Overexpression of Human Superoxide Dismutase Inhibits Oxidation of Low-Density Lipoprotein by Endothelial Cells

From the Departments of Biochemistry (X.F., A.A.S.), Internal Medicine (N.L.W., C.D.R., D.A.C., D.D.H., A.A.S.), Pharmacology (D.D.H.), and the Radiation Research Laboratory (L.W.O., T.Y.), University of Iowa College of Medicine, Iowa City, and the Institute for Human Gene Therapy (R.M.Z., J.F.E.), University of Pennsylvania Medical Center, Philadelphia.

Correspondence to Arthur A. Spector, MD, Department of Biochemistry, 4-403 BSB, University of Iowa, Iowa City, IA 52242.

	Abstract

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Abstract—Oxidation of LDL in the subendothelial space has been proposed to play a key role in atherosclerosis. Endothelial cells produce superoxide anions (O₂^.-) and oxidize LDL in vitro; however, the role of O₂^.- in endothelial cell–induced LDL oxidation is unclear. Incubation of human LDL (200 µg/mL) with bovine aortic endothelial cells (BAECs) for 18 hours resulted in a 4-fold increase in LDL oxidation compared with cell-free incubation (22.5±1.1 versus 6.3±0.2 [mean±SEM] nmol malondialdehyde/mg LDL protein, respectively; P<0.05). Under similar conditions, incubation of LDL with porcine aortic endothelial cells resulted in a 5-fold increase in LDL oxidation. Inclusion of exogenous copper/zinc superoxide dismutase (Cu/ZnSOD, 100 µg/mL) in the medium reduced BAEC-induced LDL oxidation by 79%. To determine whether the intracellular SOD content can have a similar protective effect, BAECs were infected with adenoviral vectors containing cDNA for human Cu/ZnSOD (AdCu/ZnSOD) or manganese SOD (AdMnSOD). Adenoviral infection increased the content and activity of either Cu/ZnSOD or MnSOD in the cells and reduced cellular O₂^.- release by two thirds. When cells infected with AdCu/ZnSOD or AdMnSOD were incubated with LDL, formation of malondialdehyde was decreased by 77% and 32%, respectively. Two other indices of LDL oxidation, formation of conjugated dienes and increased LDL electrophoretic mobility, were similarly reduced by SOD transduction. These data suggest that production of O₂^.- contributes to endothelial cell–induced oxidation of LDL in vitro. Furthermore, adenovirus-mediated transfer of cDNA for human SOD, particularly Cu/ZnSOD, effectively reduces oxidation of LDL by endothelial cells.

Key Words: low density lipoprotein • superoxide anion • superoxide dismutase • gene transfection • endothelial cell

	Introduction

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Elevated concentrations of LDL are thought to promote the development of atherosclerosis.^{1 2 3 4} Cholesterol derived from LDL accumulates in macrophages residing within the vessel wall, resulting in the formation of foam cells and fatty streaks, cardinal features of atherosclerotic lesions.⁵ Whereas macrophages in culture do not take up substantial quantities of native (unmodified) LDL, they avidly take up LDL that has been modified by oxidation and, consequently, are converted into foam cells.^{1 2 3 4} These and other experimental studies suggest that oxidative modification of LDL greatly enhances its atherogenic potential. In vivo, little LDL is oxidized within the circulation; rather, LDL is thought to become oxidatively modified within the arterial wall, perhaps in the subendothelial space,¹ where LDL particles are sequestered in a pro-oxidant environment. In this regard, 3 types of cells within the vessel wall, endothelial cells, smooth muscle cells, and macrophages, have all been shown to oxidatively modify LDL in vitro.¹

Despite intensive investigations, the mechanism(s) by which vascular cells oxidatively modify LDL has not been definitively established. Cell-induced oxidation of LDL in vitro requires the presence of low concentrations of copper or iron in the medium and can be inhibited by metal chelators.^{6 7} However, the nature of the free radical species responsible for the cell-induced LDL oxidation is controversial. A role for the superoxide anion (O₂^.-) was proposed by some investigators on the basis of reports that endothelial cells generate O₂^.- and that addition of SOD to incubations of LDL with cells prevented LDL oxidation.^{8 9 10} In contrast, others have reported that the addition of SOD did not inhibit endothelial cell–induced LDL oxidation.¹¹ However, the use of exogenous SOD to investigate the role of O₂^.- in mediating cell-induced LDL oxidation has several limitations. For example, unless it is highly purified or is obtained by recombinant DNA techniques, exogenous SOD may contain impurities that could nonspecifically alter LDL oxidation. Moreover, exogenous SOD, which remains principally in the extracellular fluid, can inhibit copper-induced LDL oxidation (in the absence of cells), possibly by chelating metal ions in a redox-inactive state.⁴ Finally, since both endothelial cells and SOD are negatively charged, extracellular SOD may be repelled from the cells, thus preventing it from dismutating O₂^.- near the cell surface, where interactions with LDL particles may occur. Therefore, the effects produced by addition of exogenous SOD do not firmly establish the role of O₂^.- in mediating LDL oxidation.

