© 1999 American Heart Association, Inc.
Original Contributions |
Uptake of 13-Hydroperoxylinoleic Acid by Cultured Cells
From the Department of Gynecology and Obstetrics, Emory University, Atlanta, Ga. Current addresses for Nathalie Augé, PhD, Unité INSERM 466, Rangueil, Université de Médecine, 31403 Toulouse Cedex 4, France; and Channa Keshava, PhD, Toxicology and Molecular Biology Branch, HELD, CDC, NIOSH, M/S 3014, 1095 Willowdale Road, Morgantown, WV 26505.
Correspondence to Sampath Parthasarathy, PhD, Department of Gynecology and Obstetrics, Emory University, Atlanta, GA 30322. E-mail spartha{at}emory.edu
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Abstract—Oxidized free fatty acids have profound effects on cultured cells. However, little is known about whether these effects depend on their uptake and metabolism by cells or primarily involve their interaction with cell-surface components. We determined the uptake and metabolism of unoxidized (linoleic or oleic acid) and oxidized linoleic acid (13-hydroperoxyoctadecadienoic acid, 13-HPODE) by endothelial cells, smooth muscle cells, and macrophages. We show that 13-HPODE is poorly taken up by cells. The levels of uptake were dependent on the cell type but were independent of the expression of CD36. 13-HPODE was also poorly used by microsomal lysophosphatidylcholine acyltransferase that is involved in the formation of phosphatidylcholine. Based on these results, we suggest that most of the biological effects of 13-HPODE and other oxidized free fatty acids on cells might involve a direct interaction with cell-surface components. Alternatively, very small amounts of oxidized free fatty acids that enter the cell may have effects, analogous to those of hormones or prostanoids.
Key Words: atherosclerosis • linoleic acid • 13-hydroperoxyoctadecadienoic acid • endothelial cells • macrophages
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Introduction |
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Oxidation products of linoleic acid and other free fatty acids (FFAs) affect several different cell types in a profound manner (Table 1
).1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Oxidized FFA
(Ox-FFA) also abounds in oxidized LDLs (Ox-LDLs) and in atherosclerotic
lesions.16 17 Several cell types generate Ox-FFA via
several reactions. For example, the secretion of
lipoxygenase products by
endothelial cells, smooth muscle cells, and
macrophages has been well documented and such reactions are
activated during the atherosclerotic
process.18 19 20 21 22 In our extensive review of literature, we
noted that there is a void of information on the uptake of Ox-FFA by
these various cell types. In other words, despite their importance as
cell-signaling molecules, little is known regarding their uptake and
metabolism by cells. To understand how Ox-FFAs affect
cells, it is essential to document the extent of their uptake and
metabolism.
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FFA is readily transported across cell membranes and is predominantly metabolized by esterification reactions to form complex lipid.23 The uptake and fate of Ox-FFA, in contrast, have received little attention. A few short-term studies have looked at the incorporation of oxidized arachidonic acid derivatives (mostly lipoxygenase products) into endothelial cell lipids and have shown that hydroxyeicosatetraenoic acids (HETEs) are incorporated into both neutral lipids and phospholipids.18 Differences in incorporation among various oxidized products of arachidonic acids were also observed, suggesting that different Ox-FFAs might suffer from different intracellular fate.24 The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cholesteryl esters by the acyl-coenzyme A (acyl-CoA) cholesterol:acyltransferase (ACAT) reaction was much lower compared with those of unoxidized fatty acids.25 26 Studies on the incorporation of ricinoleic acid (12-hydroxyoleic acid) in castor phospholipids using microsome fractions also showed that the utilization of Ox-FFA was poor compared with that of oleic acid.27 Haas et al24 showed that there was specific binding of 5-3H-hydroxyeicosatetraenoic acid ([3H]HETE), but no binding of 13-hydroxyoctadecadienoic acid (13-HODE) in human umbilical vein endothelial cells.
In this study we have determined the uptake of 13-hydroperoxyoctadecadienoic acid (13-HPODE), using 1-14C–labeled 13-HPODE by macrophages, smooth muscle cells, and endothelial cells. 1-14C–labeled oleic (as oxidation-resistant FFA) and linoleic (as oxidation-susceptible FFA) acids were used as controls. Our results indicate clearly that cells take up 13-HPODE poorly and that the levels of uptake depend on the cell type.
