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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3469-3474

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3469-3474.)
© 1997 American Heart Association, Inc.

Articles

Endothelial Cells Prevent Accumulation of Lipid Hydroperoxides in Low-Density Lipoprotein

David M. Smalley; Neil Hogg; B. Kalyanaraman; ; Kirkwood A. Pritchard, Jr

From the Department of Pathology (D.M.S., K.A.P.), Cardiovascular Research Center (D.M.S., K.A.P.), Department of Pharmacology and Toxicology (K.A.P.), and the Biophysics Research Institute (N.H., B.K.), Medical College of Wisconsin, Milwaukee.

Correspondence to Kirkwood A. Pritchard, Jr, PhD, Medical College of Wisconsin, Cardiovascular Research Center, 493D, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail kpritch{at}post.its.mcw.edu


*    Abstract
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 *Abstract
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Abstract A variety of cell types, including endothelial cells, oxidize low-density lipoprotein (LDL). To investigate the mechanisms by which endothelial cells modulate LDL oxidation states, endothelial cell cultures were incubated with LDL (240 mg cholesterol/dL) for 24 hours in M199 supplemented with fetal bovine serum (FBS, 16.7%). These conditions were not toxic to endothelial cells over the time frame of the study. Changes in LDL oxidation were monitored by measuring thiobarbituric acid–reactive substances (TBARS), lipid hydroperoxide (LOOH), and conjugated dienes (A234nm). LDL medium incubated in the absence of endothelial cells contained higher TBARS than did LDL medium incubated with endothelial cells (0.35±0.08 versus 0.23±0.08 nmol MDA/mg, respectively). LOOHs were higher in LDL medium incubated without endothelial cells than in LDL medium incubated with endothelial cells (6.8±4.4 versus 0.49±0.89 nmol/mg, respectively). Conjugated diene formation, based on changes in absorbance at 234 nm, increased to a greater extent in LDL medium incubated in the absence of endothelial cells than when endothelial cells were present. To increase oxidative stress on the endothelial cell cultures, increasing concentrations of Cu2+ (0 to 4 µmol/L) were added to LDL medium. Endothelial cells prevented LOOH accumulation until the concentration of Cu2+ exceeded 0.75 µmol/L. At 1.5 and 4 µmol/L Cu2+, endothelial cells enhanced LOOH formation nearly 3 and 2.5 times the LOOH values in the corresponding medium incubated in the absence of endothelial cells. This loss of protective function however, was not permanent. Endothelial cells, preincubated for 24 hours with Cu2+-containing LDL medium, were still able to prevent LOOH accumulation in fresh LDL medium. Endothelial cells prevented LOOH accumulation even when exposed to LDL medium that contained low concentrations of LOOHs (<22 nmol/mg). However, endothelial cells accelerated the accumulation of LOOHs in LDL when exposed to LDL medium that contained slightly higher concentrations of preexisting LOOHs ({approx}33 nmol/mg). These data indicate that endothelial cells have a limited capacity for preventing LOOH formation and that small increases in LOOHs may play a critical role in enhancing the potential of endothelial cells for oxidative modification of LDL.


Key Words: LDL • endothelial cells • lipid peroxides


*    Introduction
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 up arrowAbstract
 *Introduction
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Since 1984, it has been well recognized that endothelial cells promote oxidative modification of LDL in culture.1 The majority of studies were performed with low LDL concentrations and in the absence of serum. In this study, we exposed cultured human umbilical vein endothelial cells to high concentrations of LDL (240 mg/dL) in the presence of FBS (16.7%). Native LDL and oxidatively modified LDL are important mediators in the premature development of atherosclerosis (see References 2, 32 3 for review). The prevailing theory of hypercholesterolemia-induced atherosclerosis is that LDL entry into the vessel wall increases in direct response to increases in plasma LDL concentrations.4 Once there, LDL can be trapped and then oxidatively modified.2 Such modifications increase the electronegative character of the LDL particle by blocking the positively charged lysine residues of apolipoprotein B.5 The effects of oxidatively modified LDL on endothelial cells, smooth muscle cells, and monocyte function are believed to play important roles in accelerating atherosclerosis.6 Moreover, these cells have been shown to stimulate oxidative modification of LDL in culture.7 8 9

