Hindawi Publishing Corporation
International Journal of Hypertension
Volume 2012, Article ID 740203, 11 pages
ReciprocalEffectsof Oxidative Stress onHeme
OxygenaseExpressionand Activity Contributes to Reno-Vascular
Tomoko Kawakami,1NitinPuri,2Komal Sodhi,2LarsBellner,3Toru Takahashi,1
Kiyoshi Morita,1RitaRezzani,4TimD.Oury,5and Nader G.Abraham2,3
1Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Japan
2Department of Physiology & Pharmacology, University of Toledo College of Medicine, Toledo, OH, 43614, USA
3Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA
4Division of Human Anatomy, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy
5Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, USA
Correspondence should be addressed to Nader G. Abraham, firstname.lastname@example.org
Received 12 August 2011; Accepted 19 September 2011
Academic Editor: David E. Stec
Copyright © 2012 Tomoko Kawakami et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Heme oxygenase (HO) system is one of the key regulators of cellular redox homeostasis which responds to oxidative stress (ROS)
reports, this study was designed to evaluate effects of chronic oxidative stress on HO system and its role in contributing towards
patho-physiological abnormalities observed in extracellular superoxide dismutase (EC-SOD, SOD3) KO animals. Experiments
mice exhibited oxidative stress, renal histopathological abnormalities, elevated blood pressure, impaired endothelial function,
reduced p-eNOS, p-AKT and increased HO-1 expression; although, HO activity was significantly (P < 0.05) attenuated along
with attenuation of serum adiponectin and vascular epoxide levels (P < 0.05). CoPP, in EC-SOD(−/−)mice, enhanced HO activity
(P < 0.05)andreversedaforementionedpathophysiologicalabnormalitiesalongwithrestorationofvascularEET,p-eNOS,p-AKT
and serum adiponectin levels in these animals. Taken together our results implicate a causative role of insufficient activation of
heme-HO-adiponectin system in pathophysiological abnormalities observed in animal models of chronic oxidative stress such as
Oxidative stress induces NRF2-dependent antioxidant en-
zymes including the heme-HO system , whose two iso-
forms HO-1 (inducible) and HO-2 (constitutive) catabolizes
free heme to equimolar concentrations of biliverdin (BV),
carbon monoxide (CO), and iron. Excess-free heme, due to
its pro-oxidant and proinflammatory properties, contributes
to an increase in free radical formation and cellular injury
[1, 2], thus necessitating its catabolism by HO. Apart from
restricting accumulation of pro-oxidant heme, antioxidant
properties of heme-HO system arise from production of BV
[5, 6] (reviewed by ), antiapoptotic , and vasomodula-
tory properties. These properties of the heme-HO system are
pertinent to redox balance and its associated physiological
ramifications, especially in the cardiovascular-renal systems
[1, 8]. Further evidence of HO-mediated sustenance of ren-
ovascular homeostasis is provided by studies demonstrating
HO-dependent activation of adiponectin release , which
has antioxidant and anti-inflammatory properties  in
addition to its renoprotective effects . An increase in
adiponectin has also been shown to lead to increased levels
via an increase in Bcl-XL . The Bcl-2 family of proteins,
2International Journal of Hypertension
consisting of anti- and proapoptotic proteins, along with
serine-threonine kinase (Akt) (protein kinase B), are critical
in cell death/survival pathways . Akt is activated through
phosphorylation at either threonine-308 or serine-473 .
Activated Akt inhibits ASK-1, a proapoptotic member of
the MAP kinase kinase family, and protects against stress
induced apoptosis in endothelial cells .
Apart from inducible NRF2-dependent genes, constitu-
tive enzymes such as superoxide dismutases (SOD) regulate
basal redox and prevent excess-free radical accumulation.
