dissertations in health sciences - uefmodified structure of the infarcted area in svegfr3 mice,...

DIS

SE

RT

AT

ION

S | T

AIN

A V

UO

RIO

| VA

SC

UL

AR

EN

DO

TH

EL

IAL

GR

OW

TH

FA

CT

OR

RE

CE

PT

OR

3 AN

D IT

S... | N

o 497

uef.fi

PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND

Dissertations in Health Sciences

ISBN 978-952-61-2999-0ISSN 1798-5706



TAINA VUORIO

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

As the burden of cardiovascular diseases

remains substantially high, novel regulators of lipid metabolism and disease progression are anticipated. Lymphatic system regulates

tissue fluid homeostasis, lipid absorption and immune reactions, thereby modulating

many processes involved in the development of cardiovascular diseases. In this thesis, the function of lymphatic growth factor receptor VEGFR3 in lipoprotein metabolism as well as in atherosclerosis and myocardial infarction

was elucidated.

TAINA VUORIO

30984656_UEF_Vaitoskirja_NO_497_Taina_Vuorio_Terveystiede_kansi_19_01_02.indd 1 2.1.2019 9.03.40

Vascular Endothelial Growth Factor Receptor 3 and Its Ligands in

Cardiovascular Diseases

30984656_UEF_Vaitoskirja_NO_497_Taina_Vuorio_Terveystiede_sisus_19_01_02.indd 1 2.1.2019 9.04.35

TAINA VUORIO



To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Tietoteknia auditorium, Kuopio, on January 25th, 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


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


Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology


Associate Professor (Tenure Track) Tarja Malm, Ph.D. A.I. Virtanen Institute for Molecular Sciences


Lecturer Veli-Pekka Ranta, Ph.D. School of Pharmacy


Distributor: University of Eastern Finland

Kuopio Campus Library P.O. Box 1627

FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto

ISBN (print): 978-952-61-2999-0 ISBN (pdf): 978-952-61-2821-4

ISSN (print): 1798-5706 ISSN (pdf): 1798-5714

ISSN-L: 1798-5706

III

Author’s address: A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND E-mail: [email protected]

Supervisor: Professor Seppo Ylä-Herttuala, M.D., Ph.D.

A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND

Reviewers: 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


Grano Jyväskylä, 2019

Series Editors:

Professor Tomi Laitinen, M.D., Ph.D. Institute of Clinical Medicine, Clinical Radiology and Nuclear Medicine


Associate Professor (Tenure Track) Tarja Kvist, Ph.D. Department of Nursing Science


Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology


Associate Professor (Tenure Track) Tarja Malm, Ph.D. A.I. Virtanen Institute for Molecular Sciences


Lecturer Veli-Pekka Ranta, Ph.D. School of Pharmacy


Distributor: University of Eastern Finland

Kuopio Campus Library P.O. Box 1627

FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto

ISBN (print): 978-952-61-2999-0 ISBN (pdf): 978-952-61-2821-4

ISSN (print): 1798-5706 ISSN (pdf): 1798-5714

ISSN-L: 1798-5706

III

Author’s address: A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND E-mail: [email protected]

Supervisor: Professor Seppo Ylä-Herttuala, M.D., Ph.D.

A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND

Reviewers: 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


IV

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 497. 2019. 84 p. ISBN (print): 978-952-61-2999-0 ISBN (pdf): 978-952-61-2821-4 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706 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 thedevelopment of atherosclerosis and its manifestations myocardial infarction (MI) and stroke.Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are the majorregulators of vascular and lymphatic systems. The aim of this thesis was to study the role ofVEGFR3 and its ligand VEGF-D in lipoprotein metabolism as well as in atherogenesis andMI.

In the first study, we investigated the effects of attenuated lymphatic function on thedevelopment of hyperlipidemia and atherosclerosis. We found that the expression of solubledecoy VEGFR3 (sVEGFR3) on atherosclerotic Ldlr-/-/Apob100/100 background caused severehypercholesterolemia on mice fed a Western-type high fat diet. Furthermore, sVEGFR3 micehad fewer lymphatic vessels in their vascular wall as well as accelerated atherogenesis. Theseresults elucidated the role of the lymphatic system in the regulation of lipoproteinmetabolism and development of atherosclerosis. Subsequently, we evaluated the role ofVEGF-D in lipoprotein metabolism and atherogenesis. VEGF-D knockout (KO) mice on Ldlr-

/-/Apob100/100 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 concludedthat VEGF-D regulates chylomicron remnant uptake in the liver and its deletion leads to theaccumulation of these large lipoproteins in plasma. However, the accumulated particles arenot 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. Wefound that the structure of cardiac lymphatic network was modified in mice with attenuatedVEGFR3 signaling. Furthermore, sVEGFR3 mice had a significantly higher mortality after MIthan their littermates. Novel MRI methods and histology revealed hemorrhages and amodified structure of the infarcted area in sVEGFR3 mice, which might have predisposedthese mice to early cardiac failure. These findings suggest an important role for VEGFR3 inthe healing process after MI.

