TAINA VUORIO VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3 AND ITS LIGANDS IN CARDIOVASCULAR DISEASES

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1 PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences TAINA VUORIO VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3 AND ITS LIGANDS IN CARDIOVASCULAR DISEASES

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3 Vascular Endothelial Growth Factor Receptor 3 and Its Ligands in Cardiovascular Diseases

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5 TAINA VUORIO Vascular Endothelial Growth Factor Receptor 3 and Its Ligands in Cardiovascular Diseases To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Tietoteknia auditorium, Kuopio, on January 25 th, 2019, at 12 noon Publications of the University of Eastern Finland Dissertations in Health Sciences Number 497 A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland Kuopio 2019

6 Grano Jyväskylä, 2019 Series Editors: Professor Tomi Laitinen, M.D., Ph.D. Institute of Clinical Medicine, Clinical Radiology 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:

7 III Author s address: Supervisor: Reviewers: A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND taina.vuorio@uef.fi Professor Seppo Ylä-Herttuala, M.D., Ph.D. A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND Professor Sohvi Hörkkö, M.D., Ph.D. Research Unit of Biomedicine Faculty of Medicine University of Oulu OULU FINLAND Docent Raisa Serpi, Ph.D. Biocenter Oulu Faculty of Biochemistry and Molecular Medicine University of Oulu OULU FINLAND Opponent: Professor Hannu Järveläinen, M.D., Ph.D. Department of Internal Medicine University of Turku TURKU FINLAND Satakunta Central Hospital PORI FINLAND

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9 V Vuorio, Taina Vascular Endothelial Growth Factor Receptor 3 and Its Ligands in Cardiovascular Diseases University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences p. ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: ABSTRACT Cardiovascular diseases are one of the main causes of morbidity and mortality in the world. Lifestyle and inherited risk factors predispose patients to high lipid levels, which underly the development of atherosclerosis and its manifestations myocardial infarction (MI) and stroke. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are the major regulators of vascular and lymphatic systems. The aim of this thesis was to study the role of VEGFR3 and its ligand VEGF-D in lipoprotein metabolism as well as in atherogenesis and MI. In the first study, we investigated the effects of attenuated lymphatic function on the development of hyperlipidemia and atherosclerosis. We found that the expression of soluble decoy VEGFR3 (svegfr3) on atherosclerotic Ldlr -/- /Apob 100/100 background caused severe hypercholesterolemia on mice fed a Western-type high fat diet. Furthermore, svegfr3 mice had fewer lymphatic vessels in their vascular wall as well as accelerated atherogenesis. These results elucidated the role of the lymphatic system in the regulation of lipoprotein metabolism and development of atherosclerosis. Subsequently, we evaluated the role of VEGF-D in lipoprotein metabolism and atherogenesis. VEGF-D knockout (KO) mice on Ldlr - /- /Apob 100/100 background developed severe hyperlipidemia when exposed to a high-fat diet. Mechanstically, the deletion of VEGF-D led to the reduced levels of hepatic Syndecan 1 (SDC1) leading to the retention of chylomicron remnant particles in the blood. However, VEGF-D KO mice displayed similar levels of atherosclerosis than controls. We concluded that VEGF-D regulates chylomicron remnant uptake in the liver and its deletion leads to the accumulation of these large lipoproteins in plasma. However, the accumulated particles are not able to penetrate through the vascular endothelium and cause accelerated atherogenesis. In the third study, the role of VEGFR3 was evaluated in the healthy hearts and after MI. We found that the structure of cardiac lymphatic network was modified in mice with attenuated VEGFR3 signaling. Furthermore, svegfr3 mice had a significantly higher mortality after MI than their littermates. Novel MRI methods and histology revealed hemorrhages and a modified structure of the infarcted area in svegfr3 mice, which might have predisposed these mice to early cardiac failure. These findings suggest an important role for VEGFR3 in the healing process after MI. In conclusion, this thesis highlights the significance of VEGFR3 in the regulation of lipoprotein metabolism at least in mice. Furthermore, the attenuation of VEGFR3 signaling can lead to the aggravation of cardiovascular diseases. National Library of Medicine Classification: QU 85, QU 107, WG 120, WG 550, WH 700 Medical Subject Headings: Vascular Endothelial Growth Factor Receptor-3; Vascular Endothelial Growth Factors; Blood Vessels; Lymphatic System; Lymphatic Vessels; Cardiovascular Diseases; Hypercholesterolemia; Atherosclerosis; Myocardial Infarction; Lipoproteins/metabolism; Syndecan-1; Chylomicron Remnants; Mortality; Models, Animal; Mice

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11 VII Vuorio, Taina VEGFR3:n ja sen ligandien merkitys sydän- ja verisuonisairauksissa Itä-Suomen yliopisto, Terveystieteiden tiedekunta Publications of the University of Eastern Finland. Dissertations in Health Sciences s. ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: TIIVISTELMÄ Sydän- ja verisuonitaudit ovat merkittävimpiä kuolleisuuden aiheuttajia maailmassa. Elintavat ja perinnölliset riskitekijät altistavat hyperlipidemialle, mikä voi johtaa ateroskleroosin kehittymiseen sekä lopulta sydäninfarktiin tai aivohalvaukseen. Verisuonten endoteelin kasvutekijät (VEGF:t) ja niiden reseptorit (VEGFR:t) ovat verenkiertoelimistön ja imusuoniston tärkeimpiä säätelijöitä. Tässä väitöskirjassa tutkittiin VEGFR3-reseptorin ja sen ligandi VEGF-D:n merkitystä lipoproteiinien aineenvaihdunnassa sekä ateroskleroosin kehittymisessä ja sydäninfarktin jälkeisessä parantumisessa. Ensimmäisessä osatyössä tutkittiin heikentyneen imusuonikierron vaikutuksia hyperlipidemian ja ateroskleroosin kehittymiseen. Tutkimuksessa havaittiin, että liukoisen VEGFR3:n (svegfr3) ilmentyminen hiirissä johti merkittävään hyperkolesterolemiaan. Lisäksi svegfr3-hiirillä oli vähemmän imusuonia ateroskleroottisissa valtimoissa ja nopeutunut aterogeneesi. Toisessa osatyössä tutkittiin VEGF-D kasvutekijän merkitystä lipoproteiinien aineenvaihdunnassa ja aterogeneesissä. VEGF-D poistogeenisille hiirille kehittyi merkittävä hyperlipidemia rasvadieetillä. VEGF-D:n puuttuminen johti heikentyneeseen maksan syndekaanin 1 (SDC1) ilmentymiseen, mikä aiheutti ruokavalion rasvoja kuljettavien kylomikronien kertymiseen verenkiertoon. VEGF-D poistogeenisissä hiirissä ei kuitenkaan havaittu lisääntynyttä ateroskleroosia. Tulosten perusteella voidaan todeta, että VEGF-D säätelee kylomikronien sisäänottoa maksaan. Suuret kylomikronipartikkelit eivät kuitenkaan pysty tunkeutumaan verisuonten endoteelin läpi ja aiheuttamaan nopeutunutta ateroskleroosin kehittymistä. Kolmannessa osatyössä tutkittiin VEGFR3:n merkitystä sydäninfarktissa. svegfr3:n ilmentyminen johti laajentuneisiin imusuoniin terveissä hiirten sydämisssä. svegfr3-hiirillä oli huomattavasti suurempi kuolleisuus sydäninfarktin jälkeen kuin kontrollihiirillä. Uudenlaisilla magneettikuvausmenetelmillä havaittiin, että svegfr3-hiirten infarktialueen rakenne oli muuttunut. Lisäksi svegfr3-hiirten sydämissä havaittiin verenpurkaumia, mikä on mahdollisesti johtanut sydämen heikentyneeseen toimintakykyyn ja sydämen vajaatoimintaan. Yhteenvetona voidaan todeta, että imusuonilla on merkittävä tehtävä lipoproteiinien aineenvaihdunnan säätelyssä käytetyissä hiirimalleissa. VEGFR3-reseptorin kautta tapahtuvan signaloinnin heikentyminen voikin johtaa sydän- ja verisuonitautien pahenemiseen. Luokitus: QU 85, QU 107, WG 120, WG 550, WH 700 Yleinen Suomalainen asiasanasto: kasvutekijät; reseptorit; imusuonisto; verisuonet; sydän- ja verisuonitaudit; ateroskleroosi; sydäninfarkti; hyperkolesterolemia; hyperlipidemia; lipoproteiinit; kuolleisuus; koe-eläinmallit; hiiret

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13 IX I have never tried that before, so I think I should definitely be able to do that. Astrid Lindgren, Pippi Longstocking

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15 XI Acknowledgements This study was carried out in the A.I. Virtanen Institute for Molcular Sciences, University of Eastern Finland during the years I am sincerely thankful for all those people I was privileged to work with during all these years and who contributed to my research. First of all, my deepest gratitude belongs to my supervisor Professor Seppo Ylä-Herttuala. I am grateful for giving me the opportunity to work in his research group and for his continuous support during these years. You always have time to discuss and provide help and guidance. You have special skills to motivate your students and your positive attitude and in-depth knowledge of life and science is truly inspiring. My sincere gratitude goes to the official reviewers of this thesis, Professor Sohvi Hörkkö and Docent Raisa Serpi from the University of Oulu whose constructive comments and expert insights helped me to improve this thesis. I am also deeply grateful for Professor Hannu Järveläinen for accepting to be my opponent at the public defence. I would also like to thank Thomas Dunlop for the linguistic revision. This thesis would not have been possible without all the excellent co-authors I have been privileged to work with. First, I would like to thank Annakaisa Tirronen for collaboration and friendship. You are innovative and forward-thinking and I am fortunate that we have worked together. I think we compensated each other and ended up with an excellent study. Elias Ylä-herttuala, thank you for collaboration during the third study of my thesis. MRI stuff is almost incomprehensible to me and I was glad I could leave it with you. Thank you for being so positive and down-to-earth when my pessimistic mind took over. Our students and now colleagues Sanna Kettunen and Krista Hokkanen, thank you for your valuable help during the second study. My deepest gratitude goes to Svetlana Laidinen, your contribution to all my animal studies has been irreplaceable. I also want to thank Johanna Laakkonen, Henri Niskanen, Hanne Laakso, Minna Kaikkonen-Määttä and Timo Liimatainen for their expertise in methods I couldn t do myself. Jere Kurkipuro, Haritha Samaranayake and Tommi Heikura, thank you for your help and scientific and non-scientific discussions especially during my first years of research when I was clueless of everything. I would also like to express my deepest gratitude to the collaborators at the University of Helsinki, National Institute of Health and Welfare, University of Denver and University of California San Diego. I want to thank all my present and former colleagues in SYH group for inspirational and relaxed atmosphere. I could not have wished for any better place to spend these years. I would especially like to thank my roommates Emmi, Sanna, Tiina, Venla, Pyry and Hanna for their unconditional help, inspiring discussions and friendship. All issues are big enough to be talked over in our office. I also want to thank our office neighbour Anna-Kaisa for sharing the hurdles of atherosclerosis research and helping me with the numerous small things before the dissertation. My sincere gratitude goes to all technical personnel who have helped me during these years. Anne, Maarit, Seija and Joonas, I would have been totally lost without you. Our excellent secretaries Helena, Marjo-Riitta, Jatta and Marja, sometimes the world of money, bureaucracy and SoleTM is almost incomprehensible but you have always provided help and solutions even to the most insuperable problems. I also want to thank the personnel of the lab animal center for the excellent care of our mice. Tiina, Anssi, Minttu and Miika, thank you for sharing the student life and for your longlasting friendship. I m grateful that you have been part of my life even when we are far apart. Laura, Heidi and Emmi, thank you for being my peers in science and all the memorable gettogethers we ve had during the years. I also want to thank my lovely Venla-team Suvi, Mirva

16 XII and Petra for taking me out from the lab and into the forests. MissMä Oon? will definitely beat them all some day. My loving thanks go to my dear family. Mum and dad, you have always made me feel that I could achieve anything I want in my life. Even if the whole world is trembling, the stronghold you have created will last. My big brother Antti and sister-in-law Laura and your beautiful children Elias and Emma, thank you for all the memorable get-togethers, travels and discussions. Thank you for being my secret idols and always pushing me forward. I am grateful for my parents-in-law Tuula and Kari for taking me into their lives with open arms. Finally, I want to thank my dearest other half Tuomas. You bring so much joy, happiness and goofiness in my life. I might not have survived these years without your precious Are you ready to go home? calls. I m eagerly waiting for all the adventures we will have together. Kuopio, January 2019 Taina Vuorio This study was supported by grants from Academy of Finland, Antti and Tyyne Soininen Foundation, European Research Council, Finnish Foundation for Cardiovascular Research, Ida Montini Foundation, Kuopio University Foundation, Kymenlaakso Regional Fund, Paolo Foundation, UEF Doctoral Program in Molecular Medicine, Urho Känkänen Foundation and Veritas Foundation.

17 XIII List of the original publications This dissertation is based on the following original publications: I II III Vuorio T, Nurmi H, Moulton K, Kurkipuro J, Robciuc MR, Ohman M, Heinonen SE, Samaranayake H, Heikura T, Alitalo K, Ylä-Herttuala S. Lymphatic vessel insufficiency in hypercholesterolemic mice alters lipoprotein levels and promotes atherogenesis. Arterioscler Thromb Vasc Biol Jun;34(6): Tirronen A*, Vuorio T*, Kettunen S, Hokkanen K, Ramms B, Niskanen H, Laakso H, Kaikkonen MU, Jauhiainen M, Gordts PLSM, Ylä-Herttuala S. Deletion of angiogenic and lymphangiogenic VEGF-D leads to hyperlipidemia and delayed clearance of chylomicron remnants. Arterioscler Thromb Vasc Biol Oct;38(10): Vuorio T, Ylä-Herttuala E, Laakkonen JP, Laidinen S, Liimatainen T, Ylä-Herttuala S. Downregulation of VEGFR3 signaling alters cardiac lymphatic vessel organization and leads to a higher mortality after acute myocardial infarction. Scientific Reports : *Authors with equal contribution The publications were adapted with the permission of the copyright owners.

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19 XV Contents 1 INTRODUCTION REVIEW OF THE LITERATURE Circulatory system Cardiovascular system Lymphatic system Cardiovascular diseases Hyperlipidemias Atherosclerosis Myocardial infarction Heart failure Mouse models for cardiovascular diseases Lipids and lipoprotein metabolism Plasma lipids Lipoproteins Apolipoproteins ApoA ApoB ApoC ApoE Lipoprotein receptors LDLR LRP HSPGs Other lipoprotein receptors Exogenous pathway Endogenous pathway HDL metabolism and reverse cholesterol transport Lymphatic system in lipid and lipoprotein metabolism VEGFs and their receptors VEGF-A VEGF-B VEGF-C VEGF-D Other VEGFs VEGF Receptors VEGFR VEGFR VEGFR NRPs AIMS OF THE STUDY MATERIALS AND METHODS... 31

20 XVI 4.1 Animals Analysis of lipid, lipoprotein and glucose metabolism Imaging Surgery Histology Molecular biology and cell culture Statistical methods RESULTS Attenuated lymphatic function leads to hypercholesterolemia and accelerated atherogenesis (I) VEGF-D regulates chylomicron metabolism (II) The expression of svegfr3 leads to higher mortality after myocardial infarction (III) DISCUSSION The role of VEGFR3 mediated signaling in lipid metabolism and atherosclerosis (I) The function of VEGF-D in lipoprotein metabolism (II) Cardiac lymphatic vessels in myocardial infarction (III) CONCLUSIONS REFERENCES APPENDIX: ORIGINAL PUBLICATIONS (I-III)

21 XVII Abbreviations ABCA A ABCG ACAT ACTA ATP binding cassette subfamily ATP binding cassette subfamily G Acetyl-coenzyme A acetyltransferase Actin alpha (α-sma) ADAMTS A Disintegrin and AKT ALAT metalloproteinase with thrombospondin motifs Protein kinase B Alanine transaminase ANGPTL Angiopoietin-like Apo Apolipoprotein APOBEC Apolipoprotein B mrna editing ARH ATGL enzyme, catalytic polypeptidelike Low-density lipoprotein receptor adapter protein Adipose triglyceride lipase CALCRL Calcitonin receptor-like receptor CCBE Calcium binding epidermal CD CETP CM COL growth factor domains Cluster determinant Cholesteryl ester transfer protein Chylomicron Collagen CVD Cardiovascular disease DLL Delta-like protein ECG Electrocardiography ECM Extracellular matrix EF Ejection fraction enos Endothelial nitric oxide synthase ER Endoplasmic reticulum ERK Extracellular signal regulated kinase FATP Fatty acid transport protein FC Fragment crystallizable FDA U.S. Food and Drug Administration FFA Free fatty acid FPLC Fast protein liquid chromatoraphy GPIHBP Glycosylphosphatidylinositol anchored high density lipoprotein binding protein GTT Glucose tolerance test HDL High-density lipoprotein HF Heart failure HIF Hypoxia inducible factor HMGB High mobility group box HMGCoA 3-Hydroxy3-methylglutaryl coenzyme A HMGCR HMG-CoA reductase