SOD enzymes are known to exist in three isoforms: Cu-Zn
SOD (SOD1), predominately located in the cytoplasm, Mn-
SOD (SOD2) in the mitochondria, and EC-SOD (SOD3)
in the extracellular space. Although SOD1 accounts for
60% to 80% of SOD activity in vivo [16, 17], SOD3 is
highly expressed in renal and vascular tissues, particularly
in the arterial wall, and its activity constitutes almost half
of the total SOD activity in the human aorta . Gene
deletion of EC-SOD results in chronic oxidative stress,
endothelial dysfunction, and increased blood pressure [17,
19, 20], implicating this enzyme in the regulation of redox
homeostasis and preservation of cardiovascular and renal
function. Where, in an event of increased oxidative stress
HO-1 is rapidly induced, recent reports have emerged
suggesting an inhibitory effect of the same on HO activity
. In this contradictory contexture, the present study was
designed to examine the effects of chronic oxidative stress,
as observed in EC-SOD(−/−)mice, on HO expression and
activity with regards to the pathophysiological abnormalities
observed in these animals. The study was performed in WT
and EC-SOD(−/−)mice in the absence and in the presence
of HO inducer, CoPP. Our results demonstrate that EC-
SOD deficiency, although accompanied by oxidative stress
and induction of HO-1, is characterized by attenuation
of HO activity with the resultant attenuation of vascular
epoxide and adiponectin levels. Phenotypic analysis of
EC-SOD knockout mice revealed renal microvascular and
corticomedullary damage along with elevated blood pressure
and vascular endothelial dysfunction. Induction of HO-
1 in SOD3-deficient mice not only restored HO activity
and redox homeostasis, but also prevented renovascular
injury and offset endothelial dysfunction and elevated blood
pressure. These events are accompanied by the restoration
of vascular epoxide and serum adiponectin levels with a
concomitant increase in p-AKT and p-eNOS expression.
male, EC-SOD(−/−)and C57BL/6 genetic background EC-
SOD(+/+)mice were used for this study. EC-SOD(−/−)
mice were a gift from University of Pittsburgh, PA, USA.
These mice had been backcrossed to C57BL/6 mice and
are congenic with this line of mice ; as such, C57BL/6,
purchased from The Jackson Laboratories, were used as
WT controls. All animals were maintained on a standard
laboratory diet and water, and every effort was made
to minimize animal suffering according to the National
Institutes of Health (NIH), Institutional Animal Care and
Use Committee guidelines. The studies were approved by
the animal use committee at New York Medical College.
Mice, both WT and KO, were divided into two groups
(6mice/group): controls, injected with saline solution, and
the treatment group, treated with cobalt protoporphyrin
(CoPP) 5mg/Kg i.p. weekly for 6 weeks. CoPP was dissolved
in trisma base as previously described . The last injection
of CoPP was made three days before sacrificing the animals.
Mice were followed for 6 weeks (duration of the experi-
ments), at which time they were sacrificed with CO2 gas.
Blood and tissues were collected and stored, as described, for
2.2. Morphology and Proteins Analysis. Renal cross-sections
histology using standard techniques. Tissue sections were cut
at 3-4 micron thickness and stained with H&E and periodic
acid Schiff (PAS). A portion of the tissues was snap-frozen
and kept at −80◦for proteins determinations and Western
2.3. Tissue Preparation for Western Blot, HO Activity, and O2−
Levels. Sections of femoral arteries and whole kidneys were
homogenized in homogenization buffer (255mM sucrose,
20mM tris-hcl, 1mM EDTA, 0.1mM PMSF, and 0.5%
Nonidet P-40 at pH 7.4) containing a cocktail of protease
inhibitors (Roche, Indiana, IN) and Halt, a phosphatase
inhibitor cocktail (Pierce Biotechnology, Rockford, IL). The
homogenates were centrifuged at 14000g for 10min at 4◦C,
supernatant was isolated and protein levels assayed using
a Bio-Rad kit on the basis of the Bradford dye binding
2.4. Measurement of O2−Levels. Employing previously
described methods , kidney and arterial samples were
placed in glass scintillation mini vials containing 5μM
lucigenin, for the detection of O2−in a final volume of
1mL of air-equilibrated Krebs solution buffered with 10mM
HEPES-NaOH (pH 7.4). Lucigenin chemiluminescence was
measured in a liquid scintillation counter (LS6000IC, Beck-
as counts/minute/mg protein after background subtraction.
2.5. Measurement of HO Activity. Tissue HO activity was
assayed as described previously  using a technique in
which bilirubin, the end product of heme degradation,
was extracted with chloroform, and its concentration was
determined spectrophotometrically (dual UV/VIS beam
spectrophotometer lambda 25; PerkinElmer Life and Ana-
lytical Sciences, Wellesley, MA, USA) using the difference in
absorbance at a wavelength from λ460 to λ530 nm with an
absorption coefficient of 40mM−1cm−1. Under these con-
ditions, HO activity was linear with protein concentration,
time-dependent, and substrate-dependent .
