(Received for publication, November 14, 1994; and in revised form, January 6, 1995)
From the
There is strong experimental evidence that oxidized low density
lipoprotein (Ox-LDL) plays an important role in atherosclerosis.
However, the mechanisms by which Ox-LDL is formed in vivo are
unknown. To test whether 15-lipoxygenase (15-LO) could play a role in
oxidation of LDL by cells, we expressed 15-LO activity in murine
fibroblasts, which do not normally have 15-LO activity, and tested
their ability to modify LDL. Using a retroviral vector, we prepared
fibroblasts that expressed 2- to 20-fold more 15-LO activity than
control fibroblasts infected with a vector containing
galactosidase (lacZ). Compared with LDL incubated with lacZ cells,
LDL incubated with 15-LO-containing cells were enriched with lipid
hydroperoxides. When these LDL samples were subsequently subjected to
oxidative stress, they were more susceptible to further oxidative
modification, as judged by increased conjugated diene formation and by
increased ability to compete with
I-Ox-LDL for uptake by
macrophages. These findings establish that cellular 15-LO can
contribute to oxidative modification of LDL, but the quantitative
significance of these findings to the in vivo oxidation of LDL
remains to be established.
Oxidatively modified low density lipoprotein (LDL) ()is potentially much more atherogenic than native
LDL(1) , and there is strong evidence that it plays a role in
experimental atherosclerosis(2) . While a causative role in
human atherosclerosis has not yet been established, there is indirect
evidence to support that possibility (reviewed in Refs. 1, 3). A number
of clinical trials to test the effectiveness of antioxidant supplements
in the prevention of coronary heart disease are either underway or in
the planning stages. These studies are predicated on the introduction
into the LDL particle itself of chain-breaking antioxidants (e.g. vitamin E) that prevent or limit the extent of LDL oxidation. If
we knew the mechanisms involved in oxidation of LDL in vivo,
it should become possible to design novel forms of intervention in
addition to or instead of the introduction of antioxidants into LDL.
For example, if, as has been suggested, 15-lipoxygenase (15-LO) plays a
role in oxidation of LDL by cells(4, 5) , then the use
of appropriate 15-LO inhibitors might offer a new complementary mode of
treatment. If, on the other hand, oxidation in vivo depends on
the generation of superoxide anion (6, 7, 8, 9) , interventions that
inhibit NADPH oxidase or other sources of superoxide anion would be of
potential benefit, as would interventions that enhance the activity of
superoxide dismutase or other systems quenching the action of the
superoxide anion. Unfortunately, little or nothing is known about the
sites and the mechanisms of LDL oxidation in vivo. Studies in
cell culture allow us to identify a large number of candidate systems
that could participate in oxidative modification of LDL. These include
not only 15-LO and NADPH oxidase but also the P450 system, the
mitochondrial electron transport chain, myeloperoxidase,
cyclooxygenase, and others. From studies in vitro, it is
already clear that different cells may utilize different systems and
that individual cell types probably use multiple systems in the
oxidative modification of LDL. For example, monocytes and smooth muscle
cells do not express 15-LO(10) , but they do oxidatively modify
LDL, showing that 15-LO is not necessary for oxidative
modification of LDL. Similarly, freshly isolated monocytes express
little or no NADPH activity, but they can oxidize LDL at a low rate.
After activation to induce expression of NADPH oxidase and the
accompanying production of superoxide anion, the rate of LDL oxidation
is considerably enhanced. Again, the NADPH oxidase system is not necessary but it can certainly contribute to oxidative
modification. Secretion of H
O
by some cells
could also play a role especially if thiols are concurrently secreted (11, 12) . We are presently trying to identify which
candidate systems are worth further study, especially in vivo.
