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(Circulation. 2001;104:1814.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Mildly Oxidized LDL Induces Activation of Platelet-Derived Growth Factor ß-Receptor Pathway

Isabelle Escargueil-Blanc, PhD; Robert Salvayre, MD PhD; Nathalie Vacaresse, PhD; Günther Jürgens, PhD; Benoit Darblade, MS; Jean-François Arnal, MD PhD; Sampath Parthasarathy, PhD; Anne Nègre-Salvayre, Dr Pharm, PhD

From INSERM U-466 and the Biochemistry Department, IFR-31, CHU Rangueil, Toulouse, France (I.E.-B., R.S., N.V., A.N.-S.); Institute of Medical Biochemistry, Karl-Franzens Universität, Graz, Austria (G.J.); INSERM U-397, IFR-31, CHU Rangueil, Toulouse, France (B.D., J.-F.A.); and the Department of Gynecology and Obstetrics, Emory University, Atlanta, Ga (S.P.).

Correspondence to Dr A. Negre-Salvayre, Biochimie and INSERM U-466-CHU Rangueil, Avenue Jean-Poulhès 31403-Toulouse Cedex-4 France. E-mail anesalv{at}rangueil.inserm.fr

	Abstract

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Background— Mildly oxidized LDL (moxLDL) is thought to play a role in atherogenesis. MoxLDL induces derivatization of cell proteins and triggers a variety of intracellular signaling. We aimed to investigate whether moxLDL-induced protein derivatization may influence the activity of platelet-derived growth factor receptor ß (PDGFRß), a tyrosine kinase receptor of major importance in vascular biology and atherogenesis.

Methods and Results— In cultured rabbit arterial smooth muscle cells, moxLDL induces activation of the PDGFRß signaling pathway, as shown by PDGFRß tyrosine phosphorylation on Western blot and coimmunoprecipitation of SH2-containing proteins. The cellular events involved in the moxLDL-induced PDGFRß activation can be summarized as follows. Oxidized lipids from moxLDL trigger two phases of PDGFRß activation involving two separate mechanisms, as shown by experiments on cultured cells (in situ) and on immunopurified PDGFRß (in vitro): (1) the first phase may be mediated by 4-hydroxynonenal, which induces PDGFRß adduct formation and subsequent PDGFRß activation (antioxidant-insensitive step); (2) the second phase involves ceramide-mediated generation of H₂O₂ (these steps being inhibited by tosylphenylalanylchloromethylketone, an inhibitor of ceramide formation, and by antioxidant BHT, exogenous catalase, or overexpressed human catalase). Because 4-hydroxynonenal–PDGFRß adducts are also detected in atherosclerotic aortas, it is suggested that this novel mechanism of moxLDL-induced PDGFRß activation may occur during atherogenesis.

Conclusions— MoxLDL acts as a local autoparacrine mediator in the vascular wall, and PDGFRß acts as a sensor for both oxidized lipids and oxidative stress. This constitutes a novel mechanism of PDGFRß activation in atherosclerotic areas.

Key Words: lipoproteins • platelet-derived factors • atherosclerosis

	Introduction

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Atherogenesis involves a complex sequence of events associating endothelial activation, transendothelial migration of mononuclear cells, lipid accumulation, and smooth muscle cell proliferation, leading finally to fibroatheroma plaque formation.¹ LDL is thought to become atherogenic after undergoing oxidative modifications,^2,3 characterized by oxidized lipid formation and structural alterations of apoB.^3,4

Oxidized LDL (oxLDL) is present in atherosclerotic areas³ and elicits changes in (1) lipoprotein metabolism (foam cells), (2) gene expression (adhesion molecules, cytokines, growth factors, coagulation proteins), (3) cell migration, motility, and contractility, (4) cell proliferation; (5) cell viability/death, and (6) local immune response.³

OxLDL triggers or alters various signaling pathways,⁵ namely calcium,⁶ trimeric G proteins and cAMP,⁷ phospholipase D,⁸ ceramide,⁹ PPAR,¹⁰ and epidermal growth factor receptor (EGFR activation being associated with 4-hydroxynonenal [4-HNE] derivatization).¹¹ We have examined whether this concept may be extended to platelet-derived growth factor receptor ß (PDGFRß), a tyrosine kinase receptor of importance in atherogenesis.¹²

PDGFR and PDGFRß belong to the tyrosine kinase receptor family. PDGFRß binds only PDGF B-chain, whereas PDGFR binds A and B PDGF isoforms.^13–15 PDGFR is involved in smooth muscle cell (SMC) hypertrophy, and PDGFRß is involved in the migration and proliferation of SMC,^13–15 a major event in atherosclerotic plaque formation.¹² PDGFRß activation triggers tyrosine phosphorylation, which creates binding sites for SH2-containing proteins, including Src, phospholipase C-1, phosphatidylinositol-3'-kinase, SHP2 protein tyrosine phosphatase, GTPase-activating protein of p21ras (rasGAP), Nck, Grb2, and Shc.^13–15

