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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3639-3645.)
© 1997 American Heart Association, Inc.

Articles

Fibroblasts That Overexpress 15-Lipoxygenase Generate Bioactive and Minimally Modified LDL

Farhad Sigari; Chris Lee; Joseph L. Witztum; ; Peter D. Reaven

From the Division of Endocrinology and Metabolism, Department of Medicine, University of California at San Diego, La Jolla, Calif.

Correspondence to Peter Reaven, Division of Endocrinology and Metabolism, Department of Medicine, 0682, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0682.

	Abstract

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Abstract Several lines of evidence suggest that the cellular enzyme 15 lipoxygenase (15-LO) may be important in promoting the oxidation of lipoproteins in vivo. In previous studies we have shown that fibroblasts transfected with 15-LO "seed" LDL with lipoperoxides such that subsequent oxidation readily generates an LDL that is taken up by macrophages through scavenger receptors. We now demonstrate that LDL incubated with 15-LO cells is "minimally modified" and has bioactive properties. Characterization of LDL incubated with 15-LO cells reveals that lipid peroxidation is modest, with low levels of TBARS generated (12.6±4.7 nmole MDA per mg protein) and small amounts of 18:2 lost as a result of oxidation (7%, compared with extensive loss [82%] with copper oxidation). The 15-LO–conditioned LDL showed mildly increased electrophoretic mobility on agarose gels, and on polyacrylamide gels it showed only mild protein degradation compared with copper-oxidized LDL. Additionally 15-LO–conditioned LDL competed very well for the LDL receptor of fibroblasts but did not compete for macrophage uptake of ¹²⁵I-acetylated LDL. Importantly, compared with LDL incubated on ß-galactosidase (lac Z)–transfected control cells, LDL incubated on 15-LO cells stimulated monocyte chemotaxis (15-LO–LDL, 6.9±1.2 monocytes per field versus lac Z-LDL, 0±0.9 monocytes per field) and when added to endothelial cells enhanced adhesion (15-LO–LDL, 31.1±5.0 monocytes per field versus lac Z–LDL, 0±2.0 monocytes per field). Preincubation of 15-LO cells with 15-LO inhibitors significantly inhibited the generation of bioactive LDL. Lipid extracts of LDL conditioned on 15-LO cells showed chemotactic activity not related to lysophosphatidylcholine levels. Preincubation of target endothelial cells with several different platelet-activating factor receptor antagonists prevented stimulation of monocyte adhesion by 15-LO–conditioned LDL. When probucol- or vitamin E–enriched LDL was incubated with 15-LO cells it was less oxidized and less bioactive, which suggests that these cells seed LDL with LOOH, which then requires further propagation of lipid peroxidation to yield bioactivity. These studies demonstrate that fibroblasts expressing 15-LO reliably produce a bioactive "minimally modified" LDL, which may explain in part how cellular 15-LO activity may generate atherogenic LDL in vivo.

Key Words: chemotaxis • monocyte adhesion • modified LDL • lipid peroxidation • autoantibodies

	Introduction

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Strong evidence now suggests that oxidation of LDLs may play an important role in early atherosclerotic lesion formation.^{1 2 3} Recently it has become clear that the extent of LDL oxidation may determine which of a multitude of potential atherogenic properties of oxidized LDL predominate at a given point in time. When LDL is less oxidized, ie, mm-LDL, it stimulates monocyte chemotaxis, transmigration, and adherence to endothelial cells.^{4 5 6 7} mm-LDL also stimulates expression of a number of growth factors such as M-CSF.^{7 8} These and other properties of mm-LDL are undoubtedly important in initiating the earliest steps of atherosclerosis. On the other hand, when LDL is more extensively oxidized it is taken up more rapidly by macrophage scavenger receptors,^{1 9} which forms foam cells. Extensively oxidized LDL is also toxic to cells, which could contribute to artery wall inflammation and injury.¹⁰ Presumably, in the very early stages of lesion formation predominantly mm-LDL exists, whereas in more advanced lesions there is a broad spectrum of oxidized LDL, ranging from mm-LDL to extensively oxidized LDL.

