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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1583-1590.)
© 1997 American Heart Association, Inc.

Articles

Both Intracellular and Extracellular Vitamin C Inhibit Atherogenic Modification of LDL by Human Vascular Endothelial Cells

Antonio Martin; ; Balz Frei

From the Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Mass.

Correspondence to Balz Frei, PhD, Linus Pauling Institute, Oregon State University, 571 Wenger Hall, Corvallis, OR 97331. E-mail balz.frei.{at}orst.edu

	Abstract

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Abstract Oxidative modification of LDL by vascular cells has been proposed as a mechanism by which LDL becomes atherogenic. Antioxidants that can prevent LDL oxidation may therefore act as antiatherogens. We used endothelial cells (ECs) from human aortas (HAECs), human saphenous veins (HSECs), and bovine aortas (BAECs) to investigate the role of intracellular and extracellular vitamin C (ascorbate) in EC-mediated LDL modification. Incubation of LDL (0.1 mg protein per milliliter) with confluent HAECs in Ham's F-10 medium led to time-dependent modification of the lipoprotein. In contrast, incubation of LDL with HAECs in medium 199, which does not contain redox-active transition metal ions, did not lead to LDL modification. Both HAEC-mediated and cell-free LDL modifications in Ham's F-10 medium were strongly inhibited in a time- and dose-dependent manner by physiological concentrations of ascorbate. Confluent HAECs cultured under conventional conditions contained very little intracellular ascorbate (<0.5 nmol/mg protein) but could be loaded with up to 20 nmol ascorbate per milligram protein in a time- and concentration-dependent manner. Ascorbate-loaded HAECs exhibited a lower capacity to modify LDL than did non–ascorbate-loaded control cells. When LDL was incubated with HSECs instead of HAECs, similar time- and concentration-dependent inhibitory effects on LDL modification of intracellular and extracellular ascorbate were observed. In contrast to human ECs, BAECs did not take up vitamin C and therefore only coincubation but not preincubation with ascorbate inhibited BAEC-mediated LDL modification. Our data show that enrichment of human vascular ECs with vitamin C lowers their capacity to modify LDL. In addition, extracellular vitamin C strongly inhibits EC-mediated, metal ion–dependent atherogenic modification of LDL.

Key Words: modified LDL • metal ions • ascorbate • atherosclerosis • human endothelial cells

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The pathological hallmark of the early human atherosclerotic lesion is the appearance of macrophage-derived, lipid-laden foam cells in the subendothelial space of the vascular wall.¹ Macrophages have the capacity to accumulate modified LDL in contrast to native LDL.^{2 3} Free radical–mediated oxidation has been proposed as a mechanism by which LDL becomes modified in the vascular wall, leading to increased uptake by macrophages via the scavenger receptor pathways.^{2 3 4 5} OxLDL may promote atherosclerosis by additional mechanisms, such as chemoattraction of monocytes and SMCs, cytotoxicity, inhibition of endothelium-derived relaxing factor, and stimulation of SMC proliferation.^{3 6 7} OxLDL also may modulate thrombosis and fibrinolysis by stimulating EC synthesis of procoagulant tissue factor⁸ and plasminogen activator inhibitor-1.⁹ All major cell types present in the arterial wall, including ECs, SMCs, and macrophages, can modify LDL in vitro by a metal ion–dependent mechanism.^{5 10 11 12} Cell-mediated modification of LDL initially involves lipid peroxidation. The lipid hydroperoxides subsequently decompose to aldehydic products that can covalently modify apolipoprotein B-100, leading to an increased net negative surface charge of the LDL particle and altered receptor recognition.^{4 13}

