Redox regulation of resveratrolmediated switching of death signal into survival signal

1
Redox regulation of resveratrol-mediated
switching of death signal into survival signal
Das S, Khan N, Mukherjee S, Bagchi D, Gurusamy N,
Swartz H, Das DK. Cardiovascular Research Center,
University of Connecticut School of Medicine,
Farmington, CT 06030, USA.
Tad Kawashima, Kevin Roy, Letian Xie, Yuchen Shi,
and Carolina Zapata
Survival
Resveratrol Preconditioning
Intracellular Oxidation
Ischaemia/ Reperfusion
ROS
Apoptosis
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The French Paradox and Polyphenols
-Ethanol ingestion enhances ROS production and
lipid peroxidation, which are regarded
as underlying factors in cardiovascular disease
-The French, despite having high-fat diets
enriched with red wine, have exceptionally low
incidences of coronary heart disease and reduced
injury due to ischemia-reperfusion

• Because French wine is rich in polyphenols,
• these compounds have been presumed to be
• responsible for the protective benefits

3
Resveratrol an abundant polyphenol
-found in a variety of dietary sources, including
grapes, plums, and peanuts -resveratrol is
mainly found in the skins of grapes, thus it is
enriched in red wine which is fermented with the
skin. -red wine can contain 2-12 mg/L
resveratrol.
Polyphenols are generally thought to be
protective due to antioxidant activity. However,
every antioxidant is a reduction- oxidation agent
and can be pro-oxidant under certain conditions.
Antioxidant Potential as a Free Radical Scavenger
.
(3,4,5-trihydroxystilbene)
R
RH
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Resveratrol the all-protective polyphenol?
Das, D. and Maulik N. Resveratrol in
Cardioprotection. 2006. Mol Interventions.
636-47.
5
Resveratrol in cardioprotection
Das, D. and Maulik N. Resveratrol in
Cardioprotection. 2006. Mol Interventions.
636-47.
6
Ischaemia-Reperfusion
Ischaemia shortage in blood supply to an organ,
resulting in hypoxia, or lack of oxygen.
Reperfusion resumption of blood flow resulting
in reoxygenation
Cells made hypoxic can survive variable lengths
time depending on the tissue. However,
reperfusion often results in reoxygenation
injury. Due to excessive ROS production, the
reperfusion phase can be more harmful than
ischaemic phase.
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Thioredoxin system plays a crucial role in
defense against oxidative stress
Main function in cytoprotection against ROS is to
reduce intracellular disfulides to free thiols
8
Thioredoxin system plays a crucial role in redox
signal transduction
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Preconditioning potentiates the survival of
cardiac tissue after ischaemia/reperfusion
Classic Preconditioning short cycles of
reversible ischaemia/reperfusion
Previous studies in rodents have shown that
classic preconditioning renders the heart more
resistant to subsequent lethal ischemic/reperfusio
n injury. This resistance has been shown to be
the result of an increase in endogenous defense
mechanisms.
Classic preconditioning results in an immediate
resistance effect lasting several hours and a
delayed resistance effect appearing after 12-24
hours and lasting up to 72 hours.

• The mechanism of cardiac preconditioning is
  COMPLEX and debated,
• But appears to involve
• - upregulation of antioxidant defenses
• activation of adenosine receptors (A1), kinases
  (PKC, MAPK, and Y-kinase),
• the mitochondrial KATP channel
• - upregulation of HO and iNOS/eNOS during
  resveratrol preconditioning

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Aim Determine the role of thioredoxin 1 and 2
in resveratrol-mediated cardioprotection
Results

• Relative to untreated controls, resveratrol
  treatment
• - lowered damaged heart tissue
• lowered apoptotic cells
• lowered MDA level
• increased GSH/GSSG
• upregulated anti-apoptosis through Akt-P and
  Bcl-2
• upregulated thioredoxin system, including trx2
• reduced free radicals introduced during ischaemia
  faster

All of these resveratrol-mediated effects were
abolished by inhibition of both Trx-1 and Trx-2
but not by inhibition of Trx-1 alone. These
results implicate Trx-2 as having a crucial role
in preconditioning-mediated cardioprotection.
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Animal Treatment

