Vitamin C Protects Human Arterial Smooth Muscle Cells Against Atherogenic Lipoproteins

Effects of Antioxidant Vitamins C and E on Oxidized LDL–Induced Adaptive Increases in Cystine Transport and Glutathione

From the Vascular Biology Research Centre, Biomedical Sciences Division, King's College London, UK (R.C.M.S, H.S., J.D.P., G.E.M.); the Biochemistry Department, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan (H.S., S.B.); and the School of Animal and Microbial Sciences, University of Reading, Reading, UK (D.S.L.).

Correspondence to Prof Giovanni E. Mann, Vascular Biology Research Centre, Biomedical Sciences Division, King's College London, Campden Hill Road, London, W8 7AH, UK. E-mail giovanni.mann{at}kcl.ac.uk

	Abstract

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Abstract—Glutathione (GSH) plays a key role in cellular antioxidant defenses by scavenging reactive oxygen species and reducing lipid peroxides. Intracellular GSH levels are regulated by transport of its precursor L-cystine via system x_c^-, which can be induced by oxidant stress. As oxidatively modified low density lipoproteins (LDLs) contribute to impaired vascular reactivity and the formation of atherosclerotic lesions, we have examined the effects of oxidized LDL and the antioxidant vitamins C and E on the L-cystine–GSH pathway in human umbilical artery smooth muscle cells (HUASMCs). Oxidized LDL, but not native LDL, elevated intracellular GSH levels and L-cystine transport via system x_c^- in a time-dependent (up to 24 hours) and dose-dependent (10 to 100 µg · mL^-1) manner. These increases were dependent on protein synthesis and the extent of LDL oxidation, but the induction of L-cystine transport activity was independent of GSH synthesis. Pretreatment of HUASMCs for 24 hours with vitamin E (100 µmol/L) attenuated oxidized LDL–mediated increases in GSH, whereas pretreatment with vitamin C depressed basal levels and abolished oxidized LDL–induced increases in GSH and L-cystine transport in a time-dependent (3 to 24 hours) and dose-dependent (10 to 100 µmol/L) manner. Pretreatment of cells with dehydroascorbate had no effect on oxidized LDL–mediated increases in L-cystine transport and only marginally attenuated increases in GSH. Our findings provide the first evidence that vitamin C spares endogenous adaptive antioxidant responses in human vascular smooth muscle cells exposed to atherogenic oxidized LDL.

Key Words: amino acid transport • ascorbic acid • -tocopherol • atherosclerosis • oxidative stress

	Introduction

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Atherogenesis is associated with an elevated level of lipid peroxidation and increased formation of reactive oxygen species within the vascular wall, which could overwhelm cellular antioxidant defense mechanisms such as glutathione.¹ As depletion of intracellular reduced glutathione (L--glutamyl-L-cysteinylglycine; GSH) in cultured human umbilical vein endothelial cells² and murine macrophages³ reduces NO biosynthesis and activity of soluble guanylyl cyclase,⁴ alterations in GSH levels may contribute to the impaired vascular reactivity observed during atherogenesis.^{5 6} Reduced GSH also plays an important role in the protection of cells against oxidant stress by being complexed to electrophilic agents via glutathione transferase or by reducing H₂O₂ and lipid peroxides via glutathione peroxidase to form oxidized glutathione disulfide (GSSG), which can be reduced back to GSH by the NADPH-dependent glutathione reductase.⁷ GSH is also involved in the recycling of the dietary antioxidants vitamin E (-tocopherol), vitamin C (L-ascorbic acid), and ß-carotene to their reduced forms to maintain the reducing milieu within cells.^{8 9}

Synthesis of GSH is dependent on availability of intracellular L-cysteine, and cellular transport of L-cystine has been proposed to be rate-limiting for GSH synthesis.¹⁰ L-Cystine is incorporated into cells via the Na⁺-independent anionic amino acid transporter system x_c^-, previously characterized in human fibroblasts,¹¹ umbilical vein endothelial cells,¹² and murine macrophages,¹³ in which exposure to oxidative stress results in an initial decrease in intracellular GSH and subsequent adaptive increases in GSH and L-cystine transport.¹⁴ Within cells, L-cystine is rapidly reduced to the sulfhydryl form L-cysteine, which is used for GSH and protein synthesis, whereas extracellularly, L-cysteine is autoxidized to the disulfide L-cystine. To maintain intracellular L-cysteine levels, there is a continuous cycling of L-glutamate efflux and L-cystine influx via system x_c^-, driven not only by the cellular metabolism of L-cysteine but also by the extracellular redox state.¹³

Oxidatively modified LDLs have been implicated in the pathogenesis of atherosclerosis and impaired vascular reactivity,^{5 6} and exposure of macrophages to oxidized LDL (oxLDL) elicits an increase intracellular glutathione levels and rates of L-cystine transport.^{15 16 17} In addition to their important role in regulating the redox state of cells,¹⁸ vitamins C and E have been reported to reverse endothelial dysfunction in patients with coronary artery disease¹⁹ and cultured human aortic endothelial cells exposed to oxLDL.²⁰

We have previously shown that oxLDL induces expression of the antioxidant stress protein heme oxygenase-1 (HO-1) in smooth muscle cells²¹ and that vitamin C pretreatment attenuates HO-1 induction in cultured umbilical artery smooth muscle cells.²² As enhanced expression of HO-1 and other antioxidant enzymes in cultured vascular endothelial cells²³ and in rat brain²⁴ has been associated with a reduction in intracellular GSH levels, we have investigated (1) modulation of the cystine-GSH pathway by oxidatively modified LDL and (2) the cytoprotective effects of vitamins C and E in cultured human umbilical artery smooth muscle cells. Pretreatment of vascular smooth muscle cells with vitamin C significantly decreased basal and oxLDL–stimulated increases in L-cystine transport and GSH levels, while -tocopherol and dehydroascorbate were less effective antioxidants. Our findings demonstrate that adaptive antioxidant responses are induced in human vascular smooth muscle cells in response to oxidative stress and that dietary antioxidant vitamins may protect the vessel wall from oxidative injury.

