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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:141-148.)
© 1997 American Heart Association, Inc.

Articles

Oxidized LDL Stimulates Mitogen-Activated Protein Kinases in Smooth Muscle Cells and Macrophages

Masatoshi Kusuhara; Alan Chait; Angelina Cader; Bradford C. Berk

the Cardiology Division (M.K., B.C.B.) and the Metabolism, Endocrinology, and Nutrition Division (A. Chait, A. Cader), Department of Medicine, University of Washington (Seattle).

Correspondence to Bradford C. Berk, Division of Cardiology, Box 357710, University of Washington, Seattle, WA 98195. E-mail bcberk@u.washington.edu.

	Abstract

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It has been proposed that oxidized LDL is more atherogenic than native LDL. However, the mechanisms by which native LDL and oxidized LDL alter function of cells in the vessel wall remain undefined. A signal transduction pathway that mediates many changes in cell function is the mitogen-activated protein (MAP) kinase cascade. We therefore examined the effect of native LDL and oxidized LDL on MAP kinase activity in cultured vascular smooth muscle cells (VSMC), endothelial cells, and macrophages by using an in-gel-kinase assay and anti-phosphotyrosine MAP kinase antibodies. Native LDL and LDL oxidized by the addition of Cu²⁺ (Cu²⁺-oxidized LDL) stimulated MAP kinase in a time- and dose-dependent manner in baboon and rat VSMC but not in bovine endothelial cells. Cu²⁺-oxidized LDL stimulated MAP kinase in human monocyte-derived macrophages, but the effect was much greater in cells cultured for 7 days compared with 1 day, suggesting dynamic regulation of the cellular response to oxidized LDL. In rat VSMC, the maximal MAP kinase response to Cu²⁺-oxidized LDL was significantly greater than the response to native LDL. Cu²⁺-oxidized LDL was more potent, with half-maximal activation at 15 µg/mL versus 30 µg/mL for native LDL. Stimulation of MAP kinase appeared to involve protein kinase C, since phorbol ester pretreatment for 24 hours blocked MAP kinase activation. Oxidation of LDL by other methods showed that activation of MAP kinase was not well correlated with lipid peroxides or aldehydes, suggesting that other components present in oxidized LDL were responsible. The active moiety appeared to be lipid based on extraction of oxidized LDL with organic solvents. These data indicate that LDL stimulates MAP kinase in VSMC, oxidation of LDL potentiates the effect, a lipid moiety is involved, and Cu²⁺-oxidized LDL activation of MAP kinase is cell-type specific. These findings suggest a role for MAP kinase in the pathways by which oxidized LDL contributes to altered cellular function associated with atherogenesis.

Key Words: atherosclerosis • signal transduction • smooth muscle

	Introduction

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A high concentration of circulating LDL is a major risk factor for atherosclerosis. Oxidized LDL appears to play a critical role in atherogenesis, in part because it is taken up by tissue macrophages to form foam cells that accumulate in the atherosclerotic lesion. Oxidized LDL also influences several processes involved in atherogenesis, including leukocyte attachment and chemotaxis, cytotoxicity, and expression of cytokines and growth factors.^{1 2 3} As determined by immunohistochemical and biochemical analyses, oxidized lipoproteins accumulate in atherosclerotic lesions in human and various hyperlipidemic animal models.⁴ In addition, studies with hyperlipidemic animal models have shown that antioxidants such as probucol, which inhibit LDL oxidation in vitro, reduce lesion development in animal models.^{5 6 7 8}

Proliferation of VSMC in the intima of arteries has also been viewed as a key mechanism in atherogenesis.⁹ Both native LDL and oxidized LDL have been shown to stimulate VSMC growth.^{10 11 12 13} The growth-promoting effects of native LDL and oxidized LDL may be indirect, via induction of growth factors (eg, PDGF) from VSMC themselves^{14 15 16} or from other cells in the vessel wall.^{13 17} However, several direct growth-related events have been described for native LDL, including calcium mobilization and activation of Na⁺/H⁺ exchange.^{18 19 20 21} Although oxidized LDL has been shown to induce autocrine growth mechanisms such as PDGF A chain expression by arterial smooth muscle cells,^{14 15} the effects of oxidized LDL on intracellular signal transduction related to VSMC growth have been studied to a limited extent.¹⁸

Phosphorylation of cellular proteins by kinases plays an important role in regulating cell growth, migration, differentiation, and responses to extracellular stimuli. Among these kinases, the family of enzymes known as the MAP kinases appears to be nearly universal. Although initially characterized by their rapid activation in response to mitogens that bind to receptors with intrinsic tyrosine kinase activity, MAP kinases are activated by many other stimuli, including cytokines, antigens that bind to T- and B-cell receptors, hormones that bind to G protein–coupled receptors, and physical forces such as fluid shear stress and stretch.²² Two studies have shown that unsaturated fatty acids such as arachidonic acid and linoleic acid can activate MAP kinase in VSMC.^{23 24} Thus, the MAP kinase pathway represents a common signaling mechanism for many stimuli that alter cell function.