In conclusion, this thesis highlights the significance of VEGFR3 in the regulation oflipoprotein metabolism at least in mice. Furthermore, the attenuation of VEGFR3 signalingcan 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


VI

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 497. 2019. 84 s. ISBN (print): 978-952-61-2999-0 ISBN (pdf): 978-952-61-2821-4 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706 TIIVISTELMÄ

Sydän- ja verisuonitaudit ovat merkittävimpiä kuolleisuuden aiheuttajia maailmassa.Elintavat ja perinnölliset riskitekijät altistavat hyperlipidemialle, mikä voi johtaaateroskleroosin kehittymiseen sekä lopulta sydäninfarktiin tai aivohalvaukseen. Verisuontenendoteelin 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 vaikutuksiahyperlipidemian ja ateroskleroosin kehittymiseen. Tutkimuksessa havaittiin, että liukoisenVEGFR3:n (sVEGFR3) ilmentyminen hiirissä johti merkittävään hyperkolesterolemiaan.Lisäksi sVEGFR3-hiirillä oli vähemmän imusuonia ateroskleroottisissa valtimoissa janopeutunut aterogeneesi. Toisessa osatyössä tutkittiin VEGF-D kasvutekijän merkitystälipoproteiinien aineenvaihdunnassa ja aterogeneesissä. VEGF-D poistogeenisille hiirillekehittyi merkittävä hyperlipidemia rasvadieetillä. VEGF-D:n puuttuminen johtiheikentyneeseen maksan syndekaanin 1 (SDC1) ilmentymiseen, mikä aiheutti ruokavalionrasvoja kuljettavien kylomikronien kertymiseen verenkiertoon. VEGF-D poistogeenisissähiirissä ei kuitenkaan havaittu lisääntynyttä ateroskleroosia. Tulosten perusteella voidaantodeta, että VEGF-D säätelee kylomikronien sisäänottoa maksaan. Suuretkylomikronipartikkelit eivät kuitenkaan pysty tunkeutumaan verisuonten endoteelin läpi jaaiheuttamaan nopeutunutta ateroskleroosin kehittymistä. Kolmannessa osatyössä tutkittiinVEGFR3:n merkitystä sydäninfarktissa. sVEGFR3:n ilmentyminen johti laajentuneisiinimusuoniin terveissä hiirten sydämisssä. sVEGFR3-hiirillä oli huomattavasti suurempikuolleisuus sydäninfarktin jälkeen kuin kontrollihiirillä. Uudenlaisillamagneettikuvausmenetelmillä havaittiin, että sVEGFR3-hiirten infarktialueen rakenne olimuuttunut. Lisäksi sVEGFR3-hiirten sydämissä havaittiin verenpurkaumia, mikä onmahdollisesti johtanut sydämen heikentyneeseen toimintakykyyn ja sydämenvajaatoimintaan.

Yhteenvetona voidaan todeta, että imusuonilla on merkittävä tehtävä lipoproteiinienaineenvaihdunnan säätelyssä käytetyissä hiirimalleissa. VEGFR3-reseptorin kauttatapahtuvan signaloinnin heikentyminen voikin johtaa sydän- ja verisuonitautienpahenemiseen. 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


VIII

IX

“I have never tried that before, so I think I should definitely be able to do that.”

Astrid Lindgren, Pippi Longstocking


X

XI

Acknowledgements

This study was carried out in the A.I. Virtanen Institute for Molcular Sciences, University of Eastern Finland during the years 2010-2018. 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 long-lasting 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 get-togethers we’ve had during the years. I also want to thank my lovely Venla-team Suvi, Mirva


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 SoininenFoundation, European Research Council, Finnish Foundation for Cardiovascular Research,Ida Montini Foundation, Kuopio University Foundation, Kymenlaakso Regional Fund, PaoloFoundation, UEF Doctoral Program in Molecular Medicine, Urho Känkänen Foundation and Veritas Foundation.

XIII

List of the original publications

This dissertation is based on the following original publications:

I 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. 2014 Jun;34(6):1162-1170.

