doi: 10.1161/hq0701.092092
© 2001 American Heart Association, Inc.
Vascular Biology |
Changes in Expression of Antioxidant Enzymes Affect Cell-Mediated LDL Oxidation and Oxidized LDL–Induced Apoptosis in Mouse Aortic Cells
From the Department of Physiology (Z.G., H.V., H.Y., J.V., A.R.) and the Department of Cellular and Structure Biology (X.C., J.M.), University of Texas Health Science Center at San Antonio, and the Geriatric Research, Education and Clinical Center (Z.G., H.V., A.R.), South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio, Tex; the Department of Pediatrics (C.J.E.), University of California, San Francisco; and the Institute of Chemical Toxicology and Department of Biochemistry (Y.-S.H.), Wayne State University, Detroit, Mich.
Correspondence to Dr ZhongMao Guo, Department of Physiology, UTHSCSA, 7703 Floyd Curl Dr, San Antonio, TX 78229. E-mail GUO{at}uthscsa.edu
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Abstract—Transgenic mice overexpressing Cu/Zn superoxide dismutase (hSod1Tg+/0) or catalase (hCatTg+/0) and knockout mice underexpressing manganese superoxide dismutase (Sod2+/-) or glutathione peroxidase-1 (Gpx1-/-) were used to study the effect of antioxidant enzymes on cell-mediated low density lipoprotein (LDL) oxidation and oxidized LDL (oxLDL)-induced apoptosis. Incubation of LDL with mouse aortic segments or smooth muscle cells (SMCs) resulted in a significant increase in LDL oxidation. However, LDL oxidation was significantly reduced when LDL was incubated with aortic segments and SMCs obtained from hSod1Tg+/0 and hCatTg+/0 mice compared with those obtained from wild-type mice. In contrast, LDL oxidation was significantly increased when LDL was incubated with aortic segments and SMCs obtained from Sod2+/- and Gpx1-/- mice. CuSO4-oxidized LDL increased DNA fragmentation and caspase activities in the primary cultures of mouse aortic SMCs. However, oxLDL-induced DNA fragmentation and caspase activities were reduced 50% in SMCs obtained from hSod1Tg+/0 and hCatTg+/0 mice compared with wild-type control mice. In contrast, oxLDL-induced DNA fragmentation and caspase activities were significantly increased in SMCs obtained from Sod2+/- and Gpx1-/- mice. These findings suggest that overexpression of Cu/Zn superoxide dismutase or catalase reduces cell-mediated LDL oxidation and oxLDL-induced apoptosis, whereas underexpression of manganese superoxide dismutase or glutathione peroxidase-1 increases cell-mediated LDL oxidation and oxLDL-induced apoptosis.
Key Words: LDL • transgenic mice • aortas • antioxidant enzymes
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Introduction |
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Accumulation of oxidized LDL (oxLDL) in the arterial wall has been proposed to play a key role in the development of atherosclerosis.1 2 However, how oxLDL is formed and how oxLDL damages the arterial wall are still a matter of debate. In vivo, little LDL is oxidized in the circulation; therefore, it is thought that the arterial pool of oxLDL is, at least in part, derived from LDL that enters the intima from plasma as native LDL but is then oxidized locally by cells in the arterial wall.1 In this regard, 3 types of cells within the arterial wall, ie, endothelial cells (ECs),3 4 smooth muscle cells (SMCs),3 and macrophages,5 have all been shown to mediate LDL oxidation. In vitro, oxLDL can injure or kill a variety of cell types, including ECs,6 SMCs,6 and macrophages.7 Recently, studies have shown that the degree of LDL oxidation is directly proportional to the rate of superoxide production8 and that oxLDL increases superoxide formation in cultured vascular cells.9 Thus, cell-mediated LDL oxidation and oxLDL-induced cell death appear to be a reactive oxygen species (ROS)-dependent cycle. Free radicals released from vascular cells mediate oxidation of LDL that enters the arterial wall as a native form; the oxidized forms of LDL, in turn, induce cells to generate more free radicals. When lesions induced by free radicals reach a critical level, cell death occurs.