We found that oxidized lipids of mildly oxidized LDL (moxLDL) induce derivatization of PDGFRß (among other cell proteins¹⁶) and trigger activation of the PDGFRß signaling pathway. Two separate mechanisms are involved: a first antioxidant-independent early phase resulting possibly from PDGFRß derivatization by 4-HNE and a second antioxidant-sensitive phase mediated (at least in part) by ceramide and reactive oxygen species (ROS).

	Methods

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Cell Culture
Rabbit arterial SMC and SMCcat+ (transduced with catalase cDNA and overexpressing catalase by 350%)¹⁷ and human ECV-304 EC (from ATCC) and LDL receptor–negative fibroblasts (GM-486A from NIGMD) were grown in RPMI-1640 containing 10% FCS. Subconfluent cells were starved for 24 hours before experiments in 0.5% FCS.¹¹

Rabbits
New Zealand White male rabbits (2.5 kg) were randomly assigned to regular (n=3) and hypercholesterolemic (1% cholesterol for 8 weeks) (n=8) diets. After euthanasia (pentobarbital 50 mg/kg), thoracic aortas were removed and fatty streaks were stained with Sudan IV. Experiments were performed according to French Accreditation of Laboratory Animal Care.

LDL and Lipids
Human LDL was oxidized by UV-C irradiation (moxLDL)¹⁸ or by overnight incubation with ECV-304 EC (cell-moxLDL).⁹ MoxLDL contained 3.2 to 4.9 nmol TBARS and 6.5 to 8.7 nmol 4-HNE/mg apoB) and minor apoB alterations (TNBS-reactive amino groups were 89% to 95% of nonoxidized LDL; relative electrophoretic mobility ranged between 1.1 and 1.3).

[³H]4-HNE was prepared from [³H]4-HNE diethylacetal (a generous gift of J.P. Cravedi, Toulouse)¹⁹ and allowed to react with moxLDL for 1 hour. Cells (4 culture flasks of 80 cm², containing 1.5 mg cell protein) were preincubated with inhibitors, incubated with [³H]4-HNE-moxLDL (250 000 dpm/100 µg apoB/mL for 1 hour), washed, harvested, and used for PDGFRß immunoprecipitation and counting the PDGFRß-associated radioactivity.

Sphingomyelinase (Smase) treatment of LDL was performed with B cereus Smase (Sigma) (40 mU/mL, 1 hour, 37°C in 5 mmol/L MgCl₂, 0.1 mol/L tris/HCl, pH 7.5); Smase-treated LDL was separated from sphingomyelinase on a Sephadex G75 column (Pharmacia). Lipids were extracted in chloroform/methanol by the Folch procedure, as previously used.¹¹

Western Blot Analysis
Cells or tissue samples were lysed, PDGFRß was immunoprecipitated, and Western blots were performed as reported.¹¹

Determination of PDGFRß-Free Amino Groups
The free amino group content of PDGFRß was evaluated after immunoprecipitation by [³H]NSP ([³H]N-succinimidyl propionate (Amersham) labeling.¹¹

Intracellular ROS Determination
Intracellular ROS were determined fluorometrically with the use of dichlorodihydrofluorescein diacetate (H2DCFDA) or dihydrorhodamine (DHR) from Molecular Probes, incubated with cells 30 minutes before determination.²⁰

Determination of Enzyme Activities
Protein tyrosine phosphatase (PTPase) activity was determined according to methods described in Reference ²¹.²¹ Tyrosine kinase activity was evaluated as previously reported.¹¹

Statistical Analysis
Data are given as mean±SEM. Estimates of statistical significance were performed by ANOVA (Student-Newman-Keuls multiple comparison test).

Chemicals and Biological Reagents
Antiphosphotyrosine (4G10) and anti–PDGF-AB antibodies were from UBI, monoclonal anti-PDGFRß from Transduction Laboratories, human PDGF-BB, anti-SHP2, anti-Grb2, anti-ERK1/ERK2, and polyclonal anti-PDGFRß from Santa Cruz, anti-Src, anti–P-serine from Calbiochem, anti–P-threonine from Chemicon, antiactivated MAPK from Promega, anti–4-HNE-protein polyclonal (K5 to 4412) from G. Jürgens Laboratory,²² 4-HNE from Tebu-Biomol, H2DCFDA and DHR from Molecular Probes, cell culture reagents from Gibco, and other chemicals from Sigma.