To our knowledge, the utilization of 13-HPODE for acylation reactions has not been demonstrated. We observed that 13-HPODE is poorly used by microsomal enzymes as a substrate for the formation of phosphatidylcholine (PtdCho) from lysophosphatidylcholine (lysoPtdCho).
Based on these results, we propose that the biological effects of 13-HPODE could be because of its interaction with specific components of cell-surface membrane.
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Methods |
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Materials
RPMI medium (RPMI 1640), MEM and DMEM, penicillin, streptomycin, glutamine, trypsin, and Hanks' balanced salt solution (HBSS) were purchased from Mediatech Inc. Linoleic and oleic acid, soybean lipoxygenase, lysoPtdCho, anti-guinea pig IgG conjugated with horseradish peroxidase, epidermal growth factor, hydrocortisone acetate, dibutyryl cAMP were obtained from Sigma Chemicals. [1-14C]Linoleic (53 mCi/mmol) and oleic acid (58 mCi/mmol) were obtained from New England Nuclear. Other reagents and solvents were obtained from Fisher Chemicals. Guinea pig CD36 polyclonal antibody was a generous gift from Drs Mysore Ramprasad and Daniel Steinberg (University of California San Diego).
Cell Culture
Rabbit femoral artery smooth muscle cells (RFASMCs) were
obtained from the American Type Culture Collection (ATCC, Rockville,
Md) and were routinely grown in MEM medium, 10% FCS containing 1%
L-glutamine, 1% penicillin, and 1% streptomycin. Human
umbilical vein endothelial cells (HUVECs, Clonetics
Corp) were grown in M199 medium, 10% FCS containing 1%
L-glutamine, 1% penicillin, and 1% streptomycin. RAW
macrophage cells (RAW 264.7 from ATCC) were cultured in DMEM
medium, 10% FCS, 1% L-glutamine, 1% penicillin, and 1%
streptomycin. Human microvascular endothelial cells
(MVECs) were obtained from the Dermatology core cell service facility,
Emory University, and were grown routinely in
endothelial basal media (Clonetics Corp) with epidermal
growth factor 5 ng/mL, hydrocortisone acetate 1 µmol/L, 30%
human serum, 5x10-5 dibutyryl cAMP, 1%
L-glutamine, 1% penicillin, and 1% streptomycin. MVECs
express CD36 after 4 days when cultured in the absence of growth factor
and in the presence of cAMP.28
Swiss–Webster mice (22 to 25 g) were purchased from Harlan (Birmingham, Ala) and euthanized after anesthesia (ether) by CO2 asphyxiation. Mouse peritoneal macrophages (MPMs) were recovered by peritoneal lavage with 5 mL of cold RPMI 1640 medium and isolated by centrifugation. The cells were seeded in 12-well culture plates and cultured in RPMI 1640 medium, 1% L-glutamine, 1% penicillin, and 1% streptomycin. The medium was replaced each day for 3 days to remove nonadherent cells.
Isolation of LDL and the Preparation of Acetyl-LDL
Blood was collected from healthy donors, and LDL
(d=1.019 to 1.063) was isolated by
ultracentrifugation as previously
described,29 using a TL-100 tabletop
ultracentrifuge. The isolated LDL was dialyzed against PBS, pH
7.4, for 6 hours. Ac-LDL was prepared as described by Pitas et
al.30
Preparation of Labeled Solution of Fatty Acids
The stock solution of radioactive linoleic or oleic acid (5000
dpm/nmol) was prepared in absolute ethanol and diluted in HBSS. The
linoleic acid solution was oxidized with immobilized
soybean lipoxygenase (100 U/mL, 1 hour, 37°C) to
prepare 13-HPODE. The formation of 13-HPODE was monitored
spectrophotometrically by scanning the absorption between 200 and 300
nm (Model DB-3500; SLM-Aminco), using HBSS as reference. Under these
conditions, the conversion into 13-HPODE is observed as an increase in
absorbance at the optical density of 234 nm. Usually, >90% conversion
of linoleic acid to 13-HPODE was achieved. Identity of the 13-HPODE was
confirmed by using HPLC.
