Oxidative Stress and Atherosclerosis

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

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

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 1O2 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)

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

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

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

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

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

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

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

Antioxidant Mechanisms

Glutathione System Selenium

In RBCS

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

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

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-

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

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

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)

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

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

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

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

Athersclerotic Plaque Formation Un-regulated LDL Uptake

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