	Results

Top Abstract Introduction Methods Results Discussion References

 
MoxLDL Induces PDGFRß Tyrosine Phosphorylation in Cultured SMC Independent of Any Autocrine Effect
MoxLDL (at mitogenic concentration) elicited tyrosine phosphorylation of PDGFRß, identified by immunoprecipitation and Western blot (Figure 1). MoxLDL-induced PDGFRß tyrosine phosphorylation is sustained for 5 hours (Figure 1A), is dose-dependent (maximal PDGFRß tyrosine phosphorylation at 1 hour and 5 hours being obtained with 100 to 150 and 50 to 75 µg apoB/mL, respectively) (Figure 1B), and is associated with the lipid fraction (Figure 1C).

View larger version (29K): [in this window] [in a new window]   Figure 1. MoxLDL induces tyrosine phosphorylation of PDGFRß in SMC independent of any autocrine effect. A, Time course of PDGFRß tyrosine phosphorylation induced by moxLDL (75 µg/mL) or PDGF (10 ng/mL, 20 minutes, as positive control). PDGFRß was immunoprecipitated and Western blots revealed by antiphosphotyrosine (anti-PY) and anti-PDGFRß antibodies. B, PDGFRß tyrosine phosphorylation induced by increasing concentration of oxidized LDL (same conditions as in A) at 1 or 5 hours (B and C, respectively). C, PDGFRß tyrosine phosphorylation in cells incubated for 1 hour with lipid extracts (prepared as in Reference 11, solubilized in DMSO and used at 0.15 nmol cholesterol/mL) of moxLDL or native LDL. D, Western blots of coimmunoprecipitates of PDGFRß and SH2-containing proteins from SMC incubated with UV-moxLDL or cell-moxLDL (75 µg/mL for 5 hours) (revelation by the indicated antibodies). E, Effect of neutralizing anti-PDGF antibody on PDGFRß tyrosine phosphorylation elicited by moxLDL (75 µg apoB/mL) or PDGF (10 ng/mL, 20 minutes), with (+) or without (-) neutralizing anti-PDGF antibody (25 µg/mL). F, Transfer of preconditioned medium. SMC were pulsed for 1, 2, or 4 hours with moxLDL (75 µg apoB/mL); after medium change, cells were "chased" in fresh basic (lipoprotein- and serum-free) medium for 1 hour. This preconditioned "chase" medium was transferred on and incubated with unstimulated "reporter" SMC for 1 hour. In control, SMC were incubated for 1 hour without (0) or with moxLDL.

Coimmunoprecipitation experiments showed that SH2-containing relay proteins, such as Grb-2 (24 kDa), Src (60 kDa), and SHP2 (69 kDa), associated to PDGFRß phosphotyrosines (Figure 1D), thus demonstrating that UV-moxLDL or cell-moxLDL triggers effective activation of the PDGFRß signaling pathway. We prefered to use UV-moxLDL because cell-moxLDL preparations may contain potentially interfering bioactive compounds (released from ECV-304 cells).

A role for autocrine PDGF secretion was excluded because neutralizing anti-PDGF antibody (inhibiting PDGF-induced PDGFRß autophosphorylation) did not block the moxLDL-induced PDGFRß tyrosine phosphorylation (Figure 1E). Other autocrine mediators were also excluded by transfer of preconditioned medium on reporter cells (Figure 1F).

Mechanisms Implicated in moxLDL-Induced PDGFRß Activation
Antioxidants Inhibit the Late Phase of moxLDL-Induced PDGFRß Activation
The antioxidants BHT (100 µmol/L) or catalase (1500 U/mL, permitting effective loading of SMC with catalase²³) were inactive in the first phase (1 hour) but strongly inhibited the second sustained phase (5 hours) of moxLDL-induced PDGFRß activation (Figure 2, A and B). In SMCcat+ overexpressing catalase (by 350%), this second phase was abolished, in contrast to parental SMC (Figure 2, C and D). Altogether, the two phases are apparently mediated by two separate mechanisms, the first one involving an antioxidant-insensitive mechanism and the second one involving H₂O₂ (catalase sensitive).

View larger version (35K): [in this window] [in a new window]   Figure 2. Effect of antioxidants on time course of moxLDL-induced PDGFRß activation. A and B, SMC were preincubated (for 30 minutes) with antioxidants BHT (100 µmol/L) (A) or catalase (1500 U/mL) (B), then incubated with moxLDL (75 µg apoB/mL) for the indicated time. C and D, SMCcat+ (overexpressing catalase) or (parental nontransfected) SMC were incubated with moxLDL (75 µg apoB/mL), without antioxidant. Western blots were performed and labeled as in Figure 1.