In vitro, numerous modalities have been used to generate moderately and more extensively oxidized LDL, including transition metals, hemin, thiols, ceruloplasmin, myeloperoxidase, and a variety of cell types and smooth muscle–endothelial cell cocultures.^{2 5 7 9 11 12} However, the mechanisms underlying cell-mediated oxidation of LDL in vivo are still not fully understood. It is likely that several different mechanisms are involved. Among these possibilities, we and others have proposed that 15-LO plays an important role in vivo, and there are many lines of evidence in support of this concept: (1) purified soybean lipoxygenase and mammalian 15-LO can oxidize LDL^{13 14} ; (2) the macrophage, a predominant cell in early atherosclerosis lesions, contains 15-LO, and endothelial cells have 12-LO or 15-LO activity via a leukocyte type 12-LO^{15 16} ; (3) murine fibroblasts transfected with human 15-LO have an enhanced capacity to enrich LDL with lipid hydroperoxides^{17 18} ; (4) Activity of 15-LO is present in animal and human fatty streak lesions, and 15-LO mRNA and protein have also been demonstrated in high levels in atherosclerotic lesions, frequently colocalized with oxidized LDL^{14 19 20} ; (5) transfer of the human 15-LO gene to rabbit iliac arteries results in the appearance of oxidized LDL epitopes²¹ ; and (6) the specific stereoisomer products of 15-LO–induced oxidation of esterified linoleate have been demonstrated in both rabbit and human lesions in greater excess than would be expected by nonenzymatic oxidation.^{14 22}

Recently we prepared murine fibroblasts that overexpressed 15-LO and demonstrated that LDL incubated with these cells was enriched with lipid hydroperoxides and was uniquely susceptible to further oxidative stress, such as exposure to copper for short intervals, eventually resulting in apo B-100 modification and subsequent recognition and uptake by macrophage scavenger receptors.¹⁷ The present study demonstrates that incubation of LDL with 15-LO fibroblasts generates an mm-LDL that is bioactive and stimulates both monocyte chemotaxis and monocyte adhesion to endothelial cells.

	Methods

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LDL Isolation
LDL (d=1.022 to 1.063) was isolated by density-gradient ultracentrifugation from pooled human plasma containing a final concentration of gentamicin 0.22 mmol/L, chloramphenicol 0.15 mmol/L, D-phenylalanyl-L-prlyl-L-arginine chloro-methyl ketone 1 µmol/L, benzamidine 2 mmol/L, and 300 µmol/L EDTA.⁹ For selected experiments, LDL containing probucol or vitamin E was isolated from plasma of patients taking probucol (1 g/d) or vitamin E (800 IU/d). After isolation, LDL was stored at 4°C in the dark and was used within 2 weeks. EDTA was removed from the LDL just before each experiment by dialysis for 20 hours at 4°C in the dark with two 6-L changes of PBS.

LDL Oxidation
EDTA-free LDL was incubated in the presence of Cu²⁺ 5 µmol/L at 37°C in PBS for 4 hours or on fibroblasts for 20 hours, and TBARS were measured by the method of Yagi.²³

Cell Culture Procedures
Murine fibroblasts expressing high levels of intracellular 15-LO or control fibroblasts expressing ß-galactosidase (lac Z) were established by infection with a retroviral vector as previously described^{17 18} and were used between passages 10 and 15. Cells were grown in DMEM with high glucose (10 mmol/L), containing 10% FCS and G418 sulfate 50 µg/mL at 37°C and in 5% CO₂. The activity of 15-LO in cells was previously determined by the rate of conversion of [¹⁴C]linoleic acid to 13-HODE^{17 18} and found to be 10-fold to 20-fold greater than in control transfected fibroblasts. Fibroblasts were plated on 96-well plates at 35 000 cells per well and grown for 2 days until approximately confluent. The cells were washed free of serum, and LDL (250 µg/mL) was then incubated with the fibroblasts at 37°C for 20 hours in Ham's F-10 medium. In some experiments the 15-LO inhibitors PD 146176 (a generous gift from Joe Cornicelli, Park Davis) and EYTA (Cayman Chemical Co) were added over a range of concentrations to the fibroblasts for 5 hours and the cells were washed before LDL was added in fresh medium. In other experiments EDTA was added (10 or 50 µmol/L) with the LDL to the fibroblasts. As described below, even 50 µmol/L EDTA did not cause detachment of cells. The extent of LDL modification was determined by measures of lipid peroxidation, changes in protein structure, and bioactivity (described below).