	Methods

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Cell Culture
Human umbilical artery smooth muscle cells (HUASMCs) were cultured from explants in MCDB131 medium (Clonetics) supplemented with 10% (vol/vol) FCS (Sigma), 100 U · mL^-1 penicillin, and 100 µg · mL^-1 streptomycin at 37°C in a 5% CO₂, 95% air atmosphere. Cells were confirmed as smooth muscle by their typical "hill-and-valley" morphology and positive immunofluorescent staining for smooth muscle -actin. HUASMCs between passages 3 and 7 were used in all experiments. Cells were seeded into either 6- or 96-well microtiter plates at a density of 10⁵ cells mL^-1. Confluent cell monolayers were cultured for 6 to 48 hours in complete serum containing medium in the absence or presence of human LDLs (10 to 100 µg protein · mL^-1): native (nLDL), mildly oxidized (moxLDL), or highly oxidized (oxLDL) LDL. To examine whether preexposure of HUASMCs to LDL in FCS influences subsequent actions of human LDL, cells in some experiments were deprived of serum for 24 hours (in the presence of 0.5% BSA, Sigma) and then exposed to nLDL for 24 hours in the absence of FCS. Exposure to LDL was terminated by gently washing the cells twice with ice-cold Dulbecco's PBS.

The viability of cells after the LDL treatments was determined by measuring LDH release into the culture media²⁵ and mitochondrial dehydrogenase activity by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.²⁶ The effects of antioxidants were examined in cells that were pretreated for 24 hours in medium containing 10% FCS, supplemented with 0 to 100 µmol/L of either D--tocopherol (Sigma) (in 100% ethanol; final concentration, <0.1%), L-ascorbic acid (BDH), or dehydroascorbate (Aldrich) before exposure to LDL in the continued presence of the antioxidants.

LDL Preparation
LDLs (density=1.019 to 1.063 g · mL^-1) were isolated from blood from normal subjects by sequential ultracentrifugation in the presence of EDTA, followed by dialysis against the following (in µmol/L): NaCl 154, NaH₂PO₄ 16.7, Na₂HPO₄ 21.1, and EDTA 0.1, pH 7.4. LDL was oxidized by incubation at 100 µg protein · mL^-1 (in µmol/L): NaCl 137, KCl 2.68, Na₂HPO₄ 8.10, and KH₂PO₄ 1.47, pH 7.4, with CuSO₄ (net concentration 5 µmol/L above the EDTA present) at 37°C,²⁷ and the absorbance of LDL at 234 nm was monitored to measure the formation of conjugated dienes.²⁸ EDTA (1 mmol/L) was added to stop oxidation when absorbance had increased by 0.2 U to obtain moxLDL. OxLDL was formed by incubating LDL as above for 24 hours before adding 1 mmol/L EDTA. The density of the solution was then raised to 1.2 g · mL^-1 with KBr (in the presence of Chelex-100), and LDL was concentrated by ultracentrifugation and dialyzed against several changes of the following (in µmol/L): NaCl 154, NaH₂PO₄ 16.7, Na₂HPO₄ 21.1, and EDTA 0.1, pH 7.4. LDL was then sterilized by membrane filtration (0.2 µm), and protein, lipid hydroperoxide content,²⁹ and electrophoretic mobility were determined. The lipid hydroperoxide levels in nLDL, moxLDL, and highly oxLDL were 43±18, 64±20, and 107±35 nmol · µg protein)^-1 respectively, and the relative electrophoretic mobilities of moxLDL and oxLDL (compared with nLDL) were 1.19±0.1 and 3.5±0.9, respectively.

Amino Acid Transport Assays
Transport of L-cystine, L-arginine, L-serine, and L-leucine in HUASMCs was determined as described previously.¹¹ Confluent HUASMCs cultures in 96-well microtiter plates were washed with PBS containing the following (in µmol/L): CaCl₂ 1.0, MgC1₂·6H₂O 0.5, and D-glucose 5.5, at 37°C. Cells were then incubated at 37°C with the same transport buffer containing either 2 µCi · mL^-1 L-[¹⁴C]cystine (New England Nuclear), 1 µCi · mL^-1 L-[³H]arginine (New England Nuclear), 1 µCi · mL^-1 L-[³H]serine (Amersham), or 1 µCi · mL^-1 L-[³H]leucine (Amersham). In experiments where L-cystine transport was determined under low extracellular sodium, the sodium-containing compounds in the buffer were replaced by their choline equivalents. Transport was terminated by rapid removal of transport buffer from the cells followed by 3 washes with ice-cold PBS. After addition of 0.5 mol/L NaOH to each well, total cellular protein concentrations were determined by using the BCA protein assay (Pierce). Disintegrations per minute (dpm) in each sample were determined by liquid scintillation, and rates of amino acid transport were expressed as pmol · µg protein^-1 · min^-1.

Measurement of Intracellular Glutathione
Intracellular GSH and oxidized GSH (GSSG) levels were determined by high-performance liquid chromatography³⁰ (HPLC) and total GSH levels by spectrophotometry.³¹ Unless stated otherwise, GSH denotes total intracellular glutathione levels, since oxidized GSSG levels were not detectable in HUASMCs (data not shown). Confluent HUASMC cultures in 6-well plates were washed with ice-cold PBS. For HPLC analysis, cells were then lysed with ice-cold 6% perchloric acid containing 1 mmol/L bathophenanthrolinedisulfonic acid (Sigma). Supernatants were then mixed with 100 µmol/L -glutamyl-glutamate (Sigma, internal standard). Derivatives of 2,4-dinitrophenyl were separated on a 3-aminopropyl column (Jones Chromatography) by reversed-phase ion-exchange HPLC and detected by monitoring absorbance at 365 nm (Kontron Instruments HPLC system). For determination of total glutathione, cells were lysed with ice-cold 5% (vol/vol) trichloroacetic acid. The rate of catalytic reduction of 5,5'-dithiobis-2-nitrobenzoic acid by glutathione reductase (Sigma), utilizing the extracted total intracellular glutathione as the substrate, was measured by monitoring absorbance changes at 412 nm in glutathione standards and samples.