II 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. 2018 Oct;38(10):2327-2337.

III 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 2018 8:16709.

*Authors with equal contribution

The publications were adapted with the permission of the copyright owners.


XIV

XV

Contents

1 INTRODUCTION ........................................................................................................................... 1 2 REVIEW OF THE LITERATURE ................................................................................................. 3

2.1 Circulatory system ..................................................................................................................... 3 2.1.1 Cardiovascular system ...................................................................................................... 3 2.1.2 Lymphatic system .............................................................................................................. 3

2.2 Cardiovascular diseases ............................................................................................................ 4 2.2.1 Hyperlipidemias ................................................................................................................ 5 2.2.2 Atherosclerosis ................................................................................................................... 5 2.2.3 Myocardial infarction ........................................................................................................ 6 2.2.4 Heart failure ....................................................................................................................... 7 2.2.5 Mouse models for cardiovascular diseases .................................................................... 7

2.3 Lipids and lipoprotein metabolism ......................................................................................... 8 2.3.1 Plasma lipids ...................................................................................................................... 8 2.3.2 Lipoproteins ....................................................................................................................... 9 2.3.3 Apolipoproteins ............................................................................................................... 10

2.3.3.1 ApoA ........................................................................................................................... 10 2.3.3.2 ApoB ........................................................................................................................... 10 2.3.3.3 ApoC ........................................................................................................................... 11 2.3.3.4 ApoE ........................................................................................................................... 11

2.3.4 Lipoprotein receptors ...................................................................................................... 13 2.3.4.1 LDLR ........................................................................................................................... 13 2.3.4.2 LRP1 ............................................................................................................................ 13 2.3.4.3 HSPGs ......................................................................................................................... 14 2.3.4.4 Other lipoprotein receptors ..................................................................................... 14

2.3.5 Exogenous pathway ........................................................................................................ 15 2.3.6 Endogenous pathway ..................................................................................................... 15 2.3.7 HDL metabolism and reverse cholesterol transport .................................................. 17 2.3.8 Lymphatic system in lipid and lipoprotein metabolism............................................ 18

2.4 VEGFs and their receptors ...................................................................................................... 19 2.4.1 VEGF-A ............................................................................................................................. 20 2.4.2 VEGF-B .............................................................................................................................. 22 2.4.3 VEGF-C ............................................................................................................................. 22 2.4.4 VEGF-D ............................................................................................................................. 23 2.4.5 Other VEGFs ..................................................................................................................... 24 2.4.6 VEGF Receptors ............................................................................................................... 24

2.4.6.1 VEGFR1 ...................................................................................................................... 25 2.4.6.2 VEGFR2 ...................................................................................................................... 25 2.4.6.3 VEGFR3 ...................................................................................................................... 26 2.4.6.4 NRPs ........................................................................................................................... 26

3 AIMS OF THE STUDY ................................................................................................................ 29 4 MATERIALS AND METHODS ................................................................................................. 31


XVI

4.1 Animals ...................................................................................................................................... 31 4.2 Analysis of lipid, lipoprotein and glucose metabolism ...................................................... 32 4.3 Imaging ...................................................................................................................................... 33 4.4 Surgery ....................................................................................................................................... 34 4.5 Histology ................................................................................................................................... 34 4.6 Molecular biology and cell culture ........................................................................................ 34 4.7 Statistical methods ................................................................................................................... 36

5 RESULTS ........................................................................................................................................ 37 5.1 Attenuated lymphatic function leads to hypercholesterolemia and accelerated atherogenesis (I) ............................................................................................................................. 37 5.2 VEGF-D regulates chylomicron metabolism (II) ................................................................. 41 5.3 The expression of sVEGFR3 leads to higher mortality after myocardial infarction (III) .................................................................................................................................................... 46

6 DISCUSSION ................................................................................................................................ 51 6.1 The role of VEGFR3 mediated signaling in lipid metabolism and atherosclerosis (I) ... 51 6.2 The function of VEGF-D in lipoprotein metabolism (II) .................................................... 52 6.3 Cardiac lymphatic vessels in myocardial infarction (III) ................................................... 54

7 CONCLUSIONS ........................................................................................................................... 57 8 REFERENCES ................................................................................................................................ 59 APPENDIX: ORIGINAL PUBLICATIONS (I-III)

XVII

Abbreviations ABCA ATP binding cassette subfamily A ABCG ATP binding cassette subfamily

G

ACAT Acetyl-coenzyme A

acetyltransferase

ACTA Actin alpha (α-SMA)