Uptake of Unoxidized and Oxidized Fatty Acids by Cells
Cells were seeded at a final density of
2x105 cells per well in routine culture medium
in 6-well plates (RFASMCs or HUVECs) or 12-well plates (RAW or
peritoneal macrophages) for 16 hours. The medium was then
removed, the cells were washed twice with HBSS, and the experiments
were performed in the absence of serum by using 50 µmol/L (5000
dpm/nmol) of labeled solution of FFA. As soon as the labeled FFA was
added to the medium, an aliquot was mixed with EcoLume (ICN) for the
determination of radioactivity. After 30 minutes of incubation, the
medium was collected and the cells were washed twice with HBSS
containing 0.5% BSA. A serum-free medium was used to prevent excess
lipids. As the duration of the experiment was very short, lack of serum
did not result in cytotoxicity or cell death. Radioactivity in an
aliquot of the mixture (culture medium and cell washings) was
determined to establish the remaining labeled FFA in the medium. The
cells were solubilized in 1.9 mL of deoxycholic acid solution (0.5
mg/mL) and the radioactivity was determined in 100 µL of the cell
extract. After acidification by adding 20 µL of 6 N HCl, 4 mL
of chloroform/methanol (1:1, vol/vol) was added to the 1.8 mL of cell
lysate to extract the cellular lipids.31 All extraction
procedures had 10 mmol/L of butylated hydroxytoluene included to
prevent further oxidation. After centrifugation (10
minutes, 3000 rpm), 500 µL of the upper phase was gently dried
(37°C, under nitrogen) and the radioactivity was determined. The
lower chloroform phase was collected and 100 µL was gently dried
(37°C, under nitrogen) and the radioactivity of FFA associated with
cellular lipids was determined. The remaining chloroform phase was
gently dried (37°C, under nitrogen) and saved for the identification
of cellular lipids by thin layer chromatography
(TLC).
Incorporation of [1-14C]FFA Into Cellular
Lipids
The dried chloroform phase was dissolved in 100 µL of
chloroform/methanol (2:1, vol/vol) and the neutral lipids were
separated by TLC on silica gel G60 analytical plates, using a solvent
system containing n-hexane/diethyl ether/acetic acid
(90:18:1.5, by volume). The neutral lipids and the phospholipids were
identified by using iodine in the presence of standards. The lipid
spots at the origin, corresponding to the phospholipids, were scraped
off, suspended in 500 µL of water, and extracted according to the
method of Folch et al.32 After gently drying the
chloroform phase (37°C, under nitrogen), the phospholipids were
separated by using TLC silica gel G60 analytical plates and a solvent
system of chloroform/methanol/water/acetic acid (65:25:3:1, by volume).
The phospholipids were identified by exposure to iodine in the presence
of standards. Spots were scraped off and the radioactivity was
determined.
Isolation of Microsomes From Rat Liver and RAW Macrophages
Microsomes were isolated from rat liver and RAW cells by
differential centrifugation as previously
described.33 Microsomal fractions were
homogenized in 1 mL of sucrose/phosphate buffer (0.25 mol/L
sucrose, 0.05 mol/L
Na2H2PO4,
pH 7.4) and an aliquot was saved for the protein determination
according to the method described by Smith et al.34
Microsomal Acyltransferase Activity
Microsomal acyltransferase activity was performed in a final
volume of 800 µL of Tris-HCl (0.1 mol/L, pH 7.4) as described by
Parthasarathy et al.35 In brief, 100 µg of rat liver or
RAW macrophage microsomal protein was incubated in the presence
of 50 µmol/L [1-14C]oleic acid or
[1-14C]linoleic acid (500 dpm/nmol) or 13-HPODE
(500 dpm/nmol) as a substrate, 100 µmol/L lysoPtdCho
(acyl receptor), 200 µmol/L ATP, 200 µmol/L magnesium
chloride, and 1 µmol/L of CoA for 1 hour at 37°C. After this
incubation, lipids were extracted and the phospholipids were separated
by TLC as described earlier.