Statistical Analysis
The statistical variance from the mean was determined by using the normal distribution and represented by mean (SE; n=number of different cell cultures). When comparing 2 values, confidence limits were established by using unpaired Student's t test. ANOVA was used when comparing multiple groups. Statistical significance between data sets was accepted when P<0.05. Least-squares analyses of the kinetics of L-cystine transport were performed by using the computer programs Enzfitter and Ultra Fit (Elsevier, Biosoft).

	Results

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Cell Viability
Treatment of cultured HUASMCs with 10 to 100 µg protein · mL^-1 nLDL, moxLDL, or oxLDL for up to 48 hours did not result in significant cytotoxicity, as release of LDH and mitochondrial dehydrogenase activity were affected negligibly (data not shown). Subsequent experiments were performed with a maximal LDL concentration of 100 µg protein · mL^-1.

Effects of LDL on L-Cystine Transport and Intracellular Glutathione Levels
The time course of L-cystine transport (50 µmol/L) was measured in untreated confluent HUASMC monolayers over 10 minutes. First-order nonlinear least-squares regression analyses of total L-cystine transport data revealed that transport of L-cystine was linear for up to 2 minutes, reaching a plateau after 6 to 10 minutes. Incubation of HUASMCs in a nominally sodium-free buffer did not significantly alter L-cystine transport over the time course investigated (data not shown), suggesting that transport was mediated predominantly by a sodium-independent pathway. Subsequent studies of L-cystine transport were performed in sodium-containing buffer over 2-minute incubation periods.

OxLDL induced a concentration-dependent increase in cystine transport and intracellular glutathione levels, with significant increases detected in HUASMCs treated with 25 µg protein · mL^-1 for 24 hours (P<0.05, data not shown). To determine whether the low concentration of LDL present in FCS (9.9 µg protein · mL^-1)³² affects the sensitivity of HUASMCs to human LDL, cells were deprived of serum for 24 hours (+0.5% BSA) and then exposed to 100 µg protein · mL^-1 nLDL, moxLDL, or oxLDL for another 24 hours. Under these experimental conditions, nLDL had no effect on total intracellular GSH levels (16±1 versus 15.4±0.5 nmol · µg protein^-1, n=3), whereas moxLDL (16±1 versus 19.4±2 nmol · µg protein^-1, n=3, P<0.05) and oxLDL (15±2 versus 28±1.4 nmol · µg protein^-1, n=3, P<0.05) increased GSH levels significantly. These findings suggest that preexposure of HUASMCs to low concentrations of LDL in FCS does not alter their sensitivity to human LDL.

The specificity of L-cystine transport and its role in GSH synthesis were investigated by assessing the inhibitory effects of putative competitor amino acids (2.5 mmol/L) on transport of L-cystine (50 µmol/L, 2 minutes) and intracellular reduced GSH levels in confluent HUASMCs pretreated for 24 hours with 100 µg protein · mL^-1 nLDL or oxLDL. Amino acids were added to the L-cystine transport buffer or, in the case of the GSH determinations, added for 24 hours together with oxLDL to the cell monolayers. Both L-cystine transport and GSH levels were significantly increased after treatments with oxLDL, whereas nLDL had no effect (Figure 1). L-Glutamate and L-homocysteate (substrates for the amino acid transport system x_c^-), significantly reduced L-cystine transport and GSH levels in control and oxLDL-treated HUASMCs, whereas L-aspartate and L-arginine (substrates for amino acid transport systems x_G^- and y⁺, respectively) had no effect on oxLDL-stimulated increases in L-cystine transport or GSH (Figure 1). L-Serine, a substrate for Na⁺-dependent and Na⁺-independent neutral amino acid transport systems ASC and asc, respectively, had no effect on L-cystine transport or GSH levels in control cells and inhibited L-cystine transport by only 27% in oxLDL-treated HUASMCs (Figure 1). Inhibition of L-cystine transport by selected amino acids was not altered by removal of sodium from the incubation buffer (data not shown), suggesting that elevation of both GSH levels and L-cystine transport by oxLDL is mediated predominantly by influx of L-cystine via a sodium-independent, x_c^--like amino acid transporter. Basal transport rates for L-leucine (5.3±0.2 versus 5.2±0.1 pmol · µg protein^-1 · min^-1, n=4) and L-arginine (3.5±0.2 versus 3.2±0.2 pmol · µg protein^-1 · min^-1, n=4) were unaffected by any of the LDL treatments (24 hours, 100 µg protein · mL^-1), although L-serine influx (4.2±0.2 versus 3.4±0.2 pmol · µg protein^-1 · min^-1, n=4, P<0.05) was reduced in cells treated with oxLDL.

View larger version (26K): [in this window] [in a new window]   Figure 1. Effect of LDL and amino acid inhibitors on L-cystine transport and reduced glutathione levels. HUASMCs were treated with 100 µg protein · mL-1 nLDL or oxLDL for 24 hours and washed, and (A) L-cystine transport (50 µmol/L, 2 minutes) was measured in transport buffer in the absence or presence of 2.5 mmol/L L-glutamate (L-Glu), L-homocysteate (L-HCys), L-aspartate (L-Asp), L-arginine (L-Arg), or L-serine (L-Ser). B, GSH levels were measured by HPLC after treatment of cells for 24 hours with LDL in the absence or presence of the putative amino acid inhibitors. Values denote mean±SE (n=4 different cell cultures), *P<0.05 relative to control, +P<0.05 relative to oxLDL.

To determine the effects of LDL on the kinetics of L-cystine transport, influx was measured over a range of extracellular L-cystine concentrations (10 to 500 µmol/L, 2 minutes) in confluent HUASMCs pretreated for 24 hours with 100 µg protein · mL^-1 of nLDL, moxLDL, or oxLDL. As summarized in Table 1, kinetics of L-cystine transport were not altered by nLDL treatment, whereas moxLDL and oxLDL increased the V_max for L-cystine transport with no significant changes in K_m. L-Cystine transport (50 µmol/L, 2 minutes) and intracellular GSH levels were also measured in HUASMCs pretreated for 0 to 48 hours in the absence or presence of 100 µg protein · mL^-1 nLDL or oxLDL. As shown in Figure 2, oxLDL increased both L-cystine transport and GSH levels in a time-dependent manner. L-Cystine influx and GSH levels were elevated significantly above basal values after 8 hours' incubation with oxLDL, with maximal levels achieved by 24 hours and sustained for up to 48 hours. Over the same time period, L-cystine transport and GSH levels were unaffected by nLDL. GSSG levels in HUASMCs were not detectable under basal conditions or in cells treated with oxLDL for 24 to 48 hours.