22 XVIII HPLC High performance liquid chromatography MRI mtorc Magnetic resonance imaging Mammalian target of rapamycin HSPG Heparan sulfate proteoglycan complex HTGL Hepatic triglyceride lipase MTP Microsomal triglyceride transfer ICAM Intercellular adhesion molecule protein IDOL E3 ubiquitin ligase inducible NDST N-deacetylase/N- degrader of LDLR sulfotransferase IHC Immunohistochemistry NF-kB Nuclear factor kappa-light-chain- IL Interleukin enhancer of activated B cells i.p. intraperitoneal NPC Niemann-Pick disease, type C i.v. intravenous NRP Neuropilin INSIG Insulin induced gene PCR Polymerase chain reaction ITT Insulin tolerance test PCSK Proprotein convertase KLF Krueppel-like factor subtilisin/kexin type KO LAD LAM Knockout Left anterior descending artery Lymphangioleiomyomatosis PGC Peroxisome proliferatoractivated receptor gamma coactivator LCAT Lecithin cholesterol PI3K Phosphatidylinositide 3-kinase acyltransferase PKC Protein kinase C LDL Low-density lipoprotein PLAGL Pleomorphic adenoma gene-like LDLR Low-density lipoprotein receptor PlGF Placental growth factor LEC Lymphatic endothelial cell p.o. per os (orally) LIPA Lysosomal acid lipase POSTN Periostin LMF Lipase maturation factor PROX Prospero homeobox protein LPL Lipoprotein lipase RCT Reverse cholesterol transport LRP LDL receptor related protein SEMA Semaphorin LYVE Lymphatic vessel endothelial hyaluronan receptor SMC SNP Smooth muscle cell Small nuclear polymorphism LVW MI Left ventricle wall Myocardial infarction SR-BI Scavenger receptor class B type 1

23 XIX SREBP Sterol regulatory elementbinding protein TGF TNF TRL VCAM Transforming growth factor Tumor necrosis factor Triglyceride rich lipoprotein Vascular cell adhesion protein VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor VHD VLDL VEGF homology domain Very low-density lipoprotein VLDLR Very low-density lipoprotein receptor WB WHO WM Western blot World Health Organization Whole mount

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25 1 Introduction Cardiovascular diseases (CVDs), such as myocardial infarction (MI) and stroke, are the leading causes of morbidity and mortality in the world despite extensive research, efforts for early prevention and advanced treatment options. Atherosclerosis is the main culprit of cardiovascular disease and it is characterized by the accumulation of lipids, inflammatory cells and necrotic material in the arterial wall. The development and aggravation of atherosclerosis can be prevented by pursuing a healthy lifestyle, such as eating healthy diet, increasing the amount of exercise and refraining from smoking. However, the prevention of the disease is challenging because the disease progression occurs without any major clinical symptoms and in many cases elevated plasma cholesterol, triglyceride and glucose levels are the only indications of the disease risk. While mortality from cardiovascular events has declined in Western societies, it remains high in middle- and low income countries. (Benjamin et al., 2017, Townsend et al., 2016). Lipids, such as cholesterol and triglycerides, are essential for normal physiological functions. They have a high calorie content that can be utilized as energy and they also serve as the structural components of cellular membranes and function as signaling molecules. In plasma, both diet-derived and endogenously produced cholesterol, triglycerides and phospholipids are transported in lipoprotein particles that are comprised of lipid-filled cores and hydrophilic outer shells. Several factors affect lipoprotein metabolism, such as the composition of diet, physical activity, gender and inherited traits. The excessive amount of plasma lipoproteins can lead to hyperlipidemia, which is one of the major risk factors for atherosclerosis. (Thompson, 1989). Vascular Endothelial Growth Factors (VEGFs) are strong inducers of angiogenesis and lymphangiogenesis. As they are major activators and regulators of vascular function they may also control plasma lipoprotein metabolism (Heinonen et al., 2013). VEGF-C and VEGF- D are ligands for VEGF Receptor (VEGFR) 3 and they regulate the development, growth and maintenance of lymphatic vessels (Yla-Herttuala et al., 2007). Emerging evidence suggests that lymphatic vessels participate in lipoprotein metabolism by transporting diet-derived large lipoproteins and mediating reverse cholesterol transport through High-density lipoproteins (HDL) (Randolph, Miller, 2014). Furthermore, lymphatic vessels are required for the maintenance of fluid balance and inflammatory reactions, both critical factors in pathological conditions such as MI (Huang, Lavine & Randolph, 2017). The aim of this thesis was to investigate the role of VEGFR3 as well as its ligands VEGF-C and VEGF-D in lipoprotein metabolism and CVDs. In the first part of the thesis, the effects of lymphatic deficiency on plasma lipid levels and atherogenesis were explored as well as the function of VEGF-D in lipoprotein metabolism. Subsequently, the role of lymphatic vessels was studied in healthy murine hearts and after MI.

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27 3 2 Review of the literature 2.1 CIRCULATORY SYSTEM Cardiovascular system The cardiovascular system is an essential circulatory network required for the transport of oxygen, nutrients, immune cells and antigens as well as waste products in the body. It is a closed network comprised of the heart, arteries, veins and capillaries. The heart is a central muscular pump located in the thoracic cavity. It sits in the pericardial sac filled with pericardial fluid, which lubricates the heart allowing it to pump efficiently. The heart is formed by three layers: the endocardium, which lines the inside of the heart, the myocardium formed by cardiac muscle cells and a serous membrane called the epicardium, which is the innermost layer of pericardium. Cardiac muscle is composed of elongated cardiomyocytes, which are able to contract with the aid of actin and myosin fibers in a response to action potentials generated by the sinoatrial node. The heart is composed of four chambers: the left and right atria and the left and right ventricle. Ventricular walls have thick muscle layers enabling strong muscle contractions, whereas the walls of atria are thinner. During diastole, blood flows from the atria into the ventricles, where it continues to blood circulation in the periphery during systole. Atrioventricular valves prevent blood flowing backwards from the ventricles to the atria: the tricuspid valve is located between the right atrium and ventricle whereas the mitral valve controls the blood flow from the left atrium to the left ventricle. Two cup-shaped semilunar valves, the pulmonary and aortic valve, control the blood flow from the right ventricle to pulmonary circulation and from the left ventricle to the aorta, respectively. (Rhoades, Pflanzer, 2003). The vascular system is divided into pulmonary and systemic circulation. Pulmonary circulation carries deoxygenated blood from right ventricle to the lungs where it is reoxygenated in the pulmonary alveoli. Blood is then returned to the left atrium through the pulmonary vein. Systemic circulation transports oxygen and nutrient-rich blood from the left ventricle through the aorta to the peripheral tissues and returns it back via veins and vena cava to the right atrium for re-oxygenation in the lungs. Arteries have strong muscular walls that can control blood flow and pressure by contractions and dilations, whereas veins are thinner enabling dilatation and thus serving as reservoir for blood. Both large arteries and veins consist of three layers: intima, media and adventitia. The intima is formed by a single layer of endothelium that lines the vessel lumen, the smooth muscle cells (SMCs), collagen and the internal elastic lamina. The media consists of SMCs and elastic and collagenous fibers, whereas the adventitia is primarily composed of collagen. Nutrients and oxygen are supplied to the outer layers of large arteries by the vasa vasorum, small arteries, veins and lymphatic vessels that reach into the adventitia as well as the outer layers of media. Unlike large arteries and veins, capillaries are thin and permeable, which facilitates the exchange of oxygen, fluids and macromolecules between blood and tissues. (Rhoades, Pflanzer, 2003) Lymphatic system The lymphatic system is a blind-end circulatory system, which is formed by lymphatic capillaries, collecting lymphatic vessels, lymph nodes as well as lymphatic organs such as the tonsils and the spleen. In adults, lymphatic vessels are found in almost every vascularized tissue except bone marrow. They are classified into lymphatic capillaries, precollecting vessels and collecting lymphatics according to their hierarchy, function and morphology. Lymphatic flow begins from small, blind-end lymphatic capillaries that absorb extravasated

28 4 fluid, immune cells and macromolecules from tissues. They are formed by a single layer of oak-leaf-shaped lymphatic endothelial cells (LECs) that are connected to collagen fibers in the extracellular matrix (ECM) with anchoring filaments. Since LECs are interconnected with discontinuous button-like junctions, fluid, macromolecules and immune cells can easily enter the lymphatic capillary through the flap-like openings. From capillaries, lymph moves into precollecting vessels that are characterized by a sparse SMC layer and finally to lymphatic collecting vessels characterized by thicker SMC layer, a basement membrane, tight zipperlike endothelial junctions and bileaflet valves thus resembling small venous structures. The pulsatations of the heart and the contraction of SMCs and surrounding skeletal muscles help the lymph to move forward in the lymphatic collector while lymphatic valves prevent the backflow. (Swartz, 2001, Alitalo, 2011). The lymphatic system is an important regulator of tissue fluid balance and participates actively in other physiological processes such as the regulation and trafficking of immune cells and the absorption of dietary fats from the intestine. Lymphatic vessels transport immune cells and soluble antigens from tissues to lymph nodes for antigen presentation to adaptive immune system. LECs express chemokines and adhesive molecules that attract immune cells, such as dendritic cells into the lymphatic system. Additionally, activated lymphangiogenesis and lymphatic vessel remodeling are associated with inflammatory diseases, such as rheumatoid arthritis. Furthermore, the lymphatic system provides a way for tumor cells to escape the primary tumor, encouraging metastases to occur. Thus, many tumors express factors that promote the growth of lymphatic vessels. (Alitalo, 2011, Aspelund et al., 2016, Randolph et al., 2017). The heart has a wide lymphatic network covering all layers of the myocardium and the cardiac valves (Kholova et al., 2011). The cardiac lymphatic system is crucial for correct fluid balance in the heart. Experiments in canine models propose that cardiac lymph flow starts from the endocardium, passes through the myocardium into the epicardium, where lymph is collected to larger pre-collecting lymphatic vessels that finally drain to mediastinal lymph nodes. The movement of the heart enhances lymph flow within the cardiac lymphatics. During diastole, the increased pressure of the chambers drives lymph from endocardium to myocardium. Subsequently, contractions of the myocardium during systole pushes the lymph flow from the myocardium to epicardial lymphatics. (Norman, Riley, 2016, Huang, Lavine & Randolph, 2017). 2.2 CARDIOVASCULAR DISEASES CVDs are one of the most common causes of morbidity and mortality globally. Around 17.7 million people die from CVDs annually, which accounts for 31.5 % of all deaths worldwide. From these, 7.4 million deaths are due to coronary artery disease and 6.7 million are caused by stroke. In Finland, around 13 % of the population have heart or circulation problems and around deaths are caused by coronary artery disease annually (Official Statistics of Finland (OSF): Causes of death). There is an uneven distribution of CVDs mortality in the world: advanced industrialized countries have the lowest mortality whereas countries in Eastern Europe as well as low and middle-income countries in Asia and Africa have the highest death rates. The prevalence, incidence and mortality of CVD are generally higher for men than for women at younger ages but this equalizes after menopause. (Townsend et al., 2016, Joseph et al., 2017, Roth et al., 2017). According to World Health Organization (WHO), the most important risk factors for CVD are an unhealthy diet, lack of physical activity, the usage of tobacco products and the harmful consumption of alcohol ( These factors predispose individuals to increased plasma lipid and glucose levels, elevated blood pressure and obesity, which trigger the development of coronary artery disease. One of the most established risk factors for CVDs is increased cholesterol levels in low-density lipoproteins (LDL) (Joseph et al., 2017). Additionally, positive family history is strongly associated with

29 5 the risk of CVDs and heritability of coronary artery disease has been estimated to be between 40% and 60%. Even though some specific risk genes have been identified, the analysis of genome-wide association studies have shown that in most cases coronary artery disease derives from the cumulative effect of multiple common risk alleles rather than single highrisk alleles (McPherson, Tybjaerg-Hansen, 2016) Hyperlipidemias Major risk factor for CVDs is hyperlipidemia, which is characterized by the elevated levels of plasma cholesterol (hypercholesterolemia), triglycerides (hypertriglyceridemia) or both (mixed hyperlipidemia). Hyperlipidemia is extremely common in Western countries, for example 48% of Americans have elevated cholesterol levels and 24% have hypertriglyceremia (Benjamin et al., 2017). Several factors affect the levels of lipids and lipoproteins in plasma, such as the composition of diet, age, gender, race, diet, body weight, physical activity and genetic factors. Therefore, it is often difficult to determine the underlying cause of hyperlipidemia and high plasma lipid levels that can result from a combination of many predisposing factors. Familial hyperlipidemias are caused by genetic mutations in factors regulating lipid metabolism, such as lipoprotein lipase (LPL) in type I hyperlipidemia or LDL receptor (LDLR) in familial hypercholesterolemia (Type IIa). Hyperlipidemia can also occur secondary to or in combination with other conditions, such as diabetes, obesity, renal dysfunction or hormonal factors. (Thompson, 1989). Hyperlipidemias are typically treated with lifestyle management interventions, such as changing the diet towards low-fat and fiber-rich modalities often in combination with increasing physical activity. Furthermore, the treatment of hypercholesterolemia often requires lipid-lowering medications. Statins are drugs that lower plasma cholesterol levels by inhibiting cholesterol production in the liver and activating cholesterol uptake into hepatocytes. Multiple statins are on the market and they have been shown to effectively reduce the risk of CVDs. (LaRosa, He & Vupputuri, 1999). Additionally, ezetimibe decreases intestinal and biliary cholesterol absorption by inhibiting the function of cholesterol receptor Niemann-Pick C1-like 1 (NPC1L1). Ezetimibe can be combined with statins when statin treatment does not achieve a sufficient enough reduction in LDL-cholesterol levels. (Phan, Dayspring & Toth, 2012). Alternatively, novel drugs for hypercholesterolemia are Proprotein convertase subtilisin/kexin type (PCSK) 9 inhibitors, monoclonal antibodies which increase the cholesterol uptake into hepatocytes by enhancing LDLR recycling in cells. (Piepoli et al., 2016, Chaudhary et al., 2017). Antibodies against PCSK9, such as Evolocumab (Blom et al., 2014) or Alirocumab (Robinson et al., 2015), have shown to decrease plasma cholesterol levels by up to 70% and have been approved for clinical use by U.S. Food and Drug Administration (FDA) Atherosclerosis Atherosclerosis is the underlining cause of coronary artery disease. It is characterized by the accumulation of lipids, cells and fibrous material in the arterial wall, which results in the narrowing of the arterial lumen. The disease is extremely common; atherosclerotic lesions can be found in nearly all individuals reaching the age of 65 years old. The early atherosclerotic lesions, the so called fatty streaks, can already be found in children under ten years of age (Yla-Herttuala et al., 1986). These small lesions are not clinically significant but they can function as the precursors of more advanced, fibrous lesions. In most cases, atherosclerosis is asymptomatic for decades and the first signs of the disease are highly lethal cardiovascular events such as MI or stroke. (Lusis, 2000, Ross, 1999). The development of atherosclerosis begins from endothelial dysfunction especially in the curvature regions of the coronary arteries. LDL and other apolipoprotein (Apo) B containing lipoproteins enter through the vascular wall and accumulate in the subendothelial space (Tabas, Williams & Boren, 2007). LDL is then oxidized, which triggers the pro-inflammatory reaction in endothelial cells (Yla-Herttuala et al., 1989, Berliner et al., 1990). The activation of

30 6 pro-inflammatory factors and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) leads to the upregulation of cell adhesion molecules such as Vascular cell adhesion protein (VCAM) 1 and Intercellular adhesion molecule (ICAM) 1, which mediate the entry of monocytes and T-cells to the subendothelium (Moore, Freeman, 2006, de Winther et al., 2005). In the vascular wall, monocytes differentiate into macrophages, take up oxidized LDL via their scavenger receptors and become foam cells. This leads to the formation of foam cell-rich fatty streaks that can either regress or develop into larger atherosclerotic plaques. (Steinberg et al., 1989). SMCs in the medial layer of the artery are also activated during atherosclerotic lesion progression. Multiple factors, such as the accumulation of cholesterol, the breakdown of the ECM and factors such as myocardin and Kruppel-like factor (KLF4) 4 stimulate so called phenotype switching in the SMCs, which start to proliferate and migrate from the media towards the intima. Activated SMCs produce excess amounts of ECM proteins that accumulate under the intima and create a fibrous cap that protects the developing plaque from a rupture. (Bennett, Sinha & Owens, 2016, Campbell &Campbell, 1985). If the cholesterol flux into the macrophages is prolonged, excess free cholesterol can promote the apoptotic cell death of the foam cells. The clearance of these apoptotic cells by other nearby macrophages is efficient in the early stages of atherosclerosis but becomes disrupted in more advanced lesions. This can lead to the necrotic death of foam cells, which then release their lipid-contents into the extracellular space and initiate the formation of a necrotic core within the atherosclerotic plaque, one of the characteristics of advanced lesions. Furthermore, small blood capillaries are often found in the large advanced lesions. These newly formed vessels are often enlarged and leaky and can lead to intraplaque hemorrhages, which predisposes plaques to vulnerability. In addition, calcification is often found in advanced lesions. If the production of fibrous material by SMCs is reduced or the degradation of ECM is increased, the fibrous cap might become thinner and break down, which exposes the plaque to the platelets of the blood and this can lead to the formation of a thrombosis. (Stary et al., 1995, Lusis, 2000) Myocardial infarction Acute MI is a devastating disease with high mortality and disability rates. In many cases, it is the first manifestation of coronary artery disease. MI refers to acute myocardial ischemia caused by the lack of oxygen and nutrients to the affected myocardium. MI typically results from atherosclerotic plaque rupture and thrombus formation causing an occlusion within a coronary artery. (Thygesen et al., 2012). The lack of oxygen in the heart muscle causes the necrotic death of cardiomyocytes. This induces a sequence of events aimed at the restraining further damage and prevention of the myocardial rupture subsequently enhancing the healing of the affected region. Firstly, necrotic cell death induces an innate inflammatory reaction via Toll-like receptor (TLR) mediated pathways (Arslan et al., 2010) and the upregulation of High mobility group (HMGB1) B1 (Andrassy et al., 2008, Mann, 2011). Furthermore, the synthesis of chemokines and cytokines, such as Tumor necrosis factor (TNF) α and Interleukins (IL) 1β and IL-6 attract inflammatory cells to the infarcted heart. Neutrophils have been shown to migrate to the myocardium within the first hour after MI, and they are followed by monocytes, macrophages, dendritic cells and T lymphocytes. Inflammatory cells clear the infarcted area from the debris of dead cells and ECM. (Frangogiannis, 2014). Subsequently, proinflammatory signaling is suppressed and the activation of the proliferative stage of the MI healing is initiated. Macrophages secrete growth factors that attract resident fibroblast to acquire the proliferative and secretive myofibroblast phenotype. Additionally, other cell types, such as endothelial cells, pericytes and SMCs may also differentiate into myofibroblasts. Activated myofibroblasts migrate to the infarct border zone and produce large quantities of ECM proteins such as collagens I and II starting the formation of collagen-based matrix at the infarct site. The cross-linking of collagen fibers increases the tensile strength of the scar and