Evidence that supports a role of 15-LO in oxidative modification of LDL can be summarized as follows: 1) purified 15-lipoxygenases can oxidatively modify LDL in cell-free systems(4, 13, 14) ; 2) oxidation of LDL by endothelial cells and by macrophages in culture is suppressed by known inhibitors of lipoxygenase(4, 5) ; 3) tissue samples from atherosclerotic lesions, but not from normal aortas, exhibit measurable levels of 15-LO mRNA and protein(15, 16) , as well as 15-LO enzyme activity(17) ; 4) transfer of the 15-LO gene into rabbit iliac arteries results in the appearance of epitopes of oxidized LDL(18) ; 5) most importantly, increased amounts of stereospecific products of the 15-LO reaction have been demonstrated in early atherosclerotic lesions (14) but not in later lesions, where non-enzymatic oxidation reactions due to later steps of the propagation reactions of lipid peroxidation predominate(19) .
However, interpretation of studies using LO inhibitors must be made with caution because most of these inhibitors are also nonspecific antioxidants. Consequently, their presence in the medium could suppress LDL oxidation and yet that inhibition might not be related to inhibition of 15-LO activity. The acetylenic analog of arachadonic acid, eicosatetrayonic acid (ETYA), on the other hand, is devoid of nonspecific antioxidant activity, but it does inhibit 15-LO activity and LDL oxidation in cultured cells(5) . Sparrow et al.(20) point out, however, that the concentrations needed to inhibit LDL oxidation appear to be much higher than the concentrations needed to inhibit 15-LO activity. Because of this apparent dissociation, they suggested the possibility that the ETYA inhibition of LDL oxidation might be nonspecific, possibly the result of cell toxicity. Indeed, because inhibitors are rarely specific to one and only one target, the use of inhibitors to implicate an enzyme system may yield misleading results. Indeed, ETYA inhibits many other cellular functions as well (21) . For these reasons we decided to explore further the role of 15-LO in LDL oxidation by expressing 15-LO activity in murine fibroblasts, which do not normally express 15-LO, and to assess the effect of this overexpression on the ability of these modified cells to oxidize LDL.
A parallel construct which contains the
-galactosidase cDNA (lacZ) instead of 15-LO cDNA was designated
pLZRNL (25) and was also used to produce stably transformed
PA317 cells (lacZ cells). Because lacZ cells differ from 15-LO cells
only in their production of
-galactosidase instead of 15-LO, they
were used as controls for the 15-LO cells.
Quantification of 15-LO mRNA in selected PA317 clonal populations
was performed using competitive polymerase chain reaction amplification (26) . The oligonucleotide primers used were specific for human
reticulocyte 15-LO, as described previously(27) . Briefly, 100
ng of poly(A) was reverse transcribed, and 10 ng of
the product was amplified with the 15-LO primers for 30 cycles in the
presence of a range of known concentrations of an internal 15-LO
standard which was the 15-LO cDNA with a 115-bp deletion. PCR products
were electrophoresed and resolved in 8% polyacrylamide gels and then
silver-stained. The destained gels were dried, photographed, and the
intensity of the bands quantified on an image processor. When the
intensities of both bands in the same lane were equal, the
concentration of the unknown was assumed to be equal to the known
concentration of the internal standard(26) .
In other experiments, aliquots of LDL-containing media that had been
preconditioned by incubation with lacZ or 15-LO cells for 20 h were
subsequently incubated with cultures of mouse peritoneal macrophages in
DME/RPMI/(50:50 mixture) for 20 h to effect an oxidative stress
(instead of exposure to copper as described above). Then aliquots
containing 25 µg of unlabeled LDL were added to a new set of
macrophage cultures in DME containing 2.5 µg of I-ox-LDL and extent of degradation of
I-ox-LDL determined as described above.
In some
experiments, hydrogen peroxide (HO
) in the
media was removed by incubating aliquots for 10 min at 37 °C with
gentle shaking (80 revolutions/min) in the presence of catalase (EC
1.11.1.6) immobilized on 4% agarose beads (2.4 units) (Sigma) which
selectively destroys H
O
but not LOOH. Aliquots
were chilled to 4 °C, centrifuged at 1,500 revolutions/min for 5
min to remove immobilized catalase, and aliquots (<2.5 µg) of
LDL obtained for fluorescence study. To measure the LOOH specifically
bound to LDL, modified LDL were reisolated from the media as described
below.