As an important corollary of the "oxidative modification hypothesis of atherosclerosis," antioxidants that can inhibit LDL oxidation may slow the progression of atherosclerosis. Our previous studies have demonstrated that vitamin C (ascorbate) is the most effective water-soluble antioxidant in inhibiting lipid peroxidation in human plasma and also very effectively protects isolated LDL against oxidative modification.^{14 15 16 17} We and others have shown that physiological concentrations of ascorbate effectively inhibit LDL oxidation under many different oxidizing conditions, including vascular cells in culture (rabbit aortic ECs, human monocyte–derived macrophages, and murine macrophages), stimulated human neutrophils, myeloperoxidase-derived hypochlorous acid or tyrosyl radicals, aqueous peroxyl radicals, Cu²⁺, and heme iron.¹⁸

However, it is not known whether vitamin C can inhibit atherogenic modification of LDL by human vascular ECs. Furthermore, while much is known about the inhibition of LDL oxidation by LDL-associated antioxidants (eg, -tocopherol) or extracellular water-soluble antioxidants (eg, ascorbate),^{6 13 18} little is known about the effects of these antioxidants at the cellular level. Specifically, intracellular antioxidants may play an important role in limiting a cell's capacity to oxidize LDL. In support of this hypothesis, enrichment with ß-carotene or -tocopherol of cocultured human aortic ECs and SMCs, but not of LDL, has been shown to result in decreased LDL modification by these cells and consequent decreased monocyte adhesion.^{19 20} Similarly, Parthasarathy²¹ reported that loading of ECs or macrophages with a water-soluble derivative of probucol markedly decreased LDL oxidation by these cells.

Interestingly, nothing is known about the role of intracellular ascorbate in cell-mediated LDL modification. Effects of ascorbate at the cellular level may be of major importance, as cells can accumulate large amounts of ascorbate against a concentration gradient and physiological concentrations of ascorbate in cells are usually considerably higher (in the low millimoles per liter range) than those in extracellular fluids (30 to 150 µmol/L).^{15 22 23} Therefore, in the present study we investigated atherogenic modification of LDL by human vascular ECs and the role of both extracellular and intracellular vitamin C in this process.

	Methods

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EC Culture
HAECs were purchased from Clonetics Laboratories, and HSECs were obtained from surgical patients at the New England Medical Center, Boston, Mass. All procedures were approved by the institutional review committee. ECs were removed from saphenous veins under sterile conditions by using 0.1% type II collagenase (Sigma Chemical Co.). Both HAECs and HSECs were cultured in M199 (GIBCO Laboratories) under humidified 5% CO₂ in room air. The medium also contained 10% FBS, 5 µg/mL EC-derived growth factor prepared from bovine retina, 100 µg/mL EDTA, 100 U/mL penicillin, 100 U/mL streptomycin, and 1.25 µg/mL amphotericin B (Sigma). ECs were cultured in 1% gelatin–coated flasks in six-well plates (Corning Inc). The medium was replaced every 2 days until the cells attained confluence, at which time the FBS content of the medium was reduced to 5%. The cells were maintained for another 24 hours under these conditions before experiments were initiated. Cells of the fourth to seventh passage were used for experiments. Under phase-contrast microscopy, confluent ECs displayed a "cobblestone" morphology typical of quiescent ECs. Identity was further confirmed by the presence of von Willebrand factor antigen by immunofluorescent microscopy. Cell viability was determined by the trypan blue exclusion assay subsequent to treatment of the cells with 0.05% trypsin for 1 to 3 minutes or until 80% of the cells had become detached.

BAECs were obtained from segments of bovine aorta from freshly slaughtered animals at a local meat-processing plant. Aortas were immediately immersed in cold, sterile HBSS containing 100 U/mL penicillin, 100 U/mL streptomycin, and 1.25 µg/mL amphotericin B in a sterile stainless steel container and transported to the laboratory at 4°C. After being rinsed in cold HBSS, one end of the vessel was clamped, the vessel filled with 0.1% collagenase in HBSS and clamped at the other end. The vessels were placed in HBSS and incubated at 37°C for 2 hours, after which the contents were decanted into a sterile 50-mL centrifuge tube. The tube was centrifuged at 800 rpm at 4°C for 5 minutes, and the resulting pellet was resuspended in DMEM containing 15% FBS. Cells were seeded in T-75 flasks, grown in M199 containing 10% FBS, supplemented as described above until they reached confluence, and then split into six-well plates. Cells in culture medium were maintained at 37°C under humidified 5% CO₂ in room air, and the medium was changed every 2 days. When the cells reached confluence, the FBS content of the medium was reduced to 5% and the cells were maintained for another 24 hours under these conditions before experiments were initiated.