31 7 thereby aid the contraction of the myocardium. When this process is completed, most of the myofibroblasts are cleared from the infarcted area but some remain even after decades. (Talman, Ruskoaho, 2016). The infiltration of inflammatory cells and the activation of myofibroblasts are both highly important for the healing of the myocardium but they can also contribute to the remodeling of the heart which can eventually lead to heart failure (HF). Hypoxia-inducible factor (HIF) 1α, cytokines and chemokines activate the production of angiogenic factors, such as VEGF, and induce the growth of new blood vessels in the infarcted myocardium (Lee et al., 2000, Banai et al., 1994). Additionally, lymphangiogenesis is activated in the heart to clear inflammatory cells as well as the excess fluid resulting from the leakage from the damaged vasculature (Kholova et al., 2011, Sun, Wang & Guo, 2012). Edema has shown to increase cardiac fibrosis and if fluid accumulation is prolonged, it can lead to decreased myocardial contraction and increased stiffness in the heart (Davis et al., 2000) Heart failure HF refers to a complex clinical condition resulting from the decreased pumping efficacy of the heart. HF is a major health issue that affects 23 million people Worldwide. It is common especially in patients with previous myocardial injuries, such as MI as well as in patients having conditions like hypertension and diabetes. (Bui, Horwich & Fonarow, 2011, Kemp, Conte, 2012). The central feature of HF is ventricular dysfunction that leads to a hemodynamic overload and myocardial remodeling. HF is classified in two types: HF with reduced ejection fraction (HFrEF) and HF with restored EF (HFpEF). If EF is decreased, the patient typically has systolic dysfunction, a dilated left ventricle cavity and low cardiac output. If EF is normal, the patient typically has diastolic dysfunction and the myocardium is thickened and stiffened. (Braunwald, 2013). Several cellular processes are involved in the development of HF, such as the hypertrophy of myocytes, accelerated apoptosis, the excessive accumulation of ECM and neurohumoral activation. The central feature of HF is the widening and elongation of myocytes as a response to factors such as angiotensin, endothelin and inflammatory stimulus. The resulting cardiac hypertrophy can eventually lead to an increase in the mass of the myocardium. Furthermore, myocardial necrosis enhances the release of growth factors that can also increase the number of ECM-producing myofibroblasts in the healthy regions of the heart (Davis et al., 2000). This excess of ECM can lead to stiffness of the myocardium and can cause reduced contraction as well as the relaxation of the ventricular wall. This can eventually lead to the pathological remodeling of the heart, causing hypertrophy in the healthy myocardium and the dilatation of the chambers. (Colucci, 1997, Jarvelainen et al., 2009) Mouse models for cardiovascular diseases The generation of transgenic mice has allowed researchers to study various mechanisms behind the complex and multifactorial CVDs and to develop new potential treatment options. However, unlike humans, mice do not synthesize Cholesteryl ester transfer protein (CETP), a protein that transports cholesterol from HDL to Very-low density lipoprotein (VLDL). Therefore, cholesterol is mainly carried in HDL particles in mice and they do not develop atherosclerosis spontaneously. Thus, the deletion of either apolipoproteins or lipoprotein receptors is required to generate phenotypes that resemble human CVDs. Furthermore, the role of other mediators can be studied by cross breeding common proatherosclerotic strains with other genetically modified mice. (Getz, Reardon, 2012). The first genetically modified mouse model for atherosclerosis research was an ApoE knockout (KO) mouse that was developed in 1992 (Plump et al., 1992, Zhang et al., 1992). Apoe -/- mice exhibit hypercholesterolemia and atherosclerosis on regular chow diet and disease progression can be accelerated by exposing the mice to a high-fat diet. Since ApoE is a primary apolipoprotein for CM and VLDL uptake into the liver, Apoe -/- mice display an accumulation of these ApoB48-containing cholesteryl ester-rich lipoproteins and low levels

32 8 of HDL particles. Apoe -/- mice spontaneously develop atherosclerotic lesions that resemble human plaques, predominantly in the aortic root, the aortic arch and the branch points of the aorta. (Zhang et al., 1994). The drawback of this model is that the lipoprotein profile does not resemble the human lipoprotein distribution that is usually characterized by high levels of LDL. Furthermore, functional ApoE has anti-oxidative, anti-proliferative and antiinflammatory potential, all major contributors of atherosclerosis. Therefore, the development of atherosclerotic plaques in Apoe -/- mice might not be caused by increased plasma cholesterol levels but rather the deletion of ApoE itself. (Getz, Reardon, 2016). To overcome the obstacles observed with the Apoe -/- mouse model, the LDLR KO mouse was developed a year later (Ishibashi et al., 1993). LDLR is responsible for the uptake of ApoB100 containing particles and the lack of LDLR impairs hepatic lipoprotein uptake. Therefore, the main circulating lipoprotein in Ldlr -/- mice on a chow-diet is cholesterol-rich LDL. Since young Ldlr -/- mice have only mildly increased plasma cholesterol levels, a highfat diet is typically used to induce the development of atherosclerosis in this model. The highfat diet also increases the amount of cholesterol in triglyceride-rich VLDL particles. (Getz, Reardon, 2012, Gleissner, 2016). As mice are capable of synthisizing ApoB48 in the liver and incorporate it into VLDL particles, these lipoproteins can be cleared through another lipoprotein receptor, LDL receptor related protein (LRP) 1. To create humanized apolipoprotein distribution in mice, the Ldlr -/- mice were crossed with ApoB48 deficient mice. The resulting Ldlr -/- /ApoB 100/100 mice have equal levels of plasma cholesterol than Apoe -/- mice, but their lipoprotein profile resembles human lipoprotein distribution as cholesterol is mainly carried in ApoB100 containing VLDL and LDL particles. (Veniant et al., 1998). 2.3 LIPIDS AND LIPOPROTEIN METABOLISM Plasma lipids The major lipids found in human plasma are cholesterol esters, triglycerides and phospholipids that comprise the lipid moiety of lipid-carrying lipoproteins. Additionally, plasma transports albumin-bound free fatty acids (FFA), non-esterified fatty acids that result from the hydrolysis of triglycerides in adipose tissue as well as through the lipolysis of lipoproteins. Cholesterol is a sterol and it is the main element of cell membranes. Additionally, it functions as a precursor molecule for bile acids, adrenal and gonadal steroid hormones and vitamin D. Cholesterol is mostly in its free form in cells but in plasma it occurs esterified with a long-chain fatty acid. Cholesterol biosynthesis occurs mainly in the hepatocytes and it is a tightly controlled multistep process. Synthesis starts when acetyl-coa and acetoacetyl-coa are converted into 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) through the mevalonate pathway. HMG-CoA is then reduced to mevalonate by the enzyme HMG-CoA reductase (HMGCR). Through multiple steps, mevalonate is further converted through isopentenyl pyrophosphate, farnesyl pyrophosphate, squalene and lanosterol to cholesterol. The synthesis of cholesterol is regulated by the levels of available cholesterol within the cell. The main regulator is Sterol regulatory element-binding protein (SREBP) 2, a transcription factor that in the presence of cholesterol is bound to SREBP2 cleavage-activating protein (SCAP) and Insulin induced gene (INSIG) 1. When cholesterol levels fall, INSIG1 dissociates from the complex allowing SREBP2 to migrate to the Golgi apparatus, where it is cleaved by proteases, allowing for its entry into the nucleus. In the nucleus, cleaved SREBP2 functions as a transcription factor for multiple genes, such as HMGCR and LDLR. (Brown, Sharpe, 2016). Cholesterol-lowering drugs, statins, function by inhibiting the production of cholesterol. Statins have similar structural HMG moiety to that of HMG-CoA and thus compete for the binding sites in HMG-CoA reductase with endogenous HMG-CoA, and this leads to the blockage of the cholesterol synthesis. Furthermore, as a response to lowered

33 9 cholesterol synthesis, cells enhance the synthesis of LDLR and increase the LDL cholesterol uptake to the hepatocytes thus lowering the levels of plasma cholesterol. (Liao, Laufs, 2005, Istvan, Deisenhofer, 2001). Fatty acids are carboxylic acids with an aliphatic chain and they are divided into saturated fatty acids that have only single bonds between carbon atoms and unsaturated fatty acids that display one or multiple double bonds. The size of a fatty acid varies according to the carbon atoms in the aliphatic chain. Fatty acids have a high energy content and they are utilized in tissues as energy sources in a process called β-oxidation. Acute stress, prolonged fasting and the lack of insulin promote the release of FFAs into plasma. FFAs are released into the blood stream from adipose tissue as a response to the activation of hormone sensitive lipase (HSL) and Adipose triglyceride lipase (ATGL). Additionally, FFAs can escape from the internalization to the adipocytes during the LPL-mediated lipolysis of lipoproteins. The released FFAs covalently bind to serum albumin. Plasma FFAs are transported to the liver, skeletal muscle, heart and renal cortex for utilization as energy. Triglycerides are composed of three fatty acids that are bound to a single glycerol molecule with an ester bond. Triglycerides function as the major storage molecules for fatty acids. (Lehner, Quiroga, 2016). Phospholipids form lipid bilayers that are the major components of all cell membranes. Additionally, they form the outer shell of lipoproteins forming a lipophilic core for cholesterol and triglycerides. The most distinguishable characteristic of phopsholipids is their amphiphilic structure: they consist of hydophic fatty acid tails and a hydrophilic negatively charged phosphate group containing head. (Ridgway, 2016) Lipoproteins Cholesterol and triglycerides are insoluble in the plasma and have to be transported as spherical lipoprotein particles. Lipoproteins have a central core, where hydrophobic lipids are stored and a hydrophilic outer shell consisting of phospholipids and apolipoproteins. Plasma lipoprotein particles are classified according to their size and density to chylomicrons (CMs), CM remnants, VLDL, Intermediate density lipoproteins (IDL), LDL and HDL (Table 1). CMs are produced in intestinal enterocytes and contain mainly triglycerides. These large lipoprotein particles ( nm) carry dietary triglycerides and cholesterol from the intestine to peripheral tissues. After LPL-mediated lipolysis, CMs are depleted of most of the triglycerides and are called CM remnants, which are rapidly endocytosed by the liver. Another large lipoprotein particle, VLDL is produced in the liver and it resembles CM in its composition and structure. However, VLDLs are smaller, ranging in size from nm. LPL hydrolyzes triglycerides from VLDL particles that turn to IDL or VLDL remnants. These nm particles are either taken up by the liver or further lipolyzed. When most of the triglyceride content in IDL particle is hydrolyzed, the remaining particle is called LDL (20-25 nm), which is the major cholesterol carrying particle in plasma. LDL particles are efficiently removed from plasma by the LDLR which is expressed in hepatocytes. HDL is the smallest lipoprotein particle (5-25 nm). It is required for the recycling of cholesterol from peripheral cells to the liver. (Hegele, 2009, Rodewell et al., 2018).

34 10 Table 1. The composition of main lipoprotein subclasses in humans. Modified from (Hegele, 2009). Lipoprotein Origin Diameter (nm) Lipid (% by weight) Protein (% by weight) Main lipid component CM Intestine Triglyceride CM remnant Lipolysis of CM Triglyceride, Cholesterol VLDL Liver Triglyceride IDL Lipolysis of VLDL Triglyceride, Cholesterol LDL Lipolysis of VLDL Cholesterol HDL Liver, intestine Cholesterol Apolipoproteins Apolipoproteins are the main structural protein components in the outer hydrophilic shell of lipoproteins. In addition to their structural function in lipoproteins, apolipoproteins are required for the binding of lipoproteins to their cell-surface receptors and some function as cofactors for enzymes. Apolipoproteins are mainly synthetized in the liver and intestine. They also shuttle between different lipoprotein particles. (Dominiczak, Caslake, 2011) (Table 2) ApoA ApoAs are the main protein components of HDL. ApoA-I is produced in the liver and intestine. Intestinally produced ApoA-I is incorporated into CMs but is quickly transferred to HDL particles in plasma. ApoA-I is a necessary structural component of all HDL particles. It is a primary target for the lipidation of HDL by ATP-binding cassette subfamily A (ABCA) 1 (Oram, 2003) and ATP-binding cassette subfamily G (ABCG) 1 (Gelissen et al., 2006). ApoA- I is also a cofactor for Lecithin-cholesterol acyltransferase (LCAT), an enzyme responsible for the formation of cholesteryl esters from free cholesterol (Jonas, 2000). In addition, ApoA-I is capable of interacting with the Scavenger receptor class B type I (SR-BI) and mediate the transport of cholesterol from plasma HDL for bile excretion (Xu et al., 1997). ApoA-II is produced in the liver as a homodimer and it associates with lipids within hepatocytes. ApoA-II particle interacts with ApoA-I in plasma and generates an HDL particle with both ApoAI and ApoA-II (LpA-I/A-II). ApoA-II accounts for 20% of the protein component contained in HDL particles. (Pownall, Gillard & Gotto, 2013). ApoA-II might play multiple roles in HDL metabolism. It has been shown to control HDL particle size by decreasing LCAT function (Marzal-Casacuberta et al., 1996) and facilitate the binding of HDL to SR-BI (de Beer et al., 2001). Alternatively, it has been shown to regulate plasma VLDL concentration (Blanco-Vaca et al., 2001). ApoA-IV is mainly produced in the intestine and it is incorporated into CM particles. After the lipolysis of CMs, ApoA-IV dissociates from CMs and is either associated with HDL or it remains free in the plasma. (Kohan et al., 2015, Bisgaier et al., 1985). The deletion of ApoA-IV gene in mice leads to larger CM size and slower remnant catabolism (Kohan et al., 2012) indicating that ApoA-IV is an essential structural protein of CMs. Furthermore, HDL bound ApoA-IV can activate LCAT (Steinmetz, Utermann, 1985) and lipid-free ApoA-IV can promote reverse cholesterol transport from cells, at least in vitro, (Steinmetz et al., 1990) suggesting that ApoA-IV might mediate atheroprotection (Cohen et al., 1997) ApoB ApoB is the major structural component of all plasma lipoproteins except HDL. Each lipoprotein particle contains a single copy of ApoB protein and therefore, the amount of

35 11 ApoB can serve as a marker of lipoprotein particles. (Dominiczak, Caslake, 2011). ApoB occurs in two isoforms: Full sized ApoB100 (4536 amino acids) that is found in VLDL, IDL and LDL particles as well as truncated ApoB48 (2152 amino acids), which is the main structural protein of CMs (Powell et al., 1987, Chen et al., 1987, Davidson, Shelness, 2000). ApoB48 and ApoB100 are produced from the same gene but two isoforms are generated through the mrna editing facilitated by apolipoprotein B mrna editing enzyme catalytic polypeptide-like (APOBEC) 1 (Teng, Burant & Davidson, 1993). RNA editing introduces a stop codon into the ApoB mrna, which results in the truncated ApoB48 protein produced during the translation (Powell et al., 1987, Chen et al., 1987). APOBEC1 is expressed exclusively in the intestine in humans whereas both the intestine and liver produce APOBEC1 in mice. Therefore, liver produce exclusively ApoB100 and the intestine ApoB48 in humans, whereas ApoB100 and ApoB48 are produced both in liver and intestine in mouse. (Greeve et al., 1993, Hirano et al., 1997). The isoforms of ApoB differ in their function and receptor binding: although ApoB48 can bind to LRP1, it is mainly considered a structural protein of CMs (Willnow et al., 1994, Veniant et al., 1998), whereas ApoB100 is essential for binding to LDLR (Goldstein, Brown, 1974, Segrest et al., 2001). Several ApoB variants have been discovered in humans (Young, 1990). For example, mutations in the LDLR binding regions of ApoB100 delay LDL clearance and cause increased plasma LDL levels (Innerarity et al., 1987, Boren et al., 1998) ApoC ApoCs are mainly produced in liver and intestine and they shuttle back and forth between CMs, VLDLs and HDLs. In the fasted state, ApoCs are bound to HDL whereas they are mostly redistributed to CMs and VLDL after feeding. (Jong, Hofker & Havekes, 1999). The role of ApoCs in lipoprotein metabolism has been extensively studied in transgenic mice. ApoC-I transgenic mice displayed severe hypertriglyceridemia and increased triglyceride levels in VLDL particles but only mild increase in plasma cholesterol levels (Shachter et al., 1996). First studies suggested that ApoC-I can inhibit lipoprotein remnant clearance by inhibiting the binding of the lipoprotein particle to its receptors (Jong et al., 1996) but later studies have revealed that ApoC-I mainly inhibits LPL mediated lipolysis by displacing LPL from lipoprotein particles (Berbee et al., 2005). Another member of the ApoC family, ApoC-II, is a cofactor for LPL (Kinnunen et al., 1977, Goldberg et al., 1990). Several studies have shown that the defects in the production or structure of ApoC-II in humans leads to hypertriglyceridemia, which corresponds the phenotype of LPL deficiency (Jong, Hofker & Havekes, 1999). Interestingly, transgenic mice with increased levels of ApoC-II levels have also severe hypertriglyceridemia and accumulation of triglyceride-rich VLDL particles (Shachter et al., 1994) indicating that maintaining a correctly balanced level of ApoC-II in lipoproteins is essential for the efficient function of LPL. Similarly to ApoC-I transgenic mice, the overexpression of ApoC-III leads to increased triglyceride levels (Ito et al., 1990). In vitro studies demonstrated that ApoC-III inhibits LPL mediated lipolysis (Jong, Hofker & Havekes, 1999) but the findings from in vivo studies have been unclear. Transgenic ApoC-III mice display delayed hepatic clearance of lipoprotein remnants (Aalto-Setala et al., 1992, de Silva et al., 1994) that could result from the inhibition of lipolysis mediated by Glycosylphosphatidylinositol anchored high density lipoprotein binding protein (GPIHBP) 1 bound LPL (Larsson et al., 2017). On the other hand, the deletion of ApoC-III causes reduction in triglyceride levels by 70% (Maeda et al., 1994) and enhances LDLR/LRP1 mediated hepatic uptake of lipoprotein remnants suggesting that ApoC-III mainly functions in the hepatic clearance of these particles (Gordts et al., 2016) ApoE ApoE is a multifunctional protein that is produced in various organs such as liver, brain, spleen and kidney. It is a structural protein of CM remnants, VLDL, IDL and HDL. ApoE