Porcine aortic endothelial cells, a generous gift from Dr Mohamad Navab (UCLA), were cultured at 37°C and in high-glucose DMEM (4.5 g/mL) with 15% FCS, penicillin-streptomycin (100 µg/mL), and 2 mmol/L L-glutamine. The endothelial cells were used up to passage 10.

Human monocytes were isolated from blood collected in 4 mmol/L EDTA. A monocyte-enriched fraction was isolated by density ultracentrifugation at 22°C using Histopaque 1077 (Sigma Chemical Co). The cells were then plated in RPMI 1640 (Biowhittaker)+10% homologous serum for 3 hours at 37°C. Nonadherent cells were washed off, and the adherent monocytes were released by use of PBS containing 0.18% EDTA and were then washed twice in PBS.

Monocyte Chemotaxis Assay
Assays were performed in chemotaxis chambers (Neuro Probe Inc.) with a polycarbonate filter (Poretics) that had a 5-µm pore size used to separate the upper and lower chambers. The lower wells were filled with 28 µL of supernatant (diluted 1:20 in 0.1% BSA–Tyrode's salt solution) from the fibroblast incubation experiments, and the chambers were treated as previously described by Berliner et al.⁴ The monocytes that migrated from the upper chamber to the lower surface of the filter were then counted using a light microscope and expressed as cells per high power field. The results of at least 4 to 8 wells were averaged for each experimental condition. In some experiments we separated conditioned LDL from the aqueous supernatant by passing the incubation mixture through a membrane cone with a molecular weight cutoff of 25 000 (Amicon). Both the LDL (resuspended in Tyrode's salt solution) and ultrafiltrate were then tested individually for chemotaxis activity. In additional experiments we extracted the lipid from conditioned LDL samples using a chloroform-methanol extraction.²⁴ The chloroform-methanol extract was dried down under nitrogen and resuspended in ethanol to test for chemotactic activity as described above. An aliquot was also subjected to thin-layer chromatography to measure lysoPC content as described.²⁵ LysoPC was identified by comparison to a known standard and quantified by determination of phosphorous content.²⁶

Monocyte Adhesion Assay
The assay, with minimal modifications, was carried out as described by Navab et al.⁶ LDL conditioned in F-10 medium alone or by fibroblasts in F-10 medium for 20 hours was transferred to confluent porcine aortic endothelial monolayers in 96-well tissue-culture plates, and the plates were incubated for 4 hours at 37°C. In some experiments PAF-receptor antagonists were added at 10 µmol/L in an ethanol-DMSO solution (<1%) to endothelial monolayers 30 minutes before conditioned LDL was added. Specific PAF-receptor antagonists that were tested included Lau 0203, Lau 0603, and BN 50730. After the 4-hour incubation the supernatant was removed and the endothelial monolayers were washed twice with RPMI 1640. THP-1 cells (a monocyte-like cell line) were placed on the endothelial cells at 40 000 cells per well, and the plates were incubated for 20 minutes at 37°C. The suspension was removed, and the cells were vigorously washed (at least three times) to remove all but the firmly adherent THP-1 cells. The number of adherent THP-1 cells was determined in four high-power fields per well, and the results of 6 to 8 separate wells were averaged for each experiment.