ADAMTS A Disintegrin and

metalloproteinase with

thrombospondin motifs

AKT Protein kinase B

ALAT Alanine transaminase

ANGPTL Angiopoietin-like

Apo Apolipoprotein

APOBEC Apolipoprotein B mRNA editing

enzyme, catalytic polypeptide-

like

ARH Low-density lipoprotein receptor

adapter protein

ATGL Adipose triglyceride lipase

CALCRL Calcitonin receptor-like receptor

CCBE Calcium binding epidermal

growth factor domains

CD Cluster determinant

CETP Cholesteryl ester transfer protein

CM Chylomicron

COL 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


XVIII

HPLC High performance liquid

chromatography

HSPG Heparan sulfate proteoglycan

HTGL Hepatic triglyceride lipase

ICAM Intercellular adhesion molecule

IDOL E3 ubiquitin ligase inducible

degrader of LDLR

IHC Immunohistochemistry

IL Interleukin

i.p. intraperitoneal

i.v. intravenous

INSIG Insulin induced gene

ITT Insulin tolerance test

KLF Krueppel-like factor

KO Knockout

LAD Left anterior descending artery

LAM Lymphangioleiomyomatosis

LCAT Lecithin–cholesterol

acyltransferase

LDL Low-density lipoprotein

LDLR Low-density lipoprotein receptor

LEC Lymphatic endothelial cell

LIPA Lysosomal acid lipase

LMF Lipase maturation factor

LPL Lipoprotein lipase

LRP LDL receptor related protein

LYVE Lymphatic vessel endothelial

hyaluronan receptor

LVW Left ventricle wall

MI Myocardial infarction

MRI Magnetic resonance imaging

mTORC Mammalian target of rapamycin

complex

MTP Microsomal triglyceride transfer

protein

NDST N-deacetylase/N-

sulfotransferase

NF-kB Nuclear factor kappa-light-chain-

enhancer of activated B cells

NPC Niemann-Pick disease, type C

NRP Neuropilin

PCR Polymerase chain reaction

PCSK Proprotein convertase

subtilisin/kexin type

PGC Peroxisome proliferator-

activated receptor gamma

coactivator

PI3K Phosphatidylinositide 3-kinase

PKC Protein kinase C

PLAGL Pleomorphic adenoma gene-like

PlGF Placental growth factor

p.o. per os (orally)

POSTN Periostin

PROX Prospero homeobox protein

RCT Reverse cholesterol transport

SEMA Semaphorin

SMC Smooth muscle cell

SNP Small nuclear polymorphism

SR-BI Scavenger receptor class B type 1

XIX

SREBP Sterol regulatory element-

binding protein

TGF Transforming growth factor

TNF Tumor necrosis factor

TRL Triglyceride rich lipoprotein

VCAM Vascular cell adhesion protein

VEGF Vascular endothelial growth

factor

VEGFR Vascular endothelial growth

factor receptor

VHD VEGF homology domain

VLDL Very low-density lipoprotein

VLDLR Very low-density lipoprotein

receptor

WB Western blot

WHO World Health Organization

WM Whole mount


XX

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.


2

3

2 Review of the literature

2.1 CIRCULATORY SYSTEM

2.1.1 Cardiovascular systemThe 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 aclosed 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 pericardialsac 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, themyocardium formed by cardiac muscle cells and a serous membrane called the epicardium,which is the innermost layer of pericardium. Cardiac muscle is composed of elongatedcardiomyocytes, which are able to contract with the aid of actin and myosin fibers in aresponse to action potentials generated by the sinoatrial node. The heart is composed of fourchambers: the left and right atria and the left and right ventricle. Ventricular walls have thickmuscle 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 bloodcirculation in the periphery during systole. Atrioventricular valves prevent blood flowingbackwards from the ventricles to the atria: the tricuspid valve is located between the rightatrium 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. Pulmonarycirculation carries deoxygenated blood from right ventricle to the lungs where it isreoxygenated in the pulmonary alveoli. Blood is then returned to the left atrium through thepulmonary vein. Systemic circulation transports oxygen and nutrient-rich blood from the leftventricle through the aorta to the peripheral tissues and returns it back via veins and venacava to the right atrium for re-oxygenation in the lungs. Arteries have strong muscular wallsthat can control blood flow and pressure by contractions and dilations, whereas veins arethinner enabling dilatation and thus serving as reservoir for blood. Both large arteries andveins consist of three layers: intima, media and adventitia. The intima is formed by a singlelayer of endothelium that lines the vessel lumen, the smooth muscle cells (SMCs), collagenand the internal elastic lamina. The media consists of SMCs and elastic and collagenousfibers, whereas the adventitia is primarily composed of collagen. Nutrients and oxygen aresupplied to the outer layers of large arteries by the vasa vasorum, small arteries, veins andlymphatic vessels that reach into the adventitia as well as the outer layers of media. Unlikelarge arteries and veins, capillaries are thin and permeable, which facilitates the exchange ofoxygen, fluids and macromolecules between blood and tissues. (Rhoades, Pflanzer, 2003).