2.6. RT-PCR Analysis. Frozen arterial and renal segments
were pulverized under liquid nitrogen and RNA extracted
International Journal of Hypertension3
out using the Advantage RT-for-PCR Kit (Clontech). Poly-
d (T) n was used as the reverse transcription primer.
employed. PCR was performed using a Taq PCR kit (Roche,
Indianapolis, IN, USA). For each RT-PCR, a sample without
reverse transcriptase was processed in parallel and served
as a negative control. Cycling parameters for amplifying RT
products were as follows: 95◦C, 1?, 60◦C, 1?, and 72◦C, 1–3?,
for 30 cycles, and then extended at 72◦C for another 5?. After
amplification, PCR products were electrophoresed on 1.2%
agarose gel, stained with ethylene bromide, and visualized
under UV light.
removed and placed in cold oxygenated Krebs-bicarbonate
solution, cleaned of fat and loose connective tissue, and
sectioned into rings of approximately 3mm length. Two
rings per artery were obtained, and each ring was mounted
myograph bath (DMT, Atlanta, GA) filled with Krebs solu-
tion, pH 7.4, gassed with 95% O2/5% CO2, and maintained
at 37◦C. The rings were incubated under a passive tension
of 0.2g for 1 hour. The Krebs buffer solution was replaced
every 15 minutes and the tension readjusted each time. Force
was recorded from force displacement transducers via AD
Instrument’s Powerlab system, running Chart 5 software.
At the end of the equilibration period, the maximal force
generated by the addition of a depolarizing solution of
KCl (60mM) was determined. To evaluate acetylcholine-
induced vasorelaxation, the rings were preconstricted with
phenylephrine to obtain maximal contraction followed by
cumulative dose-response curves to acetylcholine.
2.8. Blood Pressure Measurements. Blood pressure was mea-
sured by the tail cuff method before the start and at
the completion of the experiment (6wks). Prior to the
experiment, mice were all acclimated to the tail cuff method.
Mice were placed in a heat controlled box (36◦C–38◦C)
for approximately 10min. before applying the tail cuff.
The mean of a minimum of 5 measurements was obtained
from each mouse. All measurements were determined at the
same time of day, between 9:00 and 13:00hr. Systolic blood
pressure is reported in mm of Hg.
2.9. Adiponectin Measurements. Adiponectin was deter-
mined using an ELISA assay (Pierce Biotechnology, Inc.,
Woburn, MA, USA).
2.10. Western Blot Analysis. The supernatant from tissue
homogenates was used for evaluation of protein expression
via Western blot analysis. Primary antibodies used are listed
as following: HO-1 and HO-2 (Assay Designs, Inc.), ASK1
(Abcam), eNOS, Bcl-Xl and AKT (Cell Signaling Technolo-
gies, Inc.), p-eNOS-Thr495 (Cell Signaling Technologies,
Inc.), and p-Akt-Ser473 (Cell Signaling Technologies, Inc.).
B-actin (Sigma-Aldrich) was used as housekeeping gene for
normalization. Antibodies were prepared in the following
dilutions: HO-1 and HO-2 (1:1000); eNOS, p-eNOS, Bcl-xl,
ASK1, and p-AKT antibodies (1:5000); β-actin (1:10000).
Experimental protocol in brief: 20μg of lysate supernatant
was separated by 12% SDS-polyacrylamide gel electrophore-
sis and transferred to a nitrocellulose membrane (Amersham
Biosciences, Uppsala, Sweden) with a semidry transfer
apparatus (Bio-Rad, Hercules, CA, USA). The membranes
were incubated with 10% milk in tris-buffered saline with
tween 20 (10mM Tris-HCl, pH 7.4, 150mM NaCl, and
0.05% Tween 20) at 4◦C, overnight. After incubation, the
membranes were washed with tris-buffered saline with
tween 20, and the membranes were then incubated with
secondary antibodies for 1h at room temperature with
constant shaking. The filters were washed and subsequently
rabbit or anti-mouse IgG (Amersham Biosciences). Chemi-
luminescence detection was performed with the Amer-
sham Biosciences enhanced chemiluminescence detection
kit, according to the manufacturer’s instructions.