Cell Cholesterol Esterification Assay
MPMs were seeded at the final density of
2x105 cells per well in RPMI 1640 medium
containing 10% FCS, 1% glutamine, penicillin, and streptomycin for 48
hours in 12-well culture plates. The medium was removed, and the cells
were washed twice with HBSS and incubated for 16 hours in complete
media without serum. The experiments were performed in RPMI 1640, 1%
glutamine, penicillin, streptomycin in the presence of 25 µg/mL of
acetyl-LDL (Ac-LDL) and 50 µmol/L of labeled solution of FFA
(5000 dpm/nmol). As soon as the labeled FFA was added to the medium,
the radioactivity in the culture media was determined. After 30 minutes
or 6 hours of incubation, the medium was collected and the cells were
washed twice with HBSS. The lipids were extracted and separated by TLC,
using a neutral lipid solvent system as described earlier. The
1-14C–cholesteryl ester was identified by using
iodine in the presence of standards. The corresponding spot were
scraped off and the radioactivity was determined.
CD36 Expression
Cells (2x106 per dish) were solubilized
in a lysis buffer by sonication and centrifuged
(12 500g, 10 minutes, 4°C). CD36 was identified in the
proteins from the supernatant (100 µg) by using western blot
(SDS-PAGE 10%). In brief, after electrotransfer, the nitrocellulose
membrane was incubated for 2 hours in the blocking buffer of 20
mmol/L Tris-buffered saline (TBS) pH 7.4 and 0.1% Tween 20
containing 10% nonfat milk at room temperature and then 2 hours in the
blocking buffer containing 1% nonfat milk and anti-CD36 antibody
(1:150 dilution). After extensive washing, the secondary antibody was
added to the membrane (anti-guinea pig IgG conjugated with horseradish
peroxidase; dilution, 1:1500) for 1 hour at room temperature. The
protein was identified by chemiluminescence, using the ECL detection
kit available from Amersham.
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Results |
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Uptake of FFA by Different Cell Types
Cells (HUVECs, MVECs, RFASMCs, RAW, and MPMs) were incubated with [1-14C]oleic acid, [1-14C]linoleic acid, or HPODE at a final concentration of 50 µmol/L at 37°C for 30 minutes, and cell-associated radioactivity was determined. Because some of the cell types used in the study (RAW and MPMs) may convert linoleic acid into oxidized products during incubation,36 we used oleic acid (an oxidation resistant fatty acid) as a control. To our knowledge, this is the first study that compares the uptake of oxidation-resistant, oxidation-susceptible, and an Ox-FFA by different cell types.
After 30 minutes of incubation, 14.5% to 50% of unoxidized FFA (oleic
and linoleic acids) were associated with cells (Figure 1). Of the cell types studied, RFASMCs
showed the least uptake of fatty acids (Figure 1A, 14.5% or
42±4-nmol/mg of protein). In HUVECs and MVECs 
30% (81±6.5 nmol/mg
of protein) of the 1-14C–unoxidized fatty acid
added to the media were found associated with cellular lipids (Figure 1B and 1C). The percentage of lipid-associated radioactivity was
highest with macrophages (40% or 86±7.2 nmol/mg of
protein) (Figure 1D and 1E). The incorporation of lipids was
comparable between macrophages and endothelial
cells. Although the uptake of oleic and linoleic acids was comparable
in smooth muscle cells and endothelial cells, the
uptake of linoleic acid was disproportionately high in
macrophages. This could reflect the ability of these cells to
accumulate large quantities of lipids or their ability to convert
linoleic acid into 13-HPODE.36 However, we do not have an
explanation as to the difference noted in the uptake of linoleic acid
compared with that of oleic acid.
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In contrast to unoxidized FFA, the uptake of 13-HPODE was significantly
low (3% to 12%) by all cell types (Figure 1
). The uptake of
HPODE was higher in macrophage cell lines (10%, 13±1.8
nmol/mg of protein) compared with endothelial or smooth
muscle cells (2.5%, 5±1.2 nmol/mg of protein for smooth muscle
cells and 3±0.5 nmol/mg of protein for endothelial
cells). In these experiments, we had directly added the labeled HPODE
from lipoxygenase incubations. As there was a
possibility that these preparations contained some FFA, we also tested
the uptake of TLC-purified 13-HPODE and observed similar results (data
not shown).
