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 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)

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 & 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) 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

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)

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

Enzymatic Antioxidant Mechanisms

Glutathione System Selenium

Glutathione system in RBCS

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

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

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)

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

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

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

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

Athersclerotic Plaque Formation