Effects of Oxidative Stress on the Expression and Function of L

Regulation of the Inducible L-arginine-Nitric Oxide Pathway by Oxidative Stress and Statins M A B de V N Costa PhD 2009 Regulation of the Inducible L-arginine-Nitric Oxide Pathway by Oxidative Stress and Statins Maria Alexandra Barata de Vasconcelos Nunes Costa Submitted to the University of Hertfordshire in partial fulfilment of the requirements of the degree of PhD June 2009 Abstract Oxidative stress (OS) plays a critical role in the pathogenesis of atherosclerosis potentially through interaction with nitric oxide (NO) generated by the inducible nitric oxide synthase (iNOS) pathway. Although considerable literature supports a pro-atherogenic role for iNOS-induced NO, recent evidence suggest an anti-atherogenic property for this enzyme where iNOS-induced NO attenuates atherosclerotic lesions after immune injury, enhancing endothelial integrity, survival, protecting against OS-induced apoptosis and necrosis. We therefore hypothesize that iNOS may have a cardio-protective role in the atherosclerotic vessel and that under conditions of OS, expression and function of this enzyme may be impaired, thus contributing to the deleterious consequences of OS. Experiments have therefore been conducted to establish whether pro-oxidants regulate iNOS expression/function in rat cultured aortic smooth muscle cells (RASMCs). These cells were induced for 24 hours with LPS and IFN-γ to mimic inflammatory conditions. Oxidative stress inducers may modulate iNOS-induced NO production through alteration of the expression and/or function of the inducible L-arginine-NO pathway. We examined the effects of hydrogen peroxide (H2O2), antimycin A and diethyl maleate (DEM) on this pathway in vascular smooth muscle cells. H2O2 had little effect on NO production or L-arginine transport while antimycin A and DEM independently caused a concentration dependent inhibition of both processes. Only DEM induced heme- oxygenase-1 (HO-1) expression, monitored by western blotting as a marker of OS. The effects of statins on NO synthesis and L-arginine transport in the presence and absence of OS were also investigated. The benefits of statins therapy in cardiovascular medicine are ascribed in part to their lipid-lowering effect by inhibiting 3-hydroxy-3-methoxyglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme for cholesterol synthesis. However, statins may possess anti-inflammatory properties and are able to improve endothelial function, stabilize atherosclerotic plaque, and inhibit platelet aggregation, vascular smooth muscle cells proliferation and vessel wall inflammation. These effects may be exerted through novel actions of statins that include interaction with specific signalling pathways in cells which may be associated with the induction of iNOS and/or cationic amino acid transporters (CATs). Thus, we have extended our investigations to include an examination of the effects of statins on both iNOS and CAT function and expression under control conditions and following exposure of cells to OS. Atorvastatin caused a bell shaped response on NO production and iNOS expression and also enhanced L-arginine transport but in a non-concentration dependent manner. Simvastatin only affected NO synthesis without altering transporter activity. Pravastatin was without effect on either system. Further studies demonstrated that that atorvastatin was able to reverse the effects of antimycin A and DEM but only on NO production. These findings confirm that the inducible L-arginine-NO pathway can be down- regulated by pro-oxidants. This mechanism may therefore contribute to the deleterious effects observed in disease states associated with OS. Moreover, statins (in particular atorvastatin) appear to be effective in reversing the inhibition of NO production caused by inducers of OS. This, together with the fact that atorvastatin and simvastatin can potentiate iNOS-induced NO production and indeed L-arginine transport (with atorvastatin), highlights a potential novel mechanism through which the cardio-protective actions of these compounds could be mediated. Acknowledgements I would here like to express my thanks to the people who have been very helpful to me during the time it took me to complete my PhD. It’s with great honour and joy that I thank my supervisors, I have been extremely fortunate with them. This thesis would not have been possible without the guidance, support, enthusiasm and patience of my principal supervisor Professor Anwar R. Baydoun, not to mention his advice and unsurpassed knowledge. The good advice, support and friendship of my second supervisor, Dr. Shori Thakur, has been invaluable on both an academic and personal levels, for which I am extremely grateful. I would also like to thank the valuable insight of my supervisor, Professor