2.11. Measurement of EETs. Femoral arterial segments (two
segments per tube) were incubated in 1mL of oxygenated
Krebs’ buffer containing 1mM NADPH at 37◦C for 1h.
Thereafter, internal standards were added to each sample
followed by acidification to pH ≈ 4.0 with glacial acetic acid.
Eicosanoids were extracted with twice the volume of ethyl
acetate (performed three times), dried under gentle stream
of nitrogen, and stored at −80◦C until further analysis.
Identification and quantification of EETs and DiHETs was
performed with a Q-trap 3200 linear ion trap quadruple
liquid chromatography-tandem mass spectrometry system
tro spray mode (Applied Biosystems, Foster City, CA, USA).
Extracted samples were suspended in 10μL of methanol and
injected into the high-performance liquid chromatography
via an Agilent 1200 standard series auto sampler equipped
with a thermostat set at 4◦C (Agilent Technologies). The
high-performance liquid chromatographic component con-
sisted of an Agilent 1100 series binary gradient pump
equipped with an Eclipse plus C18 column (50 × 4.6mm;
1.8mm) (Agilent Technologies). The column was eluted
at a flow rate of 0.5mL/min with 100% mobile phase A
[methanol/water/acetic acid (60:40:0.01, v/v/v)] from 0 to
2min and a gradient increasing to 100% B (100% methanol)
at 13min. Multiple reaction monitoring was used with a
dwell time of 25 or 50ms for each compound, with the
following source parameters: ion spray voltage, −4500V,
curtain gas, 40U, ion source gas flow rate 1, 65U, ion
source gas flow 2, 50U, and temperature, 600◦C. Synthetic
standards were used to obtain standard curves (5–500pg)
for each compound. These standard curves were used and
extrapolated to calculate the final EET concentrations, which
are presented as nanograms per milligram of protein per
2.12. Statistical Analysis. The data are presented as mean ±
SE for the number of experiments. Statistical significance
(P ≤ 0.05) was determined by the Fisher method of multiple
comparisons. For comparison between treatment groups,
the Null hypothesis was tested by a single-factor analysis of
4International Journal of Hypertension
ecSOD/18S mRNA ratio
Bilirubin (nmol/mg protein)
Figure 1: (a) Detection of EC-SOD mRNA transcripts by RT-PCR analysis in renal tissues. EC-SOD(−/−)animals demonstrated significant
(P < 0.05) reduction in EC-SOD mRNA as compared to their WT counterparts, and the administration of CoPP had no significant effect on
SOD3 mRNA levels in both WT and KO animals. Results are expressed as mean ± SE,∗P < 0.01 versus WT mice. (b) Western blot analysis
of HO-1 and HO-2 proteins in kidney homogenates of WT and EC-SOD(−/−)mice. Immunoblots were performed using antibodies against
mice HO-1 and HO-2 proteins. Blots are representative of six separate experiments. Results are expressed as mean ± SE,∗P < 0.01 versus
WT,+P < 0.05 versus WT + CoPP,‡P < 0.05 versus EC-SOD(−/−). (c) Bilirubin production in WT, EC-SOD(−/−)mice and EC-SOD(−/−)
mice treated with CoPP. Values are expressed as the mean ± SD of 4 experiments,∗P < 0.05 versus WT mice,‡P < 0.01 versus EC-SOD(−/−).
variance (ANOVA) for multiple groups or unpaired t-test for
3.1. Effect of SOD3 Gene Deletion on HO Expression and
Activity. The effectiveness of EC-SOD gene knockdown was
analyzed in renal homogenates using R.T-PCR. SOD3 KO
animals demonstrated significant (P < 0.05) reduction in
EC-SOD mRNA as compared to their WT counterparts (WT
−2.55 ± 0.5 versus KO −0.36 ± 0.04). The administration of
CoPP had no effect on SOD3 mRNA levels in both WT and
KO animals (Figure 1(a)). Oxidative stress was confirmed in
renal homogenates from SOD3 KO animals using lucigenin
detectable chemiluminescence (WT −0.78 ± 0.1 versus KO
−1.85±0.14cpm × 1000/mg, P < 0.05). Renal homogenates
from EC-SOD(−/−)animals demonstrate significant (P <
0.05) induction of HO-1 which was further accentuated in
this HO-1 induction was accompanied by attenuation (P <
0.05) of HO activity in SOD3 KO animals, as measured
by total bilirubin generation (WT −0.53 ± 0.12 versus
KO −0.28 ± 0.09nmol/mg protein). EC-SOD(−/−)mice
concurrently exposed to CoPP demonstrated increased HO-
1 expression which was accompanied by a corollary increase
in (P < 0.05) HO activity in these animals (Figure 1(c)).
