ANNA-KAISA RUOTSALAINEN THE ROLE OF TRANSCRIPTION FACTOR NRF2 IN ATHEROGENESIS AND HEPATIC STEATOSIS IN HYPERCHOLESTEROLEMIC MOUSE MODELS

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1 PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences ANNA-KAISA RUOTSALAINEN THE ROLE OF TRANSCRIPTION FACTOR NRF2 IN ATHEROGENESIS AND HEPATIC STEATOSIS IN HYPERCHOLESTEROLEMIC MOUSE MODELS

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3 The Role of Transcription Factor Nrf2 in Atherogenesis and Hepatic Steatosis in Hypercholesterolemic Mouse Models

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7 ANNA-KAISA RUOTSALAINEN The Role of Transcription Factor Nrf2 in Atherogenesis and Hepatic Steatosis in Hypercholesterolemic Mouse Models To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Medistudia Auditorium MS301, Kuopio, on Friday, November 23 th 2018, at 12 noon Publications of the University of Eastern Finland Dissertations in Health Sciences Number 494 A.I.Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland Kuopio 2018

8 Grano Oy Jyväskylä, 2018 Series Editors Professor Tomi Laitinen, M.D., Ph.D. Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences Associate professor (Tenure Track) Tarja Kvist, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences Associate Professor (Tenure Track) Tarja Malm, Ph.D. A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Lecturer Veli-Pekka Ranta, Ph.D. School of Pharmacy Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O.Box 1627 FI Kuopio, Finland ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L:

9 III Author s address: Supervisors: A.I.Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland P.O. Box KUOPIO FINLAND Professor Anna-Liisa Levonen, MD, Ph.D. A.I.Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland KUOPIO FINLAND Adjunct Professor Eija Pirinen, Ph.D. Research Program for Molecular Neurology Biomedicum University of Helsinki HELSINKI FINLAND Reviewers: Docent Katariina Öörni, Ph.D. Wihuri Research Institute HELSINKI FINLAND Professor Peppi Karppinen, MD, Ph.D. Faculty of Biochemistry and Molecular Medicine University of Oulu OULU FINLAND Opponent: Professor Eriika Savontaus, MD, Ph.D. Institute of Biomedicine Research Center for Integrative Physiology and Pharmacology University of Turku TURKU FINLAND

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11 V Ruotsalainen, Anna-Kaisa The Role of Transcription Factor Nrf2 in Atherogenesis and Hepatic Steatosis in Hypercholesterolemic Mouse Models University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences Number p. ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: ABSTRACT Global expansion of unhealthy lifestyle has increased the incidence of obesity and metabolic disorders that lead to diseases such as atherosclerosis and hepatic steatosis by promoting lipid accumulation, chronic inflammation and oxidative stress in the vasculature system and liver. Nrf2 (NF-E2 related factor 2) is a transcription factor, activated by oxidative stress, which is known to protect cells against reactive oxygen species via its antioxidant functions and subsequently prevents inflammation and mitochondrial dysfunction. Thereby, Nrf2 has become an interesting target for drug development to treat different chronic inflammatory diseases. The aim of this study was to investigate the macrophage-specific and systemic effects of Nrf2 on the progression of atherosclerosis and hepatic steatosis by using gene modified Nrf2-deficient mice crossbred with the mouse models of human hyperlipidemia and atherosclerosis. This thesis demonstrates that the loss of Nrf2 increased the cholesterol uptake and the expression of pro-inflammatory cytokines in mouse peritoneal macrophages that subsequently enhanced foam cell formation in vitro and increased the size of early lesion in the mouse aorta. In addition, Nrf2 deficiency increased local vascular inflammation and oxidative stress that likely impaired the stability of atherosclerotic plaque and made the plaque prone to rupture, which possibly led to myocardial infarction and sudden death in hypercholesterolemic mice. In contrast, global Nrf2 deficiency was shown to protect against atherogenesis and hepatic steatosis by lowering plasma lipid levels due to systemic and peripheral metabolism. In the liver, Nrf2 deficiency improved mitochondrial oxidative metabolism that led to reduced lipid accumulation and inflammation. To conclude, Nrf2 has protective effects against atherosclerosis and its complications locally in the vascular wall, but it promotes atherosclerotic plaque development and liver lipid accumulation via the regulation of systemic and peripheral lipid metabolism. This thesis provides novel information on the cardiometabolic effects of Nrf2. This is important, as Nrf2 activators have been developed for the long-term treatment of chronic diseases. National Library of Medicine Classification: QU 55.97, QY 58, WG 550, WI 710 Medical Subject Headings: Transcription Factors; NF-E2-Related Factor 2; Blood Vessels; Atherosclerosis; Liver; Mitochondria/metabolism; Non-alcoholic Fatty Liver Disease; Inflammation; Oxidative Stress; Cholesterol; Hypercholesterolemia; Lipids; Lipid Metabolism; Cytokines; Foam Cells; Disease Models, Animal; Mice

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13 VII Ruotsalainen, Anna-Kaisa The Role of Transcription Factor Nrf2 in Atherogenesis and Hepatic Steatosis in Hypercholesterolemic Mouse Models University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences Number p. ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: TIIVISTELMÄ Maailmanlaajuisesti lisääntyneet epäterveelliset elämäntavat johtavat ylipainoon ja aineenvaihdunnan häiriöihin, jotka johtavat ateroskleroosin, eli valtimonkovettumataudin ja alkoholista riippumattoman rasvamaksan kehittymiseen edistämällä rasvan kertymistä, kroonista tulehdusta ja hapetusstressiä verisuonistossa ja maksassa. Nrf2 (NF-E2 related factor 2) on hapetusstressissä aktivoituva tumatekijä, jonka tiedetään suojaavan soluja hapetusstressiltä antioksidanttisäätelyn kautta, mikä vähentää myös kudosten tulehdusreaktiota ja parantaa mitokondrioiden toimintaan. Tämän vuoksi Nrf2:sta on tullut kiinnostava lääkekehityksen kohde monien kroonisten tulehdussairauksien hoitoon. Tämän väitöstutkimuksen tarkoituksena oli selvittää Nrf2:n makrofagispesifisiä ja systeemisiä vaikutusmekanismeja valtimonkovettumataudin ja rasvamaksataudin kehittymisessä käyttäen Nrf2 poistogeenisiä hiiriä yhdistettynä erilaisiin ihmisen hyperlipidemian ja ateroskleroosin hiirimalleihin. Väitöstutkimus osoitti, että Nrf2:n puutos lisäsi kolesterolin kuljetusta ja tulehdusta edistävien sytokiinien ilmentymistä hiiren makrofageissa, mikä edisti vaahtosolumuodostusta ja varhaisten ateroskleroottisten plakkien mudostumista myös hiirimallissa. Nrf2:n puutos myös altisti sydäninfarktille ja äkkikuolemalle ihmisen hyperlipidemiaa mallintavissa hiirissä lisäämällä valtimoplakin paikallista tulehdusta ja oksidatiivista stressiä, ja siten edistämällä valtimoplakin repeämäherkkyyttä, joka voi johtaa sydän- tai aivoninfarktiin. Toisaalta, Nrf2:n kokonaisvaltaisella puutoksella osoitettiin olevan ateroskleroosilta ja rasvamaksalta suojaavia vaikutuksia, sillä Nrf2- puutos madalsi hiirten veren rasva-arvoja mahdollisesti systeemisen lipidimetabolian säätelyn kautta ja tehosti maksan mitokondrioiden metaboliaa, mikä osaltaan vähensi rasvan kertymistä ja tulehdusta maksassa. Yhteenvetona voidaan todeta, että Nrf2:lla on paikallisesti verisuonen seinämässä ateroskleroosilta suojaavia vaikutuksia, mutta myös valtimoplakkien kasvua ja maksan rasvoittumista edistäviä vaikutuksia metabolian ja mitokondrioiden toiminnan säätelyn kautta. Tämä tutkimus tarjoaa uutta tietoa Nrf2:n metabolia- ja verisuonivaikutuksista, jotka ovat tärkeitä kehitettäessä Nrf2:ta aktivoivia lääkeaineita pitkäaikaisten kroonisten sairauksien hoitoon. Luokitus: QU 55.97, QY 58, WG 550, WI 710 Yleinen Suomalainen asiasanasto: transkriptiotekijät; verisuonet; ateroskleroosi; maksa; mitokondriot; rasvamaksa; tulehdus; oksidatiivinen stressi; kolesteroli; hyperkolesterolemia; lipidit; sytokiinit; koeeläinmallit; eläinkokeet; hiiret

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15 IX Sometimes the right path is not the easiest one. - Pocahontas, Disney

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17 XI Acknowledgements This thesis was carried out in the research group of Professor Anna-Liisa Levonen in the A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland during the years I express my sincere gratitude to my supervisors, Professor Anna-Liisa Levonen and Adjunct Professor Eija Pirinen. I am grateful to Anna-Liisa for giving me an opportunity to perform both my bachelor s and master s thesis under your guidance that kindled my interest in science and my will to begin doctoral studies. During these years, you provided great opportunities to gain skills and knowledge and create networks. You have taught and encouraged me many ways, but still allowed me to find my own interests and paths. I am deeply grateful to Eija, as you have hands-on taught me how to perform mouse experiments, especially metabolic studies, and I treasure and obey those advices still today. Thank you for the opportunity to visit and perform the mitochondrial lab work in your lab in Helsinki. You have introduced me to the world of mitochondria, which has been challenging, but still fascinating for me. Moreover, I appreciate your guidance in scientific writing and several valuable discussions regarding my projects. I wish to thank the official reviewers of this thesis, Professor Peppi Karppinen and Docent Katariina Öörni for your valuable time to pre-evaluate and comment my thesis. I would like to thank Professor Eriika Savontaus for agreeing to be my opponent in the public examination of the thesis. I want to thank Nihay Laham Karam for kindly performing linguistic revision of my thesis. I am sincerely grateful to all the skilful co-authors of the original articles and the manuscript. Especially, I wish to thank Jari Lappalainen, who has kindly helped and supported me with the constant animal work and histology and shared the ideas, knowledge and inspiration in the field of atherosclerosis research. I want to express my gratitude to Matti Jauhiainen for great collaboration. Thank you for your help with lipid measurements and sharing your expertise and enthusiasm regarding my projects and science in general. I wish to thank Emilia Kansanen, as you have helped and kind of supervised me in many ways during this process. Thank you for your friendship and understanding. I also wish to thank Virve Sihvola and Suvi Kuosmanen for their collaboration, support and friendship. A real lifesaver, Arja Korhonen, I am truly grateful to you for all your help with practical laboratory work. Without you this work would not have been done. The old ALL-group, it was a pleasure to work with you all! I am deeply grateful to Seppo Ylä-Herttuala for your support and the valuable collaboration with your helpful and skilful research group. I express my gratitude to all the past members of SYH-group, thank you for your support and bad jokes. You are inspiring and awesome people, and I am happy that many of you have become my friends also outside the science. My special thanks go to my dear friend Line. I am sincerely thankful for your friendship and support both in scientific and personal life. I am thankful also to all the current SYH-group members, who have supported me at the chalk lines of the thesis process. Especially Ella and Taina, thank you for your support, many discussions in science and sharing the thoughts and advices regarding the last steps in the thesis process. I would like to thank Anne Karppinen, Tuula Reponen, Seija Sahrio and Helena Pernu for your assistance and helpfulness. My gratitude is extended to Jouko Mäkäräinen and Jari Nissinen for assisting with various practical issues during the years. My sincere thanks

18 XII belongs also to the staff of the Lab Animal Center, especially Auli Nissinen and Eveliina Kyllönen, who have taken good care of my sensitive Nrf2-mice. It is essential to take time out from work sometimes and I am lucky to have such great people around me. My lovely friend Sini, I am grateful for your friendship, support, understanding and sharing the thoughts both in and outside the scientific life. I would like to thank Kaisu and Esko for your peer support in everyday life and taking care of Eelis at many critical moments, when I have multitasked and worked slightly desperately on my thesis at home. I express my gratitude to Petri and Marja for helping me with technical problems regarding the layout of the thesis and delighting my family with your freshly baked goodies. My life would be nothing without horses and thereby, I want to express my sincere gratitude to my dear co-equestrians, especially Tiina, Kati, Virpi, Saija, Kata, Vilma, Marja and Juha-Pekka. Thank you for your friendship and many great moments we have spent together. You have made it possible for me to do what I love the most, which has provided valuable counterbalance to science and kept me mentally (more) balanced. My dear friends Jenna, Eetu, Hanna-Maija, Marika, Jussi, Outi, Ville, Paula, Jussi, Katri, Nanna, Auni, Maria, Sanna and Pekka, not in any order, thank you for your friendship and bringing the happiness into my life. Most importantly, my loving thanks belongs to my family. My parents Ari and Pirjo, thank you for your never-ending love and support through my life, and the encouragement whatever I have chosen to do. Mom, I wish you were here! I am thnakful to my dear little brother Olli and his other half Jenni. It s always my happy moment, when you are visiting us or we can spend time together at summer cottage. Thank you for your support and always being there for me. I would also like to thank my mother-in-law Marketta and father-in-law Pertti for supporting me and taking me as a part of your family, it means a lot to me. I am also grateful to my grandparents, Eeva and Katri, for your love, encouragement and being always interested in my work. Finally, I express my deepest gratitude to Jarno. Thank you for your love and understanding. Jarno and our precious Eelis, you are my everything and I love you to the moon and back. This thesis is dedicated to the loving memory of my mother. Kuopio, November 2018 Anna-Kaisa Ruotsalainen This study was supported by grants from Ida Montin Foundation, Aarne Koskelo Foundation, North Savo Regional Foundation of Finnish Cultural Foundation, Finnish Foundation for Cardiovascular Research, Faculty of Health Sciences, the Foundation of Kuopio University, Foundation of Helena Vuorenmies and Sigrid Juselius Foundation.

19 XIII List of the original publications This dissertation is based on the following original publications: I Ruotsalainen A-K, Inkala M, Partanen M, Lappalainen J, Kansanen E, Mäkinen P, Heinonen S, Laitinen H, Heikkinen J, Vatanen T, Hörkkö S, Yamamoto M, Ylä- Herttuala S, Jauhiainen M, Levonen A-L. The absence of macrophage Nrf2 promotes early atherogenesis. Cardiovasc Res 98: , II Ruotsalainen A-K, Lappalainen J, Heiskanen E, Sihvola V, Näpänkangas J, Merentie M, Lottonen-Raikaslehto L, Kansanen E, Ylä-Herttuala S, Jauhiainen M, Pirinen E, Levonen A-L. Nrf2 deficiency impairs atherosclerotic lesion development but promotes features of plaque instability in hypercholesterolemic mice. Cardiovasc Res, 2018 Jun 18. III Ruotsalainen A-K, Sihvola V, Ryytty S, Kärjä V, Jauhiainen M, Pirinen E, Levonen A-L. Nrf2 deficiency alleviates hepatic steatosis via improved hepatic mitochondrial oxidative metabolism in LDL-receptor deficient mice. Manuscript. The publications were adapted with the permission of the copyright owners. Unpublished results are also presented.

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21 XV Contents 1 INTRODUCTION REVIEW OF LITERATURE Transcription factor Nrf The structure of transcription factor Nrf The activation of Nrf Nrf2 in oxidative stress Nrf2 in inflammation Nrf2 in mitochondrial metabolism Nrf2 in hepatic lipid metabolism Atherosclerosis Arterial anatomy Lipoprotein metabolism Early atherogenesis (type I-II lesions) Macrophages in early atherogenesis Foam cell formation and cholesterol trafficking in macrophages Macrophage inflammatory actions Advanced atherosclerosis and plaque maturation (type III-IV lesions) Plaque vulnerability Non-alcoholic fatty liver disease and steatohepatitis Pathogenesis of hepatic steatosis and steatohepatitis The role of Nrf2 in atherogenesis and hepatic steatosis The role of Nrf2 in atherogenesis Systemic metabolic effects of Nrf Nrf2 function in hepatic steatosis Hyperlipidemic mouse models Atherosclerotic plaque development in mice compared to human Mouse models of atherosclerosis Spontaneous plaque rupture in mouse models of atherosclerosis Spontaneous myocardial infarction in mouse models of atherosclerosis Hyperlipidemic mice as models of NAFLD and NASH AIMS OF THE STUDY MATERIALS AND METHODS... 33

22 XVI 5 RESULTS Nrf2 deficiency in bone marrow cells promotes early atherogenesis Global deficiency of Nrf2 protects against early and advanced atherogenesis Global deficiency of Nrf2 predisposes to myocardial infarction Nrf2 deficiency alleviates hepatic steatosis DISCUSSION Nrf2 - a double-edged sword in atherogenesis The role of Nrf2 in spontaneous myocardial infarction and sudden death The role of Nrf2 in regulation of metabolism and hepatic steatosis Nrf2 as a therapeutic target and future perspectives SUMMARY AND CONCLUSION REFERENCES... 55

23 XVII Abbreviations 3-NT 3-nitrotyrosine CCR2 Chemokine (C-C motif) 4-HNE 4-hydroxynoneal receptor 2 AAV Adeno associated virus CD36 Cluster of differentiation 36 ABCA1 ATP binding cassette A1 CETP Cholesterol ester transfer ABCG1 ATP binding cassette G1 protein ACC1 Acetyl-CoA carboxylase 1 ChREBP Carbohydrate-responsive ACAT1 Acetyl-coenzyme A element-binding protein cholesterol acetyltransferase 1 CNC Central nervous system ACAT2 Acetyl-coenzyme A CoA Co-enzyme A acetyltransferase 2 COX2α Cyclo-oxygenase 2α AcLDL Acetylated LDL CPT1α Carnitine ACOX1 Acyl-coenzyme A oxidase 1 palmitoyltransferase 1α ADP Adenosine diphosphate CVD Cardiovascular diseases ApoAI Apolipoprotein AI CXCL16 Chemokine (C-X-C motif) ApoB100 Apoliporotein B100 ligand 16 ApoB48 Apolipoprotein B48 DNA Deoxyribonucleic acid Apobec Apolipoprotein B editing ELISA Enzyme-linked complex immunosorbent assay ApoE Apolipoprotein E ER Endoplasmic reticulum ARE Antioxidant response element ETC Electron transport chain ATF3 Activating transcription FAS Fatty acid synthase factor 3 FAD Flavin adenine dinucleotide ATP Adenosine triphosphate FBN1 Fibrillin-1 ATP5G Adenosine triphosphate FFA Free fatty acid synthase lipid-binding FGF21 Fibroblast growth factor 21 protein 5G FH Familial ATP8B Phospholipid-transporting ATPase 8B hypercholesterolemia

