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Chronic hyperbaric oxygen treatment elicits an anti-oxidant response and attenuates atherosclerosis in apoE knockout mice

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We previously demonstrated that hyperbaric oxygen (HBO) treatment inhibits diet-induced atherosclerosis in New Zealand White rabbits. In the present study we investigate the mechanisms that might be involved in the athero-protective effect of HBO treatment in a well-accepted model of atherosclerosis, the apoE knockout (KO) mouse. We examine the effects of daily HBO treatment (for 5 and 10 weeks) on the components of the anti-oxidant defense mechanism and the redox state in blood, liver and aortic tissues and compare them to those of untreated apoE KO mice. HBO treatment results in a significant reduction of aortic cholesterol content and decreased fatty streak formation. These changes are accompanied by a significant reduction of autoantibodies against oxidatively modified LDL and profound changes in the redox state of the liver and aortic tissues. A 10-week treatment significantly reduces hepatic levels of TBARS and oxidized glutathione, while significantly increases the levels of reduced glutathione, glutathione reductase (GR), transferase, Se-dependent glutathione peroxidase and catalase (CAT). The effects of HBO treatment are similar in the aortic tissues. These observations provide evidence that HBO treatment has a powerful effect on the redox state of relevant tissues and produces an environment that inhibits oxidation. The anti-oxidant response may be the key to the anti-atherogenic effect of HBO treatment.
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Atherosclerosis 193 (2007) 28–35
Chronic hyperbaric oxygen treatment elicits an anti-oxidant response
and attenuates atherosclerosis in apoE knockout mice
Bhalchandra J. Kudchodkar, Anson Pierce1, Ladislav Dory
Department of Molecular Biology & Immunology, The University of North Texas Health Science Center, Fort Worth, TX 76104, USA
Received 10 March 2006; received in revised form 6 July 2006; accepted 3 August 2006
Available online 14 September 2006
Abstract
We previously demonstrated that hyperbaric oxygen (HBO) treatment inhibits diet-induced atherosclerosis in New Zealand White rabbits.
In the present study we investigate the mechanisms that might be involved in the athero-protective effect of HBO treatment in a well-accepted
model of atherosclerosis, the apoE knockout (KO) mouse. We examine the effects of daily HBO treatment (for 5 and 10 weeks) on the
components of the anti-oxidant defense mechanism and the redox state in blood, liver and aortic tissues and compare them to those of
untreated apoE KO mice.
HBO treatment results in a significant reduction of aortic cholesterol content and decreased fatty streak formation. These changes are
accompanied by a significant reduction of autoantibodies against oxidatively modified LDL and profound changes in the redox state of the
liver and aortic tissues. A 10-week treatment significantly reduces hepatic levels of TBARS and oxidized glutathione, while significantly
increases the levels of reduced glutathione, glutathione reductase (GR), transferase, Se-dependent glutathione peroxidase and catalase (CAT).
The effects of HBO treatment are similar in the aortic tissues. These observations provide evidence that HBO treatment has a powerful effect
on the redox state of relevant tissues and produces an environment that inhibits oxidation. The anti-oxidant response may be the key to the
anti-atherogenic effect of HBO treatment.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Hyperbaric oxygen; Atherosclerosis; Apoe knock-out mice; Oxidative stress; Anti-oxidant enzymes; Liver; Aorta
1. Introduction
There is mounting evidence that atherosclerosis is a
chronic, immune-mediated inflammatory response in the
arterial intima to tissue damage [1]. Tissue damage may
result through increased formation of reactive oxygen
species (ROS) in response to altered shear stress, ischemia/
reperfusion or exposure to modified (oxidized) LDL [2].
Superoxide (O2•−) production is the first step in the gen-
eration of ROS. Potential sources of ROS in the vasculature
include NAD(P)H oxido-reductases, xanthine oxidase, nitric
oxide synthases, cyclooxygenases, lipoxygenases, mitochon-
Corresponding author. Tel.: +1 817 735 0180; fax: +1 817 735 2118.
E-mail address: ldory@hsc.unt.edu (L. Dory).
1Present address: Department of Cellular & Structural Biology, UT HSC
at San Antonio, TX, USA.
drial oxidation and autooxidation of tissue metabolites [3].