In the present study, we investigated the role of O₂^.- in endothelium-induced LDL oxidation by infecting endothelial cells with replication-deficient adenoviral vectors containing genes for human copper/zinc SOD (Cu/ZnSOD) and manganese SOD (MnSOD). This approach enabled us to selectively increase the intracellular concentration of purified human SOD. Our results suggest that O₂^.- production contributes to oxidation of LDL by endothelial cells in vitro. Moreover, adenovirus-mediated transfer of cDNA for human SOD, particularly Cu/ZnSOD, is highly effective in reducing endothelial cell–induced LDL oxidation. Portions of these results have been published in abstract form.¹²

	Materials and Methods

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DMEM, Ham F-10 medium, MEM nonessential amino acid, MEM vitamin solution, Cu/ZnSOD, the ferric-sodium salt of EDTA (iron-EDTA), fluorescein-conjugated goat anti-rabbit antibody, cytochrome c, Tween 20, and tetramethoxypropane were obtained from Sigma Chemical Co. FBS was purchased from HyClone Laboratories, and gentamicin was obtained from Schering Corp. Medium 199, HEPES, trypsin, and L-glutamine were originally obtained from Sigma Chemical Co and prepared by the University of Iowa Cancer Center, Iowa City. The LDL electrophoresis system was purchased from CIBA-Corning.

Cell Culture
BAECs were isolated and suspended in medium 199 supplemented with MEM nonessential amino acids, MEM vitamin solution, 15 mmol/L HEPES, 2 mmol/L L-glutamine, 50 µmol/L gentamicin, and 20% FBS.¹³ The suspended cells were counted with a hemocytometer and plated into 25-cm² flasks at the density of 4x10⁴ cells/mL, and the cultures were maintained until confluent at 37°C in a humidified atmosphere containing 5% CO₂. Stocks were subcultured weekly by trypsinization, and the cells were passaged into 6-well plates before use in all experimental protocols. Cultures were used between passage numbers 3 and 12. PAECs, cultured as described previously,¹⁴ were used between passage numbers 3 and 10.

Infection of Endothelial Cells With Adenoviral Vectors Containing Human MnSOD and Cu/ZnSOD cDNA
MnSOD and Cu/ZnSOD recombinant adenoviral constructs were generated using previously described methods. Briefly, MnSOD constructs were generated by cloning of an EcoRI/PvuII fragment from the pRK5 MnSOD construct.¹⁵ Cu/ZnSOD constructs were generated from human placental mRNA by reverse transcription with polyT followed by polymerase chain reaction with Cu/ZnSOD-specific primers harboring restriction sites for direct cloning into adenoviral constructs. Recombinant adenoviral plasmid constructs were generated by cloning transgenes into pAd.CMVlink, which contains the CMV enhancer/promoter and an SV40 polyadenylation site for efficient expression of the transgenes.¹⁶ Recombinant viruses were generated by cotransfection of NheI-cut pAd plasmid with ClaI-cut Ad5.sub360 (E3-deleted) viral DNA.¹⁷ After transfection, plates were overlaid with agar, and initial plaques were harvested for screening by enzymatic activity. These recombinant viruses were screened for MnSOD and Cu/ZnSOD activity by secondary infection on 293 cells. Initial plaques that expressed functional enzyme were further purified through 2 subsequent rounds of plaque purification. Recombinant viruses expressing MnSOD and Cu/ZnSOD are designated AdCMVMnSOD and AdCMVCu/ZnSOD, respectively. Adenovirus containing no foreign cDNA, designated as AdBgII, was used as a negative control. All viral titers were determined by assessing pfu on 293 cells. All viral stocks were essentially free from replication-competent adenovirus, as assessed by the absence of a cytopathic effect on IB3 cells (MOI=1000 pfu/cell) after serial passage.¹⁶

Subconfluent endothelial cells were incubated with various MOIs of AdCu/ZnSOD, AdMnSOD, or AdBgII in serum-free medium 199. After 2 hours, FBS was added (final concentration, 10%), and the incubation was continued for an additional 22 hours. Afterward, the medium was removed, and the cells were either washed twice with medium 199 and incubated with LDL, or they were washed with cold PBS (mmol/L: KH₂PO₄ 1.9, KCl 2.7, NaCl 138, and Na₂HPO₄ 8.0, pH 7.4), harvested by scraping into microtubes, and centrifuged at 3500 rpm for 15 minutes for determination of SOD protein content and activity (see below). In some cases, the medium was also examined for SOD protein content.

