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 R3C. Carbon-centered R3N. Nitrogen-centered
R-O. Oxygen-centered
R-S. Sulfur-centered
Free Radicals:
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
H2O2 Hydrogen peroxide
HOCl- Hypochlorous acid
O3 Ozone
1O Singlet oxygen
ONOO- Peroxynitrite
Men+ Transition metals
Radicals are highly reactive species
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 and 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)
O2-. , H2O2 , OH-. By partial reduction of molecular oxygen in electron transport chain in mitochondria
Oxygen-derived Free radicals: e.g., Superoxide and hydroxyl radicals
Non-free radical: Hydrogen peroxide

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

13. Antioxidant Mechanisms

14. Glutathione System
Selenium

15. In RBCS

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

18. NOS ( nitric oxide synthase)
2 constitiutive NOS:
are calcium-calmodulin dependent and
constantly produce low level of NO
( primarly in endothelium=eNOS, neural=nNOS).
1 inducible NOS :
calcium independent
can be expressed in many cells including hepatocytes, macrophages and neutrophils.
The inducers include bacterial toxins, tumor-necrosis factor and inflammatory cytokines
It can produce large amounts of NO over hours or even days

19. NO• signaling in physiology
Nitric Oxide Synthase
NO•
Binds to heme moiety of guanylate cyclase
Conformational change of the enzyme
Increased activity (production of cGMP)
Modulation of activity of other proteins (protein kinases, phospho- diesterases, ion channels)
Physiological response (relaxation of smooth muscles, inhibition of platelet aggregation, etc.)
O2-•
ONOO-

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. Oxidative Stress Cell Injury Lipids Proteins DNA DNA damage
Altered gene
expression
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. Consequences of lipid peroxidation (polyunsaturated fatty acids)
Structural changes in membranes
alter fluidity ( increase rigidity)
altered activity of membrane receptors
alter membrane-bound signaling proteins
alter channels& ion permeability
Lipid peroxidation products form adducts/crosslinks with non lipids
e.g., proteins( affects the membrane functions)

23. Vascular effects of ROS:
Altered vascular tone
Increased endothelial cell permeability

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

25. Regulated LDL Uptake high-affinity, specific & tightly regulated
LDL-Receptor

26. Pathogenesis of Atherosclerosis
Modified (oxidized) LDL … Oxidative stress (imbalance between oxidants and antioxidants)
Endothelial injury of arterial wall
Adherence of monocytes to endothelial cells and their movement into intima where it becomes macrophages
Uptake of oxLDL by macrophage scavenger receptor: Scavenger receptor class A (SR-A)
Low-affinity, non-specific receptor
Un-regulated receptor
Foam cell transformation: Accumulation of excess lipids inside the cells (unregulated receptor)
Atherosclerotic plaque formation

27. Athersclerotic Plaque Formation Un-regulated LDL Uptake

28. Thank You