Competition for LDL Receptor–Mediated Cell Association and Degradation
Competition assays for total cell-association (binding and internalization) of ¹²⁵I-LDL with human fibroblasts and degradation of ¹²⁵I-LDL by human fibroblasts was performed as previously described.²⁷ Iodination of a standard LDL preparation was performed by the method of Salacinski et al²⁸ using carrier-free ¹²⁵Nal and iodogen (Pierce Chemical). Subsequent acetylation of the labeled LDL was performed as described by Goldstein et al.²⁹ Fibroblasts were grown to near confluence in DMEM and 10% FCS on 24-well plates (Nunc). The cells were washed 3 times and then plated at 37°C overnight in DMEM and 1% lipoprotein-deficient serum (LPDS) and 20 mmol/L HEPES. The following morning the cells were washed 3 times with the same medium and the final wash was left on the cells for 30 minutes. We then determined binding of ¹²⁵I-labeled LDL in the absence or presence of various amounts of unlabeled LDL competitors. The ¹²⁵I-LDL and cold competitors (15-fold to 20-fold excess) were mixed and then added to the cells or the no-cell control wells. After 4 hours at 37°C the supernatant was removed and the cells were washed three times with PBS containing 1% BSA and two times with PBS only. The washed cells were solubilized with 0.2 N NaOH, and the total amount of "cell associated" ¹²⁵I-LDL was counted in the gamma counter. The supernatant was also harvested, and the amount of ¹²⁵I-LDL degradation was determined from TCA-soluble iodine free counts in the medium.³⁰ We thus tested for competition for both LDL receptor–mediated cell association and degradation.

Competition for Scavenger Receptor Binding
Mouse resident peritoneal macrophages were isolated by peritoneal lavage and plated in RPMI with 10% FCS.³⁰ After being incubated overnight at 37°C, nonadherent macrophages were removed by washing with DMEM containing 20 mmol/L HEPES and 1% human LPDS. The competition assay was performed in the manner described above except that the labeled LDL was Ac-LDL and the competition assay was performed at 0°C for 4 hours. We determined binding of ¹²⁵I-labeled Ac-LDL in the absence or presence of various amounts of unlabeled LDL competitors. The cells were dissolved overnight in 0.2 N NaOH and removed from the plate, and bound ¹²⁵I–Ac-LDL was counted using a LKG Wallace 1282 Universal Gamma Counter. Counts were corrected for cell protein per well and nonspecific binding of labeled LDL.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Because previous studies suggested that LDL modified by 15-LO overexpressing fibroblasts was only modestly oxidized, we set out to establish whether these cells might in fact generate a form of mm-LDL. This necessitated showing that the modified LDL had both modest lipid oxidation and limited protein modification.² Most importantly, we needed to determine whether LDL incubated with 15-LO cells was proinflammatory. We therefore compared the properties of LDL incubated with 15-LO cells with LDL incubated with control (lac Z) cells.

Lipid Peroxidation
While native LDL yielded values of <2 nmole MDA per milligram LDL protein when measured in the TBARS assay, values of LDL samples incubated on 15-LO cells (12.6±4.7 nmoles MDA per milligram protein) were slightly but not significantly higher than LDL from lac Z incubations (10.1±4.2 nmoles MDA per milligram protein). By comparison, LDL incubated with copper (5 µmol/L) for as little as 4 hours consistently generated TBARS in the range of 40 to 60 nmole MDA per milligram LDL protein. In some experiments LDL conditioned on 15-LO or lac Z cells was also analyzed for the extent of reduction in polyunsaturated fatty acids. As shown in the Table, LDL incubated for 20 hours on either set of fibroblasts led to modest decreases in arachidonic acid (<35%) and to a minimal loss in linoleic acid (<7%). In contrast, copper-oxidized LDL showed extensive reductions in both of these polyunsaturated fatty acids. As demonstrated previously,^{17 18} incubating LDL along with increasing amounts of EDTA on these fibroblasts led to progressively greater inhibition of lipid peroxidation in the LDL (data not shown). This was not a result of EDTA-induced cell toxicity or release of cells, as they were healthy appearing and remained firmly adhered throughout the incubation periods as assessed by cell protein assay determinations. This is consistent with previous work, which suggested that 15-LO cells seeded LDL with low levels of lipid peroxides, which then required subsequent proton abstraction and propagation of lipid peroxidation facilitated by transition metals.¹⁷