2.1.2 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


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 zipper-like 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 10 000 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 (http://www.who.int/cardiovascular_diseases/en/). 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

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 high-risk alleles (McPherson, Tybjaerg-Hansen, 2016).

2.2.1 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).

2.2.2 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


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).

2.2.3 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

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).

2.2.4 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).

2.2.5 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 pro-atherosclerotic 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


8

of HDL particles. Apoe-/- mice spontaneously develop atherosclerotic lesions that resemblehuman plaques, predominantly in the aortic root, the aortic arch and the branch points of theaorta. (Zhang et al., 1994). The drawback of this model is that the lipoprotein profile does notresemble the human lipoprotein distribution that is usually characterized by high levels ofLDL. Furthermore, functional ApoE has anti-oxidative, anti-proliferative and anti-inflammatory potential, all major contributors of atherosclerosis. Therefore, the developmentof atherosclerotic plaques in Apoe-/- mice might not be caused by increased plasma cholesterollevels but rather the deletion of ApoE itself. (Getz, Reardon, 2016).

To overcome the obstacles observed with the Apoe-/- mouse model, the LDLR KO mousewas developed a year later (Ishibashi et al., 1993). LDLR is responsible for the uptake ofApoB100 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-richLDL. Since young Ldlr-/- mice have only mildly increased plasma cholesterol levels, a high-fat diet is typically used to induce the development of atherosclerosis in this model. The high-fat 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 VLDLparticles, these lipoproteins can be cleared through another lipoprotein receptor, LDLreceptor related protein (LRP) 1. To create humanized apolipoprotein distribution in mice,the Ldlr-/- mice were crossed with ApoB48 deficient mice. The resulting Ldlr-/-/ApoB100/100 micehave equal levels of plasma cholesterol than Apoe-/- mice, but their lipoprotein profileresembles human lipoprotein distribution as cholesterol is mainly carried in ApoB100containing VLDL and LDL particles. (Veniant et al., 1998).

2.3 LIPIDS AND LIPOPROTEIN METABOLISM

2.3.1 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

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).

2.3.2 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 (90-1000 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 30-90 nm. LPL hydrolyzes triglycerides from VLDL particles that turn to IDL or VLDL remnants. These 25-35 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).


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 90-1000 98-99 1-2 Triglyceride

CM remnant Lipolysis of CM 45-150 92-94 6-8 Triglyceride, Cholesterol

VLDL Liver 30-90 90-93 7-10 Triglyceride

IDL Lipolysis of VLDL 25-35 89 11 Triglyceride, Cholesterol

LDL Lipolysis of VLDL 20-25 79 21 Cholesterol

HDL Liver, intestine 5-25 43-78 32-57 Cholesterol

2.3.3 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).

2.3.3.1 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).

2.3.3.2 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

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).

2.3.3.3 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).

2.3.3.4 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


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).

Apolipoprotein Lipoprotein particle Primary Source Function

ApoA-I HDL, CM Liver, Intestine

Structural protein of HDL. Interacts with ABCA1, ABGC1,

LCAT and SR-BI.

ApoA-II HDL Liver Structural protein of HDL.

Interacts with LCAT and SR-BI. Inhibits HL.

ApoA-IV CM, HDL Intestine 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.

13

2.3.4 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).

2.3.4.1 LDLR

A receptor for LDL was postulated in 1974 (Goldstein, Brown, 1974) and the human LDLreceptor (LDLR) was cloned in 1984 (Yamamoto et al., 1984). LDLR is a key receptor inlipoprotein metabolism. It is the main receptor for LDL via ApoB100 but it can also bind toApoE and internalize CM remnants, VLDL and VLDL remnants. LDLR is ubiquitouslyexpressed, but it mainly functions in the liver. (Brown, Goldstein, 1986). Mutation in bothLDLR alleles causes familial hypercholesterolemia, characterized by highly increasedcholesterol levels, up to ten-fold elevation in LDL levels and premature atherosclerosis thatleads to early MI in many cases. Heterozygous mutations are relatively common and causetwo-fold increase plasma LDL. (Marks et al., 2003, Goldstein, Brown, 2009).