The effect of LDL on endothelial cell function depends strongly on the degree of oxidative modification, although the definition of this parameter is variable. Cu2+-oxidized LDL, which has a high degree of oxidation, promotes cell injury and death.10 Oxidized LDL scavenges nitric oxide11 and inhibits synthesis of nitric oxide synthase.12 Oxidized LDL also increases the thrombotic character of the endothelium by increasing the production of type 1 plasminogen activator inhibitor.13 Minimally modified LDL increases monocyte adherence by induction of an adhesion molecule that is not ICAM-1.14 More recently, electronegative LDL and MDA-lysine-modified LDL have been detected in the plasma of individuals who are at increased risk for atherosclerosis.15 16 17 18 However, the effects of LDL are not restricted to oxidized forms alone, as native LDL has also been shown to perturb endothelial cell function in vitro in ways that are consistent with early changes in vascular function in hypercholesterolemia. In vitro, native LDL increases endothelial cell ICAM-1 production,19 endocytotsis,20 and permeability21 and induces nitric oxide synthase to generate superoxide anion.22 Such changes in function coincide with increased monocyte adherence,2 vascular permeability,23 24 and increased vascular tone.25 26 27 28 29 These reports suggest that native LDL and oxidized LDL likely play distinct but interdependent roles in the progression of atherosclerosis and that endothelial cell responses are influenced by the degree of LDL oxidative modification.30

In this study, we have investigated the ability of endothelial cells to affect lipid hydroperoxide(LOOH) accumulation in LDL as a function of LOOH content. We demonstrate that endothelial cells are very effective at limiting LOOH accumulation in LDL and that small increases in LOOH content of "native" LDL may represent a critical trigger for increasing endothelial cell oxidation of LDL.


*    Methods
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 *Methods
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Materials
M199, FBS, heparin, HEPES, NaCl, EDTA, cholesterol oxidase kit (Cat. No. 352-20), potassium iodide, antibiotics, and mycotics were from Sigma Chemical Company. Butylated hydroxytoluene (BHT), thiobarbituric acid, MDA, chloroform, methanol, acetic acid, copper dichloride, potassium iodide, and cadmium acetate were from Aldrich Chemical Company. The Limulus amebocyte assay kit (Cat. No. QCL-1000) was from Whittaker Biochemical. Primeria culture dishes were purchased from Falcon Becton-Dickinson. Dulbecco's Phosphate-Buffered Saline (DPBS) was from Gibco BRL. Human recombinant basic fibroblastic growth factor (bFGF) was kindly provided by John A. Thompson (University of Alabama at Birmingham).

Methods
Endothelial Cell Isolation and Culture
Human umbilical vein endothelial cells were extracted by using collagen digestion and cultured as described.19 22 31 32 Endothelial cell cultures were maintained in M199 media containing FBS (16.7%), heparin (90 µg/mL), HEPES (20 mmol/L), pH 7.4, antibiotics and mycotics, and human recombinant bFGF (10 ng/mL).19 22

Low-Density Lipoprotein Isolation
Fresh, nonfrozen human plasma (3 to 4 units) was obtained from the Blood Center of Southeastern Wisconsin. LDL was isolated by sequential density ultracentrifugation (d=1.019 to 1.063 g/mL) using sterile techniques.19 22 31 32 LDL was isolated in the absence of added antioxidants except where noted. LDL was dialyzed against two changes of NaCl (150 mmol/L) containing EDTA (0.27 mmol/L, 2 L) and one change of M199 (1 L). Experiments were conducted within 5 days of isolation. In some studies, the pooled plasma was divided into two equal portions, and BHT was added to one portion at a final concentration of 20 µmol/L. BHT-protected LDL was dialyzed against 2 changes of NaCl (150 mmol/L) containing EDTA (0.27 mmol/L, 2 L) and 20 µmol/L BHT and one change of M199 (1 L) without BHT. Each pool was processed independently to prevent cross-contamination of BHT. Cholesterol was determined with the cholesterol oxidase kit from Sigma Chemical Company. References to LDL concentrations in the present work are in terms of milligrams of cholesterol. Endotoxin levels were measured with the limulus amebocyte assay kit from Whittaker Biochemical. Endotoxin concentrations were found to be lower than the concentrations required to activate endothelial cells (<0.01 EU/mL).19

Thiobarbituric Acid–Reactive Substances
The oxidation state of LDL was determined by a modified thiobarbituric acid–reactive substances (TBARS) assay, as was previously described.19 31 Briefly, apo B–containing lipoproteins were isolated by precipitation with phosphotungsate-MnCl2 before the addition of thiobarbituric acid. MDA equivalents were quantified on a CytoFluor II (PerSeptive Biosystems). Results are expressed as nanomoles of MDA per milligram.