View this table: [in this window] [in a new window]   Table 1. Percent Distribution of Fatty Acids in LDL

Protein Modification
The extent of protein modification in 15-LO–conditioned LDL was also modest; electrophoretic mobility on agarose gels was at most slightly greater than that of LDL incubated in medium alone and was substantially less than that of LDL treated with copper for as little as 4 hours. Electrophoresis of 15-LO–modified LDL on polyacrylamide gels revealed a slight breakdown of apo B-100 compared with native LDL, whereas copper-oxidized LDL was extensively degraded and was no longer effectively stained by Coomassie brilliant blue, as has been noted previously.³¹ A silver stain of a polyacrylamide gel containing the same copper-oxidized LDL documented many new lower molecular weight bands, consistent with extensive breakdown (data not shown). To determine whether cell-mediated 15-LO modification of LDL decreased its binding and uptake by the LDL receptor or enhanced its binding by the scavenger receptor, we measured the ability of 15-LO–conditioned LDL to compete for native ¹²⁵I-LDL binding to fibroblasts and ¹²⁵I–Ac-LDL binding to macrophages. As shown in Fig 1A, native LDL, LDL incubated in medium alone, and 15-LO–modified LDL were able to extensively compete for native LDL cell association (binding plus internalization) with human fibroblasts. In contrast, Ac-LDL competed poorly for native LDL binding and/or internalization by the LDL receptor. LDL conditioned on 15-LO cells was also very effective in competing for fibroblast degradation of native LDL (Fig 1B), whereas Ac-LDL was not. On the other hand, 15-LO–modified LDL competed poorly for macrophage scavenger receptor binding of Ac-LDL, with minimal competition observed only when added at the highest concentration (Fig 2). Native LDL and LDL incubated in media alone also failed to compete for scavenger receptor uptake, whereas unlabeled Ac-LDL competed as expected. These experiments confirm that 15-LO–modified LDL has undergone only mild protein modification, consistent with a mm-LDL.^{2 3}

View larger version (27K): [in this window] [in a new window]   Figure 1. Competition experiments to determine the extent of LDL receptor recognition of LDL incubated with 15-LO cells. Cell association (binding and internalization) and degradation by human fibroblasts of various modified LDL samples was determined by competition for labeled native LDL as described in "Methods." Each modified LDL competitor was added in increasing amounts up to 90 µg/mL or 15-fold to 20-fold the 125I-LDL tracer amount. Unlabeled competitors included native LDL (circle), LDL conditioned in media alone (square), 15-LO LDL (triangle), and acetyl-LDL (diamond). Shown is a representative experiment. Each value is the average of 2 to 3 replicate determinations. Panel A shows results of LDL cell association; panel B, the extent of LDL degradation.

View larger version (17K): [in this window] [in a new window]   Figure 2. Competition experiments to determine the recognition of 15-LO–modified LDL by the scavenger receptor. The binding to mouse macrophages of modified LDL was determined by competition for labeled Ac-LDL as described in "Methods." Each modified LDL competitor was added in increasing amounts up to 90 µg/mL or 15-fold to 20-fold the 125I–Ac-LDL tracer amount. Unlabeled competitors included native LDL (circle), LDL conditioned in medium alone (triangle), 15-LO LDL (square), and Ac-LDL (diamond). Shown is a representative experiment. Each value is the average of two replicate determinations.