Isolation of LDL
LDL (1.019 to 1.063 g/mL) was isolated from the plasma of a healthy, normolipidemic (<160 mg LDL cholesterol per deciliter) male volunteer by single vertical discontinuous density gradient ultracentrifugation.^{16 24} Heparinized blood was collected and centrifuged for 15 minutes at 2000 rpm. The density of the plasma was adjusted to 1.21 g/mL by the addition of solid KBr (0.365 g/mL). Centrifuge tubes were loaded by layering 1.5 mL of density-adjusted plasma under 3.5 mL of 0.154 mol/L NaCl, placed in a near-vertical tube-90 rotor, and centrifuged in a Beckman L7-80M ultracentrifuge at 70 000 rpm and 7°C for 45 minutes with slow acceleration and deceleration modes. The yellow LDL band located in the upper middle portion of the tube was collected into a syringe by puncturing the tube. Residual KBr and plasma-derived small-molecular-weight contaminants present in the LDL preparation were removed by passage through a Sephadex G–25M PD-10 column (Pharmacia) equilibrated with medium. LDL protein was determined by a modified Lowry assay²⁵ using BSA as the standard. LDL isolated in this manner is free of detectable amounts of lipid hydroperoxides (<0.005 molecule of lipid hydroperoxides per LDL particle)²⁶ and contains <3% albumin contamination as assessed by both SDS–polyacrylamide gel electrophoresis and size-exclusion HPLC.²⁷ Isolated LDL was used for experiments immediately after preparation.

Experiments
Confluent HAECs, HSECs, or BAECs in six-well plates were washed twice with HBSS; serum-free Ham's F-10 medium, LDL (0.1 mg protein per milliliter), and the indicated concentrations of vitamin C were added. Vitamin C was added from a freshly prepared 5 mmol/L stock solution of L-ascorbic acid (Sigma) in Ham's F-10 medium. After incubation for various periods of time (0 to 48 hours), modification of LDL was assessed as described below.

To determine the cellular uptake and intracellular concentration of vitamin C, confluent ECs were incubated in M199 containing 6% FBS and various concentrations of vitamin C (added from a fresh 5 mmol/L stock solution in M199). After 0 to 24 hours of incubation, the supernatant was removed and the cell monolayer washed twice with HBSS. Cells were trypsinized (0.05% trypsin), collected into HBSS containing 1 mmol/L EDTA, and spun for 5 minutes at 800 rpm and 4°C. The supernatant was discarded and the cell pellet resuspended in 300 µL of HBSS containing 1 mmol/L EDTA. A 200-µL aliquot of this cell suspension was used for ascorbate determination as described below, and a 50-µL aliquot was used for protein determination.²⁵

To examine the role of intracellular ascorbate in EC-mediated LDL modification, cells were incubated for 16 hours in M199 containing 6% FBS and the indicated concentrations of vitamin C. The cells were then washed twice with HBSS, and serum-free Ham's F-10 medium containing LDL (0.1 mg protein per milliliter) was added. After incubation for various periods of time (0 to 48 hours), modification of LDL was assessed as described below.