The indicating reagent was prepared as follows: (i) 2 ml of
0.01 N NaOH was added to 0.5 ml of a solution of
2`,7`-dichlorofluorescein diacetate dissolved in ethanol (1
mM), and after 30 min at room temperature, the reaction
solution was diluted with 10 ml of 25 mM sodium phosphate
buffer (pH 7.2), yielding 2`,7`-dichlorofluorescein solution; (ii) 1 mg
of hematin was dissolved in 0.5 ml of 0.2 N NaOH, diluted with
100 ml of 25 mM sodium phosphate buffer, and a 14-ml aliquot
of this hematin solution was boiled for 15 min with 100 ml of 25 mM sodium phosphate buffer, yielding an activated hematin solution;
and (iii) the 2`,7`-dichlorofluorescein solution (2 ml) and the
activated hematin solution were mixed at 4 °C, yielding the
indicator reagent, which was kept under N gas to prevent
oxidation.
Reactions between media samples and the indicator reagent were carried out at 50 °C for 50 min and, after cooling in a room temperature water bath, the fluorescence intensity (excitation = 440 nm, emission = 550 nm) of each sample was measured within 30 min. The standard curve for the assay was generated using fluorescence intensities obtained from known quantities (0-2 nmol) of oxidized linoleic acid (1 µmol of linoleic acid was dissolved in 1 ml phosphate-buffered saline, and oxidized with 500-1000 units of soybean lipoxygenase for 60 min at 37 °C). The concentrations of oxidized linoleic acid was confirmed in the standard samples by monitoring absorbance at 234 nm.
In some experiments, LDL was isolated from the
media prior to determination of LOOH content according to a
modification of the procedure of Lynch and Frei(29) . In brief,
media were subjected to concentration by centrifugal ultrafiltration
(Amicon) and isolation by gel filtration on Sephadex-G25 M columns (PD-10, Pharmacia) equilibrated with phosphate-buffered
saline at a flow rate of 1.2-1.5 ml/min. Fractions were collected
at 1.5 min and monitored by absorbance at 280 nm. Total lipids in
0.5-ml aliquots were extracted, according to a modified procedure of
Bligh and Dyer (using 1 ml of CHCl, MeOH, and 0.5 ml of
0.03 N HCl)(30) , and the CHCl
layer was
dried under N
, redissolved in 0.1 ml of MeOH and 0.9 ml of
distilled H
O, and used for fluorescence measurement.
Another aliquot of media was used for determination of protein, which
was measured by the procedure of Lowry et al.(31) .
Ebselen is a seleno-organic compound (2-phenyl-1,2-benzoisoselenozol-3(2H)-on) that can directly reduce lipoperoxides and can mimic glutathione peroxidase(32, 33, 34) . To further document that the fluorescence measured in these experiments was indeed LOOH, we pretreated LDL previously incubated with 15-LO cells with ebselen and then measured LOOH fluorescence. Subsequent to its incubation with 15-LO cells (clone 12), LDL was reisolated by gel chromatography using PD-10 columns, concentrated by ultrafiltration, and the protein concentration adjusted to 0.5-1 mg/ml with 0.1 M Tris-HCl, 1 mM EDTA, pH 7.4. Specific additions of ebselen (added in ethanol) were made, and the mixture was then incubated at 37 °C in Tris-HCl/EDTA under nitrogen for indicated times. Ebselen was then removed from the incubation by gel filtration, and LOOH was measured as described above. In some experiments, GSH (3 mM) which enhances the ability of ebselen to reduce LOOH (34) was also added.
Figure 1:
Expression of retroviral RNA by stably
transformed populations of LLORNL-infected PA317 cells. Left
panel, ethidium bromide-stained gel containing poly(A) RNA from PA317 cells infected with pLZRNL and pLLORNL, prior to
capillary transfer to nitrocellulose membrane. Right panel,
Northern analysis of samples from left panel using a randomly
labeled, 1.5-kb cDNA probe (MstII fragment from human 15-LO). Lane 1, poly(A)
RNA from lacZ-infected clone (LZ); lanes 2-4, 15-LO clones 2, 8, and 12,
respectively.