Several studies have shown that an increase in superoxide dismutase (SOD) activity is capable of preventing LDL oxidation in culture. For example, Heinecke et al10 have reported that addition of SOD to the culture medium inhibits the LDL oxidation induced by human and monkey SMCs. Steinbrecher4 has observed that SOD attenuates the LDL oxidation mediated by rabbit ECs and SMCs in culture. More recently, Fang et al8 have reported that transduction of the Sod1 or Sod2 gene into ECs reduces LDL oxidation. The effect of antioxidant enzymes on oxLDL-induced cell death has also been investigated. For example, Galle et al9 have observed that the presence of exogenous SOD and catalase in culture medium inhibits oxLDL-induced apoptosis in human ECs and rabbit aortic segments.
In the present study, we examined the effect of antioxidant enzymes on cell-mediated LDL oxidation and oxLDL-induced apoptosis by using aortic segments or SMCs obtained from transgenic mice with elevated activities of Cu/Zn-SOD or catalase and from knockout mice with reduced activities of Mn-SOD or glutathione peroxidase (Gpx)1. Our data clearly demonstrate that an increase in the activity of either Cu/Zn-SOD or catalase reduces cell-mediated LDL oxidation and oxLDL-induced apoptosis. In contrast, a decline in the activity of either Mn-SOD or Gpx1 elevates cell-mediated LDL oxidation and oxLDL-induced apoptosis.
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Animals
Transgenic mice overexpressing either Cu/Zn-SOD (hSod1Tg+/0 mice) or catalase (hCatTg+/0 mice) were respectively generated with 80-kb P1 clones containing either the entire human Sod1 gene (10 kb) or the entire human catalase gene (30 kb) in the Nathan Shock Center of Excellence in Basic Biology of Aging at the University of Texas Health Science Center at San Antonio. The hemizygous transgenic mice were identified by polymerase chain reaction with the use of primers specific for human Sod1 or the catalase gene. The knockout mice with reduced levels of Mn-SOD (Sod2+/-) were developed by deletion of the third exon of the Sod2 gene,11 which resulted in a loss in enzyme activity. Lack of the Sod2 gene in the mouse germ line resulted in a lethal phenotype, and homozygous Sod2+/- mice died within 3 weeks from cardiomyopathy11 or neurodegeneration,12 depending on the genetic background. The heterozygous Sod2+/- mice used in the present study were obtained from Epstein’s laboratory (Li et al11 ) and were backcrossed into C57BL/6 for 18 generations. The Sod2+/- mice, which appeared normal and fertile, showed reduced Mn-SOD activity in all the tissues studied.13 The Gpx1 knockout mice used in the present study were developed by disrupting the second exon of the Gpx1 gene with a neomycin resistance cassette14 and were backcrossed to 57BL/6 for 12 generations. Homozygous Gpx1-/- mice deficient in Gxp1 activity were healthy and fertile.14 The Sod2+/- and Gpx1-/- mice were identified by polymerase chain reaction and Southern blots, as described by Li et al11 and Ho et al,14 respectively. The wild-type controls used in the present study were the littermates of the aforementioned transgenic/knockout mice. Mice at 4 to 6 months of age were euthanized by cervical dislocation for the collection of tissues. All procedures for handling the animals were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and the Subcommittee for Animal Studies at the Audie L. Murphy Memorial Veterans Hospital.
Measurement of Antioxidant Enzyme
Activities
The activities of Cu/Zn-SOD, Mn-SOD, and
extracellular SOD (EC-SOD) in mouse aortas and SMCs were measured by
using SOD activity gels as previously described by Van Remmen et
al.13 Briefly, aorta and SMC
extracts containing 15 µg of protein were separated on a 10%
polyacrylamide gel. The gel was soaked in a solution containing
nitro blue tetrazolium, riboflavin, and
N,N,N',N'-tetramethyl-ethylenediamine.
The gel was then illuminated at 560 nm in a light box for 15 minutes.