View larger version (15K): [in this window] [in a new window]   Figure 2. Time-dependent increases in L-cystine transport and GSH levels in HUASMCs treated with LDLs. Cells were cultured in the absence () or presence of 100 µg protein · mL-1 nLDL () or oxLDL () for the specified times. A, L-Cystine transport (50 µmol/L, 2 minutes) and (B) GSH content were then determined. Details as in the legend to Figure 1. Values denote mean±SE (n=3 to 5 different cell cultures).

Effects of Inhibiting GSH Synthesis
To examine the effects of inhibiting glutathione synthesis on L-cystine transport and intracellular GSH levels, HUASMCs were pretreated with LDL or diethylmaleate (DEM), an electrophilic agent that at 100 µmol/L elevates GSH levels in endothelial cells,⁷ in the absence or presence of L-buthionine sulfoximine (BSO), an inhibitor of -glutamylcysteine synthetase. Transport of L-cystine (50 µmol/L, 2 minutes) and GSH levels were measured in HUASMCs pretreated for 24 hours with 100 µg protein · mL^-1 of nLDL, moxLDL, oxLDL, or DEM (100 µmol/L) in the absence or presence of 100 µmol/L BSO. Transport of L-cystine and intracellular GSH levels were increased significantly by moxLDL (30%), oxLDL (80%), and DEM (110%), whereas nLDL did not alter basal rates of L-cystine transport or GSH levels (Figure 3). Treatment of cells with BSO did not alter L-cystine transport but reduced intracellular GSH content to <20% of basal levels in both untreated and LDL-treated cells. Inhibition of protein synthesis with 1 µmol/L cycloheximide reduced L-cystine influx (control 292±14 versus cycloheximide 160±13 pmol · µg protein^-1 · min^-1), n=4, P<0.05) and GSH levels (control 16.4±1.1 versus cycloheximide 5.3±0.6 nmol · µg protein^-1, n=4, P<0.05) in untreated cells and abolished oxLDL– or DEM-mediated increases (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 3. Effects of inhibiting glutathione synthesis on L-cystine transport and GSH levels. HUASMCs were cultured with 100 µg protein · mL-1 nLDL, moxLDL, oxLDL, or 100 µmol/L DEM for 24 hours in the absence () or presence () of 100 µmol/L L-BSO. L-Cystine transport (A) (50 µmol/L, 2 minutes) and GSH content (B) were then determined. Values denote mean±SE (n=3 to 5 different cell cultures), *P<0.05, +P<0.01 relative to control in the absence of BSO.

Modulation of L-Cystine Transport and GSH by Ascorbic Acid and -Tocopherol
The time and concentration dependence of ascorbic acid–mediated decreases in both L-cystine transport and total intracellular GSH levels were investigated in untreated and oxLDL-treated HUASMCs. Cells were pretreated for 0 to 24 hours in the presence or absence of increasing concentrations (10 to 100 µmol/L) of L-ascorbic acid and then exposed for a further 24 hours to 100 µg protein · mL^-1 oxLDL in the continued absence or presence of vitamin C. Pretreatment significantly reduced basal and oxLDL-stimulated L-cystine transport and total intracellular GSH levels in a time-dependent (Figure 4) and concentration-dependent (Figure 5) manner. Reduced rates of L-cystine transport and total GSH levels were observed in untreated and oxLDL-treated cells at concentrations of L-ascorbic acid 50 µmol/L, while inhibitory effects of L-ascorbic acid pretreatment were observed even after 1 hour of pretreatment. Moreover, pretreatment of cells for 24 hours with L-ascorbic acid (100 µmol/L) reduced total intracellular GSH below basal values in untreated cells and abolished moxLDL-induced (data not shown) or oxLDL-induced (Figures 4B and 5B) increases in total GSH. Although pretreatment of cells with L-ascorbic acid did not prevent moxLDL-induced (data not shown) or oxLDL-induced (Figures 4A and 5A) increases in L-cystine transport, rates of L-cystine transport in LDL-challenged cells were reduced significantly after vitamin C pretreatment.

View larger version (14K): [in this window] [in a new window]   Figure 4. Time-dependent effects of L-ascorbic acid pretreatment on LDL-mediated changes in L-cystine transport and total glutathione content. HUASMCs were pretreated for the specified times with 100 µmol/L L-ascorbic acid before incubation for a further 24 hours in the continued presence of L-ascorbic acid and the absence () or presence () of 100 µg protein · mL-1 oxLDL. A, L-Cystine transport (50 µmol/L, 2 minutes) and (B) total intracellular glutathione levels were determined spectrophotometrically as described in Methods. Values denote mean±SE (n=3 different cell cultures). *P<0.05 relative to values in the absence of L-ascorbic acid.

View larger version (15K): [in this window] [in a new window]   Figure 5. Dose-dependent decrease in L-cystine transport and total glutathione levels in HUASMCs pretreated with L-ascorbic acid. Cells were pretreated with the specified concentration of L-ascorbic acid for 24 hours and then cultured for a further 24 hours in the continued presence of L-ascorbic acid and the absence () or presence () of 100 µg protein · mL-1 oxLDL. A, L-Cystine transport (50 µmol/L, 2 minutes) and (B) total intracellular glutathione levels were determined as described in Methods. Values denote mean±SE (n=3 to 5 different cell cultures). *P<0.05 relative to values in the absence of L-ascorbic acid.

The effects of antioxidants on LDL-mediated changes in L-cystine transport and total intracellular GSH were also investigated in HUASMCs pretreated for 24 hours in the absence or presence of 100 µmol/L -tocopherol or 100 µmol/L dehydroascorbate and then for a further 24 hours with 100 µg protein · mL^-1 nLDL, moxLDL, or oxLDL in the continued absence or presence of a given dietary antioxidant. Increases in total GSH levels induced by oxLDL, but not nLDL or moxLDL, were attenuated by -tocopherol, although increases in L-cystine transport were unaffected (Table 2). Treatment of HUASMCs with 100 µmol/L dehydroascorbate for up to 24 hours had negligible effects on basal or oxLDL-stimulated L-cystine transport and only marginally attenuated oxLDL-induced increases in total intracellular GSH (data not shown).