The expression of the LDLR gene is regulated by cellular cholesterol levels, via thetranscription factor SREBP2. As a response to decreased intracellular cholesterol levels,LDLR is synthesized on the ER, glycosylated in the Golgi apparatus, before being transportedto the plasma membrane. LDLR binds to the apolipoprotein via its ligand binding domainfollowed 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, Low-density lipoprotein receptor adapter protein (ARH) 1 and clathrin-coated pit proteinsClathrin 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 anacidic environment that enables the detachment of lipoprotein from the LDLR (Rudenko etal., 2002). Subsequently, cholesterol is then released from the lipoprotein particle byLysosomal acid lipase (LIPA) (Goldstein et al., 1975), bound to Nieman-Pick disease, type Cproteins (NPC1 and NPC2) and released to the cytosol of the cell (Higgins et al., 1999). LDLRis efficiently recycled to the plasma membrane, but when LDLR is not required, proteins suchas PCSK9 (Horton, Cohen & Hobbs, 2007) and the E3 ubiquitin ligase inducible degrader ofthe LDLR (IDOL) (Zelcer et al., 2009) target it for degradation.

2.3.4.2 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


14

atherosclerosis in mice, suggesting that LRP1 is also essential in the regulation of inflammatory reactions during atherosclerosis (Overton et al., 2007).

2.3.4.3 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).

2.3.4.4 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).

15

2.3.5 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 re-esterification 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).

2.3.6 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


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).

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).

2.3.7 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 anti-atherogenic 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


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).

2.3.8 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

19

in Prospero homeobox protein (Prox1)+/- mice (Harvey et al., 2005) and in Chy mice with aninactivating point mutation in Vegfr3 gene (Rutkowski et al., 2010). Lymph containsadipogenic 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 lipidmetabolism is bidirectional, as high plasma lipid levels and obesity seem to deteriorate thefunction 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 lymphaticdysfunction in these mice are not yet clear. An activated inflammatory reaction is a strongcandidate, but other factors have also been suggested, such as those involving the proteinsleptin, adiponectin, apelin and CD36 (Escobedo, Oliver, 2017).

Additionally, lymphatic vessels have shown to participate in the trafficking of HDL. Asearly as the 1970s and 1980s, the analysis of lymph collected from cannulated lymphaticvessels showed that lipoprotein distribution in lymph differs from blood. Lymph is almosttotally 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 cholesterolthan could be explained by the filtration of plasma HDL across the endothelium (Nanjee etal., 2000, Nanjee et al., 2001) indicating that HDL enters tissues in order to collect excessivecholesterol 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 theexpression of SR-BI on the surface of LECs (Lim et al., 2013). Therefore, the activation oflymphatic transport could be a potential treatment option for lowering cholesterolaccumulation 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.


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 E9.5-10.5 Vascular defects (Carmeliet et al., 1996)

Vegfa+/- Lethal at E11-12 Vascular defects (Ferrara et al., 1996)

VEGF-A TG (adipose) Viable Increased vascularity in adipose

tissue, decreased lipid levels (Elias et al., 2012)

Vegfb-/- Viable Reduced heart size, decreased accumulation of lipids in heart (Hagberg et al., 2010)

VEGF-B TG (cardiac) Viable Cardiac hypertrophy (Karpanen et al., 2008)

Vegfc-/- Lethal at E16.5 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 E8.5-9.0 Vascular defects, endothelial cell overgrowth (Fong et al., 1995)

Vegfr1 TK-/- Viable Suppressed macrophage migration (Hiratsuka et al., 1998)

Vegfr2-/- Lethal at E8.5-9.5 Vascular defects (Shalaby et al., 1995)

Vegfr2 TK-/- Lethal E8.5-9.5 Vascular defects (Sakurai et al., 2005)

Vegfr3-/- Lethal at E10.5 Vascular defects, heart failure (Dumont et al., 1998)

K14-sVEGFR3 Viable Lymphedema, lack of cutaneous lymphatic vessels (Makinen et al., 2001)

Vegfr3+/- (Chy) Viable Lymphedema, lack of cutaneous lymphatic vessels (Karkkainen et al., 2001)

Nrp1-/- Lethal at E10.5-12.5 Vascular defects (Kawasaki et al., 1999, Kitsukawa et al., 1997)

Nrp2-/- Viable Neuronal anomalies, lymphatic defects

(Giger et al., 2000, Yuan et al., 2002)

E: embryonic day, TG: Transgenic, TK: Tyrosine kinase

2.4.1 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)

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 adeno-associated 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.