Oxidized Lipids and 4-HNE Induce Derivatization and Activation of PDGFRß
MoxLDL (but not natLDL or PDGF) induces 4-HNE derivatization of PDGFRß, as shown by evaluating [³H]NSP-reactive free amino groups of immunoprecipitated PDGFRß and 4-HNE–PDGFRß adducts (Figure 3, A and B) and by transfer of [³H]4-HNE to PDGFRß (and other cell proteins) in SMC incubated for 1 hour with [³H]4-HNE–labeled moxLDL. This transfer was reduced by chloroquine and concanamycin A (inhibitors of endolysosomal acidification and lipoprotein receptor recycling) and by cold (4°C) (Figure 3C). Chloroquine also inhibited moxLDL-induced PDGFRß activation (Figure 3D): In these experiments, cold and concanamycin A cannot be used because they induced per se PDGFRß autophosphorylation. Moreover, it may be noted that moxLDL elicited PDGFRß activation and MAPK activation in normal fibroblasts but no PDGFRß nor MAPK activation in LDL receptor–negative fibroblasts (data not shown). Altogether, these data suggest that a small part of moxLDL-associated 4-HNE derivatize PDGFRß through a chloroquine-sensitive process.

View larger version (46K): [in this window] [in a new window]   Figure 3. MoxLDL-induced derivatization and activation of PDGFRß is mimicked by 4-HNE in intact SMC in situ (A through G) and in vitro (H and I). A and B, Cells were incubated with native LDL or moxLDL (75 µg apoB/mL for 2 hours), 4-HNE (0.5 µmol/L for 2 hours), or PDGF (10 ng/mL for 20 minutes), then PDGFRß was immunoprecipitated. In A, free amino group content of PDGFRß was determined by [3H]NSP labeling (mean±SEM of 3 experiments; **P<0.01). In B, 4-HNE–PDGFRß adduct formation was visualized by Western blot. C, After preincubation in 0.5% FCS without or with chloroquine (10 µmol/L, 16 hours) or concanamycin A (10 nmol/L, 1 hour), cells were incubated with [3H]4-HNE–labeled moxLDL (100 µg apoB/mL, 250 000 dpm/100 µg apoB for 1 hour), then washed and harvested; PDGFRß was immunoprecipitated (from 1.5 mg cell proteins) and [3H]4-HNE radioactivity counted. D, Cells were incubated with or without moxLDL (75 µg apoB/mL for 5 hours) in the presence or absence of chloroquine (10 µmol/L); PDGFRß tyrosine phosphorylation was visualized on Western blot. E, Experiment under conditions of A, B, then coimmunoprecipitation of PDGFRß and SH2-containing proteins (revealed by the indicated antibodies). F, Time course of ROS formation monitored by fluorogenic probe H2DCFDA (fluorescent when oxidized) in SMC incubated with natLDL or moxLDL (75 µg apoB/mL) or 4-HNE (0.5 µmol/L). G, PDGFRß activation in SMC incubated with 4-HNE (0.5 µmol/L, 2 hours) or PDGF (10 ng/mL, 20 minutes) without or with BHT (100 µmol/L, preincubated for 30 minutes). H and I, In vitro experiments. Immunopurified PDGFRß, incubated in vitro for 30 minutes with PDGF (10 ng/mL), 4-HNE (1 nmol/L), or lipids extracted from native or moxLDL (equivalent to 75 µg apoB/mL), in phosphorylation buffer (Reference 11), was used for evaluating free amino groups of PDGFRß by [3H]NSP-labeling (H) and autophosphorylation by Western blot (I).

Consistently, incubation of SMC with 4-HNE induced 4-HNE–PDGFRß adduct formation, PDGFRß tyrosine phosphorylation, and phosphotyrosine-binding SH2-protein recruitment (Figure 3, A, B, and E). 4-HNE–induced PDGFRß activation was not associated with ROS generation and was antioxidant-insensitive (Figure 3, F and G), like the first phase of moxLDL-induced PDGFRß activation.

To investigate whether 4-HNE acts directly on PDGFRß, we designed in vitro experiments with the use of immunopurified PDGFRß. 4-HNE or moxLDL lipid extracts induced PDGFRß derivatization (decreased [³H]NSP-reactive amino group content) and PDGFRß tyrosine phosphorylation, whereas lipid extracts of native LDL were inactive (Figure 3, H and I). This suggests that 4-HNE and oxidized lipids mimic and may participate in the first (antioxidant-insensitive) phase of moxLDL-induced PDGFRß activation.

