1. Oxidative Stress and Atherosclerosis

2. Objectives What is “free radical” ?
Reactive oxygen and nitrogen species (RONS)
Are the RONS always dangerous?
Well known term “oxidative stress” - what is it?
Antioxidants - types
Disorders Associated with Oxidative stress
Oxidative stress and atherosclerosis

3. Basics of Redox Chemistry
Term Definition
Oxidation Gain in oxygen
Loss of hydrogen
Loss of electrons
Reduction Loss of oxygen
Gain of hydrogen
Gain of electrons
Oxidant Oxidizes another chemical by taking
electrons hydrogen or by adding oxygen
Reductant Reduces another chemical by supplying
electrons, hydrogen or by removing oxygen

4. Reactive Species Free Radicals Non Radicals R3C. Carbon-centered
A molecule with an unpaired electron in an outer valence shell
Tend to reach equilibrium, plucks an electron from the nearest intact molecule.
Most of biomoleculs are not radicals
Radicals are highly reactive species
R3C. Carbon-centered
R3N. Nitrogen-centered
R-O. Oxygen-centered
R-S. Sulfur-centered
H2O2 Hydrogen peroxide
HOCl Hypochlorous acid
O3 Ozone
1O2 Singlet oxygen
ONOO- Peroxynitrite
Men+ Transition metals
Non Radicals
Species that have strong oxidizing potential
Species that favor the formation of strong oxidants (e.g. transition metals)

5. Reactive Oxygen Species (ROS)
Radicals
O2.- Superoxide
OH .- Hydroxyl
RO2. Peroxyl
RO. Alkoxyl
HO2. Hydroperoxyl
Non-Radicals
H2O2 Hydrogen peroxide
HOCl- Hypochlorous acid

6. Reactive Nitrogen Species (RNS)
Radicals as:
NO. Nitric Oxide
NO2. Nitrogen dioxide
Non-Radicals as:
Peroxynitrite

7. Oxidative Stress Antioxidants Oxidants
“An imbalance favoring (pro) oxidants and/or disfavoring antioxidants, potentially leading to damage”

8. Oxidative Stress
It is a state in the cells in which there is increased concentration of reactive species which is not counterbalanced by increased levels of antioxidants.
This imbalance was implicated in production of different diseases as atherosclerosis

9. Endogenous sources of ROS & RNS
Mitochondria
Lysosomes
Peroxisomes
Endoplasmic Reticulum
Cytoplasm
Microsomal Oxidation,
Flavoproteins, CYP enzymes
Myeloperoxidase
(phagocytes)
Electron transport
Oxidases,
Flavoproteins
Plasma Membrane
Lipoxygenases,
Prostaglandin synthase
NADPH oxidase
Xanthine Oxidase,
NOS isoforms Fe Cu
Transition metals

10. Reactive Oxygen Species (ROS)
ROS are formed continuously by partial reduction of molecular oxygen in electron transport chain in mitochondria
ROS are:
Free radicals: e.g. Superoxide & hydroxyl radicals
Non radicals: Hydrogen peroxide

11. Sometimes, ROS is useful for humans
For example:
Oxygen dependent pathway for microbial killing
In phagocytic cells (neutrophils & monocytes),
microorganisms are phagocytosed
NADPH oxidase in these cells converts
molecular oxygen into superoxide (Respiratory
Burst).
Superoxide + chloride ions are converted into
hypochlorous acid that kills bacteria
(by help of the lysosomal enzyme myeloperoxidsase
MPO)

12. Antioxidants Enzymes that catalyze antioxidant reactions: Vitamins
1- Superoxide dismutase
2- Catalase
3- Glutathione system (glutathione, NADPH, reductase,
peroxidase & selenium).
Vitamins
1- Vitamin A & β-carotenes
2- Vitamin C (ascorbic acid)
3- Vitamin E
Trace elements
Selenium

13. Enzymatic Antioxidant Mechanisms

14. Glutathione System
Selenium

15. Glutathione system in RBCS

16. Nitric Oxide (NO) Nitric oxide (NO): Synthesis Effects
Free radical gas
Very short half-life (seconds)
Metabolized into nitrates & nitrites & perooxynitrite
Synthesis
Enzyme: NO synthase Precursor: L-Arginine
Effects
Relaxes vascular smooth muscle
Prevents platelet aggregation
Bactricidal & tumoricidal effects
Neurotransmitter in brain

18. Nitric Oxide Synthase (NOS)
Two constitiutive NOSs
in endothelium = eNOS neural = nNOS
constantly produce low level of NO
calcium-calmodulin dependent
One inducible (iNOS)
Can be expressed in many cells including hepatocytes, macrophages & neutrophils.
Inducers include bacterial toxins, tumor-necrosis factor & inflammatory cytokines
It can produce large amounts of NO over hours or even days
Calcium independent

19. NO effect on vascular endothelium
Endotheilal Nitric Oxide Synthase (eNOS)
NO production
Diffuses to smooth muscles
Activates guanylate cyclase
production of cGMP from GTP
Activation of protein kinase G
Phosphorylstes myosin light-chain kinase (becomes inactive)
Decrease smooth muscular contraction
(RELAXATION)

20. Oxidative Stress: Role of Nitric Oxide (NO)
NO produced by endothelial NOS (eNOS)  improving
vascular dilation and perfusion (i.e. beneficial).
Vasodilators such as nitroglycerin is metabolized into NO and
causes vasodilatation
Increased iNOS activity is generally associated with
inflammatory processes

21. Activation/deactivation of various enzyme systems
Cellular Damage
caused by Oxidative Stress
Lipids Proteins DNA
Oxidation of
vitamin E
Thiol oxidation
Carbonyl formation
DNA damage
Mutations
Lipid peroxidation
Damage to Ca2+ and
other ion transport
systems
Membrane damage
Disruption of normal
ion gradients
Depletion of ATP
and NAD(P)H
Activation/deactivation
of various enzyme systems
Cell Injury
Adapted from: Kehrer JP, 1993

22. Pathological conditions that involve
oxidative stress
Inflammation
Atherosclerosis
Ischemia/reperfusion injury
Cancer
Aging
Obesity

23. ATHEROSCLEROTIC PLAQUES
Oxidative stress & Atherosclerosis
Oxidation of the lipid components & apo B in LDL
Oxidized LDL
Injury of endothelium (inner lining of blood vessels)
Monocytes adhere to endothelial cells
Move to (subendothelium) intima
Monocytes are converted to Macrophages
Macrophages uptakes oxidized LDL through scavenger receptor class A (SR-A)
Macrophages are converted to Foam cells
Release cytokines
& participates in formation of
ATHEROSCLEROTIC PLAQUES

24. Athersclerotic Plaque Formation