Oxidative Stress and Atherosclerosis

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 biomoleculsare not radicals H2O2Hydrogen peroxide HOCl- Hypochlorous acid O3Ozone 1O2 Singlet oxygen ONOO- Peroxynitrite Men+ Transition metals Radicals are highlyreactivespecies • 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: H2O2Hydrogen 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” -H. Sies

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

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: 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 Selinium

15. In RBCS

16. Nitric Oxide (NO) NO: Freeradicalgas Very short half-life (seconds) Metabolized into nitrates&nitrites and perooxynitrite Synthesis: Enzyme: NO synthasePrecursor: 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 Lipids Proteins DNA Oxidation of vitamin E Thiol oxidation Carbonyl formation DNA damage Altered gene expression Damage to Ca2+ and other ion transport systems Lipid peroxidation 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 and channels • alter membrane-bound signaling proteins • increases ion permeability • Lipid peroxidation products form adducts/crosslinks with non lipids • e.g., proteins and DNA • Cause direct toxicity of lipid peroxidation products • Disruptions in membrane-dependent signaling

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

24. Pathologicalconditions that involve oxidative stress • Inflammation • Atherosclerosis • Ischemia/reperfusion injury • Cancer • Aging • Obesity

25. 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

26. Athersclerotic Plaque Formation Un-regulated LDL Uptake

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

28. Thank You