Western Blot Analysis
The cells were sonicated in 0.05 mol/L potassium phosphate buffer (pH 7.8) on ice with three 30-second bursts by use of a Vibra Cell sonicator (Sonics and Materials, Inc) at 10% output and 80% duty cycle. The protein concentrations were measured by the method of Bradford¹⁸ with a Bio-Rad Protein Assay kit according to manufacturer's instructions. Bovine serum albumin was used as a protein assay standard. Samples were denatured with SDS loading buffer at 95°C for 5 minutes and then separated on an SDS/12% polyacrylamide gel with a 5% stacking gel in SDS/Tris/glycine running buffer.¹⁹ The protein was electrophoretically transferred to a nitrocellulose membrane, which was then blocked with 5% (wt/vol) nonfat milk in TTBS buffer (0.02 mol/L Tris/0.15 mol/L NaCl buffer, pH 7.45, and 0.1% Tween 20) for 1 hour at room temperature on an orbital shaker. The membrane was then incubated with specific rabbit anti-serum raised against either human MnSOD (1:1000) or human Cu/ZnSOD (1:500) in TTBS buffer for 1 hour. The use of these anti-sera, which do not cross-react with other antioxidant enzymes, has been described previously.²⁰ The blot was incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG (1:10 000, Boehringer-Mannheim Corp) for 1 hour at room temperature. The anti-SOD antibodies were then detected using an ECL detection system (Bio-Rad Laboratories) and exposed to x-ray film.

Immunohistochemical Localization of SOD Protein
Infected cells (100 MOI) were washed with PBS and fixed with 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.2, for 30 minutes at room temperature. The cells were then incubated with rabbit anti-serum against either human MnSOD (1:300 dilution in PBS/0.5% Triton X-100) for 3 hours or human Cu/ZnSOD (1:500 dilution in PBS/0.5% Triton X-100) for 2 hours at room temperature. After rinsing with PBS, the cells were incubated with fluorescein-conjugated goat anti-rabbit antibody (1:200 dilution, Molecular Probe, Inc) for 3 hours at room temperature and then visualized by laser confocal microscopy.

Determination of Antioxidant Enzyme Activity
Cell extracts were prepared by sonication as described above. SOD activity was measured by the modified NBT method as described by Oberley and Spitz.²¹ Briefly, xanthine/xanthine oxidase was used to generate O₂^.-, which was detected by the reduction of NBT to blue formazan. Spectrophotometric measurement of the rate of blue formazan formation in the presence of increasing amounts of cellular protein was performed. Total SOD activity was determined from the amount of inhibition of NBT reduction. MnSOD activity was determined in the presence of 5 mmol/L sodium cyanide. Cu/ZnSOD activity was calculated from the difference between total SOD activity and MnSOD activity. The activities of 2 other antioxidant enzymes, catalase and glutathione peroxidase, were also measured spectrophotometrically as previously described.²²

Isolation of LDL and Modification by the Cells
Plasma LDL (density, 1.02 to 1.05 g/mL) was prepared by sequential ultracentrifugation of plasma from fasted healthy human subjects and quantified as described previously.²³ The subjects had the most common apolipoprotein E phenotype (E3/3). Endothelial cells were incubated with 200 µg protein/mL of human LDL in 1 mL Ham F-10 medium containing 5 µmol/L iron-EDTA for the stated durations. After incubation, lipid peroxidation products in the medium were determined by measuring TBARS using a fluorometric assay.²⁴ Values are expressed as nanomolar equivalents of MDA per milligram of LDL protein. Formation of conjugated dienes in the 1 mL incubation medium was determined spectrophotometrically (absorbance at 234 nm)²⁵ immediately after incubation; the absorbance obtained with Ham F-10 medium alone (in the absence of incubation with cells) was set as the baseline value before assaying the incubation medium. The concentration of conjugated dienes was calculated with the assumption that absorbance at 234 nm equals 29 500 mol^-1 · cm^-1. Agarose gel electrophoresis was performed on samples of media diluted with equal volumes of defatted albumin at pH 8.6 by use of a CIBA-Corning system.²³ Control samples of oxidized LDL were produced by incubating LDL with 5 µmol/L CuSO₄ in Ham F-10 medium for 24 hours. In some experiments, lactate dehydrogenase activity in the incubation medium was measured by a colorimetric assay using diagnostic kits (catalog No. 500-C) obtained from Sigma Chemical Co.

Measurement of Superoxide Release
Secretion of O₂^.- by the endothelial cells was determined by SOD-inhibitable reduction of cytochrome c.²⁶ Cells were incubated for 1 hour in phenol red–free DMEM in the presence of 20 µmol/L cytochrome c. O₂^.- release was calculated from the difference of absorbance at 550 nm in the absence and presence of SOD (100 µg/mL); a molar extinction coefficient of 21 000 was used.

Statistical Analyses
All data are expressed as mean±SEM. Differences between mean values of 2 groups were analyzed by Student t tests. Differences between mean values of multiple groups were analyzed by 1-way ANOVA with a Newman-Keuls post hoc analysis. Values of P0.05 were considered to be statistically significant.

	Results

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Oxidation of LDL by Endothelial Cells
After incubation of LDL for 18 hours with BAECs, TBARS formation in the medium was increased 4-fold compared with LDL incubated in the absence of cells (Table). Under similar conditions, increases in TBARS were also observed after incubation of LDL with PAECs.