Bioactivity
These mild changes in the extent of lipid oxidation and protein modification of 15-LO–conditioned LDL were strikingly similar to those previously demonstrated for LDL that was minimally modified either by cell coculture or by other means.^{6 7 32 33} To assess whether 15-LO–modified LDL also had biological properties previously ascribed to mm-LDL, we tested conditioned LDL for bioactivity with two assays. LDL was either added directly to chemotaxis chambers to assess its chemotactic activity for monocytes or added to endothelial cells to determine whether it stimulated subsequent THP-1 cell adhesion. LDL incubated with the control lac Z cells was not more chemotactic than native LDL or LDL incubated in medium in the absence of cells. In contrast, LDL conditioned on 15-LO–transfected cells was significantly more chemotactic (Fig 3A). Media conditioned on 15-LO cells in the absence of LDL did not contain chemotactic activity. To further demonstrate that 15-LO activity was primarily responsible for generating bioactive LDL, we pretreated 15-LO cells with 15-LO inhibitors over a range of concentrations. In three different experiments both PD 146176 and ETYA nearly completely inhibited the formation of chemotactic activity in LDL conditioned on the treated cells (a representative experiment is shown in Fig 4). Levels of TBARS in LDL conditioned on cells pretreated with 15-LO inhibitors decreased as the concentration of 15-LO inhibitors increased. In the experiments described above, aliquots of the entire cell supernatant containing LDL were used for subsequent chemotaxis assays. Therefore, to demonstrate that the bioactivity resulted from LDL modification, we performed several additional experiments. We separated LDL conditioned on 15-LO cells or LDL conditioned in medium alone from the remaining aqueous supernatant using ultrafiltration. In each instance, the vast majority of the chemotactic activity resided within the LDL fraction. In further experiments lipid extracts from LDL conditioned on 15-LO cells but not lac Z cells contained as much chemotactic activity as the whole LDL particle (Fig 5). This was not due to greater lysoPC content in LDL conditioned on 15-LO cells. In three separate experiments lysoPC levels were equally low in both LDL conditioned on 15-LO cells (33.2±10.5 µg/mg LDL protein) and on lac Z (44.7±18.9 µg/mg LDL protein) cells but high in copper-oxidized LDL (118±51.9 µg/mg LDL protein, P<.05).

View larger version (42K): [in this window] [in a new window]   Figure 3. LDL stimulation of monocyte chemotaxis and adhesion. A, LDL was incubated on either 15-LO cells or control fibroblasts (lac Z) for 20 hours as described in "Methods" and then added to chemotaxis chambers. The lower wells were filled with 28 µL of supernatant (diluted 1:20 in 0.1% BSA–Tyrode's salt solution) from the fibroblast incubation experiments, and the chambers were treated as previously described by Berliner et al.4 The monocytes that migrated from the upper chamber to the lower surface of the filter were then counted using a light microscope and expressed as cells per high-power field (after subtracting values obtained from LDL incubated in medium in the absence of cells). The results of at least 4 to 8 wells were averaged for each experimental condition. Shown are mean±SE from seven experiments. B, Porcine aortic endothelial cells were treated for 4 hours with LDL (at 250 µg/mL in F-10) that had been incubated for 20 hours in medium alone or with 15-LO or lac Z fibroblasts. The cells were washed and then incubated with THP-1 cells for 20 minutes. Nonadherent THP-1 cells were vigorously washed off, and cells were fixed and counted by light microscopy. Values reflect mean±SE from six different experiments. *Significant at P<.01.

View larger version (47K): [in this window] [in a new window]   Figure 4. Effect of 15-LO inhibitors on LDL stimulation of monocyte chemotaxis. The 15-LO cells were pretreated with 15-LO inhibitors PD 146176 and ETYA for 5 hours as described in "Methods." LDL was incubated on 15-LO cells for 20 hours and added to chemotaxis chambers, and the assay was performed as described in Fig 3. The results of at least 4 to 8 wells were averaged for each experimental condition. NCC indicates LDL incubated in media in the absence of cells. Shown are mean±SD from a representative experiment.