Assessment of LDL Modification
Modification of LDL was assessed by agarose gel electrophoresis. LDL was electrophoresed at 100 V for 30 minutes on a 0.5% agarose gel (Lipo gels, Beckman) using 0.05 mol/L barbital buffer, pH 8.6. Gels were fixed, stained, and destained according to the manufacturer's instructions. Anodic electrophoretic mobility of modified LDL was expressed as mobility relative to native LDL, which was defined as 1.0. Increased anodic electrophoretic mobility of LDL has been previously shown to correlate with increased recognition of LDL by the scavenger receptors on macrophages, resulting in formation of lipid-laden foam cells characteristic of the early atherosclerotic fatty streak.^{4 13}

Quantification of Ascorbate
Ascorbate was measured by HPLC with electrochemical detection.^{15 16} In brief, the sample was mixed with an equal volume of cold 5% (wt/vol) metaphosphoric acid containing 1 mmol/L of the metal ion chelator diethylenetriaminepentaacetic acid (Sigma) and centrifuged to remove the precipitated proteins. An aliquot of the supernatant was chromatographed on an LC8 column (150x4.6 mm inner diameter, 3-µm particle size; Supelco) using 99% deionized water and 1% methanol containing 40 mmol/L sodium acetate and 1.5 mmol/L dodecyltriethylammonium phosphate (Q12 ion-pair cocktail, Regis) as the mobile phase. Ascorbate was detected at an applied potential of +0.6 V by an LC 4B amperometric electrochemical detector (Bioanalytical Systems). Ascorbate eluted as a single peak with a retention time of 5.5 minutes.

Quantification of Intracellular H₂O₂
Concentrations of H₂O₂ in HAECs were measured using 2',7'-diacetyldichlorofluorescin (Polysciences Inc.), a fluorescent probe that diffuses across cell membranes and is enzymatically deacetylated by cellular esterases to the more hydrophilic nonfluorescent reduced dye 2',7'-dichlorofluorescin.²⁸ Intracellular H₂O₂ rapidly oxidizes 2',7'-dichlorofluorescin to the fluorescent metabolite 2',7'-dichlorofluorescein. Cultured HAECs in 96-well plates were washed with HBSS and then with 100 µL of the assay solution containing 10 mmol/L 2',7'-diacetyldichlorofluorescin, 1 mmol/L HEPES in HBSS without phenol red was then added. After incubation for 30 minutes, the fluorescence intensity was measured in a Citofluor II (Per Septive Biosystems) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.²⁸

	Results

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Incubation of LDL (0.1 mg protein per milliliter) for up to 48 hours with confluent HAECs in Ham's F-10 medium led to time-dependent modification of LDL, as assessed by relative anodic electrophoretic mobility (Fig 1A). Because oxLDL can be cytotoxic to ECs,²⁹ the number of remaining cells attached to the flask and their viability were assessed. Only 71±4% (mean±SD, n=3 experiments) of HAECs remained attached after 24 hours of incubation with LDL, in contrast to 98±5% in three control incubations (P<.05 by two-tailed, paired Student's t test). The viability of the attached cells in the presence or absence of LDL was 84±6% and 94±4%, respectively (P<.05). These data indicate that the LDL modified by HAECs exerted cytotoxic effects.

View larger version (21K): [in this window] [in a new window]   Figure 1. Inhibition by ascorbate of LDL modification by HAECs (A) and in a cell-free system (B). In A, LDL (0.1 mg protein per milliliter) was incubated with confluent HAECs in Ham's F-10 medium for up to 48 hours either in the absence of ascorbate () or in the presence of 65 (), 130 (), or 260 µmol/L () ascorbate. At selected times, the anodic electrophoretic mobility of LDL was assessed on an agarose gel. LDL modification is expressed as electrophoretic mobility relative to native LDL. In B, LDL (0.1 mg protein per milliliter) was incubated in Ham's F-10 medium alone (ie, without cells) for up to 48 hours either in the absence of ascorbate (, solid line) or in the presence of 65 (), 130 (), or 260 µmol/L () ascorbate. HAEC-mediated LDL modification (, dashed line) is also shown in B for comparison with cell-free LDL modification (, solid line). Results shown are representative of three experiments.

