Expression of 12/15-Lipoxygenase Attenuates Intracellular Lipid Deposition During In Vitro Foam Cell Formation
Objectives— Lipoxygenases with different positional specificity have been implicated in atherogenesis, but the precise roles of the various isoforms remain unclear. Because of its capability of oxidizing low-density lipoprotein (LDL) to an atherogenic form, 12/15-lipoxygenases have been suggested to initiate LDL oxidation in vivo; thus, these enzymes may exhibit pro-atherogenic activities. However, in several rabbit atherosclerosis models, the enzyme appears to act atheroprotective.
Methods and Results— To test the impact of 12/15-lipoxygenase expression on early atherogenesis, we established an in vitro foam cell model, which is based on the uptake of acetylated LDL by murine macrophages. In this system, we found that 12/15-lipoxygenase expression protects the cells from intracellular lipid deposition. This effect was related to an attenuated uptake of modified LDL, as indicated by impaired expression of scavenger receptor A and to accelerated intracellular lipid metabolism.
Conclusions— Our results indicate that the role of 12/15-lipoxygenase in atherogenesis may not be restricted to oxidative LDL modification. Expression of this lipid-peroxidizing enzyme may impact both lipid uptake and intracellular lipid turnover. These data provide a plausible explanation for the antiatherogenic effect of 12/15-LOX in rabbit atherosclerosis models.
A key step in early atherogenesis is the formation of lipid-laden foam cells. According to the lipid oxidation hypothesis,1 oxygenation of low-density lipoprotein (LDL) is a critical step in foam cell formation. There are multiple mechanisms for in vivo LDL oxidation, and the lipid-peroxidizing 12/15-lipoxygenase (12/15-LOX) may contribute. The enzyme is capable of directly oxidizing LDL to an atherogenic form and functional disruption of its gene protected mice from lesion formation.2,3 Transgenic mice that overexpress the human 12/15-LOX in vascular endothelium have more lesions develop.4 These data suggested a pro-atherogenic activity of the enzyme. However, antiatherogenic activities have been reported in 12/15-LOX–overexpressing rabbits.5,6 The response to injury hypothesis emphasizes that atherosclerosis constitutes an inflammatory disease.7 Because 5-LOXs are involved in the biosynthesis of pro-inflammatory leukotrienes,8 this LOX isoform has recently been implicated in atherogenesis. In fact, 5-LOX is high-level expressed in human atherosclerotic lesions,9 and atheroresistant CON6 mice express 5-LOX at significantly lower levels than a corresponding B6 control strain.10
In the past, the pathophysiological role of 12/15-LOXs has been related mainly to extracellular LDL oxidation. However, intracellular activity of the enzyme may enhance lipid metabolism and, thus, may deplete lipid stores. For immature red blood cells, it has been reported that the 12/15-LOX initiates breakdown of intracellular organelles by oxidizing membrane lipids.11 A similar lipid-catabolizing role has recently been suggested for lipid body LOXs during early stages of plant development.12
Foam cell formation is an imbalance of intracellular lipid turnover. When lipid-catabolizing processes cannot counteract excessive lipid internalization, an intracellular lipid deposition results. In this study, we investigated the impact of the 12/15-LOX expression on intracellular lipid metabolism and found that 12/15-LOX–transfected J774A.1 cells deposit significantly less lipids in intracellular stores than the corresponding mock-transfected controls. These results provide a plausible explanation for the antiatherogenic effect observed in several rabbit atherosclerosis models.5,6
The chemicals used were from the following sources: arachidonic acid and high-performance liquid chromatography (HPLC) standards of HETE-isomers from Cayman Chem. (Ann Arbor, Mich); free cholesterol, cholesteryl oleate, cholesteryl linoleate, cholesteryl arachidonate, and chloroquine from Sigma (Taufkirchen, Germany); acetic acid anhydride from Merck (Darmstadt, Germany); anti-murine SR-A antibody (CD 204, RPE-conjugated) from Serotec (Düsseldorf, Germany) and anti-murine CD36 antibody from Cascade BioSciences, (Winchester, Mass); anti-murine IgA (fluorescein isothiocyanate-conjugated) from Sigma (Taufkirchen, Germany); HPLC solvents from Baker (Deventer, the Netherlands); cell culture media from Bio Whittaker (Verviers, Belgium); penicillin–streptomycin solution, geneticin (G418) and l-glutamine from PAA Laboratories GmbH (Colbe, Germany); fetal calf serum from GIBCO (Grand Island, NY); M-MLV reverse-transcriptase and agarose from Promega (Mannheim, Germany); Pyra TaqDNA polymerase from Q BIO gene (Illkirch Cedex, France); dNTPs from Carl Roth GmbH (Karlsruhe, Germany); ciglitazone and PGJ2 from Biomol (Hamburg, Germany); oligonucleotides from BIOTEZ GmbH (Berlin, Germany); and high-density oligonucleotide microarrays (U74Av2) from Affymetrix (Santa Clara, Calif).