Under these conditions, the area where the SOD activity was located
showed an achromatic band on the gel. The gel image was captured with a
digital camera imager system (ImageMaster VDS, Amersham
Pharmacia Biotechnology) and analyzed by using
ImageQuant software (Molecular Dynamics). The
activities of catalase and Gpx1 in mouse aortas and SMCs were measured
by using the catalase and Gpx activity gels as described by Sun et
al,15 with slight
modifications. Aorta and SMC extracts containing 50 µg of protein
were separated on an 8% native polyacrylamide gel with a 5%
stacking gel. For detection of Gpx1 activity, the gel was incubated in
a solution containing 0.008% cumene hydroperoxide and 1.5 mmol/L
reduced glutathione for 10 minutes and then stained with a solution
containing 1% ferric chloride and 1% potassium ferricyanide until the
gel became dark green with yellow activity bands. For detection of
catalase activity, the gel was soaked in a solution containing 0.003%
hydrogen peroxide for 10 minutes and then stained with the same
staining solution used for the Gpx activity gel. The gel images were
recorded and analyzed by using the image-acquiring system
and software as described above.
Isolation and Culturing of SMCs
Mouse aortic SMCs were isolated and cultured as
described by Wang et al,16
with modifications. After removal of the connective tissue and blood,
mouse aortas were incubated in an enzyme solution containing 1.5 mg/mL
collagenase/dispase (Boehringer-Mannheim), 0.5
mg/mL elastase type II-A (Sigma Chemical
Co), 1 mg/mL trypsin inhibitor type I-S
(Sigma), and 2 mg/mL BSA
(Sigma). After 30 minutes of incubation, the
medium was changed to the second enzyme solution that contained 1 mg/mL
collagenase type II (Sigma), 0.3
mg/mL trypsin inhibitor type I-S, and 2 mg/mL BSA. The
aorta was incubated in this solution for 30 minutes. All incubations in
the enzyme solutions were carried out in 95% air/5%
CO2 at 37°C. The aorta was then triturated by
using a fire-polished Pasteur pipette to obtain a cell suspension. This
procedure gave cell viabilities >95%, and the cells contracted in the
presence of 10 µmol/L norepinephrine, as observed under a
phase-contrast optical microscope. Cells were cultured in DMEM
containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin.
The cells were cultured at 37°C under 5% CO2
and used within 16 to 24 hours after they were
plated.
Determination of LDL Oxidation Induced by
Aortic Segments and SMCs
Human LDL (200 µg protein/mL) was incubated with 10
mg of mouse aorta or 1 million SMCs at 37°C under 5%
CO2 in 0.5 mL of F-10 Ham medium (serum-free)
containing 5 µmol/L iron EDTA. An equal amount of LDL was incubated
in the above medium in the absence of cells, or equal numbers of SMCs
were incubated in the above medium in the absence of human LDL as
controls. After 16 hours of incubation, LDL oxidation in the medium was
determined by using a thiobarbituric acid–reactive substances (TBARS)
assay as described by Zhu et
al,17 with slight
modifications. Briefly, the cell/tissue suspension was
centrifuged at 1000g to
remove cells/tissues, and the supernatant was mixed with 0.5 mL of 10%
trichloroacetic acid and 0.5 mL of 0.6% thiobarbituric acid. The
mixture was incubated at 80°C for 15 minutes and cooled on ice for 5
minutes. After mixing with 1.5 mL of butanol, the mixture was
centrifuged at 2000g
for 10 minutes, and the absorbance of the supernatant was determined at
532 nm by using a DU 7400 spectrophotometer (Beckman Instrument Inc). A
standard curve was generated by using the optical density of
1,1,3,3-tetraethoxypropane, which was converted quantitatively to
malondialdehyde in the thiobarbituric acid reaction. Nanomolar
equivalents of TBARS per milligram of LDL protein in the culture
supernatant were calculated on the basis of the optical density of the
supernatant and the absorption coefficient produced from the standard
curve.