LDL incubated in Ham's F-10 medium in the absence of cells (cell-free control) also became modified in a time-dependent manner, albeit at a lower rate than in the presence of cells (Fig 1B). In contrast, LDL incubated in M199 for up to 48 hours in the presence or absence of HAECs did not become modified to a detectable degree (relative electrophoretic mobility=1.0; data not shown). Ham's F-10 medium contains 1.7 to 3.9 µmol/L iron and 0.2 to 0.6 µmol/L copper,^{10 30} whereas M199 is free of detectable amounts of transition metal ions (0.2 µmol/L).¹⁰ These data, therefore, suggest that HAEC-mediated and cell-free LDL modifications are metal ion dependent, as has been reported previously for rabbit aortic ECs^{5 11} and cell-free systems.^{4 5 10}

HAEC-mediated (Fig 1A) and cell-free (Fig 1B) LDL modifications in Ham's F-10 medium were time- and dose-dependently inhibited by ascorbate, with no detectable LDL modification occurring in the presence of 260 µmol/L ascorbate for up to 24 and 48 hours of incubation, respectively, in the presence or absence of HAECs. Interestingly, 65 µmol/L ascorbate exerted an antioxidant effect during the initial 6 hours of incubation of LDL in the cell-free system but a slight pro-oxidant effect during longer incubation times (Fig 1B). In agreement with this observation, we found that after 24 hours LDL incubation in the cell-free system, 120 µmol/L ascorbate increased LDL modification by up to 60%, whereas 130 µmol/L ascorbate strongly inhibited or completely prevented LDL modification (Table 1).

View this table: [in this window] [in a new window]   Table 1. Pro-oxidant and Antioxidant Effects, Respectively, of Low and High Concentrations of Ascorbate on LDL Modification in a Cell-Free System

To investigate the role of intracellular vitamin C in LDL modification by HAECs, we first studied the uptake of the vitamin by these cells. As shown in Fig 2, the HAECs used in our experiments, which were primary cells of the fourth to seventh passage cultured under conventional conditions, contained very little endogenous ascorbate (0.4±0.1 nmol/mg protein, n=3 cell preparations). This nonphysiological state most likely resulted from the subphysiological concentration of ascorbate in M199, which we measured to be 0.3 to 3.0 µmol/L. Incubation of HAECs in M199 containing 6% FBS and 100 µmol/L ascorbate led to a time-dependent accumulation of ascorbate by the cells (Fig 2A). Intracellular ascorbate reached a maximal level of 9.5±1.6 nmol/mg protein (n=3) after 16 hours of incubation. Cellular accumulation of ascorbate was dependent on the extracellular concentration and was nonsaturable up to 250 µmol/L (Fig 2B).

View larger version (16K): [in this window] [in a new window]   Figure 2. Time- and dose-dependent uptake of ascorbate by HAECs. A, Confluent HAECs in M199 containing 6% FBS were incubated with 100 µmol/L ascorbate for up to 24 hours. At selected times cells were washed twice with HBSS, and intracellular ascorbate was determined as described in "Methods." B, HAECs were incubated with increasing concentrations of ascorbate, and after 16 hours of incubation intracellular ascorbate was determined. Results shown are the mean±SD of three experiments. Intracellular ascorbate levels are given in nmol/mg protein (p).

HAECs loaded with ascorbate, as described above, oxidized LDL (0.1 mg protein per milliliter) less extensively than did non–ascorbate-loaded control cells. As shown in Fig 3, when LDL was incubated for 18 hours in Ham's F-10 medium with HAECs loaded previously with 65, 130, or 260 µmol/L ascorbate, the degree of LDL modification was dose-dependently decreased compared with control LDL. These results were confirmed in a second experiment in which preincubation of HAECs with 0, 130, or 260 µmol/L ascorbate and subsequent incubation for 18 hours with LDL resulted in its relative electrophoretic mobility of 2.7, 2.0, and 1.8, respectively. To assess whether intracellular ascorbate was released from the cells during incubation with LDL, we collected media from the experiments shown in Fig 3 but did not detect ascorbate (<0.1 µmol/L) in any of these samples.