22

2.4.2 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 adeno-associated 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).

2.4.3 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

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 pre-clinical 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, https://clinicaltrials.gov/ct2/show/NCT02994771). 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).

2.4.4 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-DΔNΔC) 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


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-DΔNΔC 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-DΔNΔC 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).

2.4.5 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.

2.4.6 VEGF Receptors VEGFs bind with high affinity to VEGFRs, tyrosine kinase receptors that have a ligand-

binding 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.

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). 2.4.6.1 VEGFR1

VEGFR1, also called as FLT1, was cloned in 1990 (Shibuya et al., 1990) and it functions asa receptor for VEGF-A (de Vries et al., 1992), VEGF-B (Olofsson et al., 1998) and PlGF (Parket al., 1994). VEGFR-1 is expressed on vascular endothelium at considerably high levels andit can be found also on non-endothelial cells such as monocytes, macrophages and vascularSMCs (Grosskreutz et al., 1999, Sawano et al., 2001). The expression of VEGFR1 is regulatedby 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 excessiveproliferation of angioblasts (Fong et al., 1995). However, mice with inactivated tyrosinekinase 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 ofvasculature, VEGFR1 mediated signaling is dispensable. VEGFR1 can be considered as adecoy receptor for VEGF-A as it directs VEGF-A binding away from the more angiogenicVEGFR2 (Hiratsuka et al., 1998). Indeed, VEGF-A binds to VEGFR1 with high affinity butcauses low tyrosine-kinase activation (Waltenberger et al., 1994) and deletion of VEGFR1 inadults leads to the upregulation of VEGFR2 signaling and increased angiogenesis (Ho et al.,2012). VEGFR1 can form heterodimers with VEGFR2 and mediate endothelial cell functionsby transphosphorylating VEGFR2 (Huang et al., 2001). Although VEGFR1 mediatedsignaling seems not to be important for vascular development, it might have an essential rolein other biological processes. For example, VEGFR1 has been shown to regulate the migrationand survival of macrophages (Hiratsuka et al., 1998) and to mediate the production oflymphangiogenic VEGF-C from macrophages (Murakami et al., 2008).

2.4.6.2 VEGFR2

VEGFR2, also called KDR in humans (Terman et al., 1991) and Flk1 in mice (Matthews et al., 1991), was cloned in 1991. 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-


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 E8.5-9.5 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 E8.5-9.5, highlighting the importance of VEGFR2 signaling for the endothelial and hematopoetic development (Sakurai et al., 2005).

2.4.6.3 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).

2.4.6.4 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 E10.5-12.5 (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

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).


28

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) To study the role of attenuated VEGFR3 signaling on lipoprotein metabolism and atherosclerosis in transgenic sVEGFR3 and Chy mice.

(II) To determine the role of VEGF-D in lipoprotein metabolism in VEGF-D KO mice.

(III) 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.


30

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-/-/Apob100/100 mice were originally obtained from the Jackson Laboratory (stock no 003000). 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-/-/Apob100/100 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.


32

Table 4. Study groups.

Article Mouse strain Gender Age Diets Follow-up I

sVEGFR3 (FVB) Male

Female

3-4 mo. 6-7 mo.

11-12 mo.

Chow diet HFD

14 days (HFD) 42 days (HFD) 84 days (HFD)

WT littermates (FVB)

Chy (NMRI)

WT littermates (NMRI)

sVEGFR3 x Ldlr-/-/Apob100/100

(C57Bl/6JOlaHsd) Chy x Ldlr-/-/Apob100/100

(C57Bl/6JOlaHsd) Ldlr-/-/Apob100/100 littermates

(C57Bl/6JOlaHsd) II

VEGF-D KO x Ldlr-/-/Apob100/100

(C57Bl/6JOlaHsd) Male

4-5 mo. 12 mo.

Chow diet HFD

42 days (HFD) 84 days (HFD)

Ldlr-/-/Apob100/100

(C57Bl/6JOlaHsd) III



Female

3-4 mo. 12 mo.

Chow diet HFD

4 days (MI) 8 days (MI) 42 days (MI)

84 days (HFD) Chy x Ldlr-/-/Apob100/100


(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.

33

Table 5. Methods used in the analysis of plasma lipids and lipoprotein and glucose metabolism.