NAD(P)H oxidases present in various arterial cell types may
also be a critical source of oxygen radicals [3]. Although
O2•− can spontaneously form hydrogen peroxide, this reac-
tion is greatly accelerated by the action of superoxide dismu-
tases (SODs). Hydrogen and lipid peroxides are then reduced
by catalase (CAT) or glutathione peroxidases (GPx), respec-
tively. GPx iso-enzymes are expressed ubiquitously and play
an important role in reducing H2O2, and lipid hydroperox-
ides. Glutathione-S-transferase (GST) facilitates the excre-
tion of oxidized lipids and other biomolecules out of the cell,
thus preventing cytotoxicity.
Hyperbaric oxygen (HBO) therapy is approved by the
Undersea and Hyperbaric Medical Society for a limited num-
ber of diseases/conditions, including air or gas embolism,
carbon monoxide poisoning, clostridial myonecrosis, excep-
tional anemia resulting from blood loss, necrotizing soft
0021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.atherosclerosis.2006.08.018
B.J. Kudchodkar et al. / Atherosclerosis 193 (2007) 28–35 29
tissue infections, osteoradio-necrosis and thermal burns. It
is associated with a number of positive effects: it improves
transcutaneous oxygen pressure, diabetic foot ulcers and
other characteristics seen in peripheral atherosclerosis. It is
also protective in several animal models of organ ischemia
and endotoxemia [4,5]. HBO treatment may also reverse the
subendothelial hypoxia known to be associated with ischemia
and atherosclerosis [6]. On the other hand, the increased avail-
ability of oxygen in tissues as a result of HBO treatment
leads to increased formation of ROS. Generally, HBO ther-
apy with moderate pressures (2–2.5 ATA) of short duration
(60–90 min) is safe, without serious complication. The bulk
of the studies that investigate the effect of HBO treatment on
the anti-oxidant enzymes in experimental animals involve a
single dose of high-pressure HBO until convulsions or death
are observed. Repeated, short exposures to HBO at pressures
of <3 atm are safe and reduce oxidative stress, possibly by a
combined effect on the pro- and anti-oxidant enzyme activi-
ties [7].
We previously demonstrated that HBO treatment prevents
the progression and accelerates the regression of diet-induced
atherosclerosis in New Zealand White rabbits [8]. This effect
appears to be mediated by, at least in part, reducing the extent
of lipid oxidation in the plasma and tissue compartments,
rather than by changes in plasma lipid and lipoprotein pro-
files. Based on these results we hypothesized that chronic
and intermittent treatment with HBO induces components
of the anti-oxidant defense response, leading to decreased
formation of oxidized substrates (including oxLDL) and an
attenuated immune response. In the present study we test this
hypothesis and demonstrate that HBO reduces atherosclero-
sis without affecting the elevated plasma cholesterol levels
in female apoE knockout (KO) mice. The decrease in fatty
streak lesions is accompanied by a decrease in the titer of
autoantibodies against oxidatively modified LDL, an increase
in the activities of a number of anti-oxidant enzymes and
a concomitant increase in tissue glutathione levels. Over-
all, and perhaps paradoxically, HBO treatment shifts the
tissue environment to a more reduced redox state, resem-
bling that of the WT, normal mice and in direct contrast to
the oxidative environment seen in untreated apoE KO mice.
Our results suggest that the induction of anti-oxidant enzyme
activities (or their restoration) by HBO treatment is strongly
atheroprotective.
2. Materials and methods
2.1. Animals
Four weeks old female apolipoprotein E KO mice, back-
crossed for 10 generations to a C57BL/6 background and
C57BL/6 mice (wild-type, WT) were purchased from Jack-
son laboratories (Bar Harbor, Maine). Mice were maintained
in a pathogen-free environment on a 12 h light, 12 h dark
cycle. They were provided with standard rodent chow and
water ad libitum. After 5 weeks of age, the WT mice were
randomly assigned to two groups (n= 12 each) and the apoE
KO mice were assigned to four groups. One group of apoE
KO mice received HBO treatment for a period of 5 weeks
(n= 10), while the other for a period of 10 weeks (n= 14).
The other two groups of apoE KO mice and the two groups
of WT mice remained untreated and were compared to the
HBO-treated mice. While the untreated apoE KO mice were
used as a control group for the comparison with the HBO-
treated mice, we used WT mice to determine the baseline,
age-matched values for tissue anti-oxidant components we
were examining. The effects of the apoE gene deletion on the
redox state of the various tissues have not been reported.