presses HO activity, is substantiated by these results which
also suggest that porphyrin-induced overexpression of HO
overwhelms this inhibitory effect and restores HO activity.
International Journal of Hypertension5
Figure 2: Representative images of renal parenchyma (haematoxylin-eosin staining, 200X) in EC-SOD(−/−)and WT mice. WT mice and WT
mice treated with CoPP showed no significant morphologic alterations both in cortex (arrow’s head for proximal tubules and black point for
distal tubules) and medulla (asterisk for vasa recta) ((a), (b), (e), (f)). EC-SOD(−/−)mice displayed tubular damage, hyperproliferation in
the glomerulii (red arrows) with Bowman’s capsule dilatation (red point), infiltrates (black arrows) and breakage of vasa recta in outer and
inner medulla (double asterisks). ((c), (g)). Tubular interstitial and microvascular pathology are abrogated in CoPP-treated EC-SOD(−/−)
mice ((d), (h)). Western blot analysis of (i) ASK1 (j) BCL-xl protein expression in kidney of WTmic, EC-SOD(−/−)mice, and mice treated
with CoPP. Quantitative densitometry evaluation of Bcl-xl, ASK-1-to-β-actin ratio was determined. Blots are representative of four separate
experiments. Results are expressed as mean ± SE,∗P < 0.05 versus WT,+P < 0.01 versus WT + CoPP,‡P < 0.05 versus EC-SOD(−/−).
3.2. Effects of HO Induction on Renal Histopathology in EC-
SOD(−/−)Animals. HO deficiency, as seen in HO-2(−/−)
animals, has already been shown to exacerbate renal
histopathological abnormalities in streptozotocin- (STZ-)
induced model of oxidative stress . SOD3(−/−)mice also
exhibit chronic oxidative stress with insufficient stimulation
of the heme-HO system; as such, we examined renal mor-
phology in these mice (and WT controls) with and without
pharmacological induction of HO. Histological examination
of renal sections revealed no significant abnormalities in
WT mice treated or not with CoPP, showing no dilatation
of glomeruli, proximal and distal tubules, and absence of
inflammatory infiltration (Figures 2(a), 2(b), 2(e), and 2(f)).
In contrast, EC-SOD(−/−)mice exhibited corticotubular
damage, characterized by dilatation and loss of epithelial
cells in the tubular structures with the presence of cellular
infiltrate (Figure 2(c)). A considerable number of Bowman’s
capsules showed dilatation with hyperproliferation of cap-
illary tufts and cellular infiltration (Figure 2(c)). Inner and
outer medulla showed breakage of vasa recta (Figure 2(g)).
6International Journal of Hypertension
CoPP treatment abrogated this glomerular-tubulointerstitial
and microvascular pathology in EC-SOD(−/−)mice (Figures
Renal morphological abnormalities, in EC-SOD(−/−),
were accompanied by alterations in apoptotic and antiapop-
totic signals. ASK1 was significantly (P < 0.05) elevated
in renal homogenates of SOD3 KO animals as compared
to WT animals. Similarly, antiapoptotic protein Bcl-xl was
attenuated (P < 0.05) in renal homogenates from SOD3 KO
as compared to their WT counterparts. The expression levels
of ASK1 and Bcl-xl proteins were restored in EC-SOD(−/−)
mice treated with CoPP (Figures 2(i), 2(j)).
3.3. Effects of HO Induction, in SOD3 KO Animals, on
Systolic Blood Pressure and Vascular Endothelial Function.