36 12 mediates lipolysis by activating LPL and Hepatic triglyceride lipase (HTGL). Furthermore, ApoE binds to LDLR, LRP1 and Heparan sulfate proteoglycans (HSPGs) with high affinity and thereby mediates endocytosis of lipoprotein particles to the liver. (Mahley, 1988, Mahley, Weisgraber & Huang, 2009). Furthermore, ApoE controls cholesterol efflux from cells to HDL particles and activates LCAT (Mahley, Huang & Weisgraber, 2006). Three common isoforms of ApoE are found in humans: Apo E2, E3 and E4, from which the ApoE3 isoform is the most common with the frequency of 65-70% (Mahley, Weisgraber & Huang, 2009, Phillips, 2014a). The ApoE2 isoform can be found from 5-10% of individuals and its homozygous form (ApoE2/E2) is associated with type III hyperlipoproteinemia, characterized by increased accumulation of lipoprotein remnants in plasma resulting from defective LDLR binding site in ApoE2 (Mahley, Huang & Rall, 1999). The ApoE4 isoform is found in 15-20% of individuals. It is a risk factor for Alzheimer s disease and 60 80% of the patients with Alzheimer s disease have at least one ApoE4 allele. The ApoE4 isoform is also associated with the increased risk of atherosclerosis, resulting from increased plasma cholesterol and LDL levels. (Huang, Mahley, 2014, Corder et al., 1993, Saunders et al., 1993). Table 2. Main apolipoproteins in human plasma and their primary functions. Modified from (Dominiczak, Caslake, 2011). Lipoprotein Apolipoprotein Primary Source Function particle ApoA-I HDL, CM Liver, Intestine Structural protein of HDL. Interacts with ABCA1, ABGC1, LCAT and SR-BI. ApoA-II HDL Liver ApoA-IV CM, HDL Intestine Structural protein of HDL. Interacts with LCAT and SR-BI. Inhibits HL. Structural protein of CM and HDL. Interacts with LCAT. ApoB-48 CM Intestine Structural protein of CM. ApoB-100 VLDL, IDL, LDL Liver Structural protein of VLDL, IDL and LDL. Ligand for LDLR. ApoC-I CM, VLDL, HDL Liver Inhibition of LPL. ApoC-II CM, VLDL, HDL Liver Regulation of LPL. ApoC-III CM, VLDL, HDL Liver Regulation of remnant clearance: inhibition of LPL and/or remnant binding to receptor. ApoE CM, VLDL, HDL Liver Brain Ligand for LDLR, LRP and HSPGs. Regulation of LPL, CETP and LCAT. Antioxidant molecule.

37 Lipoprotein receptors The main function of lipoprotein receptors is to clear lipoproteins from the circulation, body fluids and interstitial spaces. Most of them belong to the LDLR gene family and share structural similarities: LDLR type A repeats that are responsible for ligand binding, an epidermal growth factor (EGF)-like domain that regulates ligand-receptor interaction and a transmembrane domain that anchors the receptor to the cell membrane. Many of the receptors, like LDLR and LRP1 also contain a cytoplasmic domain that is required for the targeting of receptors to coated pits and mediating signal transduction. (Dieckmann, Dietrich & Herz, 2010). In addition to the members of LDLR family, HSPGs play an important role in mediating the trapping of the lipoproteins onto endothelial cell surfaces as well as endocytosis of CM and VLDL remnants (Foley, Esko, 2010). Furthermore, SR-BI mediates the internalization of HDL particles (Acton et al., 1996) LDLR A receptor for LDL was postulated in 1974 (Goldstein, Brown, 1974) and the human LDL receptor (LDLR) was cloned in 1984 (Yamamoto et al., 1984). LDLR is a key receptor in lipoprotein metabolism. It is the main receptor for LDL via ApoB100 but it can also bind to ApoE and internalize CM remnants, VLDL and VLDL remnants. LDLR is ubiquitously expressed, but it mainly functions in the liver. (Brown, Goldstein, 1986). Mutation in both LDLR alleles causes familial hypercholesterolemia, characterized by highly increased cholesterol levels, up to ten-fold elevation in LDL levels and premature atherosclerosis that leads to early MI in many cases. Heterozygous mutations are relatively common and cause two-fold increase plasma LDL. (Marks et al., 2003, Goldstein, Brown, 2009). The expression of the LDLR gene is regulated by cellular cholesterol levels, via the transcription factor SREBP2. As a response to decreased intracellular cholesterol levels, LDLR is synthesized on the ER, glycosylated in the Golgi apparatus, before being transported to the plasma membrane. LDLR binds to the apolipoprotein via its ligand binding domain followed by the endocytosis of the lipoprotein-receptor complex via clathrin-coated pits. Endocytosis requires an interaction of the NPxY motif in the cytoplasmic tail of LDLR, Lowdensity lipoprotein receptor adapter protein (ARH) 1 and clathrin-coated pit proteins Clathrin and Adaptin 2 (Chen, Goldstein & Brown, 1990, Stolt, Bock, 2006, He et al., 2002). Once inside the cell, clathrin-coated vesicles are fused with early endosomes, which have an acidic environment that enables the detachment of lipoprotein from the LDLR (Rudenko et al., 2002). Subsequently, cholesterol is then released from the lipoprotein particle by Lysosomal acid lipase (LIPA) (Goldstein et al., 1975), bound to Nieman-Pick disease, type C proteins (NPC1 and NPC2) and released to the cytosol of the cell (Higgins et al., 1999). LDLR is efficiently recycled to the plasma membrane, but when LDLR is not required, proteins such as PCSK9 (Horton, Cohen & Hobbs, 2007) and the E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) (Zelcer et al., 2009) target it for degradation LRP1 LRP1 is a ubiquitously expressed multifunctional protein belonging to the LDLR gene family. It is essential in many biological processes as it binds to multiple ECM proteins, proteases, viruses, cytokines and growth factors and mediates both endocytosis and signal transduction pathways (Gonias, Campana, 2014). It is vital during embryonic development as the deletion of LRP1 is embryonically lethal (Herz, Clouthier & Hammer, 1992). In lipoprotein metabolism, LRP1 has a partly overlapping function with LDLR as it binds to ApoE containing lipoproteins, namely CM and VLDL remnants. However, the inactivation of LRP1 does not lead to hyperlipidemia, presumably due to LDLR compensation (Rohlmann et al., 1998). The function of LRP1 has shown to be useful in atherosclerosis, as LRP1 controls the proliferation of vascular SMCs and thereby maintains the structural integrity of the vascular wall (Boucher et al., 2003). Furthermore, the deletion of LRP1 specifically in macrophages leads to the increased expression of inflammatory cytokines and accelerated

38 14 atherosclerosis in mice, suggesting that LRP1 is also essential in the regulation of inflammatory reactions during atherosclerosis (Overton et al., 2007) HSPGs HSPGs are glycoproteins that reside on the cell surface (syndecans and glypicans), in the ECM (perlecans, agrin, type XVIII collagen) or in secretory vesicles (serglycin). They are characterized by long heparan sulfate chains and a core protein, which varies according to the localization and function of the HSPG. Heparan sulfate chains are composed of glycosaminoglycans, whose organization and length vary between different cell types. Furthermore, GlcNac N-deacetylase/N-sulfotransferase (NDST) attaches variable amounts of sulfate groups to the heparan sulfate chain that form negatively charged binding sites for ligands. Interestingly, heparan sulfate chains are more highly sulfated in hepatocytes compared to most other cell types creating unique ligand binding properties of HSPGs in the liver. (Sarrazin, Lamanna & Esko, 2011, Gordts, Esko, 2018). HSPGs can bind to various ligands and they have multiple functions in physiology. For example, LPL (Saxena, Klein & Goldberg, 1991) as well as apolipoproteins ApoA-V (Lookene et al., 2005), ApoB (Weisgraber, Rall, 1987, Flood et al., 2002) and ApoE (Saito et al., 2003) are capable of binding to heparan sulfate chains. Therefore, hepatic HSPGs, mainly Syndecan (SDC) 1, can facilitate the uptake of large lipoproteins such as CM and VLDL remnants into the hepatocytes (MacArthur et al., 2007, Stanford et al., 2009). Indeed, the deletion of SDC1 leads to high plasma cholesterol and triglyceride levels, especially when LDLR is not present (Stanford et al., 2009). In vitro studies have shown that HSPGs can directly mediate the endocytosis of lipoprotein particles (Fuki et al., 1997, Deng et al., 2012). Alternatively, HSPGs can trap lipoproteins on the cell surface of the hepatocytes and introduce them to the other lipoprotein receptors, such as LDLR or LRP1, enhancing the endocytosis of lipoproteins through these receptors (Mahley, Huang, 2007). Additionally, HSPGs can modulate VEGF signaling and they are required for efficient angiogenic sprouting (Jakobsson et al., 2006) Other lipoprotein receptors Another member of LDLR family, VLDL receptor (VLDLR) was cloned in 1994 (Sakai et al., 1994). In contrast to the other lipoprotein receptors, VLDLR is not expressed in hepatocytes but it functions widely in other tissues such as the heart, skeletal muscle, adipose tissue and the brain. VLDLR mediates the endocytosis of ApoE containing lipoproteins but is unable to bind LDL. (Takahashi et al., 1992). Furthermore, VLDLR can bind and internalize LPL thus regulate the activity of LPL on lipoproteins (Argraves et al., 1995). Additionally, VLDLR might contribute to the delivery of fatty acids across the endothelial cell membrane (Yamamoto et al., 1993). Indeed, mice deficient of VLDLR have normal plasma lipid levels (Frykman et al., 1995) and are protected from obesity induced by high fat diet (Goudriaan et al., 2001) indicating that VLDLR is important in delivering fatty acids into tissues (Takahashi et al., 2004). SR-BI was identified in 1993 (Calvo, Vega, 1993). The primary function of SR-BI is to mediate the uptake of HDL cholesterol into hepatocytes (Acton et al., 1996). In addition, SR- BI mediates the bidirectional exchange of cholesterol between cells and HDL (Yancey et al., 2000) thus reducing foam cell formation in atherosclerotic plaques by mediated cholesterol removal from macrophages (Yancey et al., 2007). Additionally, SR-BI can mediate VLDL clearance from plasma (Van Eck et al., 2008). The deletion of SR-BI in mice leads to elevated plasma cholesterol levels, an increase in the size of HDL particles (Rigotti et al., 1997) and results in increased atherosclerosis (Van Eck et al., 2003) as well as spontaneous MIs (Braun et al., 2002). In humans, genetic variants in the SR-BI gene cause increased plasma HDL levels but, unexpectedly, also a higher risk for CVDs (Zanoni et al., 2016, Vergeer et al., 2011).

39 Exogenous pathway Dietary fats provide the densest caloric source and are efficiently absorbed by the intestine. While amino acids, carbohydrates and short- and medium-chain fatty acids are taken up directly by blood capillaries, long-chain fatty acids and cholesterol are transported to the blood circulation and finally to the liver via the exogenous lipoprotein pathway presented in Figure 1. (Mansbach, Siddiqi, 2016). Dietary triglycerides are first emulsified by bile and then hydrolyzed by the pancreatic lipase in the intestinal lumen (Buttet et al., 2014). The resulting fatty acids are then transported into the enterocytes that line the intestinal microvilli, either by passive diffusion (Shiau, 1990) or possibly by Cluster determinant 36 (CD36) (Nauli et al., 2006) or Fatty acid transport protein 4 (FATP4) (Stahl et al., 1999). After internalization into the enterocyte, FAs are transported to endoplasmic reticulum (ER) by Fatty acid binding proteins (FABPs) for reesterification into triglycerides (Gajda, Storch, 2015). In parallel, dietary cholesterol is transported to enterocytes by the specific cholesterol transporters, NPC1L1 (Sane et al., 2006) and SR-BI (Bietrix et al., 2006). Cholesterol dedicated to CM production is esterified by enzyme Acetyl-coenzyme A acetyltransferase 2 (ACAT2) (Nguyen et al., 2012). Triglycerides, cholesteryl esters and phospholipids are assembled with ApoB48 by microsomal triglyceride transfer protein (MTP) to produce pre-cms (Iqbal, Rudel & Hussain, 2008, Dash et al., 2015) and newly formed CMs obtain ApoA-IV during the lipidation step (Kohan et al., 2015). Pre- CMs are transported to the Golgi where they acquire ApoA-I and ApoA-II. Subsequently, mature CMs are exported from Golgi to the surface of the enterocyte, from where they are transported into the lacteals, small lymphatic vessels within the villus. From the lacteals, CMs are transported via the lymphatic system to the thoracic duct, a major collecting lymphatic trunk, which finally drains CMs to the left subclavial vein (Dash et al., 2015, Dixon, 2010). Once they have entered the blood, CMs further acquire apolipoproteins, namely ApoC-II, ApoC-III and ApoE from other lipoproteins, such as HDL. Once CMs reach the capillary endothelium they undergo lipolysis through the action of LPL. LPL is produced in parenchymal cells, such as adipocytes and cardiomyocytes (Li et al., 2014). LPL function is regulated by several proteins such as Lipase maturation factor (LMF) 1, GPIHBP1, angiopoietin-like proteins (ANGPTLs), Apo-AV and ApoCs (Kersten, 2014). LMF1 regulates LPL production translationally by creating a catalytically active LPL from its precursor (Peterfy et al., 2007). GPIHBP1 transports active LPL through the endothelial cell to the capillary lumen and provides a binding site for LPL and CMs on endothelial cells (Fong et al., 2016, Goulbourne et al., 2014). Furthermore, ANGPTL4 regulates lipolysis by dissociating active LPL dimers into inactive monomers (Sukonina et al., 2006). Additionally, CM-bound ApoC-II is required for the LPL function whereas ApoC-III inhibits LPL as described in the previous chapter. After lipolysis, the size of the CM particles has decreased and the resulting CM remnants release their ApoAs and ApoC-II back to HDL. The remnants are small enough to pass through the small caps within the hepatic endothelial cells, called sinusoidal fenestrae and enter the presinusoidal Space of Disse (Fraser, Dobbs & Rogers, 1995). In the space of Disse, CM remnants are enriched with ApoE and modified by LPL and HTGL. The remnants are then cleared rapidly by LDLR, LPR1 and HSPGs, most notably SDC1 (Mahley, Ji, 1999, Stanford et al., 2009). Interestingly, particle size and dietary conditions affect the receptor binding profiles of the lipoproteins. HSPGs prefer smaller remnants enriched with ApoE and ApoA-V, while LDLR and LRP1 internalize larger particles (Foley et al., 2013) Endogenous pathway The liver is the major regulator of lipoprotein metabolism as it controls the biosynthesis, storage and secretion of cholesterol and fatty acids, as well as converts cholesterol to bile acids. The endogenous pathway refers to the hepatic VLDL production and secretion as well as the lipolysis of VLDL particles on the endothelial surface of capillaries to IDL and subsequently to cholesterol-rich LDL particles. The endogenous pathway terminates when

40 16 LDL particles are taken up back to the liver. The endogenous pathway is presented in Figure 1. VLDL particles are formed in the hepatocytes and secreted into the blood stream as a response to increased cellular fatty acid content. FFAs are supplied to the hepatocytes from adipose tissues and the intestine as well as by CM remnants. (Tiwari, Siddiqi, 2012). As in a similar way to the production of CMs, MTP assembles phospholipids, cholesterol and triglycerides with newly synthetized ApoB100 in the ER (Shelness et al., 1999, Hussain, Shi & Dreizen, 2003, Sundaram, Yao, 2010). Nascent VLDL particles are transported from the ER to the Golgi, where the particles gain more lipids and ApoA-I. Mature VLDL particles are then transported to the plasma membrane and released into the blood for circulation. (Tiwari, Siddiqi, 2012). The amount of triglycerides in VLDL particles can vary significantly depending on available cellular sources (Sundaram, Yao, 2010). However, if sufficient amount of lipids is not available, newly formed ApoB100 is degraded. (Zhou, Fisher & Ginsberg, 1998). After VLDL particles are secreted to the blood stream, they gain ApoE and ApoC-II from other lipoprotein particles. LPL hydrolyzes triglycerides from the VLDL particles forming IDL or VLDL remnants. Furthermore, the cholesterol content of IDL particles is increased through the function of CETP, which mediates the exchange of cholesterol esters and triglycerides between ApoB-containing particles and HDL (Inazu et al., 1990). VLDL remnant particles are either sent back to liver and endocytosed or further hydrolyzed by HTGL to even smaller, cholesterol-rich LDL particles containing only ApoB100. Subsequently, LDL particles can be taken up to the liver by LDLR. (Daniels et al., 2009).