Inclusion of 100 µg/mL Cu/ZnSOD during incubation of LDL with BAECs resulted in a 79% reduction in TBARS formation compared with control (6.0±0.1 [CuZnSOD] versus 29.1±2.6 [control] nmol MDA/mg protein, P<0.01). Formation of conjugated dienes in the medium, another indicator of lipid peroxidation, was reduced by 57% in the presence of 100 µg/mL Cu/ZnSOD (data not shown). Inclusion of Cu/ZnSOD exerted a similar inhibitory effect on PAEC-induced LDL oxidation (TBARS formation during incubation of LDL with PAECs was decreased by 38% in the presence of 100 µg/mL Cu/ZnSOD compared with the control value). These results indicate that under these experimental conditions, exogenous SOD inhibits oxidation of LDL by BAECs and PAECs.

Transfection of Human SOD Genes to Endothelial Cells
To determine the effects of intracellular concentration of SOD on oxidation of LDL by endothelial cells, BAECs were infected with various doses of AdMnSOD, AdCu/ZnSOD, or, as a control, AdBgII. After 24 hours, the incubation medium was removed, and cell lysates were prepared and subjected to Western blot analysis using polyclonal antibodies reactive with either human MnSOD or human Cu/ZnSOD. Expression of cellular Cu/ZnSOD (Figure 1, top) and MnSOD (Figure 1, bottom) proteins was increased in a dose-dependent manner after infection with AdCu/ZnSOD and AdMnSOD, respectively, compared with infection with AdBgII. A single 16-kDa band corresponding to authentic human Cu/ZnSOD was detected in lysates prepared from AdCu/ZnSOD-infected cells. In lysates from AdMnSOD-infected cells, a 22-kDa band corresponding to authentic human MnSOD was detected, as well as an unidentified 24-kDa band, which might be a precursor form of MnSOD. In contrast, significant amounts of SOD proteins were not detected in the incubation medium after infection with 300 MOI of AdCu/ZnSOD or AdMnSOD (data not shown).

View larger version (58K): [in this window] [in a new window]   Figure 1. Effects of SOD gene transfer on expression of Cu/ZnSOD and MnSOD proteins. BAECs grown to subconfluence in 6-well plates were incubated with 10 to 300 MOI AdCu/ZnSOD, AdMnSOD, or the AdBgII control in serum-free medium 199. After 2 hours, FBS was added (final concentration, 10%), and the incubation was continued for an additional 22 hours. The medium was then removed, and the cells were washed with cold PBS, harvested, and sonicated. Human Cu/ZnSOD (top) and MnSOD (bottom) proteins in the cell lysates were detected by Western-blot analysis. Cell lysate (10 µg protein) was loaded for both Cu/ZnSOD and MnSOD blots. The numbers refer to the MOI. Results from control (noninfected) cells are shown in lane C, and authentic human Cu/ZnSOD (0.2 µg) and MnSOD (0.2 µg) protein are shown in lane +.

To investigate the intracellular location of the transduced SOD proteins, cells that were infected with AdCu/ZnSOD or AdMnSOD were incubated with specific rabbit anti-sera against human SOD. The cells were then labeled with anti-rabbit fluorescein-conjugated antibody and examined by immunohistochemical confocal microscopy. In Cu/ZnSOD-transfected cells, the immunofluorescence was localized diffusely throughout the cytoplasm (Figure 2A). In contrast, in MnSOD-transfected cells, the immunofluorescence was confined to discrete cytoplasmic particles (Figure 2B), an observation that is consistent with previous findings indicating that transduced MnSOD is localized within mitochondria.²⁷ No significant fluorescence was observed in AdBgII-transfected cells incubated with rabbit anti-serum against either human MnSOD or Cu/ZnSOD (data not shown).

View larger version (91K): [in this window] [in a new window]   Figure 2. Immunohistochemical localization of SOD proteins in endothelial cells after infection with AdCu/ZnSOD (A) or AdMnSOD (B). The cells were incubated with rabbit anti-serum against human Cu/ZnSOD (A) or human MnSOD (B), followed by a fluorescein-conjugated goat anti-rabbit antibody. The cells were then examined by laser confocal microscopy.