Preparation of Human LDL and Lipoprotein Modification
Human LDL was isolated from EDTA plasma of healthy volunteers by sequential floating ultracentrifugation.13 Acetylation was achieved by incubating isolated LDL with acetic acid anhydride and subsequent dialysis.
Cell Culture, Maintenance, and Transfection
J774A.1 cells were obtained from the German Tissue and Cell Culture Collection (Braunschweig, Germany) and cultured as recommended by the vendor. Cells were stably transfected with the porcine leukocyte-type 12-LOX14 and 12-LOX positive clones were selected. Mock-transfected cells were established according to the same protocol using the wild-type pBOSNeo vector.
In Vitro Foam Cell Assay
J774A.1 cells (wild-type or transfectants) were cultured to near confluence (80% to 90%), and 24 hours before the experiment fetal calf serum was removed. On the day of the experiment, the medium was exchanged, different amounts of acetylated low-density lipoprotein (acLDL) were added, and the cells were incubated for 24 hours. After this incubation period, the medium was removed, the cells were harvested, and they were washed 3 times with phosphate-buffered saline (PBS). The intracellular lipids were extracted and analyzed by reverse phase HPLC to quantify the cellular content of cholesterol derivatives. HPLC was performed on a Nucleosil column–C18 column (Macherey/Nagel, KS-system; 250×4 mm, 5-μm particle size) and compounds were eluted at 45°C with the solvent system acetonitrile/2-propanol (75:25; v/v) at a flow rate of 1 mL/min.
Oligonucleotide Microarray Analysis
Total RNA isolated from the various cell types (see http://atvb.ahajournals.org) was quantified by ultraviolet spectroscopy. RNA quality was checked by analyzing the samples on a LabChip (BioAnalyzer; AGILENT Technologies, Santa Clara, Calif). cDNA synthesis, nucleic acid labeling, microarray hybridization (murine genome U74Av2 chip; Affimetrix, Santa Clara, Calif), and data evaluation were performed as recommended by the vendor.
Fluorescence-Associated Cell Sorting
To quantify the expression of scavenger receptors [CD36 and SR-A (CD204)], the cells were analyzed by fluorescence-associated cell sorting (FACS) using commercial antibodies. Cells were washed with cold PBS containing 0.25% EDTA and stained for 45 minutes on ice with monoclonal antibodies. For staining of CD204, we used a FITC-labeled anti-CD204 antibody. For CD36 quantification, FITC-conjugated secondary antibody (anti-mouse Ig A) was used and the samples were incubated for an additional 45 minutes on ice. Here the anti-CD36 antibody was diluted 1:200; the fluorescein isothiocyanate-conjugated secondary antibody was diluted 1:16. After labeling, the cells were washed twice and resuspended in PBS containing 1% fetal calf serum and 0.1% Na-azide. Immunostains were analyzed on a flow cytometer (FACS Calibur; Becton Dickinson). For each sample, 10 000 cells were quantified using the CellQuest software package.
Oxidized LDL used in comparative studies was prepared by incubating human LDL for 20 hours at room temperature with copper sulfate at a molar apoB/copper ratio of 1:10. Then, the solution was dialyzed against PBS containing 0.25% (wt/vol) EDTA. Protein concentrations were determined with the Roti-Quant detection system (Roth, Karlsruhe, Germany). To quantify expression of 12/15-LOX in the transfected cell lines, LOX activity assays and immunoblotting were performed.
J774 Cells Deposit Intracellular Lipids When Incubated With acLDL
To investigate the impact of 12/15-LOX expression on foam cell formation, we established an in vitro foam cell model, which is based on HPLC quantification of intracellular cholesterol esters after a 24-hour incubation of monocyte/macrophages with acLDL. To select a useful cellular system, we first screened several monocyte/macrophage cell lines (U937, THP1, J774, P388.D1) and found J7741.A cells to be most suitable. Incubating these cells with acLDL, they accumulated large amounts of intracellular cholesterol esters (Figure 1). From Table 1, it can be seen that the cellular content of cholesteryl linoleate, cholesteryl arachidonate, and cholesteryl oleate was very low when J774 cells were incubated for 24 hours in the absence of acLDL. In contrast, the lipid content was significantly higher when acLDL was present. For all subsequent experiments, we assayed the 2 major cholesterol esters (cholesteryl linoleate and cholesteryl oleate) to quantify foam cell formation because the differences were most pronounced for these compounds.