24 XVIII GCLC Glutamate-cysteine ligase LRP Low density lipoprotein catalytic subunit receptor related protein GCLM Glutamate-cysteine LXR Liver X receptor regulatory subunit MARCO Macrophage receptor with GSH Glutathione collagenous structure GST Glutathione S-transferase MCP-1 Monocyte chemoattractant HDL High-density lipoprotein protein 1 HFD High fat-diet M-CSF Macrophage colony HIF1α Hypoxia-inducible factor 1α stimulating factor HL HO-1 Hepatic lipase Hemeoxygenase-1 MDA-LDL Malondialdehyde modified low-density lipoprotein ICAM-1 Intercellular adhesion MmLDL Minimally modified low- molecule-1 density lipoprotein IFN γ Interferon γ MMP Matrix metalloproteinase IHC Immunohistochemistry MTCO1 Cytochrome c oxidase IL Interleukine subunit 1 KEAP1 Kelch-like ECH-associated MTTP Microsomal triglyceride protein 1 transfer protein LAD Ligation of the left anterior NAD Nicotinamide adenine descending coronary artery dinucleotide LCAT Lecithin cholesterol NAFLD Non-alcoholic fatty liver acyltransferase deficient disease LDL Low-density lipoprotein NASH Non-alcoholic steatohepatitis LDLR Low-density liprotein NECH1 Neutral cholesterol ester receptor hydrolase 1 LOX-1 Lectin-like oxidized low- NEFA Non-esterified fatty acids density lipoprotein receptor 1 NEH Nrf2 ECH-homology LPDS Lipoprotein deficient serum NF-κB Nuclear factor κb LPL Lipoprotein lipase NFE2L2 Nuclear factor (erythroid- LPS Lipopolysaccharide derived 2)-like 2

25 XIX NLRP3 Nucleotide-binding domain SMC Smooth muscle cell and Leucine-rich repeat SR-A Scavenger receptor class A Receptor containing a Pyrin SR-B Scavenger receptor class B domain 3 SRBP1c Sterol regulated binding NOS Nitric oxide synthase protein 1c NQO1 NAD(P)H:quinone TCA Tricarboxylic acid oxidoreductase 1 TGF-β Transforming growth factor β NRF2 Nuclear factor-e2-related TLR4 Toll-like receptor 4 factor 2 TNFα Tumor necrosis factor alpha OxLDL oxidized low-density lipoprotein UCP-1 Uncoupling protein 1 USF1 Upstream stimulatory factor 1 OXPHOS Oxidative phosphorylation VCAM-1 Vascular cell adhesion PCSK9 Pro-protein convertase molecule 1 subtilisin/kexin type 9 VDAC-1 Voltage-dependent anion PGC1α Peroxisome proliferator- channel 1 activated receptor gamma VEGF Vascular endothelial growth coactivator 1-alpha factor PPAR Peroxisome proliferator- WB Western blot activated receptor WT Wild-type PPP Pentose phosphate pathway PRDX Peroxiredoxin qpcr Quantitative polymerase chain reaction RNA Ribonucleic acid ROS Reactive oxygen speace RXRα Retinoic X receptor α SDHB Succinate dehydrogenase subunit B SIRT1 Sirtuin 1 SMHC Smooth muscle cell heavy chain

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27 1 Introduction Global expansion of unhealthy lifestyle, such as unhealthy diet and physical inactivity, have increased the incidence of obesity and metabolic disorders that lead to serious pathologies by promoting lipid accumulation, chronic inflammation and oxidative stress in the cardiovascular system and liver. A chronic inflammatory process within the arterial wall leads to the formation of plaques that narrow the arterial lumen and impair blood flow to the target tissue. This process is called atherosclerosis, and it is the most common cause of cardiovascular diseases (CVDs) including coronary artery disease, heart failure and stroke. These diseases have been the leading causes of death worldwide for several decades, despite the progress in treatment practices. Lipid accumulation and mitochondrial dysfunction in the liver lead to a non-alcoholic liver disease (NAFLD) that may further progress to steatohepatitis, cirrhosis and hepatocellular carcinoma (Koenig et al. 2016, Benedict et al. 2017). NAFLD itself is considered to be an independent risk factor for CVDs as it has harmful effects on vascular endothelial function via dyslipidemia and proinflammatory stimulus (Bhatia et al. 2012, Vlachopoulos 2010, Sookoian 2008). The incidence of NAFLD is continuously increasing, and currently 20-30% of individuals worldwide suffer from the disease. One of the main characteristics in the progression of atherosclerosis and NAFLD is oxidative stress, which is an imbalance between the production and disposal of reactive oxygen species (ROS) (Birben et al. 2012). ROS are formed in normal physiology as byproducts of mitochondrial respiration and enzymatic oxidation-reduction reactions, but also by environmental factors, like cigarette smoking. The balance between the formation and disposal of ROS is critical, as excessive amounts of ROS have several harmful effects in cells and organ systems. The transcription factor Nuclear factor E2-related factor 2 (Nrf2), which is the master regulator of several antioxidant, detoxification and anti-inflammatory genes, is activated by oxidative stress, providing cytoprotection (Kensler et al. 2007, Taguchi et al. 2011, Levonen et al. 2007, Jyrkkänen et al. 2008, Chambel et al. 2015). As Nrf2 is perceived to be a multi-organ protector in several pathologies via its anti-inflammatory and antioxidative actions, it has become an interesting target for developing novel therapies against diseases in which oxidative stress and inflammation take part (Lee et al. 2005). The aim of this thesis was to clarify the role of Nrf2 in the pathogenesis of atherosclerosis and hepatic steatosis in mice. Local vascular and systemic effects of Nrf2 on early and advanced atherosclerosis were observed by using mice deficient of Nrf2 in bone marrow derived cells and global deletion of Nrf2 in two different hypercholesterolemic mouse models mimicking human atherosclerosis. Moreover, the role of systemic Nrf2 deficiency in hepatic steatosis was examined in a mouse model of NAFLD. This thesis interrogates the physiological actions of Nrf2 in atherosclerosis and fatty liver disease, and brings out novel targets for the development of Nrf2 mediated therapies and future diagnostics.

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29 3 2 Review of literature 2.1 TRANSCRIPTION FACTOR NRF2 The human body and organ systems are exposed to stress by different environmental and endogenous factors. At the cellular level, different mechanisms have developed to maintain the homeostasis and intercept these stressors, such as fluctuating environmental factors, toxicants, xenobiotics or ROS. These stressors can interrupt systemic homeostasis, causing oxidative stress and cellular protein and lipid damage, as well as mutations and epigenetic perturbation by damaging DNA and chromatin modifying proteins, further leading to the onset of disorders and diseases (Halliwell & Whiteman 2004). A master regulator of stress defense to counteract environmental and endogenous stressors in mammalian cells is the redox-activated transcription factor Nrf2, encoded by Nuclear Factor Erythroid-2-Like 2 (NFE2L2) gene, located in chromosome 2, 34,8 kb in size (Moi et al. 1994). Transcription factors regulate the initiation of transcription and hence the production of RNA molecules destined for protein translation. Nrf2 is expressed in several tissues and it seems to have bidirectional effects as it affords cellular protection, but in cancers these properties become harmful, as uncontrolled accumulation of Nrf2 improves cancer cell survival and the resistance of cytostatic drugs (Kansanen et al. 2013, Leinonen et al 2014). Malfunction of Nrf2 by genetic polymorphism of the NFE2L2 gene, such as varying SNPs and haplotypes, has been connected to different disorders and chronic diseases, such as cancer, autoimmune, neurodegenerative, vascular, respiratory and renal diseases (Hartikainen et al. 2012, Figarska et al. 2014, Kunnas et al. 2016). Somatic mutations of Nrf2 or its repressor protein Kelch-like ECH-associated protein 1 (KEAP1) that lead to persistent Nrf2 activation are linked to different cancers (Shibata et al. 2009, Singh et al. 2006) or may cause an early onset multisystem disorder with severe immunodeficiency and neurological symptoms (Huppke et al. 2017) The structure of transcription factor Nrf2 Nrf2 belongs to the stress activated cap n collar (CNC) basic leucine zipper (bzip) transcription factor family (Moi et al. 1994), which share a typical, conserved structural domain CNC, first discovered in the Drosophila cap and collar (CNC) gene (Mohler et al. 1991, Mohler et al. 1995). The family of CNC transcription factors includes p45-nfe2, Nrf1, Nrf3 and small Maf proteins (Itoh et al. 1997, Katsuoka et al. 2016) all of which have different developmental and homeostatic functions. Nrf2 protein is a heterodimer composed of a p45 protein and a small Maf protein. Nrf2 consists of 589 amino acids and seven Neh (Nrf2-ECH homology) domains (Neh1-Neh6), from these Neh1 contains a DNA binding site, termed CNC basic region, and a leucine zipper structure for binding and heterodimerization site of small Maf proteins (Figure 1). Nrf2 inhibitor protein KEAP1, which consists of 624 amino acid residues and five domains, binds to and DLG motifs in Neh2 domain. Three other domains Neh3-Neh5 are transactivation domains (Itoh et al. 1997, Padmanabhan et al. 2006) and Neh6 mediates the degradation of Nrf2 in the cell nucleus (McMahon et al. 2004). The recently characterized Neh7 domain interacts with the DNA-binding domain of retinoic X receptor α repressing transcription of Nrf2 target genes (Wang et al. 2013).

30 4 Figure 1. The structure of transcription factor Nrf2 that consists of 589 amino acids and has seven domains, Neh1-6. Neh1 contains the DNA binding site, CNC basic region and a leucine zipper (L-Zip) structure, which mediates dimerization with small Maf proteins. Nrf2 inhibitor protein Keap 1 binds to ETGE and DLG motifs in Neh2 domain. Domains Neh3-Neh5 are transactivation domains and Neh6 mediates the degradation of Nrf2 in the cell nucleus. Neh 7 represses the transcription of Nrf2 target genes by interacting with the DNA-binding domain of retinoic X receptor α (RXRα). Style adapted from the original figure of Emilia Kansanen The activation of Nrf2 In basal conditions, only a small amount of Nrf2 protein is present in the cytoplasm and it is rapidly ubiquitylated and proteasomaly degraded by its interaction with two KEAP1- proteins, which are adaptor proteins in the CUL3-based E3 ligase system (Figure 2) (Kansanen et al. 2013). The inhibition of Nrf2 by KEAP1-CUL3 complex is not complete, as a small proportion of Nrf2 protein is translocated into the nucleus to maintain basal transcription. The transcription of Nrf2 gene, NFE2L2, seems to be autoregulated by its own expression through an antioxidant response element-like (ARE-like) element (Kwak et al. 2002). Ubiquitously expressed Nrf2 protein is activated by different exogenous factors, such as different xenobiotics, phytochemicals, heavy metals, ionizing radiation and cigarette smoke. In addition, Nrf2 is modulated by varying endogenous stressors, like reactive oxygen and nitrogen species, electrophiles, lipid peroxidation products and vascular shear stress (Kansanen et al. 2013, Levonen et al 2014, Kensler et al. 2007). Nrf2 activity is regulated via Nrf2 protein stability, post-translational modifications and other binding partners of KEAP1. Nevertheless, the precise mechanism(s) of Nrf2 activation is currently unknown, but there are two prevailing models, the hinge-and-latch model and the KEAP1-CUL3 dissociation model that demand its activation mechanism. In the presence of activators, the interaction of Nrf2 with the KEAP1- ubiquitin ligase complex is disrupted, resulting in stabilization of de novo synthesized Nrf2 and its subsequent translocation to the nucleus. Other alternative models have also been proposed.

31 5 Figure 2. The activation of Nrf2-KEAP1 pathway. Nrf2 is activated by different exogenous or endogenous inducers. In the presence of activators, the interaction of Nrf2 with the KEAP1-complex is disrupted, resulting in a stabilization of de novo synthesized Nrf2 and its subsequent translocation to the nucleus. In nucleus, Nrf2 form heterodimers with small MAF proteins, further binding to the antioxidant response element (ARE) to activate the transcription of its target genes. Style adapted from the original figure of Emilia Kansanen Nrf2 in oxidative stress ROS are produced from molecular oxygen by normal metabolic reactions or environmental factors in living organisms. ROS can be free radicals or nonradicals, but they avidly react with cellular macromolecules to instigate oxidative modifications. ROS are essential in normal physiological processes and can act as signaling molecules. However, the balance between the formation and disposal of ROS is critical as excessive ROS cause oxidative stress, which has harmful effects on lipids, proteins and DNA, consequently promoting the progression of several diseases including atherosclerosis, non-alcoholic steatohepatitis (NASH), diabetes and neurodegenerative diseases (Valko et al. 2006, Wu et al. 2014, Takaki et al. 2013, Birben et al. 2012) When Nrf2 is activated by oxidative stress, it translocates into the nucleus and binds to the cis-acting sequence termed the Antioxidant Responsive Element (ARE) with the small Maf proteins, directly influencing ARE-mediated gene expression of antioxidant enzymes (Itoh et al. 1997) (Figure 2). Nrf2 target gene NAD(P)H:quinone oxidoreductase1 (NQO1) is a master regulator of ROS defense, as it reduces superoxide levels and regulates protein proteasomal degradation (Jaiswal et al. 1996, Ross et al. 2018). NADPH is used as an electron donor, when oxidized glutathione is reduced by glutathione reductase. Modulation of glutathione metabolism is one example on, how Nrf2 mediates protective

32 6 actions in cells. Nrf2 regulates NADPH and glutathione levels by regulating enzymes like glutathione S-transferase (GST), glutamate-cysteine ligase modifier subunit (GCLM) and glutamate-cysteine ligase catalytic subunit (GCLC) (Bea et al. 2003, Sherman et al 2001, Glickman et al 2002). Nrf2 antioxidative properties also stem from regulation of the thioredoxin system (Thimmulappa et al. 2002, Itoh et al. 1997, Hayes et al 2000) Nrf2 in inflammation Inflammation is a biological response to cell injury or different pathogens, aiming to eliminate detrimental factors and to initiate the tissue repair process. Inflammation is classified into the acute, subacute and chronic inflammation on the basis of duration and the dominating cell type characteristic for the phase of the inflammatory process. Chronic inflammation is typical for several disease pathologies, in which injured cells produce cytokines to recruit lymphocytes and plasma cells into the injured area to initiate healing, with additional contribution of macrophages and fibroblasts in forming granulation tissue (scar formation). This healing reaction can be seen in wound healing, but also in several chronic diseases where cellular injury is involved, like in atherosclerosis and steatohepatitis (Glass & Witztum 2001). Oxidative stress and inflammation forms a vicious cycle, as ROS increase the expression of pro-inflammatory mediators, but on the other hand, inflammation itself promotes oxidative stress. Nrf2 controls and inhibits inflammation via antioxidant defense, but it also directly suppresses inflammation via transcriptional regulation of pro-inflammatory cytokines, such as interleukins 6 (IL-6) and 1β (IL-1β) (Kobayashi et al 2016). Nrf2 inhibits the migration and proliferation of inflammatory cells, as the activation of Nrf2 prevents lipopolysaccharide (LPS) -induced transcription of certain cytokines and chemokines, like monocyte chemoattractant protein-1 (MCP-1), and cell adhesion molecules by downregulating the activation of classical nuclear factor κb (Nf-κB) inflammatory pathway (Kobayashi et al. 2016, Levonen et al. 2007, Kuhn et al. 2011). Mouse studies support the notion that the loss of Nrf2 elevates pro-inflammatory cytokine expression in vivo (Thimmulappa et al. 2006). However, the anti-inflammatory role of Nrf2 is ambiguous, as it has been reported to enhance Nucleotide-binding domain and Leucine-rich repeat Receptor containing a Pyrin domain 3 (NLRP3) inflammasome activation, which is a multiprotein complex that recognizes microbial and oxidative stress signals and mediates secretion of IL- 1β and other pro-inflammatory cytokines (Freigang et al. 2011, Zhao et al 2014) Nrf2 in mitochondrial metabolism Mitochondria are essential cellular machineries for the production of ATP to fuel metabolism. Mitochondria synthesize ATP from glucose, amino acids and free fatty acids (FFAs) during cellular respiration in the presence of oxygen. Glucose and FFAs are the main source for energy production, as amino acids are secondary and used only if other sources are not available. Glucose breakdown by glycolysis produces pyruvate and the energy released in this process is used to produce two ATP molecules. FFAs are oxidized in β-oxidation by mitochondria, providing acetyl CoA for fueling the tricarboxylic acid (TCA) cycle. Pyruvate is converted to acetyl CoA in mitochondrial matrix and further oxidized in the TCA cycle. Excess amounts of pyruvate, which are not used in the TCA cycle, are then converted to lactate and secreted from cells. In the TCA cycle, acetyl CoA is oxidized to carbon dioxide, as well as reducing nicotinamide adenine dinucleotide (NAD) and flavin

33 7 adenine dinucleotide (FAD) to NADH and FADH2, respectively, further feeding protons to the electron transport chain (ETC). The majority of ATP molecules are synthesized via oxidative phosphorylation in the ETC in the inner mitochondrial membrane. ETC is formed by four separate transmembrane protein complexes, each of them constructed from several subunits that are encoded by nuclear- and mitochondrial DNA. In the ETC, electrons are released from NADH and FADH2 in the inner mitochondrial membrane via complex I and II, transferring electrons for the electron carriers, ubiquinone and cytochrome c (complex III). Finally, electrons are released to oxygen, which is reduced to H2O via cytochrome c oxidase (complex IV) (Gao et al. 2009). Electron transport releases energy, which is used for pumping the protons back from the mitochondrial matrix to the inner mitochondrial membranes, thus allowing ATP synthase (complex V) to phosphorylate ADP to ATP. Oxidative phosphorylation is a side of ROS production in healthy cells, as mitochondrial respiratory chain produces superoxide anion (O2 - ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH. ), as byproducts of the aerobic metabolism process (Schieber & Chandel 2014). Molecular oxygen can undergo an one-electron reduction to generate superoxide anion in the ETC by complex I and III (Holmström & Finkel 2014). As Nrf2 is a regulator of multiply antioxidant and cytoprotective genes, it is been proposed to indirectly govern mitochondrial antioxidant defense, and thereby to regulate mitochondrial capacity. In addition, in mice Nrf2 regulates respiration efficiency as loss of Nrf2 increases oxidative stress induced uncoupling via uncoupling protein 1 (UCP-1) (Schneider et al. 2016). UCP-1 increases the permeability of the inner mitochondrial membrane, which leads to the decreased proton gradient in oxidative phosphorylation and generate heat instead of ATP production (Schneider et al. 2016). Mitochondrial function of Nrf2 is investigated more in the context of neurobiology rather than in metabolic diseases. Nrf2 expression is known to be declined in diseases with mitochondrial dysfunction, such as Parkinson s disease and Friedrich s ataxia (Matigian et al. 2010, Lo et al. 2009), whereas Nrf2 activation is suggested to support mitochondrial function by protecting against oxidative stress (Kovac et al. 2015, Holmström et al. 2016, Itoh et al. 2015). To conclude, varying effects of Nrf2 on energy metabolism and mitochondrial function have been reported, but the exact mechanism still remains unknown and requires further studies Nrf2 in hepatic lipid metabolism The major classes of lipids that are essential for normal physiological processes and for building cellular structures, are fatty acids, glycerolipids (triglycerides), glycerophospholipids (phospholipids) and sterols (cholesterol). Lipids have two main pathways for their generation and transport, exogenous and endogenous that act on lipids that are either derived from the diet or produced in the liver, respectively. In the exogenous pathway, dietary lipids are absorbed from the small intestine. In enterocytes, lipids form chylomicrons that are large lipid particles consisting of triglycerides, phospholipids and cholesterol, as well as apolipoprotein B48 (ApoB48). In the endogenous pathway, fatty acids, cholesterol, triglycerides and phospholipids can be synthesized from acetyl-coa in de novo lipogenesis mainly in the liver and in small quantities in other cells. Lipogenesis is tightly regulated by transcription factors, including upstream stimulatory factor 1 (USF1), sterol regulatory element binding protein 1c (SREBP-1c), liver X receptor (LXR), fatty acid synthase (FAS), peroxisome proliferator-activated receptors (PPARs) and carbohydrateresponsive element-binding protein (ChREBP) (Wang et al. 2015). Some papers have reported that loss of Nrf2 reduces lipogenesis by downregulating transcription of lipogenic genes, like SREBP1C, FAS and PPARγ (Huang et al. 2010). This corresponded to reduced