Second Antioxidant-Sensitive Phase of moxLDL-Induced PDGFRß Activation Is Mediated by Ceramide and H₂O₂
The second phase (3 to 5 hours) of moxLDL-induced PDGFRß activation is catalase-sensitive, thus possibly mediated by H₂O₂. This hypothesis was compatible with moxLDL-induced ROS generation (fluorescence of oxidized DHR) (Figure 4A) and the inhibitory effect of BHT and catalase (under loading conditions²³). H₂O₂ also triggered a strong PDGFRß autophosphorylation that was inhibited by BHT and catalase (Figure 4B).

View larger version (31K): [in this window] [in a new window]   Figure 4. Role of H2O2 and ceramide in moxLDL-induced PDGFRß activation of SMC. A, Time course of ROS formation in SMC preincubated with BHT (100 µmol/L), catalase (1500 U/mL), or TPCK (10 µmol/L) and incubated with moxLDL (75 µg apoB/mL) (under conditions of Figure 3, A and B). B, PDGFRß tyrosine phosphorylation induced by H2O2 (500 µmol/L) in the presence or absence of exogenous catalase (1500 U/mL) or BHT (100 µmol/L). C and D, SMC were incubated with Smase-treated LDL (75 µg apoB/mL, 5 hours) (C) or with C2-ceramide (10 µmol/L) or Smase (25 mU/mL) (D). PDGFRß tyrosine phosphorylation was visualized on Western blots (as in Figure 1). E, Effect of TPCK (10 µmol/L, 30 minutes of preincubation) on moxLDL-induced PDGFRß tyrosine phosphorylation (at 5 hours) (experimental conditions of Figure 5A).

View larger version (24K): [in this window] [in a new window]   Figure 5. Effect of moxLDL on PTPase activity and serine phosphorylation as mechanisms terminating PDGFRß activation. A and B, PTPase activity in moxLDL-treated (75 µg apoB/mL) SMC and comparative effects of moxLDL with or without BHT (100 µmol/L), H2O2 (500 µmol/L), and PDGF (10 ng/mL) with or without orthovanadate (OV, 10 µmol/L ). C, Serine and threonine phosphorylation of PDGFRß (immunoprecipitation and immunoblot) in SMC incubated with moxLDL (75 µg apoB/mL, 5 hours). D, Tyrosine kinase activity of PDGFRß in SMC incubated with moxLDL (75 µg apoB/mL) or PDGF (10 ng/mL) for time indicated (mean±SEM of 3 experiments).

Because moxLDL activates the sphingomyelin/ceramide pathway (ceramide peaking at 1 to 2 hours) in SMC⁹ and ceramide induces H₂O₂ generation,²⁴ we investigated the possible role of ceramide in moxLDL-induced H₂O₂ generation and PDGFRß activation. Theoretically, ceramide may be generated through sphingomyelin hydrolysis either in LDL (by secretory sphingomyelinase)²⁵ or in cell membranes.⁹ Smase-treated LDL did not trigger PDGFRß autophosphorylation (Figure 4C). In contrast, C2-ceramide and bacterial sphingomyelinase (generating ceramide at the plasma membrane⁹) trigger ROS generation (consistent with data from Reference ²⁴)²⁴ and PDGFRß autophosphorylation (Figure 4D). Interestingly, TPCK (tosylphenylalanylchloromethylketone), which blocks moxLDL-induced sphingomyelin/ceramide pathway activation,⁹ also inhibited moxLDL-induced ROS generation and PDGFRß autophosphorylation (Figure 4, A and E). Altogether, these data suggest that the second antioxidant-sensitive phase of moxLDL-induced PDGFRß activation may be mediated (at least in part) by cellular ceramide and subsequent H₂O₂ generation. A sustained PDGFRß tyrosine phosphorylation also may result from inhibition of regulatory mechanisms terminating PDGFR activation (namely PTPases and serine/threonine-phosphorylation of PDGFR).^26,27

In moxLDL-treated SMC, a transient activation of PTPases was followed by inhibition (during the second phase of moxLDL-induced PDGFR activation), which was reversed by BHT. PDGF elicited activation of PTPases, which was inhibited by orthovanadate (a PTPase inhibitor) and H₂O₂, in agreement with data from Reference ²⁶ (Figure 5, A and B).

Whereas PDGF triggered PDGFRß serine and threonine phosphorylation (serine phosphorylation being involved in terminating PDGFRß activation²⁷), incubation of SMC with moxLDL for 5 hours induced no PDGFRß serine phosphorylation (Figure 5C). Conversely, moxLDL incubated with cells for 1 hour and 5 hours did not downregulate PDGFRß kinase, in contrast to PDGF (Figure 5D). All these data suggest that the regulatory mechanisms terminating PDGFRß activation are inactive during the second phase of oxLDL-induced PDGFRß activation.