Determination of DNA Double-Strand Breaks
Induced by OxLDL
The oxLDL used in the present study was obtained
by incubation of human LDL with CuSO4 (5
µmol/L) in PBS for 24 hours at 37°C. The level of LDL oxidation was
56 to 62 nmol of TBARS per milligram protein. The native LDL showed
0.5 to 1 nmol of TBARS per milligram protein. Mouse SMCs
(5x105) were cultured with or without oxLDL
(50 µg/mL) or native LDL (50 µg/mL) for 4 hours. DNA double-strand
breaks were determined by using a comet assay as described by Olive and
Banath,18 with slight
modifications. Cultured cells were mixed with 0.5% low-melting
agarose, and the mixture was layered on a microscope slide. Cells were
lysed by immersing the slide in 2.5 mol/L sodium chloride, 0.1 mol/L
EDTA, 0.1 mol/L Tris-Cl, pH 10, 10% dimethyl sulfoxide, and 1%
Triton X-100 at 4°C for 2 hours. The slide was
then transferred into a horizontal gel electrophoresis
apparatus, and electrophoresis was carried out at 30 V
(1 V/cm) in 1x TBE buffer (89 mmol/L Tris base, 89 mmol/L
boric acid, and 2 mmol/L EDTA) for 10 minutes. Slides were stained
with SYBR green (Molecular Probes, Inc). The
fluorescence image of cells was viewed by using a
fluorescence microscope (Nikon, 100-W
mercury lamp) and analyzed by using a
Komet 4.0 SCG image analysis system
(Integrated Laboratory System). One hundred cells were analyzed
from each slide, and 3 slides were examined for each
sample.
Caspase Activity Assay
The caspase activity was determined by using an
exogenous fluorogenic peptide substrate,
carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin
(DEVD-AFC, Enzymes Systems
Products).19 The
DEVD-AFC can be cleaved by caspase-3, -6, -7, -8, and -10 after the P1
aspartate residue, resulting in release of the fluorescent
product, AFC.19 The
released AFC has been shown to be proportional to the activities of
these
caspases.19 20 In
this experiment, SMCs grown in a 6-well plate were incubated with LDL,
oxLDL (50 µg/mL), or culture medium alone. After 4-hour incubation,
cells were lysed with a lysis buffer (pH 7.4): 1%
Triton X-100, 115 mmol/L NaCl, 1
mmol/L KH2PO4, 1
mmol/L dithiothreitol, 25 mmol/L HEPES, 1 mmol/L benzamidine,
1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL
phenanthroline, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 10
µg/mL pepstatin. The resulting lysates were centrifuged at
12 000g for 5 minutes at
4°C, and the supernatants (50 µg of protein in 50 µL) were added
to 200 µL of enzyme reaction buffer containing 50 µmol/L peptide
substrate, 0.1% CHAPS, 10% sucrose, 1 mmol/L EDTA, 10
mmol/L dithiothreitol, and 100 mmol/L HEPES (pH 7.4). After
incubation at 37°C for 1 hour, fluorescence was monitored by
exciting the sample at 360 nm and measuring the emission at 530 nm on a
SpectorFluor plate reader (Tecan US Inc). The
fluorescence was normalized by subtracting the background
fluorescence, which was determined with 50 µL of lysis buffer
and 200 µL of reaction buffer. For each experiment, a standard curve
was constructed with free AFC. Based on the standard curve, the
fluorescence reading from the enzymatic reaction was converted
into the molar amount of AFC liberated by the
caspases.
Statistical Analysis
Results from multiple experiments were reported as
mean±SEM. The differences between transgenic/knockout and their
wild-type littermates and the differences between control and oxLDL
treatment were analyzed by 2-factor ANOVA followed by
Sidak’s multiple comparison test. A value of
P
0.05 was considered
statistically significant.