2.2. HBO treatment
Mice were treated in their cages, placed into a specialized
HBO chamber for animals, with 100% oxygen for 90 min at
2.4 atm, 5 days/week for 5 or 10 weeks. The desired pressure
in the chamber was reached over 15 min; after the 90 min
treatment period the pressure was released over 15 min.
Untreated apoE KO mice and WT mice were handled exactly
the same way, except they were not placed into the hyperbaric
chamber.
2.3. Tissue and sample preparation
At the end of the treatment periods, mice from each group
(untreated and HBO-treated apoE KO as well as WT mice)
were anesthetized by subcutaneous injection of ketamine
(80 mg kg1) and xylazine (10 mg kg1). Blood was col-
lected from the retro-orbital plexus into heparinized tubes.
The abdomen and chest were opened, and the organs were
perfused with ice-cold PBS. Tissues were excised, frozen in
liquid nitrogen and kept at 80 C until analysis. All animal
procedures were approved by the Institutional Animal Care
and Use Committee.
2.4. Assessment of atherosclerosis
Atherosclerosis or fatty streak formation was evaluatedby:
(a) measuring total aortic cholesterol content; and (b) en face
analysis of Oil Red O-stained sections of the aortic arc. For
cholesterol content, aliquots of aortic homogenate, prepared
as described below, were extracted with chloroform/methanol
(2:1, v/v) and cholesterol content determined by the enzy-
matic method using a microtiter plate [9].En face analyses
of lesions were carried out as described [10]. The extent of
lesioned surface area was determined by analysis with Image
Pro Plus (Media Cybernetics).
2.5. Biochemical analyses of plasma, liver and aorta
2.5.1. Plasma and plasma lipoproteins
Plasma lipoprotein distribution and cholesterol content
were determined by FPLC chromatography of plasma fol-
30 B.J. Kudchodkar et al. / Atherosclerosis 193 (2007) 28–35
lowed by determination of cholesterol content in each frac-
tion.
2.5.2. Detection of anti-oxLDL antibodies by ELISA
The levels of immunoglobulin (Ig) autoantibodies binding
to native LDL (nLDL), copper–oxidized LDL (oxLDL) and
malondialdehyde modified LDL (MDA-LDL) were deter-
mined [11,12] in pooled plasma samples using 1:80 and 1:480
dilution for oxLDL and MDA-LDL, respectively. The amount
of IgM bound to nLDL, oxLDL and MDA-LDL antigen was
detected with biotin-conjugated goat anti-mouse IgM. Data
are expressed as relative absorbance units at 630nm and are
corrected for the non-specific binding to native LDL.
2.5.3. Tissue homogenates
Frozen liver and aortic tissues were ground with mortar
and pestle in liquid nitrogen. The powdered samples were
transferred into a Dounce homogenizer and homogenized
in 10 volumes of extraction buffer (ice-cold 50 mM potas-
sium phosphate buffer pH 7.4, containing a protease inhibitor
cocktail and 1 mM EDTA) at 3–5 C. The homogenates were
centrifuged at 10,000 ×gat 4 C for 10 min and the super-
natant was divided into aliquots and stored at 80 C prior
to use.
2.5.4. Measurement of lipid oxidation
Lipid oxidation in liver tissue homogenates was mea-
sured by determining thiobarbituric acid-reactive substances
(TBARS), as described [13].
2.5.5. Assay of reduced glutathione (GSH) content
The GSH content of the liver homogenate was measured
spectrophotometricaly and that of the aortic homogenate
was determined fluorometricaly [14]. Liver and aortic tissue
GSH content was calculated using concurrently run standard
curves and expressed as nmoles of GSH/mg of sample pro-
tein.
2.5.6. Assay of oxidized glutathione (GSSG) content
N-ethylmaleimide (NEM) was added (50 mM final) to
the deproteinized supernatant of the liver homogenate. After
incubation for 1 h at RT excess NEM was removed by suc-
cessive extractions (10×) with ethyl ether (1:1, v/v). The
ether-free residue was dissolved in Tris-EDTA buffer (pH
7.6) containing 1 mM NADPH and 6 U of glutathione reduc-
tase (GR). Reduced glutathione levels were determined, as
described above. Tissue GSSG content was calculated using
concurrently run standard curves and expressed as nmoles of
GSSG/mg of sample protein.
