New Strategies

1
New Strategies To Overcome Oxidative Stress in
Vascular Cells
Joong-Yeol Park M.D., Ki-Up Lee M.D.
University of Ulsan College of Medicine, Seoul,
Korea
2
Modifying hypertension does not fully reduce CVD
risk
0.05
3
Modifying dyslipidemia does not fully reduce CVD
risk
4
Modifying diabetes does not fully reduce CVD risk
Myocardial Infarction (cumulative)
fatal or non fatal myocardial infarction, sudden
death 573 of 3867 patients (15)
5
Oxidative Stress and Atherosclerosis
Hemodynamic Forces
Diabetes
Hyperlipidemia
Hypertension
Oxidative Stress
High
Low
Normal
Redox-Sensitive Signaling Pathway, Transcriptiona
l Factors
Atherogenic Genes
Atheroprotective Genes
Inflammation Vascular Dysfunction
Anti-inflammatory Vascular Protection
Normal
Kunsch C, Circulation Research. 1999
6
Effects of antioxidants on the development and
progression of atherosclerosis in humans ?
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a-Lipoic Acid
?-Lipoic acid
Dihydrolipoic acid
SH2
SH2
S
S
H2C
H2C
H2C
H2C
CH2
CH
CH2
CH
COOH
CH2
CH2
CH2
COOH
CH2
CH2
CH2

• A universal antioxidant
• Used in diabetic neuropathy
• PDH cofactor

pyruvate
pyruvate
PDH
?-Lipoic acid
Krebs Cycle
Acetyl CoA
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Effects of a-Lipoic Acid on Vascular System
Suppression of AGE induced NFkB activation in
cultured endothelial cells Bierhaus A et al,
Diabetes, 1997 Decrease ROS generation and NFkB
activity induced by glucose
Du X et al, Free Radic Biol Med, 1999
9
Effects of ALA on endothelial function and
apoptosis in obese diabetic rats
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Hypothesis
NO ?
Vascular dysfunction
Oxidative stress ?
Apoptosis
?-Lipoic acid (ALA)
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Aim of Study

• OLETF (Otsuka Long-Evans Tokushima Fatty) rats
• good model for obese type 2 diabetes
• vascular dysfunction ()
• to assess the effects of supplement of
• ?-lipoic acid on the vascular function
• and NO amount in vivo in OLETF rats

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Animals and Methods
- LETO group nondiabetic control (n8) - OLETF
group (n8) - ALA OLETF group 0.5 ALA in
diet (n8)
12 week
40 week
treatment
aortic ring study urinary NO
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Aortic Ring Study
open the chest wall under anesthesia
remove thoracic aorta
remove blood, fat and connective tissue
equilibrate in organ bath (5 CO2 and 95 O2)
preconstrict with 3 ? 10-7 M phenylephrine
relax with acetylcholine (10-9 10-5 M)
nitroprusside (10-11 10-7 M)
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Results
Aortic ring study at 40 week
OLETF LETO

OLETF LETO

0
0
-20

-20
-40

-40


-60

-60
-80
Relaxation ()
Relaxation ()
-80
-100
-120
-100
-12
-11
-10
-9
-8
-7
-6
-10
-9
-8
-7
-6
-5
-4
Conc of nitroprusside (log M)
Conc of acetylcholine (log M)
Plt0.05 vs LETO
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Effect of ALA
OLETF ALALETO
OLETF ALALETO


0
0


-20
-20

-40


-40

-60




-60
-80



-80

Relaxation ()
-100
Relaxation ()
-120
-100
-9
-8
-7
-6
-5
-4
-12
-11
-10
-9
-8
-7
-6
-10
Conc of nitroprusside (log M)
Conc of acetylcholine (log M)
Plt0.05 vs LETO Plt0.05 vs OLETF group or ALA
group
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Summary I
- Endothelium-dependent endothelium-independent
vascular relaxation were impaired in OLETF
rats - Impaired endothelium-dependent vascular
relaxation was partially reversed by ?-lipoic
acid treatment
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Effects of ALA on plasma oxidative stress markers
LETO
OLETF
OLETF ALA
MDA (µM)
8-OHdG(ng/ml)




P lt0.05 vs. LETO, Plt0.05 vs. OLETF
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Effects of ALA on urinary NO amounts at 40 week