41 17 Figure 1. Exogenous and endogenous pathways. CMs are produced from dietary cholesterol and triglycerides in the intestinal enterocytes and transported to blood via the lymphatic system. In blood, LPL hydrolyzes FFAs from CMs for the energy needs of peripheral cells. Depleted CM remnants are taken up by the hepatocytes via the LDLR, LRP1 and HSPG receptors. During the endogenous pathway, cholesterol and triglycerides are assembled in hepatocytes as VLDL particles, which are then released to the blood. VLDLs are lipolyzed by LPL and HTGL creating a dense, cholesterol-rich LDL particle that can enter tissues, such as the vascular wall, directly through vascular endothelium. LDL can also be taken up by hepatocytes via the LDLR. Modified from (Dominiczak, Caslake, 2011) HDL metabolism and reverse cholesterol transport Since the overload of cholesterol is toxic for cells and most cells are unable to catabolize cholesterol, excess cholesterol needs to be removed from cells via extrinsic mechanism. Reverse cholesterol transport (RCT) refers to a pathway in which cholesterol is secreted and transported from tissues to circulation in HDL particles and subsequently excreted from the body as bile. RCT from macrophages residing in atherosclerotic plaques is an essential antiatherogenic mechanism. (Lewis, Rader, 2005, Phillips, 2014b). RCT is represented in Figure 2. RCT is intiated in the hepatocytes and enterocytes, which produce ApoA-I and release it into the blood. On the peripheral cell membrane, ApoA-I interacts with the ATP binding cassette subfamily A (ABCA1) 1 protein that transfers phospholipids and free cholesterol from the cell to ApoA-I (Oram, 2003). Lipidated ApoA-I forms a nascent discoidal pre-hdl enriched with unesterified cholesterol. Subsequently, LCAT esterifies the free cholesterol and thus prevents its release back to the cells (Jonas, 2000) and the pre-hdl becomes spherical mature HDL particle. Mature HDL can interact with ABCG1, which also transfers cholesterol from the tissues to HDL (Wang et al., 2004). Cholesterol in mature HDL can be internalized by hepatocytes via SR-BI (Acton et al., 1996). In addition, around 50% of RCT in humans occurs via the transfer of cholesterol esters and triglycerides by CETP between HDL and ApoB containing lipoproteins (Inazu et al., 1990, Adorni et al., 2007). As a result, HDL becomes depleted of cholesterol and enriched with triglycerides. Subsequently, lipolysis through HTGL (Collet et al., 1999) generates lipid-poor ApoA-I, which is degraded in the

42 18 kidneys and HDL remnants, which are internalized by hepatocytes and subsequently degraded (Lewis, Rader, 2005). Figure 2. Reverse cholesterol transport (RCT). ApoA-I is produced in hepatocytes and transported to blood circulation. It gains free cholesterol from peripheral cells through ABCA1 and ABCG1 - mediated transport. The enzyme LCAT esterifies free cholesterol to cholesterol esters, which increases the size of HDL particles. Mature HDL can be internalized by hepatocytes via SR-BI. Alternatively, HDL particles exchange cholesterol and triglycerides with ApoB-containing particles, such as VLDL. Cholesterol depleted HDL is then taken up by the liver, whereas ApoA-I can be degraded by kidneys. Modified from (Dominiczak, Caslake, 2011) Lymphatic system in lipid and lipoprotein metabolism Most of the lipid and lipoprotein metabolism occurs in the blood or inside cells. However, emerging evidence shows that lymphatic vessels also play an essential role in the regulation of lipid and lipoprotein metabolism. The best known function of lymphatic vessels in the lipoprotein metabolism is the transport of dietary lipids from the intestine to blood circulation. Nearly all intestinal lipids are packaged as CMs in the intestinal enterocytes and transported through the lymphatic system to the thoracic duct and finally into blood stream. The intestinal villus contains a single lymphatic vessel, a lacteal that absorbs CMs produced in the enterocytes. It is still not completely understood how CMs are transported from the enterocyte cell membrane into the lacteal, and several possible mechanisms have been proposed, such as the paracellular flux through the junctions between LECs, an intracellular pathway through the LEC cytoplasm or through the tip of the villus (Dixon, 2010). Furthermore, several factors have shown to be essential for the maintenance and functionality of the lacteals, and thus the absorption of lipids, such as Pleomorphic adenoma gene-like 2 (PLAGL2) (Van Dyck et al., 2007), VEGF-C (Nurmi et al., 2015), the Delta-like protein (DLL) 4 (Bernier-Latmani et al., 2015), the Calcitonin receptor-like receptor (CALCRL) (Davis et al., 2017) and VEGFR2 (Zhang et al., 2018). In addition to controlling CM absorption and transport, lymphatic vessels are closely connected with the function of adipose tissue. Lymph node resection in breast cancer patients leads often to lymphedema as well as the deposition of lipids into the areas of fluid accumulation (Stanton et al., 2009, McLaughlin et al., 2008). Lipid accumulation has also been reported in the mice with genetical modifications leading to lymphatic dysfunction, such as

43 19 in Prospero homeobox protein (Prox1) +/- mice (Harvey et al., 2005) and in Chy mice with an inactivating point mutation in Vegfr3 gene (Rutkowski et al., 2010). Lymph contains adipogenic factors, such as FFAs, that can promote the growth of adipocytes (Escobedo et al., 2016, Harvey et al., 2005). Interestingly, the crosstalk between lymphatic vessels and lipid metabolism is bidirectional, as high plasma lipid levels and obesity seem to deteriorate the function of lymphatic vessels on high fat diet, both in C57Bl/6 mice (Weitman et al., 2013) and in hyperlipidemic Apoe -/- mice (Lim et al., 2009). The mechanisms leading to lymphatic dysfunction in these mice are not yet clear. An activated inflammatory reaction is a strong candidate, but other factors have also been suggested, such as those involving the proteins leptin, adiponectin, apelin and CD36 (Escobedo, Oliver, 2017). Additionally, lymphatic vessels have shown to participate in the trafficking of HDL. As early as the 1970s and 1980s, the analysis of lymph collected from cannulated lymphatic vessels showed that lipoprotein distribution in lymph differs from blood. Lymph is almost totally devoid of VLDL and all ApoB present in lymph is found in LDL particles. Interestingly, human lymph contains large HDL particles with around 30 % more cholesterol than could be explained by the filtration of plasma HDL across the endothelium (Nanjee et al., 2000, Nanjee et al., 2001) indicating that HDL enters tissues in order to collect excessive cholesterol and is absorbed by lymphatic capillaries from interstitial fluid (Randolph, Miller, 2014). The absorption of HDL from tissues to lymphatic vessels is actively regulated by the expression of SR-BI on the surface of LECs (Lim et al., 2013). Therefore, the activation of lymphatic transport could be a potential treatment option for lowering cholesterol accumulation in atherosclerotic plaques. 2.4 VEGFS AND THEIR RECEPTORS The VEGF family of growth factors are important regulators of the physiological and pathological growth of blood and lymphatic vessels. The family consists of dimeric glycoproteins VEGF-A, -B, -C, -D and the placenta growth factor (PlGF) as well as VEGF homologues from parapoxvirus (VEGF-E) and snake venom (VEGF-F). VEGFs are characterized by a central VEGF homology domain (VHD) that contains the receptor binding portion of the growth factor. VEGFs primarily function through tyrosine kinase receptors VEGFR1, VEGFR2 and VEGFR3 and the neuropilin co-receptors (NRP) 1 and 2, which are expressed on the vascular endothelium. (Ferrara, Gerber & LeCouter, 2003). In the following chapters, an overview of the structure and function of VEGFs will be given. Furthermore, their therapeutical potential in CVDs as well as in the lipid and lipoprotein metabolism will be discussed. Current mouse models for studying VEGFs, VEGFRs and NRPs are presented in Table 3.

44 20 Table 3. Summary of gene targeting of VEGFs, VEGFRs and NRPs in mice. Modified from (Olsson et al., 2006). Genotype Survival Phenotype Reference Vegfa -/- Lethal at E Vascular defects (Carmeliet et al., 1996) Vegfa +/- Lethal at E11-12 Vascular defects (Ferrara et al., 1996) VEGF-A TG (adipose) Vegfb -/- VEGF-B TG (cardiac) Viable Viable Vegfc -/- Lethal at E16.5 Increased vascularity in adipose tissue, decreased lipid levels Reduced heart size, decreased accumulation of lipids in heart (Elias et al., 2012) (Hagberg et al., 2010) Viable Cardiac hypertrophy (Karpanen et al., 2008) Lymphedema, lack of lymphatic vessels (Karkkainen et al., 2004) Vegfc +/- Viable Lymphedema (Karkkainen et al., 2004) K14-VEGF-C Viable Lymphatic vessel hyperplasia, obesity (Jeltsch et al., 1997, Karaman et al., 2016) Vegfd -/- Viable (Baldwin et al., 2005) Plgf -/- Viable (Carmeliet et al., 2001) Vegfr1 -/- Lethal at E Vascular defects, endothelial cell overgrowth (Fong et al., 1995) Vegfr1 TK -/- Viable Suppressed macrophage migration (Hiratsuka et al., 1998) Vegfr2 -/- Lethal at E Vascular defects (Shalaby et al., 1995) Vegfr2 TK -/- Lethal E Vascular defects (Sakurai et al., 2005) Vegfr3 -/- Lethal at E10.5 K14-sVEGFR3 Vegfr3 +/- (Chy) Viable Viable Vascular defects, heart failure Lymphedema, lack of cutaneous lymphatic vessels Lymphedema, lack of cutaneous lymphatic vessels Nrp1 -/- Lethal at E Vascular defects Nrp2 -/- Viable E: embryonic day, TG: Transgenic, TK: Tyrosine kinase Neuronal anomalies, lymphatic defects (Dumont et al., 1998) (Makinen et al., 2001) (Karkkainen et al., 2001) (Kawasaki et al., 1999, Kitsukawa et al., 1997) (Giger et al., 2000, Yuan et al., 2002) VEGF-A VEGF-A is the best-characterized member of the VEGF family (Senger et al., 1983, Ferrara, Henzel, 1989). It is a strong inducer of angiogenesis, but it also regulates many other functions such as the survival of endothelial cells, vascular permeability, inflammatory cell functions and vasodilation (Ferrara, Gerber & LeCouter, 2003). In humans, alternative splicing generates at least six different transcripts of the VEGFA gene, which have different binding properties to the receptors VEGFR1, VEGFR2 and NRPs (Robinson, Stringer, 2001). The expression of VEGF-A is strongly regulated by hypoxia through the interaction of HIF- 1α (Forsythe et al., 1996) and HIF2α (Blancher et al., 2000) with the hypoxia-response elements in the VEGFA gene promoter regions. Additionally, many growth factors, hormones, cytokines, and cellular stress regulate the expression of VEGF-A. VEGF-A is the main angiogenic factor during development and both homozygous (Carmeliet et al., 1996)

45 21 and heterozygous (Ferrara et al., 1996) deletions are embryonically lethal due to defects in the vascular system. VEGF-A is also crucial for post-natal development but is not required for the survival of adult organs (Gerber et al., 1999). Since VEGF-A is a strong inducer of angiogenesis, it has been used in several pre-clinical work and clinical studies to induce therapeutical growth of blood vessels in ischemic tissues (Yla-Herttuala et al., 2017). Pre-clinical studies using plasmids, adenoviruses and adenoassociated viruses have been successful in inducing the transient sprouting of blood vessels and capillary enlargement (Gowdak et al., 2000, Su, Lu & Kan, 2000, Lopez et al., 1998, Schwarz et al., 2000). However, most of the clinical trials using VEGF-A have failed due to either low dose of therapeutic agents or insufficient gene expression time (Zachary, Morgan, 2011, Giacca, Zacchigna, 2012). Some animal studies have indicated that the upregulation of VEGF-A increases atherosclerotic plaque size (Celletti et al., 2001b, Celletti et al., 2001a, Lucerna et al., 2007, Heinonen et al., 2013) but this has not been verified in other animal studies (Leppanen et al., 2005) or in clinical trials (Henry et al., 2003, Zachary, Morgan, 2011, Hedman et al., 2009). Generally, VEGF-A therapies have been safe and well tolerated and the only major side effect reported is fluid accumulation within tissues due to increased vascular permeability (Baumgartner et al., 1998). Pathological conditions such as metastatic tumors, intraocular vascular diseases (diabetic retinopathy being an example) and inflammatory conditions, including psoriasis and rheumatoid arthritis, are characterized by an undesired upregulation of VEGF-A driven angiogenesis and thus anti-angiogenic therapy has been shown to be beneficial in these conditions (Vasudev, Reynolds, 2014, Zhang, Zhang & Weinreb, 2012, Szekanecz, Koch, 2007). Five anti-angiogenic biological therapeutics aimed at blocking the function of VEGF- A are currently approved by the FDA: aflibercept, bevacizumab and ramucirumab for the treatment of cancer and aflibercept, ranibizumab and pegaptanib for the treatment of ocular diseases. (Gotink, Verheul, 2010). Recent reports suggest that in addition to the activation of hypoxia-induced angiogenesis, VEGF-A also plays a role in lipid and lipoprotein metabolism. VEGF-A mediated cross-talk between the endothelium and adipocytes has been suggested (Cao, 2013) and, indeed, obesity increases circulating VEGF-A levels in both mice and humans (Gomez-Ambrosi et al., 2010). On the other hand, mice overexpressing VEGF-A in the adipocytes had increased vascularity in white adipose tissue leading to enhanced energy expenditure and decreased serum triglyceride, cholesterol and HDL-cholesterol levels (Elias et al., 2012, Sun et al., 2012, Sung et al., 2013), whereas the deletion of VEGF-A lead to metabolic defects and increased plasma cholesterol levels (Sung et al., 2013). Therefore, increased tissue specific vascularity could be beneficial for the management of obesity and improving metabolic health. Futhermore, VEGF-A concentration is increased in the plasma of hypercholesterolemic patients, and while cholesterol levels are decreased during statin treatment, serum VEGF-A levels decline as well (Trape et al., 2006, Blann et al., 2001). Significant associations between small nucleotide polymorphisms (SNPs) in the close vicinity of the VEGFA gene and plasma HDL-cholesterol and LDL-cholesterol levels have been found (Stathopoulou et al., 2013). VEGF-A can activate SR-BI and enhance the endothelial binding, uptake and transport of HDL, which could be beneficial in the treatment of atherosclerotic plaques (Velagapudi et al., 2017). On the other hand, therapeutically induced VEGF-A overexpression has been shown to decrease plasma LPL activity, leading to increased amounts of large lipoproteins (Heinonen et al., 2013), which could lead to accelerated atherogenesis. Interestingly, HDL is capable of inducing ischemia- and inflammation-driven angiogenesis via the activation of the HIF-1α/VEGF-A pathway (Tan, Ng & Bursill, 2015), whereas LDL has been shown to cause the internalization of VEGFR2 and lead to the attenuation of angiogenesis (Jin et al., 2013) further suggesting an interplay between lipoproteins and VEGF-A signaling.