2.5.7. Glutathione redox state
The redox state of the liver was determined as
the ratio of oxidized glutathione/reduced glutathione ×2
(GSSG/2GSH). The glutathione redox couple is two to four
orders of magnitude more abundant than any other redox cou-
ple in the cell [15].
2.6. Enzyme assays
2.6.1. Plasma paraoxonase 1 (PON-1) and arylesterase
activities
Paraoxonase (PON1) activity towards paraoxon is spe-
cific for PON-1 and was assayed by measuring p-nitrophenyl
release from the substrate paraoxon [8]. Plasma arylesterase
activity, shared by PON-1 and PON-3 was measured using
phenylacetate as substrate [16]. Blanks were included
to correct for the nonenzymatic hydrolysis of the sub-
strate. The enzyme activity was calculated from the molar
extinction coefficients: 17,100 M1cm1for PON-1 and
1310 M1cm1for arylesterase. One unit of the enzyme
activity equals 1 mol of substrate hydrolyzed (L min1).
2.6.2. Catalase activity
Catalase activity was measured by the method of Aebi
[17]. The decomposition of H2O2was monitored at 240 nm,
at 25 C for 1 min. One unit of catalase activity equals 1 mol
of H2O2consumed (min mg1) of sample protein.
2.6.3. Oxidized glutathione reductase (GR) activity
GR activity was measured, as described, using blanks
that did not contain GSSG [18]. The oxidation of NADPH
was followed at 25 C at 340 nm for 3 min. GR activ-
ity was calculated using the extinction coefficient of
6.22 mM1cm1, and expressed as nanomoles of NADPH
consumed (min mg1) of sample protein.
2.6.4. Glutathione S-transferase (GST) activity
Tissue GST activity was measured, as described by Habig
and Jakoby [19]. The increase in the absorbance due to the for-
mation of the conjugate between 1-chloro-2,4-dinitrobenzene
(CDNB) and reduced glutathione (GSH) was monitored at
340 nm at 25 C. GST activity was calculated using the
extinction coefficient of 9600 M1cm1, and expressed as
nanomoles of GSH–CDNB conjugate formed (min mg1)of
sample protein.
2.6.5. Glutathione peroxidase (GPx) activity
GPx activity was determined using a glutathione
reductase-coupled assay [20]. The total GPx activity was
determined using cumin hydroperoxide as the substrate and
the activity of Se-dependent GPx was measured with H2O2
as substrate. The activity of non-Se-dependent GPx was
obtained by calculation. NADPH consumption was moni-
tored at 340 nm for 4 min at 25 C. Blanks were run using
homogenization buffer in place of the tissue homogenate.
GPx activity was calculated using the extinction coefficient
of 6.22 mM1cm1and expressed as nanomoles of NADPH
consumed (min mg1) of sample protein.
2.6.6. Protein
Tissue homogenate protein content was determined by the
Lowry method [21], using bovine serum albumin as stan-
dard.
B.J. Kudchodkar et al. / Atherosclerosis 193 (2007) 28–35 31
2.6.7. Statistical analysis
Values reported in the text and in the table represent
means ±SEM. Data obtained from apoE KO mice (untreated
or HBO-treated) are expressed as percent of those mea-
sured in age-matched WT mice. One-way ANOVA (Bon-
ferroni/Dunn test) and Student’s t-test were performed to
see whether any significant differences occurred within and
between groups. A probability p0.05 was accepted as sta-
tistically significant. All analyses were performed with the
use of Stat View 4.5 software (Abacus Concepts, Berkley,
CA). Although apoE KO mice were treated for 5- and 10-
week periods, data are shown only for the 10-week period.
Unless otherwise stated, the 5-week data are similar to the
10-week data.