4-HNE Derivativatization Occurs in Rabbit Aortic Atherosclerosis
To check the relevance of the data reported above to in vivo atherosclerosis, 4-HNE–PDGFRß adduct formation was investigated in atherosclerotic rabbits. Cholesterol-fed rabbits exhibit hypercholesterolemia (25.10±3.46 g/L versus 1.04±0.10 g/L in normals) and large fatty streaks in thoracic aorta (Figure 6A). 4-HNE–PDGFRß adducts were clearly detected in atherosclerotic aorta (exhibiting extensive fatty streaks) but not in normal aorta (Figure 6B).

View larger version (56K): [in this window] [in a new window]   Figure 6. Derivatization by 4-HNE of PDGFRß from atherosclerotic aortas. A, Aortas from rabbits fed with normal (left) or lipid-rich (right) diets. Lipid-rich areas are stained in red. B, PDGFRß immunoprecipitated from aorta homogenates of control (Co) or atherosclerotic (Ath) rabbits was immunoblotted and revealed with anti-4-HNE.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The data suggest that (1) PDGFRß is a target of moxLDL and is activated through two novel mechanisms involving oxidized lipids and ceramide/H₂O₂ (Figure 7); and (2) moxLDL may be considered as a local autoparacrine mediator (generated in vascular wall and acting on neighboring cells).

View larger version (23K): [in this window] [in a new window]   Figure 7. Schema of mechanisms potentially involved in moxLDL-induced PDGFRß activation.

4-HNE may constitute a first mediator of moxLDL-induced PDGFRß activation because 4-HNE evokes in vitro derivatization and tyrosine phosphorylation of immunopurified PDGFRß (the fine molecular mechanism, eg, conformational changes, linking both events remains to be elucidated). In intact living cells, 4-HNE–induced PDGFRß derivatization and activation is antioxidant-insensitive and independent of any ROS production, like the first phase of moxLDL-induced PDGFRß activation. HNE exists in two forms in oxLDL: (1) the major part is 4-HNE covalently bound to amino groups of proteins or phospholipids; (2) a minor part of 4-HNE remains free, is poorly diffusible toward the water environment, and is probably dissolved in the phospholipid phase of moxLDL because of its hydrophobicity.^4,28 Experiments with [³H]4-HNE–labeled moxLDL show that a small part of moxLDL-associated [³H]4-HNE is transferred to PDGFRß (and other cell proteins). The mechanism of transfer of 4-HNE from moxLDL to PDGFRß is yet unknown. Diffusion of free 4-HNE from moxLDL toward PDGFRß through the culture medium is not probable because 4-HNE is hydrophobic (poorly diffusible)²⁸ and because [³H]4-HNE trace amounts are rapidly scavenged by thiols (glutathione, cysteine) of culture medium (data not shown). Therefore, in moxLDL and during its transfer to PDGFRß, free 4-HNE is probably prevented to react with thiols, hypothetically because of direct contact of moxLDL with PDGFRß or diffusion through a hydrophobic environment, such as membrane lipids. Finally, our data suggest that 4-HNE (and possibly other oxidized lipids) may play a role in the first antioxidant-insensitive phase of the moxLDL-induced PDGFRß activation.

The second antioxidant-sensitive phase (3 to 5 hours) of moxLDL-induced PDGFRß activation is mediated by ceramide and ROS. Cellular moxLDL-induced ceramide does not originate in LDL-associated ceramide but is generated through hydrolysis of cellular sphingomyelin (step blocked by TPCK).⁹ The reported data suggest that moxLDL-induced ceramide⁹ triggers ROS generation, which evokes PDGFRß activation: (1) TPCK, a nonantioxidant inhibitor of moxLDL-induced ceramide generation,⁹ blocks ROS formation and PDGFRß activation; (2) C2-ceramide and ceramide generated by bacterial sphingomyelinase treatment triggers ROS generation, consistent with data from Reference ²⁴, and PDGFRß activation; (3) BHT and catalase block both moxLDL-induced ROS formation and PDGFRß activation but not ceramide formation (data not shown); and (4) H₂O₂ triggers PDGFRß activation (which is blocked by catalase and BHT).