34 8 liver lipid content in obese Lep ob/ob mouse model (Xu et al. 2015, Huang et al. 2010, Barajas et al. 2011). In contrast to these reports, the mouse liver microarray analysis demonstrated that Nrf2 deficiency increased the expression of several fatty acid synthesis regulating genes, elevating plasma fatty acid levels (Kitteringham et al. 2010, Chartoumpekis et al. 2013, Tanaka et al. 2008, Tanaka et al. 2012). Interestingly, Nrf2 activation by deletion of KEAP1 or triterpenoids reduce plasma and liver lipid content and downregulate the expression of fatty acid synthesis genes, as well as inhibit white adipose tissue expansion in a mouse model of obesity (Zhang et al. 2013, Xu et al. 2012, Furusawa et al. 2014, Shin et al. 2009). However, further studies are needed in tissue specific mouse models to clarify the metabolic effects of Nrf ATHEROSCLEROSIS Atherosclerosis is a progressive chronic inflammatory disease leading to the accumulation of cholesterol containing plasma lipoproteins in the vascular wall of medium and large size arteries, causing vascular wall thickening, stiffening and obstruction of the lumen (Glass & Witztum 2001). The most notable risk factors for atherosclerosis are high blood low density lipoprotein (LDL) and low high density lipoprotein (HDL) content, hypertension, smoking, obesity and diabetes (Marais 2004, Rader et al. 2003). Atherosclerosis is the most common cause of CVDs, such as coronary artery disease, heart failure and stroke that cause millions of deaths every year worldwide (Naghavi et al. 2003, Bentzon et al. 2014). As a consequence of an increasing prevalence of obesity, unhealthy diet (rich in saturated fat and cholesterol), physical inactivity and increased average life span, the number of deaths are predicted to increase in the following decades. Current treatments of CVDs consist of the treatment of underlying metabolic disorders, treatment for hypertension and lowering body weight. In addition, invasive operations, such as coronary angioplasty, coronary bypass grafting and stenting can be used for the treatment of severe coronary artery disease. The incidence of chronic heart failure has increased during recent years as a result of chronic coronary disease, although the treatment of acute myocardial infarction has improved (Cassar et al. 2009). Atherosclerosis takes decades to develop, initiating the development of early lesions even in early childhood. Atherosclerotic lesions are classified to five different categories as follows: I, initial lesion/intimal thickening; II, fatty streak; III. intermediate lesion; IV, atheroma; V fibroatheroma; and VI, advanced lesion (Figure 3) (Virmani et al. 2000, Koenig & Khuseyinova 2005). Type I-III plaques are relatively small in size and clinically silent, and do not cause complications (Virmani et al. 2000). The complexity of plaque morphology is characteristic for type IV (Libby et al. 2002, Bentzon et al. 2014) and V plaques (Hansson & Hermansson 2011, Libby 2012). Complications are the result of type VI lesions, which are highly vulnerable and have plaque rupture, intraplaque hemorrhage, hematoma or thrombosis. The growth of the plaque cause clinical manifestations when the arterial lumen is narrowed enough to limit blood flow that leads to ischemia in target tissue, or by provoking thrombi formation that can interrupt blood flow locally or cause distal embolization of the artery. Advanced plaques possess features such as a thin and collagenpoor fibrous cap with a few vascular smooth muscle cells (SMCs) but a substantial number of macrophages. These features make the plaque prone to rupture, the most common complication of atherosclerosis, accounting for ~70% of fatal acute myocardial infarctions, sudden coronary deaths and strokes (Naghavi et al. 2003). Thus, the most risky plaques do not occur at the site of the most severe arterial narrowing but at the region of fibrous cap

35 9 and plaque shoulder areas after physical disruption or erosion, when the inner procoagulant material of the plaque is exposed to thrombotic factors in the bloodstream, triggering thrombosis (Virmani et al. 2000). Figure 3. Classification of atherosclerotic plaques to six different categories. Modified from (Koenig & Khuseyinova 2005) Arterial anatomy Large and medium sized arteries are anatomically composed of three layers (Figure 4) (Stary et al. 1992). The outermost layer is termed tunica adventitia, which consists of type I collagen and elastic fibers. The external elastic lamina separates adventitia from the tunica media, a thick muscle layer, where smooth muscle cells are helically arranged with several layers of elastic laminaes between them. The internal elastic lamina separates the tunica media from the subendothelial space, forming fenestraes that allow the diffusion of substances to nourish arterial cells. The innermost layer is called tunica intima, composed of a monolayer of endothelial cells and a basement membrane, in which atherosclerosis initiates causing intimal thickening.

36 10 Figure 4. The cross-section of mouse aorta to demonstrate the arterial anatomy. Medium and large size arteries are formed from three layers, as follows: the outermost layer is the adventitia, containing mainly connective tissue (blue), underneath it is the media, which is formed of smooth muscle cells (SMCs; pink), and the innermost layer is the intima (subendothelial space), which is under the endothelium. The space inside the tubular structure of vessels, is the lumen Lipoprotein metabolism Cholesterol is transported in lipoprotein particles that enable cholesterol to move within the aqueous environment. Lipoproteins are divided into five groups from the biggest to the smallest in size and by lipoprotein density: chylomicrons, VLDL, intermediate density lipoprotein (IDL), LDL and HDL (Hegele 2009). Lipoproteins have a central core of neutral lipids, such as triglycerides and cholesteryl esters that are surrounded by phospholipids, free cholesterol and different apolipoproteins. Chylomicrons circulate in lymphatic vessels, throught the liver circulation and end up in the bloodstream, where the particles become more mature as those receive apolipoproteins C and E from HDL. Chylomicrons are hydrolyzed to chylomicron remnants by lipoprotein lipase (LPL), releasing glycerol and fatty acids, which are used as an energy source in pheripheral tissues (Nakajima et al. 2011). The liver secretes surplus endogenous and exogenous lipids via VLDL particles into the circulation. VLDL particles in blood stream can either be taken up back into the liver or are hydrolyzed by LPL to IDL and further to LDL, which transports cholesterol to all tissues including arterial wall. VLDL particles further release cholesteryl esters and triglycerides to HDL particles by cholesterol ester transfer protein (CETP), and VLDL remnants become IDL particles. IDL particles are converted to cholesteryl ester rich LDL particles by LPL, hepatic lipase (HL) and CETP. HDLs are the smallest lipoprotein particles, which delivers cholesterol from tissues into the liver, thereby having atheroprotective properties Early atherogenesis (type I-II lesions) Atherosclerotic lesions develop in the arterial tree at branches, bifurcations and curvatures, where the mechanical stress is enhanced by turbulent blood flow and low shear stress. These hemodynamic stresses increase endothelial permeability and adaptive intimal thickening (eccentric thickening) as the vasculature attempts to maintain normal conditions

37 11 in response to mechanical stress (Stary et al. 1992, Bentzon et al. 2014). Endothelial dysfunction, the first step in the initiation of the atherogenic process, can be caused by increased oxidative stress, excessive amount of LDL cholesterol, hypertension and/or infectious agents (Gimbrone & García-cardeña 2016, Falk 2006). The dysfunctional endothelium secretes pro-inflammatory cytokines, chemotactic molecules and adhesion molecules that capture leukocytes on the endothelial cell surface and promotes migration into subendothelial space. The dysfunctional endothelium allows excessive LDL to penetrate the endothelial layer, then to adhere to intimal negatively charged proteoglycans via apolipoprotein B100 (ApoB100) and finally accumulate into the intimal layer (Libby et al 2011, Borén et al. 1998, Öörni et al. 1997, Camejo et al. 1992). In the intima, LDL undergo oxidative modification, as the unsaturated fatty acids of LDL are decomposed by lipoxygenases and myeloperoxidases, producing ROS in addition to malondialdehyde and 4-hydroxynonenal as lipid peroxidation byproducts (Taniyama & Griendling 2003, Ylä- Herttuala et al. 1989, Palinski et al. 1989). Modified LDL can provoke cytokine secretion from monocytes and endothelial cells, increases the expression of chemotactic proteins such MCP-1 and its receptor chemokine (C-C motif) receptor 2 (CCR2), as well adhesion molecules such as ICAM-1, VCAM-1, P- and E-selectin, thus facilitating leukocyte aggregation in the vascular wall (Libby et al. 2002). Circulating monocytes migrate through the endothelium and differentiate into mononuclear phagocytic macrophages in intimal layer (Libby et al. 2002). Intimal macrophages engulf modified LDL particles in cytoplasmic lipid droplets and transform into foam cells that accumulate in arterial intima forming fatty streaks (Libby et al. 2002, Glass & Witztum 2001, Bentzon et al. 2014, Virmani et al. 2000). As macrophages become apoptotic, they are removed via efferocytosis, an eating mechanism of apoptotic cells by phagocytes like lesional macrophages, which decreases the lesional cellularity and can lead to the resolution of the lesion in the early phase of the atherogenesis (Seimon & Tabas 2009, Tabas et al. 2010) Macrophages in early atherogenesis Macrophages differentiate from monocytes that originate from the bone marrow-derived common myeloid progenitor cells or are also produced in the spleen in a process called splenic extramedullary myelopoiesis. Macrophages are crucial for the pathogenesis of atherosclerosis, as macrophages gorge themselves on lipids, like LDL, in the vascular wall initiating the intimal thickening (Figure 5). The role of monocyte-derived cells, such as macrophages and dendritic cells in atherogenesis has been extensively studied both in humans and in murine models. (Tabas & Bornfeldt 2016). Macrophages maintain different phenotypes supporting the pro-inflammatory process (M1) or having anti-inflammatory functions (M2) (Figure 6) (Tabas & Bornfeldt 2016, Adamson & Leitinger 2011). Macrophages are very plastic as they express several pro-inflammatory cytokines in response to modified LDL particles and other inflammatory stimuli (e.g. LPS), but macrophages can also resolve inflammation by producing anti-inflammatory cytokines (Yang et al. 2014) Foam cell formation and cholesterol trafficking in macrophages Cholesterol can be transported to and from macrophages by several mechanisms (Figure 5) (Moore et al. 2013). Native LDL is taken up by micropinocytosis via LDL receptors that are downregulated in the early phase of foam cell formation by elevated intracellular

38 12 cholesterol. If LDL particles are modified by free radicals or enzymes, such as 12/15- lipoxygenase and myeloperoxidase, these are then recognized by pattern recognition receptors (PRRs) expressed in macrophages and in other cells of the innate immune system. (Plainski et al 1990, Takeuchi & Akira 2010). Macrophages express several PRRs, like scavenger receptor class A type 1 (SR-A1), macrophage receptor with collagenous structure (MARCO or SR-A2), CD36, scavenger receptor class B type 1 (SR-B1), lectin-like oxidized LDL receptor 1 (LOX-1) and CXCL16 that are scavenger receptors for phosphatidylserine and oxidized LDL (Kunjathoor et al. 2002). SR-A1 and CD36 mediate as much as 75-90% of modified LDL uptake in in vitro conditions. However, the deletion of SR-A1 and CD36 in mouse macrophages has no effect on atherosclerotic lesion development, leading to the conclusion that macrophages have other compensatory mechanisms in vivo for modified LDL uptake. When lipoproteins are internalized via endocytosis, cholesterol esters are hydrolyzed to free cholesterol and fatty acids (Moore et al. 2013). Cholesterol esters are digested to free cholesterol and fatty acids in lysosomes, transferred to the endoplasmic reticulum (ER) and then re-esterified by acetyl-coenzyme A:cholesterol acetyltransferase 1 (ACAT1) to cholesteryl fatty acid esters. These then form lipid droplets and provide the foamy phenotype for macrophages. Esterified cholesterol containing lipid droplets are transferred to the plasma membrane either after lysosomal degradation or from the ER, in which this can occur either via lipolysis by neutral cholesterol ester hydrolase 1 (NCEH1) or via lipophagy, which in turn delivers lipid droplets back to lysosomes (Chistiakov et al. 2016). Intracellular cholesterol induces Toll-like receptor (TLR) signaling, NF-κB pathway and NLRP3 inflammasome activation in macrophages (Curtiss & Boisvert 2000, Sheedy et al. 2013). Extracellular cholesterol can precipitate as microcrystals, which can be taken up by macrophages and activate an inflammasome by cleaving a proform of IL-1β and converting it into a bioactive IL-1β that can be secreted by macrophages (Duewell et al. 2010, Latz et al. 2013). IL-1β induces the production of several other pro-inflammatory molecules, such as leukocyte adhesion molecules, IL-6, prostaglandin E2 and matrixdegrading metalloproteinases. In addition, intracellular cholesterol regulates the expression of sterol regulated transcription factors liver X receptors α and β activated ATP binding cassette (ABC) transporters ABCA1 and ABCG1 that mediate reverse cholesterol transport in macrophages. Free cholesterol is transported to lipid-poor Apolipoprotein A1 (ApoAI) via ABCA1 or to mature HDL particles via ABCG1 (Moore et al. 2003).

39 13 Figure 5. Cholesterol trafficking in foam cell macrophages. Native and modified or aggregated LDL are transported to macrophages via different receptors, micropinocytosis and phagocytosis. Intracellular cholesterol is digested in lysosomes and the free cholesterol is transported for HDL particles via reverse cholesterol receptors. In addition, intracellular cholesterol may induce the expression of inflammatory and apoptotic pathways. Style adapted from the original figure of Moore et al Macrophage inflammatory actions Several pro- and anti-inflammatory stimuli regulate macrophage differentiation and actions in different stages of atherogenesis. Classically, inflammatory responses activate macrophages, but macrophage differentiation and the number of circulating monocytes is regulated also by cellular cholesterol content, as impaired reverse cholesterol trafficking in mouse macrophages increases the number of circulating monocytes and macrophages (Yvan-charvet et al. 2010). In contrast, enhanced cholesterol efflux and elevated plasma HDL content decrease plasma monocyte number, and reduce the risk of atherosclerosis in the human population (Deo et al. 2004). There are several macrophage populations, but M1 and M2 are two major groups, and they have different features in normal physiology and disease pathogenesis (Figure 6) (Bisgaard et al. 2016). Interferon γ (IFNy), microbial products, such as LPS, cholesterol crystals, ROS and oxidized LDL, trigger a Th1 inflammatory response directing macrophage differentiation to CD14 + CD16 subset in human, corresponding LY6C hi macrophages in mice that are thought to be the precursors of pro-atherogenuc M1 macrophages. M1 macrophages secrete high levels of proinflammatory cytokines, such as IL-6, interleukin 12 (IL-12), inducible NOS (inos) and TNFα and CCR2, which is the main receptor regulating monocyte migration into the plaque (Lawrence & Natoli 2011). Anti-inflammatory cytokines interleukine 4 (IL-4) and IL- 10 guide macrophages to differentiate into CD14 - CD16 + in human, corresponding LY6C low

40 14 monocytes in mice that promote a Th2 response and are proposed to be the precursors of anti-atherogenic M2 macrophages. It has been proposed that oxidized phospholipids and nitrosylated fatty acids, for example in atherosclerotic plaques, are capable to induce Moxphenotype in murine macrophages that characteristically express high levels of ROS, HO-1 and Nrf2 (Kadl et al. 2011). Figure 6. Macrophage polarization in response to different cytokines, lipids and inflammatory mediators. M1, M2 and Mox are three major groups of macrophage populations, and they have different features in normal physiology and disease pathogenesis Advanced atherosclerosis and plaque maturation (type III-IV lesions) Many fatty streaks do not develop further, but plaque progression continuously disrupt the normal intimal structures at predilection sites. Excessive accumulation of foam cells and proliferation of macrophages increase plaque cellular apoptosis, which promotes the formation of lipid pools and necrotic cores inside the plaque (Figure 7). As apoptosis is highly accelerated, efferocytosis is not efficient enough to clean apoptotic cells, leading to secondary necrosis and release of cellular lipids that form the plaque necrotic core (Tabas 2005). These processes provoke chronic inflammation locally in the plaque and the development of necrotic areas (Liao et al. 2012, Marsch et al. 2014). At this stage, a proatherogenic environment stimulates smooth muscle cell phenotype switching, proliferation and migration into the plaque. SMCs promote synthesis and secretion of extracellular matrix proteins, such as elastin, collagen and proteoglycans and form a fibrous cap to cover the atheroma plaque (Clarke & Bennett 2006). However, local inflammatory stimulus within plaques, e.g. secretion of TNF, induces macrophages to secrete different matrix metalloproteinases such as MMP-1, -2, -3, -7, -8, -9 and -13 that destabilize plaque by aggravating fibrous cap thinning, hence predisposing to plaque rupture (Sukhova et al. 1999, Sluijter et al. 2006, Newby 2007). In addition, apoptosis of smooth muscle cells causes plaque fibrous cap thinning and allows the expansion of the necrotic core due to reduced synthesis of extracellular matrix in response to a pro-inflammatory environment. Smooth muscle cell apoptosis itself promotes plaque inflammation and monocyte infiltration by increasing IL-1, IL-6 and MCP-1 expression, further regulating plaque calcification and negative remodeling (vessel shrinkage) (Clarke & Bennett 2006, Clarke et al 2008). Apoptotic cells, extracellular matrix and necrotic core material can act as a nidus for calcification in the plaque (Doherty et al. 2003, Shioi & Ikari 2018). Calcium granules are microscopic, and can spread as scattered or subsequently expand to large calcium deposits.