Iodometric Assay
LOOH was determined by the iodometric assay as described by Girotti and coworkers.33 34 In this assay, care was taken to minimize exposure of extracted samples to oxygen. All solutions were purged with nitrogen before use, and the assay was performed under dimmed lights. LOOH measurements were determined on LDL in complete medium. Briefly, 0.001 mL of EDTA (0.5 mol/L) and 0.8 mL of chloroform:methanol (2:1) were added to 0.5 mL of sample in a 1.5-mL microfuge tube. The tubes were vortexed at high speed for 45 seconds and then centrifuged at 14,000 RPM for 5 minutes to separate organic and aqueous phases. A 0.3-mL aliquot of the organic phase (containing LDL lipids) was placed into a new centrifuge tube and then evaporated to dryness under a constant stream of nitrogen. Next, 0.02 mL of potassium iodide (1.2 g/mL) was added to the dried samples, followed by 0.3 mL of chloroform:acetic acid (2:3). Each tube was then purged with nitrogen, vortexed, and incubated for 10 minutes at room temperature. Next, 0.9 mL of cadmium acetate (0.25%) was added, and the tubes were vortexed. The tubes were centrifuged for 5 minutes at 14,000 RPM to separate aqueous and chloroform layers. LOOH stoichiometrically reduces iodide to tri-iodide, which can be quantified in the aqueous layer by using the 353-nm extinction coefficient (22.5 mmol/L-1 cm-1). Results are reported as nanomoles of LOOH per milligram.

Conjugated Diene Formation
Oxidation of polyunsaturated lipids rearranges isolated double bonds to form conjugated dienes that absorb strongly at 234 nm. Aliquots of LDL in complete medium were mixed with 1 mL of phosphate buffered saline (final concentration of 5 mg/dL). Absorbances were read in a quartz cuvette on a Beckman DU-640 UV/VIS spectrophotometer.

Experimental Protocols
LDL was incubated under standard tissue culture conditions (5% CO2, 95% air, 100% humidity at 37°C) in M199 containing FBS (16.7%) in Primeria 100-mm dishes, unless otherwise noted. The dishes contained either no cells or confluent endothelial cell monolayers. For the studies here, LDL was added at a final concentration of 240 mg/dL.

To increase rates of oxidation during culture, confluent endothelial cell cultures in six well cluster plates were exposed to LDL medium containing increasing concentrations of CuCl2 (0 to 4 µmol/L). After 24 hours, aliquots of the Cu2+/LDL medium were removed and analyzed for changes in LOOH. To determine whether Cu2+/LDL exposure permanently impaired the ability of endothelial cells to limit LOOH accumulation, the cultures were preconditioned with LDL alone or with LDL containing 4 µmol/L Cu2+ for 24 hours. To control for the possibility that residual Cu2+ may have remained bound to endothelial cell proteins in the experimental dishes, control monolayers, incubated with LDL medium, were briefly supplemented with 4 µmol/L Cu2+. Next, all cultures were washed four times with DPBS and then fed fresh LDL in medium without Cu2+. After 18 hours, aliquots were removed for LOOH measurements.

To determine whether the initial concentrations of LOOHs in LDL played a role in modulating the ability of endothelial cells to limit LOOH accumulation, monolayers were exposed to mildly oxidized LDL. Fresh LDL medium was incubated under standard tissue culture conditions for 24 hours to increase LOOH content. The next day, this oxidized LDL and fresh LDL were mixed to yield interrelated pools containing a range of LOOH concentrations. After mixing, aliquots were removed for LOOH measurements. The interrelated pools were then incubated in the absence and presence of endothelial cells. After 24 hours, aliquots were removed and assayed for LOOH content.