Cell lysates were also examined for antioxidant enzyme activities. Infection with either AdMnSOD or AdCu/ZnSOD resulted in dose-dependent increases in total cellular SOD activity (Figure 3, top). After infection with AdCu/ZnSOD or AdMnSOD, cellular SOD activity (determined by a modified NBT inhibition assay) was increased as much as 8- to 10-fold over that observed in control (uninfected) cells. In contrast, SOD activity was not altered in cells infected with AdBgII (10 to 300 MOI) compared with control cells (data not shown). Furthermore, after infection with 300 MOI of either AdMnSOD or AdCu/ZnSOD, the increases in total SOD activity were solely related to increases in MnSOD or Cu/ZnSOD activity, respectively, which in turn accounted for >95% of the total cellular SOD activity (Figure 3, bottom). Similar results were observed in cells infected with 10 or 100 MOI of either AdCu/ZnSOD or AdMnSOD (data not shown). To investigate whether SOD activity in the medium was increased after infection with 300 MOI of AdCu/ZnSOD or AdMnSOD, cells were infected with 300 MOI of the adenoviral vectors as described previously, and the incubation medium was collected for analysis; however, the amount of protein present in the medium was too low to perform the SOD activity assay, even when concentrated 200-fold. We also examined the effects of adenoviral infection on the activities of 2 other antioxidant enzymes, catalase and glutathione peroxidase. In contrast to the effects of adenoviral infection on cellular SOD activity, catalase activity (expressed as k units per gram protein) did not differ among the lysates of cells infected with 100 MOI of any of the 3 adenoviral vectors (895±79 [AdBgII], 935±159 [AdCu/ZnSOD], and 751±73 [AdMnSOD] k units per gram protein, n=3, P>0.05). Measurable levels of glutathione peroxidase activity were not detected in cell lysates prepared from cells infected with 100 MOI of any of the adenoviral vectors. These results suggest that infection with AdCu/ZnSOD or AdMnSOD markedly increased cellular SOD activity without significantly affecting the activities of other antioxidant enzymes.

View larger version (31K): [in this window] [in a new window]   Figure 3. Effects of SOD gene transfer on cellular SOD activity. BAECs were infected with 0 to 300 MOI of AdCu/ZnSOD or AdMnSOD for 24 hours, as described in Figure 2. Afterward, the medium was removed, and the cells were washed with cold PBS, harvested, and sonicated. For each group, aliquots of cell lysates were incubated with NBT in the presence of chemically generated O2.-, and SOD activity was determined by the amount of inhibition of NBT reduction. MnSOD activity was determined in the presence of 5 mmol/L sodium cyanide. Cu/ZnSOD activity was calculated from the difference between total SOD activity and MnSOD activity. Total SOD activity is shown in the top panel, and Cu/ZnSOD and MnSOD activities are shown in the bottom panel. Each bar represents the mean±SEM of values obtained from 3 or 4 separate cultures. *P<0.05 vs control cells (0 MOI) and corresponding result at 10 MOI; +P<0.05 vs corresponding result at 100 MOI.

Effects of Transfection of SOD Genes on O₂^.- Release and Oxidation of LDL
Both noninfected BAECs and BAECs infected with AdBgII released 4 nmol of O₂^.-per well into the medium per hour (Figure 4). Infection with either AdMnSOD or AdCu/ZnSOD greatly reduced the rate of O₂^.- release into the medium.

View larger version (48K): [in this window] [in a new window]   Figure 4. Effects of SOD gene transfer on O2.- release from BAECs. Endothelial cells were infected with 300 MOI of an adenoviral vector containing cDNA for human MnSOD, Cu/ZnSOD, or no cDNA (BgII) for 24 hours, as described in Figure 1. The cells were washed and then incubated in 1 mL serum-free phenol red–free DMEM containing 20 µmol/L cytochrome c for 1 hour. Superoxide accumulation in the medium was calculated from the amount of cytochrome c reduction. Results obtained from noninfected (control) cells are also shown. Each bar represents the mean±SEM of values obtained from 3 separate cultures. *P<0.05 vs control and BgII.

Effects of transduction of SOD on endothelial cell–induced LDL oxidation were determined. TBARS formation was decreased by 31% and 81%, respectively, in cells infected with AdMnSOD or AdCu/ZnSOD compared with control cells infected with AdBgII (Figure 5, top). Formation of conjugated dienes (expressed as nanomoles per milligram LDL protein) was similarly reduced after infection with AdMnSOD or AdCu/ZnSOD compared with AdBgII (117±14 nmol/mg LDL protein [AdBgII] versus 70±5 nmol/mg LDL protein [AdMnSOD], P<0.05; 39±8 nmol/mg LDL protein [AdCu/ZnSOD], P<0.05 versus AdBgII and AdMnSOD).

View larger version (54K): [in this window] [in a new window]   Figure 5. Effects of SOD gene transfer on BAEC- and PAEC-induced LDL oxidation. Endothelial cells were incubated with 300 MOI of an adenoviral vector containing cDNA for human MnSOD, Cu/ZnSOD, or no cDNA (BgII) for 24 hours, as described in Figure 1; after which, the cells were incubated with LDL, as described in the Table. After incubation, TBARS formation in the medium (expressed as nmol MDA/mg LDL protein) was determined. Each bar represents the mean±SEM of values obtained from 3 separate cultures. The TBARS values obtained from cell-free controls were 4.2±0.03 and 6.6±0.2 nmol MDA/mg LDL protein for BAECs (top) and PAECs (bottom), respectively. *P<0.05 vs BgII; +P<0.05 vs MnSOD.