41 15 For instance, necrotic core can be completely calcified and form large fibrocalcific nodules with time. In addition, osseous metaplasia (chondroid metaplasia in murine arteries) can occur in very heavily calcified arteries. However, calcification can also take place without necrotic areas, but the mechanism of this is not fully understood. Figure 7. Development of an atherosclerotic plaque. Endothelial dysfunction and turbulent blood flow initiate the development of atherosclerotic plaque by increasing endothelial permeability and subsequently allowing substantial penetration of circulating monocytes and LDL particles into the endothelium and eventually into the intima. LDL particles are oxidized (oxldl) in the intima by ROS. Intimal macrophages engulf modified LDL macrophages and transform into foam cells, subsequently promoting the secretion of pro-inflammatory cytokines, chemokines and adhesion molecules that induce the migration of circulating monocytes into the intima. Accumulation of foam cells forms fatty streaks in the arterial wall. The pro-inflammatory stimuli and the accumulation of macrophages induce the migration of smooth muscle cells from the medial layer to the intima. Smooth muscle cells secrete extracellular matrix proteins that form a fibrous cap on the top of the plaque. As the plaque grows in size, the persistent inflammation and hypoxia increase the apoptosis of smooth muscle cells and macrophages in advanced plaques that cause the formation of a lipid pool and a necrotic core. When the plaque is expanding, the fibrous cap becomes thinner, thus the plaque becomes prone to erosion and rupture that reveal the inside of the plaque to blood, which predispose to thrombus formation Plaque vulnerability The mortality and morbidity from atherosclerosis is mainly due to extreme luminal narrowing or the thrombi, which obstruct the blood flow to the target tissue causing ischemia, infarction or stroke (Bentzon et al. 2014, Naghavi et al. 2003, Ylä-Herttuala et al. 2013). Unstabile plaques are characterized by extremely thin fibrous cap (<65 µm), neovascularization, intraplaque hemorrhages, expansive vessel remodeling, enhanced accumulation of inflammatory cells and elevated amounts of proteolytic enzymes (Ylä- Herttuala et al. 2011, Bentzon et al. 2014). Moreover, vessel wall shear stress can induce plaque rupture, especially in macrophage-rich shoulder areas. As plaque ruptures, it

42 16 exposes a highly thrombogenic lipid-rich and necrotic plaque core to blood erythrocytes, platelets and fibrin resulting in thrombosis. Different mechanisms that promote rupture of vulnerable plaque have been recognized. Inflammatory stimulus is one of the major enhancers of plaque destabilization as it has both structural and functional effects in the plaque (Hansson et al 2015). First, inflammation activates macrophages, mast cells and T cells to release cytokines that have effect on plaque structure by inhibiting fibrotic cap formation, decreasing collagen content and increasing the amount of proteases that digest matrix proteins (Libby et al. 2012, Libby et al. 2002). IFNγ, secreted by Th1-type T cells and natural killer cells, has multiple effects as it inhibits smooth muscle cell differentiation, pro-collagen I gene expression and collagen crosslinking enzyme (Ranjbaran et al. 2007). However, anti-fibrotic effects are counterbalanced by regulatory T cells (Treg) and Th17 cells (Taleb et al. 2014). Treg cells secrete fibrogenic transforming growth factor β (TGF-β), which inhibits macrophage activity and plaque inflammation. Th17 cells are involved in wound healing and have powerful fibrogenic properties by promoting formation of collagen fibers stimulated by IL-17A to strengthen the plaque against hemodynamic forces. Nevertheless, thrombi without plaque rupture, due to plaque erosion (absence of endothelium) is also common in pathological intimal thickening and fibroatheromas (Bentzon et al. 2014). Moreover, endothelial denudation or dysfunction can promote procoagulative properties of endothelium. In addition, expansion of calcified nodules through the fibrous cap have been proposed as a separate mechanism of thrombosis. However, immune signals also promote atherothrombosis by increasing the platelet aggregation and clotting via the expression of CD40 ligands, thromboxane A2, and formation of prostaglandin-i2 and E2 (Davi & Patrono 2007). Intraplaque neovascularization, which is sprouting angiogenesis from vasa vasorum of large and medium size arteries, predisposes to intraplaque hemorrhage, increasing the risk of plaque rupture and thrombus formation, and thereby it is a novel target for plaque stabilizing therapies (Parma et al. 2017, Virmani et al. 2000, Virmani et al. 2005). Intraplaque neovessels grow into the base of advanced and mature atherosclerotic plaques in necrotic and calcified regions via cellular malnutrition and hypoxia, which stimulate angiogenesis by stabilization of hypoxia-inducible factors (HIFs), and expression of vascular endothelial growth factors (VEGFs), angiopoietin-2 and other different growth factors, such as platelet derived growth factor and fibroblast growth factors (Vink et al. 2007, Carmeliet & Jain 2011, Fong 2008). Neovessels in vulnerable plaques are immature and fragile, as they lack supporting cells, and thereby very leaky, allowing extravasation of plasma proteins and erythrocytes into the plaque, hence promoting plaque inflammation and plaque necrosis (Virmani et al. 2005, Kolodgie et al. 2007, Rademakers et al 2013). 2.3 NON-ALCOHOLIC FATTY LIVER DISEASE AND STEATOHEPATITIS NAFLD is the most distinctive cause of chronic liver disease in Western countries and it is one of the key risk factors for CVDs such as atherosclerosis (Sookoian & Pirola 2008, Misra et al. 2009). It associates closely with unhealthy lifestyle and metabolic syndrome, but is also influenced by some genetic factors. About 20-30% of people have fatty liver and it is most commonly diagnosed in obese middle-aged people, but it affects also some young adults and children as well as non-obese individuals % of the people suffering from NAFLD will develop NASH, which can further progress to cirrhosis, hepatocellular carcinoma and death. NASH is the third most common cause for liver transplantation in

43 17 individuals over 65 years old and the number is expected to increase due to increased prevalence of metabolic diseases and average life span (Zezos & Renner 2014). Most people suffering from NAFLD are asymptomatic, thereby impeding the diagnostics. However, diagnosis of NASH is even more challenging, because the liver inflammation and fibrosis can be diagnosed only from liver biopsy (Vuppalanchi & Chalasani 2009). There is currently no efficient pharmacological treatment for NAFLD and NASH, hence the patients are encouraged to lose weight, increase physical activity and follow healthy eating habits Pathogenesis of hepatic steatosis and steatohepatitis Hepatic triglycerides are synthesized from FFAs, which can be derived from white adipose tissue lipolysis that release non-esterified fatty acids (NEFA) into the plasma (60%), from liver de novo lipogenesis (25%) or from dietary fatty acids (15%) (Donnelly et al. 2005). In obese individuals, the storage capacity of adipose tissue is exceeded, which promotes lipolysis and the release of fatty acids into the plasma (Lambert et al. 2014). Furthermore, insulin resistance, common in obese and NAFLD patients, drives the influx of fatty acids into the liver and enhanced liver lipogenesis. In simple steatosis, which is the first phase in the progression of NAFLD, excessive triglycerides accumulate into hepatocytes and form intracellular lipid droplets due to an imbalance in recycling the FFAs and triglycerides in the liver. By definition, NAFLD exists when more than 5% of hepatocytes contain lipid droplets (Brunt & Tiniakos 2010, Kleiner et al. 2005). Lipid droplet formation in hepatocytes can be seen as a self-protecting mechanism against lipotoxicity, as excessive amount of fatty acids, diacylglycerides, phospholipids and free cholesterol induce oxidative stress and stimulate the disease progression (Feldstein et al. 2004). To protect cells from lipid accumulation, free cholesterol is stored in lipid droplets in the liver macrophages and the liver packs triglycerides into VLDL particles via microsomal triglyceride transfer protein (MTTP) and secrete those from the liver, but this elevates plasma triglyceride level aggravating atherosclerosis (Cases et al. 2001). In addition, mitochondria oxidize liver fatty acids, therefore effective mitochondrial oxidation capacity has a critical role in the limitation of liver lipid accumulation (Nassir & Ibdah 2014). Simple steatosis alone is often a benign process, but it is detrimental for some individuals. Susceptible individuals develop NASH, which requires multiple hits (multiple hit theory) such as increased oxidative stress, mitochondrial dysfunction, imbalanced adipocytokines, dysregulated apoptosis and phagocytosis or autophagy for pathogenesis (Buzzetti et al. 2016, Paschos & Paletas 2009). All these processes are linked to each other and form a vicious cycle, which lead to cell injury, chronic inflammation and disease progression (Figure 8). In NAFLD, mild inflammation, characterized by accumulation of lymphocytes, eosinophils and neutrophils, is present in the lobular area, later on spreading to the portal area (Brunt & Tiniakos 2010). When NAFLD is progressing into NASH, fibrosis is a key factor in a separating these two stages. In the early phases of NASH, fibrosis can be absent, but is spreading in a reticulated pattern to a portal area, later on into bridging fibrosis and cirrhosis. In addition, microvesicular steatosis, megamitochondria, glycogenated hepatocyte nuclei and Mallory Denk bodies, apoptotic bodies and polymorphonuclear infiltrates are common in NASH, associating with the severity of the disease. A characteristic feature for the pro-inflammatory or stress stimulus in the liver is the recruitment of circulating monocytes and the expansion of liver resident macrophages, Kupffer cells, subsequently increasing the hepatic macrophage number and activation (Bilzer et al. 2006). Kupffer cells are important scavengers, expressing multiple toll like receptors, such as TLR4 and TLR9 that mediate cell injury response in the liver. Injured

44 18 cells activate Kupffer cells to secrete inflammatory cytokines, such as TNFα, IL-1β and IL-6 that further activate liver stellate cells (Ito-cells) to proliferate and transform into myofibroblast, secreting extracellular matrix proteins and growth factors (TGFβ1 and platelet derived growth factor), thereby promoting liver fibrinogenesis and scarring. However, Kupffer cells have also anti-inflammatory properties by secreting IL-10 and IL-1 receptor antagonist, triggering the phagocytosis of apoptotic cells, thereby alleviating inflammation and restoring the liver tissue. Figure 8. Pathogenesis of hepatic steatosis and steatohepatitis. Triglycerides and free fatty acids are either derived in the liver via white adipose tissue lipolysis and liver de novo lipogenesis or from the diet. Excessive triglycerides accumulate in hepatocytes and form intracellular lipid droplets that promote the secretion of proinflammatory cytokines and oxidative stress, further inducing the recruitment of circulating monocytes to the liver and activate the proliferation of liver resident macrophages, Kupffer cells. Injured hepatocytes and other hepatic cells, like macrophages, secrete inflammatory cytokines and ROS that cause mitochondrial dysfunction, dysregulated apoptosis and phagocytosis. These processes activate liver stellate cells (Ito-cells) to proliferate and transform into myofibroblast, which secrete extracellular matrix proteins and growth factors, subsequently promoting liver fibrinogenesis and scarring. 2.4 THE ROLE OF NRF2 IN ATHEROGENESIS AND HEPATIC STEATOSIS Nrf2 has been considered protective in physiological processes, but also in the pathological conditions such as atherogenesis and hepatic steatosis, as it plays an important role in the regulation of oxidative stress and inflammation The role of Nrf2 in atherogenesis In the vasculature, Nrf2 is activated in regions where the blood flow is laminar and unidirectional, which are not prone to atherosclerotic lesion development (Fledderus et al 2008). Laminar blood flow stimulates the release of nitric oxide from endothelial cells, which is known to protect against atherogenesis. In contrast, in curvatures and arterial branches the blood flow is turbulent, which suppresses Nrf2 activity and nitric oxide production, increses superoxide production and oxidative stress that have unfavorable

45 19 effects on vascular homeostasis and function (Hsieh et al. 2014). Nrf2 has local vasculoprotective effects also via its target genes, such as GCLM, HO-1, PRDX1 (peroxiredoxin 1) and NQO1 that are expressed in response to oxidized phospholipids and oxidized LDL in human vascular endothelial cells (Bea et al. 2003, Collins et al. 2012, Cho et al 1999, Bretscher et al. 2015), and in murine arteries (Jyrkkänen et al. 2008). Several Nrf2-regulated genes, such as HO-1, GCLM, PRDX1 AND PRDX2 have shown to protect against atherosclerosis in different mouse models of dyslipidemia (Park et al. 2011, Kisucka et al. 2008, Callegari et al. 2011, Orozco et al. 2007, Cheng et al. 2009). For example, overexpression of HO-1 inhibited atherosclerosis in apolipoprotein E deficient (ApoE -/- ) mice, whereas systemic and macrophage-specific HO-1 deficiency promoted atherosclerosis development and lesion maturation (Juan et al 2001, Yet et al 2003, Orozco et al 2007, Cheng et al 2009). Macrophage-specific Gclm expression protects against atherosclerosis in ApoE -/- mice, by increasing endogenous levels of GSH, which lower the risk for coronary disease (Bea et al. 2003, Callegari et al. 2011). PRDX1 and PRDX2 mediate atheroprotection via endothelial activation, regulation of inflammatory cell adhesion and infiltration, as well as by maintaining macrophage cholesterol homeostasis under oxidative stress (Kisucka et al. 2008, Park et al. 2011). However, the loss of activating transcription factor 3 (ATF3 -/- ), a target gene of Nrf2, was shown to increase macrophage foam cell formation in vivo, as well as atherosclerotic lesion development in ApoE -/- mice, correlating with increased levels of cholesterol-25-hydroxylase in ATF3 / mouse aorta (Gold et al. 2012). Nrf2 target genes have several beneficial functions, but also Nrf2 itself mediates actions that affect positively in the initiation and the progression of atherosclerosis (Figure 9). For example, macrophage-specific deletion of Nrf2 promotes advanced atherogenesis and plaque maturation by increasing pro-inflammatory cytokine expression and reducing antioxidant defense in LDL receptor deficient (LDLR -/- ) mice (Collins et al. 2012). Notably, Nrf2 was considered to have anti-atherogenic properties only via its redox-regulation, but recently, it was shown to suppress the transcription of pro-atherogenic cytokines, such as IL-6 and Il-1β, in murine macrophages (Kobayashi et al 2016). Supporting the antiatherogenic role of Nrf2, Nrf2 activation by gene transfer reduced vascular inflammation and oxidative stress in rabbit aorta after angioplasty (Levonen et al 2007). Controversial with several beneficial effects of Nrf2 on vascular wall, macrophage specific deficiency of Nrf2 reduced advanced atherogenesis in an ApoE -/- model by reduced macrophage proinflammatory stimuli (Harada et al. 2012). Four different studies have also demonstrated that global Nrf2 deficiency alleviates atherosclerosis in ApoE -/- mice (Sussan et al. 2008, Barajas et al. 2011, Freigang et al. 2011, Harada et al. 2012) via reduced foam cell formation and plasma total cholesterol. Sussan et al and Barajas et al reported that deletion of Nrf2 reduced the uptake of modified LDL in mouse macrophages via downregulation of CD36 scavenger receptor (Sussan et al. 2008, Barajas et al. 2011). Freigang et al suggested that Nrf2 activates inflammasome Nlrp3 mediated Il-1β production, subsequently promoting atherogenesis in ApoE -/- mice (Freigang et al. 2011).

46 20 Figure 9. Pro- and anti-atherogenic effects of Nrf2 studied in atherosclerotic ApoE -/- mice. Nrf2 was proposed to increase the formation of foam cells due to enhanced expression of CD36 and to induce Nlrp3-mediated inflammasome activation in macrophages, leading to aggravated atherosclerosis. Nevertheless, Nrf2 mediates atheroprotective effects in vascular wall via its anti-inflammatory and antioxidative effects. In addition, Nrf2 was reported to regulated lipogenesis regulating genes, thereby providing both pro- and antiatherogenic effects Systemic metabolic effects of Nrf2 Nrf2 mediates cellular protection via local tissue effects, but it contributes to pathogenesis of atherosclerosis and hepatic steatosis also via systemic metabolic effects. Nrf2 seems to regulate body weight gain and lipid content in plasma and other tissues. However, varying results and mechanisms have been reported, as follows. Nrf2 deficiency seem to protect at least partially against HFD-induced obesity via three different mechanisms. Firstly, Nrf2 deficiency impairs adipogenesis via downregulation of PPARγ (Pi et al. 2010, Chorley et al. 2012), secondly, it increases energy expenditure via oxidative stress-induced UCP-1 expression in white adipose tissue in C57Bl mice (Schneider et al. 2016). Thirdly, Nrf2 deficiency diminishes liver and white adipose tissue mrna expression and plasma levels of fibroblast growth factor 21 (FGF21), the hormone that regulates fatty acid oxidation in the liver and glucose metabolism in white adipose tissue, and is known to be upregulated in obesity and NAFLD in human (Chartoumpekis et al. 2011, Dushay et al. 2010). Reduced white adipose tissue mass and liver lipid content possibly improve insulin sensitivity in Nrf2 deficient mice. Since the loss of Nrf2 has been investigated in metabolic regulation in mice, Nrf2 activators have been studied as well. Nrf2 activation whether by deletion of KEAP1 or triterpenoids inhibited white adipose tissue accumulation in obese and in wildtype mice (Shin et al. 2009, Zhang et al. 2013, Xu et al. 2012). In addition, Nrf2 activation by triterpenoid reduced liver lipid content and plasma lipid levels by downregulating fatty acid synthesis genes (Shin et al. 2009) and by increasing the expression of FGF21 in diabetic db/db mice (Furusawa et al. 2014). Substantial in the protection against atherosclerosis, the loss of Nrf2 is reported to lower plasma LDL cholesterol level due to reduced cholesterol synthesis by downregulating transcription of Srebp1 and its target genes (Huang et al. 2010, Barajas et al. 2011). Nevertheless, some paper demonstrated the opposite, as Nrf2 deficiency promoted the expression of cholesterol synthesis regulating genes, studied by microarray analysis