We considered the possibility that the low uptake of 13-HPODE by cells
could be because of insufficient time of incubation or limiting
concentration of HPODE used in these incubations. However, the uptake
of 13-HPODE by smooth muscle cells reached maximum levels at
concentrations <20 µmol/L (Figure 2B) whereas there was a
concentration-dependent increase in the uptake of linoleic acid even up
to 100 µmol/L levels (Figure 2A). Furthermore, although
the uptake of 13-HPODE increased during longer incubations, its levels
never reached those of unoxidized FFA (Figure 2C). These results
suggest that there is an intrinsic difference in the mechanism(s) by
which FFA and Ox-FFA are taken up by cells.
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Competition for the Uptake of 13-HPODE
Cells may take up 13-HPODE by passive diffusion through the
membrane or via a regulated mechanism. To determine the potential
involvement of a receptor-mediated mechanism in the uptake of 13-HPODE,
we evaluated the ability of unlabeled 13-HPODE or oleic acid to compete
with the uptake of a tracer amount of
[1-14C]13-HPODE. A 9-fold excess of oleic acid
did not inhibit the uptake of 5 µmol/L
[1-14C]13-HPODE. In contrast, the addition of a
9-fold excess of 13-HPODE resulted in an inhibition of 80% of the
uptake. It is interesting that the addition of linoleic acid also
generates substantial inhibition, suggesting perhaps the formation of
unlabeled 13-HPODE and release into the medium in these incubations, as
observed by others.36 37 38 The results indicate that only
13-HPODE competes with its own uptake in smooth muscle cells (Figure 3). Similar results were obtained by
using RAW cells (data not shown). This result may be interpreted to
suggest an uptake of 13-HPODE different from that involved with
FFA.
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Incorporation of [1-14C]FFA Into Cellular
Lipids
Most of the radioactivity associated with cells in these
incubations could be accounted for by FFA and phospholipid fractions
(Figure 4A and 4B, respectively) in all
cell types. The major phospholipid that was labeled was PtdCho (Figure 4C). There was much lower radioactivity associated with the
other phospholipid fractions (sphingomyelin, 17±2%;
lysoPtdCho, 3±0.7%; and phosphatidylethanolamine, 6±1%),
expressed as percentages of radioactivity incorporated into the
phospholipid fraction (data not shown) with all fatty acids used.
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When the cells were incubated with 13-HPODE, most of the radioactivity
incorporated into cellular lipids was associated with the phospholipid
fraction, compared with cells incubated with unoxidized fatty acids.
The results presented in Figure 4B were expressed as
percentages of radioactivity incorporated into the cells and suggested
that 13-HPODE is a better substrate for phospholipid acyltransferase
reaction than unoxidized FFA. However, it should be remembered that the
Ox-FFA taken up by cells is very low in comparison with unoxidized FFA.
For this reason we determined the efficiency of the acyltransferase
reaction by using rat liver or RAW microsome as the source of the
enzyme and labeled oxidized and unoxidized FFA as substrate.
Oleic and Linoleic Acid Are Better Substrates for Microsomal
Acyltransferase Reaction Than 13-HPODE
Both de novo synthesized fatty acids and exogenous fatty acids
must be activated to acyl-CoA derivatives before they can enter
a metabolic reaction. The formation of fatty acyl-CoA
derivatives is catalyzed by the microsomal enzyme acyl-CoA synthetase.
The product, acyl-CoA is also efficiently used for complex lipid
synthesis by microsomal acyltransferases. Dominant acyltransferases
that determine the fate of fatty acid are the lysoPtdCho
acyltransferase, the diacylglycerol acyltransferase, and the
acyl-CoA–cholesterol acyltransferase, which generate
phosphatidylcholine, triacylglycerol, and
cholesteryl esters, respectively.
