1, B and C). The lipid extract of native LDL had no effect on PDGF activity in either the presence or absence of cells (data not shown).
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To determine which component(s) of the ox-LDL extract was (were) responsible for the inactivation of PDGF, the lipids of n-LDL and ox-LDL were separated by reverse-phase HPLC (Fig. 2). Although several differences between the two chromatograms were apparent, the peak from the ox-LDL extract eluting at approximately 25 min (fraction 5) was notable because of its virtual absence in the lipid profile of n-LDL extract (Fig. 2). Fractions corresponding to the peaks numbered in Fig. 2B were collected and identified by thin layer chromatography (data not shown). Fractions 5 and 9 were shown to be cholesteryl esters. Saponification or cholesteryl esterase treatment of these two fractions produced free cholesterol and fatty acid as determined by both thin layer chromatography and HPLC, further supporting the identification of cholesteryl esters. TBARS analysis determined that fraction 5 was oxidized, but fraction 9 was not (data not shown). The effect of all of the HPLC fractions of the ox-LDL extract on PDGF activity after a 72-h incubation is shown in Fig. 3. Although some of the lipid fractions inactivated PDGF to a small extent, only fraction 5 (oxidized cholesteryl ester) completely inactivated PDGF. Unoxidized cholesteryl ester (fraction 9) had no effect on PDGF activity. Fraction 5 was applied to a normal phase CN HPLC column for further purification. The fraction resolved into three distinct peaks (Fig. 4). Several doses of ox-LDL lipid extract, the C18 peak (fraction 5; Fig. 2B), and the three peaks from the CN column (peaks 1, 2, and 3 from Fig. 4) were incubated with PDGF to determine the relative PDGF-inactivating potency at each stage of purification. PDGF was inactivated in a dose-dependent manner at all stages of purification (Fig. 5). It is apparent from Fig. 5 that the dose response curves for the C18 versus the CN 1, CN 2, and CN 3 peaks are not significantly different, and it can therefore be concluded that these four fractions are nearly equally potent at inactivating PDGF. By fast atom bombardment mass spectrometry and infrared spectroscopy, the first CN peak was identified as cholesteryl (13-hydroperoxy-9Z,11E)-octadecadienoate, the second as cholesteryl (13-hydroperoxy-9E,11E)-octadecadienoate, and the third as a mixture of cholesteryl (9-hydroperoxy-10E,12Z)-octadecadienoate and cholesteryl (9-hydroperoxy-10E,12E)-octadecadienoate (data not shown). For the remainder of this report, these hydroperoxy derivatives of cholesteryl octadecadienoates will be collectively referred to as cholesteryl HPODE.
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When inactivated PDGF was subjected to SDS-PAGE (Fig. 6; lane 3) under nonreducing conditions, a broad band at approximately 30 kDa was observed. The addition of cholesteryl HPODE emulsion to active PDGF directly before gel application did not affect migration of the protein (lane 4). Radioactivity in gel slices of inactivated PDGF (Fig. 6) yielded a broad peak of slightly higher molecular mass compared with active PDGF. Under reducing conditions, the results were the same: native PDGF appeared as a band of approximately 15 kDa, whereas inactivated PDGF appeared as a diffuse band of slightly higher molecular mass (data not shown).
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We tested the specificity of the oxidized lipid for inactivating PDGF.
Pre-incubating cholesteryl HPODE with PDGF (Fig.
7A) or bFGF (Fig.
7D) blocked the mitogenic activity in human foreskin fibroblasts but had no effect on the mitogenic activity of EGF (Fig.
7B). Pre-incubating the oxidized lipid with TGF-1
abolished the antiproliferative effect of TGF-
1 in a mink lung
epithelial cell assay (Fig. 7C).
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To determine whether other cholesteryl esters could inactivate PDGF after oxidation, cholesteryls stearate, oleate, linoleate, linolenate, arachidate, and arachidonate were subjected to oxidizing conditions as described under "Materials and Methods." Lipid oxidation of cholesteryls stearate, oleate, and arachidate as determined by TBARS was essentially zero; TBARS for cholesteryls linoleate, linolenate, and arachidonate were 2.6, 83.2, and 88.1 nmol malondialdehyde/mg of cholesteryl ester, respectively. Table I shows that, in the presence of cholesteryl esters that had become oxidized, PDGF was inactivated in a dose-dependent manner and that the extent of inactivation correlated with the degree of lipid peroxidation. PDGF activity was not affected when conditioned media was incubated with cholesteryl esters that had not become oxidized.
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We also examined whether nonlipid hydroperoxides could inactivate PDGF. Even though cumene hydroperoxide, t-butyl hydroperoxide, and hydrogen peroxide were incubated with PDGF for 72 h, these nonlipid hydroperoxides did not inactivate PDGF (Table II) even at concentrations greatly exceeding that required of cholesteryl HPODE (0.01-0.03 mM) to inactivate PDGF. When a noninactivating cholesteryl ester emulsion (cholesteryl oleate; experiment B in Table II) was added to the incubations, thereby providing a lipid surface for the nonlipid hydroperoxides, no effect on PDGF activity was observed. It therefore can be concluded that nonlipid hydroperoxides do not affect PDGF activity.