View this table: [in this window] [in a new window]   Table 2. Effect of -Tocopherol Pretreatment on L-Cystine Transport and Intracellular GSH Levels in HUASMCs Treated With LDL

	Discussion

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The current study demonstrates that moxLDL and oxLDL, but not nLDL, induce oxidative stress in HUASMCs, resulting in adaptive increases in intracellular GSH levels and L-cystine transport. Ascorbic acid afforded protection against moxLDL and oxLDL and markedly attenuated adaptive increases in cystine transport and GSH levels, whereas -tocopherol selectively reduced oxLDL-induced increases in GSH. Our results provide the first evidence that physiologically relevant concentrations of vitamin C spare endogenous adaptive antioxidant responses in human vascular smooth muscle cells exposed to atherogenic lipoproteins.

The kinetics and Na⁺ independence of L-cystine transport are characteristic of the anionic amino acid transport system x_c^-.^{9 10 11 12 13 33} The significant inhibition of L-cystine transport in HUASMCs by 50-fold excess concentrations of glutamate and homocysteate suggests that the membrane-bound enzyme -glutamyl transpeptidase, which transfers the glutamate moiety of GSH at the outer cell surface to extracellular amino acids forming the corresponding -glutamyl amino acid,^{34 35 36} may account for only a small fraction of L-cystine influx in HUASMCs. The specificity of cystine transport was largely unaffected by LDL, although L-serine inhibited L-cystine influx in HUASMCs exposed to oxLDL. We have no explanation for the inhibitory actions of serine on cystine transport in oxLDL-treated cells and cannot exclude the possibility that oxLDL induced expression of a dibasic amino acid transport system b^o,+ capable of transporting cystine.³⁷

Reduced glutathione was the predominant form of glutathione in HUASMCs, while (oxidized) GSSG was not detectable basally or after treatment of cells with LDLs or the GSH-conjugating electrophilic agent DEM. These findings are consistent with recent reports on the effects of oxLDL in macrophages¹⁵ and DEM or hyperoxia in vascular endothelial cells³⁸ and may reflect the rapid reduction of GSSG back to GSH via glutathione reductase or cellular efflux of GSSG.⁷ However, it is worth noting that in bovine aortic endothelial cells, exposure for 48 hours to cytotoxic doses of oxLDL depletes intracellular ATP and GSH levels after an initial increase in intracellular glutathione.³⁹ The toxicity of oxidized LDL to cells depleted of glutathione^{39 40} suggests that GSH and its dependent enzymes play an important role in the cellular antioxidant defense against aging and atherogenesis.¹ In the current study, oxLDL (100 µg protein · mL^-1) was not cytotoxic and induced larger increases in GSH and L-cystine transport than moxLDL (Figure 3). Increases in GSH in response to oxLDL treatment were dependent on L-cystine transport, since GSH levels were reduced to below basal values when cells were cotreated with high concentrations of the acidic amino acids L-glutamate or L-homocysteate (Figure 1). The apparent affinity (K_m) of L-cystine transport in HUASMCs was not altered significantly by LDL treatments, but the graded increase in the maximal rate of transport (V_max) in response to moxLDL and oxLDL (Table 1) suggests an increased activity or synthesis of transporter protein in oxidative stress.

Inhibition of protein synthesis depressed basal and oxLDL-stimulated rates of L-cystine transport and intracellular GSH in HUASMCs, suggesting a continual turnover of transporter protein basally and an elevated synthesis in oxLDL-treated cells. Decreased GSH synthesis and recycling of GSSG back to GSH, together with a reduced availability of L-cysteine, may account for the low GSH content in HUASMCs treated with cycloheximide. Inhibition of de novo GSH synthesis in HUASMCs by BSO significantly reduced GSH levels and inhibited the adaptive increases in GSH induced by oxLDL or DEM. In contrast, basal and stimulated rates of L-cystine transport in HUASMCs were not altered significantly after inhibition of GSH synthesis with BSO (Figure 3). These results are similar to findings in endothelial cells^{36 37} and suggest that induction of L-cystine transport activity is not exclusively dependent on the increased synthesis or depletion of GSH per se, but also on the redox state of cells.

GSH and vitamins C and E are central to cellular antioxidant defense systems and interact in a series of coupled reduction-oxidation reactions to efficiently quench oxidants normally produced within or outside cells.⁷ The GSH cycle is involved in the regeneration of ascorbate from dehydroascorbate and also helps to keep -tocopherol in its reduced form, either by a direct reaction or by a pathway involving ascorbic acid.⁸ In the current study, pretreatment of HUASMCs with ascorbic acid resulted in a time- and dose-dependent decrease in L-cystine transport and total intracellular GSH levels in both untreated and oxLDL-treated HUASMCs (Figures 4 and 5). Maximal effects were observed after pretreatment with 50 to 100 µmol/L ascorbic acid, concentrations well within normal physiological plasma levels⁴¹ and in the optimal range for L-ascorbic acid uptake into human endothelial cells.⁴² As vascular smooth muscle cells were exposed to a constant hyperoxic stress during culture, basal rates of L-cystine transport and GSH levels may have been elevated, as shown previously in endothelial cells and peritoneal macrophages.^{12 13}

Our observation that ascorbic acid reduced L-cystine transport and cellular GSH levels in untreated HUASMCs is consistent with its ability to spare GSH,⁸ as reported previously in human red blood cells⁴³ and rat astrocytes.⁴⁴ Cultured human umbilical vein endothelial cells have low intracellular levels of ascorbic acid, which increase rapidly after addition of physiological concentrations of the vitamin to the culture medium⁴⁵ and protect cells against the cytotoxic effects of H₂O₂. Ascorbic acid auto-oxidizes in solution to dehydroascorbate, which is more readily taken up by cells via glucose transporters.^{46 47 48} Within cells, dehydroascorbate is rapidly reduced back to ascorbate by processes that may involve the oxidation of glutathione⁸ or by glutathione-independent enzymatic mechanisms.⁴⁸ However, pretreatment of HUASMCs with dehydroascorbate did not significantly alter basal GSH levels and only marginally attenuated oxLDL-mediated increases in GSH (data not shown). Interestingly, when HUASMCs were pretreated with ascorbic acid and then exposed to moxLDL or oxLDL, GSH levels remained depressed, despite a continued elevation in L-cystine transport in response to oxLDL. The contribution of vitamin C to decreasing GSH synthesis may not necessarily be accompanied by a parallel inhibition of the induction of L-cystine transport, as observed during the inhibition of GSH synthesis by BSO (Figure 3). It remains to be elucidated whether vitamin C acts directly on GSH-synthesizing enzymes or indirectly by regulating the GSH requirement of cells.