View larger version (24K): [in this window] [in a new window]   Figure 5. Stimulation of monocyte chemotaxis by lipid extracts of LDL. Total lipids were extracted as described in "Methods" from LDL incubated on 15-LO or lac Z cells for 20 hours. The extract was redissolved in ethanol and added to chemotaxis chambers, and the assay was performed as described in Fig 3. The results of at least 4 to 8 wells were averaged for each experimental condition (after subtracting values obtained from LDL incubated in medium in the absence of cells). Values reflect the mean±SE from three different experiments. *Significant at P<.01.

When LDL conditioned on lac Z cells was added to endothelial monolayers, it failed to stimulate THP-1 cell adhesion to a greater degree than that of LDL incubated in medium alone (Fig 3B). In contrast, LDL incubated on 15-LO cells and then added to endothelial cells induced significantly greater adhesion of THP-1 cells above that of LDL incubated on lac Z cells or in F-10 media alone (Fig 3B).

However, development of bioactivity was dependent on propagation of lipid peroxidation in LDL. When EDTA was present during incubation of LDL with the 15-LO cells, generation of TBARS was inhibited, as was subsequent stimulation of THP-1 adhesion (Fig 6). Similarly, if LDL enriched with vitamin E or probucol was added to the fibroblasts, both lipid peroxidation (data not shown) and THP-1 adhesion was reduced (Fig 6). To rule out the possibility that this effect could be due to a direct inhibitor effect on endothelial cells by antioxidants transferred from LDL, we added vitamin E or probucol (10-25 µmol/L) in ethanol in the medium along with LDL conditioned on 15-LO cells. This did not reduce THP-1 cell adhesion.

View larger version (43K): [in this window] [in a new window]   Figure 6. Effect of EDTA and antioxidants on the ability of 15-LO–conditioned LDL to stimulate monocyte adhesion. Porcine aortic endothelial cells were treated for 4 hours with LDL (at 250 µg/mL in F-10) that had been preincubated for 20 hours on 15-LO fibroblasts. In some experiments EDTA at 10 or 50 µmol/L was added during the initial incubation on 15-LO cells; in other experiments LDL added to the fibroblasts was isolated from subjects taking either probucol (1 g/d) or vitamin E (800 IU/d). The cells were washed and incubated with THP-1 cells for 20 minutes. Nonadherent THP-1 cells were vigorously washed off, and cells were fixed and counted by light microscopy. Values reflect the mean±SE (as a percent of inhibition of monocyte binding compared with pooled LDL modified on 15-LO cells without EDTA or antioxidant enrichment) from 3 to 5 different experiments.

To determine whether the proinflammatory activity of 15-LO–conditioned LDL was at least in part related to the generation of PAF-like particles during oxidation, we tested whether PAF receptor antagonists could inhibit 15-LO–conditioned LDL from stimulating monocyte adhesion. Addition of the compound Lau 0203 to the target endothelial cells before the addition of 15-LO–conditioned LDL reduced monocyte adhesion to background levels of binding (Fig 7). PAF receptor antagonists BN 50730 and Lau 0603 were also tested and induced similar levels of inhibition of monocyte adhesion.

View larger version (34K): [in this window] [in a new window]   Figure 7. Inhibition of monocyte adhesion to endothelial cells by the PAF-receptor antagonist Lau 203. The assay was performed as described in "Methods." PAF-receptor antagonists (PAF-A) were added at 10 µmol/L in an ethanol-DMSO solution (<1%) to endothelial cells 30 minutes before LDL conditioned on 15-LO cells was added. The supernatant was then removed, the endothelial cells were washed, and THP-1 cells were placed on the endothelial cells at 40 000 cells per well. After 20 minutes at 37°C the suspension was removed and the cells were vigorously washed (at least three times) to remove all but the firmly adherent THP-1 cells. The number of adherent THP-1 cells was determined in four high-power fields per well, and the results of 6 to 8 separate wells were averaged for each experiment. Values represent the mean±SE of three experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
There is substantial evidence that cellular 15-LO may play a role in the development of atherosclerotic lesions in vivo, presumably through its ability to seed LDL with hydroperoxides and thus render it more susceptible to oxidation. The current study provides additional insight into the potential atherogenic properties of 15-LO–modified LDL by demonstrating its similarity to mm-LDL. Even after 20 hours of incubation with cells overexpressing 15-LO, LDL was only minimally oxidized, as demonstrated by the low levels of TBARS and preserved content of PUFAs in conditioned LDL. Furthermore, as shown by agarose and polyacrylamide-gel electrophoresis the extent of protein modification that resulted was very mild. Moreover, 15-LO–modified LDL resembled native LDL in its binding to the LDL-receptor and did not develop significant binding to the acetyl receptor. Despite the absence of major structural changes, the 15-LO–conditioned LDL developed "proinflammatory properties," as evidenced by its ability to stimulate monocyte chemotaxis and adhesion. These characteristics of 15-LO–modified LDL are consistent with those attributed to mildly oxidized LDL (whether achieved by iron- or coculture-induced oxidation) which has been referred to as mm-LDL by Fogelman and colleagues.^{3 6 34 35} Minimally modified LDL has been shown to possess a variety of important pro-atherogenic properties.³ The data from this study suggest that cellular 15-LO has the ability to generate an LDL particle with similar proatherogenic potential.