We measured the formation of PtdCho by using lysoPtdCho as acyl
acceptor in the presence of RAW cell microsome (Figure 5A) or rat liver microsomes (Figure 5B). As seen in Figure 5, oleic and linoleic acids were
efficiently incorporated into phosphatidylcholine. In contrast 13-HPODE
was poorly used by the enzyme to generate PtdCho. The enzyme was
pretreated with cold 13-HPODE and purified by acetone precipitation.
This pretreated enzyme was catalytically active and could incorporate
13-HPODE (data not shown). Increasing the concentration of 13-HPODE or
the concentration of CoA had no effect on the rate of formation of
phosphatidylcholine (data not shown). This suggested that the poor
incorporation of 13-HPODE was not because of an inhibition of the
enzyme or because of depletion in CoA by 13-HPODE. The decreased
formation was also not because of differences in the mobility of PtdCho
and oxidized PtdCho on the TLC system. In the solvent system used
chloroform/methanol/water/acid acetic (65:25:3:1, by volume), both
phospholipids comigrate as a single spot.
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Cholesterol Esterification in MPMs
Foam cells of atherosclerotic lesions accumulate large amounts of
Ox-FFA, predominantly in the esterified form.39
Endocytosis of modified lipoprotein is suggested to involve hydrolysis
of the lipoprotein-derived cholesterol esters in the
lysosomes and the reesterification of free
cholesterol with the liberated fatty acids in the
endoplasmic reticulum to generate cytoplasmic cholesteryl
esters.40 41 AcLDL delivers lipoprotein
cholesterol to macrophages by scavenger pathways
and stimulates cholesterol esterification in
MPMs.42 Linoleic acid is the predominant unsaturated fatty
acid associated with LDL and considerable 13-HPODE is formed during the
oxidation of LDL.43 If the cholesteryl ester associated
with Ox-LDL were to undergo hydrolysis and the reesterification
reaction, the released 13-HPODE would be expected to be incorporated
readily into the cholesterol ester. The increased
incorporation of exogenously added [1-14C]oleic
acid into cellular cholesteryl esters under these conditions has been
well documented.44 45 To our knowledge, the uptake and
incorporation of 13-HPODE during the uptake of modified lipoproteins by
macrophages have not been reported before. Because of our
finding that 13-HPODE was taken up and incorporated poorly into
cellular PtdCho by macrophages, we determined the uptake of
13-HPODE in the presence and absence of Ac-LDL by
macrophages.
After incubation of cells with 25 µg/mL of Ac-LDL and
[1-14C]FFA or
[1-14C]13-HPODE for 30 minutes and 6 hours, we
counted the radioactivity associated with cholesteryl esters. As the
level of uptake of each fatty acid differs in MPMs, we expressed the
results as percentages of the total radioactivity incorporated in
cells. At 30 minutes of incubation there was very little incorporation
of fatty acids. After 6 hours in the presence of Ac-LDL, cholesteryl
esterification was greater when cells were incubated in presence of
oleic or linoleic acid than in incubations performed in the presence of
Ac-LDL and 13-HPODE (Figure 6A and 6B).
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CD36 Expression
CD36 has been suggested to be a receptor for Ox-LDL.
However, recent evidence seems to suggest that CD36 might recognize the
lipid components of Ox-LDL.46 47 48 In fact, studies by
Endemann et al48 showed that moderately Ox-LDL
(which presumably would have more undegraded peroxides compared with
that of fully oxidized LDL) was more readily recognized by CD36.
Besides, a protein that bears substantial sequence homology to CD36 has
been detected as a fatty acid transport protein in fibroblasts and
adipocytes.49 50 In these studies, the possibility that
CD36 or its homolog could bind to Ox-FFA has been suggested. As our
finding suggested a difference in the levels of uptake of 13-HPODE
among different cell types studied, we considered the possibility that
the uptake could be linked to the differences in the expression of
CD36. The results, as demonstrated by western blot analysis,
indicate that RAW cells that take up Ox-FFA more than smooth muscle
cells or endothelial cells express the 88-kDa
CD36 at increased levels, whereas the other cell types only express the
54-kDa form of the protein (Figure 7). It
is noteworthy that we observed only a poor expression of the 88-kDa
CD36 protein in MPMs, which in contrast internalized much more 13-HPODE
compared with smooth muscle cells or endothelial cells.