To investigate whether adenovirus-mediated transfer of the genes for human SOD would attenuate LDL oxidation produced by a different endothelial cell line, PAECs were infected with 300 MOI of AdBgII, AdMnSOD, or AdCu/ZnSOD and incubated with 200 µg/mL of LDL under the same conditions described previously. Infection with either AdCu/ZnSOD or AdMnSOD also decreased the LDL oxidation compared with infection with AdBgII (Figure 5, bottom). Similar to the results obtained with BAECs, infection of PAECs with AdCu/ZnSOD was more effective in reducing LDL oxidation than was infection with AdMnSOD.

Effects of adenoviral infection on BAEC-induced alterations in LDL electrophoretic mobility were also examined (Figure 6). Incubation of LDL with cells infected with the control virus, AdBgII (lane 1), resulted in increased LDL electrophoretic mobility relative to LDL incubated in medium alone (lane 5), consistent with oxidative modification of the LDL. Infection of BAECs with adenoviral vectors containing cDNA for human SOD, particularly Cu/ZnSOD (lane 3), reduced the LDL electrophoretic mobility compared with control (lane 1). Similar results were observed when BAECs were incubated with LDL in the presence of exogenous Cu/ZnSOD (lane 4). Increased LDL electrophoretic mobility from CuSO₄-induced LDL oxidation also occurred, as shown in lane 6.

View larger version (29K): [in this window] [in a new window]   Figure 6. Effects of SOD gene transfer on BAEC-induced changes in LDL electrophoretic mobility. Cells were infected with 300 MOI of AdMnSOD, AdCu/ZnSOD, or the AdBgII control for 24 hours as described in Figure 1; after which, the cells were incubated with LDL, as described in the Table. The medium was removed, and LDL electrophoretic mobility was assayed by agarose gel electrophoresis. One typical gel result is shown here, but similar results were obtained from 2 other identically treated sets of cultured cells. From left to right, the lanes show results obtained from AdBgII-infected cells (lane 1), cells infected with AdMnSOD (lane 2), cells infected with AdCu/ZnSOD (lane 3), and noninfected cells in the presence of 100 µg/mL exogenous Cu/ZnSOD (lane 4). The final 2 lanes represent LDL incubated in medium alone (lane 5) and LDL incubated in medium containing 5 µmol/L CuSO4 (lane 6).

To investigate whether the inhibition of endothelial cell–induced LDL oxidation by SOD transduction might have been dependent on the presence of iron-EDTA in the incubation medium, 18-hour incubations were carried out in Ham F-10 medium without iron-EDTA in the absence or presence of noninfected BAECs or BAECs infected with 300 MOI of AdMnSOD or AdBgII. Under these conditions, incubation of LDL with noninfected endothelial cells resulted in a small but significant increase in TBARS formation compared with cell-free incubations (12.5±0.3 nmol MDA/mg LDL protein [noninfected cells] versus 7.9±0.3 nmol MDA/mg LDL protein [cell-free incubation], P<0.05). Despite omission of iron-EDTA, incubation of LDL with AdMnSOD-infected cells still resulted in reduced TBARS formation compared with AdBgII-infected cells (10.2±0.1 nmol MDA/mg LDL protein [AdMnSOD] versus 13.6±0.9 nmol MDA/mg LDL protein [AdBgII], P<0.05).

To investigate whether infection with AdCu/ZnSOD might have prevented BAEC-induced LDL oxidation by causing cell toxicity, we determined the total protein concentration and lactate dehydrogenase activity in the medium. Neither total protein concentration nor lactate dehydrogenase activity (98±11 U/mL [control] versus 106±7 U/mL [AdCu/ZnSOD], n=3, P>0.05) differed between control and AdCu/ZnSOD-infected cells (300 MOI) at the end of a 24-hour incubation with LDL. Furthermore, infection with the adenoviral vectors did not alter the numbers of cells or their morphological appearance by phase-contrast microscopy (data not shown). Thus, the reduction in BAEC-induced LDL oxidation resulting from infection with AdCu/ZnSOD and AdMnSOD most likely did not result from cytotoxicity.

Time- and Dose-Dependent Effects of SOD Gene Transfection on LDL Oxidation by BAECs
The time course of LDL oxidation by BAECs and the time-dependent effects of infection with AdCu/ZnSOD on BAEC-induced LDL oxidation were examined. After infection with the virus for 24 hours, the cells were incubated with 200 µg/mL of LDL for 0 to 24 hours. Incubation of AdBgII-infected cells with LDL resulted in time-dependent increases in LDL oxidation (detected by TBARS formation), which were decreased by infection with AdCu/ZnSOD at both 8 and 24 hours (Figure 7, top).

View larger version (16K): [in this window] [in a new window]   Figure 7. Time- and dose-dependent effects of human SOD gene transfer on BAEC-induced LDL oxidation, as determined by TBARS formation. In the top panel, cells were infected with 300 MOI of AdCu/ZnSOD or the control vector (AdBgII) for 24 hours, as described in Figure 1. The cells were then incubated with 200 µg/mL of LDL for 2 to 24 hours, as described in the Table. TBARS values obtained from cell-free controls (expressed as the average of 2 samples) were 7.9 and 14.6 at 2 and 24 hours, respectively. In the bottom panel, cells were infected with 0 to 300 MOI of AdCu/ZnSOD for 24 hours, as described in Figure 1; after which, the cells were incubated with 200 µg/mL LDL, as described in the Table. The results are expressed as the mean±SEM of values obtained from 3 separate cultures. *P<0.05 versus BgII.