EC-SOD(−/−)mice have been documented to have elevated
blood pressure and exhibit vascular endothelial dysfunction
. In this regard, we examined the effect of HO induction
on vascular endothelial function, and factors regulating it, in
EC-SOD(−/−)mice. First, vascular redox status was assessed
that SOD3 gene deletion was associated with increased
(P < 0.05) vascular O2−levels; attenuated by concurrent
exposure to CoPP (P < 0.05) (Figure 3(a)). EC-SOD(−/−)
mice demonstrated a significantly (P
ated vasorelaxation response to increasing concentrations
of acetylcholine. CoPP treatment enhanced acetylcholine-
induced vasodilation (P < 0.05) in these mice (Figure 3(b))
and in complimentary experiments, CoPP rescued the
attenuative effects (P < 0.05) of SOD3 gene knockdown on
the expression of p-eNOS (P < 0.05) (Figure 3(d)). Blood
pressure measurements, performed in WT and EC-SOD-
deficient mice, revealed elevated baseline blood pressure in
EC-SOD(−/−)mice (WT −112.4 ± 1.8versus KO −120.4 ±
0.9mm Hg, P < 0.05). This elevated blood pressure was
successfully normalized in mice exposed to CoPP (KO +
CoPP −109.8 ±2.4) (Figure 3(c)).
3.4. Effects of HO Induction, in SOD3 KO Animals, on Epoxide
and Serum Adiponectin Levels. In addition to endothelial
dysfunction, vascular levels of epoxides were significantly
reduced in EC-SOD(−/−)mice (WT −2.15 ± 0.2versus KO
−0.89 ± 0.3ng/mg protein/hr, P < 0.05). This effect of
SOD3 KO on EET levels was reversed in CoPP treated
animals (2.35 ± 0.2ng/mg/protein/hr, P < 0.05 versus KO)
Stimulation of the heme-HO system, along with
enhancement of epoxides, has been shown to be associated
with increased serum and tissue levels of adiponectin.
Attenuation of HO activity, in EC-SOD(−/−)mice, was
accompanied by significant (P < 0.05) attenuation of serum
adiponectin levels (WT −6.10 ± 0.3 versus KO −0.93 ±
0.45μg/mL). WT mice treated with CoPP exhibited serum
adiponectin levels similar to those in controls; however,
CoPP treatment restored serum adiponectin levels in SOD3
deficient mice (KO + CoPP −8.05 ± 0.9μg/mL, P < 0.05)
(Figure 4(b)), indicative of its association with HO activity.
In addition, expression of p-AKT, an adiponectin-dependent
regulatory pathway, was also attenuated (P < 0.05) in
vascular tissues from EC-SOD(−/−)mice and was restored
in animals concurrently exposed to CoPP (P < 0.05)
The present study demonstrates that chronic oxidative stress,
as seen in EC-SOD(−/−)mice, has reciprocal effects on HO
expression and activity and that pharmacological induction
of HO restores redox homeostasis and reverts pathophys-
iological abnormalities observed in these animals. These
effects of HO induction are associated with concomitant
increases in epoxide and adiponectin levels, which may
contribute towards the HO-induced reversal of renovascular
abnormalities in EC-SOD(−/−)mice.
The first key finding presented in this study is the
observation that mice expressing reduced levels EC-SOD
have increased renovascular oxidative stress which, although
results in a significant (P < 0.05) attenuation in HO activity.
This discordance in HO-1 expression and activity is in
line with recent reports showing that oxidative stress, in
in vitro systems, inhibits HO activity by a yet unidentified
post-translational modification [21, 29, 30]. In addition,
investigators have also shown an insufficient induction of
HO-1 in conditions of severe prolonged oxidative stress,
where levels of HO are unable to provide adequate cellular
antioxidant protection . Accordingly, irrespective of the
underlying mechanisms involved, accumulating evidence
now suggests attenuation of HO activity by chronic redox
imbalances in face of an increased HO-1 expression. Such an
effect of prolonged oxidative stress, though counterintuitive,
may explain the need and protective role of therapeutic
HO induction in various models of chronic oxidative stress
, including the current model of SOD3 KO animals.
How pharmacological HO-1 induction does overcome the
inhibitory effect of chronic oxidative stress and restores HO
activity cannot be fully explained at this time but may entail
suppressive effect of cellular redox.