In this study, we investigated whether there were differences in the activation of MAP kinase by native LDL compared with oxidized LDL. We report three findings: (1) activation of MAP kinase differs in VSMC, macrophages, monocytes, and endothelial cells, with more potent responses to oxidized LDL in VSMC and macrophages; (2) activation of MAP kinase by oxidized LDL appears to be PKC dependent; and (3) the active moiety in oxidized LDL is a lipid.

	Methods

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Cell Culture
Rat VSMC were isolated by collagenase-elastase digestion as described previously²⁵ and maintained in DMEM supplemented with 10% calf serum. Cells between passages 5 and 13 were growth arrested at 70% to 80% confluence by incubation in 0.1% calf serum/DMEM for 48 hours.

Baboon VSMC were isolated by the explant method and maintained in DMEM supplemented with 5% calf serum and 0.1% MITO⁺ serum extender (Collaborative Biomedical Products). Between passages 6 and 15, cells were growth arrested at 70% to 80% confluence by incubation for 72 hours in serum-free medium (DMEM/Ham's F-12 medium [1:1] containing 6 µg/mL insulin, 5 µg/mL transferrin, 1 mg/mL ovalbumin, 100 U/mL penicillin, and 100 µg/mL streptomycin).²⁶

Bovine aortic endothelial cells were isolated by collagenase digestion as described previously²⁷ and maintained in Medium-199 with Earle's salts supplemented with 10% fetal bovine serum. Cells between passages 6 and 10 were growth arrested by contact inhibition for 1 day after the cells reached 100% confluence; they were then incubated in Medium-199 supplemented with 0.5% fetal bovine serum for 24 hours.

Human monocytes and monocyte-derived macrophages were isolated by density gradient centrifugation by the method of Boyum,²⁸ as described previously,²⁹ and maintained in RPMI supplemented with 20% autologous serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine. Monocytes were used 24 hours after isolation and plating. Monocyte-derived macrophages were fed twice weekly and used 7 days after plating. Cells were preincubated in RPMI containing 0.5% autologous serum for 24 hours before the addition of LDL.

LDL Preparation
LDL (d=1.019 to 1.063 g/mL) was separated from normal human plasma by preparative ultracentrifugation as described previously³⁰ and stored in 1 mmol/L EDTA to prevent oxidation. Oxidative modification was undertaken by several methods. Extensive oxidation was achieved by incubation of dialyzed LDL (300 µg/mL) in PBS in the presence of 5 µmol/L copper sulfate for 18 hours at 37°C in air³¹ (Cu²⁺-oxidized LDL). The bound metal in the Cu²⁺-oxidized preparation was not removed. Oxidation by the thermally dependent free radical generator AAPH was achieved by incubation of LDL (500 µg/mL) with AAPH (2 mmol/L) for 18 hours at 37°C in the presence of Chelex-treated PBS.³² Oxidative modification of LDL by hypochlorite was achieved by incubation of 400 µL LDL (1.5 mg/mL) with 5 µL reagent-grade HOCl for 20 minutes on ice, after which the lipoprotein was dialyzed extensively against PBS to remove the HOCl.³³ LDL also was oxidatively modified by incubation with soybean lipoxygenase Sepharose 4B beads as described by Parhami et al.¹ In brief, 125 mg of the enzyme-coupled beads (100 U/µL soybean lipoxygenase) was incubated with 1 mg LDL in 2 mL PBS containing 2 µL linoleic acid for 48 hours at room temperature with very gentle agitation on a mechanical rocker. All incubations were terminated by the addition of butylated hydroxytoluene (25 µmol/L final concentration). The extent of modification was assessed by measurement of TBARS,³⁴ lipid peroxides,³⁵ conjugate dienes by absorbance at 234 nmol/L,³¹ electrophoretic mobility on agarose gels in barbital buffer at pH 8.6,²⁹ and TNBS reactivity (a measure of free amino groups³⁶ ).