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Results |
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An important concern in using transgenic mice that overexpress or underexpress an antioxidant gene is whether the mice have compensated for the increased or reduced expression of the gene by upregulating or downregulating other components of the antioxidant defense system. Previous studies have shown that the mice with reduced expression of Mn-SOD13 or Gpx114 did not exhibit significantly altered expression or activities of other major antioxidant genes in all tissues studied, including brain, heart, lung, liver, kidney, spleen, and muscle. In the present study, we measured the activities of Cu/Zn-SOD, Mn-SOD, EC-SOD, catalase, and Gpx1 in aortas obtained from hSod1Tg+/0, hCatTg+/0, Sod2+/-, and Gpx1-/- mice. Figure 1
shows representative photographs of
SOD, catalase, and Gpx activity gels. The 3 isoenzymes of SOD were
separated by molecular weight in the SOD activity gel. Cu/Zn-SOD is a
64-kDa protein composed of 2 identical 32-kDa subunits. Mn-SOD is a
tetrameric protein with a molecular mass of 88 kDa. It has been
reported that rat EC-SOD, with a molecular mass of 68 kDa, is composed
of 2 subunits.21 Thus, the
top, middle, and bottom bands of the SOD activity gel correspond to the
activity of mouse Mn-SOD, EC-SOD, and Cu/Zn-SOD, respectively. As shown
in
Table 1, the Cu/Zn-SOD activity in the aortas of
hSod1Tg+/0
mice was 2.5-fold higher than that in the aortas of their wild-type
littermates; however, the activities of Mn-SOD, EC-SOD, catalase, and
Gpx1 were not significantly altered in the aortas of
hSod1Tg+/0
mice. The catalase activity in the aorta of
hCatTg+/0
mice was 2.3-fold higher than that in the wild-type control mice,
and the activities of other antioxidant enzymes studied were not
significantly different from those in the wild-type mice
(Table 1). The Mn-SOD activity in the aortas of
Sod2+/-
mice was 50% lower than that in the aortas of their wild-type
littermates. The activities of Cu/Zn-SOD, EC-SOD, catalase, and Gpx1
were slightly higher in the aortas of
Sod2+/-
mice compared with the wild-type control mice; however, this difference
was not statistically significant. The Gpx1 activity in the aortas of
Gpx1-/-
mice was barely detectable, and no significant compensation from other
antioxidant enzymes studied was observed in the aortas of
Gpx1-/-
mice compared with their wild-type littermates. To establish whether
oxLDL induces a compensatory alteration in the activities of
antioxidant enzymes, we measured the activities of Cu/Zn-SOD, EC-SOD,
catalase, and Gpx1 in the primary cultures of aortic SMCs that were
incubated with or without oxLDL. As data in
Figure 2 illustrate, aortic SMCs isolated from
hSod1Tg+/0,
hCatTg+/0,
Sod2+/-,
and
Gpx1-/-
mice showed changes in the activities of the antioxidant enzymes
similar to those observed in the aortas of these mice. For example, the
activities of Cu/Zn-SOD and catalase were increased 2.5-fold in the
SMCs obtained from
hSod1Tg+/0
and
hCatTg+/0
mice, respectively. The Mn-SOD activity was reduced 50%, and the
Gpx1 activity was barely detectable in SMCs obtained from
Sod2+/-
and Gpx1 mice, respectively. No significant compensatory alterations
from other antioxidant enzymes were observed in the SMCs incubated with
or without oxLDL
(Figure 2). Therefore, the transgenic/knockout mice used in
the present study showed altered activity of 1 of the antioxidant
enzymes in the aorta and aortic SMCs without compensation by other
major antioxidant enzymes.
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One of the goals of the present study was to determine
the effect of overexpression or underexpression of various antioxidant
enzymes on vascular cell–induced LDL oxidation. Our results showed
that incubation of human LDL with mouse aortic segments obtained from
wild-type mice resulted in a significant increase in LDL oxidation
compared with incubation in a tissue-free system (24±1 versus 1.5±0.4
nmol TBARS/mg protein, respectively). Under similar conditions,
incubation of LDL with aortic segments of
hSod1Tg+/0
and
hCatTg+/0
mice resulted in a 2.5- and 6-fold decrease, respectively, in the
formation of TBARS compared with incubation of LDL with aortic segments
of their wild-type littermates
(Figure 3A). In contrast, incubation of LDL with aortic
segments from
Sod2+/-
and
Gpx1-/-
mice resulted in increases of 35% and 26%, respectively, in the
formation of TBARS compared with incubation of LDL with aortic segments
from their wild-type littermates
(Figure 3A). When LDL was incubated with SMCs, the effect of
overexpressing Cu/Zn-SOD or catalase and of underexpressing Mn-SOD or
Gpx1 on cell-induced LDL oxidation was similar to that observed with
aortic segments
(Figure 3B).
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The comet assay (single-cell microgel electrophoresis) has
been used by many laboratories as a sensitive method for assessing
apoptosis in transformed cell
lines,22 tumor
cells,18 and cells isolated
from human23 and
animal24 tissues. In the
present study, we used the comet assay to evaluate
apoptosis in mouse aortic SMCs.