2
Urinary NO (pmol/100g weight/day)
1
0
ALA OLETF
LETO
OLETF
Plt0.05 vs OLETF
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Effects of NO inhibitor on improvement in
endothelium dependent vasodilation by ALA
Reversing effect of ALA is partly mediated by NO.
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Summary II
-Plasma oxidative markers were elevated in OLETF
rats and decreased by ALA administration. -Urinary
amount of NO was decreased in OLETF rats and
increased by ALA administration. -NO inhibitor
partially blocked ALA-induced improvement of
vascular relaxation.
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Effects of ALA on endothelial apoptosis
LETO
OLETF
OLETF ALA
? plt0.001 OLEFT or Lipoic acid vs LETO ?? plt0.001
OLEFT vs Lipoic acid
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LETO
OLETF 1
OLETF ALA
OLETF 2
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Effects of ALA on endothelial function and
superoxide production in diabetic patients
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Methods
Participants 31 postmenopausal women with
diabetes
Endothelial function test 1)
Non-invasive technique 2) Sonos 5500
Echocardiograph and 7.5 MHz probe (
Hewlettpackard, USA)
Measurement of Superoxide anion 1)
Neutrophil isolation 2) Measurement of
Superoxide anion by NBT reduction
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-10 to 15min
Rest at supine position
Occlusion of brachial artery by BP cuff Measure
arterial diameter and flow
Baseline study
0
Deflation of BP cuff
5
Measure arterial diameter and flow
6
Endothelial dependant vasodilation
20 min
Sublingual NTG (0.6mg)
Endothelial independent vasodilation
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Measure arterial diameter and flow
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Reactive Hyperemia ( cuff deflation)
Brachial artery occlusion For 5 min
Baseline
1 minutes
Media
NO
NO
Endothelium
Hyperemic Blood Flow
Vasodilatation
Diameter
NO
Shear stress
NO
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Lipoic acid treatment protocol
TIME
0
1WK
3WK
2WK
ALA
Treatment 1200mg/day
WASHOUT
Treatment 1200mg/day
Baseline
1WK F/U
2WK F/U
3WK F/U
FMD EID Superoxide,etc
FMD EID Superoxide,etc
FMD EID Superoxide,etc
FMD EID Superoxide,etc
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Results
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Effect of ?-lipoic acid on Continuous
monitoring of flow-mediated vasodilation
()
?
6
??
4
2
Washout
(second)
60
60
60
0
? p lt 0.05 vs basal,
?? p lt 0.05 vs basal
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Effect of ?-lipoic acid on flow-mediated
vasodilation
()
?
??
8
6
FMD
4
2
0
Baseline
1 wk
2 wk
3 wk
? p lt 0.05 vs basal,
?? p lt 0.05 vs basal
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Effect of ?-lipoic acid on Endothelial
independent vasodilation
()
12
8
EID
4
0
240
(second)
170
90
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Effect of alpha-lipoic acid treatment on
formation of superoxide in neutrophils
12
?
8
??
Superoxide(nmol/107 cells/min)
4
0
Baseline
1 wk
2 wk
3 wk
? p lt 0.05 vs baseline
,?? p lt 0.003 vs baseline
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Summary
1. ?-Lipoic acid(ALA) treatment
decreased superoxide formation of
neutrophil. 2.
?-Lipoic acid(ALA) treatment increased FMD ,
improved endothelial dysfunction.
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Conclusion
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Newer modality to overcome intracellular oxidative
stress and vascular dysfunction - Uncoupling
protein2
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• Mitochondrial ROS generation

Glucose/FFA
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• Uncoupling protein (UCP) intracelluar ROS ?

Glucose/FFA
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Introduction
FFA
PKC
ET-1
NF-kB
Endothelial cell
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Aim of Study
? The effect of UCP-2 gene transfection 1.
In Endothelial Cell
1) ET-1 mRNA expression 2) NFkB
activity 3) ROS generation
2. Contractile response of aortic ring
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Materials Methods
Materials Methods

• Primary cultured human aortic endothelial cells
  (HAEC)

• UCP-2 gene transfection with adenoviral vector

• ET-1 expression Nothern blot analysis

• NF-kB activity
  
• Electrophoretic Mobility Shift Assay
  (EMSA)

• ROS generation
• dichlorofluorescin (DCFH) oxidation assay

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• Effect of UCP-2 transfection on ET-1 mRNA
  expression

ET-1mRNA
GADPH
160
UCP-2 ()
UCP-2 (-)


120


Optical Density ( of control)
80
40
Control
P lt0.05 vs. control P lt0.05 vs. UCP-2 (-)
Linoleic Acid (450uM)
Oleic Acid (300uM)
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• Effect of UCP-2 transfection on NF-kB activity

UCP-2 ()
UCP-2 (-)

160

120

Optical Density ( of control)

80
40
UCP-2
0
Control
Linoleic Acid (450uM)
Oleic Acid (300uM)
P lt0.05 vs. control P lt0.05 vs. UCP-2 (-)
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• Effect of UCP-2 transfection on ROS generation

UCP-2 ()
UCP-2 (- )

160
120

80
of highly fluorescent cells
40
0
Control
Control
Lipid
Lipid
Control
P lt0.05 vs. control P lt0.05 vs. UCP-2 (-)
Intralipid
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Materials Methods

• Aortic ring contraction

- SD rat
- Throracic aortic ring
- Transfection with adenovirus containing UCP-2
gene (1.0 ?105 PFU/ml) for 30minutes -
Pretreatment with or without 10ug/ml oxidized LDL
- Contraction Phenylephrine 3 ? 10-7 M
Relaxation Acetylcholine Chloride Sodium
Nitroprusside 10-9, 10-8,
10-7, 10-6, 10-5 M
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• Effect of UCP-2 transfection on Aortic Ring
  Contraction

oxLDL(10ug/ml) UCP-2 (-)
oxLDL(10ug/ml) UCP-2 ()
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Conclusion
Fatty acid
Fatty acyl CoA
Ox LDL
Mitochondria
NFkB ?
UCP2 ?
ROS ?
Vascular dysfunction
Endothelin ?
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These results suggest that antioxidant, such as
?-lipoic acid and drugs which enhance UCP2
expression be new, effective modalities to
overcome oxidative stress in vascular cells and
atherosclerosis.
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Acknowledgement
University of Ulsan, College of
Medicine Endocrinology and Metabolism Asan
Institute for Life Sciences
University of Ulsan, College of Medicine Division
of Cardiology
Seong-Wook Park, Seung-Jung Park Ki Hoon Han
Ki-Up Lee Sung-Kwan Hong Min Sun Kim Jung-Min
Koh, Il-Sung Namkung, Kyoung Soo Kim, Ki Ho
Song Hye-Sun Park, Do Sook Choi Hyun-Sik Kim,
Jae-Won Yoo Ki-Young Park, Hae Sun Song Ji-Young
Yoon, Min-Jung Shin Chan-Hee Kim, Yoon Mi
Kim Cheol Namkung, Jung-Won Song
Inha University
In Sun Park
Kyemyong University
In Kyu Lee
KNIH
In Ho Jo, Sung Won Bae
Harvard Medical School
Young Bum Kim