We assayed for LPS in our LDL preparation because LPS has been reported to stimulate MAP kinase.³⁷ We could not detect any LPS, and furthermore LPS did not activate MAP kinase in rat VSMC (not shown). Addition of Cu²⁺ (1 to 100 µmol/L), linoleic acid (1 µmol/L), AAPH (2 mmol/L), or butylated hydroxytoluene (25 µmol/L) alone had no effect on MAP kinase activity of rat VSMC under our assay conditions.

Preparation of LDL Lipid Extracts
LDL (0.8 mL at 300 µg/mL) was added to a clean conical glass tube. Chloroform (1 mL) and methanol (2 mL) were added and vortexed for 5 minutes. Chloroform (1 mL) was added and vortexed for 1 minute, followed by water (1 mL) that was vortexed for 1 minute. The sample was then centrifuged for 10 minutes at 2000 rpm in a clinical centrifuge. The upper methanol/water phase extract was removed and placed in a clean tube, and the lower chloroform phase extract was removed and placed in a second tube. Both extracts were dried under N₂ and immediately dissolved in 40 µL DMSO (equivalent to 6 µg protein per microliter). Routinely, 12.5 µL extract was added to 3 mL medium, resulting in final concentrations of 0.4% DMSO and 25 µg protein per milliliter.

MAP Kinase Activity Assay
Growth-arrested cells were stimulated with native LDL and oxidized LDL for various time periods, then harvested for MAP kinase activity using an in-gel-kinase assay exactly as described previously.³⁸ After SDS-PAGE and the in-gel-kinase assay, the gel was dried and subjected to autoradiography. Autoradiographic signal intensity was quantified by densitometry in the linear range of film exposure using NIH Image 1.49. We have compared three methods of measuring MAP kinase activity: in-gel-kinase assay, Western blot band shift, and immune-complex kinase assay.³⁹ We demonstrated that there was an excellent correlation (R²=.95) among the three techniques. Immunodepletion of ERK1/2 using specific antibodies demonstrates that 95% of the autoradiographic density at 42 and 44 kD is due to these MAP kinase isozymes. In the present study, we also used the anti-phosphotyrosine MAP kinase antibody from New England Biolabs to measure MAP kinase activation. Cell extracts were prepared, and SDS-PAGE was performed as described above. After electrophoresis, proteins were transferred to nitrocellulose, and Western blot analysis was performed as previously described³⁹ using the anti-phosphotyrosine antibodies at 1:1000 dilutions. Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL, Amersham) and quantified by densitometry.

Cell Labeling and Immunoprecipitation
Growth-arrested cells were rinsed twice with DMEM lacking methionine and then labeled for 3 hours with 600 µCi [³⁵S]methionine per milliliter in DMEM minus methionine with 100 nmol/L angiotensin II, 25 µg/mL native LDL, or 25 µg/mL oxidized LDL.³⁷ After a brief wash with cold PBS, cells were lysed in denaturing buffer (50 mmol/L Tris-HCl, pH 7.4, 0.5% SDS, 70 mmol/L ß-mercaptoethanol), boiled for 10 minutes, and then diluted by adding 4 volumes of assay buffer (10 mmol/L Tris-HCl, pH 7.4, 1% sodium deoxycholate, 1% Nonidet P-40, and 150 mmol/L NaCl).⁴⁰ Cell lysates were incubated with anti–c-Fos antibody (Santa Cruz) for 2 hours at 4°C and then incubated with 20 µL protein A–Sepharose CL-4B (Sigma) for 1 hour on a roller system at 4°C. The immunocomplexes with protein A–Sepharose beads were washed five times with assay buffer with 0.1% SDS. The samples were separated by SDS-PAGE, and the gel was dried and subjected to autoradiography.

Data Analysis
Data are reported as mean±SEM. Results were analyzed by one-way ANOVA (control and LDL treatment). The statistics were computed with the program Systat. To compare group means after computation of the ANOVA, a contrast was set up to yield F and probability values. Post hoc analysis was performed using the Tukey-Kramer honestly significant difference test of pairwise mean comparisons. A value of P<.05 was considered significant.