View larger version (76K): [in this window] [in a new window]   Figure 3. Inhibition by intracellular ascorbate of LDL modification by HAECs. Confluent HAECs (indicated by +) were loaded with ascorbate as described in "Methods" ("Preincubation" with 65, 130, or 260 µmol/L) or incubated in the absence of ascorbate (0 µmol/L), washed, and incubated with LDL (0.1 mg protein per milliliter) in Ham's F-10 medium for 18 hours. The anodic electrophoretic mobility of LDL was assessed on an agarose gel. For comparison, native LDL (nLDL) and LDL incubated for 18 hours without cells (indicated by -, last lane to the right) are also shown. The arrowhead on the right side of the gel indicates the origin of LDL loading.

To assess the possibility that the decreased capacity of ascorbate-loaded HAECs to modify LDL is due to decreased production of reactive oxygen species by these cells, we measured intracellular concentrations of H₂O₂. As shown in Fig 4, H₂O₂ levels were significantly lower (P<.001) in HAECs loaded with 120 µmol/L ascorbate compared with non–ascorbate-loaded control cells.

View larger version (40K): [in this window] [in a new window]   Figure 4. Decrease in intracellular H2O2 concentration in HAECs by ascorbate. Confluent HAECs were preincubated without (control) or with ascorbate (120 µmol/L), and intracellular H2O2 was measured as described in "Methods." Results shown are the mean±SD of three independent experiments. *P<.001 vs control by two-tailed, paired Student's t test.

In addition to HAECs, we used HSECs and BAECs to investigate the role of intracellular and extracellular vitamin C in EC-mediated LDL modification. LDL incubated with confluent HSECs in Ham's F-10 medium became modified in a time-dependent manner (Fig 5, A and B). In contrast, LDL incubated with HSECs in M199 for 24 hours did not become modified to a measurable degree (Fig 5A); however, after 48 hours of incubation, a slight increase in the electrophoretic mobility was observed (Fig 5B). Coincubation with ascorbate strongly inhibited HSEC-mediated LDL modi- fication in Ham's F-10 medium in a time- and concentration-dependent manner (Fig 5, A and B). Although the inhibitory effects of added ascorbate appear somewhat greater than those observed in the presence of HAECs (Fig 1A), it should be noted that LDL was modified at a lower rate in the experiments using HSECs. Thus, the relative electrophoretic mobility of LDL after 24 hours of incubation with HAECs or HSECs was 5.5 and 3.3, respectively (cf Figs 1A and 5A). Correspondingly, intracellular ascorbate also appeared more effective at inhibiting LDL modification by HSECs than HAECs. LDL modification by HSECs preincubated with 60, 180, or 360 µmol/L ascorbate was decreased dose-dependently compared with LDL modification by non–ascorbate-loaded control cells (Fig 5A). After 48 hours of incubation, these inhibitory effects of intracellular vitamin C were no longer observed (Fig 5B).

View larger version (94K): [in this window] [in a new window]   Figure 5. Inhibition by extracellular and intracellular ascorbate of LDL modification by HSECs. LDL (0.1 mg protein per milliliter) was incubated for 24 (A) or 48 (B) hours with confluent HSECs (indicated by +) in Ham's F-10 medium without ascorbate (0 µmol/L, lane 2 from left) or with the indicated concentrations of ascorbate ("Coincubation" with 60, 180, or 360 µmol/L). To assess the role of intracellular ascorbate, confluent HSECs were loaded with ascorbate as described in "Methods" ("Preincubation" with 60, 180, or 360 µmol/L), washed, and incubated with LDL (0.1 mg protein per milliliter) in Ham's F-10 medium for 24 (A) or 48 (B) hours. The anodic electrophoretic mobility of LDL was assessed on agarose gels. For comparison, native LDL (nLDL), LDL incubated without cells (indicated by -, lane 3), and LDL incubated with HSECs in M199 (lane 4) are also shown. The arrowhead on the right side of the gel indicates the origin of LDL loading.

