1. Complex, multisystemic features of preeclampsia
Hypertension
Proteinuria
Eclampsia
HELLP syndrome
Intra-uterine growth restriction
Multi-organ disease
Cerebral vessels
Fetus
Liver
Systemic blood vessels
Kidneys

2. Oxidative stress Antioxidant capacity ROS synthesis Vitamin C SOD O2._
H2O2
ONOO_
Antioxidant capacity
ROS synthesis

3. 2-Stage Model of Preeclampsia
Placental events:
Abnormal Implantation
Placental hypoperfusion
Maternal constitution:
Environment/Behavior/
Genetics -> syndrome X
Adaptation to pregnancy
STAGE 1
Oxidative stress
Maternal endothelial cell dysfunction
Preeclampsia
Bilodeau and Hubel J Obstet Gynaecol Can 2003;25:742;
Roberts and Hubel LANCET 1999; 354:788
STAGE 2
Maternal constitution:
Environment/Behavior/
Genetics -> syndrome X
Later life endothelial disease
(Hypertension,
Ischemic heart disease)
Oxidative stress

4. Spiral Arteries
Non-pregnant Normal Pregnancy

5. Spiral Arteries
Normal Pregnancy Preeclampsia

6. Xanthine Oxidase/Dehydrogenase
Xanthine dehydrogenase (XD)
Xanthine
NADH + Uric Acid
Xanthine oxidase (XO)
O Uric Acid
Xanthine
Hypoxia
Cytokines XD XO XD XO
Posthypoxic reperfusion:
O2.-

7. Xanthine Oxidase/ Dehydrogenase Placental bed curettings
Many, Hubel, Fisher, Roberts, Zhou Am J Pathol 2000

8. Nitrotyrosine residues: a “footprint” of ROS/RNS damage
OH- O O
NO. O N
ONOO-
peroxynitrite +
O2.- R R
tyrosine nitrotyrosine

9. Nitrotyrosine Cytokeratin Nitrotyrosine Normal, Villous placenta
Invasive cytoytophoblast
Preeclampsia,
Villous placenta
Preeclampsia,
Invasive cytotrophoblast
Many A et al
Also reported by Myatt et al. 1996; 1998

10. Nitrotyrosine Immunoreactivity in Villous Tissues w/wo Hypoxia-Reoxygenation
Hypoxia 20 min. +
2 hrs. reoxygenation
(air / 5%CO2)
Immediately after delivery
After hypoxia
(95%N2/5%CO2)
2 hrs.
Hypoxia 20 min. +
2 hrs. reoxygenation
(air / 5%CO2)
Hypoxia 20 min. +
2 hrs. reoxygenation
(5%O2/90%N2/5%CO2)
Negative control
Hung T-H, Skepper JN, Burton GJ. Am J Pathol 2001; 159:

11. NAD(P)H oxidase O ._ O membrane Mox O ._ cytosol 2 NAD(P)H NAD(P)+ 2
Cytochrome b558
p22
membrane
Mox
rac
p47 O 2 ._
p67
NAD(P)H
NAD(P)+
cytosol

12. Endothelial mediated relaxation to methacholine is blunted by preeclampsia plasma
100 80 60
% Constriction
Normal Pregnancy 40
Plasma (n=7) 20
Preeclamptic Pregnancy
Plasma (n=7)
1E-09
1E-08
1E-07
1E-06
1E-05
Methacholine Dose (M)
Gandley and Hubel, 2003

13. Endothelial mediated relaxation to methacholine is restored by losartan
p<0.05
(20)
(10) 10 20 30 40 50 60 70 80 90
100
1E-09
1E-08
1E-07
1E-06
1E-05
Preeclamptic Plasma
+Losartan (n=5)
Normal Plasma
Preeclamptic Pregnancy
Plasma (n=7)
Normal Pregnancy
% Constriction
Methacholine Dose (M)

14. Ascorbate is an outstanding antioxidant:
• Ascorbate is the “first line” water-soluble antioxidant.
• Ascorbate radical, formed from quenching of more powerful oxidants, is unreactive.
• Vitamin C regenerates
vitamin E (synergistic action).
From: Carr AC. Circ Res 2000;87:349