Transfected J774 Cells Express 12/15-LOX at Similar Levels as Other Native Cells
Wild-type J774 cells do not express 12/15-LOX, as concluded from activity assays and reverse-transcriptase polymerase chain reaction. To study the impact of this enzyme on foam cell formation, wild-type J774 cells were transfected with the porcine leukocyte 12/15-LOX. Activity assays and immunoblotting confirmed expression of the enzyme in transfected cells and its lack in the corresponding mock transfectants. Comparative activity assays [transfected J774 cells, 0.79±0.11 μg HETE/106 cells (n=4); IL4-treated human monocytes, 2.1 μg HETE/106 cells; IL4-treated A549 cells, 0.5 μg HETE/106 cells; murine peritoneal lavage cells, 1.8 μg HETE/106 cells] indicated that our 12/15-LOX–transfected J774 cells express the enzyme at similar levels as primary mammalian cells.
Overexpression of 12/15-LOX Attenuates Intracellular Lipid Deposition
When 12/15-LOX–transfected J774 cells were incubated for 24 hours with acLDL, we observed significantly lower intracellular deposition of cholesterol esters when compared with the corresponding mock-transfected controls. Related to cellular protein, 12/15-LOX–transfected cells accumulated 23.5±4.9 nmol cholesterol linoleate/mg protein (n=4). In contrast, for the corresponding mock transfectants, we analyzed 39.3±3.2 nmol/mg protein (n=4; Student t test; P=0.009). Similar results were obtained for cholesterol oleate (39.4±2.6 nmol/mg protein for 12/15-LOX transfectants versus 69.6±10.7 nmol/mg protein for the mock controls; n=4; Student t test; P=0.002). Such impaired lipid deposition was consistently observed at all time points of the incubation period and at variable acLDL concentrations. Moreover, this difference was independent of the duration of the preculturing period of the cells (Figure 2). In our kinetic studies (Figure 2A), we observed an almost linear increase in intracellular lipid deposition, and this was the case with both LOX-transfected and mock-transfected cells. In contrast, we found clear saturation kinetics when the dependence of lipid deposition on acLDL concentration was tested (Figure 2B). When cultured cells grow to confluence, they gradually alter their gene expression pattern and, thus, their functionality. We found that duration of the preculturing period (degree of confluence) did also impact the degree of intracellular lipid deposition and maximal cholesterol ester accumulation was observed after 3 days (Figure 2C). However, independent of the duration of the preculturing period 12/15-LOX–transfected cells always accumulated less intracellular cholesterol esters than the corresponding mock transfectants.
To obtain more detailed information of the mechanistic reasons for the reduced lipid deposition in 12/15-LOX–transfected cells, we performed oligonucleotide-based microarray studies to compare the gene expression profiles of the 2 cell types.
Expression of Scavenger Receptor-A (SR-A) but not of CD36 Is Downregulated in 12/15-LOX–Transfected J774A.1 Cells
SR-A and CD36 together account for ≈75% to 90% of uptake of modified LDL in murine macrophages.15 Searching our microarray database for SR-A signals, we did not find significant differences between the 2 cell types (766 versus 903 GAPDH-normalized fluorescence units in mock-transfected and LOX-transfected cells, respectively; P=0.858, paired t test), and these results were confirmed with semiquantitative reverse-transcription polymerase chain reaction (data not shown). However, FACS analysis indicated that 12/15-LOX–transfected cells express lower levels of SR-A than the corresponding mock transfectants (Figure 3). Similar results were obtained in 7 independent experiments and statistic evaluation revealed a 41% lower expression (P<0.001). Next, we searched our microarray database for CD36 but did not find significant differences (P=0.986). These data were confirmed on the protein level by 3 independent FACS experiments (Figure 3B).
12/15-LOX–Transfected J774 Cells Degrade Internalized LDL Lipids More Rapidly
Next, we explored the possibility of whether accelerated intracellular lipid degradation may contribute to impaired lipid deposition. For this purpose, we loaded 12/15-LOX and mock transfectants with acLDL and quantified the decay kinetics of intracellular cholesterol esters after removal of the extracellular lipid source. To adjust comparable starting conditions, the mock-transfected cells were pre-incubated with acLDL for a shorter time period to reach similar initial lipid loading (Figure 4). With both cell types, we observed a steep decline of the intracellular cholesterol esters during the first 5 hours of the decay period. Interestingly, the decay kinetics were different for the 2 cell types: (1) initial decay was more rapid for 12/15-LOX–transfected cells; and (2) after 24 hours, the lipid content of the mock-transfected cells was reduced to ≈50% of initial loading. In contrast, in the 12/15-LOX transfectants, ≈75% of the internalized cholesterol esters were degraded. These data indicate that 12/15-LOX–transfected cells apparently catabolize internalized cholesterol esters more rapidly than the corresponding mock-controls.