In contrast to human ECs, BAECs could not be loaded with vitamin C (data not shown). Therefore, preincubation of BAECs with ascorbate had no effect on the subsequent modification of LDL by these cells in Ham's F-10 medium (data not shown). However, extracellular vitamin C inhibited BAEC-mediated LDL modification in a concentration-dependent manner, with 170 µmol/L ascorbate providing complete protection for 24 hours of incubation (Table 2).

View this table: [in this window] [in a new window]   Table 2. Inhibition by Extracellular Ascorbate of LDL Modification by BAECs

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The data presented in this article demonstrate that human aortic and saphenous vein ECs modify LDL in culture media that contain transition metal ions and that both intracellular and extracellular vitamin C inhibit EC-mediated LDL modification. Furthermore, the data show that human ECs cultured under conventional conditions contain grossly subphysiological levels of intracellular vitamin C but can readily be enriched with the vitamin by in vitro incubation. The absence of significant amounts of vitamin C in cultured ECs has been previously observed³¹ and represents a nonphysiological state that may critically affect results from in vitro studies of cell-mediated LDL modification and related oxidation reactions.

Most previous investigations of LDL modification by ECs^{5 11 12 32} have used a cell line of rabbit aortic ECs³³ and have shown that these cells oxidatively modify LDL by a mechanism dependent on both transition metal ions^{5 11} and thiol compounds released from the cells.¹² It has been suggested that metal ions cause oxidation of thiols in the medium to thiyl radicals, which may initiate lipid peroxidation in LDL, either directly or indirectly via production of reactive oxygen species.¹² Interestingly, previous studies of LDL oxidation by human ECs have all used M199,^{19 34 35 36} which does not contain detectable amounts of transition metal ions.¹⁰ These studies have shown very modest LDL modification, usually a relative electrophoretic mobility of about 1.4 to 1.7,^{34 36} and sometimes no detectable modification at all.^{19 36} We were unable to detect LDL modification by HAECs in M199, in contrast to metal ion–containing Ham's F-10 medium. However, we did observe a relative electrophoretic mobility of 1.5 for LDL incubated with HSECs in M199 (Fig 5B). Thus, our data are in line with those of others,^{34 36} and at the same time show that significant LDL modification by human ECs requires metal ions.

The inhibition of EC-mediated LDL oxidation by antioxidants has not been widely studied.^{5 11 36} Addition of the lipid-soluble antioxidant -tocopherol to incubations with ECs was shown to inhibit LDL oxidation.^{5 36} However, in those experiments, -tocopherol was added at concentrations corresponding to 1000 and 2300 nmol/mg LDL protein,^{5 36} which is two orders of magnitude higher than the physiological range of 8 to 24 nmol/mg protein.³⁷ In contrast, addition of supraphysiological levels of ß-carotene (200 nmol/mg protein, compared with physiological levels of 0.5 nmol/mg protein¹³ ) had no inhibitory effect on EC-mediated LDL oxidation.³⁶ The only experiment that assessed the effect of vitamin C on EC-mediated LDL modification used the rabbit aortic EC line mentioned above³³ and showed that LDL modification was strongly inhibited by 50 µmol/L ascorbate.¹¹ The data presented here demonstrate that physiological concentrations of ascorbate strongly inhibit LDL modification by human ECs in a concentration- and time-dependent manner. These findings extend our previous observations of very strong protective effects of endogenous ascorbate against lipid peroxidation in human plasma^{14 15} and exogenous ascorbate against LDL oxidation by Cu^{2+16 17} or activated human neutrophils.²⁷ Other researchers have shown equally potent effects of ascorbate against LDL modification by human monocyte–derived macrophages.³⁸ Because vitamin C is present in human plasma,^{15 22 23} interstitial fluid,²³ and arterial walls³⁹ at concentrations comparable to those used in the present study, our data strongly suggest that vitamin C can prevent vascular cell–mediated LDL modification in vivo.