3. Results
3.1. Body and spleen weights, plasma cholesterol levels
and paraoxonase activities
As shown in Table 1, HBO treatment has no effect on
body weight or plasma cholesterol concentrations of the
apoE KO mice. On the other hand, HBO treatment signif-
icantly reduces spleen weight (expressed as percent of body
weight) and increases plasma PON-1 and arylesterase activ-
ities. These findings are in agreement with our previous
findings in cholesterol-fed rabbits [8]. It may be of interest
that apoE KO mice, when compared to the WT mice, have a
significantly reduced body weight, increased spleen weight
and significantly suppressed plasma PON-1 and arylesterase
activities. It is also of interest that HBO treatment of the
apoE KO mice “corrected” these values to be similar to and
in some cases indistinguishable from those of the WT mice.
Plasma cholesterol levels were markedly higher in apoE KO
mice when compared to WT mice (p< 0.0001), and HBO
treatment had no effect on them or on the distribution of
cholesterol among the lipoprotein fractions, as analyzed by
FPLC chromatography (data not shown).
The hypercholesterolemia of the apoE KO mice is asso-
ciated with a profound decrease in plasma PON-1 activity (a
28% decrease), an observation similar to the one we made in
cholesterol-fed rabbits [8]. HBO treatment restores plasma
PON-1 activities of the apoE KO mice to normal, WT lev-
els, but it is less effective in restoring plasma arylesterase
activity.
3.1.1. Autoantibodies to oxidized LDL
The levels of autoantibodies to oxidized LDL have been
shown to reflect the levels of oxLDL in circulation and cor-
relate positively with the progression and the regression of
experimental atherosclerosis in mouse models [11,12].In
accord with these reports the circulating levels of antibodies
to both, anti-oxLDL (Fig. 1A) and anti- MDA-LDL (Fig. 1B)
were significantly higher (p< 0.0001) in apoE KO mice when
compared to WT mice. After 10 weeks of HBO treatment the
levels of circulating autoantibodies against both oxLDL and
MDA-LDL declined by 45% (p< 0.001) and 50% (p< 0.001),
respectively and were not significantly different from those
of WT mice.
3.2. Oxidation products and anti-oxidant enzymes of the
liver
There is no single measure of the intracellular net oxidant
load under conditions of oxidative stress. Lipids as well as
other biomolecules, such as glutathione, are oxidized and
accumulate in plasma and tissues under oxidative stress. The
data shown in Figs. 2 and 3 are expressed as percent of the
age-matched control (WT) values. This allows for a direct
comparison of the effects of HBO treatment on the variables
of the apoE KO mice, as well as an immediate comparison of
the apoE KO mice to WT animals. Although only the results
after the 10 week treatment period are shown, changes in
the same direction, albeit of lower magnitude were observed
after 5 weeks of treatment.
3.2.1. Lipid oxidation and redox environment
As expected and shown in Fig. 2, hepatic TBARS levels
are significantly higher in the apoE KO mice, when compared
to WT mice (34%, p< 0.0001). HBO treatment results in a
marked reduction in the TBARS formation in the livers of
apoE KO mice to levels not different from those of WT mice.
Cellular levels of oxidized glutathione (GSSG) are known
to increase under oxidative stress. Like TBARS, the GSSG
content of the liver is markedly higher in apoE KO mice
(39%, p< 0.0001), but HBO treatment completely reverses
this accumulation to levels indistinguishable from those of
Table 1
Body and spleen weights, plasma cholesterol concentrations and paraoxonase activities in wild type (WT) and apoE KO mice
Wild-type ApoE KO ApoE KO + HBO
Body weight (g) 21.2 ±0.3 19.8 ±0.4*20.1 ±0.4*
Spleen weight (%BW) 0.40 ±0.01 0.47 ±0.01*0.43 ±0.01*,#
Plasma chol. (mg dL1) 80.7 ±0.8 411 ±11*454 ±26*
PON-1 (mmol L1min1) 147.7 ±7.3 105.7 ±4.4*145.9 ±7.5#
Arylestrase (mmoles L1min1) 72.6 ±4.2 46.6 ±0.7*60.3 ±2.8*,#
Values represent averages ±SEM of 12 WT, 9 apoE KO and 10 apoE KO +HBO-treated mice for body and spleen weight values. The remaining values are
averages ±SEM of 5 animals in each group.
*Significantly different (p< 0.05) from WT values.
#Significantly different (p< 0.05) from untreated apoE KO values.