It may be noted that although the fluorogenic oxidant–sensitive probes detect various ROS,²⁰ H₂O₂ is very probably the prominent ROS because increased catalase level (induced by cell loading²³ or overexpression) inhibited both moxLDL-induced ROS generation and the second phase of PDGFRß activation. This is consistent with the requirement of H₂O₂ in PDGF signal transduction,²³ ROS-induced PDGFRß tyrosine-phosphorylation,²⁹ and ROS-induced SMC proliferation.³⁰

Moreover, moxLDL-induced inhibition of PTPases and serine phosphorylation of PDGFRß (two regulatory mechanisms involved in terminating PDGFRß activation) may be implicated in the second phase of moxLDL-induced PDGFRß activation. In agreement with data from Reference ²⁶, this PTPase inhibition may be mediated by H₂O₂ generated during the second phase of moxLDL-induced PDGFRß activation. BHT (which blocks H₂O₂ generation) also prevented PTPase inhibition and the subsequent second phase of PDGFRß activation.

The reported data suggest that in atherosclerotic areas, PDGFRß can be activated by moxLDL, in addition to PDGF. However, in contrast to growth factors, moxLDL is a nonspecific mediator that triggers a broad spectrum of cellular signals.^5–11 For instance, moxLDL-induced PDGFRß activation is similar to that of EGFR,¹² thus extending the concept that moxLDL activates various tyrosine kinase receptors and suggesting that besides their classic role of transducers of growth factors signaling, PDGFRß and EGFR may act as cellular sensors for oxidative stress and oxidized lipids.

From a pathophysiological point of view, the presence of 4-HNE–PDGFRß adducts in atherosclerotic areas is direct evidence that oxidized lipids (originating from moxLDL or generated inside the cell during oxidative stress) may react in vivo with PDGFRß, as in vitro and in cultured cells. Although tyrosine phosphorylation cannot be detected in aorta extracts (probably because of a rapid postmortem loss of phosphotyrosine in tissues), it may be speculated that oxidized lipids may interact with membrane tyrosine kinase receptors of vascular cells, thereby disturbing cellular responses such as migration, proliferation, and gene expression^13–15 and playing a role in intimal changes occurring during atherogenesis.

	Acknowledgments

 
This work was supported by grants from INSERM, Université Paul Sabatier-Toulouse III, Fondation pour la Recherche Médicale, European Community (Biomed-2 BMH4-CT983181) to U-466, the Austrian Research Council special research center "Biomembranes" project F (11) 710, and Jubiläumsfonds der Österreichischen Nationalbank project 6941 to Dr Jürgens. Dr Escargueil-Blanc was the recipient of a fellowship from SFA and VML. The authors wish to thank Dr J.P. Cravedi (INRA, Toulouse) for the generous gift of [³H]4-HNE diethylacetal.

Received March 5, 2001; revision received June 22, 2001; accepted July 5, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Stary HC. The sequence of cell and matrix changes in atherosclerotic lesions of coronary arteries in the first forty years of life. Eur Heart J. 1990; 11 (suppl E): 3–19.[Free Full Text]
   
2. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem. 1983; 52: 223–261.[Medline] [Order article via Infotrieve]
   
3. Steinberg D, Parthasarathy S, Carew T, et al. Beyond cholesterol: modification of LDL that increase its atherogenicity. N Engl J Med. 1989; 320: 915–924.[Medline] [Order article via Infotrieve]
   
4. Esterbauer H, Dieber-Rotheneder M, Waeg G, et al. Biochemical, structural and functional properties of oxidized LDL. Chem Res Toxicol. 1990; 3: 77–92.[Medline] [Order article via Infotrieve]
   
5. Hajjar DP, Haberland ME. Lipoprotein trafficking in vascular cells. J Biol Chem. 1997; 272: 22975–22978.[Free Full Text]
   
6. Nègre-Salvayre A, Fitoussi G, Reaud V, et al. A delayed and sustained rise of cyosolic calcium is elicited by oxidized LDL in cultured bovine endothelial cells. FEBS Lett. 1992; 299: 60–65.[Medline] [Order article via Infotrieve]
   
7. Parhami F, Fang ZT, Yang B, et al. Stimulation of Gs and inhibition of Gi proteins by minimally oxidized LDL. Arterioscler Thromb Vasc Biol. 1995; 15: 2019–2024.[Abstract/Free Full Text]
   
8. Natarajan V, Scribner WM, Hart CM, et al. Oxidized LDL-mediated activation of phospholipase D in smooth muscle cells. J Lipid Res. 1995; 36: 2005–2016.[Abstract]
   
9. Auge N, Escargueil-Blanc I, Lajoie-Mazenc I, et al. Potential role for ceramide in MAPK activation and proliferation of vascular smooth muscle cells induced by oxidized LDL. J Biol Chem. 1998; 273: 12893–12900.[Abstract/Free Full Text]
   
