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. TermDefinition OxidationGain in oxygen Loss of hydrogen Loss of electrons ReductionLoss of oxygen Gain of hydrogen Gain of electrons OxidantOxidizes another chemical by taking electrons, hydrogen, or by adding oxygen ReductantReduces another chemical by supplying electrons, hydrogen, or by removing oxygen Basics of Redox Chemistry

4. Free Radicals 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 R 3 C. Carbon-centered R 3 N. Nitrogen-centered R-O. Oxygen-centered R-S. Sulfur-centered Reactive Species Non-Radicals Non-Radicals:  Species that have strong oxidizing potential  Species that favor the formation of strong oxidants (e.g., transition metals) H 2 O 2 Hydrogen peroxide HOCl - Hypochlorous acid O 3 Ozone 1 O 2 Singlet oxygen ONOO - Peroxynitrite Me n+ Transition metals Radicals are highly reactive species

5. Reactive Oxygen Species (ROS) Radicals: O 2.- Superoxide. OH Hydroxyl RO 2. Peroxyl RO. Alkoxyl HO 2. Hydroperoxyl Non-Radicals: H 2 O 2 Hydrogen peroxide HOCl - Hypochlorous acid

6. Reactive Nitrogen Species (RNS) Radicals as: NO. Nitric Oxide NO 2. Nitrogen dioxide Non-Radicals as: Peroxynitrite

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

8. 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) Oxygen-derived Free radicals: e.g., Superoxide and hydroxyl radicals Non-free radical: Hydrogen peroxide O2., H2O2, OH. By partial reduction of molecular oxygen in electron transport chain in mitochondria

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 : 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. 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.) NO signaling in physiology O2-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. Lipids ProteinsDNA Oxidation of vitamin E Lipid peroxidation Membrane damage Thiol oxidation Carbonyl formation Damage to Ca 2+ and other ion transport systems DNA damage Cell Injury Disruption of normal ion gradients Activation/deactivation of various enzyme systems Altered gene expression Depletion of ATP and NAD(P)H Adapted from: Kehrer JP, 1993 Oxidative Stress

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. Pathological conditions 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