The second key finding presented in this study is the
modulatory effect of heme-HO system on vascular epoxide
and serum adiponectin levels in EC-SOD(−/−)mice. In these
mice, a chronic oxidative state is characterized by insufficient
HO activity and where both epoxide and adiponectin levels
are attenuated. Recovery of vascular epoxide and serum
adiponectin levels with CoPP-induced HO stimulation
implicates the role of HO in modulating these pathways.
These results are in line with our earlier report  showing
interplay between HO and epoxides in animal models
of metabolic syndrome. Recent reports have also shown
that induction of HO-1 expression and activity leads to
enhancement of adiponectin levels along with activation of
adiponectin directed signaling pathways [31, 33, 34]. HO-
mediated stimulation of adiponectin release from adipose
tissues [31, 35] appears to involve the HO-induced restora-
inflammation, and increased release of protective adipokines
International Journal of Hypertension7
Vehicle Vehicle CoPPCoPP
SBP (mm Hg)
Figure 3: (a) Superoxide levels in femoral arteries of WT mice, EC-SOD(−/−)mice, and mice injected with CoPP. Superoxide levels were
determined as described in methods. Results are mean ± SE, n = 4;∗P < 0.01 versus WT,‡P < 0.01 versus EC-SOD(−/−);+P < 0.05 versus
WT + CoPP. (b) Dose-response curves for acetylcholine-induced vascular relaxation after preconstriction with phenylephrine. Results are
mean ± SE, n = 4;∗P < 0.05 versus WT,‡P < 0.05 versus EC-SOD(−/−),+P < 0.05 versus WT + CoPP, and (c) systolic blood pressure
measurements at the completion of the study (n = 4). Mice were injected with CoPP once a week as described in Methods. Blots are
representative of four separate experiments. Results are expressed as mean ± SE,∗P < 0.05 versus WT,‡P < 0.01 versus EC-SOD(−/−),
+P < 0.05 versus WT + CoPP mice, and Western blot and densitometry analysis of (d) eNOS and p-eNOS.
. In addition, epoxides, whose synthesis was enhanced
by HO-induction in EC-SOD(−/−)mice, have been shown
in the past to attenuate dysfunctional adipogenesis and
dependent manner. Thus, our results presented here support
the existence of an interdependent physiological axis formed
by the heme-HO, epoxide, and adiponectin systems, which
regulates cardiovascular-renal function, and where redox-
induced attenuation of HO activity negatively affects the
other two components.
Physiological implications of attenuation of HO-
adiponectin axis are evident as renal corticomedullary
lesions in EC-SOD(−/−)mice, amenable to reversal with
CoPP. Abrogation of these renal histopathological changes,
in mice treated with CoPP, was accompanied by attenuation
of apoptotic and enhancement of antiapoptotic pathways,
negatively regulated by cellular redox , where reduced
thioredoxin binds and inhibits ASK1. This inhibitory effect
is overcome under conditions of oxidative stress , thus
8International Journal of Hypertension
Vehicle VehicleCoPP CoPP
Figure 4: (a) EETs levels in WT and EC-SOD(−/−)mice and mice treated with CoPP. EETs levels were determined as described in Methods.
Results are mean ± SE, n = 4;∗P < 0.05 versus WT mice,‡P < 0.05 versus EC-SOD(−/−)and+P < 0.05 versus WT + CoPP mice. (b)
Adiponectin levels in WT and EC-SOD(−/−)mice and mice treated with CoPP. Adiponectin levels were determined as described in methods.
Results are mean ± SE, n = 4;∗P < 0.05 versus WT mice,‡P < 0.05 versus EC-SOD(−/−)mice, and+P < 0.05 versus WT + CoPP mice. (c)
AKT and pAKT proteins in arteries of WT and EC-SOD(−/−)mice. Mice were injected with CoPP once a week as described in Methods. Blots
are representative of four separate experiments. Results are expressed as mean ± SE,∗P < 0.05 versus WT,‡P < 0.01 versus EC-SOD(−/−),
+P < 0.05 versus WT + CoPP mice.
increasing ASK1 expression. Increased cellular expression
of ASK1 has been linked to recruitment of inflammatory
components, tumorogenesis, and endothelial dysfunction
. Reduced HO activity, in EC-SOD(−/−)mice, could
facilitate oxidative stress in turn increasing ASK1 expression,
and contributing towards pathological alterations observed
in these animals. Induction of HO-1 expression and activity,
via CoPP administration, restores cellular redox, attenuates
ASK1 expression and prevents renal damage. In addition,
EC-SOD(−/−)animals also demonstrate reduced tissue
expression of Bcl-XL and p-AKT/AKT proteins. AKT
interacts with HSP-90 and is involved in inhibition of
ASK1 and associated pathways . Increased HO-1 and
adiponectin levels enhance p-AKT/AKT expression with
resultant cardio-reno-protective effects [36, 40] including
suppression of ASK1. These results are also in line with
earlier reports [11, 41] demonstrating renoprotective effects
of adiponectin in patients with chronic kidney disease.