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DISCUSSION |
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Previous studies showed that oxidized LDL could completely inhibit
the production of active PDGF from bovine aortic EC (10). We now report
that the lipid moiety of ox-LDL causes inactivation of PDGF in a
cell-free system. The inactivating molecules of ox-LDL have been
identified as the 9- and 13-hydroperoxy derivatives of cholesteryl
linoleate. Additional experiments demonstrated that these cholesteryl
hydroperoxides were also capable of inactivating TGF-1 and bFGF, but
not EGF.
The mechanism by which cholesteryl HPODE inactivates PDGF and why the
oxidized lipid inactivates PDGF, bFGF, and TGF-1, but not EGF, are
not yet known. Inactivated PDGF, whether nonreduced or reduced, has a
slightly higher molecular weight than active PDGF, which would suggest
a lack of aggregation of the PDGF molecules, and more likely a covalent
attachment of the oxidized lipid to PDGF. Such a modification might
cause global conformational changes to the growth factor or an
obstruction such that PDGF no longer binds to its receptors. Free HPODE
has been reported previously to modify an essential methionine in
15-lipoxygenase and alter enzymatic activity (22), thus setting
precedence for HPODE-mediated protein alteration.
The biochemistry and pathology of protein oxidation have been recently reviewed (23). Inactivation of FGF, presumably via oxidative processes, has been reported by Engleka and Maciag (24) who demonstrated that acidic FGF, but not bFGF, was inactivated by copper-catalyzed oxidation and that the inactivation involved the formation of dimers from monomeric acidic FGF. Grant et al. (25) have recently shown that the growth of human endothelial cells was inhibited by increasing levels of O2 exposure and that this inhibition was mediated through the inactivation of acidic FGF, bFGF, and endothelial cell growth factor by O2 exposure.
That the inactivating molecules of ox-LDL were identified as isomers of cholesteryl HPODE is of physiological significance: cholesteryl linoleate comprises 50% of the total cholesteryl esters in normal human LDL (26), suggesting that the potential for a significant concentration of these oxidized cholesteryl esters may exist under pathological conditions. Although cholesteryl linolenate and cholesteryl arachidonate comprise less than 10% of the total cholesteryl esters in normal human plasma, our studies show that they are highly oxidizable and very potent inactivators of PDGF. Oxidized derivatives of cholesteryl esters, particularly cholesteryl linoleate, have been found in both human plasma (27) and human atheromas (28-33). In recent studies reported by Folcik et al. (12), cholesteryl HPODE specifically was identified in all surgical specimens of atherosclerotic lesions from carotid, aortic, femoral, and abdominal arteries analyzed, regardless of lesion progression suggesting that the formation of these primary oxidation products of cholesteryl linoleate may be a continual process in vivo. Aortic quantities of cholesteryl linoleate and its oxidation products have been shown to increase with the progression of vascular disease (28, 32, 34, 35). Our data suggest that it is possible that these molecules could inactivate certain growth factors under physiological conditions. In this regard, Shen et al. (36) have recently reported that expression of 15-lipoxygenase, the enzyme capable of oxidizing free or esterified linoleate to HPODE, protected transgenic rabbits from developing atherosclerosis. The results presented here, if operative in vivo, may participate in this protective process. It will be important to determine whether oxidized lipids can inactivate PDGF, as well as other growth factors and proteins in vivo, and what the impact of the inactivation is on the processes of aortic lesion development, inflammation, and wound healing.
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ACKNOWLEDGEMENTS |
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We thank Earl Poptic and Drs. Philip Howe, Julie Tebo, Guy Chisolm, and Thomas Hamilton (Cleveland Clinic) for helpful discussions during the course of these studies. We also acknowledge Vicki Chlanda for infrared analytical support. Human umbilical vein EC and human foreskin fibroblasts were prepared from tissue provided by the Perinatal Clinical Research Center (National Institutes of Health GCRC Award RR-00080), Cleveland Metrohealth Hospital.
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FOOTNOTES |
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* These studies were supported in part by grant HL-29582 from the National Institutes of Health (to P. E. D).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Schering-Plough Research Institute, Kenilworth,
NJ 07033.
§ Present address: Hoechst Marion Roussel, Kansas City, MO 63134.
¶ To whom correspondence and reprint requests should be addressed: Dept. of Cell Biology, Lerner Research Institute, NC10, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-5849; Fax: 216-444-9404; E-mail: dicorlp{at}cesmtp.ccf.org.
1
The abbreviations used are: PDGF,
platelet-derived growth factor; HPODE, hydroperoxyoctadecadienoate;
EGF, epidermal growth factor; FGF, fibroblast growth factor; bFGF,
basic FGF; TGF-1, transforming growth factor
1; LDL, low density
lipoprotein; n-LDL, native LDL; ox-LDL, oxidized LDL; EC, endothelial
cells; TBARS, thiobarbituric acid-reacting substances; HPLC, high
performance liquid chromatography; DMPC,
dimyristoylphosphatidylcholine; CL, cardiolipin; PAGE, polyacrylamide
gel electrophoresis.
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REFERENCES |
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