Statistics
Unless otherwise indicated, results are mean±SD. The Student's one-tailed t test was used to determine whether data were significantly different.


*    Results
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 *Results
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Endothelial Cells Prevent Oxidative Modification of LDL
To determine the effect of endothelial cells on LDL oxidation, LDL (240 mg/dL) was incubated in the presence and absence of endothelial cells. In the absence of endothelial cells, the concentrations of TBARS, LOOHs, and conjugated dienes markedly increased in comparison to starting concentrations (Table 1Down). The presence of endothelial cells prevented the accumulation of TBARS, LOOH, and conjugated dienes, a result indicating that endothelial cells are either preventing or removing LOOH from LDL.


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  		Table 1. Effect of Endothelial Cells on LDL Oxidation

These changes in LDL oxidative modification, induced by incubation under standard tissue culture conditions, can be attenuated by adding BHT, a chain-breaking antioxidant, to the plasma before LDL isolation. As can be seen in Table 2Down, BHT markedly decreased LOOH accumulation but did not eliminate it.


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  		Table 2. Effects of BHT on the Accumulation of LOOHs in LDL

Modulation of LOOH Accumulation in LDL by Cu2+
It has previously been demonstrated that the oxidation of LDL by cell culture medium depends on the content of redox-active transition metal ions, ie, Fe3+ and Cu2+.1 To enhance the pro-oxidant nature of the culture medium, M199 was supplemented with increasing concentrations of Cu2+. LOOH content increased as a function of Cu2+ concentration in both cell and cell-free conditions (Fig 1Down). At low Cu2+ concentrations (<0.75 µmol/L), cell-free incubations generated higher concentrations of LOOHs than were generated when cells were present. However, at higher concentrations of Cu2+, the presence of cells increased the yield of LOOHs (Fig 1Down).



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  		Figure 1. Effect of Cu2+ on endothelial cell–dependent LOOH accumulation. LDL (240 mg/dL) was incubated in M199 containing FBS (16.7%) for 24 hours with Cu2+ in the presence (solid bars) or absence (open bars) of endothelial cells. Samples were removed and analyzed for LOOH content. Data represent the mean of duplicate experiments. Bars indicate the spread of raw data points from the mean.

Effects of Cu2+/LDL Preconditioning on Accumulation of LOOHs in LDL
To determine whether preconditioning endothelial cells with Cu2+/LDL permanently altered the ability of the cells to limit LOOH accumulation, cultures were incubated with LDL in the presence and absence of Cu2+ (4 µmol/L) for 24 hours. The monolayers were washed and then incubated with fresh LDL medium without added Cu2+. After preconditioning in Cu2+/LDL, endothelial cells were still able to limit LOOH formation at 18 hours in a manner that was indistinguishable from that of endothelial cells exposed to LDL alone (Fig 2Down). These data indicate that the ability of endothelial cells to limit LOOH accumulation is not lost by preincubation with Cu2+/LDL.



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  		Figure 2. Effect of Cu2+ on endothelial cell–dependent LOOH accumulation. LDL (240 mg/dL) was incubated with M199 containing FBS (16.7%) for 18 hours in the absence of endothelial cells (solid bars) or in the presence of endothelial cells that had been pretreated for 24 hours with LDL (240 mg/dL, open bars) or with LDL (240 mg/dL) medium containing Cu2+ (4 µmol/L, hatched bars). Samples were removed, and LOOH concentrations were determined. Data represent mean±SD of three experiments performed in triplicate. Comparisons were to LDL incubated without endothelial cells (**P<.01).

Effects of LOOH Content in LDL Medium on the Ability of Endothelial Cells to Limit LOOH Accumulation
To determine whether the initial concentration of LOOHs in LDL played a role in modulating the ability of endothelial cells to limit LOOH accumulation, LDL was oxidized by incubation in the absence of cells. This increased the LOOH content in LDL to {approx}33 nmol/mg. Oxidized LDL was mixed with fresh LDL to yield interrelated pools that contained a range of LOOH concentrations ({approx}2.5 to 33 nmol/mg; solid bars, Fig 3Down). After 24 hours, the difference between the initial and postincubation LOOH concentrations in the presence of cells was nearly equal (hatched bars, Fig 3Down). Endothelial cells prevented LOOH accumulation in pools 1, 2, and 3 (open bars, Fig 3Down). Interestingly, endothelial cells were unable to reduce LOOHs below what was initially present. In contrast to data for pools 1 to 3, endothelial cells incubated with pool 4 increased rather than prevented LOOH formation. These data show that endothelial cells attenuated increases in LOOH accumulation as long as the initial LOOH concentrations did not exceed a critical value, in this case somewhere between 22 and 33 nmol/mg. If the starting concentration of LOOHs was higher than this critical value, then endothelial cells promoted LOOH accumulation.