	Results

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Cu²⁺-Oxidized LDL Stimulates MAP Kinase to a Greater Extent Than Native LDL
The ability of native LDL and Cu²⁺-oxidized LDL to stimulate MAP kinase activity was determined in growth-arrested rat VSMC. In response to 25 µg protein per milliliter of Cu²⁺-oxidized LDL, there was a rapid stimulation of both ERK 1 and ERK 2, which are p44 and p42 MAP kinase, respectively, with peak activity at 5 minutes and a return to near baseline by 60 minutes (Fig 1). In contrast to Cu²⁺-oxidized LDL, 25 µg protein per milliliter of native LDL caused a smaller increase in MAP kinase activity, although the time course was similar. Therefore, in all subsequent experiments cells were stimulated for 5 minutes by native LDL and Cu²⁺-oxidized LDL. To determine the relative magnitude of LDL activation of MAP kinase, we compared the results with activation by 200 nmol/L PMA and 10% serum (Fig 2). As shown previously,⁴¹ PMA and serum were equipotent, causing near stoichiometric activation of MAP kinase. Because the magnitude of increase by PMA was more reproducible, subsequent comparisons used PMA only. The increase in MAP kinase activity relative to 200 nmol/L PMA was 35.2±5.4% in response to native LDL and 54.3±9.1% in response to Cu²⁺-oxidized LDL (Fig 2A; n=6, P<.05). In comparison, MAP kinase activity in control unstimulated cells was 17.1±5.2%. To confirm the results of the in-gel-kinase assay, MAP kinase activation was measured by determining phosphotyrosine content using a specific anti-phosphotyrosine MAP kinase antibody. Analysis of three experiments showed that Cu²⁺-oxidized LDL stimulated MAP kinase to a significantly greater extent than native LDL (1.92±0.25-fold greater, P<.05, n=3).

View larger version (41K): [in this window] [in a new window]   Figure 1. Time course: Cu2+-oxidized LDL stimulates MAP kinase to a greater extent than native LDL. Growth-arrested rat VSMC were stimulated for 5 minutes with 200 nmol/L PMA, 25 µg protein per milliliter of native LDL, or 25 µg protein per milliliter of Cu2+-oxidized LDL for the indicated times. Cells were harvested in lysis buffer as described in "Materials and Methods," and in-gel-kinase assays were performed using myelin basic protein as substrate. Results are representative of three experiments.

View larger version (38K): [in this window] [in a new window]   Figure 2. Comparison to PMA: Cu2+-oxidized LDL stimulates MAP kinase to a greater extent than native LDL. A, In-gel-kinase assays from six experiments performed as in Fig 1 were quantified by autoradiographic density using a visible light scanner and NIH Image 1.49 software. Results were calculated as the % maximum based on the activation of MAP kinase by 200 nmol/L PMA at 5 minutes for each experiment (autoradiographic intensity, 1.0). Values are mean±SEM. All treatments differed significantly from control and from each other. n LDL indicates native LDL; ox LDL, Cu2+-oxidized LDL. B, In-gel-kinase assay demonstrating comparable activation of p42 and p44 MAP kinases in growth-arrested rat VSMC stimulated by 200 nmol/L PMA, 200 nmol/L angiotensin II (AngII), and 10% serum.

Both native LDL and Cu²⁺-oxidized LDL stimulated MAP kinase activity in a concentration-dependent manner (Fig 3). An increase in MAP kinase activity was first detected at 2.5 µg protein per milliliter of Cu²⁺-oxidized LDL and 10 µg protein per milliliter of native LDL. The maximal response to Cu²⁺-oxidized LDL occurred at 150 µg protein per milliliter (49.8% of PMA), with half-maximal response at 15 µg/mL. In contrast, the maximal response to native LDL occurred at 150 µg protein per milliliter (35.9% of PMA), with half-maximal response at 30 µg/mL.

View larger version (24K): [in this window] [in a new window]   Figure 3. Dose response: Cu2+-oxidized LDL stimulates MAP kinase to a greater extent than native LDL. Growth-arrested rat VSMC were stimulated with 200 nmol/L PMA for 5 minutes or with the indicated concentrations of native LDL and Cu2+-oxidized LDL for 5 minutes. Quantification of % maximum MAP kinase activity was as described in Fig 2.