Finally, significant improvement of endothelial function
and attenuation of elevated blood pressure, in EC-SOD(−/−)
mice, by induction of heme-HO system underscores the role
homeostasis. Induction of HO via CoPP provides excess CO
and BV, where BV acts as an endogenous chain breaking
antioxidant  and increases NO bioavailability and CO has
been shown to induce vasodilation via activation of vascular
International Journal of Hypertension9
Chronic oxidative stress
Cardiovascular-renal protective effects
Figure 5: The schematic outlines the salient points discussed in the paper. Chronic oxidative stress upregulates HO-1 expression whose
products, CO & BV have antioxidant properties and help restore redox homeostasis. In addition, HO induction stimulates epoxide synthesis
and these pathways combined enhance adiponectin release. Antioxidant, antiapoptotic, vasomodulatory, and renoprotective properties
of HO-EET-adiponectin axis restore cardiovascular-renal homeostasis often disrupted in conditions of oxidative stress. HO induction
overcomes a concurrent inhibition of HO activity by redox-dependent mechanisms which offset the protective effects of enhanced HO
expression thus contributing towards pathophysiological abnormalities observed in conditions of chronic oxidative stress.
Kcachannels [1, 42]. Another aspect of the HO-1 induction-
mediated amelioration of vascular dysfunction may involve
upregulation of eNOS and stimulation of epoxide synthesis.
Epoxides are one of the candidates for endothelium derived
hyperpolarizing factor (EDRF) and induce vasodilation
along with inhibition of inflammatory response and stim-
ulation of epithelial cell growth [43, 44]. In addition, HO
stimulation is accompanied by increased adiponectin release
and adiponectin has been shown to induce generation of
NO in vascular endothelial cells  and has also been
shown to mediate AKT-dependent vasodilation in the rat
aorta . Thus, current evidence, in light of previous
reports, implicates the interaction between the HO, epoxide
and adiponectin systems in governing vascular endothelial
function. The antioxidant and vasomodulatory properties of
HO products are complimented by upregulation of vascular
p-eNOS, increase in vasodilatory epoxides and stimulation
of adiponectin-AKT pathway. These vascular and hemody-
namic effects EC-SOD gene deletion may also contribute
towards renal histopathological alteration observed in these
mice and hemodynamic improvement, observed in CoPP-
treated SOD3 KO mice, could thus contribute towards
attenuation of renal pathology in these animals.
In conclusion (Figure 5), this study puts in perspective
two emerging concepts in cardiovascular pathophysiology.
First, it demonstrates a reciprocal effect of chronic oxida-
tive stress on HO-1 expression and activity and secondly,
the present study suggests interdependence amongst three
epoxide systems. Where chronic oxidative stress conditions,
such as SOD3 deficiency potentiate HO-1 expression, a
concurrent inhibitory effect on HO activity is observed.
This effect is accompanied by renovascular abnormalities
and suppression of epoxide and adiponectin synthesis.
Restoration of HO activity, via pharmacological modulators
of the heme-HO system, not only restores cellular redox, but
also recovers vascular epoxide and serum adiponectin levels
along with abrogation of renal pathology, improvement of
vascular function, and attenuation of blood pressure.
Cu/Zn SOD: Copper-zinc superoxide dismutase
EC-SOD: Extracellular superoxide dismutase
HO: Heme oxygenase
ROS: Reactive oxygen species
SE: Standard error
Serine/threonine protein kinase
Apoptotic signaling kinase-1
This work was supported by National Institutes of Health
grants DK068134, HL55601 and HL34300 (N. G. Abraham).
The authors declare no competing financial interests. T.
Kawakami and N. Puri contributed equally to this work.
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