32 B.J. Kudchodkar et al. / Atherosclerosis 193 (2007) 28–35
Fig. 1. Titers of autoantibodies against Cu–oxLDL and MDA-LDL. Mouse anti-oxLDL or MDA-LDL IgM was quantified by an ELISA using goat anti-mouse
IgM conjugated with biotin, followed by incubation with avidin-conjugated horseradish peroxidase. Optical density at 630 nm was corrected for native LDL.
Open bars represent the WT animals, while the stippled and full bars represent apoE KO mice in the absence or presence of HBO treatment, respectively
(average ±SEM of 5 animals in each group). (*) Significantly different from WT mice. (#) Significantly different from untreated apoE KO mice.
WT mice. Conversely, hepatic GSH levels in the apoE KO
mice are significantly lower (16%, p< 0.005) when com-
pared to the WT mice. Treatment with HBO restores GSH
to normal, WT levels. These results indicate that the liver
glutathione redox state in the apoE KO mice is shifted pro-
foundly to an oxidative environment: a 67% increase in the
GSSG/2GSH (p< 0.0001), when compared to age-matched
WT mice. HBO treatment completely reverses this trend.
3.2.2. Enzymes
HBO treatment completely restores (raises) the hepatic
glutathione reductase and catalase activities in apoE KO mice
to normal (WT) values. HBO treatment also increases hepatic
Fig. 2. Oxidation products and anti-oxidant enzymes of the liver. Enzymes,
metabolites or metabolite ratios are expressed as a percent of those values
found in age-matched WT mice (n= 12). Stippled bars represent untreated
apoE KO mice (n= 9), while the full bars the HBO-treated mice (n= 10).
Each bar is an average ±SEM. (*) Significantly different from WT mice
(represented by the line through 100%). (#) Significantly different from
untreated apoE KO mice.
glutathione S-transferase activity by 40%, to levels indis-
tinguishable from the values seen in WT mice. Similarly,
selenium-dependent GPx-1 (Se-GPx) activity is increased by
HBO treatment to nearly WT values. In contrast, the activ-
ity of the selenium-independent GPx (nonSe-GPx) is not
affected by HBO treatment or the apoE gene ablation.
3.3. Levels of reduced glutathione and the activities of
anti-oxidant enzymes in the aorta
3.3.1. Redox environment
As shown in Fig. 3, a 10-week HBO treatment increases
aortic GSH levels by 44% (p< 0.001) to levels significantly
higher than even those of the WT mice (by 20%, p< 0.04).
Fig. 3. Levels of reduced glutathione and the activities of anti-oxidant
enzymes in the aorta. Reduced glutathione and the enzymes are expressed
as a percent of those values found in age-matched WT mice (n= 12). Stip-
pled bars represent untreated apoE KO mice (n= 9), while the full bars the
HBO-treated mice (n= 10). Each bar is an average ±SEM. (*) Significantly
different from WT mice (represented by the line through 100%). (#) Signif-
icantly different from untreated apoE KO mice.
B.J. Kudchodkar et al. / Atherosclerosis 193 (2007) 28–35 33
Fig. 4. Total aortic cholesterol content. Total aortic cholesterol content was
determined in extracts of aortic homogenate, as described in the Materials
and Methods. The effects of a 10-week HBO treatment of apoE KO mice
(full bar, n= 10) is compared to the untreated apoE KO mice (stippled bar,
n= 9) and WT mice (open bar, n=7). (*) Significantly different from WT
mice. (#) Significantly different from untreated apoE KO mice.
We were unable to measure the levels of oxidized (GSSG)
glutathione levels due to the scarcity of the aortic tissue.
3.3.2. Enzymes
HBO treatment has a profound effect on the aortic anti-
oxidant enzymes: a 26, 52, 66 and 49% increase for GR,
GST, Se-GPx and CAT, respectively. The levels of all of these
enzymes in the aortic tissue of the HBO-treated apoE KO
mice are significantly higher than even those of the WT mice.
The decreased levels of these enzymes in the untreated apoE
KO mice are consistent with the increased oxidative stress
observed in the apoE KO genotype.
3.4. Aortic fatty streaks and cholesterol
Fatty streak lesions in HBO-treated or untreated apoE KO
mice were assessed by two independent means: (a) aortic
cholesterol content; and (b) by en face analyses of the aortic
arch. As shown in Fig. 4, the levels of cholesterol in the aortic
tissue of the 15-week-old apoE KO mice are 2.4-fold higher
(p< 0.0001) than those of age-matched WT mice, reflecting
significant fatty streak formation and atherosclerosis. HBO
treatment leads to a marked decrease in aortic cholesterol
content (37%, p< 0.0001), but it is still substantially higher
than the cholesterol content of the WT aorta.