To determine the extent to which oxidation of native LDL during preparation and storage may have contributed to MAP kinase activation, LDL was isolated rapidly (within 4 hours) by density gradient ultracentrifugation and molecular sieve chromatography. This freshly prepared LDL was immediately tested for its effect on MAP kinase activation. As shown in Fig 4, even very fresh preparations of LDL stimulated MAP kinase, but it did so to a lesser extent than LDL that had been isolated by slower preparative ultracentrifugal techniques and had been stored for up to 2 weeks. These findings are consistent with the notion that mild degrees of LDL oxidation, such as might occur with careful preparation and storage of LDL or which might occur in vivo, could account for the activation of MAP kinase observed with native LDL. In sum, progressive oxidation of LDL is associated with progressive increases in MAP kinase activation, suggesting that oxidation increases the degree to which LDL can activate upstream signal events responsible for MAP kinase activation.

View larger version (44K): [in this window] [in a new window]   Figure 4. MAP kinase is activated slightly by freshly prepared native LDL. Growth-arrested rat VSMC were stimulated for 5 minutes with 200 nmol/L PMA and the indicated concentrations of freshly prepared native LDL ("fresh LDL"), native LDL stored for 2 weeks ("old LDL"), and Cu2+-oxidized LDL. Cells were harvested in lysis buffer as described in "Materials and Methods," and in-gel-kinase assays were performed using myelin basic protein as substrate.

Activation of MAP Kinase by Oxidized LDL Is Dependent on Mechanism of Oxidation
Modification of LDL by different techniques yields LDL with different biological properties. As examples, HOCl-mediated modification preferentially modifies protein components of LDL, Cu²⁺ and soybean lipoxygenase preferentially oxidize lipid components, and AAPH preferentially oxidizes lipid components gently (under conditions used for this study).^{29 32 33} The effects of these modifications on several properties of LDL were determined (Table). As expected, Cu²⁺-oxidized LDL exhibited the highest levels of TBARS and electrophoretic mobility, whereas HOCl-modified LDL had very low levels of TBARS. The highest levels of conjugated dienes were present in soybean lipoxygenase–modified LDL. High levels of TNBS reactivity were observed in AAPH-modified and soybean lipoxygenase–modified LDL.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Modified LDL and Their Effect on MAP Kinase Activity

The effects of these modified LDL particles on MAP kinase activation then were determined (Table). Normalized to native LDL (MAP kinase activity, 1.0), Cu²⁺-oxidized LDL exhibited a 1.37-fold increase. In contrast, AAPH-modified LDL and soybean lipoxygenase–modified LDL were more effective at stimulating MAP kinase (1.69±0.42 and 1.73±0.44 of native LDL response, respectively). HOCl-modified LDL (1.17-fold greater than native LDL) was not different from native LDL. Comparison of the results in the Table indicates that there was not a good correlation between lipid oxidation and MAP kinase activation. Both TBARS and lipid peroxide content are indices of the extent of oxidation of the LDL. However, since neither correlated well with stimulation of MAP kinase activity, it is unlikely that either aldehydes, which are measured in the TBARS assay, or lipid peroxides themselves are responsible for the activation of MAP kinase. Rather, other components of LDL that increase during their oxidative modification are more likely to be involved. There was also no relationship between reduction in TNBS reactivity (a measure of protein modification) and stimulation of MAP kinase activity, since Cu²⁺-oxidized and HOCl-modified LDL caused similar reductions in TNBS reactivity but had very different effects on MAP kinase activation.

MAP Kinase Activating Factor in Cu²⁺-Oxidized LDL Is Lipid Soluble
Because changes in lipid peroxidation appeared to correlate with the ability to stimulate MAP kinase, we evaluated the effect of chloroform/methanol extracts from native LDL and Cu²⁺-oxidized LDL on MAP kinase activity.⁴² The MAP kinase–activating material was relatively enriched in the chloroform extract compared with the methanol extract, indicating that the active moiety was a lipid (Fig 5). We also examined the effect of 7-ketocholesterol, the major oxysterol present in Cu²⁺-oxidized LDL, and found that 7-ketocholesterol (up to 100 µg/mL) did not stimulate MAP kinase activity (data not shown). Another prominent component of LDL is lysophosphatidyl choline.⁴³ Addition of lysophosphatidyl choline at concentrations up to 1 µg/mL failed to stimulate MAP kinase in VSMC (data not shown), although there was a small effect at 10 µg/mL.