Free radicals within oxLDL may have been preferentially quenched by ascorbic acid,⁴⁹ which would spare intracellular GSH. In the current study HUASMCs were preincubated with ascorbic acid for 24 hours, and cells were then exposed to oxLDL for another 24 hours. Under these conditions, the concentration of ascorbic acid in the culture medium may be low⁴² and unlikely to have quenched free radicals within oxLDL. The field remains controversial, with ascorbic acid and dehydroascorbate reported to increase or inhibit LDL oxidation.^{50 51 52} On the basis of the low transition-metal ion concentrations in our culture media and the observed attenuation of increased rates of L-cystine transport levels and intracellular glutathione, we suggest that ascorbic acid may be acting intracellularly to spare GSH from oxidation.^{8 9 53} Moreover, ascorbic acid increases LDL degradation in cultured vascular smooth muscle cells⁵⁴ and alters their production of extracellular matrix basally⁵⁵ and after treatment with oxLDL.⁵⁶ These actions may explain the antiatherogenic potential of ascorbic acid in reducing intimal thickening and LDL levels in the subendothelial space.

-Tocopherol has previously been reported to protect vascular endothelial^{20 39} and smooth muscle^{57 58} cells against oxidant stress induced by oxidized LDL. Treatment of HUASMCs with -tocopherol resulted in a significant attenuation of oxLDL-induced increases in total GSH, whereas transport of L-cystine was unaffected (Table 2). -Tocopherol stabilizes membranes against lipid peroxidation and may thus afford protection against oxidized LDL, either by scavenging lipid peroxide radicals within LDL⁵⁹ or by protecting lipids within the cell membrane from oxidation.⁶⁰ GSH is able to reduce tocopheroxyl radicals to regenerate -tocopherol in a GSH-consuming process, and this may contribute to the attenuated increase in GSH detected in cells treated with oxidatively modified LDL. As L-cystine transport was unaltered after -tocopherol treatment, this suggests that system x_c^- may be less sensitive to the small changes in oxidant stress in cells treated with this vitamin. The significant protective effects of ascorbic acid may result from its potential action as a more effective inhibitor of lipid peroxidation than vitamin E.⁶¹ Moreover, ascorbic acid can also preserve and enhance the antioxidant activity of vitamin E within cell membranes.⁶² In addition, the activation of the transcription factors NF-B and AP-1 in smooth muscle cells by oxidized LDL^{63 64} may be dependent on intracellular GSH levels, since their induction has been suggested to be influenced by the intracellular redox state.⁶⁵

Although plasma levels of atherogenic lipoproteins and antioxidant vitamins may vary considerably within a population, the sparing of GSH by ascorbic acid may have clinical implications for the protection of the vessel wall from oxidant injury in subjects at increased risk of cardiovascular disease.^{1 18 19} Our present findings support the current consensus that both GSH and antioxidant vitamins play a central role in the antioxidant defenses of vascular smooth muscle cells and may help to prevent oxidized LDL-mediated alterations in cell signaling, gene transcription, and vascular reactivity occurring during atherogenesis.

	Acknowledgments

 
This work was supported by the "Antioxidants in Food Program" from the Ministry of Agriculture Fisheries and Food, UK (AN413) (G.E.M., J.D.P.), the British Council, and the Ministry of Education, Science and Culture, Japan (G.E.M., S.B.). D.S.L. was the recipient of a Wellcome Trust Research Leave Fellowship. We thank Dr Luis Sobrevia and Dr Jacob Sweiry for their helpful discussions and Justin Richards for his skillful preparation of oxLDLs used in this study.

Received January 14, 1998; accepted June 29, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Halliwell B. The role of oxygen radicals in human disease, with particular reference to the vascular system. Haemostasis. 1993;23:118–126.

2. Ghigo D, Alessio P, Foco A, Bussolino F, Costamagna C, Heller R, Garbarino G, Pescarmona GP, Bosia A. Nitric oxide synthesis is impaired in glutathione depleted human umbilical vein endothelial cells. Am J Physiol. 1993;256:C728–C732.

3. Stuehr DJ, Kwon NS, Nathan CF. FAD and GSH participate in macrophage synthesis of nitric oxide. Biochem Biophys Res Commun. 1990;168:558–565.[Medline] [Order article via Infotrieve]

4. Marczin N, Ryan US, Catravas JD. Effects of oxidant stress on endothelium-derived relaxing factor induced and nitrovasodilator-induced cGMP accumulation in vascular cells in culture. Circ Res. 1992;70:326–340.[Abstract/Free Full Text]

5. Cox DA, Cohen ML. Effects of oxidized low-density lipoprotein on vascular contraction and relaxation: clinical and pharmacological implications in atherosclerosis. Pharmacol Rev. 1996;48:3–19.[Abstract]

6. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]

7. Deneke SM, Fanburg BL. Regulation of cellular glutathione. Am J Physiol. 1989;257:L163–L173.[Abstract/Free Full Text]

8. Winkler BS, Orselli SM, Rex TS. The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radic Biol Med. 1994;17:333–349.[Medline] [Order article via Infotrieve]

9. Meister A. Glutathione-ascorbic acid antioxidant system in animals. J Biol Chem. 1994;269:9397–9400.[Free Full Text]

10. Bannai S, Tateishi N. Role of membrane transport in metabolism and function of glutathione in mammals. J Membrane Biol. 1986;89:1–8.[Medline] [Order article via Infotrieve]