• - Rats were randomly assigned to one of the
  following groups
• I.  Control Group
•   II. Ischemia/Reperfusion (I/R)
•   III. Resveratrol I/R
•   IV. Resveratrol I/R shRNA-Trx-1
•   V. Cisplatin Resveratrol I/R

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Cisplatin and shRNA-Trx-1 Treatments

• - For group 2-5, resveratrol (2.5 mg/kg body
  wt/day) was fed by gavaging for 10 days before
  the experiment.
• For group 5, cisplatin, which inhibits both
  Trx-1 and Trx 2, was injected on days 1, 3, 5, 7
  and 9 intravenously during the gavaging of
  resveratrol.
• For group 4, a single dose of 100ul shRNA
  Trx-1 was
• injected into the anterior wall of the left
  ventricle.
• After 10 days, isolated rat and mouse hearts
  were subject to ischaemia for 30 mins followed
  by 2 hour of reperfusion.

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Cisplatin, but not shRNA-Trx-1, abolishes the
effects of resveratrol on percentage of damged
heart tissue and myocyte apoptosis.
Resveratrol
Resveratrol
Treated with I/R
Treated with I/R
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Measurement of malonaldehyde for assessment of
oxidative stress

• Malonaldehyde is an end product of lipid
  oxidation, and was measured as MDA-DNPH
  derivative by HPLC

15
GSH/GSSG ratio is an indicator of intracellular
oxidative stress

• Concentration of GSH and GSSG were determined in
  vitro by a glutathione reductase cycling
  procedure.

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Cisplatin, but not shRNA-Trx-1, abolishes the
effects of resveratrol on MDA formation and
GSH/GSSG levels of the heart
Resveratrol
Resveratrol
1 2 3 4 5 1 2 345
1 2 3 4 5 1 2 345
Increased survival is accompanied by
reduced oxidative stress
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Survival Signaling of Resveratrol
Bcl-2 is outer mitochondrial membrane
protein Bcl-2 /Bad heterodimer induces
apoptosis Akt dependent Bad phosphorylation
disrupts dimer Destabilized heterodimer is
anti-apoptotic

Apoptosis
Am J Physiol Heart Circ Physiol. 2005
Jan288(1)H328-35. Epub 2004
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Sample Preparation for Western Blot Analysis

• Interested in looking at anti-apoptotic pathway
  markers Akt-P and Bcl-2
• Left ventricles from hearts homogenized in
  buffer containing
• Sodium Orthovanadate (Na3VO4) Inhibitor of
  phosphatases
• Okadaic Acid Inhibits Serine/Threonine
  Phosphatases
• PMSF Serine Protease Inhibitor
• Purpose to protect phosphoylation states
  and protein degradation
• Blotted with antibody against
• NF-kB, Akt, Akt-P, Bcl-2, GAPDH (loading
  control)

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Cisplatin and not Trx-1 Decreases Survival Markers
shRNA-Trx1
Cisplatin
I/R Resveratrol
-
- -

• I/R treatment decreases Survival Marker levels
• Resveratrol enhances levels of Akt-P Bcl-2
• Addition of shRNA-Trx1 had no effect on markers
• Addition of Cisplatin negates enhancement of
  RSV

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Cisplatin Decreases Protein Levels of Trx-2
Cisplatin
-
I/R Resveratrol
- -

• Trx-2 protein expression levels dropped during
  I/R
• Resveratrol was able to restore Trx-2 levels
• Cisplatin abolished the effects of resveratrol
• Trx-1 protein could not be detected

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Cisplatin abolishes resveratrol-mediated increase
in Trx-1/2 RNA levels after I/R
Cisplatin
-
I/R Resveratrol
- -

• Trx-1 and Trx-2 transcripts were reduced after
  I/R
• Resveratrol treatment increased expression
  levels
• Cisplatin diminished Trx-1/2 even in presence
  of RSV

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Role of thioredoxin system in resveratrol-mediated
preconditioning

• So far the data indicated that resveratrol
  provided cardioprotection by triggering a
  survival signal through phosphorylation of Akt
  and activation of Bcl-2.
• Thioredoxin system seems to have a role because
  cisplatin abolished the survival signal and
  cardioprotection generated with resveratrol.
• However, the inability of shRNA directed against
  Trx-1 to block the effects of resveratrol
  indicated that Trx-1 had no role in
  resveratrol-mediated cardioprotection.