Figure 4 shows examples of photographic images of the comet
assay. In the cells that possess no or few DNA double-strand breaks,
DNA fluorescence was shown as class I nuclei. However, in the
cells with DNA double-strand breaks, a fluorescence tail along
the electric field was observed (class II and III nuclei) because small
DNA fragments migrated from the nuclei. The image pattern shown in
class III nuclei has been generally accepted to be associated with the
DNA fragmentation that occurs in
apoptosis.24 The
data in
Table 2 show the percentage of class III cells in mouse
aortic SMCs incubated with or without native LDL or
CuSO4-oxidized LDL. When cells were incubated
only with culture medium, 2% of SMCs showed class III nuclei, and
no significant difference was observed between wild-type mice and all
the transgenic/knockout mice. After the primary cultures of SMCs were
incubated with oxLDL, a substantial increase in the percentage of cells
with class III nuclei was observed. Native LDL did not significantly
increase class III nuclei
(Table 2). The increase in cells with class III nuclei
varied in transgenic/knockout mice and their wild-type littermates. For
example, the percentage of cells with class III nuclei in SMCs obtained
from
hSod1Tg+/0
and
hCatTg+/0
mice was 50% lower than that from their wild-type littermates. In
contrast, SMCs obtained from
Sod2+/-
and
Gpx1-/-
mice had 30% more cells with class III nuclei compared with SMCs
obtained from their wild-type littermates
(Table 2).
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Although DNA fragmentation is one of the hallmark
characteristics of apoptotic cell death, recent studies have
shown that DNA fragmentation also occurs in early
necrosis.19 To establish
whether the observed DNA fragmentation induced by oxLDL in aortic SMCs
is related to apoptosis, we measured the caspase activity by
using DEVD-AFC, a peptide substrate of caspase-3, -6, -7, -8, and
-10.19 20 As data
in
Table 3 show, when cells were incubated with culture medium
or native LDL, the caspase activity was similar in all
transgenic/knockout mice and their wild-type controls. In contrast,
after the primary cultures of SMCs were incubated with oxLDL, a
significant increase in the caspase activity was observed. The increase
in caspase activity varied in transgenic/knockout mice and their
wild-type littermates. For example, the caspase activity in SMCs
obtained from
hSod1Tg+/0
and
hCatTg+/0
mice was 50% lower than the caspase activity in SMCs obtained from
their wild-type littermates. In contrast, compared with SMCs obtained
from their wild-type littermates, SMCs obtained from
Sod2+/-
and
Gpx1-/-
mice had 50% higher caspase activity
(Table 3). Thus, data in
Tables 2
and 3 indicate that oxLDL induces apoptosis
in mouse aortic SMCs, that overexpression of catalase or Cu/Zn-SOD
reduces oxLDL-induced apoptosis, and that underexpression of
Mn-SOD or Gpx1 enhances oxLDL-induced
apoptosis.
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In the present study, we observed that the activity of either Cu/Zn-SOD or catalase was significantly increased in the aortas and aortic SMCs obtained from hSod1Tg+/0 and hCatTg+/0 mice, respectively, compared with wild-type mice. In contrast, the activity of either Mn-SOD or Gpx1 was significantly reduced in the aortas and aortic SMCs obtained from Sod2+/- and Gpx1-/- mice, respectively, compared with their wild-type littermates. Aortas and aortic SMCs obtained from these transgenic/knockout mice did not show any evidence of compensation for the altered activity of the antioxidant enzyme by upregulating or downregulating other major components of the antioxidant defense system. Thus, the hSod1Tg+/0, hCatTg+/0, Sod2+/-, and Gpx1-/- mice are valuable models for studying the role of oxidative stress in vascular biology because the antioxidant defense system has been altered in these mice. In the present study, we used these transgenic/knockout mice to evaluate the effect of changes in expression of the antioxidant enzymes on cell-mediated LDL oxidation and oxLDL-induced apoptosis in aortic cells.