47 21 (Kitteringham et al. 2010, Chartoumpekis et al. 2013, Tanaka et al. 2008, Tanaka et al 2012). To support the notion, Nrf2 activation by triterpenoid CDDO-Im reduced plasma lipid content (Furusawa et al. 2014). However, further studies are needed to characterize the role of Nrf2 in metabolic regulation Nrf2 function in hepatic steatosis Nrf2 has been hypothesized to protect against progression of steatohepatitis by improved antioxidative properties (Tang et al. 2014, Hayes et al 2000), although the precise mechanisms how Nrf2 affects the development of NAFLD and NASH remains unknown. Several papers have shown the protective role of Nrf2 in liver injury models (Xu et al 2008, Liu et al 2013, Kudoh et al 2014), and the deletion of Nrf2 leads to rapid onset and exacerbation of NASH in C57BL mice after methionine-choline deficient diet. In these studies, Nrf2 improved liver unfolded protein response in the ER, which is activated via unfolded or misfolded proteins due to downregulation of Nfκβ-pathway and protection against HFD-induced oxidative stress (Sugimoto et al. 2013, Chowdhry et al. 2010, Meakin et al. 2014). In addition, macrophage-specific deletion of Nrf2 accelerated hepatic inflammation and fibrosis in LDLR -/- mice by promoting macrophage migration and increasing pro-inflammatory cytokine expression and oxidative stress signaling (Collins et al. 2012). Several Nrf2 activators have been studied in hepatic steatosis as well. Bardoxolone methyl, an activator of Nrf2, restored HFD-repressed Nrf2 protein expression and subsequently prevented the development of hepatic steatosis, insulin resistance, and inflammation after long-term HFD in C57Bl mice (Camer et al. 2015). Nrf2 activation by deletion of KEAP1 accomplished the same outcome in leptin deficient mice (Xu et al. 2012), but the mechanism remains unclear. Finally, Nrf2 may partially promote the progression of hepatic steatosis and steatohepatitis by dysregulating the cellular autophagy, which is an essential cellular self-eating and cleaning mechanism, as there is a positive reciprocal regulation of Nrf2 and autophagy-related protein p62. p62 competes with Nrf2 for binding to KEAP1 resulting in dissociation of Nrf2 from KEAP1 and activation of Nrf2 (Kansanen et al. 2013). Interestingly, Nrf2 activation promotes liver fibrosis and tumorigenesis in autophagy related 5 deficient mice with defective autophagy (Ni et al 2014). 2.5 HYPERLIPIDEMIC MOUSE MODELS The mouse (Mus musculus) was used as a laboratory animal first time in the 16 th century, and has now achieved a central role as a genetically adaptable model organism for research on human biology and disease (Perlman 2016). The laboratory mouse offers a close view of humankind in terms of similarity in the physiology, tissue structure and organization, as well as many common diseases. Despite of the evolutionary divergence, human has a close kinship with the mouse, as human encoded genome secuence reflects the 99% similarity with the mouse genome (Waterston et al. 2002) The usage of mice is reasonable, as cell culture and in vitro experiments offer precise information in controlled experimental conditions, but these are lacking relevant physiological conditions and interactions between cells and tissues. Thus, several experimentally-induced and gene modified mouse models have been developed to study human disease pathologies and to define new therapies. The mouse model can be a homologous or a predictive model with human disease, having the same cause and symptoms as human disease or display symptoms without a known cause,

48 22 respectively. Most mouse models are isomorphic where the etiology and pathogenesis of experimentally-induced disease differs from the one spontaneously existing in human, but has similar symptoms and treatments thereby provide a valuable tool in experimental and translational research (Perlman 2016). When aiming to mimic human diseases in mice, genetic, dietary and/or chemical modification is often required, as mice naturally have some metabolic and physiological differences compared to humans (Table 1). Mice are naturally very resistant to diet-induced hyperlipidemia and atherosclerosis, as mice are so called HDL-animals and ~90% of cholesterol is mainly carried in anti-atherogenic HDL particles, whereas in humans, cholesterol is also carried in proatherogenic LDL and VLDL particles (Figure 10) (Farese et al. 1996, Murielle M Véniant et al. 1998). Furthermore, subclasses of HDL exhibit varying atheroprotective characteristics and the clearance of LDL particles is much faster in mice than in humans, making mice resistant to hypercholesterolemia. Mice also do not express CETP, which in humans lowers HDL levels due to transportation of cholesterol esters from HDL to ApoB containing LDL and VLDL particles (Masson et al. 2009). Apolipoproteins differ between humans and mice, as mice express both ApoB100 and ApoB48, an aminoterminal portion of ApoB100, in the liver due to the presence of mrna editing activity, whereas only ApoB100 is expressed in the human liver (Farese et al. 1996, Murielle M Véniant et al. 1998). ApoB48 containing VLDL particles are hydrolyzed by LPL and remodeled to very small and dense ApoB48-VLDL remnants that are more rapidly cleared via apolipoprotein E (ApoE) receptors compared to ApoB100, which is taken up by LDL receptors. Lastly, the cardiovascular anatomy and physiology, such as arterial vessel hemodynamics, is different in mice in comparison to human, resulting in different preferential sites for the development of atherosclerotic lesions. Table 1. Physiological differences between mouse and human related to atherogenesis. Modified from Goldberg & Dansky (2006), Bentzon & Falk (2010) and Perlman (2016). Characteristic Human Mouse Life span (years) ~80 ~2 Mass (kg) ~70 ~0.030 Normal diet High fat, omnivore Low, omnivore Hypertension Yes No Major plasma lipoprotein LDL HDL Plasma cholesterol (mmol/l) ~5 ~2 Cholesterol synthesis (mg/d/kg) Hepatic LDL clearance (ml/d/kg) ApoB subtypes in liver B100 B48 and B100 CETP Yes No Aortic sinus atherosclerosis No Common Coronary artery atherosclerosis Common Rare Plaque rupture and thrombosis Common Rare

49 Atherosclerotic plaque development in mice compared to human Atherosclerotic mouse models are great tools for investigating human atherosclerosis. Nevertheless, there is some notable differences in the development and maturation of human and mouse atherosclerotic plaques. Atherosclerosis is a multifactorial disease and this complexity is not simple to mimic in a mouse, not in the least because it takes decades for atherosclerosis to develop in humans, whereas the timeframe is much shorter in mice, especially if accelerated with western type diet (Bentzon & Falk 2010). The benefit of shorter timeframe is the ability to monitor atherogenesis in a reasonable time. Moreover, human and mouse have some differences in normal vessel architecture, which has an impact on atherosclerotic plaque morphology (Icardo & Colvee 2001, Boon & Horrevoets 2009). Human artery is made of thick medial and intimal layers, especially at atherosclerosisprone regions of vasculature bifurcations (adaptive intimal thickening). The normal human arterial intima and media contain thick layer of SMCs and connective tissue fibers, which have remarkable deposits already in the early atherosclerotic lesion and are pronounced also in advanced plaques in human (Fisher & Miano). In mice, the medial layer consists only of a few layers of SMCs, a small number of lamellaes and fibrotic fibers, and intima is almost non-existent, formed only of an endothelial monolayer. This reflects also to atheroma plaque morphology, as lesions contain only a few smooth muscle cells and minor fibrotic components (Bentzon & Falk 2010). Lesion distribution is not identical in mice and human, as lesions are present more often in coronary arteries, carotids and peripheral vesseles in human, but in aortic root, aortic arch and brachiocephalic artery in mice (Jackson et al. 2007). Nonetheless, in human familial hypercholesterolemia, lesions occur in the aortic valves and in some autopsy studies also in the aortic root. In mice, lesions rarely progress up to the stage of atheroma (i.e. a thick fibrous cap overlying a necrotic core in human), but mostly consist of intimal foam cells without a fibrous cap, which is most often clinically silent if the plaque does not occlude the arterial lumen (Williams et al. 2002, Matoba et al. 2013b). However, aged or prolonged HFD-fed mice may develop unstable plaque morphology, but almost invariably without secondary thrombosis (see detailed discussion of this in chapter 2.4.3). In contrast, plaque calcification is more dependent on aging than the atherogencity of the diet or plasma hyperlipidemia (Heinonen et al. 2007). Notably, different diets and different genetic modifications to induce hypercholesterolemia have also other metabolic properties, such as the ability to induce obesity, insulin resistance or glucose tolerance. Opposite to humans, female mice are more prone to atherogenesis than males in several genetically modified atherosclerotic mouse models. In addition, sexual dimorphism has been demonstrated in many hepatic genes involved in lipid metabolism in murine models (Yang et al. 2006, Caligiuri et al 1999).

50 24 Figure 10. Plasma lipid profile in healthy wild-type mouse and human. Adapted from: Steenbergen et al Mouse models of atherosclerosis Different mouse strains might have some physiological differences for example in blood pressure, heart rate and plasma lipids, resulting in some strains being more susceptible to certain diseases, for example C57Bl/6J strain is prone to hyperlipidemia-induced atherosclerosis and other CVDs due to lower plasma HDL level in comparison to other wild-type strains (Paigen et al. 1987). Various genetic mouse models have been created to replicate human dyslipidemia and atherosclerosis. Gene inactivation has been the most common approach to create atherosclerotic mouse models, LDL receptor and ApoE genes being the most commonly inactivated ones. Mice have only a single ApoE isoform, but in humans ApoE exists in 3 isoforms, E2, E3 and E4, with varying effects of each on atherosclerosis. ApoE is a ligand for numerous lipoprotein receptors and is a key player in the regulation of cholesterol homeostasis. Nevertheless, it regulates adipose tissue biology, as well as the inflammation and cholesterol trafficking in macrophages. Rarely, homozygous null mutations in the ApoE gene have been recognized in humans, causing familial dysbetalipoproteinemia (Schaefer et al. 1986). The apolipoprotein E3-Leiden genetic mutation causes hyperlipidaemia in human. ApoE3-Leiden transgenic mice have been developed to model atherosclerosis, but plasma cholesterol and triglyceride levels are quite low on a chow diet and thereby this strain does not develop atherosclerotic lesions without a HFD, which elevates plasma lipid levels in this model (Zadelaar et al. 2007, Lutgens et al. 2005). LDL receptor is a master transport receptor of ApoB-100 containing LDL particles in both hepatic and extrahepatic tissues, thereby maintaining the plasma cholesterol levels. In familial hypercholesterolemia (FH), the most common genetic cause for extreme hypercholesterolemia, the primary defect is a mutation of the LDL receptor gene, causing cholesterol accumulation in the arterial wall and other tissues. The production of LDL receptor deficient mice (LDLR -/- ) was reported in 1993 by Ishibashi et al and thereafter became the most widely used hyperlipidemic mouse model together with ApoE -/- mice, which had been generated one year earlier (Ishibashi et al. 1993, Piedrahita et al. 1992, Plump et al 1992). Both models develop mild hypercholesterolemia on a chow diet compared to wild type mice, but do not reach the levels measured in human patients suffering from FH, because of the differences in lipoprotein metabolism between mouse and human (Marais 2004). In addition, LDLR -/- mice have a compensatory mechanism to regulate plasma cholesterol as LDLR related protein (LRP) receptor is able to take up lipoproteins (Ishibashi et al. 1993, Ishibashi et al. 1994). Thus, both models need a high fat or high cholesterol diet to induce prominent elevation of plasma cholesterol and

51 25 triglycerides. The main plasma lipoprotein fraction in LDLR -/- mice is IDL/LDL and both ApoB48 and B100 levels are elevated, whereas VLDL fraction dominates in plasma of ApoE - /- mice, and only ApoB48 is increased. The atherosclerotic phenotype is milder in LDLR -/- mice than ApoE -/- mice, since LDLR -/- mice do not develop remarkable atherosclerosis on a chow diet, whereas ApoE -/- mice develop spontaneous atherosclerotic lesions on a regular diet (Véniant et al. 2001, Getz & Reardon 2016). However, the high fat feeding accelerates the process in both models. Long-term western-type diet promotes lesion progression and maturation from early initial lesion to more advanced and complex lesion, furthermore, spontaneous plaque rupture and secondary thrombosis have been reported in both models, albeit at low incidence (Williams et al. 2002). LDLR -/- and ApoE -/- mice have also been combined, but the model does not provide significant advantages compared to single ApoE -/-, except more aggressive and spontaneous atherosclerotic lesion development without a HFD (Kampschulte et al. 2014). Moreover, ApoB-transgenic mice, overexpressing human or mouse ApoB100 have been developed to model human atherosclerosis. In these models, plasma HDL levels are reduced and LDL cholesterol levels increased, but the atherosclerotic phenotype is quite mild and a HFD is needed to induce atherosclerotic plaques (Purcell-Huynh et al. 1995, McCormick et al. 1996). LDLR -/- and ApoE -/- have been coupled with a mouse model expressing ApoB100 only (Powell-Braxton et al. 1998, Véniant et al. 1998, Farese et al. 1996). In this model, enzymatic editing of ApoB mrna is limited, resulting in lack of ApoB48 expression and exclusively the synthesis of ApoB100 (Veniant et al 1998, Veniant et al 2000, Powell-Braxton et al 1998, Osuga et al. 1997). The mice expressing ApoB100-only develop atherosclerotic plaques even on a chow diet as they have more aggressive and more human-like atherosclerotic phenotype in comparison to single LDLR or ApoE knockout (Figure 11) (Powell-Braxton et al. 1998). Thus, combined LDLR and ApoB48 deficiency is considered to mimic human hypercholesterolemia most closely. In addition to these commonly used mouse models of atherosclerosis, also other genetic dyslipidemic models exist, but these do not resemble closely to human proatherogenic plasma lipid profile. For instance, impaired cholesterol reverse transport in Abca -/- mice led to reduced plasma total cholesterol and lipid accumulation in tissues and macrophages, but did not induce atherosclerosis in ApoE -/- or LDLR -/- mice (Aiello et al 2002). Low level or lack of HDL cholesterol was hypothesized to increase atherosclerosis, but surprisingly this was not seen in single knock-out mice, such as ApoAI -/-, apolipoprotein B editing complex deficient (APOBEC -/- ) or lecithin cholesterol acyltransferase deficient (LCAT -/- ) mice that have low HDL levels (Moore et al. 2003, Hughes et al 1997, Nakamuta et al 1998, Lambert et al 2001). In addition, transgenic mice expressing CETP have reduced plasma HDL levels and increased LDL and VLDL plasma levels, but the atherosclerotic phenotype is mild (Marotti et al. 1993). However, this model develops severe atherosclerosis if crossbred with ApoE3-Leiden mice (Westerterp et al. 2006). Moreover, HL -/-, LPL -/- and SR-B -/- mice have mildly increased plasma triglycerides or total cholesterol, including HDL, but these do not develop atherosclerotic lesions, unless they are combined with ApoE -/- or LDLR -/- mice (Mezdour et al. 1997, Weinstock et al. 1995, Huszar et al 2000). In addition to genetic models, a novel mouse model of atherosclerosis without germline genetic engineering has been developed, when the pro-protein convertase subtilisin/kexin type 9 (PCSK9) adeno associated virus (AAV) mice was reported by two different research groups in 2014 (Björklund et al 2014, Roche-Molina et al 2015). PCSK9 is a serine protease, which is expressed in the liver, it binds to LDL receptors and reduces hepatic uptake of LDL via enhanced lysosomal degradation of LDL receptors (Li et al 2007, Akram et al 2010). PCSK9-AAV treated mice have a two-fold plasma total cholesterol levels and similarly increased LDL and VLDL fractions compared to wild type mice (Björklund et al 2014, Roche-Molina et al 2015). Their phenotype resembles that of LDLR -/- mice, the

52 26 hypercholesterolemia induces mild atherosclerotic lesion development, which can be induced to more advanced lesions by HFD. This model is rapid, versatile and cost-effective compared to genetically modified models and crossbreeding, in addition, there are no biosafety concerns. Figure 11. Comparison of aortic root cross-sectional atherosclerotic lesion area and plaque development between female and male LDLR -/- and LDLR -/- ApoB 100/100 mice on the different diets and timelines Spontaneous plaque rupture in mouse models of atherosclerosis Most human atherosclerotic plaques are asymptomatic, but some become vulnerable and prone to rupture and thrombosis formation, leading to acute myocardial infarction or stroke (Ylä-Herttuala et al. 2011). Different genetic and mechanical injury models for these clinical manifestations have been developed during the last decades, as the need for plaquestabilizing therapies is notable. Plaque rupture as a process can be divided into the three steps: plaque destabilization, injury of fibrous cap resulting in plaque rupture and thrombotic occlusion of the artery (Bentzon et al. 2014). Induction of plaque rupture or spontaneous myocardial infarction has been challenging in hypercholesterolemic mouse models, as a very long period of time is required to induce complex plaques, and even then the plaque is not necessarily vulnerable. Thus, plaque rupture with a superimposed occlusive thrombus is very rare in mouse models of atherosclerosis and occurs only sporadically (Majesky et al. 2002, Matoba et al. 2013). Moreover, mice develop plaques in aortic sinus and brachiocephalic artery, and only rarely in coronary arteries, which is the most common site for atherosclerotic plaque in human (Jackson et al. 2007). Changes in arterial shear stress induce atherogenesis locally in the branching points of arteries, such as brachiocephalic artery and proximal coronary artery (Boon & Horrevoets 2009). Notably, high shear stress is associated with vulnerable plaque phenotype in coronary arteries in human and in brachiocephalic artery in mice (Samady et al. 2011, Hartwig et al. 2015). Due to the location of plaques, the clinical complications of possible plaque rupture as myocardial infarction and stroke are almost never seen in mice (Jackson et al. 2007). In addition, it is noteworthy that the scale of the arteries is different in mouse and human, affecting the susceptibility to thrombus formation. Moreover, the propensity to develop thrombi is different between these two organisms, as the plasma level of plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor are 5 to 12 fold and 2 to

53 27 7 fold lower in mice than in human, respectively. Thus, fibrinolysis is enhanced and accelerated, and thrombi are rapidly cleared in mice (Jackson et al. 2007). Mouse atherosclerotic plaque development in brachiocephalic artery was identified by Russell Ross s laboratory for the first time in 1994, and plaque morphology, including intraplaque hemorrhage, was described in more detail by Rosenfeld et al six years later (Rosenfeld et al. 2000, Nakashima et al. 1994). This was a remarkable finding, as aortic sinus is widely used to analyze atherosclerosis in mice, but intraplaque hemorrhage or other indications of plaque rupture do not occur in the aorta even after prolonged HFD (Johnson et al. 2005). Nevertheless, buried fibrous caps and layering can be identified in brachiocephalic artery of aged ApoE -/- mice, as a possible hallmark of repaired plaque rupture (Mann & Davies 1999). In addition, other hallmarks of a unstable plaque, such as a highly necrotic landscape, large fibrofatty nodules with lateral xanthomas, infiltration of inflammatory cells, thinned fibrous cap and scattered or nodular calcification, are demonstrated in ApoE -/- and LDLR -/- ApoB 100/100 mouse models (Heinonen et al. 2007, Rosenfeld et al. 2000). So far, the most promising model for atherosclerotic plaque rupture is fibrillin-1 C1039G transgenic mice, created by Herck et al, and crossbred with ApoE -/- mice (FbnC1039 +/- ApoE -/- ) (Van Herck et al. 2009). FbnC1039 +/- ApoE -/- mice develop advanced atherosclerosis and increased plaque instability due to enhanced intraplaque neovascularization and degradation of elastin (Van Der Donckt et al. 2015). Intraplaque neovascularization is rarely seen in atherosclerotic mouse models, but in FbnC1039 +/- ApoE -/- mice it is similar to human plaque pathology (Van Der Donckt et al. 2015). Plaque vulnerability and rupture has also been induced with different chemical and technical approaches. For example, intraplaque hemorrhage has been induced in ApoE -/- mice by macrophage specific retrovirus, encoding active form of MMP-9, as it increased elastin degradation leading to disruption of fibrous cap (Gough et al. 2006). In addition, angiotensin II infusion combined with HFD accelerates development of atherogenesis, promotes abdominal aneurysm formation and increases the incidence of plaque rupture in ApoE -/- model (Daugherty et al. 2000). Thrombus is very rarely seen in mice, but plaque rupture accompanied by thrombus have been induced by ligation of the common carotid artery and cuff placement proximal to the ligation site in order to halt blood flow, this causes intimal hyperplasia, disruption of neointima and thrombus formation in chow-fed ApoE -/- mice (Sasaki et al. 2004). Other options to induce thrombus formation, are ligation of both carotid arteries or the partial ligation of unilateral renal artery, resulting in increased neointima and macrophage infiltration. By this method, 50% of ligated arteries contained a thrombus. Nevertheless, the biomechanical induction might not resemble human-like spontaneous plaque rupture and its complications very closely Spontaneous myocardial infarction in mouse models of atherosclerosis The common consequence of coronary artery plaque rupture is myocardial infarction or sudden death in humans, but this phenomenon is rarely observed in atherosclerotic mouse models. Hence, myocardial infarction is induced by surgical methods, such as by ligation of the left anterior descending coronary artery (LAD), but this requires specialized microsurgical skills and facilities to provide high quality performance to minimize deviation within the study group and loss of animals (Wang et al. 2006). Currently, the only genetic mouse model, demonstrating possible complications of plaque rupture, such as stroke and myocardial infarction, is the FbnC1039G +/- ApoE -/- mice, from which ~50% died suddenly by 35 weeks on a HFD (Van Der Donckt et al. 2015). The mice also showed increased coronary artery stenosis, perivascular fibrosis, myocardial hypertrophy,