View larger version (49K): [in this window] [in a new window]   Figure 5. The MAP kinase activating factor in Cu2+-oxidized LDL is lipid soluble. Growth-arrested rat VSMC were stimulated for 5 minutes with 200 nmol/L PMA or with extracts from native LDL (n LDL) and Cu2+-oxidized LDL (ox LDL) prepared as described in "Materials and Methods." Extracts were solubilized in DMSO, and 12.5 µL extract was added to 3 mL medium, resulting in final concentrations of 0.4% DMSO and 25 µg protein per milliliter. MAP kinase activity was measured by in-gel-kinase assay as described in Fig 1. Control VMSC were also exposed to 0.4% DMSO. Results are representative of two experiments.

Cu²⁺-Oxidized LDL Stimulation of MAP Kinase Is Predominantly PKC Dependent
Activation of MAP kinase in VSMC occurs by both PKC-dependent and PKC-independent pathways.⁴⁴ To determine whether Cu²⁺-oxidized LDL stimulation of MAP kinase was PKC dependent, we downregulated PKC by pretreatment with 1 µmol/L phorbol 12,13-dibutyrate for 24 hours. This treatment inhibited PMA stimulation of MAP kinase by >90%, indicating effective downregulation of PKC (Fig 6). Phorbol 12,13-dibutyrate pretreatment also blocked MAP kinase activation by both native LDL and Cu²⁺-oxidized LDL by >90%, indicating that LDL stimulation of MAP kinase is predominantly PKC dependent (Fig 6).

View larger version (36K): [in this window] [in a new window]   Figure 6. Cu2+-oxidized LDL stimulation of MAP kinase is PKC dependent. Growth-arrested rat VSMC were treated for 24 hours with 1 µmol/L phorbol 12,13-dibutyrate (PDBu) or vehicle alone. The medium was removed, and cells were stimulated for 5 minutes with 200 nmol/L PMA, 25 µg protein per milliliter of native LDL (n LDL), or 25 µg protein per milliliter of Cu2+-oxidized LDL (ox LDL). MAP kinase activity was measured by in-gel-kinase assay as described in Fig 1. Results are representative of two experiments.

MAP Kinase Activation by Cu²⁺-Oxidized LDL Is Cell Specific
To determine whether there are significant differences in the ability of Cu²⁺-oxidized LDL to activate MAP kinase in various types of cells present in the vessel wall, we compared monocytes/macrophages, endothelial cells, and VSMC. Monocytes were isolated from normal human volunteers and were studied after being placed in culture for 1 day. These cells showed minimal activation of MAP kinase by native LDL or Cu²⁺-oxidized LDL despite responding well to PMA (Fig 7A). In contrast, human monocyte-derived macrophages cultured for 7 days exhibited significant MAP kinase activity in response to Cu²⁺-oxidized LDL (Fig 7B) but not to native LDL (Fig 7B). Neither Cu²⁺-oxidized LDL nor native LDL stimulated MAP kinase activity in cultured bovine aortic endothelial cells (Fig 7C).

View larger version (39K): [in this window] [in a new window]   Figure 7. Effect of Cu2+-oxidized LDL on MAP kinase activity in various vascular wall cells. Cells were isolated, cultured, and passaged as described in "Materials and Methods." Monocytes were cultured for 24 hours before treatment (A); monocyte-derived macrophages were cultured for 7 days, the last 24 hours in 0.5% autologous serum (B); endothelial cells were cultured until confluent (5 to 7 days), the last 24 hours in 0.5% serum (C); and VSMC were cultured until 80% confluent, the last 24 hours in 0.1% serum (D through F). Each cell was maintained in the indicated culture medium, and the cells were stimulated for 5 minutes with 200 nmol/L PMA, 25 µg protein per milliliter of native LDL (n LDL), or 25 µg protein per milliliter of Cu2+-oxidized LDL (ox LDL). MAP kinase activity was measured by in-gel-kinase assay as described in Fig 1. Results are representative of two experiments.

Because there are significant differences in the susceptibility of rodents and primates to the atherogenic effects of hyperlipidemia, we compared activation of MAP kinase by Cu²⁺-oxidized LDL in VSMC from rats and baboons. Rat VSMC showed MAP kinase activation in response to both native LDL and Cu²⁺-oxidized LDL, with Cu²⁺-oxidized LDL being more effective (Fig 7D). Similar results were observed with VSMC derived from baboon vessels (Fig 7E).

c-Fos Induction by Cu²⁺-Oxidized LDL
MAP kinase activation leads to an increase in transcription of many immediate early genes. MAP kinase phosphorylation of p62^TCL/Elk-1 induces formation of the serum response element ternary complex and c-fos mRNA expression.⁴⁵ Therefore, we examined induction of c-Fos protein by native and Cu²⁺-oxidized LDL. Neither native LDL nor Cu²⁺-oxidized LDL induced c-Fos protein expression in rat aortic VSMC (Fig 8). In contrast, angiotensin II, which has been reported to induce c-fos mRNA expression,⁴⁶ caused a significant increase in c-Fos protein expression.