Our findings that HAEC-mediated LDL modification is metal ion dependent and strongly inhibited by vitamin C are remarkable, given the known pro-oxidant effect of vitamin C in vitro in the presence of transition metal ions.^{40 41} We had also observed strong protective rather than exacerbating effects of ascorbate on LDL oxidation by Cu²⁺ or heme iron.^{16 17} The mechanism by which extracellular vitamin C protects against metal ion–dependent LDL oxidation likely involves inhibition of initiation of lipid peroxidation^{16 17} due to decreased Cu²⁺-binding to LDL (K. Retsky et al, unpublished observations) and destruction of lipid hydroperoxides in LDL¹⁷ and possibly, reduction of the transition metal ions to the fully reduced state.⁴² It has been suggested that the simultaneous presence of reduced and oxidized forms of iron is required for initiation of lipid peroxidation and that no lipid peroxidation occurs when the metal ion is either completely reduced or completely oxidized.^{42 43} Therefore, our observation that low concentrations of ascorbate exert a pro-oxidant effect on cell-free LDL modification in Ham's F-10 medium (Table 1) may be explained by the partial reduction of transition metal ions in this medium. In contrast, high concentrations of ascorbate may completely reduce these metal ions, thus preventing LDL modification (Table 1). Interestingly, ascorbate exerted only antioxidant and no pro-oxidant effects when LDL was incubated with ECs (Figs 1A and 4 and Table 2).

A further novel finding of the present study is the reduced capacity of vitamin C–enriched human ECs to modify LDL. Only a few previous studies have investigated the role of cell-associated antioxidants in LDL modification, as opposed to LDL-associated or extracellular antioxidants. These studies have shown that cultured vascular cells enriched with -tocopherol, ß-carotene, or a water-soluble probucol derivative exhibit a lowered capacity to modify LDL.^{19 20 21} The present study shows that human ECs take up vitamin C from the extracellular fluid and as a consequence exhibit a lowered capacity to modify LDL. The amounts of intracellular ascorbate accumulated by HAECs were comparable to those previously measured in human umbilical vein ECs.³¹ If one assumes an intracellular volume of 1 µL/10⁶ HAECs and a protein content of 0.4 mg/10⁶ cells (A.M. and B.F., unpublished observations), then the concentrations of intracellular ascorbate in HAECs (Fig 2B) were in the range of 3 to 8 mmol/L, comparable to the levels in human neutrophils, monocytes, and lymphocytes.^{22 44 45}

The effects of intracellular vitamin C on EC-mediated LDL modification were not as strong as those of extracellular vitamin C. However, it should be kept in mind that in vivo intracellular ascorbate levels are constantly replenished, in contrast to the in vitro experiments presented here. Although we cannot completely exclude the possibility that the effects of intracellular ascorbate on LDL modification were mediated by ascorbate that was released into the medium, we were unable to detect extracellular ascorbate during incubations of LDL with ascorbate-enriched ECs. It is possible that intracellular vitamin C acted by lowering the production rate of reactive oxygen species by these cells (see Fig 4), thus lowering their capacity to modify LDL. Further studies will be required to clarify the mechanisms by which intracellular ascorbate and other cellular antioxidants lower the capacity of vascular cells to oxidatively modify LDL, thereby potentially slowing the initiation and progression of atherosclerosis.

	Selected Abbreviations and Acronyms

 

BAEC = bovine aortic endothelial cell EC = endothelial cell FBS = fetal bovine serum HAEC = human aortic endothelial cell HBSS = Hanks' balanced salt solution HPLC = high-performance liquid chromatography HSEC = human saphenous vein endothelial cell M199 = medium 199 oxLDL or OxLDL = oxidatively modified LDL SMC = smooth muscle cell

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL-49954 and HL-56170 (to B.F.).

Received July 22, 1996; accepted November 25, 1996.

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