54 28 pronounced inflammation and fibrosis compared to survivors. Although it is not known if the sudden death was due to plaque rupture or pronounced coronary artery plaque formation and coronary artery stenosis, these findings are remarkable because coronary artery stenosis, following spontaneous myocardial infarction, almost never develop in ApoE -/- mice or other atherosclerotic models. Thus, this model is a unique tool to investigate mechanisms of vulnerable plaque and to develop appropriate therapies. Nevertheless, coronary artery disease and spontaneous myocardial infarction without plaque vulnerability has been described in ApoE deficient mice, if it is combined with deficiency of SR-B1 or disruption of its adaptor protein PDZK1, causing approximately two-fold increase in plasma unesterified HDL cholesterol and subsequently a two-fold elevation in plasma total cholesterol (Fuller et al 2014). SR-B1 -/- ApoE -/- mice demonstrate occlusive coronary artery disease and spontaneous myocardial infarction also on a normal chow diet. In contrast, PDZK1 deficiency in ApoE background promotes cardiac fibrosis, but it does not predispose to myocardial infarction. SR-BI deficiency combined with LDLR -/- mice resulted in lethal coronary atherosclerosis, following specific diets, such as 2% cholesterol diet or two different modified Paigen diets. In addition, SR-BI deficiency in hypoe mice model, in which ApoE expression is only 2-5% of its normal levels, induced myocardial infarction after 4 weeks of Paigen diet. Other, non-traditional means to evoke coronary disease and myocardial infarction have been demonstrated in ApoE -/- mice. When ApoE -/- mice were combined with LDLR deficiency, Akt1 deficiency, or macrophage specific overexpression of urokinase, coronary artery stenosis and myocardial infarction were induced by mental stress, hypoxia, diabetes and myocardial fibrosis, respectively (Fernandez-Hernando et al. 2007), Braun et al. 2008, Cozen et al. 2004, Moriwaki et al. 2004). Vascular endothelium regulates vascular tone and thrombosis formation via nitric oxide, which is synthesized and released by endogenous NO synthase, including its three isoforms (endothelial, inducible and neuronal NOS) (Nakata et al. 2008). Blocking endothelial NOS in ApoE -/- mice resulted in coronary atherosclerosis, lethal myocardial ischemia, heart failure and aortic aneurysms after WD feeding (Kuhlencordt et al 2001). If all isoforms of NOS are blocked, then mice generated severe cardiovascular complications, including myocardial infarction, but this might have been due to coronary spasm rather than occlusive thrombus formation (Nakata et al. 2008) Hyperlipidemic mice as models of NAFLD and NASH Knowledge of the mechanisms and specific genes that promote simple steatosis progression to NASH, is of the utmost importance, as the diagnostic of this disease need to be improved and there is no specific medical treatment available. Different animal models have been used to mimic human pathology, but these have been incomplete as none demonstrate both obesity and features of metabolic syndrome with spontaneous steatohepatitis (Ka et al. 2017, Starter & Herck 2017). Different genetic and nutritional mouse models reflect separate stages of human pathogenesis, with certain limitations and tightly depending on the diet, its duration and the age of mice. Essentially, genetic models of hepatic steatosis can be separated into two groups: first, those in which steatohepatitis is developed with no or minimal metabolic syndrome and second, those in which steatosis without disease progression is developed with obesity and insulin resistance (Larter & Yeh 2008). Mouse models of atherosclerosis have been utilized in fatty liver research, as pathology of NASH and atherosclerosis is quite similar. The most popular hypercholesterolemic mouse models are LDLR -/- and ApoE -/- mice. In addition to these two traditional models, ApoE2ki (knockin) mice, resembling human type III hyperlipoproteinemia and abnormal lipid

55 29 accumulation, have been used as a NASH model (Bieghs et al. 2012). The advantage of hypercholesterolemic mouse models is a human-like lipoprotein profile, unlike in wild type mice (Ishibashi et al. 1993, Farese et al. 1996). However, these mice do not develop insulin resistance, hepatic steatosis or steatohepatitis without feeding a HFD. However, ApoE -/- and ApoE2-knock-in models have a critical disadvantage related to steatohepatitis, as proinflammatory phenotype is reduced over time and anti-inflammatory genes are upregulated after prolonged feeding of HFD unlike in LDLR -/- mice, which maintain increased hepatic inflammation (Bieghs et al. 2012). In fact, ApoE deficiency develops even milder HFD-induced hepatic steatosis compared to LDLR -/- and wild-type mice, due to delayed post-prandial triglyceride clearance from plasma (Karavia et al. 2011). However, combined ApoE and LDLR -/- deficiency results in a remarkably more aggressive liver phenotype with inflammation, fibrosis and even tumor formation, after 35 weeks of HFD (Kampschulte et al. 2014).

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57 31 3 Aims of the study Transcription factor Nrf2 is the master regulator of antioxidant defense in many tissues and organ systems. The aim of this thesis was to investigate local vascular and systemic effects of Nrf2 on atherogenesis and hepatic steatosis in two different hyperlipidemic mouse models. The specific aims for this thesis were as follows: I To investigate the effect of Nrf2 on foam cell formation and early atherogenesis in LDLR -/- mice. II To study the role of global Nrf2 deficiency in early atherogenesis in the LDLR -/- mice and in advanced atherogenesis and plaque composition in LDLR -/- mice expressing ApoB-100 only. III To study the role of global Nrf2 deficiency in high-fat diet induced hepatic steatosis in LDLR -/- mice.

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59 33 4 Materials and methods The materials and methods used in this thesis are summarized in the following tables. The detailed description of the methods are presented in the original publications (I-II) and in the manuscript (III). Table 2. Summary of the methods used in the thesis. Method Description Original article Modification of LDL Isolation of LDL I, II Oxidation, acetylation or malondialdehyde modification I, II Primary cells Isolation of bone marrow derived macrophages I, II Isolation of thiglycollate elicited peritoneal macrophages I Cell biology methods Lipid uptake and reverse cholesterol transport I Lipid extraction I Amount of mitochondrial DNA III In vivo methods Bone marrow transplantation I Euthanasia, collection of tissues, tissue processing I, II, III Echocardiography II Paraffin and frozen tissue sections I, II, III Blood sample collection I, II, III Methods to study gene expression RNA isolation, quantitative real-time polymerase chain reaction (qpcr), polymerase chain reaction (PCR) I, II, III Methods to study protein expression Isolation and measurement of total protein ELISA Western blot Immunohistochemistry I, III I I, III I, II, III Histological stainings Hematoxylin-eosin I, II, III Movat Pentachrome Masson Trichrome Picro Sirius Red Alizarin Red S Oil Red O I, II II, III II II I, III Histopathology Lesion size and composition in aorta and brachiocephalic artery Liver I, II III Clinical chemistry Plasma cholesterol and triglycerides I, II, III

60 34 Table 3. Mouse models used in the thesis. Strain Sex Age Diet Original article C57Bl/6J Male 2-3 months Normal chow I Nrf2 -/- Male 2-3 months Normal chow I LDLR -/- Female and male 4-6 months HFD I, II, III Nrf2 -/- LDLR -/- Female and male 4-6 months HFD II, III LDLR -/- ApoB 100/100 Female and male 3-12 months Normal chow II Nrf2 -/- LDLR -/- ApoB 100/100 Female and male 3-12 months Normal chow II Table 4. Cells and culture conditions used in the thesis. Primary cells Culture conditions Source Original article Mouse peritoneal macrophages RPMI medium with 5 % bovine serum albumin Isolated after thioglycollate irritation I Mouse bone marrow derived macrophages RPMI medium with 5 % bovine serum albumin Isolated bone marrow cells were differentiated by macrophage colony stimulating factor (M-CSF) I, II Table 5. Key compounds used in the thesis. Compound Description Origin Original article natldl Native LDL Ruotsalainen AK, University of Eastern Finland oxldl Oxidized LDL Ruotsalainen AK, University of Eastern Finland acldl Acetylated LDL Ruotsalainen AK, University of Eastern Finland I I I, II MDA-LDL Malondialdehyde modified LDL S. Hörkkö, University of Oulu, Finland I LPDS Lipoprotein-deficient serum By ultracentrifugation from FBS LPS Lipopolysaccharide Sigma Aldrich I, II Table 6. Primary antibodies Antibody Directed against Application Manufacturer Original article mmq Mouse macrophage IHC Accurate Chemicals I, II αsma Smooth muscle cell actin IHC Abcam III SMHC Smooth muscle cell heavy chain IHC Abcam II CD68 Macrophage marker IHC Merck III CD36 Scavenger receptor WB Abcam I

61 35 MCP-1 Monocyte chemoattractant protein 1 IHC Abcam I, II WB I TNFα Tumor necrosis factor α IHC Abcam II OXPHOS VDAC1 Oxidative phosphorylation complex subunits WB Abcam III Voltage-dependent anion channel 1 WB Abcam III 4-HNE 4-hydroxynonenal IHC Thermo Scientific II 3-NT Tyrosine nitration mediated by reactive nitrogen species IHC EMD Millipore II β-actin Loading control WB Santa Cruz I, III

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63 37 5 Results Selected results from the original publications and unpublished data are presented. The results section highlights the key findings of two original publications and one manuscript combined. 5.1 NRF2 DEFICIENCY IN BONE MARROW CELLS PROMOTES EARLY ATHEROGENESIS As Nrf2 is known to mediate the anti-inflammatory and antioxidative actions in vascular wall, we investigated the impact of the deletion of Nrf2 in bone marrow derived cells on early atherogenesis by transplanting WT or Nrf2 -/- bone marrow to LDLR -/- mice, which were fed with a HFD for 6 weeks to induce early atherogenesis. Interestingly, Nrf2 deficiency in bone marrow derived cells increased atherosclerotic plaque area in LDLR -/- mice in comparison to WT bone marrow recipients (Figure 12). Figure 12. Macrophage specific deletion of Nrf2 promotes early atherogenesis in LDLR -/- mice after 6 weeks of HFD (study I). Cross-sectional lesion area is measured from aortic root with representative pictures of hematoxylin-eosin stained sections. Representative pictures are shown with original magnification 40, scale bar of 100 μm. Values are presented as total surface area. Each symbol represents one mouse and the line represents the mean, *P<0.05. Since deletion of Nrf2 in bone marrow derived cells promoted atherogenesis in LDLR -/- mice, we next examined the lipid uptake of WT and Nrf2 -/- mouse peritoneal macrophages incubated with modified LDL to induce foam cell formation (Figure 13A). Nrf2 deficiency enhanced lipid accumulation in macrophages as it increased cholesterol content and the amount of cellular neutral lipids stained by Oil Red O in comparison to WT controls after incubation with either acldl or MDA-LDL (Figure 13B-C). Next, we investigated whether increased foam cell formation was a result of increased uptake of modified LDL or a reduction in reverse cholesterol transport. Nrf2 deficiency did not have effect on early stage cholesterol efflux either to ApoAI or HDL2. Interestingly, the expression of many scavenger receptors such as SR-A, LOX-1 and CXCL16 was increased in Nrf2 -/- macrophages, as well as was the expression of TLR4, which implicated enhanced lipid accumulation via micropinocytosis (Figure 14A-D). In addition, deletion of Nrf2 enhanced pro-inflammatory

64 38 cytokine mrna expression in response to modified LDL, indicating more a M1-like macrophage phenotype (Figure 14E-H). These results suggest that Nrf2 deficiency increases foam cell formation by increasing lipid influx likely due to increased expression of several scavenger receptors and promotes pro-inflammatory macrophage phenotype, further affecting negatively the early atherosclerotic lesion development. Figure 13. Nrf2 deficiency promotes lipid accumulation in mouse thioglycollate elicited peritoneal macrophages (study I). A, Representative pictures of Oil Red O stained macrophages after treatment with acetylated LDL (acldl) or malondialdehyde modified LDL (MDA-LDL) to induce foam cell formation. B, Oil red O stains lipids with red color and lipid area (µm 2 ) was quantified per cell. C Lipid content was analyzed by cholesterol extraction in relation to cellular total protein. The data are depicted as mean±sd, *P< 0.05, **P< 0.01, ***P<0.001.

65 39 Figure 14. Nrf2 deficiency upregulated the mrna expression of several scavenger receptors and proinflammatory cytokines in peritoneal macrophages (study I). Macrophages were treated with native (natldl), oxidized (oxldl) or acetylated LDL (acldl). The mrna expression of scavenger receptors A, SR-A, B, LOX- 1, C, CXCL16 and D, TLR4 and pro-inflammatory cytokines E, MCP-1 at mrna and F, protein level, as well as G, IL-6 and H, TNFα mrna as a fold change and in normalization to Gapdh. The data are depicted as mean±sd, *P< 0.05, **P< 0.01, ***P< GLOBAL DEFICIENCY OF NRF2 PROTECTS AGAINST EARLY AND ADVANCED ATHEROGENESIS Nrf2 seems to have a dual role in the atherogenesis, and both systemic and local vascular effects have been proposed. Thus, we determined the role of global Nrf2 deficiency in atherogenesis in two different atherosclerotic mouse models, LDLR -/- and LDLR -/- mice expressing ApoB-100 only, mimicking more closely to human hypercholesterolemia compared to ApoE -/- mice. Nrf2 -/- LDLR -/- and LDLR -/- mice were fed a HFD for 6 or 12 weeks to induce formation of early fatty streak lesions and intermediate lesions, respectively. In addition, Nrf2 -/- LDLR -/- ApoB 100/100 and LDLR -/- ApoB 100/100 mice were aged for 6 or 12 months, to investigate the effect of Nrf2 on atherogenesis on a chow diet. Nrf2 deficiency reduced plaque area in the aortic root and entire aorta after 6 and 12 weeks of HFD. Nrf2 -/- LDLR -/-

66 40 exhibited early atheromas/fatty streaks (type I-II), whereas in LDLR -/- mice lesions were more mature, containing small necrotic areas including cholesterol crystals (type II-III) (Figure 15A) (Virmani et al. 2005). We found that Nrf2 deficiency reduced the size of atheroma plaques in LDLR -/- ApoB 100/100 female mice at 6 months of age in the aortic root, and after 12 months, in the descending aortas (Figure 15B). Figure 15. Loss of Nrf2 reduced early and advanced atherogenesis in two different atherosclerotic mouse models (study II). A, Cross-sectional lesion area measured from the aortic root with representative pictures of hematoxylin-eosin stained sections from LDLR -/- mice after 6 and 12 weeks of HFD, and B in LDLR -/- ApoB 100/100 female mice after 6 and 12 months on a chow diet. B, Lesion area from entire aorta was measured by en face analysis from LDLR -/- ApoB 100/100 female mice at 12 months of age. Lesions were stained with red color (Sudan IV). Representative pictures are shown with original magnification 40, scale bar of 100 μm. Values are presented as total surface area or a percentage. Each symbol represents one mouse (white dots control, red dots Nrf2 -/- ) and the line represents the mean, *P<0.05. As Nrf2 deficiency alleviated atherosclerosis in both mouse models, we measured plasma lipid levels, and found that Nrf2 deficiency reduced plasma total cholesterol (29.3±5.9 vs. 25.5±7.8 mmol/l, p>0.05) after 6 weeks of HFD in LDLR -/- mice, but no longer after 12 weeks of HFD (32.7±3.2 vs. 34.3±6.0 mmol/l). In LDLR -/- ApoB 100/100 females, Nrf2 deficiency had no effect on plasma total cholesterol (6 months; 7.5±3.0 vs. 6.5±2.4 mmol/l, 12 months; 8.1±2.8 vs. 8.2±2.7 mmol/l), but it reduced plasma triglycerides significantly at 6 (1.8±0.3 vs. 1.2±0.3 mmol/l) and 12 (1.4±0.4 vs. 1.1±0.4 mmol/l) months of age. Supporting the favourable metabolic effects of Nrf2 deficiency, Nrf2 -/- LDLR -/- and Nrf2 -/- LDLR -/- ApoB 100/100 mice did not gain weight on the HFD or on a chow diet, when compared to their controls LDLR -/- and LDLR -/- ApoB 100/100 mice.