11. Bannai S. Exchange of cystine and glutamate across plasma membrane of human fibroblasts. J Biol Chem. 1986;261:2256–2263.[Abstract/Free Full Text]

12. Miura K, Ishii T, Sugita Y, Bannai S. Cystine uptake and glutathione level in endothelial cells exposed to oxidative stress. Am J Physiol. 1992;262:C50–C58.[Abstract/Free Full Text]

13. Watanabe H, Bannai S. Induction of cystine transport activity in mouse peritoneal macrophages. J Exp Med. 1987;165:628–640.[Abstract/Free Full Text]

14. Bannai S, Sato H, Ishii T, Sugita Y. Induction of cystine transport activity in human fibroblasts by oxygen. J Biol Chem. 1989;264:18480–18484.[Abstract/Free Full Text]

15. Sato H, Takenaka Y, Fujiwara K, Yamaguchi M, Abe K, Bannai S. Increase in cystine transport activity and glutathione level in mouse peritoneal macrophages exposed to oxidized low density lipoprotein. Biochem Biophys Res Commun. 1995;215:154–159.[Medline] [Order article via Infotrieve]

16. Darley-Usmar V, Severn A, Oleary VJ, Rogers M. Treatment of macrophages with oxidized low density lipoprotein increases their intracellular glutathione content. Biochem J. 1991;278:429–434.

17. Gotoh N, Graham A, Niki E, Darley-Usmar V. Inhibition of glutathione synthesis increases the toxicity of oxidized low density lipoprotein to human monocytes and macrophages. Biochem J. 1993;296:151–154.

18. Sies H, Stahl W, Sundquist AR. Antioxidant functions of vitamins—vitamin E and vitamin C, beta carotene, and other carotenoids. Ann N Y Acad Sci. 1992;669:7–20.[Medline] [Order article via Infotrieve]

19. Levine G, Frei B, Koulouris S, Gerhard M, Keaney J Jr, Vita J. Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation. 1996;93:1107–1113.[Abstract/Free Full Text]

20. Keaney J Jr, Guo Y, Cunningham D, Shwaery G, Xu A, Vita J. Vascular incorporation of -tocopherol prevents endothelial dysfunction due to oxidized LDL by inhibiting protein kinase C stimulation. J Clin Invest. 1996;98:386–394.[Medline] [Order article via Infotrieve]

21. Siow RCM, Ishii T, Sato H, Taketani, S, Leake DS, Sweiry JH, Pearson, JD, Bannai S, Mann GE. Induction of the antioxidant stress proteins heme oxygenase-1 and MSP23 by stress agents and oxidized LDL in cultured vascular smooth muscle cells. FEBS Lett. 1995;368:239–242.[Medline] [Order article via Infotrieve]

22. Siow RCM, Mann GE. Vitamin C attenuates induction of heme oxygenase-1 by oxidized LDL in human smooth muscle cells. J Physiol. 1998;506:37P–38P.

23. Jornot L, Junod A. Variable glutathione levels and expression of antioxidant enzymes in human endothelial cells. Am J Physiol. 1993;264:L482–L489.[Abstract/Free Full Text]

24. Ewing J, Maines M. Glutathione depletion induces heme oxygenase-1 (hsp32) messenger RNA and protein in rat-brain. J Neurochem. 1993;60:1512–1519.[Medline] [Order article via Infotrieve]

25. Amador E, Dorfman L, Wacker W. Serum lactic dehydrogenase. Clin Chem. 1963;9:391–395.[Abstract]

26. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival: modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986;89:271–277.[Medline] [Order article via Infotrieve]

27. Rice-Evans C, Leake D, Bruckdorfer R, Diplock A. Practical approaches to low density lipoprotein oxidation: whys, wherefores and pitfalls. Free Radic Res. 1996;25:285–311.[Medline] [Order article via Infotrieve]

28. Esterbauer H, Striegl G, Phul H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun. 1989;6:67–75.[Medline] [Order article via Infotrieve]

29. El-Saadani M, Esterbauer H, El-Syed M, Goher M, Nassar A, Jürgens G. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res. 1989;30:627–630.[Abstract]

30. Farris MW, Reed DJ. High-performance liquid chromatography of thiols and disulfides. Methods Enzymol. 1987;143:101–109.[Medline] [Order article via Infotrieve]

31. Tietze H. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969;27:502–522.[Medline] [Order article via Infotrieve]

32. Forte TM, Bell-Quint JJ, Cheng F. Lipoproteins of fetal and newborn calves and adult steer: a study of developmental changes. Lipids. 1981;16:240–245.[Medline] [Order article via Infotrieve]

33. Makowske M, Christensen H. Contrasts in transport systems for anionic amino acids in hepatocytes and a hepatoma cell line HTC. J Biol Chem. 1982;257:5663–5670.[Free Full Text]

34. Thompson G, Meister A. Interrelationships between the binding sites for amino acids, dipeptides. and -glutamyl donors in -glutamyl transpeptidase. J Biol Chem. 1977;39:357–368.

35. Sweiry J, Sastre J, Viña J, Elsasser H, Mann G. A role for -glutamyl transpeptidase and the amino acid transport system x_c^- in cystine transport by a human pancreatic duct cell line. J Physiol. 1995;485:167–177.[Abstract/Free Full Text]

36. Cotgreave I, Schuppe-Koistinen I. A role for -glutamyl transpeptidase in the transport of cystine into human endothelial cells: relationship to intracellular glutathione. Biochim Biophys Acta. 1994;1222:375–382.[Medline] [Order article via Infotrieve]

37. Van Winkle LJ, Campione AL, Gorman JM. Na⁺-independent transport of basic and zwitterionic amino acids in mouse blastocysts by a shared system and by processes which distinguish between these substrates. J Biol Chem. 1988;263:3150–3163.[Abstract/Free Full Text]

38. Deneke S, Baxter D, Phelps D, Fanburg B. Increase in endothelial cell glutathione and precursor amino acid uptake by diethyl maleate and hyperoxia. Am J Physiol. 1989;257:L265–L271.[Abstract/Free Full Text]