Lysosomal Hydrolysis Does Not Contribute to the Difference in Lipid Metabolism
Lysosomal hydrolysis is one of the major metabolizing reactions of internalized cholesterol esters.16 When we analyzed the lipid composition of our acLDL preparations, we found that cholesteryl linoleate was the major cholesterol ester (cholesteryl linoleate/cholesteryl oleate ratio of 5.1±0.8; n=5). In contrast, when wild-type J774 cells were loaded with acLDL, intracellular cholesteryl oleate was dominant (cholesteryl linoleate/cholesteryl oleate ratio 0.42±0.07; n=9). For 12/15-LOX–transfected and mock-transfected cells, we obtained similar values (Table 2), indicating that the transfection procedure has not altered intracellular cholesterol transesterification.
To find out whether upregulation of lysosomal degradation is a major reason for the differences in intracellular lipid accumulation between 12/15-LOX–transfected and mock-transfected cells, we performed experiments with the lysosome inhibitor chloroquine.17 From Table 2, it can be seen that for both 12/15-LOX–transfected and mock-transfected cells, the cholesteryl linoleate/cholesteryl oleate ratio was higher in the presence of chloroquine, indicating that in both cell types lysosomal hydrolysis is involved in intracellular cholesterol transesterification. To test whether there are quantitative differences between the 2 cell types, we divided the cholesteryl linoleate/cholesteryl oleate ratio of mock-transfected cells in the presence of chloroquine (2.72) by the corresponding ratio in its absence (0.55). This ratio (4.94, last column of Table 2) may be considered a suitable measure for the effectiveness of lysosomal hydrolysis. When a similar calculation was performed for the 12/15-LOX–transfected cells, a very similar ratio (4.91) was obtained, suggesting that expression of 12/15-LOX did not significantly impact lysosomal metabolism of internalized acLDL lipids.
It has been reported before that oxidized cholesterol esters are preferred substrates for macrophage-neutral cholesterol ester hydrolases.18 Thus, cytosolic oxygenation of unsaturated cholesterol esters may speed up cytosolic hydrolysis of cholesterol esters. To test whether 12/15-LOX–transfected cells contain increased levels of oxygenated cholesterol esters, we analyzed the intracellular lipids for the presence of such derivatives and obtained the following results: mock transfectants, 0.1±0.17 μg oxidized cholesterol esters/107 cells; and 12/15-LOX transfectants, 3.0±1.92 μg oxidized cholesterol esters/107 cells (n=3). These data indicate that 12/15-LOX oxygenates internalized cholesterol esters and, thus, may render them prone for accelerated cytosolic hydrolysis.18
Impact of LOX Products on Foam Cell Formation
12/15-LOX metabolites are capable of regulating cellular lipid metabolism via activation of transcription factors.19 The LOX-transfected cells used in this study convert linoleic acid and arachidonic acid mainly to 13S-HODE and 12S-HETE, respectively. To test whether these metabolites may impact foam cell formation we pre-incubated mock-transfected J774 cells with 13S-HODE and 12S-HETE in a broad concentration range (50 nM to 10 μmol/L) and then initiated foam cell formation. We found that these LOX metabolites failed to induce reduction of intracellular cholesterol ester accumulation. In contrast, we even observed a tendency for increased lipid deposition. However, this increase did not reach a high degree of significance and was not dose-dependent over a lager concentration range. It should be stressed that our data do not exclude the possibility that other LOX products may be involved in the regulation of intracellular lipid deposition. It may well be that decomposition products of free and/or esterified hydroperoxy lipids or oxysterols formed as secondary products of LOX-catalyzed fatty acid oxygenation may exhibit such activities.