46 VEGF-B VEGF-B, a second member of the VEGF family, was characterized in 1996 (Olofsson et al., 1996) and it is a ligand for VEGFR1 and NRP1 (Olofsson et al., 1998). Two isoforms of VEGF- B are generated through alternative splicing: freely soluble VEGF-B186 and VEGF-B167, which has a heparin-binding domain and therefore can interact with HSPGs (Makinen et al., 1999). VEGF-B is expressed in the tissues that have a high metabolic activity, such as in the heart and skeletal muscle (Olofsson et al., 1996). Unlike VEGF-A, VEGF-B seems not to be a strong angiogenic factor and it is not regulated by hypoxia (Enholm et al., 1997, Zhang et al., 2009). However, it has been successfully used to induce therapeutic angiogenesis in the myocardium of mice (Huusko et al., 2010), rabbits and pigs (Nurro et al., 2016, Lahteenvuo et al., 2009). The function of VEGF-B is not irreplaceable, since VEGF-B KO mice are healthy and fertile and display only minor heart phenotypes such as size reduction (Bellomo et al., 2000) and atrial conduction abnormalities (Aase et al., 2001). However, VEGF-B transgenic mice display cardiac hypertrophy suggesting the importance of VEGF-B in the heart (Karpanen et al., 2008). As VEGF-B is not capable of activating angiogenesis through VEGFR1, it is likely that VEGF-B functions as a regulator of VEGF-A mediated angiogenesis by directing VEGF-A to VEGFR2 (Anisimov et al., 2013). There is some clinical evidence that high plasma VEGF-B levels might correlate with hyperlipidemia (Sun et al., 2014), but more studies are needed to confirm this finding. Experimental studies during the past ten years have indicated that VEGF-B regulates glucose metabolism and participates in the regulation of lipid metabolism, especially in the heart and adipose tissue, but the results have been contradictory. The first study, published in 2008, showed that VEGF-B transgenic mice had decreased triglyceride levels in cardiomyocytes (Karpanen et al., 2008). However, another study showed that adenovirus-mediated VEGF-B overexpression in pig hearts resulted in elevated FATP4 expression and increased lipid content in cardiomyocytes (Lahteenvuo et al., 2009). A year later, Hagberg et al. (Hagberg et al., 2010) showed that the deletion of the Vegfb gene in mice led to decreased uptake and accumulation of lipids in the muscle, heart and brown adipose tissue. In vitro analysis indicated that VEGF-B regulates the expression of FATP3 and FATP4 in endothelial cells. Additionally, the deletion of the Vegfb gene in a diabetic mouse model improved insulin sensitivity and glucose metabolism (Hagberg et al., 2012) indicating that the downregulation of VEGF-B could be beneficial in treating insulin resistance. However, a later study using two separate VEGF-B KO models was unable to verify the improved glucose tolerance or insulin resistance (Dijkstra et al., 2014). Furthermore, Robciuc and colleagues (Robciuc et al., 2016) showed contradictory results suggesting that overexpression of VEGF-B within an adenoassociated virus vector could improve glucose metabolism and lead to decreased plasma triglyceride levels. The authors concluded that the overexpression of VEGF-B targets VEGF- A to signal through the pro-angiogenic VEGFR2 thus leading to increased vascularity in adipose tissue and enhanced insulin delivery. Recent studies have shown that the activation of VEGF-B might function directly to regulate cell energy metabolism (Zafar et al., 2017) as VEGF-B is a downstream target of the Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α)/oestrogen-related receptor α (ERRα) signaling pathway, which is activated during exercise, fasting and cold exposure (Mehlem et al., 2016) VEGF-C VEGF-C was cloned in 1996 and it is a strong VEGFR2 and VEGFR3 ligand (Joukov et al., 1996). It is produced as a propeptide that contains a VHD and cleavable N- and C-terminal sequences (Joukov et al., 1997). VEGF-C is processed by a complex of protease A disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and a secreted factor collagen and Calcium binding EGF domains 1 (CCBE1), which removes the N-terminal domain of VEGF-C and releases the fully mature VEGF-C protein (Bui et al., 2016, Jeltsch et al., 2014). The long form of VEGF-C binds only to VEGFR3, but the mature form can bind both VEGFR2 and VEGFR3. The cleavage of propeptides increases the affinity of VEGF-C to

47 23 VEGFR2, VEGFR3 and NRP-2 as well as to HSPGs (Joukov et al., 1997). Although VEGF-C can bind to VEGFR2 and mediate angiogenesis, the effect of VEGF-C on the blood vasculature is low due to the localization of uncleaved VEGF-C on LECs. Therefore, VEGF- C functions mainly through VEGFR3 and is primarily a lymphangiogenic factor. (Joukov et al., 1997). VEGF-C KO mice fail to develop lymphatic vasculature and die around E16.5 (Karkkainen et al., 2004). In humans, the genetic frameshift of VEGF-C leading to truncated VEGF-C causes a Milroy-like disease characterized by mild lymphedema (Gordon et al., 2013). As VEGF-C is a strong inducer of lymphangiogenesis, it has been used in several preclinical studies to induce lymphatic growth. Most promisingly, VEGF-C therapy was capable of inducing lymphangiogenesis after lymph node dissection in a large animal model (Lahteenvuo et al., 2011, Honkonen et al., 2013) and a clinical trial is currently ongoing using VEGF-C therapy which intends to improve secondary lymphedema after breast cancer surgery (A Phase I Study With Lymfactin in the Treatment of Patients With Secondary Lymphedema, Furthermore, the administration of VEGF-C was successfully used to induce lymphangiogenesis and improve cardiac parameters after MI in mice (Klotz et al., 2015) and rats (Henri et al., 2016). Although VEGF-C therapy has been shown to produce beneficial effects, it has also faced some obstacles. Some pre-clinical reports were not able to confirm that VEGF-C could induce stable, permanent lymphatic growth (Goldman et al., 2005) and VEGF-C overexpression might induce undesired lymphedema (Gousopoulos et al., 2016). VEGF-C has also been shown to be involved in the growth of metastatic tumors (Skobe et al., 2001) emphasizing the importance of highly controlled delivery and expression of lymphangiogenic factors for therapeutic purposes. Only a few studies have analyzed the role of VEGF-C in lipid or lipoprotein metabolism. Like VEGF-A and VEGF-B, circulating VEGF-C levels are also increased in obesity (Gomez- Ambrosi et al., 2010). In contrast to VEGF-A overexpressing mice, however, the upregulation of VEGF-C lead to a more obese phenotype and increased plasma glucose levels (Karaman et al., 2016), whereas the blockage of both VEGF-C and VEGF-D lead to the smaller adipocyte size and decreased accumulation of triglycerides in the liver (Karaman et al., 2014). VEGF-C is required for the maintenance of lacteals in the intestine and its downregulation leads to defective lipid absorption and subsequently to a leaner phenotype (Nurmi et al., 2015) VEGF-D VEGF-D was cloned in 1996 and it shares many features with VEGF-C (Orlandini et al., 1996, Yamada et al., 1997). Similarly to VEGF-C, VEGF-D is produced as a precursor protein comprising of a VHD and cleavable N- and C-terminal propeptides (Achen et al., 1998). Proteolytic processing generates multiple forms of mature VEGF-D (VEGF-D ΔNΔC ) that have different receptor binding affinities and specificities (Stacker et al., 1999). Human VEGF-D can promote angiogenesis and lymphangiogenesis by mediating signal transduction by binding to VEGFR-2 and VEGFR-3 as well as to NRP-1, NRP-2 and heparin (Achen et al., 1998, Karpanen et al., 2006a, Harris et al., 2011). In contrast to the human orthologue, mouse VEGF-D has been suggested to be a VEGFR-3 specific ligand and capable of inducing only lymphangiogenesis (Baldwin et al., 2001). However, a later study indicated that mouse VEGF-D does have angiogenic potential, at least when delivered with gene transfer vectors (Anisimov et al., 2009). Although VEGF-D can bind to VEGFR3 and induce lymphangiogenesis, it cannot replace VEGF-C during embryonic development (Karkkainen et al., 2004). However, it is capable of rescuing lymphatic hypoplasia in mice with heterozygous VEGF-C deletion (Haiko et al., 2008). VEGF-D deficient mice are healthy, viable and fertile and do not display any major defects in the blood or lymphatic vasculature (Baldwin et al., 2005). As VEGF-D seems to be dispensable for the development and maintenance of blood and lymphatic vasculature, VEGF-D might mediate lymphangiogenesis especially during local inflammatory reactions (Bui et al., 2016). In

48 24 humans, a rare polycystic lung disease lymphangioleiomyomatosis (LAM) is characterized by the abnormal proliferation of SMCs and increased serum VEGF-D levels (Seyama et al., 2006). VEGF-D ΔNΔC has been successfully used in several preclinical studies to induce therapeutical angiogenesis (Nurro et al., 2016, Rissanen et al., 2003, Rutanen et al., 2004) and to reduce neointimal thickening in restenosis (Rutanen et al., 2005). Furthermore, a recent clinical trial with adenovirus expressing VEGF-D ΔNΔC was shown to improve myocardial blood flow in refractory angina patients (Hartikainen et al., 2017). Only a few studies have focused on VEGF-D in lipid metabolism. Obese individuals (Silha et al., 2005, Gomez- Ambrosi et al., 2010) and patients with coronary artery disease (Hartikainen et al., 2017) display variable VEGF-D levels in plasma. Interestingly, high VEGF-D levels seem to predict future HF (Borne et al., 2018), atrial fibrillation and stroke (Berntsson et al., 2018) Other VEGFs PlGF was discovered in 1991 (Maglione et al., 1991) and it is a ligand for VEGFR1 (Park et al., 1994), NRP1 (Migdal et al., 1998) and NRP2 (Gluzman-Poltorak et al., 2000). Alternative RNA splicing generates at least four distinct isoforms (PlGF-1, PlGF-2, PlGF-3 and PlGF-4) in humans but only one isoform (PlGF2) in mice (Ribatti, 2008). The biological role of PlGF is still under debate. Although PlGF shares structural similarities with VEGF-A, it is only mildly mitogenic for endothelial cells (Maglione et al., 1991) and PlGF deficient mice are healthy and fertile without any major changes in their vasculature (Carmeliet et al., 2001). In addition to mediating its effects directly through VEGFR1, PlGF releases VEGF-A to signal through more angiogenic VEGFR2. Indeed, the delivery of PlGF has been shown to induce angiogenesis efficiently (Ziche et al., 1997, Luttun et al., 2002). Obese individuals display increased plasma PlGF concentrations (Pervanidou et al., 2014). Furthermore, PlGF deficient mice have a reduced number of blood vessels in the adipose tissue leading to the decreased accumulation of lipids and a leaner phenotype on high-fat diet (Lijnen et al., 2006). VEGF-E forms have been isolated from parapoxviruses (Lyttle et al., 1994). VEGF-Es are classified as VEGF family members according to their structural and functional similarity: they are ligands for VEGFR2 and can stimulate angiogenesis and vascular permeability (Meyer et al., 1999, Ogawa et al., 1998). As VEGF-E cause significantly less edema than VEGF- A, it can be considered as an alternative candidate to stimulate therapeutic angiogenesis (Shibuya, 2009). Additionally, several forms of VEGF-like proteins (VEGF-Fs) have been isolated from the venom of snakes belonging to the Viperidae family (Yamazaki et al., 2009). For example, VEGFR2 ligand Vammin has been shown to induce angiogenesis (Toivanen et al., 2017), hypotension (Yamazaki et al., 2003) and vascular permeability (Matsunaga et al., 2009) even more strongly than VEGF-A VEGF Receptors VEGFs bind with high affinity to VEGFRs, tyrosine kinase receptors that have a ligandbinding extracellular part consisting of seven immunoglobulin-like loops, a transmembrane domain, a juxtamembrane domain, a tyrosine kinase and C-terminal tail. VEGFs can bind to their receptor directly or they can be presented through co-receptors expressed on the same cell or an adjacent cell. The binding of a ligand leads to receptor dimerization, the activation of the tyrosine kinase domain and autophosphorylation of the tyrosine residues. This activates intracellular signaling cascades through various signaling molecules such as Protein kinase B (AKT), Extracellular signal regulated kinase (ERK), Phosphatidylinositide 3-kinase (PI3K) and Protein kinase C (PKC) leading to cell proliferation, migration and survival. (Koch et al., 2011). The binding characteristics of VEGFs to VEGFRs are presented in Figure 3.

49 25 Figure 3. Binding of VEGFs to VEGFRs and NRP co-receptors and their primary functions. Modified from (Olsson et al., 2006, Pellet-Many et al., 2008, Koch et al., 2011) VEGFR1 VEGFR1, also called as FLT1, was cloned in 1990 (Shibuya et al., 1990) and it functions as a receptor for VEGF-A (de Vries et al., 1992), VEGF-B (Olofsson et al., 1998) and PlGF (Park et al., 1994). VEGFR-1 is expressed on vascular endothelium at considerably high levels and it can be found also on non-endothelial cells such as monocytes, macrophages and vascular SMCs (Grosskreutz et al., 1999, Sawano et al., 2001). The expression of VEGFR1 is regulated by hypoxia (Nomura et al., 1995). The function of VEGFR1 is essential during development: Vegfr1 KO mice die at E8.5 due to the unorganized vascular channels and excessive proliferation of angioblasts (Fong et al., 1995). However, mice with inactivated tyrosine kinase part of VEGFR1 have normal life span and no vascular defects (Hiratsuka et al., 1998) indicating that although the presence of VEGFR1 is required for the development of vasculature, VEGFR1 mediated signaling is dispensable. VEGFR1 can be considered as a decoy receptor for VEGF-A as it directs VEGF-A binding away from the more angiogenic VEGFR2 (Hiratsuka et al., 1998). Indeed, VEGF-A binds to VEGFR1 with high affinity but causes low tyrosine-kinase activation (Waltenberger et al., 1994) and deletion of VEGFR1 in adults leads to the upregulation of VEGFR2 signaling and increased angiogenesis (Ho et al., 2012). VEGFR1 can form heterodimers with VEGFR2 and mediate endothelial cell functions by transphosphorylating VEGFR2 (Huang et al., 2001). Although VEGFR1 mediated signaling seems not to be important for vascular development, it might have an essential role in other biological processes. For example, VEGFR1 has been shown to regulate the migration and survival of macrophages (Hiratsuka et al., 1998) and to mediate the production of lymphangiogenic VEGF-C from macrophages (Murakami et al., 2008) VEGFR2 VEGFR2, also called KDR in humans (Terman et al., 1991) and Flk1 in mice (Matthews et al., 1991), was cloned in It is the primary receptor for VEGF-A (Terman et al., 1992) and mediates the proliferation, migration and survival of endothelial cells (Bernatchez, Soker & Sirois, 1999). In addition to VEGF-A, VEGF-C (Joukov et al., 1996), VEGF-D (Achen et al., 1998) VEGF-E (Ogawa et al., 1998) and VEGF-Fs (Yamazaki et al., 2003) are also ligands for VEGFR2. Soluble VEGFR2 (svegfr2) is formed by alternative splicing and it binds to VEGF-

50 26 C and thus inhibits lymphangiogenesis (Albuquerque et al., 2009). In addition to vascular endothelial cells, VEGFR2 is expressed in several other cell types such as hematopoetic stem cells, pancreatic duct cells and megakaryocytes (Koch et al., 2011). VEGFR2 is vital during embryonic development and its deletion leads to embryonic death at E resulting from an early defect in the development of hematopoietic and endothelial cells (Shalaby et al., 1995). In addition, the deletion of the major phosphorylation site of VEGFR2, tyrosine-1173, leads to embryonic lethality at E , highlighting the importance of VEGFR2 signaling for the endothelial and hematopoetic development (Sakurai et al., 2005) VEGFR3 The third member of the VEGFR family, VEGFR3 or FLT4 was cloned in 1992 (Aprelikova et al., 1992). VEGFR3 is a primary receptor for VEGF-C (Joukov et al., 1996) and VEGF-D (Achen et al., 1998) and it is mainly expressed on the lymphatic endothelium (Kaipainen et al., 1995). In addition, VEGFR3 is expressed in neural progenitors (Le Bras et al., 2006), osteoblasts (Orlandini et al., 2006) and macrophages (Schmeisser et al., 2006). Missense mutations in VEGFR3 gene cause primary lymphedema called Milroy s disease, which is characterized by the defective formation of cutaneous lymphatic vessels resulting in the swelling of the extremities (Karkkainen et al., 2000). During embryonic development, VEGFR3 is expressed in the primary vascular plexus at E8.5 and subsequently in venous endothelial cells in the cardinal vein, which give rise to lymphatic endothelial progenitors (Kukk et al., 1996). The expression of VEGFR3 is essential for the blood vessel development during development as the deletion of the Vegfr3 gene in mice results in abnormally organized large blood vessels, fluid accumulation within pericardial cavity and HF, leading to embryonic death at E10.5 (Dumont et al., 1998). However, the suppression of VEGFR3 in adults does not lead to lymphatic defects (Karpanen et al., 2006b). Interestingly, if either the ligand binding or the tyrosine kinase domain of VEGFR3 is inactivated during development, the growth of lymphatic vasculature is disrupted but blood vessels develop normally (Zhang et al., 2010) suggesting that the VEGFR3 expression rather than the VEGF-C/VEGFR3 signaling pathway is needed for the development of the blood vasculature. Additionally, VEGFR3 has been shown to regulate angiogenic sprouting (Tammela et al., 2008) and vascular permeability (Heinolainen et al., 2017). Thus, in addition to its function as the main receptor for lymphangiogenic signaling, VEGFR3 can control the activation of VEGFR2 (Heinolainen et al., 2017). Furthermore, VEGFR2 and VEGFR3 can form heterodimers, capable of inducing angiogenesis by binding to VEGF-A as well as lymphangiogenesis by binding to VEGF-C (Dixelius et al., 2003) NRPs NRPs were first discovered as axonal guidance molecules as they bind to class 3 semaphorin family (SEMA3) members (Kitsukawa et al., 1997, He, Tessier-Lavigne, 1997), but they were later also found on vascular endothelium (Soker et al., 1998). They function as VEGFR co-receptors and can modulate VEGFR signaling by binding to VEGFs (Soker et al., 1998, Soker et al., 2002). NRP1 has been shown to regulate endothelial cell proliferation, migration and vascular permeability (Soker et al., 1998, Becker et al., 2005). It can bind to all members of VEGF family (Wild et al., 2012) and function as a co-receptor for both VEGFR1 and VEGFR2 (Soker et al., 1998). It is essential for development since the deletion of NRP1 causes cardiovascular and neuronal defects, leading to death of murine embryos at E (Kitsukawa et al., 1997, Kawasaki et al., 1999). VEGF-A can simultaneously bind to VEGFR2 and NRP1 and induce the heterodimerization of these receptors (Soker et al., 2002). NRP1 can modify VEGFR2 signaling (Kawamura et al., 2008, Evans et al., 2011) and control VEGFR2 trafficking after ligand-induced endocytosis (Lanahan et al., 2013, Ballmer-Hofer et al., 2011). Another neuropilin, NRP2, was first identified in the neuronal system (Chen et al., 1997) and later in venous and lymphatic endothelial cells (Giger et al., 2000). It functions as a co-receptor for

51 27 VEGFRs (Favier et al., 2006, Gluzman-Poltorak et al., 2001) and binds to VEGF-A, VEGF-C and VEGF-D and PlGF2 (Karpanen et al., 2006a, Gluzman-Poltorak et al., 2000). NRP2 regulates lymphatic sprouting by modulating tip cell function in lymphatic vasculature (Karpanen et al., 2006a). The deletion of NRP2 is not lethal, but it leads to mild neuronal anomalies (Giger et al., 2000) and lymphatic defects (Yuan et al., 2002).