67 GLOBAL DEFICIENCY OF NRF2 PREDISPOSES TO MYOCARDIAL INFARCTION Although Nrf2 deficiency reduced atherogenesis in LDLR -/- ApoB 100/100 mice, Nrf2 deficiency significantly impaired the survival of LDLR -/- ApoB 100/100 female mice, as ~36% of the Nrf2 -/- mice (19/53) died suddenly or required to be euthanized between the ages of 5 to 12 months (Figure 16A). Post mortem dissection revealed spontaneous myocardial infarction (10/19) with respective dilation of the heart ventricles, pulmonary edema and coronary atherosclerosis (Figure 16B). Varied sizes of infarcted areas were observed in the left ventricle anterior wall histopathology (Figure 16C). Figure 16. The loss of Nrf2 impairs the survival of LDLR -/- ApoB 100/100 female mice, as it predisposes to myocardial infarction and sudden death (study II). A, Survival curve (%) of LDLR -/- ApoB 100/100 (n=53) and Nrf2 - /- -/- 100/100 -/- 100/100 -/- -/- 100/100 LDLR ApoB (n=44) female and LDLR ApoB (n=30) and Nrf2 LDLR ApoB (n=15) male mice up to the age of 12 months. B, Representative pictures of hematoxylin-eosin stained coronary arteries at aortic root level and in myocardium from Nrf2 -/- LDLR -/- ApoB 100/100 female mouse that died of myocardial infarction at the age of 7 months. The coronary arteries are indicated by black arrows and the aortic lumen by black star. C, Infarct scar in left ventricle (indicated by black arrows) of Nrf2 -/- LDLR -/- ApoB 100/100 female mice was stained with Masson Trichrome. Representative pictures are shown with a scale bar of 100 μm, statistical significance *P<0.05, **P<0.01 by Log-rank test. We investigate whether myocardial infarction and sudden deaths were consequences of vulnerable plaque phenotype and plaque rupture. In the aortic root, the plaque necrotic area was increased in Nrf2 -/- LDLR -/- ApoB 100/100 mice at the age of 6 months compared to LDLR -/- ApoB 100/100 mice. At the age of 12 months, the size of aortic necrotic core in relation to total lesion area grew less in Nrf2 -/- LDLR -/- ApoB 100/100 mice, but Nrf2 -/- aortic plaques showed increased plaque calcification in comparison to LDLR -/- ApoB 100/100 mice. As brachiocephalic artery is a typical site for a human-like rupture-prone plaque in mice, we analyzed the plaque morphology of brachiocephalic artery (Rosenfeld et al. 2000). Both LDLR -/- ApoB 100/100 and Nrf2 -/- LDLR -/- ApoB 100/100 mice showed advanced plaque morphology, such as large fibrocalcific and necrotic cores and lateral xanthomas (Figure 17A). Interestingly, Nrf2

68 42 deficiency increased lesion calcification (Figure 17B) and fibro fatty nodule area normalized to lesion area in LDLR -/- ApoB 100/100 mice. Collagen content (Figure 17C) or the proportion of smooth muscle cells (Figure 17D) were not significantly changed between the groups, but fibrous cap thickness was significantly reduced in Nrf2 -/- LDLR -/- ApoB 100/100 mice in comparison to LDLR -/- ApoB 100/100 mice (Figure 17E). Finally, plaque instability index was calculated as a ratio between the instability (macrophages, necrotic area) and stability (collagen, smooth muscle cells) factors. Interestingly, the ratio was increased in Nrf2 -/- LDLR - /- ApoB 100/100 mice (Figure 17F), as well as the cap to core ratio was reduced by Nrf2- deficiency in LDLR -/- ApoB 100/100 mice (Figure 17G). Figure 17. Nrf2 deficiency promotes unfavorable plaque phenotype in aged LDLR -/- ApoB 100/100 female mice (study II). LDLR -/- ApoB 100/100 (white dots) and Nrf2 -/- LDLR -/- ApoB 100/100 mice (red dots) were sacrificed at the age of 12 months and hallmarks of vulnerable plaque were analysed from brachiocephalic artery. A, The area of fibro fatty nodule was analysed from Movat s Pentachrome staining and normalized to total lesion area with representative pictures. B, Plaque calcification (%) (Alizarin Red S staining, black arrows depicit red-stained calcification) C, plaque collagen content and D, the number of plaque smooth muscle cells were measured in relation to total lesion area with representative pictures. E, Fibrous cap thickness (µm) was measured from Masson s Trichrome stained sections, with representative pictures from thinned cap (indicated by black arrows). F, Plaque instability index was measured as a ratio between the stabilizing and unstabilizing factors. G, Fibrous cap thickness was normalized to necrotic core area and indicated as a cap to core ratio. Representative pictures are shown with a scale bar of 100 μm. Each dot represents one mouse and the horizontal line represents the mean, *P<0.05, **P<0.01, ***P<0.001 by Student s t-test. Persistent chronic inflammation and oxidative stress within the atherosclerotic plaque are known to promote plaque maturation and to predispose to plaque rupture. The positive staining area of MCP-1 (Figure 18A) and 4-HNE (Figure 18B), a marker for lipid peroxidation and oxidative stress, in atherosclerotic lesions were increased in Nrf2 -/- LDLR -/- ApoB 100/100 mice compared to LDLR -/- ApoB 100/100 mice. Moreover, we analyzed the expression of pro-inflammatory cytokines in the bone marrow derived macrophages, harvested from

69 43 Nrf2 -/- LDLR -/- ApoB 100/100 and LDLR -/- ApoB 100/100 mice. Nrf2 deficiency increased the expression of MCP-1, IL-6, IL-1β and TNFα, markers of the proinflammatory state, when macrophages were exposed to modified LDL or LPS. These results support the notion that the loss of Nrf2 possibly promotes a proinflammatory phenotype in macrophages, locally found in the vascular wall that may contribute to the plaque destabilization. Figure 18. Nrf2 deficiency promotes inflammation and oxidative stress in atheroma plaque (study II). LDLR -/- ApoB 100/100 (white dots) and Nrf2 -/- LDLR -/- ApoB 100/100 mice (red dots) were sacrificed at the age of 12 months and plaque A, MCP-1 and B, 4-HNE positive staining area were quantified in relation to plaque total area. The horizontal line represents the mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 by Student s t-test NRF2 DEFICIENCY ALLEVIATES HEPATIC STEATOSIS In this thesis, we have demonstrated that Nrf2 deficiency reduces atherosclerosis due to metabolic effects like lowering of plasma cholesterol levels in LDLR -/- mice after feeding a HFD. Thus, we investigated the role of Nrf2 in HFD-induced hepatic steatosis in LDLR -/- mice. The mice were fed with a HFD for 12 weeks to gain weight and to induce hepatic steatosis with mild inflammation and fibrosis. Nrf2 deficient mice were at least partially protected against HFD-induced weight gain as Nrf2 deficiency almost halted body weight gain during the HFD. As glucose metabolism is known to be disturbed as a consequence of liver lipid accumulation and obesity, we measured plasma fasting glucose level and found that it was equal between the genotypes on a chow diet, but reduced by Nrf2 deficiency after the HFD, suggesting improved liver insulin sensitivity in Nrf2 -/- LDLR -/- mice in comparison to LDLR -/- controls. Histological assessment of liver sections revealed that steatosis was very mild in both genotypes on a chow diet (score <1), but Nrf2 -/- LDLR -/- mice displayed reduced macrosteatosis (score 1) compared to LDLR -/- controls (score 2-3) after feeding a HFD (Figure 19A,C). This was confirmed with Oil Red O staining, which demonstrated significantly reduced lipid staining in Nrf2 -/- LDLR -/- mice compared to LDLR - /- controls (Figure 19B,D).

70 44 Figure 19. Loss of Nrf2 reduced HFD-induced hepatic steatosis in LDLR -/- mice (study III). A, Representative pictures of hematoxylin-eosin and B, Oil red O stained liver sections from Nrf2 -/- LDLR -/- and LDLR -/- mice after 6 months on a chow diet and after 12 weeks of HFD. C, Hepatic macrosteatosis was scored by standard criteria and D, lipid area was measured from Oil red O stained sections. Representative pictures are shown with a scale bar of 100 μm, statistical significance *P<0.05, ***P<0.01 by Student t-test. Assessed by histology, lobular inflammation, fibrosis or drop-out necrosis were not present on a chow diet, but were observed after the HFD, but at lower stage in Nrf2 deficient mice compared to LDLR -/- controls (Figure 20A-B). As liver resident macrophages are known to be activated by lipid accumulation, subsequently promoting the migration of circulating monocytes into the liver, we stained liver macrophages with CD68 primary antibody (Figure 20C), as a marker for liver macrophages and their activation. We found that Nrf2 deficiency increased the mrna expression of macrophage marker CD68 and CD68 + liver macrophage number before and after 12 weeks of HFD (Figure 20D). Interestingly, hepatic mrna expression of pro-inflammatory cytokines, such as MCP-1 and IL-6 did not differ between the genotypes, whereas the expression of Tnfα and IL-1β was significantly reduced in Nrf2 deficient mice after 12 weeks of HFD (Figure 20E). As the liver lipid accumulation may potentially increase oxidative stress, we stained the sections with 3- NT (3-nitrotyrosine) primary antibody, which recognizes 3-NT, a byproduct of lipid peroxidation, and it is usually upregulated by oxidative stress. According to measure oxidative stress, we also measured the mrna expression of NQO1, which is Nrf2 target gene and its expression is induced by oxidative stress. Interestingly, neither 3-NT nor NQO1 were changed via Nrf2 deficiency on the HFD.

71 45 Figure 20. Nrf2 deficiency inhibits liver inflammation, macrophage activation and fibrosis (study III). Liver A, inflammation and B, necrosis were scored according to standard criteria from hematoxylin-eosin stained sections from LDLR -/- and Nrf2 -/- LDLR -/- mice on a chow diet at age of 6 months and after 12 weeks of HFD. C, Liver macrophages were stained with CD68 primary antibody and D, macrophage number was calculated. E, mrna expression of pro-inflammatory cytokines were measured from LDLR -/- (white bars) and Nrf2 -/- LDLR -/- (red bars) liver after 12 weeks of HFD. Hepatic de novo lipogenesis is the main source of liver triglycerides and it is known to be accelerated in NAFLD. We found that Nrf2 deficiency increased the expression of triglyceride synthesis regulating genes, such as SREBP1c, FAS and ACC1 mrna after the HFD, whereas SREBP1c expression was increased also on a chow diet in Nrf2 deficient mice. Fatty acid oxidation limits the lipid accumulation and protects liver against lipotoxicity. Thus, we measured the expression of liver β-oxidation regulating genes after 12 weeks of HFD, and found that Nrf2 deficiency enhanced the mrna expression of mitochondrial acetyl-coenzyme A acetyltransferase 2 (ACAT2), carnitine palmitoyltransferase 1α (CPT1α) and peroxisomal acyl-coenzyme A oxidase 1 (ACOX1), indicating possibly accelerated mitochondrial and peroxisomal fatty acid oxidation in Nrf2 - /- LDLR -/- mice liver. Moreover, mitochondrial oxidative capacity and the amount of mitochondria were measured after the HFD. The loss of Nrf2 elevated the expression of subunits of oxidative phosphorylation complexes succinate dehydrogenase subunit B (SDHB) (CII), cytochrome c oxidase subunit 1 (MTCO1, CIV), cyclo-oxygenase 2α (COX2a) (CIV), adenosine triphosphate synthase lipid-binding protein 5G (ATP5G1) (CV) and phospholipid-transporting ATPase 8B (ATP8b) (CV) mrna in comparison to LDLR -/- mice on a HFD. Moreover, the protein amounts of subunits of oxidative phosphorylation complex III, ubiquinol-cytochrome c reductase (UQCR) and complex IV subunit MTCO1 amounts were significantly increased in Nrf2 -/- LDLR -/- mice (Figure 21A), as well as the hepatic complex IV activity was significantly increased in Nrf2 deficient mouse liver after

72 46 12 weeks of HFD (Figure 21B). The amount of mitochondrial DNA did not differ between the groups in the liver after feeding a HFD, indicating that mitochondrial oxidative metabolism is not enhanced by increased mitochondrial number. Figure 21. Nrf2 deficiency improves liver mitochondrial function in LDLR -/- mice (study III). Nrf2 -/- LDLR -/- and LDLR -/- mice were fed a chow or HFD for 12 weeks. A, The protein amount of oxidative phosphorylation complex subunits were measured by western blot from Nrf2 -/- LDLR -/- (n=4) and LDLR -/- (n=4) mice livers and analyzed in relation to VDAC (checked bars) and β-actin (clear bars). B, The activity of complex IV was measured by ELISA from Nrf2 -/- LDLR -/- and LDLR -/- mice livers after 12 weeks of HFD. Each bar represents the mean ± SD, statistical significance *P<0.05, **P<0.01 and ***P<0.001 by Student s t-test.

73 47 6 Discussion 6.1. Nrf2 - A DOUBLE-EDGED SWORD IN ATHEROGENESIS Nrf2 is a master regulator of stress defense against environmental and endogenous stressors, affording cytoprotection in several conditions. Nrf2 is activated by several endogenous and exogenous stressors, such as electrophiles, ER and oxidative stress as well shear stress that contribute in atherogenesis via direct and indirect effects on vascular cells or due to modification of lipoproteins, respectively. Nevertheless, a recent discovery showed that Nrf2 has not only anti-inflammatory effects by regulation of redox homeostasis, but it also transcriptionally suppresses the proinflammatory stimulus in macrophages (Kobayashi et al. 2016). Several Nrf2 target genes afford protection against vascular injury and atherosclerosis in the mouse models. For instance, increased expression of Nrf2 and its target genes, like HO-1, reduced secretion of pro-inflammatory cytokines and oxidative stress in the vascular wall in vivo, and provided atheroprotection in ApoE -/- mice (Jyrkkänen et al. 2008, Levonen et al. 2007, Juan et al. 2001). Whereas, macrophage specific or systemic loss of HO-1 promoted atherogenesis and plaque instability in ApoE -/- mice (Orozco et al. 2007, Yet et al. 2003). Observing more closely macrophage specific effects of Nrf2, two papers surprisingly concluded that deletion of Nrf2 reduced lipid uptake and foam cell formation by reducing CD36 scavenger receptor expression in mouse peritoneal macrophages (Sussan et al. 2008, Barajas et al. 2010). In addition, reduced CD36 expression in atherosclerotic plaque was supposed to mediate declined atherogenesis in Nrf2 -/- ApoE -/- mice (Harada et al. 2012). However, contrasting results have been reported and variable mechanisms by which Nrf2 regulates lipid uptake and macrophage foam cell formation have been discussed. Contrary to the two earlier reports, our study demonstrated increased lipid uptake and foam cell formation corresponding to increased expression of several scavenger receptors, independent of reduced CD36 expression, in Nrf2 -/- mouse peritoneal macrophages in ApoE -/- background. The evidence of the protective role of Nrf2 in atherogenesis was provided when we observed that macrophage specific deletion of Nrf2 enhanced early atherogenesis in LDLR -/- mice. These findings were further corroborated by the study by Collins et al., where Nrf2-deficiency in bone marrow-derived cell promoted advanced atherosclerosis in LDLR -/- mice after 7 months on a high cholesterol diet (Collins et al. 2012). Thus, our paper and results by Collins et al are completing each other, as the loss of Nrf2 in bone marrow cells appears to promote both early and advanced atherosclerosis in LDLR -/- mice. Despite various protective functions of Nrf2 in the vasculature, total Nrf2 deficiency has been shown to reduce atherogenesis in ApoE -/- mice in three independent studies, but each of them came to a different conclusion as to what is the prevailing mechanism (Sussan et al. 2008, Barajas et al. 2010, Freigang et al. 2011). Barajas et al suggested that reduced plasma total and non-hdl inhibited atherogenesis (Barajas et al. 2010). In contrast localized effects have also been suggested, including decreased foam cell formation via reduced CD36 expression in the plaque (Harada et al. 2012, Barajas et al. 2010), and Nrf2-dependent activation of IL-1β by cholesterol crystals (Freigang et al. 2011). In our study, in line with with the earlier findings, we demonstrated that Nrf2 deficiency decreased HFD-induced

74 48 early atherogenesis in LDLR -/- mice, as well as aging induced advanced atherogenesis in LDLR -/- ApoB 100/100 mice on a chow diet. Our data supported the systemic effects of Nrf2 via regulation of lipid metabolism, as we showed reduced plasma cholesterol or triglyceride levels, depending on the model and the diet. Notably, plasma lipid profiles were vastly different in our and other two studies (Barajas et al. 2011, Harada et al. 2012), possibly due to different study design and genetic backgrounds of the mice. In combination, all these papers demonstrate the role of Nrf2 at different stages of atherogenesis, as the duration of the diet varied from chow diet to 10 or 20 weeks of HFD, causing inevitably different systemic inflammatory and metabolic alterations and varying accumulation of hepatic steatosis, which possibly alone alters the metabolic profile (Sussan et al. 2008, Barajas et al. 2010). All these papers together offer an important knowledge of Nrf2 function in atherogenesis. Importantly, the selection of mouse model used in atherosclerosis studies should be critically discussed. In particular, it is widely known that ApoE -/- mice have disconcerting drawbacks as a model of human atherosclerosis, since ApoE -/- mice have non-physiological lipoprotein profile consisting mainly of VLDL and chylomicron remnants with ApoB48 as a corresponding apolipoprotein (Getz & Reardon 2016, Farese et al. 1996). In addition, ApoE regulates cholesterol efflux in macrophages and directly regulates macrophage- and T lymphocyte-mediated immune responses, which are integral in atherogenesis (Rosenfeld et al. 2008). In our studies, to clarify the macrophage specific and systemic role of Nrf2 in early and advanced atherogenesis, we used two alternative mouse models, LDLR -/- mice fed a HFD as well as chow-fed LDLR -/- ApoB 100/100 mice that have a more human-like hypercholesteromia compared to ApoE -/- mice. Oxidative stress and inflammation have a major role in the initiation of atherosclerosis, but also in the maturation of atherosclerotic plaque (Libby et al. 2002). Global Nrf2 deficiency alleviates atherogenesis in ApoE -/-, LDLR -/- and LDLR -/- ApoB 100/100 models, but the effect on the phenotype is dependent on the stage of the disease. In our study, LDLR -/- and Nrf2 -/- LDLR -/- mice were fed with a relatively short HFD to induce early plaques. Nrf2 -/- LDLR -/- mice represented type I-II plaques (resembling fatty streak) compared to type II-III (fatty streak with small necrotic area/lipid pool in plaque core) plaques in LDLR -/- mice. Nevertheless, LDLR -/- ApoB 100/100 mice have a more aggressive atherosclerotic phenotype in comparison to LDLR -/- mice, and the plaques were far more mature, containing enlarged necrotic, fibrotic and acellular areas (type III-IV), even on a chow diet at the age of 6 months. Interestingly, plaque maturation was enhanced in Nrf2 -/- LDLR -/- ApoB 100/100 female mice, as plaques contained more enlarged necrotic areas at age of 6 months and calcification at age of 12 months (type V) compared to LDLR -/- ApoB 100/100 controls. However, it is widely known that atherosclerotic mouse models develop more diverse plaque phenotype in the brachiocephalic artery than in aortic root, and the morphology of brachiocephalic plaque is more comparable to human rupture-prone plaque characteristics (Jackson et al. 2007). Thus, we used brachiocephalic artery to observe for the first time the effect of Nrf2 deficiency in plaque stability at that site. Interestingly, Nrf2 deficiency instigated several features of plaque instability, such as enlarged fibro fatty nodule area, increased calcification, thinned fibrous caps and reduced cap to core ratio. Furthermore, the loss of Nrf2 increased the expression of MCP-1 in plaque and enhanced the positive staining of oxidative stress marker, 4-HNE. In combination these findings support a protective role of Nrf2 locally in the vascular wall and in atheroma plaque. Notably, our data supported an anti-inflammatory role of Nrf2. Nrf2 deficiency elevated the expression of pro-inflammatory M1 macrophage phenotype markers, such as MCP-1, IL-6 and TNFα in peritoneal macrophages treated with modified LDL, as well as in bone marrow derived