We obtained similar results when anti-mouse CD36 antibody was used in
western blotting (data not shown). Yet another evidence against the
role of CD36 came from uptake experiments performed in the presence of
MVECs that express CD36.28 We observed no difference in
the level of uptake of the 13-HPODE by these cells in comparison with
HUVECs (2.4%) (Figure 1C), again suggesting that CD36 may not
be involved in the uptake mechanism. However, we have not determined
the expression of CD36 in these cells.
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There is growing interest in Ox-FFA metabolism because of their ability to induce in different cell types potent biological effects and signal transduction effects (Table 1
).
The uptake of oxidized fatty acid by cells is poorly documented and
most of the studies were performed by using 3H
derivatives of arachidonic acid, using predominantly
endothelial cells as the model system. The results of
these studies may not be a true representation of the uptake
and metabolism of Ox-FFA, as 3H
radioactivity is readily displaced from fatty acids on oxidation.
Furthermore, the fate of oxidized linoleic acid that is abundant in
oxidized lipoproteins and contains only 2 double bonds would be quite
different from that of arachidonic acid
products.26 51 The results presented in this study show that (1) in contrast to FFAs, Ox-FFAs are poorly taken up by cells; (2) the level of uptake of Ox-FFAs by cells depends on the specific cell type; (3) uptake of Ox-FFA may not involve cellular expression of CD36; (4) Ox-FFAs are poorly used by microsomal acyltransferase; and (5) the mechanism of uptake of 13-HPODE is distinct from that of the uptake of FFA. More important, the results presented might suggest that the oxidized lipids that accumulate in the cholesteryl ester–rich macrophage foam cells could also originate from de novo intracellular oxidation of fatty acid components as suggested by Carpenter et al,52 as very little HPODE was incorporated in cholesterol ester during the uptake of Ac-LDL. These studies could also be interpreted to suggest that 13-HPODE is not a good substrate for acyl-CoA cholesterol:acyltransferase (ACAT) reaction. However, atherosclerosis is a slow process and externally added 13-HPODE may not mimic the fate of lipoprotein-associated 13-HPODE.
The results presented in Figure 1 suggest that very low
levels of Ox-FFAs themselves may be sufficient to induce cellular
effects, or the effect of Ox-FFAs on cells could be attributed to
extracellular events involving membrane modification. This latter
possibility is very likely, as comparable levels of uptake were noticed
in 2 types of endothelial cells (HUVECs and MVECs). The
MVECs, in contrast to HUVECs, do not show an induction of
VCAM after exposure to Ox-FFAs,53 suggesting that the
level of uptake of FFAs does not correspond to the cellular effect.
Specific receptors have been proposed for the plethora of cellular effects attributed to oxidized arachidonic acid products (the prostanoids).54 Whether HPODE could interact with any of the putative oxidized arachidonic acid receptors or other components are involved remains to be established. Results presented in the current study suggest that CD36 may not be involved in the uptake of 13-HPODE. However, whether 13-HPODE binds to CD36 could not be determined.
The cellular effects of 13-HPODE are often counteracted by added antioxidants, suggesting the involvement of additional oxidation steps.7 From the results presented in this study, it could be speculated that oxidation of specific plasma membrane targets facing the extracellular milieu by 13-HPODE could be a key event in eliciting cellular response. Whether the target is a lipid, a specific amino acid(s) component of proteins, or other components, including microdomains such as caveolae, remains to be established. Alternatively, the 13-HPODE that enters the cells could further propagate oxidation reactions (which could explain the presence of oxidized esterified lipids) or undergo catabolic reactions generating toxic products such as H2O255 or short-chain dicarboxylic acids.51
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Acknowledgments |
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This work was supported by NIH Grant HL 52628-01A3 "Molecular Mechanisms of Oxidation of LDL," and we gratefully acknowledge the support by an American Heart Association Georgia Affiliate Fellowship Grant to N.A.
Received June 19, 1998; accepted August 26, 1998.
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