To determine whether the effects of infection with AdCu/ZnSOD on LDL oxidation were dependent on the quantity of gene transferred, BAECs were infected for 24 hours with 10 to 300 MOI of AdCu/ZnSOD and then incubated with LDL for 18 hours. Infection with the lowest quantity of virus tested, 10 MOI, decreased TBARS formation by 46%. Infection with greater quantities of virus resulted in somewhat more inhibition of LDL oxidation (Figure 7, bottom).

	Discussion

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There are 3 principal findings in the present study: (1) bovine and porcine aortic endothelial cells are capable of oxidizing LDL through a mechanism involving O₂^.-; (2) adenovirus-mediated transfer of cDNA for human SOD into endothelial cells attenuates the cell-mediated oxidation of LDL; and (3) transfer of the gene for Cu/Zn SOD appears to inhibit endothelial cell–induced LDL oxidation more effectively than does transfer of the gene for MnSOD, even though the amount of enzyme produced by transduction of MnSOD is comparable.

In the present study, we found that oxidation of LDL by BAECs and PAECs was inhibited either by adding SOD to the media or by transferring the cDNA for human Cu/ZnSOD or MnSOD into the cells. SOD gene transfer resulted in large increases in the amounts and activities of purified SOD proteins within the cells. In contrast, we were unable to detect significant amounts of SOD in the incubation medium after gene transfer, suggesting that the inhibition of endothelial cell–induced LDL oxidation by adenovirus-mediated SOD gene transfer resulted from the intracellular action of SOD. These results suggest that the inhibition of LDL oxidation consequent to adenovirus-mediated SOD gene transfer is not attributable to factors such as impurities contained in the SOD or by chelation of extracellular metal ions by SOD. Accordingly, our results strongly support a role for endothelial cell–derived O₂^.- in mediating LDL oxidation under these experimental conditions.

The origin of the O₂^.- production by endothelial cells in the present study was not investigated. Superoxide anions can be generated through cyclooxygenase- or lipoxygenase-mediated metabolism of fatty acids.²⁸ A previous study indicated that lipoxygenase inhibitors markedly attenuated LDL oxidation produced by rabbit aortic endothelial cells, suggesting a role for lipoxygenase products in the LDL oxidation.²⁹ Other reports have implicated a plasma membrane NAD(P)H oxidase as a major source of production in endothelial cells.^{30 31} Recently, Mabile et al³² found that production of O₂^.- by mitochondrial respiratory-chain activity may be involved in endothelial cell–mediated LDL oxidation. LDL was also reported to uncouple L-arginine metabolism from endothelial NO synthase, thereby promoting O₂^.- production through an endothelial NO synthase–dependent-mechanism.³³ Finally, in hypercholesterolemic rabbits, circulating plasma xanthine oxidase was reported to contribute to enhanced aortic endothelial O₂^.- production.³⁴ These results suggest that O₂^.- may be generated in endothelial cells through multiple pathways. Consequently, multiple pharmacological agents might be required to completely inhibit endothelial O₂^.- production. On the other hand, our results suggest that increasing the intracellular SOD content (ie, by SOD gene transfer) could counteract the deleterious effects of O₂^.-, regardless of its source.

The mechanism(s) by which O₂^.- promotes LDL oxidation has not been definitively established. Superoxide anions do not readily react with most biological molecules; therefore, the toxicity attributed to O₂^.- may be mediated by hydroxyl radicals (HO · ), highly reactive secondary free radical species produced from superoxide anions.³⁵ Peroxynitrite, which is formed via the reaction O₂^.- with NO, also possesses strong oxidant activity.³⁶ Because the endothelium is a source of both NO and O₂^.-, the latter mechanism could contribute to endothelium-induced LDL oxidation. However, NO donors were reported to inhibit endothelial cell–induced LDL oxidation,³⁷ and inhibitors of NO synthase did not alter the endothelial cell–induced enhancement of LDL uptake and degradation by macrophages. The latter observation suggests that NO is not involved in the mechanism of LDL oxidation by cultured endothelial cells.³⁸ We also found that incubating BAECs with LDL in the presence of N-nitro-L-arginine methyl ester (100 µmol/L), an inhibitor of NO synthase, did not affect TBARS formation (data not shown). These results suggest that endothelial cell–induced O₂^.--dependent oxidation of LDL in vitro is not dependent on basal NO production and is, therefore, probably not mediated by peroxynitrite.