View larger version (31K): [in this window] [in a new window]   Figure 8. c-Fos protein induction by native and Cu2+-oxidized LDL in VSMC. Rat aortic VSMC were stimulated with 100 nmol/L angiotensin II (Ang II), native LDL (n LDL), and Cu2+-oxidized LDL (ox LDL) for 3 hours in DMEM containing 600 µCi/mL [35S]methionine. Cells were lysed, and c-Fos protein was immunoprecipitated by anti–c-Fos antibody. The immunocomplexes were separated by SDS-PAGE, and the gel was dried and subjected to autoradiography.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major finding of the present study is that Cu²⁺-oxidized LDL is a more potent activator of MAP kinase in VSMC than native LDL. The effect of Cu²⁺-oxidized LDL exhibited specificity among cells present in the vessel wall, since MAP kinase activation was demonstrated in macrophages but not in endothelial cells and only minimally in monocytes. The active moiety in Cu²⁺-oxidized LDL was found to be a lipid. Because neither TBARS nor lipid peroxide content correlated well with stimulation of MAP kinase activity, it appears that components of LDL (other than aldehydes and lipid peroxides) that increase during oxidative modification are responsible. Experiments with freshly isolated LDL showed that there was activation to a lesser extent than with native LDL stored for 2 weeks, implying that mild degrees of LDL oxidation, such as might occur in vivo, account for the activation of MAP kinase observed with native LDL. These data provide new insights into mechanisms by which oxidized LDL modulates cell function and suggest that progressive oxidation of LDL is associated with progressive increases in the levels of moieties responsible for activation of MAP kinase.

The cell specificity for Cu²⁺-oxidized LDL stimulation of MAP kinase demonstrated in the present study may have important implications for the proatherogenic effects of oxidized LDL. Cu²⁺-oxidized LDL stimulation of MAP kinase activity was readily demonstrated in VSMC and macrophages but not in endothelial cells and to only a small extent in monocytes. Similar differences in cell responses have been postulated to be involved in atherogenesis. For example, Cu²⁺-oxidized LDL inhibits the generation of PDGF B chain⁴⁷ and tumor necrosis factor-⁴⁸ by macrophages, which would inhibit chemotaxis and promote their retention in plaque and formation of foam cells.⁴⁹ In contrast, monocytes respond to Cu²⁺-oxidized LDL by increased chemotaxis.⁴⁹ VSMC have been reported to exhibit both proliferation and cytotoxicity in response to Cu²⁺-oxidized LDL,^{50 51} which may be related to changes in expression of PDGF and its receptors.^{10 11 12} The recent finding that PDGF stimulation of MAP kinase in VSMC is required for proliferation but not migration⁵² suggests further mechanisms for cell-specific atherogenic effects of oxidized LDL.

The conclusion that the component of Cu²⁺-oxidized LDL responsible for activating MAP kinase is a lipid is based on extraction with chloroform/methanol. Mediation by a lipid also is supported by the results observed with LDL modified by AAPH and HOCl. AAPH oxidizes lipids to a greater extent than proteins, whereas HOCl selectively modifies proteins. We showed that MAP kinase activity was stimulated more potently by AAPH-modified LDL than by HOCl-modified LDL, which did not differ significantly from native LDL. A candidate mediator for MAP kinase activation is 7-ketocholesterol, which is the most prevalent oxidized cholesterol in Cu²⁺-oxidized LDL.⁵³ However, 7-ketocholesterol failed to stimulate MAP kinase at concentrations as high as 100 µg/mL. Another candidate mediator is lysophosphatidyl choline, which is present in LDL at levels up to 40 µg/mg LDL protein.⁴³ However, lysophosphatidyl choline did not stimulate MAP kinase in VSMC at concentrations up to 1 µg/mL, which corresponds to the amount contained in 25 µg protein per milliliter of Cu²⁺-oxidized LDL. Thus, the nature of the lipid moiety in oxidized LDL that stimulates MAP kinase remains to be established.