At the present time, most of the published studies involving cell-mediated LDL oxidation have used cells from humans3 and cells or aortic segments from larger animals, eg, bovine,3 porcine,8 and rabbit cells or segments.25 Very little has been published involving mice. However, such information is important because of the power of using transgenic/knockout mouse models to study the molecular mechanisms of atherosclerosis. In the present study, we compared the oxidation of LDL by aortic segments or SMCs obtained from transgenic/knockout mice that had an alteration in 1 component of the antioxidant defense system. Our data demonstrated that aortas and aortic SMCs obtained from mice were capable of oxidizing LDL. However, aortic segments and SMCs from hSod1Tg+/0 mice, compared with their wild-type littermates, showed reduced LDL oxidation. In contrast, aortic segments and SMCs from Sod2+/- mice showed elevated LDL oxidation. These results, for the first time, demonstrated that increase in endogenous SOD activity inhibits vascular cell–mediated LDL oxidation and that a decrease in endogenous SOD activity promotes LDL oxidation. These data add to the reports of other investigators showing that addition of SOD to the culture medium4 10 or transfection of the SOD gene to vascular cells protects LDLs from oxidation.8
The mechanism by which vascular cells mediate LDL oxidation has not been elucidated to date. Results from previous studies showing that the degree of LDL oxidation was directly proportional to the rate of superoxide production by cells and that increased SOD activity inhibited LDL oxidation suggest a role of superoxide radicals in mediating LDL oxidation.8 However, superoxide anions do not readily react with most biological molecules9 ; therefore, it has been suggested that the toxicity attributed to superoxide radicals may be mediated, at least in part, by other radical species derived from superoxide anions. It has been known that superoxide can be converted to hydrogen peroxide by SOD in cells. Hydrogen peroxide can readily cross the cellular membrane and form hydroxyl radicals through its interaction with redox-active transitional metals.26 To test whether hydrogen peroxide plays a role in LDL oxidation, Steinbrecher4 and Heinecke et al10 incubated LDLs with ECs and SMCs in the presence or absence of catalase and measured the levels of LDL oxidation induced by these cells. They did not observe any decrease in cell-mediated LDL oxidation in the presence of catalase. However, the use of exogenous catalase to study the role of free radicals in cell-mediated LDL oxidation has several limitations. For example, exogenous catalase may contain impurities that may nonspecifically alter LDL oxidation. Moreover, catalase is negatively charged and may be repelled from the negatively charged cell surface, thus preventing it from destroying hydrogen peroxide near the cell. In the present study, using aortic segments and SMCs obtained from hCatTg+/0 and Gpx1-/- mice, we observed that an increase in intracellular catalase activity reduced LDL oxidation and that a decline in intracellular Gpx1 activity elevated LDL oxidation. Because catalase and Gpx1 catalyze the decomposition of hydrogen peroxide, changes in the activities of catalase and Gpx1 would be predicted to alter the level of intracellular hydrogen peroxide. Thus, our observations with aortic segments and SMCs obtained from mice overexpressing catalase and mice underexpressing Gpx1 suggest that hydrogen peroxide plays a role in vascular cell–mediated LDL oxidation.
Oxidized forms of LDL in vitro have been found to induce a variety of cellular responses, eg, adhesion of inflammatory cells to ECs and the death of ECs and SMCs (see review1 ). Data from recent studies suggest that oxLDL induces vascular cell death via an apoptotic pathway involving activation of the caspases,27 a family of cysteine proteases that are responsible for apoptotic cell death.27 In the present study, we observed that oxLDL, but not native LDL, increased the percentage of cells showing DNA fragmentation and induced caspase activity in the primary cultures of mouse SMCs. These data indicate that oxLDL results in apoptosis in mouse aortic SMCs.28 The importance of SMC apoptosis in the pathogenesis of atherosclerosis has been illustrated by the observation that SMC apoptosis occurs in the atherosclerotic plaques.29 The loss of SMCs will result in decreased biosynthesis of collagen fibers, which strengthens the fibrous cap of atherosclerotic plaques.30 Thus, the loss of SMCs is believed to weaken the fibrous cap and make the plaque more prone to rupture.31 Therefore, induction of SMC apoptosis could be one of the atherogenic actions of oxLDL.