52 28

53 29 3 Aims of the study The overall aim of this thesis was to gain novel insights in to the role of VEGFR3 and its ligands VEGF-C and VEGF-D in lipoprotein metabolism and in CVDs, such as atherosclerosis and MI. The specific aims of this thesis were as follows: (I) (II) (III) To study the role of attenuated VEGFR3 signaling on lipoprotein metabolism and atherosclerosis in transgenic svegfr3 and Chy mice. To determine the role of VEGF-D in lipoprotein metabolism in VEGF-D KO mice. To analyze the role of VEGFR3 signaling in the structure of cardiac lymphatic vessels and to clarify the role of cardiac lymphatic vessels in the survival after MI.

54 30

55 31 4 Materials and methods Materials and methods used in this study are summarized in following sections. Detailed descriptions of the methods are provided in the original publications (I-III). 4.1 ANIMALS Hypercholesterolemic Ldlr -/- /Apob 100/100 mice were originally obtained from the Jackson Laboratory (stock no ). These mice were crossed with svegfr3 and Chy mice in order to analyze the role of lymphatic vessels in atherosclerosis and lipid metabolism (I) and in MI (III). svegfr3 mice express a fusion protein consisting of the ligand-binding portion of the VEGFR3 extracellular domain and the fragment crystallizable (Fc) domain of immunoglobulin γ-chain under K14-keratinocyte protein (Makinen et al., 2001). Chy mice have a heterozygous inactivating point mutation in the Vegfr3 gene (Karkkainen et al., 2001). Furthermore, Ldlr -/- /Apob 100/100 mice were crossed with VEGF-D KO mice (Baldwin et al., 2001) to analyze the role of VEGF-D in lipoprotein metabolism (II). VEGF-D KO mice have a homozygous deletion of the first coding exon of Vegfd gene. Mice were fed ad libitum a normal chow diet or Western-type high-fat diet for 2, 6 or 12 weeks. Both female and male mice were used in studies I and III and only male mice were used in study II. Study groups are presented in Table 4. Mice were housed in groups or individually when necessary. Mice were maintained in the temperature- and humidity-controlled environment with a 12-hour light/dark cycle at the Laboratory Animal center of University of Eastern Finland. All animal experiments were approved by the National Animal Experimental Board of Finland.

56 32 Table 4. Study groups. Article Mouse strain Gender Age Diets Follow-up I svegfr3 (FVB) WT littermates (FVB) Male Female 3-4 mo. 6-7 mo mo. Chow diet HFD 14 days (HFD) 42 days (HFD) 84 days (HFD) Chy (NMRI) WT littermates (NMRI) svegfr3 x Ldlr -/- /Apob 100/100 (C57Bl/6JOlaHsd) Chy x Ldlr -/- /Apob 100/100 (C57Bl/6JOlaHsd) Ldlr -/- /Apob 100/100 littermates (C57Bl/6JOlaHsd) II VEGF-D KO x Ldlr -/- /Apob 100/100 (C57Bl/6JOlaHsd) Male 4-5 mo. 12 mo. Chow diet HFD 42 days (HFD) 84 days (HFD) Ldlr -/- /Apob 100/100 (C57Bl/6JOlaHsd) III svegfr3 x Ldlr -/- /Apob 100/100 (C57Bl/6JOlaHsd) Chy x Ldlr -/- /Apob 100/100 (C57Bl/6JOlaHsd) Male Female 3-4 mo. 12 mo. Chow diet HFD 4 days (MI) 8 days (MI) 42 days (MI) 84 days (HFD) Ldlr -/- /Apob 100/100 littermates (C57Bl/6JOlaHsd) HFD: High fat diet, MI: Myocardial infarction, mo.: months 4.2 ANALYSIS OF LIPID, LIPOPROTEIN AND GLUCOSE METABOLISM The analysis of basic clinical chemistry and glucose metabolism were performed from the plasma or serum of mice on chow and on high fat diet (I, II). Lipoprotein metabolism was analyzed on mice fed high fat diet for six weeks. Mice were fasted either for four hours (I, II) or overnight (II) before samples were taken for analysis. Isoflurane anesthesia (4% induction, 2% maintenance) was used during intravenous injections. The specific methods used are presented in Table 5.

57 33 Table 5. Methods used in the analysis of plasma lipids and lipoprotein and glucose metabolism. Blood chemistry Assay Administered substance Route Sample Method Article Cholesterol plasma colorimetric I, II Triglycerides plasma colorimetric I, II FFA serum colorimetric II Glucose blood enzymatic I, II ALAT plasma colorimetric II Lipoprotein metabolism Lipoprotein profiling Lipoprotein subclasses Particle size analysis plasma HPLC I, II plasma FPLC II plasma LPL activity Heparin i.v. plasma Lipid absorption In vivo RCT Tyloxapol, [ 3 H]-triolein [ 3 H]-cholesterol labeled macrophages p.o. i.p. plasma plasma LDL turnover [ 125 I]-LDL i.v. plasma Retinol excursion [ 3 H]-retinol p.o. TRL Remnant uptake plasma, liver [ 3 H]-triolein TRLs i.v. plasma light scattering liquid scintillation liquid scintillation liquid scintillation gamma counting liquid scintillation liquid scintillation II I, II I, II I I, II II II VLDL secretion Tyloxapol i.v. plasma colorimetric II Glucose metabolism GTT Glucose i.p. blood enzymatic I, II ITT Insulin i.p. blood enzymatic II ALAT: Alanine transaminase, FFA: free fatty acids, FPLC: fast protein liquid chromatography, GTT: glucose tolerance test, HPLC: high performance liquid chromatography, i.p.: intraperitoneally, ITT: insulin tolerance test i.v.: intravenously, LDL: low-density lipoprotein, LPL: lipoprotein lipase, p.o.: orally, RCT: Reverse cholesterol transport, TRL: triglyceride rich lipoprotein, VLDL: very low-density lipoprotein. 4.3 IMAGING For all imaging analysis, mice were anesthetized with isoflurane (4 % induction followed by % maintenance). The heart function of the mice was analyzed with ultrasound (II, III), electrocardiography (ECG) (III) and cardiac magnetic resonance imaging (MRI) (III). The body fat percentage was analyzed with MRI using interleaved fat and water images (II). Additionally, cardiac water content was measured with T2 weighted MRI and MI scar composition with T1ρ, TRAFF2 and TRAFF4 relaxation times (III) (Yla-Herttuala et al., 2018).

58 SURGERY Mice were anesthetized with isoflurane inhalation (4 % induction followed by 2 % maintenance) and MI was induced as described previously (Gao et al., 2010). Briefly, the heart was exposed, pushed out of the thorax and a ligation was made around the left anterior descending artery (LAD) at a site 5 mm from its origin using a 6-0 silk suture. Mice were sacrificed 4, 8 or 42 days after the operation. 4.5 HISTOLOGY For post-mortem analysis, mice were sacrificed with carbon dioxide (CO2) and perfused with phosphate buffered saline. Tissue samples were collected in 4% paraformaldehyde and fixed overnight. After fixation, samples for histological stainings were processed to paraffin and cut as 4 µm sections (I, II, III). For confocal imaging, a piece of aorta (I) or the anterior side of the heart was (III) excised and used in immunohistochemical stainings. Aortas were opened longitudinally and attached to black rubber plates, stained with Oil-red-O solution and photographed (I, II). Basic stainings were performed according to manufacturers protocols. Hematoxylin- Eosin staining was used to analyze the basic morphology of liver, heart, intestine, adipose tissue as well as the atherosclerotic plaques in aortic roots and brachiocephalic arteries (I, II, III). Masson s trichrome (I, III) and Movat s pentachrome (I) stainings were performed to analyze the structure of atherosclerotic plaques and MI scar. Collagen type I and III were analyzed from MI scars with Sirius Red staining (III). Rhodamine labeled Griffonia (Bandeiraea) Simplicifolia Lectin I staining and Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I staining were performed to detect capillaries in aortas (I) and in cardiac tissue (III), respectively. Additionally, immunohistochemical stainings were performed to analyze the presence of certain antigens in tissues (I, II, III). Primary antibodies used in immunohistochemical stainings are presented in Table 6. Histological stainings were imaged with a confocal, fluorescent or light microscope. Images were quantified with ImageJ software. 4.6 MOLECULAR BIOLOGY AND CELL CULTURE svegfr3 mice and Chy mice were genotyped with polymerase chain reaction (PCR) using DNA extracted from ear punctures (I, III). For gene expression analysis, RNA was extracted from snap-frozen tissue samples using RNA extraction kits (Qiagen) and quantitative realtime PCR (qpcr) for individual genes was performed from heart (II, III), liver (II) and intestinal (II) samples. Furthermore, the whole transcriptome of liver genes was analyzed with deep sequencing techniques (II). Western blot was performed to analyze the specific protein in plasma or tissue samples (I, II, III). Primary antibodies are presented in Table 6.

59 35 Table 6. Antibodies used in immunohistochemical stainings and Western blot. Antibody Specifity Type Method Tissue Akt ApoB ApoC-III ApoE CD138 CD3 CD31 CD45 ERK1/2 Akt Apolipoprotein B Apolipoprotein C-III Apolipoprotein E Syndecan-1 T-cells Endothelial cells Lymphocytes ERK1/2 F4-80 Macrophages LYVE1 LECs Rabbit pab Rabbit pab Rabbit pab Rat mab Rabbit mab Rat mab Rat mab Rabbit mab Rat mab Rabbit pab Product code Manufacturer Article WB liver 9272 Cell Signaling II WB plasma ab20737 Abcam II WB plasma WB plasma K23100R Ionis Pharmaceuticals Meridian Life Sciences IHC liver BD Biosciences II IHC liver ab16669 Abcam I WM aorta BD Biosciences I IHC heart BD Biosciences III WB liver 137F5 Cell Signaling II IHC heart MCA497 BioRad II, III IHC, WM skin, aortic root, heart 103-PA50 ReliaTech I, III mmq Macrophages Rabbit IHC aorta AIA31240 Accurate I mtor p-akt p-erk1/2 p-mtor Podoplanin PROX1 VEGFR2 VEGFR3 α-hfc mtor Phosporylated AKT Phosporylated ERK1/2 Phosphorylated mtor LECs LECs VEGFR2 VEGFR3 Fc domain of IgG Rabbit mab Rabbit mab Rabbit mab Rabbit mab SH mab Goat mab Rabbit mab Rat mab Goat pab WB liver 2983 Cell Signaling II WB liver 4058 Cell Signaling II WB liver 20G11 Cell Signaling II WB liver 5536 Cell Signaling II WM aorta Biolegend I WM heart AF2727 R&D Systems III WB liver 55B11 Cell Signaling II WB liver Thermo Fischer Scientific WB plasma I2136 Sigma-Aldrich I Mouse α-sma SMCs IHC heart C6198 Sigma-Aldrich III mab α-sma: α smooth muscle actin, CD: Cluster dereminant, Fc: Fragment crystallizable region, IgG: Immunoglobulin G, IHC: Immunohistochemistry, LEC: Lymphatic endothelial cell, mab: Monoclonal antibody, pab: Polyclonal antibody, SH: Syrian hamster, SMC: Smooth muscle cell, WB: Western Blot, WM: Whole mount, II II II

60 STATISTICAL METHODS Statistical analysis was performed using GraphPad Prism (version 5.04, GraphPad Software). Two-tailed unpaired t-test, one-way ANOVA, repeated measures ANOVA or two-tailed ANOVA, followed by a Bonferroni correction, or log-rank test for survival curves were used where appropriate. Data is presented as mean ± SEM and P < 0.05 was considered significant.

61 37 5 Results 5.1 ATTENUATED LYMPHATIC FUNCTION LEADS TO HYPERCHOLESTEROLEMIA AND ACCELERATED ATHEROGENESIS (I) Lymphatic vessels play a pivotal role in lipoprotein metabolism, as they are required for the transport of CMs from the intestinal enterocytes to the blood, as well as for the regulation of RCT (Randolph, Miller, 2014). However, the connection between lymphatic vessel function and arterial pathologies has remained unclear. To study the role of lymphatic vessels in lipoprotein metabolism, we used svegfr3 mice and Chy mice, which lack lymphatic vessels in their skin and have lymphatic defects in other organs. Furthermore, svegfr3 mice and Chy mice were crossed with Ldlr -/- /Apob 100/100 mice in order to study the effects of lymphatic deficiency during hyperlipidemia and in the development of atherosclerosis. Ldlr -/- /Apob 100/100 littermates served as controls in these experiments. svegfr3 mice and Chy mice in atherosclerotic background displayed significantly elevated cholesterol levels on both chow and high fat diets (Figure 4A). Furthermore, triglyceride levels were significantly increased in svegfr3 mice on the chow diet (Figure 4B). Lipoprotein fractioning was performed to analyze the distribution of lipids in lipoprotein particles (Figure 4C and 4D). The proportion of cholesterol was increased in all ApoB-containing particles in svegfr3 mice (Figure 4C), whereas triglycerides were mainly carried in the large VLDL particles (Figure 4D). Cholesterol and triglyceride levels were low in HDL particles (Figure 4C and 4D). Figure 4. Cholesterol and triglyceride levels and lipoprotein profiles in svegfr3 mice, Chy mice and control mice. (A) Cholesterol and (B) triglyceride levels were measured on a chow diet (0) and during 12 weeks on high fat diet. svegfr3 mice displayed significantly increased cholesterol levels compared to controls throughout the study, whereas triglycerides were increased in svegfr3 mice on chow diet. (C-D) Lipoprotein profiles for (C) cholesterol and (D) triglycerides were analyzed with HPLC 12 weeks after high fat diet. Excess cholesterol was carried in VLDL and LDL particles in svegfr3 mice, whereas triglycerides were found mainly in VLDLs. Data is presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.

62 38 As lymphatic vessels are known to participate in CM transport, we evaluated intestinal lipid absorption by giving an oral bolus of radiolabeled triolein and following its appearance in the plasma. We did not find any major changes in lipid absorption indicating that the function of the intestinal lacteals was normal in svegfr3 mice (Figure 5A). To evaluate the mechanisms leading to increased cholesterol levels in LDL particles in svegfr3 mice, we measured LDL turnover by injecting radiolabeled human LDL intravenously. svegfr3 mice showed a tendency towards a slower LDL uptake compared to control animals but the difference was not significant (Figure 5B). Furthermore, lymphatic vessels might contribute to RCT by mediating HDL uptake and transport from tissues to blood circulation. RCT was evaluated by loading macrophages with radiolabeled cholesterol and injecting macrophages intraperitonially. Thus, the appearance of radioactivity in plasma indicates the functionality of HDL to take up and transport cholesterol from tissue residing macrophages. However, no differences were found between svegfr3 mice and control mice, indicating that similar HDL function and transport in svegfr3 mice, at least in current study setup (Figure 5C). Figure 5. Analysis of lipoprotein metabolism in svegr3 and control mice. (A) Lipid absorption was analyzed by measuring radioactivity in plasma after an oral lipid bolus containing [ 3 H]-labeled triolein. (B) LDL turnover was analyzed by measuring radioactivity in blood after the i.v. injection of human [ 125 I]-LDL. (C) In vivo RCT was measured by loading macrophages with [ 3 H]- cholesterol. Macrophages were injected i.p. to the receiver mice and radioactivity was measured from plasma. Data is presented as mean ± SEM.

63 39 Atherosclerotic plaques were analyzed from mice aged 3-4 months (young cohort), 6-7 months (middle cohort) and months (old cohort) on a high fat diet. svegfr3 mice had increased atherosclerotic lesion sizes especially in the early stages of lesion development (Figure 6A-6F). Furthermore, atherosclerotic lesions in svegfr3 mice contained cholesterol crystals already after 2 weeks on high fat diet whereas cholesterol crystals appeared in the atherosclerotic lesions of control mice after 12 weeks on high fat diet (Figure 6G-6H). Figure 6. Analysis of atherosclerosis in svegfr3 mice, Chy mice and control mice. (A-F) Atherosclerosis was analyzed from en face preparations of aortas (A-C) and hematoxylin-eosin stainings for aortic roots (D-F). (G-H) Additionally, the structure of atherosclerotic lesions was analyzed using Movat s pentachrome staining of atherosclerotic lesions two weeks on high fat diet. svegfr3 mice displayed significantly accelerated atherogenesis and more advanced atherosclerotic lesions. Data is presented as mean ± SEM. *P<0.05, **P<0.01.