Formation of lipid-laden foam cells is a hallmark in early atherogenesis, but the mechanisms of intracellular accumulation of cholesterol esters and of counteracting activities are not well understood. Foam cells originate from monocyte/macrophages or specialized smooth muscle cells, and under certain conditions these cells express 12/15-LOXs. Human peripheral monocytes lack a functional 12/15-LOX, but when the cells are incubated with IL4/13 the enzyme is strongly induced.20 In mice, peritoneal macrophages constitutively express large amounts of the enzymes.21 In lesional foam cells, 12/15-LOX has been detected by immunohistochemistry and in situ hybridization,22 but recently expression of the enzyme has been questioned for human lesions.9
In animal atherosclerosis models there are reports about pro-atherogenic2–4 and/or antiatherogenic activities5,6 of the enzyme, and the possible reasons for these conflicting results have been discussed in detail.23 Unfortunately, in the past, the role of 12/15-LOXs in atherogenesis has been restricted to its capability to oxidize LDL to an atherogenic form. We took an alternative approach and investigated the impact of the enzyme on intracellular lipid metabolism. Surprisingly, we found that 12/15-LOX overexpression attenuated cytosolic deposition of cholesterol esters in murine J774 macrophages. The mechanistic reasons for this effect are not completely understood, but our findings suggest that 12/15-LOX may impair SR-A expression and stimulate cytosolic degradation of cholesterol esters. It has been reported before that oxidized cholesterol esters are better substrates for cytosolic cholesterol ester hydrolases18 and, thus, we propose the following scenario: the 12/15-LOX oxidizes cytosolic cholesterol esters and cholesteryl linoleate appears to be the preferred substrate. Oxidized cholesteryl linoleate is more rapidly hydrolyzed by neutral cholesterol ester hydrolases, which leads to selective removal of cholesteryl linoleate from the cytosolic pool of cholesterol esters. However, selective hydrolysis of this cholesterol ester is likely to lead to readjustment of the cellular cholesterol ester equilibrium so that the concentration of other cholesterol esters is also reduced. As a consequence, the cholesteryl linoleate/cholesteryl oleate ratio remains unaltered (Table 2).
An alternative explanation for the protective activity of 12/15-LOX in our foam cell assay is an increased cholesterol export. Although we attempted to minimize lipid efflux (lack of extracellular lipid acceptor), there was the possibility that the cells may have secreted cholesterol acceptor proteins. To address experimentally the question of whether cholesterol efflux may contribute to the impaired lipid deposition in 12/15-LOX transfectants, we loaded cells with acLDL containing 3H-cholesterol. Then, acLDL was washed away and radioactivity was assayed in the medium during the time course of the decay period. We found that only between 0.5% and 1% of the intracellular radioactivity was exported during an 18-hour decay period and there was no significant difference between 12/15-LOX–transfected and mock-transfected cells (data not shown). In contrast, in the presence of fetal calf serum as extracellular cholesterol acceptor ≈8% to 12% of the cellular radioactivity was exported. Here again, we did not observe significant differences between the 2 cell types (data not shown).
A further mechanism by which 12/15-LOX may impact foam cell formation is regulation of gene expression. Expression of this oxidizing enzyme is likely to alter the cellular redox state and, thus, expression level of redox-sensitive genes. In fact, our microarray studies indicated alterations in the gene expression pattern. We identified 10 genes that are upregulated by >1 order of magnitude and expression of >20 genes was downregulated to a similar extent. However, considering the fact that expression of 22 000 genes was quantified these alterations appear rather subtle. The products of the 12/15-LOX-pathway have been reported to coactivate the peroxysome proliferator-activated receptor γ (PPAR-γ),19 but this pathway may not be of major importance for 12/15-LOX–dependent reduction of foam cell formation. This is suggested by the failure of 13S-HODE and 15S-HETE to reduce intracellular lipid deposition but also by the lacking effect of PGJ2 and ciglitazone, which are known PPAR-γ agonists (data not shown).
In summary, our data suggest that 12/15-LOX may act as lipid-catabolizing enzymes at early stages of foam cell formation and they also provide a plausible explanation for its antiatherogenic effect in 2 independent rabbit atherosclerosis models.5,6 During early stages of foam cell formation, the enzyme appears to counteract cytosolic deposition of cholesterol esters by rendering them prone for hydrolytic cleavage. In other words, expression of the enzyme in macrophages under certain conditions may be aimed at fighting excessive intracellular lipid accumulation. In contrast, during advanced stages of plaque formation, the enzyme may change its character. At later time points, it may contribute to extracellular LDL oxidation and, thus, may act pro-atherogenic. Unfortunately, the question about the mechanistic reasons for this good-to-bad transition of the enzymes character remains unclear and more work is needed to provide satisfying answers on this topic.
Financial support for this study was provided by Deutsche Forschungsgemeinschaft (Ku961-8/1).
- Received April 8, 2004.
- Accepted January 3, 2005.
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