John Walker. Above all, I couldn’t wish better to develop as a scientist and as a person. To my parents and brother that gave me their unequivocal support throughout, even though we miss each other immensely, for which my mere expression of thanks likewise does not suffice. To my dear husband Luís, that supported me unconditionally at all times on my dream of pursuing a science career. His personal support and great patience made my path graceful and almost effortless. I thank my all my friends for their support and encouragement throughout. I thank all my colleagues in the laboratory Marzieh Zamani, Peter Humphrey, Edmund Garr, Yogesh Kamra and James Crutchley. I thank you all and will always keep you in my heart and in my life. Abbreviations Akt – Protein Kinase B Ang-II – angiotensin II AP-1 – Activator protein-1 ARE – Antioxidant responsive element ATP – Adenosine 5'-triphosphate BCA – Bicinchoninic Acid BCRP - breast cancer resistance protein BH4 – Tetrahydrobiopterin BSA – Bovine Serum Albumin CAM – Cellular Adhesion Molecule CAT – Cationic Amino acid Transporter Cdc42 - cell division cycle 42 protein CD68 – glycoprotein which binds to low density lipoprotein cDNA – Complementary Deoxyribonucleic Acid cGMP – cyclic adenosine monophosphate CNC – cap-n-collar CO – Carbon Monoxide CRP – C-reactive protein CYP3A4 – Cytochrome P450 3A4 DDW – Double Distilled Water DEM – Diethyl maleate I Dil-Ac-LDL – Dil labelled acetylated low density lipoprotein DMEM – Dulbecco’s Modified Eagle’s Medium DNA – Deoxyribonucleic acid DPM – Disintegrations Per Minute EDRF – endothelium-derived relaxation factor ECL – Enhanced chemiluminescence ECM – Extra Cellular Matrix EDTA – Ethylene Diamine Tetra Acetic Acid EGF – epidermal growth factor eNOS – endothelial Nitric Oxide Synthase ERK – Extracellular signal Regulated Kinases ESRD – End-Stage Renal Disease FAD – Flavin Adenin Nucleotide FBS – Foetal Bovine Serum FGF – fibroblast growth factor FITC – Fluorescein isothiocyanate FMN – Flavin Mononucleotide GO-1 – heterotrimeric G protein GTP – Guanosine 5'-Triphosphate gp91phox – transmembranar protein component of NAD(P)H oxidase GPx – catalases and glutathione peroxidases family GSH – Glutathione H2O2 – hydrogen peroxide HO- – hydroxyl radical II HASMC – Human Aortic Smooth Muscle Cells HDL – High Density Lipoprotein HEPES – 4-(2-hydroxyethy)-1-peperazine ethanesulphonic acid HMG-CoA – Hydroxymethyl Glutaryl - coenzyme A reductase HO-1 – Heme Oxygenase-1 HO-2 – Heme Oxygenase-2 HO-3 – Heme Oxygenase-3 HRP – Horse Readish Peroxidase HUVEC – Human Umbilical Vein Endothelial Cells ICAM – Intercellular Adhesion Molecule-1 IFN-γ – Interferon-gamma IgG – Immunoglobulin IK-BK – inhibitory protein-кB IKK-1 and 2 – Cytokine-activated IKappa B Kinases IL-1 – Interleukin-1 IL-18 – Interleukin-18 IL-1β – Interleukin-1beta iNOS – inducible Nitric Oxide Synthase IR – Ischemia Reperfusion IRP1 and 2/IRE – posttranscriptional cytoplasmic Iron Regulatory Proteins JAK2 – Janus Kinase-2 JNK – c-Jun N-terminal Kinases kDA – Kilodaltons Km – Affinity constant (half maximal saturation constant) III LDL – Low Density Protein LOX-1 – Lectin-like receptor-1 LPS – Lipopolysaccharide mA – milliamp MAPK – Mitogen Activated Protein Kinases MCP-1 – monocyte chemotactic protein-1 MIF – macrophage inhibitory factor MMP-2 – matrix metalloproteinase 2 MMP-9 – matrix metalloproteinase 9 MMPs – matrix metalloproteinases MRE – metal responsive element mRNA – messenger Ribonucleic Acid MRP2 – multidrug resistance associated protein MTF-1 – metal response element-binding transcription factor 1 MTT - 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NADPH – Nicotinamide Adenine Dinucleotide Phosphate NF-κB – Nuclear Factor-κappa B nNOS – neuronal Nitric Oxide Synthase NO – Nitric Oxide - NO2 – nitrite NOS – Nitric Oxide Synthase Nox – NADPH oxidase subunit complex Nrf2 – nuclear factor-erythroid 2-related factor 2 NTCP – sodium dependent taurocholate co-transporting polypeptide IV OATP – organic anion transporting polypeptide O2 – Dioxygen - O2 – Superoxide anion ONOO- – Peroxynitrite OS – Oxidative Stress oxLDL – oxidised Low Density Lipoprotein PAECs – Porcine Aortic Endothelial Cells PAK – P21-Activated Kinase PBS – Phosphate Buffered Saline PCR – Polymerase Chain Reaction PDGF – platelet-derived growth factor PI3K – Phosphoinositide-3 Kinase PKB – Protein Kinase B PKC – Protein Kinase C PMSF - phenyl methyl sulfonyl fluoride PS – Penicillin/Streptomycin PTPs – protein tyrosine phosphates PVDF – Polyvinyldene difluoride p38 MAPK – p38 Mitogen Activated Protein Kinase RASMC – Rat Aortic Smooth Muscle Cells rac1 – Ras-related C3 botulinum toxin substrate 1 Ras – family of genes encoding small GTPases rBAT – Broad Scope Amino acid Transporter Rho – Ras homolog

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