MAP kinase activation by LDL may be direct or indirect. Although cultured VSMC express the LDL receptor,⁵⁴ the rapidity of the MAP kinase response suggests that internalization by the LDL receptor is unlikely to mediate MAP kinase activation. Recently, several LDL and oxidized LDL receptors that may function as signal transduction receptors have been reported. For example, CD36 has two transmembrane domains and contains the carboxyl terminal sequence CXCX₅K, reported to interact with src-like kinases.⁵⁵ Another LDL receptor, SR-B1, also has two transmembrane domains and may act as a signaling receptor.⁵⁶ These reports suggest that activation of MAP kinase by oxidized LDL may be mediated directly by oxidized LDL binding to a cell-surface receptor that recognizes components of oxidized LDL and then initiates signal transduction. Tissue-specific regulation of these receptors would explain the selective activation of MAP kinase by oxidized LDL in VSMC and macrophages but not endothelial cells and monocytes. Future studies using MAP kinase activation as a physiological response for oxidized LDL binding may be useful in defining the nature of oxidized LDL receptors present in the vessel wall.

It also is possible that LDL activates MAP kinase via an indirect mechanism not requiring interaction with an LDL receptor or scavenger receptor. For example, active molecules may be released from lipoprotein particles bound to the cell surface. Hydrolysis by cell (or lipoprotein) phospholipases⁴³ may then generate lipid second messengers such as arachidonic acid or lactosylceramide. A recent report showed that components of oxidized LDL stimulated fibroblast growth factor synthesis and release of fibroblast growth factor from the extracellular matrix,¹⁶ suggesting another potential mechanism for cell activation.

Native and oxidized LDL have been reported to stimulate and inhibit VSMC growth. The MAP kinase pathway appears to integrate a wide variety of extracellular signals involved in regulation of cell growth. MAP kinase has been shown to be activated by tyrosine kinase–coupled receptors, G protein–coupled receptors, mechanical forces, and oxidative stress.²² Because MAP kinase phosphorylates p62^TCF, a component of the ternary complex that binds to the serum response element and regulates transcription, a likely candidate to mediate growth-promoting effects of oxidized LDL would be a serum response element–responsive gene such as c-fos.⁵⁷ Previous investigators have shown that LDL induced c-fos mRNA expression in human VSMC,¹⁸ and to a lesser extent in murine fibroblasts,⁵⁸ and that fatty acids such as linoleic and arachidonic acid increased both MAP kinase activity and c-fos mRNA.^{23 24} We could not detect c-Fos protein induction by native LDL or Cu²⁺-oxidized LDL in rat aortic VSMC, although angiotensin II, which stimulates MAP kinase in these cells,³⁸ induced c-Fos protein. These findings suggest that neither arachidonic acid nor linoleic acid are likely to mediate the effects of oxidized LDL. In addition, these results suggest that other signal events in addition to MAP kinase are required for c-Fos expression in these cells. Nevertheless, MAP kinase activation by LDL and oxidized LDL may have important biological effects in VSMC. Another substrate for MAP kinase is the serine/threonine protein kinase pp90^rsk,⁵⁹ which phosphorylates the S6 ribosomal protein and stimulates protein synthesis. LDL has been shown to stimulate S6 phosphorylation in VSMC.²¹ Finally, growth-factor stimulation of the Na⁺/H⁺ exchanger has been shown to be dependent on MAP kinase,⁶⁰ and the exchanger also is activated by LDL.²¹ Thus, several previously described effects of LDL on VSMC may be mediated by activation of MAP kinase. Further study is necessary to elucidate the exact mechanism of MAP kinase activation and intracellular signaling by LDL and oxidized LDL in VSMC.

	Selected Abbreviations and Acronyms

 

AAPH = 2,2,-azo-bis(2-amidinopropane)-2HCl DMEM = Dulbecco's modified Eagle medium ERK = extracellular signal regulated kinase LPS = lipopolysaccharide MAP = mitogen-activated protein PAGE = polyacrylamide gel electrophoresis PDGF = platelet-derived growth factor PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate SRE = serum response element TBARS = thiobarbituric acid–reactive substances VSMC = vascular smooth muscle cells

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health (HL49192, Dr Berk) and (HL30086, Dr Chait). Dr Berk is an Established Investigator of the American Heart Association. We thank Shari Wang for excellent technical assistance.

Received January 17, 1996; revision received May 15, 1996;

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