Figure 2: 15-LO activity in murine fibroblast clones. 15-LO activity (in nanomoles of 13-HODE/30 min/dish) was determined in broken cell preparations of lacZ and 15-LO cells (clones 2, 8, and 12) and in control dishes with no cells (NCC). Results shown are 24 ± S.D. of six separate experiments. By analysis of variance, values for lacZ were not different from NCC; values of clones 2 and 8 were greater than lacZ (p < 0.04); and values for clone 12 greater than clones 2 and 8 (p < 0.0001). Assay of 15-LO activity in intact macrophages usually yields values of 20-30 nmol of 13-HODE/30 min/dish.
The fluorometric method used to measure LOOH in these studies also
detects HO
. However, when LDL-containing media
were preincubated with catalase bound to agarose, the fluorescence
yield was not decreased, indicating that almost all of the measured
fluorescence represented LOOH rather than H
O
(data not shown). Frequently, the apparent LOOH after catalase
treatment was paradoxically modestly increased, for reasons
not known. To test whether some of the fluorescence might be
nonspecific, LDL isolated after incubation with 15-LO cells was
pretreated with ebselen, an agent known to reduce LOOH
content(32, 33, 34) . Ebselen was then
removed by gel filtration, and the content of LOOH was determined. As
shown in Table 4, nearly all of the fluorescence was eliminated
by pretreatment with 25 µM of ebselen. When ebselen and
GSH were added together, the elimination of LOOH was greatly
accelerated (see legend to Table 4).
We next tested the
ability of the different clones to oxidatively modify LDL as measured
by enhanced uptake in macrophages. In this set of experiments, I-LDL was incubated with lacZ cells or with clone 12
cells for 20 h in either Ham's F-10 or in cysteine-free RPMI and
then the rate of degradation of the conditioned
I-LDL by
mouse peritoneal macrophages was determined (Fig. 3). LDL
conditioned by lacZ cells was consistently degraded more rapidly than
native LDL or LDL incubated in the absence of cells. There was a wide
range of values from experiment to experiment, probably reflecting in
part the degree of preexisting oxidation of the LDL preparations used.
This in turn is determined importantly by the ``age'' of the
LDL preparation and the time between iodination and use in the
experiment(22) . However, the difference between clone
12 cells and lacZ cells was highly consistent, being in the same
direction in every one of 20 separate experiments. Using
Student's paired t test, clone 12 displayed a
statistically highly significant (p < 0.003), nearly 2-fold
greater ability to modify LDL than did the lacZ cells (Fig. 3).
Figure 3:
Capacity of 15-LO cells to oxidatively
modify I-LDL.
I-LDL was preincubated with
lacZ cells or clone 12 for 20 h, and then its rate of degradation by
macrophages was determined. Shown are the mean values (± S.D.),
in µg LDL degraded/5 h/mg cell protein, of results obtained in 11
separate experiments conducted in Ham's F-10 medium (
)
and 9 separate experiments conducted in cysteine-free RPMI (
).
Shown also is the rate of degradation by macrophages of native
I-LDL. For all experiments the values were corrected by
subtraction of degradation products formed in the absence of cells.
Macrophage degradation of
I-LDL modified by 15-LO clone
no. 12 is different from that of lacZ in both types of media (p < 0.003, Student's t test).
Figure 4: Time course of conjugated diene formation of LDL preincubated with murine fibroblasts. LDL were preincubated with lacZ cells or 15-LO cells (clones 2, 8, and 12) for 24 h in cysteine-free RPMI. LDL was then isolated from the medium by ultracentrifugation and conjugated diene formation measured in the presence of 2.5 µM copper in a continuously recording spectrophotometer as described(22) .