En face analyses of the aortic arches obtained from four
control (untreated) or treated apoE KO mice are shown in
Fig. 5. The affected surface area in the untreated animals at
15 weeks of age was 6.9 ±1.4%, compared to 3.2 ±0.7% in
HBO-treated mice, a significant (p< 0.03) decline of 53%.
Gross examination of the fatty streak lesions with a stere-
omicroscope also reveals that lesions in untreated mice were
more raised, when compared to lesions found in HBO-treated
mice (not shown). Thus both measurements of atherosclerosis
are in agreement and demonstrate the effectiveness of HBO
treatment in reducing the development of atherosclerosis.
4. Discussion
In the present study we extend our previous observa-
tions [8] to a well-accepted murine model of atherosclerosis:
female apoE KO mice fed a regular mouse chow. By includ-
ing age-matched WT mice we are able to examine not only
Fig. 5. Oil-Red O-stained aortic arches of 4 control (untreated apoE KO mice) and 4 HBO-treated mice. The average±SEM of the area of aorta involved
in fatty streak formation is shown below each set of pictures. The total area of aorta examined in each group was similar and statistically not different. HBO
treatment dramatically reduces the Oil-Red-O-stained lesions by 53% (p< 0.03), as determined by analysis with Image Pro Plus.
34 B.J. Kudchodkar et al. / Atherosclerosis 193 (2007) 28–35
the effects of HBO treatment on anti-oxidant enzyme activ-
ities in apoE KO mice, but also examine the effects of the
apoE gene ablation on these activities.
Relative to the WT mice, the ablation of the apoE gene
is accompanied by a substantial and statistically significant
decrease in hepatic anti-oxidant enzymes, including GST,
Se-GPx and CAT and a corresponding increase in TBARS,
GSSG and the GSSG/2GSH ratio. These changes confirm a
significant shift, in the liver, to a more oxidizing environment.
Although the data on the aortic tissue are incomplete, a similar
trend is observed. Another good indicator of the more oxidiz-
ing environment is the over 4- and 2-fold increase in the titers
of the anti-oxLDL and MDA LDL antibodies, respectively. In
agreement with these observations, others have demonstrated
that the ablation of apoE expression leads to an increase in
the oxidative stress, both in the vasculature as well as in the
neuronal tissues in this animal model [22–24].
HBO treatment of the apoE KO mice was initiated at 5
weeks of age and continued for a period of up to 10 weeks.
At 5 weeks of age they are essentially disease-free, but in
the absence of treatment measurable fatty streak formation
occurs over the next 10-week period, as shown by our results
and reported by others [25]. HBO treatment has no effect
on plasma total or individual lipoprotein cholesterol levels in
apoE KO mice.
Serum paraoxonase circulates in blood in association with
HDL and is synthesized in the liver. Both activities of PON1
(paraoxonase and arylesterase) possess anti-oxidant proper-
ties, as both reduce lipid peroxides in oxidized lipoproteins
and tissues. Oxidative stress inactivates both activities, but
can be reversed by anti-oxidants [26]. The restoration of
plasma PON1 activities in apoE KO mice by HBO is in agree-
ment with these reports.
HBO treatment significantly increases the hepatic levels of
all measured anti-oxidant enzymes and reduced glutathione.
Conversely and as expected, HBO treatment dramatically
reduces the levels of hepatic TBARS and oxidized glu-
tathione. As a result of these changes, the hepatic redox
state is dramatically shifted towards a reducing environment.
Changes of similar nature are observed in the aortic tissues.
Although we were unable to measure all of the variables that
were measured in the liver due to insufficient amounts of
tissue, significant increases in aortic reduced glutathione as
well as four anti-oxidant enzymes (GR, GST, Se-GPx and
CAT) also indicate a shift to a reducing environment. The net
and overall result of such changes is a reduction in lipid oxi-
dation and inflammatory events. The dramatic reduction of
the anti-oxLDL and MDA-LDL antibodies by the 10-week
HBO treatment to levels not different from WT mice provides
additional support for the notion of reduced oxidative stress.