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Construction of Dominate-negative (Dn) Trx-1
transgenic mice to confirm shRNA-Trx1 results

• Dn-Trx-1 transgenic mice were generated by
  mutation of Cys32 and Cys35 of hTrx-1 to Ser by
  PCR
• These transgenic mice are shown to function as a
  dominant negative for endogenous Trx-1

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Dn-Trx-1 mice confirm that Trx-1 has no role in
resveratrol-mediated cardioprotection

• LVDP left ventricular develop pressure (recovery
  of function)
• With resveratrol treatment to both wild type and
  Dn-Trx-1, recovery function in both increased
  significantly, and the infarct size for both
  decreased significantly.

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Trx-1 has no role in resveratrol-mediated
upregulation of Akt-P and Bcl-2
Akt phosphorylation levels
Bcl-2 levels

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How does resveratrol-preconditioning affect the
ability to reduce free radicals?

• Strategy determine redox status of the isolated
  perfused heart during ischaemia.
• Perfuse rat heart with 0.2 mM TEMPO nitroxide, a
  free radical used as biological probe, for 15
  minutes
• Perform ischemia for 30 minutes.
• The reduction of the nitroxide during ischemia
  results in a decrease in signal intensity over
  time.

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Electron Paramagnetic Resonance
EPR is used to detect and identify free radicals
(unpaired electrons)

• Similar to NMR, except that electron spins are
  excited and not atomic nuclei.
• Generated by exposing paramagnetic molecules to
  microwaves at a constant frequency and increasing
  the magnetic field until the gap between spin
  states matches the energy of the microwaves.
  There is a net absorption of energy and this
  absorption is measured and converted to a
  spectrum.

The difference in energy states matches the
frequency of the microwaves.
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Resveratrol-pretreatment potentiates reduction
of free radicals in myocardial tissue during
ischemia
Result Both groups of myocardial tissue had a
decrease in signal intensity with time, but
resveratrol-treated hearts exhibited a more rapid
decrease in signal intensity as compared to the
untreated heart (control).
Reduction Rates Control 4.2 0.9 s-1 RSV 7.58
1.5 s-1
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Summary and Conclusion

• Resveratrol increased the rate of decay of free
  radicals
• Relative to resveratrol only, treatment with
  cisplatin and resveratrol prior to I/R caused
• Increasing infarct size and myocyte apoptosis
• Increased oxidative state(measured via MDA level
  and GSH/GSSG ratio)
• Decrease survival signaling markers
• Trx-2 protein level decreased
• Inhibition of Trx-1 and Trx-2 RNA levels
• ResveratrolshRNA-Trx1 treated rats showed Trx-1
  plays no role in resveratrol-mediated
  cardioprotection. This is further confirmed by
  Dn-Trx-1 transgenic mice.
• Trx-2 is likely to play a role in switching
  I/R-induced death signal into survival signals.
  Mitochondria are the primary ROS source. The
  mitochondrial localization of Trx-2 suggests that
  mitochondria redox regulation and signaling is
  the primary target of resveratrol-mediated
  cardio-protection.

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Critique of Paper (Western and RT-PCR Data)

• Western
• NF-kB Ab was not used in any Westerns shown or
  stated, but indicated in protocol
• Trx-1 Western not detected suggests that Ab was
  working and no protein detected
• However no positive control was shown for Ab
  function
• RT-PCR
• Need control for RT PCR for normalization (issues
  with input variability, etc.)
• shRNA for Trx-2 data needed to get a complete
  story

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Critique of Paper Cont. (Western RT-PCR Data)
Western Blot RT-PCR
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Critique of Paper Cont. (Western Data)
Western Blot
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Critique of Paper Cont. (Western Data)
Western Blot