A noteworthy finding in this study was that the extent of lipid peroxidation of LDL incubated on 15-LO cells or lac Z (galactosidase-overexpressing) control cells was similar. Neither the levels of TBARS formed nor the oxidation induced loss of PUFA nor the extent of protein modification generated during incubations on 15-LO or lac Z cells were significantly different. Because we previously demonstrated that LDL incubated with 15-LO cells has an increased content of lipid peroxides compared with LDL incubated with lac Z cells when measured with a highly sensitive HPLC chemiluminescence detection system,¹⁸ it may be that the measures of lipid peroxidation and protein modification used in the current study are not sensitive enough to detect the subtle differences in extent of oxidation. However, the consistent increase in bioactivity in 15-LO–conditioned LDL seems out of proportion to the modestly increased levels of lipoperoxides and resulting enhanced oxidation. This suggests that 15-LO cells induce formation of unique (or at least greater levels of) bioactive compounds or their precursors that are subsequently transferred to LDL. Additional support for this possibility was provided by the 15-LO inhibitor experiments; a significant amount of the bioactivity of cell-conditioned LDL was inhibited by pretreating cells with concentrations of 15-LO inhibitors that only minimally reduced the extent of lipid peroxidation. These experiments also completed the circle of evidence that 15-LO is the agent predominantly responsible for generating mm-LDL in these transfected fibroblasts; overexpression of 15-LO in these cells enhances formation of bioactive LDL, whereas inactivation of this enzyme with 15-LO inhibitors stops this process.

Several lines of evidence demonstrate that the bioactivity resides within the cell-conditioned LDL. Medium alone from the 15-LO cells contains no measurable bioactivity, whereas LDL separated from cell-conditioned supernatant and lipid extracts from modified LDL stimulate monocyte chemotaxis. Furthermore, this process appears to require LDL oxidation. Inhibition of LDL oxidation by the presence of EDTA or antioxidants also inhibits the ability of 15-LO cell–conditioned LDL to stimulate monocyte chemotaxis.

It has been demonstrated that oxidation of LDL phospholipids generates a variety of proinflammatory particles. Although the structure of these compounds has not yet been definitively identified, some clearly have PAF-like properties.³⁶ The fact that stimulation of monocyte adhesion by 15-LO–conditioned LDL could be inhibited by PAF receptor antagonists is consistent with the concept that bioactivity results in part through the generation of particles that are sufficiently similar to PAF to activate the PAF receptor. Indirect support for this conclusion comes from our experiments, which indicate that lysoPC content in LDL conditioned on 15-LO cells does not appear higher than that in LDL conditioned on lac Z control cells. Although this does not exclude the possibility that lysoPC contributes to the development of bioactivity,²⁵ it strongly suggests that other proinflammatory compounds are generated within LDL that is minimally modified by conditioning on cells overexpressing 15-LO.

Received January 15, 1997; accepted April 23, 1997.

	References

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