These changes are associated with reduced aortic choles-
terol content by 37% and an over 50% decrease in aortic
fatty streaks. Other anti-oxidants, including vitamin E, DPPD
(N,N-diphenyl 1,4-phenylenediamine and polyphenols) have
been shown to reduce atherosclerosis in this animal model
[22,27,28].
The importance of tissue GSH levels and redox state
in modulating atherogenesis is evident from other studies.
While a reduction in tissue GSH increases the lesion size, an
increase in tissue GSH and GPx decreases the lesion size [29].
Similarly, caloric restriction which retards the pro-oxidizing
shift in the redox state of glutathione [30] has been shown
to reduce the production of superoxide and peroxides in the
arterial wall and attenuate atherosclerosis in apoE KO mice
without altering the elevated plasma cholesterol levels [31].
High levels of anti-oxidant activities have been linked, both in
human and mouse studies, to a resistance towards atheroscle-
rotic lesion development [32,33].
The present study does not investigate the molecular
mechanisms underlying the HBO-mediated elevation of the
endogenous anti-oxidants. The coordinated induction of anti-
oxidants might be mediated by ROS and lipid peroxides, as
has been demonstrated for glutathione, GPx1, catalase, GST
etc [34].
Atherosclerosis is a complex, polygenic disease in which
the involvement of oxidative stress and inflammation is well
established but the specific mechanism is not fully under-
stood. The apoE KO mice provide a particularly useful model
for the study of this disease. It is now well established that
the ablation of the apoE gene in these mice leads not only
to prolonged circulation of cholesterol-rich lipoproteins (due
to a lack of a receptor ligand) but also, and independent of
its role in lipoprotein metabolism, increase in the oxidative
stress in various tissues. It is not surprising that every vari-
able related to oxidation we measured confirms that apoE
KO mice are under increased oxidative stress relative to the
WT mice. This is true for plasma components as well liver
and aortic enzymes, markers of oxidation and glutathione
ratios. In the present report we also demonstrate that HBO
treatment dramatically reduces fatty streak formation along
with a dramatic reduction of oxidative stress, as measured
by a variety of parameters. Although the precise mecha-
nism of HBO mediated protection is not known, our studies
suggest a significant role for the induction of anti-oxidant
enzymes. We are presently in the process of addressing these
issues.
Acknowledgements
This study was supported by a grant from the National
Institutes of Health (RO1 HL70599) to L.D.
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Chapter
Publisher Summary Glutathione reductase is a flavoprotein catalyzing the NADPH-dependent reduction of glutathione disulfide (GSSG) to glutathione (GSH). The reaction is essential for the maintenance of glutathione levels. Glutathione has a major role as a reductant in oxidation–reduction processes, and serves in detoxication and several other cellular functions of great importance. A purification method of this enzyme from calf liver and rat liver is described in this chapter. Similar methods are used for the purification of the enzyme from yeast, porcine, and human erythrocytes. All the steps are carried out at about 5 ° . The purification method from calf liver consists of various steps including preparation of cytosol fraction, chromatography on DEAE-sephadex, precipitation with ammonium sulfate, and chromatography on hydroxyapatite. The purification of glutathione reductase from rat liver is usually combined with the preparation of glutathione transferases, thioltransferase, and glyoxalase I.
Chapter
Chapter
Publisher Summary All of the plasma sulfhydryl (SH) groups are protein associated. Albumin is the most abundant plasma protein (40-60 mg/ml) and is a powerful extracellular antioxidant. Plasma SH groups are susceptible to oxidative damage and are often low in patients suffering from diseases, such as coronary artery disease and rheumatoid arthritis. Additionally, protein SH (P-SH) groups, plasma contains small amounts of glutathione (GSH). Decreased plasma GSH is reported in human immunodeficiency virus (HIV) infection. A spectrophotometric assay based on 2,2-dithiobisnitrobenzoic acid (DTNB or Ellman's reagent) is commonly used for thiol assay. Most of the procedures are for cellular thiols. This chapter describes convenient assays for P-SH and GSH in plasma using spectrophotometric and spectrofluorometric methods. The P-SH level of plasma is calculated by subtracting the GSH level from the T-SH level. There normally is little difference between T-SH and P-SH because of the low GSH levels in the plasma.