Another possibility is that the I-labeled LDL used in the experiments shown in Fig. 3had already undergone oxidation secondary to the
radiation(22) , and this pro-oxidant effect masked small
differences in the abilities of cells to further oxidize LDL. To test
this idea, we incubated unlabeled LDL for 20 h with lacZ cells
and with the three different clones of 15-LO cells and then subjected
each of these LDL samples to a subsequent oxidative stress by
incubation with mouse peritoneal macrophages (cultured in DME/RPMI at
1:1 ratio) for 20 h. Each LDL was then tested for its ability to compete with
I-ox-LDL for uptake by a different
set of macrophages (Table 5). In this experiment, under the
experimental conditions used, 25 µg of unlabeled copper-oxidized
LDL was able to compete for 70% of the uptake of 2.5 µg of
I-ox-LDL. Twenty-five µg of LDL initially incubated
with lacZ cells inhibited degradation of ox-LDL by only 8%, but 25
µg of LDL previously incubated with clone 12 inhibited
I-ox-LDL uptake by 71%, i.e. it was just as
effective as copper-oxidized LDL. Twenty-five µg of LDL conditioned
by incubation with clones 2 and 8 yielded intermediate values of
inhibition.
In another set of experiments, we took unlabeled LDL
preconditioned by a 20-h incubation with lacZ cells and 15-LO cells and
then exposed the conditioned LDL samples to a brief oxidative stress by
exposure to copper for 3.5 h in the absence of cells. These LDL were
then tested for their ability to compete with I-ox-LDL
for uptake by macrophages (Table 6). In this experiment,
unlabeled copper-oxidized LDL inhibited uptake of
I-ox-LDL by 61%, and clone 12-conditioned LDL inhibited
uptake by 44%, whereas LDL conditioned by incubation with clone 2 and 8
inhibited uptake by only 15 and 12%, respectively. LDL from lacZ cells
did not inhibit degradation at all. Similar results were seen in four
other experiments.
It is of interest that despite the obvious
enhanced susceptibility of LDL conditioned by incubation with clone 12
cells to copper-mediated oxidation, as measured by conjugated diene
formation, and by its greater ability to compete with I-ox-LDL for macrophage uptake, we could not demonstrate
enhanced agarose gel electrophoretic mobility after 3, 6, or even 18 h of copper exposure (data not shown), as compared to the
lacZ conditioned LDL exposed to similar conditions. The reason for this
dissociation is not known but it implies that a greater increase in net
negative charge is not a sine qua non for recognition by
macrophage scavenger receptors.
These studies clearly show that when LDL was incubated with a cell line overexpressing 15-LO there was significantly greater seeding of that LDL with LOOH compared to LDL incubated with control cell line lacZ ( Table 2and Table 3). At the end of the first hour, the medium incubated with clone 12 showed five to six times more LOOH than was found in medium incubated with lacZ cells. However, the LOOH content of the medium did not increase linearly. Even in the incubations with clone 12, which showed the highest rate of LOOH buildup, the medium content of LOOH did not double between the end of the first hour and the end of the second hour (Table 3). In the case of the other two clones expressing 15-LO, there was actually less LOOH in the medium at the end of the second hour. The reason for this decided non-linearity is not clear, but it has a great deal of relevance to the interpretation of the studies in which LDL was incubated for longer times with these cell lines. One would anticipate that the higher content of LOOH in LDL incubated with clone 12 would lead to a greater rate of subsequent oxidative modification. Indeed, LDL conditioned by incubation with clone 12 had significantly greater rates of macrophage degradation than LDL conditioned by incubation with lacZ cells (p < 0.003) (Fig. 3). This was true despite the fact that the absolute rate of degradation of the LDL conditioned by the lacZ mouse fibroblasts was surprisingly high. In some experiments it approached that seen with LDL modified by incubation with endothelial cells or macrophages. Thus, LDL incubated for 24 h with monolayers of mouse peritoneal macrophages is generally degraded at about 7.5 µg/mg macrophage protein/5 h, and the rate for LDL conditioned by endothelial cells is only slightly less than that. In fact, it is unusual to see degradation at a rate greater than 10 µg/mg/5 h, even with copper-oxidized LDL. Thus, the values seen here with PA317 cells may be approaching a ``ceiling.'' In retrospect, the choice of these cells was not optimal for the present studies. Even though the wild-type cells contain no 15-LO activity, they clearly contain other systems that can oxidize LDL at a brisk rate. Despite this, the addition of 15-LO increased the capacity of the cells to seed LDL in the medium with LOOH and make the LDL more susceptible to oxidative modification. O'Leary et al.(36) demonstrated directly that enrichment of LDL with lipid peroxides resulting from 15-LO activity led to propagation of LDL oxidation in the presence of transition metals. Similarly, we demonstrate here that an enhanced content of LOOH in LDL incubated with 15-LO cells also made these LDL more susceptible to modification when subsequently challenged with an oxidative stress.