15. Endothelial function: Unique salubrious effects of ascorbate
• Intracellular ascorbate (mM) competes effectively with NO. for
O2 .- .
• Plasma ascorbate (30-60 µM) inhibits membrane lipid peroxidation and formation of oxLDL.
• Preservation of BH4 cofactor of NOS
• Involved in release of NO from S-nitrosothiols
From: Carr AC. Circ Res 2000;87:349

16. S-nitrosothiols are Increased in
Predeclampsia Plasma
Whole plasma
Low MW thiols
Albumin 20 15
RSNO,nmol/ml 10 5 * **
9.2 ± ± ± 2.9
4.5
± ± ± 1.2 4
.2 ± ± ± 1.4
Tyurin, Liu, Tyurina, Sussman, Hubel, Roberts, Taylor, Kagan Circ Res 2001;88:

17. Initial rate of NO release, pmol/sec
Release of NO from S-NO-albumin occurs at relevant ascorbate concentrations A B 80 3 6 9 12
+ Ascorbate 60
Current, pA
Initial rate of NO release, pmol/sec 40 20
- Ascorbate
200
400
600 40 80
120
Time, sec
Ascorbate, mM

18. The Vitamins In Pre-eclampsia Study Chappell et al., Lancet 1999
1512 women screened at weeks
242 abnormal & recruited to study
283 randomised
41 women with history of pre-eclampsia
Analysis:
Intention-to-treat
Completed study
142 assigned placebo
141 assigned mg vitamin C and 400IU vitamin E
81 participated
until delivery
79 participated

19. Clinical outcome 25 20 15 10 5 Number of women with pre-eclampsia5 10 15 20 25
Number of
women with
pre-eclampsia
Intention-
to-treat
Completed
study
Placebo
Vitamins C and E
Adjusted
odds ratio:
0.39 ( )
p=0.02
0.24 ( )
p=0.002
Chappell et al 1999

20.  Preeclampsia developed  Delivered of SGA infants
Longitudinal evaluation of indices of oxidative stress in plasma (placebo arm of antioxidant trial)
 low-risk group
 Preeclampsia developed
 Delivered of SGA infants
Chappell LC et al. Am J Obstet Gynecol :

21. Risk Factors in Common For Atherosclerosis and Preeclampsia
• Hypertension
• Diabetes
• Collagen vascular disease
• Increased plasma homocysteine
• Obesity
• Insulin resistance syndrome
Up to half of patients with coronary artery disease have serum cholesterol concentrations in the normal range…

22. The Atherogenic Lipid Phenotype in women with preeclampsia
• Increased triglycerides (TG-rich lipoproteins)
• Predominance of small, dense LDL particles
• Decreased HDL-cholesterol
• Post-prandial lipemia
• Increased circulating free fatty acids
Underlying factors: insulin resistance and increased human placental lactogen

23. Free fatty acid-mediated conformational changes in albumin increase the redox activity of albumin-associated Cu, converting albumin from an antioxidant to a prooxidant
Cu1+
Ascorbate
Radical
Cu2+
serum albumin
FFA
Dehydro-ascorbate -e
Kagan, Yturin, Borisenko, Fabisiak, Hubel, Ness, Gandley, McLaughlin, Roberts Hypertension Pregn. 2001;20:221-42

24. Potential mechanisms of decreased NO bioavailability andaltered vascular function in pre-eclampsia
GTP
cGMP
ONOO-
Vascular
Smooth
Muscle
Relaxation
02•- GC
NO•
NAD(P)H Oxidase +
ANG II
TNF
Interstitial
Space
Endothelial
Cells
CATALASE
H202
ONOO-
H20
NO•
Protein
Damage
(nitrotyrosine)
02•-
02•-
ASC +
eNOS
[ BH4 ]
NAD(P)H Oxidase
Lumen
(blood) +
02•-
ANG II
Shear Stress
TNF
Oxidized lipids
Bilodeau and Hubel, 2003