One of the important observations in the present study
is that changes in the activities of antioxidant enzymes in transgenic
and knockout mice alter the oxLDL-induced apoptosis in aortic
SMCs. For example, aortic SMCs obtained from
hSod1Tg+/0
and
hCatTg+/0
mice, compared with aortic SMCs from their wild-type littermates,
showed a 50% decrease in the percentage of cells with DNA
fragmentation and a 50% decrease in caspase activity induced by oxLDL.
These results are consistent with the observations of Galle et
al9 showing that the addition
of SOD or catalase to the culture medium blunted apoptosis
induced by oxLDL. In addition, we observed that aortic SMCs obtained
from
Sod2+/-
and
Gpx1-/-
mice showed a significant increase in the percentage of cells with DNA
fragmentation and a significant increase in caspase activity compared
with wild-type mice. Results from Galle et
al9 and from our studies
indicate that antioxidant enzymes protect vascular cells from
apoptosis induced by oxLDL. Antioxidant enzymes also have been
found to protect cells against apoptosis induced by other types
of stimuli. For example, an increase in the activities of
Cu/Zn-SOD,32
Mn-SOD,33
catalase,34 and
Gpx135 have been shown to
reduce apoptosis induced by sepsis, ionizing irradiation, tumor
necrosis factor-
, and ischemia/reperfusion in cells obtained
from humans and animals. A decrease in the activities of
Cu/Zn-SOD,32
Mn-SOD,36
catalase,37 and
Gpx135 have been shown to
increase apoptosis induced by various stimuli. These findings
suggest that the formation of intracellular superoxide and/or hydrogen
peroxide is 1 of the mechanisms by which these stimuli induce
apoptosis; ie, ROS may function as a second messenger of these
stimuli to activate caspases, leading to apoptotic cell
death (see
review38 ).
Currently, there have been 2 reports in which transgenic mice have been used to study the effect of overexpression of Cu/Zn-SOD on atherosclerosis.39 40 In these studies, Tribble et al40 fed mice overexpressing Cu/Zn-SOD and their wild-type littermates a high fat diet and measured atherosclerosis in the proximal aorta. They observed that overexpression of Cu/Zn-SOD did not reduce the formation of fatty streaks induced by the high fat diet,40 suggesting that ROS, at least superoxide radicals, do not play a role in the formation of fatty streaks induced by a high fat diet. However, in a later study, Tribble et al39 observed that the extent of atherosclerotic lesions in the proximal aorta of the transgenic mice was significantly smaller than that in the aorta of wild-type mice when these mice were exposed to a single x-ray dose and then placed on a high fat diet. These observations provide the first evidence that increase in the expression of Cu/Zn-SOD inhibits the atherogenic effect of oxidative stress, eg, ionizing radiation. Results from the present study have shown that changes in the activities of endogenous antioxidant enzymes, including Cu/Zn-SOD, alter vascular cell–mediated LDL oxidation and oxLDL-induced apoptosis of SMCs. These data suggest that alteration of the antioxidant status in the arterial wall may change the pathogenesis of atherosclerosis. For example, an increase in the activity of antioxidant enzymes may reduce the development of atherosclerosis, and a decrease in the activities of antioxidant enzymes may accelerate the development of atherosclerosis.
In summary, the present study has demonstrated that overexpression of Cu/Zn-SOD or catalase in mice protects LDL against vascular cell–mediated oxidation and reduced oxLDL-induced apoptosis in SMCs. In contrast, underexpression of Mn-SOD or Gpx1 in mice facilitates LDL oxidation and elevates the cytotoxicity of oxLDL. These data suggest that vascular cell–generated ROS, such as superoxide and hydrogen peroxide, are involved in cell-mediated LDL oxidation and oxLDL-induced apoptosis. These data have also demonstrated that mice overexpressing or underexpressing intracellular antioxidant enzymes are potentially valuable models for studying the role of oxidative stress in atherosclerosis.
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Acknowledgments |
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This research was supported by American Heart Association grant 0030239N, National Institutes of Health grants PO1 AG-13319 and AG-16998, and a Merit Review grant from the Department of Veteran Affairs.
Received February 6, 2001; accepted April 6, 2001.
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