39. Schmitt A, Salvayre R, Delchambre J, Negre-Salvayre A. Prevention by -tocopherol, and rutin of glutathione and ATP depletion induced by oxidized LDL in cultured endothelial cells. Br J Pharmacol. 1995;116:1985–1990.[Medline] [Order article via Infotrieve]

40. Kuzuya M, Naito M, Funaki C, Hayashi T, Asai K, Kuzuya F. Protective role of intracellular glutathione against oxidized low density lipoprotein in cultured endothelial cells. Biochem Biophys Res Commun. 1989;163:1466–1472.[Medline] [Order article via Infotrieve]

41. Dhariwal K, Hartzell W, Levine M. Ascorbic acid and dehydroascorbic acid measurements in human plasma and serum. Am J Clin Nutr. 1991;54:712–716.[Abstract/Free Full Text]

42. Ek A, Strom K, Cotgreave I. The uptake of ascorbic acid into human umbilical vein endothelial cells and its effect on oxidant insult. Biochem Pharmacol. 1995;50:1339–1346.[Medline] [Order article via Infotrieve]

43. Johnston C, Meyer C, Srilakshmi J. Vitamin C elevates red blood cell glutathione in healthy adults. Am J Clin Nutr. 1993;58:103–105.[Abstract/Free Full Text]

44. O'Connor E, Devesa A, Garcia C, Puertes I, Pellin A, Vina J. Biosynthesis and maintenance of GSH in primary astrocyte cultures: role of L-cystine and ascorbate. Brain Res. 1995;680:157–163.[Medline] [Order article via Infotrieve]

45. Welch R, Wang Y, Crossman A Jr, Park J, Kirk K, Levine M. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J Biol Chem. 1995;270:12584–12592.[Abstract/Free Full Text]

46. Vera J, Rivas C, Velasquez F, Zhang R, Concha I, Golde D. Resolution of the facilitated transport of dehydroascorbic acid from its intracellular accumulation as ascorbic-acid. J Biol Chem. 1995;270:23706–23712.[Abstract/Free Full Text]

47. Rumsey S, Kwon O, Xu G, Burant C, Simpson I, Levine M. Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid. J Biol Chem. 1997;272:18982–18989.[Abstract/Free Full Text]

48. Guaiquil V, Farber C, Golde D, Vera J. Efficient transport and accumulation of vitamin C in HL-60 cells depleted of glutathione. J Biol Chem. 1997;272:9915–9921.[Abstract/Free Full Text]

49. Niki E. Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am J Clin Nutr. 1991;54:1119S–1124S.[Abstract/Free Full Text]

50. Retsky K, Freeman M, Frei B. Ascorbic acid oxidation product(s) protect human low density lipoprotein against atherogenic modification: anti-rather than prooxidant activity of vitamin C in the presence of transition metal ions. J Biol Chem. 1993;268:1304–1308.[Abstract/Free Full Text]

51. Stait E, Leake DS. Ascorbic acid can either increase or decrease low-density-lipoprotein modification. FEBS Lett. 1994;341:263–267.[Medline] [Order article via Infotrieve]

52. Stait E, Leake DS. The effects of ascorbate and dehydroascorbate on the oxidation of low-density-lipoprotein. Biochem J. 1996;320:373–381.

53. Tu B, Wallin A, Moldeus P, Cotgreave I. The cytoprotective roles of ascorbate and glutathione against nitrogen dioxide toxicity in human endothelial cells. Toxicology. 1995;98:125–136.[Medline] [Order article via Infotrieve]

54. Aulinskas T, Van der Westhuyzen D, Coetzee G. Ascorbate increases the number of low density lipoprotein receptors in cultured arterial smooth muscle cells. Atherosclerosis. 1983;47:159–171.[Medline] [Order article via Infotrieve]

55. Barone L, Faris B, Chipman S, Toselli P, Oakes B, Franzblau C. Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment. Biochim Biophys Acta. 1985;840:245–254.[Medline] [Order article via Infotrieve]

56. Jimi S, Saku K, Uesugi N, Sakata N, Takebayashi S. Oxidized low density lipoprotein stimulates collagen production in cultured arterial smooth muscle cells. Atherosclerosis. 1995;116:15–26.[Medline] [Order article via Infotrieve]

57. Stauble B, Boscoboinik D, Tasinato A, Azzi A. Modulation of activator protein-1 (AP-1) transcription factor and protein kinase C by hydrogen peroxide and D--tocopherol in vascular smooth muscle cells. Eur J Biochem. 1994;226:393–402.[Medline] [Order article via Infotrieve]

58. Guyton J, Lenz M, Mathews B, Hughes H, Karsan D, Selinger E, Smith C. Toxicity of oxidized low density lipoproteins for vascular smooth muscle cells and partial protection by antioxidants. Atherosclerosis. 1995;118:237–249.[Medline] [Order article via Infotrieve]

59. Esterbauer H, Wag G, Puhl H. Lipid peroxidation and its role in atherosclerosis. Br Med Bull. 1993;49:566–576.[Abstract/Free Full Text]

60. Niki, E, Yamamoto Y, Takahashi M, Komuro E. Miyama Y. Inhibition of oxidation of biomembranes by tocopherol. Ann N Y Acad Sci. 1989;570:23–31.[Medline] [Order article via Infotrieve]

61. Frei B, England L, Ames B. Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci U S A. 1989;86:6377–6381.[Abstract/Free Full Text]

62. May J, Qu Z, Morrow J. Interaction of ascorbate and -tocopherol in resealed human erythrocyte ghosts: transmembrane electron transfer and protection from lipid peroxidation. J Biol Chem. 1996;271:10577–10582.[Abstract/Free Full Text]

63. Ares MPS, Kallin B, Eriksson P, Nilsson J. Oxidized LDL induces transcription factor activator protein-1 but inhibits activation of nuclear factor-kappaB in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1995;15:1584–1590.[Abstract/Free Full Text]

64. Maziere C, Auclair M, Djavaheri-Mergny M, Packer L, Maziere JC. Oxidized low density lipoprotein induces activation of the transcription factor NF-kappaB in fibroblasts, endothelial and smooth muscle cells. Biochem Mol Biol Int. 1996;39:1201–1207.[Medline] [Order article via Infotrieve]

65. Sen C, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J. 1996;10:709–720.[Abstract]

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