64 40 To analyze angiogenesis and lymphangiogenesis in the atherosclerotic plaques of svegfr3 mice and Chy mice, we measured the area of vasa vasorum vessels and lymphatic capillaries in the atherosclerotic aortas (Figure 7). There were no differences in CD31-positive microvasculature between groups (Figure 7A-7D). However, lymphatic vessels stained with a podoplanin antibody were almost completely absent in the atherosclerotic lesions of svegfr3 mice and Chy mice (Figure 7A-7B) and showed significantly reduced area compared to controls (Figure 7D). Figure 7. Neovascularization and lymphangiogensis in the atherosclerotic lesions. (A-D) Neovascularization was analyzed from immunoshistochemical lectin and CD31 stainings and lymphangiogenesis with podoplanin staining with confocal microscopy (A-C). svegfr3 mice and Chy mice displayed similar levels of vasa vasorum compared to those observed in controls, whereas the area of lymphatic vessels was significantly reduced (D). Data is presented as median values. *P<0.05, **P<0.01.

65 VEGF-D REGULATES CHYLOMICRON METABOLISM (II) VEGF-D has been shown to be a potential inducer of angiogenesis and lymphangiogenesis in both animal studies and clinical trials (Yla-Herttuala et al., 2017). However, its role in lipoprotein metabolism has not been examined. To study the effects of VEGF-D deficiency on plasma lipid metabolism, we crossed VEGF-D KO mice with Ldlr -/- /Apob 100/100 mice and measured plasma lipid values on regular chow and high fat diets. VEGF-D KO mice had dramatically higher plasma cholesterol and triglyceride levels than Ldlr -/- /Apob 100/100 background control mice when exposed to the high fat diet (Figure 8A and 8B). Figure 8. (A-B) Plasma triglyceride and cholesterol values in VEGF-D KO mice and controls on chow diet (0) and during 12 week Western-type high fat diet feeding. VEGF-D KO mice displayed significantly elevated triglyceride (A) and cholesterol (B) levels on the high fat diet. Data is presented as mean ± SEM. **P<0.01, ***P< According to lipoprotein profiling, cholesterol and triglycerides were mainly carried in triglyceride-rich lipoprotein (TRL) particles resembling CMs, CM remnants or large VLDL particles in VEGF-D KO mice indicating the accumulation of these large lipoproteins in the plasma (Figure 9A and 9B). Furthermore, apolipoprotein analysis revealed increased levels of ApoB100, ApoE and ApoC-III in the TRL particles of VEGF-D KO mice (Figure 9C). Figure 9. Lipoprotein profiling of triglycerides and cholesterol and apolipoprotein analysis of TRL particles in VEGF-D KO mice and controls. (A-B) Lipoprotein profiling and apolipoprotein analysis revealed the accumulation of triglycerides (A) and cholesterol (B) in large lipoprotein particles consisting of CMs, CM remnants (CR) and VLDL in VEGF-D KO mice. (C) Western blot analysis of large lipoprotein fractions revealed that these particles were enriched for ApoB100, ApoE and ApoC-III. Data is presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.

66 42 To evaluate lipoprotein metabolism in VEGF-D KO mice, we analyzed different steps in the lipoprotein processing during exogenous and endogenous pathways. The intestinal lipid absorption was measured using intragastric load of [ 3 H]-labeled triolein by following its retention in plasma. The appearance of [ 3 H]-label was similar in VEGF-D KO mice compared to the controls indicating a normal production rate for CMs in the intestine (Figure 10A). However, a longer study with [ 3 H]-labeled retinol revealed the accumulation of CMs or their remnants in plasma after 4 h (Figure 10B) and the reduced uptake of 3H-labeled retinol in the liver after 8 h (Figure 10C). Interestingly, the activity of LPL was similar in VEGF-D KO mice and control mice indicating that the accumulation of large lipoproteins was not caused by inefficient function of LPL (Figure 10D). Furthermore, the hepatic production of VLDLs was analyzed by measuring the plasma triglyceride levels in several time points after the i.v. injection of tyloxapol. VEGF-D KO mice had increased triglyceride levels 2 h after the injection indicating the accumulation of newly formed VLDL particles in the plasma (Figure 10E). However, triglycerides in VLDL particles cannot be differentiated from triglycerides in CMs with this method and increases in the triglyceride levels might have resulted from the accumulation of CMs in plasma rather than increased production of VLDL particles. Figure 10. Analysis of lipoprotein metabolism in VEGF-D KO mice and control mice. (A) Intestinal lipid absorption was analyzed after the oral fat feeding of [ 3 H]-labeled triolein. Lipid absorption was similar between VEGF-D KO mice and controls. (B) Extended study with [ 3 H]-labeled retinol revealed the accumulation of retinol in plasma after 4 h. (C) The uptake of retinol into the liver was significantly decreased in VEGF-D KO mice. (D) LPL activity was similar in VEGF-D KO mice and controls. (E) Hepatic VLDL production was analyzed by measuring plasma triglyceride levels after Tyloxapol injection. VEGF-D KO mice accumulated more triglycerides than control mice. Data is presented as mean ± SEM. *P<0.05, **P<0.01.

67 43 To clarify the function of the liver in lipoprotein metabolism, we analyzed the expression of liver genes with the aid of deep sequencing (Figure 11). A total of 424 genes were upregulated and 259 downregulated in VEGF-D KO mice compared to controls (Figure 11A). VEGF-D KO mice showed significant increases in genes regulating inflammatory responses, immune cell trafficking and cell adhesion (Figure 11B) whereas downregulated pathways were mainly involved in lipid metabolism, such as the peroxisomal β-oxidation pathway and fatty acid transport (Figure 11C and 11D). Since the VEGF-D KO mice do not express functional LDLR, other hepatic receptors take over its functions in lipoprotein internalization. Lrp1 was expressed at similar levels in VEGF-D KO mice and controls but the expression of Sdc1, the main HSPG responsible for the internalization of remnant lipoproteins, was significantly downregulated in VEGF-D KO mice (Figure 11D). Figure 11. Deep sequencing of liver genes in VEGF-D KO mice and control mice. (A) Total of 683 genes were differentially regulated in VEGF-D KO mice compared to controls. (B-D) Upregulated genes consisted mainly of factors regulating inflammatory reactions whereas downregulated genes were related to lipid metabolism.

68 44 To verify the findings from deep sequencing, we performed qpcr analysis for Scd1 and Ndst1. The gene expression of Sdc1 and Ndst1 was significantly downregulated in VEGF-D KO mice (Figure 12A). Furthermore, both immunohistochemical staining (Figure 12B) and Western blotting analysis (Figure 12C and 12D) revealed decreased levels of the SDC1 protein. To determine the pathways involved in the regulation of SDC1 during VEGF-D deficiency, we performed Western blotting analysis for VEGFR2, VEGFR3, mtor, phosphomtor, ERK1/2, phospho-erk1/2, AKT and phospho-akt. The protein levels of phospho- AKT compared to AKT levels was significantly downregulated in VEGF-D KO mice (Figure 12C and 12E), whereas there was no significant differences in the expression of the other proteins tested (Figure 12C). Figure 12. Analysis of SDC1 in VEGF-D KO mice and control mice. (A) The gene expression of Sdc1 and Ndst1 was analyzed with qpcr revealing significantly downregulated levels in VEGF-D KO mice. (B) Immunohistochemical staining of SDC1 in liver displayed patchy expression in VEGF- D KO mice. (C and D) Western blot analysis showed the downregulation of SDC1 protein. (C and E) Additionally, the phosphorylation of AKT was significantly decreased in VEGF-D KO mice. Data is presented as mean ± SEM. *P<0.05, **P<0.01.

69 45 To assess the effects of increased plasma lipid levels on atherogenesis, we measured atherosclerotic lesion sizes from the aortic root, brachiocephalic arteries and the whole aorta (Figure 13). Surprisingly, VEGF-D KO mice had significantly smaller lesions in aortic root compared to control littermates on chow diet (Figure 13A and 13D). However, lesion sizes were similar after 12 weeks on a high fat diet (Figure 13B and 13E). In contrast, lesion sizes were significantly smaller in the aortas of VEGF-D KO mice in old mice after six weeks being fed the high fat diet (Figure 13C and 13F). These results indicate that the cholesterol carried in large lipoproteins could not enter the vascular wall in VEGF-D KO as efficiently as in control mice, at least on a regular chow diet. Figure 13. Analysis of atherosclerosis in VEGF-D KO mice. (A-C) Quantification and (D-F) representative images of atherosclerotic lesions in brachiocephalic arteries, aortic roots and aortas. On the chow diet, young VEGF-D KO mice displayed decreased average size of the atherosclerotic lesions in the aortic roots compared to controls (A and D). However, there were no differences after 12 weeks on high-fat diet (B and E). Old VEGF-D KO mice had decreased atherosclerotic lesion size in en face preparation of aortas after 6 weeks on high fat diet (C and F). Data is presented as mean ± SEM. *P<0.05, **P<0.01.

70 THE EXPRESSION OF SVEGFR3 LEADS TO HIGHER MORTALITY AFTER MYOCARDIAL INFARCTION (III) The lack of oxygen during MI causes the death of cardiomyocytes and the formation of fibrotic infarction scar tissue in the affected myocardial region (Talman, Ruskoaho, 2016). The accumulation of inflammatory cells (Frangogiannis, 2014) and the formation of edema (Nilsson et al., 2001) in the left ventricle wall (LVW) are also evident during MI. To analyze the role of cardiac lymphatic vessels during MI, we used svegfr3 mice crossed into mice with the Ldlr -/- /Apob 100/100 background. For these experiments, Ldlr -/- /Apob 100/100 littermates served as the controls. Mice were followed for four days, one week or six weeks after LAD ligation. Ligation induced typical signs of MI in both svegfr3 mice and control mice, such as necrosis, the thinning of the LVW and the formation of fibrotic infarction scar (Figure 14A). Even though overall cardiac function was similar in both svegfr3 mice and the control mice, many svegfr3 mice had large infarction scars spanning more than 20 % of the LVW (Figure 14B). This led to significantly higher mortality during acute MI in svegfr3 mice (Figure 14C). Figure 14. Analysis of MI in svegfr3 mice and control mice. (A-B) Histological analysis revealed similar infarct sizes in control and svegfr3 mice 4, 8 and 42 days after the coronary artery ligation. (C) The survival of svegfr3 mice was significantly lower than in control mice. Data is presented as mean ± SEM. *P<0.05, **P<0.01.

71 47 To evaluate the effects of deficient lymphatic function on cardiac parameters, we performed functional ultrasound imaging, MRI imaging and echocardiography on healthy hearts and 3, 8 and 42 days after the infarction (Figure 15). Ultrasound and MRI images showed thinning of the ventricular wall and significant dilatation of the heart after MI (Figure 15A and B). Compared to healthy hearts, diastolic and systolic volumes quantified from MRI images were significantly increased 42 days after the infarction (Figure 15C and 15D). Furthermore, stroke volume and EF were significantly decreased indicating progressive dilatation and attenuated pumping efficacy of the heart (Figure 15E and 15F). Futhermore, elecrocardiography revealed an appearance of Q-wave and decrease of altitude of R and S waves in the infarcted hearts (Figure 15G). Figure 15. Ultrasound, functional MRI and echocardiography analysis of healthy and infarcted murine hearts. (A-B) Thinning of the left ventricular wall and dilatation of heart are clearly visible in ultrasound (A) and MRI images (B). (C-D) End diastolic (C) and end systolic volumes (D) were significantly increased 42 days after the infarction in svegfr3 mice and control mice. (E-F) Stroke volume was significantly decreased 3 and 8 days after the infarction (E) and ejection fraction was decreased in all time points after the infarction in both groups (F). (G) ECG data revealed typical changes in infarcted myocardium, such as appearance of a Q-wave. Data is presented as mean ± SEM *P<0.05, **P<0.01, ***P< The morphology of cardiac lymphatic vessels in svegfr3 mice and control mice was analyzed from whole mount immunohistochemical stainings with a confocal microscope. Lymphatic vessels formed organized lymphatic vessel networks in the epicardium of the control mice (Figure 16A). In svegfr3 mice, cardiac lymphatic vessels were significantly more dilated and they had completely lost their structure in Chy mice displaying a sheetlike morphology (Figure 16A and 16B).

72 48 Figure 16. Analysis of cardiac lymphatic vessels in healthy hearts. (A-B) Lymphatic vessels formed organized networks in the epicardium of control mice, whereas lymphatic vessels were significantly dilated in svegfr3 mice and they had completely lost their organization in Chy mice. Data is presented as mean ± SEM. *P<0.05, ***P< To resolve the accumulation of fluids and inflammatory cells within myocardium after MI, lymphangiogenesis is activated (Henri et al., 2016). We assumed that modified lymphatic vessels may lead to increased fluid accumulation within cardiac tissues and performed T2 MRI in order to detect myocardial edema (Beyers et al., 2012). However, there were no differences between svegfr3 mice and controls (Figure 17A). To analyze lymphangiogenesis in svegfr3 mice after MI, we performed qpcr gene expression analysis for Vegfc and Vegfr3 and measured the area of lymphatic vessels in the myocardium (Figure 17B-17D). Both groups displayed increased expression of Vegfc in hearts harvested 8 days after the infarction compared to healthy hearts (Figure 17B). However, svegfr3 mice had significantly less lymphatic vessels in the LVW compared to controls indicating that they may have a weaker capability to respond to lymphangiogenic signals (Figure 17C and 17D). Figure 17. Cardiac edema and lymphangiogenesis after MI in svegfr3 mice and control mice. (A) Cardiac edema was measured with T2 MR imaging. Cardiac fluid content was significantly increased after MI but there were no differences between the svegfr3 mice and control mice. (B) The expression of Vegfc was increased in infarcted hearts in both groups. (C-D) Lymphangiogenesis in LVW was analyzed from LYVE1 immunohistochemical stainings. svegfr3 mice displayed significantly less lymphatic vessels in LVW 8 days after the infarction. Data is presented as mean ± SEM *P<0.05, **P<0.01, ***P<0.001.

73 49 To evaluate the effects of svegfr3 on blood vasculature, the number of capillaries was quantified from the border zone of the infarcted area. The number of capillaries reached their highest level 4 days after the infarction and it was similar in both svegfr3 and control mice (Figure 18A and 18B) indicating equal responses to angiogenic signals. The leakiness of the newly formed vasculature can lead to hemorrhages, which have been associated with larger infarction scar size (Ghugre et al., 2017). svegfr3 mice displayed an increased number of erythrocytes in the LVW 8 days after the infarction compared those recorded in control mice hearts (Figure 18C and 18D). Interestingly, Western blot analysis revealed increased expression of VEGFR2 protein in the healthy hearts of svegfr3 mice compared to that found in the controls (Figure 18E and 18F) and the expression of Endothelial nitric oxide synthase (enos) was increased in infarcted hearts (Figure 18G). These results indicate that the newly formed vasculature might be more permeable in mice expressing svegfr3 after MI. Figure 18. svegfr3 mice displayed hemorrhages and upregulation of VEGFR2 protein. (A-B) Blood capillaries were evaluated with Lectin stainings. There were no differences between svegfr3 mice and controls. (C-D) Hemorrhage was analyzed from hematoxylin-eosin stainings 8 days after the infarction. svegfr3 mice displayed large accumulations of erythrocytes in the infarcted area. (E- F) Western blot analysis revealed significantly increased VEGFR2 protein levels in the healthy hearts of svegfr3 mice. (G) The expression of enos showed a tendency to increased levels in infarcted hearts. Data is presented as mean ± SEM. *P<0.05, **P<0.01, ***P< T1ρ MRI imaging has shown to be beneficial in determining the structure of fibrotic MI scar tissue noninvasively (Musthafa et al., 2013) and TRAFF2 and TRAFF4 are novel methods with lower specific absorption rates than T1ρ (Yla-Herttuala et al., 2018). T1ρ, TRAFF2 and TRAFF4 imaging was performed seven days after MI induction for both svegfr3 and control littermate mice (Figure 19A-19C). All methods were able to discriminate between healthy myocardium and infarcted region. No differences between groups were detected with the most conventional method T1ρ (Figure 19A). However, the novel MRI method TRAFF4 showed

74 50 a significant increase in relaxation times in svegfr3 mice compared to controls (Figure 19C). TRAFF2 also showed a trend towards increased relaxation times (Figure 19B) indicating changes in fibrotic area in svegfr3 mice. Subsequently, the expression of fibrotic proteins was analyzed with qpcr (Figure 19D). No significant changes were detected between svegfr3 and control mice one week after the infarction. Interestingly, the gene expression of Actin alpha 2 (Acta2) was significantly increased in svegfr3 mice six weeks after the infarction indicating accumulation of cells expressing α-sma in later time points after MI (Figure 19D). Furthermore, Sirius Red staining for collagens I and III showed weaker staining in some svegfr3 mice indicating changes in the accumulation of collagens in these mice (Figure 19E). However, the amount of collagen quantified from the cardiac sections of all mice did not reveal any differences in the total amount of collagen between the groups (Figure 19F). Figure 19. Analysis of fibrosis after MI. (A-C) Fibrosis was measured with T1ρ, TRAFF2 and TRAFF4 relaxation times 7 days after the infarction. Relaxation times were significantly increased in infarcted areas compared to remote areas. Additionally, TRAFF4 relaxation time (C) was significantly increased in svegfr3 mice compared to control mice. (D) The expression of genes regulating the development of fibrosis was analyzed with qpcr. Collagens (Col1a2 and Col3a1) and periostin (Postn) showed increased expression 8 days after the infarction. Acta2 was significantly upregulated in svegfr3 mice 42 days after the infarction compared to control mice. (E-F) Fibrosis was analyzed with Sirius red staining. Part of the svegfr3 mice displayed decreased amount of collagens I and II (E) but the total amount of collagen in quantified from cardiac sections was equal in svegfr3 mice and controls (F). Data is represented as mean ± SEM *P<0.05, **P<0.01, ***P<0.001.