75 49 macrophages treated with modified LDL and LPS. Moreover, macrophage specific Nrf2 deficiency increased the lesion area, but did not change the plaque composition, as 6 wk HFD-induced only fatty streaks in the LDLR -/- model. Given that continuous inflammation in the plaque is a key promoter in the progression of an atheroma to a rupture-prone plaque, the anti-inflammatory and antioxidative effects of Nrf2 in vascular macrophages possibly reduces foam cell formation in fatty streak plaque, improves survival of macrophages and inhibits vascular oxidative damage also in the late stage of the disease (Libby et al. 2002). This thesis strengthens the understanding of the dual role of Nrf2 in the regulation of atherogenesis. Further investigation need to be directed at fully characterizing the role of Nrf2 on macrophage gene expression and the possible function in relation to atherosclerotic plaque stabilization, with the help of tissue-specific gene modified mice THE ROLE OF NRF2 IN SPONTANEOUS MYOCARDIAL INFARCTION AND SUDDEN DEATH As LDLR -/- ApoB 100/100 and Nrf2 -/- LDLR -/- ApoB 100/100 mice were aged in our studies, ~40% of Nrf2 -/- LDLR -/- ApoB 100/100 female mice had myocardial infarction, died suddenly or were euthanized because of paraplegia, indicative of stroke. These findings are remarkable as coronary artery stenosis followed by spontaneous myocardial infarction almost never develop in atherosclerotic mouse models. Spontaneous plaque rupture and thrombosis, the most common life threatening complications in human atherosclerosis, are really rarely observed in mice as well. This is a consequence of physiological differences between mice and human, as the biological ability of thrombus formation is declined in mice via accelerated fibrinolysis, therefore a thrombus is almost impossible to detect, and mouse euthanasia and dissection of the tissues challenge the observation (Jackson et al. 2007). At present, the only genetic mouse model demonstrating stroke and myocardial infarction due to coronary plaque rupture and thrombosis, is the FbnC1039 +/- ApoE -/- mice, which die suddenly at the age of 16 to 23 weeks when fed with prolonged HFD (Van Herck et al 2009, Van Der Donckt et al 2015). Similar to our Nrf2 -/- LDLR -/- ApoB 100/100 mice, FbnC1039 +/- ApoE -/- mice showed coronary artery stenosis and advanced atherosclerosis in the aortic root and the brachiocephalic artery (Van der Donckt et al. 2015). Perceiving the limitations of mouse models, it is difficult to assess whether the sudden death is due to plaque rupture or pronounced coronary artery plaque formation. Nevertheless, neuronal, psychomotor and cognitive impairment are not excluded from contributing to the neurological symptoms and stroke in the Nrf2 -/- LDLR -/- ApoB 100/100 mice, as these neurological disorders have been reported in LDLR -/- ApoB 100/100 background due to hypercholesterolemia (Pineda et al. 2011), but we did not dissect or image the brains in our study. This thesis show for the first time that Nrf2 deficiency promote plaque instability in a mouse model of atherosclerosis, affording a novel concept for the treatment of Nrf2-mediated atherosclerotic plaque stabilization.

76 THE ROLE OF NRF2 IN REGULATION OF METABOLISM AND HEPATIC STEATOSIS Fatty liver disease and steatohepatitis are linked to atherosclerosis due to similar risk factors and disease pathogenesis. Nrf2 has been hypothesized to protect against these particular diseases, in which oxidative stress and inflammation are the key factors of disease progression, although the precise mechanisms remain unknown (Hayes et al 2000, Tang et al 2014). Notably, Nrf2 deficiency has also systemic metabolic effects that appear to alleviate atherosclerosis, as well as hepatic steatosis. In line with our previous results that Nrf2 deficiency reduced lipid accumulation in the arterial wall in LDLR -/- mice after 6 and 12 weeks of HFD and in LDLR -/- ApoB 100/100 mice on a chow diet, we demonstrated that it also reduced body weight gain in both models and decreased hepatic steatosis in LDLR -/- mice. The role of Nrf2 in metabolic regulation has been studied in mice, but mainly in C57Bl wild-type mice, which do not mimic human NAFLD, atherosclerosis or other metabolic disorders. Varying mechanisms explaining how Nrf2 deficiency can reduce body weight have been proposed, such as improved energy expenditure by increased UCP-1 expression via oxidative stress (Liu et al. 2016, Schneider et al. 2016) or by inhibiting adipogenesis due to impaired PPARγ signaling. In addition, Nrf2 deficiency reduced blood fasting glucose in LDLR -/- mice after the HFD, suggesting that Nrf2 deficiency improves insulin sensitivity as a result of inhibited white adipose tissue accumulation and reduced hepatic steatosis. This may partially explain the reduced atherogenesis, in addition impaired insulin signaling is known to promote atherogenesis by promoting endothelial dysfunction, increasing pro-inflammatory cytokine secretion and apoptosis in the vascular wall (DeFronzo 2010). Varying results, depending on the mouse model and the diet used, have been reported regarding the effect of Nrf2 on lipogenesis (Barajas et al. 2011, Kitteringham et al. 2010, Huang et al. 2012, Temel & Rudel 2007). Our study demonstrates increased expression of genes regulating lipogenesis in the liver of Nrf2 deficient mice either on a chow or HFD. Dysregulated mitochondrial fatty acid oxidation is known to promote the development of NAFLD (Nassir & Ibdah 2014). The role of Nrf2 in fatty acid oxidation is not clear, albeit reduced mrna expression of fatty acid oxidation enzymes by Nrf2 deficiency was previously reported (Tanaka et al. 2012, Lundtmann et al. 2014). However, our results demonstrate the opposite, as mrna expression of genes regulating β-oxidation and several oxidative phosphorylation complex subunits, protein expression of complex III and IV, and complex IV activity were increased in Nrf2 deficient mice in comparison to LDLR -/- controls after the HFD, indicating enhanced mitochondrial oxidative metabolism. Mitochondrial oxidative metabolism is possibly upregulated in response to increased fatty acid synthesis and improved insulin signaling, in addition alleviated inflammation and uniform oxidative stress stimuli have possibly improved mitochondrial function in Nrf2 deficient LDLR -/- mice, as pro-inflammatory cytokines and ROS are known to promote mitochondrial dysfunction and progression of NASH. The role of Nrf2 in mitochondrial function has not been widely studied related to metabolic disorders. Thereby, our study revealed an important role of Nrf2 in the regulation of peripheral mitochondrial function. Hepatic and vascular lipid accumulation is known to increase the secretion of proinflammatory cytokines and to promote oxidative stress. Locally in the vascular wall, Nrf2 appears to have anti-inflammatory and antioxidative properties, as well as in the liver, since the deletion of Nrf2 in bone marrow cells is reported to accelerate hepatic inflammation and fibrosis by increased inflammation and oxidative stress in LDLR -/- mice after prolonged HFD (Collins et al. 2012). In the present study, global Nrf2 deficiency reduced hepatic lobular inflammation and the expression of pro-inflammatory cytokines on

77 51 the HFD in comparison to LDLR -/- controls, this is likely due to reduced lipid accumulation. Nevertheless, Nrf2 deficiency increased the hepatic mrna expression of CD68, a marker of macrophage activation, and the number of CD68 + macrophages on a chow and HFD. CD68 has been proposed to act as a scavenger receptor, because of its ability to bind different ligands including modified LDL and apoptotic cells (Kunjathoor et al. 2002). Both increased expression of pro-inflammatory cytokines and enhanced phagocytic activity have been shown to induce the expression of CD68, whose role in disease pathogenesis is not well known. As the expression of pro-inflammatory cytokines was not increased by Nrf2 deficiency on the HFD in our study, the CD68 expression and the number of CD68 + macrophages are more likely elevated due to improved phagocytic activity, which is known to protect against the development of NAFLD. The liver phagocytic activation is usually induced via multiple mechanisms, such as enhanced inflammation and lipogenesis. Thus, one possible cause for increased liver macrophage phagocytic activity could be an increased fatty acid synthesis in Nrf2 deficient mice. In contrast, inflammation most likely does not play a role in the initiation of phagocytic activation in Nrf2 -/- mice, because the expression of chemokine MCP-1 that is known to induce the recruitment of circulating monocytes to liver was increased in Nrf2 deficient mice only on a chow diet and, not on the HFD. According to these results, we propose that the increased number of CD68 + macrophages in the liver might indicate improved phagocytic activation of liver macrophages that may resolve inflammation and restore the tissue, independent of oxidative stress, which was equal between the genotypes after the HFD. Although lipid accumulation is known to promote oxidative stress in the liver, the effect of Nrf2 on lipidinduced oxidative stress has not been much studied and requires further investigation (Rolo et al. 2012). These results reveal that Nrf2 could be an interesting mediator of liver inflammation and macrophage activation. Nevertheless, Nrf2 is associated with hepatocellular carcinoma and enhanced accumulation of CD68 + macrophages, which has been reported to provide a favorable environment for liver tumorigenesis, correlating with cancer progression and reduced patient survival (Wu et al. 2014, Wan et al. 2009, Ding et al. 2016). There is a need to investigate the effects of the global and liver-specific Nrf2 deficiency on liver inflammation after long-term HFD, as previously it was shown that at least 20 weeks of HFD is required to induce mild NASH with insulin resistance using the LDLR -/- mouse model (Merat et al. 1999). To summarize, this study provides novel information on the systemic effects of Nrf2 in the regulation of inflammation, macrophage activation and peripheral mitochondrial metabolism in the liver. These findings challenge the concept that Nrf2 is strictly a protective transcription factor, as Nrf2 seem to have a dual role via its contrasting systemic and local properties in different disease pathologies, including atherosclerosis and hepatic steatosis. Nevertheless, the precise mechanisms require further investigation NRF2 AS A THERAPEUTIC TARGET AND FUTURE PERSPECTIVES Atherosclerosis and NAFLD are both strongly associated in obesity, metabolic syndrome and hypercholesterolemia, causing life-threatening complications worldwide (Fabbrini et al. 2010, Tabas 2010). Presently, the prevention and treatment of atherosclerosis and fatty liver disease are focused on life style modification, such as healthy diet, body weight and plasma lipid lowering, as well as treatment of diabetic factors. Pathogenesis of these multifactorial diseases is quite well known, but there is still need for investigation of novel pathways to improve diagnosis and prediction of complications, as well as to develop new medical treatments. For instance, Nrf2 activators are suggested to used for the treatment of

78 52 NAFLD and NASH (Sharma et al. 2018, Vega et al. 2018). This is conflicting, as we and others have demonstrated that Nrf2 deficiency protects against atherosclerotic plaque development and hepatic steatosis in hyperlipidemic mice. Hence, Nrf2 inhibitors could be considered for the treatment of atherosclerosis and fatty liver disease as well, based on the plasma total cholesterol reducing and energy metabolism improving properties. Alternatively, the activation of Nrf2 has been tested in different animal models, and found to have beneficial metabolic effects as well, not forgetting local vascular effects, such as reduced oxidative stress, pro-inflammatory cytokine expression and smooth muscle cell migration (Levonen et al. 2007, Hur et al. 2010, Uruno et al. 2013, Chambel et al. 2015). On this account, Nrf2 activation could possibly be targeted to particular vascular cells, such as endothelial cells or monocyte-macrophages, given the stage of the disease. Several transcription factors including Nrf2, are ubiquitously expressed, and thereby global genetic deficiency or over-expression is inevitably multifold, making the causality and interpretation of the data complicated, thus highlighting the need for tissue-specific animals using cre/lox technology to investigate the exact mechanisms. Pharmacological activators of Nrf2 have been developed to improve cellular defense against oxidative and electrophilic stress that contribute to several chronic inflammatory diseases. Currently, some Nrf2 activators are commercially available, as dietary supplements, which have been recognized in the field of nutrigenomics and nutrigenetics but also as medical treatment for chronic inflammatory diseases (Eggler et al. 2008, Houghton et al. 2016). Dimethyl fumarate (Tecfidera, Biogen) was the first Nrf2 activator to be accepted into clinical use for the treatment of relapsing remitting multiple sclerosis (Schweckendiek et al 1959, Gold et al. 2012). In addition, other Nrf2 activators, such as dimethylfumarate combined with three other fumaric acid esters, and oleanolic acid derivate (semisynthetic triterpenoid), have been under development for the treatment of psoriasis and chronic kidney disease, respectively (Mrowietz et al. 2007, Madlala et al. 2015). Nevertheless, oleanolic acid derivate was not a success in clinical phase III trial as it increased cardiovascular events and did not reduce the risk of end-stage renal disease or death (Zeeuw et al. 2013). Nrf2 was originally perceived to inhibit tumor initiation and cancer metastasis, but later on several papers revealed its role as a pro-oncogenic factor since several cancers exhibit increased activation of Nrf2, which has been reported to increase the survival of cancer cells, enhance chemoresistance and increase cancer progression (Milkovic et al. 2017). In addition, accumulation of fumarate, caused by mutations in the gene coding fumarate hydratase enzyme, is known to associate strongly with aggressive renal cell carcinoma and leiomyomas (Zheng et al. 2015). Fumarate is an electrophile, which can covalently bind to cysteine residues of proteins, like the Nrf2 inhibitor KEAP1. This binding inhibits KEAP1 and subsequently increases Nrf2 activation that possibly improves the survival of cancer cells. Thus, Tecfidera or other Nrf2 activators might be risky to use for cancer patients, at least during chemotherapies. Since, Nrf2 activators are under development and have already been used for long-term treatment and in young adults, it is highly important to investigate their effects on metabolism and the cardiovascular system. Moreover, it is notable that HFD possibly regulates Nrf2 expression, suggesting a nutritional influence on Nrf2 activation. Although, not much is known about the effect of Nrf2 on lipid accumulation-induced oxidative stress, it is an important factor to consider when developing treatments for hyperlipidemia related chronic inflammatory disorders (Tanaka et al. 2008, Camer et al. 2015, Schneider et al. 2016).

79 53 7 Summary and conclusion The main findings of this thesis were: 1. Macrophage specific Nrf2 deficiency promoted foam cell formation due to increased uptake of modified LDL mediated by elevated expression of scavenger receptors and proinflammatory cytokines (M1) in vitro. These actions possibly led to an enhanced early atherogenesis in macrophage specific Nrf2 deficient mice in atherosclerotic LDLR -/- background. 2. Global Nrf2 deficiency protected against early and advanced atherogenesis, but predisposed to myocardial infarction and sudden death in LDLR -/- ApoB100-only mice, possibly due to an unstable plaque phenotype, as Nrf2 deficiency increased local plaque inflammation and oxidative stress. 3. Global Nrf2 deficiency reduced high-fat diet induced hepatic steatosis via enhanced mitochondrial oxidative metabolism in LDLR -/- mice. This thesis expands our knowledge of the effects of Nrf2 on metabolic and inflammation related diseases, such as atherosclerosis and hepatic steatosis, but also reveals novel questions about the metabolic and inflammatory actions of Nrf2. Our results demonstrated that Nrf2 deficiency has a dual role in the development of atherosclerosis and hepatic steatosis in hypercholesterolemic mice. Nrf2 regulates the formation of foam cells as Nrf2 deficiency increases lipid accumulation in macrophages due to enhanced expression of several scavenger receptors and a pro-inflammatory phenotype, further aggravating early atherogenesis in hyperlipidemic mice (Figure 22). However, systemic effects of Nrf2 on atherogenesis are partially contrasting with local vascular effects, as global Nrf2 deficiency downregulates plasma lipid levels, subsequently reducing both early and advanced atherogenesis in the hypercholesterolemic mouse models. In addition, global Nrf2 deficiency protects against hepatic steatosis, possibly via improved hepatic mitochondrial oxidative metabolism in hypercholesterolemic mice. Supporting local vascular protective effects of Nrf2, global Nrf2 deficiency promotes plaque inflammation and oxidative stress that accelerates plaque calcification and necrosis, reduces fibrous cap thickness and cap to core ratio, further leading to increased plaque instability and possibly spontaneous myocardial infarction in the atherosclerotic mice.

80 54 Figure 22. Summary of the local vascular and systemic effects of Nrf2 deficiency on atherogenesis and hepatic steatosis.

81 55 8 References Adamson, S. & Leitinger, N., Phenotypic modulation of macrophages in response to plaque lipids. Current opinion in lipidology, 22(5), pp Akram, O.N. et al, Beyond LDL cholesterol, a new role for PCSK9, Arteriosclerosis, Thrombosis, and Vascular Biology, 30(7), pp Barajas, B. et al., NF-E2-related factor 2 promotes atherosclerosis by effects on plasma lipoproteins and cholesterol transport that overshadow antioxidant protection. Arteriosclerosis, Thrombosis, and Vascular Biology, 31(1), pp Bea, F. et al., Induction of Glutathione Synthesis in Macrophages by Oxidized Low- Density Lipoproteins Is Mediated by Consensus Antioxidant Response Elements. Circulation Research, 92(4), pp Benedict, M. et al., Non-alcoholic fatty liver disease : An expanded review. World J Hepatol, 9(16), pp Bentzon, J.F. et al., Mechanisms of plaque formation and rupture. Circulation Research, 114(12), pp Bentzon, J.F. & Falk, E., Atherosclerotic lesions in mouse and man: is it the same disease? Current opinion in lipidology, 21(5), pp Bhatia, L.S. et al., Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor? European heart journal, 33(10), pp Bieghs, V. et al., LDL receptor knock-out mice are a physiological model particularly vulnerable to study the onset of inflammation in non-alcoholic fatty liver disease. PloS one, 7(1), p.e Bilzer, M., Roggel, F. & Gerbes, A.L., Role of Kupffer cells in host defense and liver disease. Liver international : official journal of the International Association for the Study of the Liver, 26(10), pp Birben, E. et al., Oxidative Stress and Antioxidant Defense. World Allergy Organ J, 5(1), pp Bisgaard, L.S. et al., Bone marrow-derived and peritoneal macrophages have different inflammatory response to oxldl and M1/M2 marker expression implications for atherosclerosis research. Scientific Reports, 6, p Boon, R.A. & Horrevoets, A.J.G., Key transcriptional regulators of the vasoprotective effects of shear stress Shear stress. Hamostaseologie, 29(10), pp Borén, J. et al., Identification of the Principal Proteoglycan-binding Site in LDL A Single-Point Mutation in apo-b100 Severely Affects Proteoglycan Interaction without Affecting LDL Receptor Binding. J Clin Invest, 101(12), pp Braun, A. et al., Myocardial infarction following atherosclerosis in murine models. Curr Drug Targets, 9(3), pp Bretscher, P. et al., Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf 2. EMBO molecular medicine, 7(5), pp Brunt, E.M. & Tiniakos, D.G., Histopathology of nonalcoholic fatty liver disease. World journal of gastroenterology : WJG, 16(42), pp

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