We found that infection with AdCu/ZnSOD inhibited LDL oxidation more effectively than did infection with AdMnSOD (Figure 5). One potential explanation for this observation is that the quantity and/or activity of intracellular SOD protein was greater after infection with AdCu/ZnSOD than with AdMnSOD. However, the SOD protein determinations and activity assays (Figures 1 and 3) suggest that this is not the case. A second possibility is that Cu/ZnSOD, which is primarily localized within the cytoplasm,³⁹ may be more effective at inhibiting O₂^.- release from the cells than is MnSOD, which is primarily localized within the mitochondria⁴⁰ (Figure 2). However, we were unable to detect significant differences in O₂^.- release from AdCu/ZnSOD- versus AdMnSOD-infected cells (Figure 4). Nevertheless, because the assay measured O₂^.- release over a 1-hour period whereas LDL oxidation occurred over 18 hours, it is possible that the slightly reduced rate of O₂^.- release from AdCu/ZnSOD-infected cells compared with AdMnSOD-infected cells noted in Figure 4 could have resulted in a substantial decrease in LDL oxidation over the 18-hour period. Finally, it is conceivable that LDL particles that are bound to the cell surface, or perhaps located immediately adjacent to cells, could be preferentially oxidized as a result of the high local concentrations of O₂^.- produced within the cytoplasm at sites near the cell surface. If there are discrete cytosolic "pools" of O₂^.- that are capable of efficiently oxidizing LDL, they might be preferentially inhibited by SOD contained within the cytoplasm (ie, Cu/ZnSOD³⁸) rather than within the mitochondria. Additional studies will be required to definitively address these possibilities.

In several earlier studies, PAECs but not BAECs were observed to oxidize LDL.^{6 10 41} More recently, however, Morgan et al³⁸ observed that BAECs oxidized LDL during incubations of 48 hours, but not 24 hours. The BAEC-induced LDL oxidation observed in the present study may have been facilitated by the addition of 5 µmol/L iron-EDTA to the Ham's F-10 medium. In preliminary studies, we observed only a modest amount of oxidation of LDL after incubation of either BAECs or PAECs for up to 24 hours in unsupplemented Ham's F-10 medium. Although Ham's F-10 medium contains trace amounts of metal ions, we found that supplementation of the medium with 5 µmol/L iron-EDTA resulted in reproducible increases in cell-induced LDL oxidation after incubations of 18 hours. The mechanism of this effect was not investigated, but it is possible that iron-EDTA facilitated the formation of HO · from cell-derived O₂^.- via the iron-catalyzed Haber-Weiss reaction.^{42 43} However, omission of iron-EDTA during incubations of LDL with BAECs still resulted in measurable increases in TBARS formation, and a significant reduction in TBARS formation was observed after infection with AdMnSOD. Thus, the presence of iron-EDTA was not obligatory for the inhibition of cell-mediated LDL oxidation by SOD gene transfer.

The pathophysiological relevance of our findings is suggested by recent reports that hypercholesterolemia increases endothelial O₂^.- production⁴⁴ and ferritin gene expression⁴⁵ in rabbit aorta. In the latter study, the time course of ferritin gene induction paralleled the time course of development of atherosclerotic lesions during the 6-week period of cholesterol feeding. Furthermore, ferritin genes were found to be highly expressed in human atherosclerotic lesions.⁴⁵ These observations suggest that the iron content within developing atherosclerotic lesions is increased concurrently with increased endothelial O₂^.- production, thereby producing an environment that may facilitate oxidation of LDL. In support of this possibility, extracts obtained from human atherosclerotic aortas contain catalytically active iron, which stimulates lipid peroxidation.⁴⁶ Taken together, these reports suggest that the iron-catalyzed oxidation of LDL through a mechanism involving endothelial cell–derived O₂^.- could contribute to the development and progression of atherosclerosis. Accordingly, increasing the intracellular SOD concentration by SOD gene transfer could potentially mitigate the atherosclerotic process.

	Selected Abbreviations and Acronyms

 

Ad = adeno BAEC = bovine aortic endothelial cell CMV = cytomegalovirus MDA = malondialdehyde MOI = multiplicity of infection NBT = nitro blue tetrazolium PAEC = porcine aortic endothelial cell pfu = plaque-forming unit(s) SOD = superoxide dismutase SV = simian virus TBARS = thiobarbituric acid-reactive substance(s)

	Acknowledgments

 
This study was supported by grants HL-49264 (Drs Spector, Chappell, and Weintraub); HL-16066, HL-14388, and NS-24621 (Dr Heistad); DK-51315 (Dr Engelhardt); CA-66081 (Drs Spector and Oberley); and DE-10758 (Dr Oberley) from the National Institutes of Health; by grants from the Department of Veteran's Affairs Research Fund (Drs Chappell and Heistad); and by an American Heart Association Grant-in-Aid (Dr Chappell) and Clinician-Scientist Award (Dr Weintraub). The authors appreciate the assistance provided by the University of Iowa Gene Transfer Vector Core, which is supported in part by a trust from the Carver Foundation. The authors also thank Christine Darby for technical assistance.

	Footnotes

 
Previously presented as preliminary results in abstract form (Circulation. 1996;94[suppl I]:I-281).

Received September 23, 1997; accepted March 23, 1998.

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