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  		Figure 3. Effect of LDL-LOOH content on endothelial cell–dependent LDL oxidation. LDL (240 mg/dL) was preincubated with M199 containing FBS (16.7%) for 24 hours in the absence of endothelial cells. The preincubated LDL, which contained an initial LOOH concentration of 33 nmol/mg, was diluted with fresh LDL medium to yield interrelated pools containing initial LOOH concentrations of 2.5, 11, 22, and 33 nmol/mg (solid bars). The interrelated pools were then incubated without endothelial cells (hatched bars) and with endothelial cells (open bars). Samples were removed after 24 hours, and LOOH content was determined. Data represent mean±SD (n=3). Comparisons were LOOH content in LDL medium incubated without endothelial cells versus LDL medium incubated with endothelial cells (*P<.05, **P<.01).


*    Discussion
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 *Discussion
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This report shows that endothelial cells play an important role in limiting LOOH accumulation in LDL. Furthermore, when the LOOH content of LDL exceeds a critical value, endothelial cells accelerate LOOH accumulation. The culture conditions that were used in the present study are in contrast to early studies using low LDL concentrations and low or no serum.1 35 It is well recognized that hypercholesterolemic concentrations of LDL (>160 mg/dL) are toxic to endothelial cells in the absence of serum. Consequently, serum was used as a supplement in all the experiments reported here. Serum, in cell-free conditions, markedly reduced the rate of LDL oxidation (data not shown) but did not prevent it (see Table 1Up). It has been previously noted that serum was able to limit the ability of cells to oxidize LDL when LDL is present in lower concentrations.36 37 As a result, most oxidation studies have been performed in the absence of serum. The mechanism by which serum prevents oxidation is unknown but may include the presence of antioxidants, chelation of transition metal ions in cell culture media, or the presence of HDL.38

In this study, cultured human umbilical vein endothelial cells were incubated with a LDL cholesterol concentration (240 mg/dL) that is associated with hypercholesterolemia. In agreement with previous reports, essentially no LDL oxidation was observed in LDL medium that was exposed to endothelial cells.19 22 The culture conditions were not found to be cytotoxic, probably because M199, in combination with high concentrations of serum (16.7%), is well known for inhibiting oxidation. Under these conditions, the ratio of LDL to endothelial cells could be increased to approximately 4 to 6 mg/106 cells, which improved our ability to determine whether the endothelial cell cultures induced subtle changes in LDL oxidation states. Paradoxically, LDL that was incubated with endothelial cells for 24 hours contained lower amounts of peroxides than did LDL incubated in cell-free conditions. Thus, endothelial cells were preventing the accumulation of LOOHs and/or increasing the removal of LOOHs from LDL particles. The fact that cell-free LDL oxidation was inhibited by BHT indicates that LOOH accumulation occurred by a propagatory mechanism.

These data suggest that endothelial cells have one or more mechanisms to limit LDL oxidation. Endothelial cells may prevent LOOH accumulation by the generation of nitric oxide39 or the release of thiols40 or may remove LOOHs by metabolism of phospholipid hydroperoxide by glutathione peroxidase.41 42 Such protective mechanisms may be overwhelmed, however, if the rate of LDL oxidation is faster than the ability of the cells to limit LOOH accumulation. For example, in Fig 3Up, endothelial cells were able to hold the accumulation of LOOHs in LDL constant as long as initial concentrations of LOOH were low. When the initial LOOH concentrations were slightly higher, oxidative modification of LDL by endothelial cells increased by what appeared to be an autocatalytic mechanism. These findings may have important implications for atherogenesis in that circulating levels of electronegative LDL16 and MDA-modified LDL18 have been detected in plasma and increased levels of "catalytic" metal ions were found in atherosclerotic "gruel"43 and mechanically injured vessels.44