Different LDL
preparations can differ widely in their susceptibility to oxidative
modification(37, 38) . Many factors contribute to that
variability, including the progressive oxidation of LDL, even when
stored at low temperatures. Khouw et al.(22) have
recently called attention to the way in which radioiodination of LDL
accentuates this oxidation during storage. LDL that has already been
``seeded'' with lipoperoxides (as a result of storage and/or
iodination) will undergo significantly more rapid modification when
subjected to oxidative stress, whether it be oxidation catalyzed by
copper or oxidation catalyzed by cells. The fact that EDTA can inhibit
macrophage-induced modification of LDL in F-10 medium (39) tells us that metal-catalyzed propagation in the medium is
essential for maximum biological modification of LDL and that this
probably rests on the presence of lipoperoxides seeded in the LDL
itself. We believe that in our first studies (Fig. 3), in which
the 15-LO cells had only a 2-fold greater ability to modify LDL, the
results were strongly affected by artifactual oxidation of the
substrate I-LDL, i.e. the radiation-induced
oxidation minimized differences that resulted from seeding by 15-LO
cells. This is supported by comparison with the data in Table 5and Table 6showing results from later studies in
which unlabeled LDL was used rather than
I-LDL. The
ability of such conditioned LDL to compete with copper-oxidized
I-LDL was studied rather than directly measuring the rate
of degradation of conditioned
I-LDL. Now the contrast
between LDL conditioned by clone 12 or by lacZ cells was much greater.
In these studies, the unlabeled LDL was first seeded with
hydroperoxides by incubation with the PA317 clones, then subjected to
an oxidative stress by incubation with macrophages, and finally tested
for competition with
I-ox-LDL. LacZ-conditioned unlabeled
LDL showed only an 8% inhibition of the degradation of oxidized
I-LDL while clone 12-conditioned LDL inhibited by 71%, a
value similar to that achieved by unlabeled LDL oxidized by exposure to
copper (Table 5). As shown in Table 6, similar results were
obtained when the cell-conditioned unlabeled LDL was incubated with
copper for 3.5 h (instead of the preliminary macrophage incubation)
before testing its ability to compete with
I-ox-LDL. In
this instance, the lacZconditioned LDL showed no detectable inhibition
while the clone 12-conditioned LDL inhibited by 44% (compared with 61%
for competition by unlabeled copper-oxidized LDL).
The precise mechanism(s) linking the increased cellular expression of 15-LO to the increased seeding of LDL with lipoperoxides is not established by these studies. One possibility is that 15-LO acts on endogenous lipids in the cell and that these are subsequently transferred to LDL in the medium. Additional possibilities are not ruled out, however. For example, while 15-LO is an intracellular enzyme, it might conceivably be able to act directly on LDL making contact with the cell's surface or there might be increased generation of reactive oxygen species linked to the action of 15-LO, as suggested by the studies of Cathcart and co-workers(40) .
These studies establish that cellular 15-LO has the potential to play a role in oxidative modification of LDL. Several lines of evidence compatible with such a role were briefly listed in the introduction. Perhaps the strongest evidence that it plays a role in vivo comes from the studies of Kühn et al.(14, 19) , showing that the lipids in fatty streak lesions of cholesterol-fed rabbits contain a disproportionately high percentage of stereospecifically derived hydroxy fatty acids. After 12 weeks of feeding, 73.7% of the 13-HODE was present in the S form (instead of the theoretical 50% if no enzymatic activity were involved). These findings, together with the previously reported finding that the 15-LO gene and its protein product are present at high levels in early lesions(15, 16) , are compatible with a role of 15-LO in vivo but do not establish it. Ultimately, it may require studies in transgenic mice to assign the proper weight to 15-LO in the atherogenic process.