10. Nagy L, Tontonoz P, Alvarez JG, et al. Oxidized LDL regulates macrophage gene expression through ligand activation of PPARgamma. Cell. 1998; 93: 229–240.[Medline] [Order article via Infotrieve]
    
11. Suc I, Meilhac O, Lajoie-Mazenc I, et al. Activation of EGF-Receptor by oxidized LDL. FASEB J. 1998; 12: 665–671.[Abstract/Free Full Text]
    
12. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362: 801–809.[Medline] [Order article via Infotrieve]
    
13. Claesson-Welsh L. Platelet-derived growth factor signals. J Biol Chem. 1994; 268: 32023–32026.
    
14. Heldin CH, Ostman A, Ronnstrand L. Signal transduction via PDGF receptors. Biochim Biophys Acta. 1998; 1378: F79–113.[Medline] [Order article via Infotrieve]
    
15. Rosenkranz S, Kazlauskas A. Evidence for distinct signaling properties and biological responses induced by the PDGF receptor alpha and beta subtypes. Growth Factors. 1999; 16: 201–216.[Medline] [Order article via Infotrieve]
    
16. Vieira O, Escargueil-Blanc I, Jürgens G, et al. Oxidized LDL alter the activity of the ubiquitin-proteasome pathway: potential role in oxidized LDL-induced apoptosis. FASEB J. 2000; 14: 532–542.[Abstract/Free Full Text]
    
17. Santanam N, Auge N, Zhou M, et al. Overexpression of human catalase gene decreases oxidized lipid-induced cytotoxicity in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1999; 19: 1912–1917.[Abstract/Free Full Text]
    
18. Negre-Salvayre A, Lopez M, Levade T, et al. Ultraviolet-treated lipoproteins as a model for the study of the biological effects of lipid peroxides on cultured cells, II: uptake and cytotoxicity of ultraviolet-treated LDL. Biochim Biophys Acta. 1990; 1045: 224–232.[Medline] [Order article via Infotrieve]
    
19. Laurent A, Perdu-Durand E, Alary J, et al. Metabolism of 4-hydroxynonenal, a cytotoxic product of lipid peroxidation, in rat precision-cut liver slices. Toxicol Lett. 2000; 114: 203–214.[Medline] [Order article via Infotrieve]
    
20. Royall JA, Ischiropoulos H. Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys. 1993; 302: 348–355.[Medline] [Order article via Infotrieve]
    
21. Buscail L, Delesque N, Esteve JP, et al. Stimulation of tyrosine phosphatase and inhibition of cell proliferation by somatostatin analogues. Proc Natl Acad Sci U S A. 1994; 91: 2315–2319.[Abstract/Free Full Text]
    
22. Jürgens G, Chen Q, Esterbauer H, et al. Immunostaining of human autopsy aortas with antibodies to modified apolipoprotein B and apoprotein (a). Arterioscler Thromb. 1993; 13: 1689–1699.[Abstract/Free Full Text]
    
23. Sundaresan M, Yu ZX, Ferrans VJ, et al. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science. 1995; 270: 296–299.[Abstract/Free Full Text]
    
24. Verheij M, Bose R, Lin XH, et al. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature. 1996; 380: 75–79.[Medline] [Order article via Infotrieve]
    
25. Schissel SL, Jiang X, Tweedie-Hardman J, et al. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH: implications for atherosclerotic lesion development. J Biol Chem. 1998; 273: 2738–2746.[Abstract/Free Full Text]
    
26. Knebel A, Rahmsdorf HJ, Ullrich A, et al. Dephosphorylation of the receptor tyrosine kinases as target of regulation by radiation, oxidants and alkylating agents. EMBO J. 1996; 15: 5314–5325.[Medline] [Order article via Infotrieve]
    
27. Bioukar EB, Marricco NC, Zuo D, et al. Serine phosphorylation of the ligand-activated ß-PDGF receptor by casein kinase I-gamma2 inhibits the receptor autophosphorylating activity. J Biol Chem. 1999; 274: 21457–21463.[Abstract/Free Full Text]
    
28. Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med. 1991; 11: 81–128.[Medline] [Order article via Infotrieve]
    
29. Stiko A, Regnstrom J, Shah PK, et al. Active oxygen species and lysophosphatidylcholine are involved in oxidized LDL activation of smooth muscle cell DNA synthesis. Arterioscler Thromb Vasc Biol. 1996; 16: 194–200.[Abstract/Free Full Text]
    
30. Gonzalez-Rubio M, Voit S, Rodriguez-Puyol D, et al. Oxidative stress induces tyrosine-phosphorylation of PDGF alpha- and beta-receptors and pp60c-src in mesangial cells. Kidney Int. 1996; 50: 164–173.[Medline] [Order article via Infotrieve]
    

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