Endothelial cell–dependent LDL oxidation has an absolute requirement for transition metal ions in the cell culture medium.1 45 Most media, including the M199 used here, are often formulated with Fe3+ salts that, in combination with reducing agents such as ascorbate and cysteine, represent a low-level oxidative stress. When the transition metal content of M199 was increased by addition of Cu2+, endothelial cell activity switched from inhibiting LOOH accumulation to promoting LDL oxidation. Endothelial cells began to promote LDL oxidation at a Cu2+ concentration of 0.75 µmol/L and above. Below 0.75 µmol/L Cu2+, endothelial cells were still able to limit LOOH accumulation. This suggests that endothelial cells have both mechanisms for LOOH removal and mechanisms for lipid oxidation. The outcome in cell function depends strongly on the Cu2+ concentration in the cell culture medium.

Two possibilities for how Cu2+ modulates the behavior of endothelial cells are that Cu2+ permanently alters the ability of endothelial cells to limit LOOH accumulation and that Cu2+ partially oxidizes LDL lipid to form LOOH and that this LOOH-loaded LDL is then more susceptible to cellular oxidation. To test the first of these hypotheses, endothelial cells were pretreated with Cu2+/LDL or LDL alone for 24 hours. We found that when the cells that had been pretreated with Cu2+/LDL were fed fresh LDL media without Cu2+, they behaved in a manner that was identical to that of control cells treated with LDL alone. To test the second hypothesis—that LOOH-loaded LDL is more susceptible to oxidation—LDL was preoxidized by incubation for 24 hours in the absence of cells. Endothelial cells promoted LDL oxidation only when they were presented with LDL that contained 33 nmol LOOH/mg. However, if cells were presented with LDL containing lower LOOHs (2.5 to 22 nmol LOOH/mg), accumulation of LOOH could still be prevented.

Endothelial cell metabolism of oxidized lipids is gaining acceptance as an important antiatherogenic mechanism. To date, endothelial cells have been shown to metabolize individual phospholipid and cholesterol ester hydroperoxides46 47 and, under certain conditions, to reduce the oxidation status of LDL.40 LOOH in LDL may be removed and metabolized by a phospholipase A2/peroxidase pathway.42 45 46 In contrast, endothelial cell oxidation of LDL has been rigorously examined. The kinetics of transition metal ion–dependent LDL oxidation have been shown to depend strongly on the starting LOOH content of LDL.33 Reducing agents generated by the endothelium can reduce transition metal ions, that will, in turn, ultimately accelerate Cu2+-dependent LDL oxidation.40 48

The physiological consequences of these observations are that endothelial cells may represent a mechanistic barrier against the buildup of LOOHs in both plasma and interstitial LDL. In the presence of an additional oxidative stress, however, endothelial cells may promote LDL oxidation, which could accelerate foam cell formation. It is intriguing to consider that the additional oxidative stress may come from the endothelium itself after chronic exposure to high LDL concentrations. We have previously demonstrated that high concentrations of LDL stimulate endothelial cells to generate superoxide anion by uncoupled endothelial nitric oxide synthase activity.22 LDL induction of nitric oxide synthase superoxide anion was also found to increase the formation of peroxynitrite, a well-recognized oxidant that is capable of lipid peroxidation,49 DNA damage,50 and nitration of LDL proteins.51 By this mechanism, LDL may play an important role in inducing endothelial cells to promote LDL oxidation. This possibility remains to be determined.


*    Acknowledgments
 
This work was supported in part by National Institutes of Health grant 48251 (to K.A.P.), HL47250 (to B.K.), and GM55792 (to N.H.) and American Heart Association, Wisconsin Affiliate grant 95-GB-56 (D.M.S.). The authors thank Dr John A. Thompson (University of Alabama at Birmingham, Birmingham, Ala) for providing recombinant human bFGF, the Birthing Center at Waukesha Memorial Hospital for umbilical cords, and the Blood Center of Southeastern Wisconsin for human plasma. We also thank Michelle Curtis and Carmen Torres for their technical assistance.

Received March 20, 1997; accepted June 11, 1997.


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 *References
 

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