Assay Administered substance Route Sample Method Article

Blood chemistry

Cholesterol plasma colorimetric I, II

Triglycerides plasma colorimetric I, II

FFA serum colorimetric II

Glucose blood enzymatic I, II

ALAT plasma colorimetric II

Lipoprotein profiling

plasma HPLC I, II

Lipoprotein subclasses

plasma FPLC II

Particle size analysis

plasma light scattering II

LPL activity Heparin i.v. plasma liquid scintillation I, II

Lipoprotein metabolism

Lipid absorption Tyloxapol, [3H]-triolein p.o. plasma liquid

scintillation I, II

In vivo RCT [3H]-cholesterol labeled macrophages i.p. plasma liquid

scintillation I

LDL turnover [125I]-LDL i.v. plasma gamma counting I, II

Retinol excursion [3H]-retinol p.o. plasma, liver

liquid scintillation II

TRL Remnant uptake [3H]-triolein TRLs i.v. plasma liquid

scintillation 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 1.5-2 % 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).


32

Table 4. Study groups.

Article Mouse strain Gender Age Diets Follow-up I

sVEGFR3 (FVB) Male

Female

3-4 mo. 6-7 mo.

11-12 mo.

Chow diet HFD

14 days (HFD) 42 days (HFD) 84 days (HFD)

WT littermates (FVB)

Chy (NMRI)

WT littermates (NMRI)


(C57Bl/6JOlaHsd) Chy x Ldlr-/-/Apob100/100


(C57Bl/6JOlaHsd) II

VEGF-D KO x Ldlr-/-/Apob100/100


4-5 mo. 12 mo.

Chow diet HFD

42 days (HFD) 84 days (HFD)

Ldlr-/-/Apob100/100

(C57Bl/6JOlaHsd) III



Female

3-4 mo. 12 mo.

Chow diet HFD

4 days (MI) 8 days (MI) 42 days (MI)

84 days (HFD) Chy x Ldlr-/-/Apob100/100


(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.

33

Table 5. Methods used in the analysis of plasma lipids and lipoprotein and glucose metabolism.

Assay Administered substance Route Sample Method Article

Blood chemistry

Cholesterol plasma colorimetric I, II

Triglycerides plasma colorimetric I, II

FFA serum colorimetric II

Glucose blood enzymatic I, II

ALAT plasma colorimetric II

Lipoprotein profiling

plasma HPLC I, II

Lipoprotein subclasses

plasma FPLC II

Particle size analysis

plasma light scattering II

LPL activity Heparin i.v. plasma liquid scintillation I, II

Lipoprotein metabolism

Lipid absorption Tyloxapol, [3H]-triolein p.o. plasma liquid

scintillation I, II

In vivo RCT [3H]-cholesterol labeled macrophages i.p. plasma liquid

scintillation I

LDL turnover [125I]-LDL i.v. plasma gamma counting I, II

Retinol excursion [3H]-retinol p.o. plasma, liver

liquid scintillation II

TRL Remnant uptake [3H]-triolein TRLs i.v. plasma liquid

scintillation 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 1.5-2 % 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).


34

4.4 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 real-time 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.

35

Table 6. Antibodies used in immunohistochemical stainings and Western blot.

Antibody Specifity Type Method Tissue Product code Manufacturer Article

Akt Akt Rabbit pAb WB liver 9272 Cell Signaling II

ApoB Apolipoprotein B Rabbit pAb WB plasma ab20737 Abcam II

ApoC-III Apolipoprotein C-III WB plasma Ionis

Pharmaceuticals II

ApoE Apolipoprotein E Rabbit pAb WB plasma K23100R Meridian Life

Sciences II

CD138 Syndecan-1 Rat mAb IHC liver 281-2 BD Biosciences II

CD3 T-cells Rabbit mAb IHC liver ab16669 Abcam I

CD31 Endothelial cells Rat mAb WM aorta 553370 BD Biosciences I

CD45 Lymphocytes Rat mAb IHC heart 550539 BD Biosciences III

ERK1/2 ERK1/2 Rabbit mAb WB liver 137F5 Cell Signaling II

F4-80 Macrophages Rat mAb IHC heart MCA497 BioRad II, III

LYVE1 LECs Rabbit pAb

IHC, WM

skin, aortic root, heart 103-PA50 ReliaTech I, III

mMQ Macrophages Rabbit IHC aorta AIA31240 Accurate I

mTOR mTOR Rabbit mAb WB liver 2983 Cell Signaling II

p-Akt Phosporylated AKT

Rabbit mAb WB liver 4058 Cell Signaling II

p-Erk1/2 Phosporylated ERK1/2

Rabbit mAb WB liver 20G11 Cell Signaling II

p-mTOR Phosphorylated mTOR


Podoplanin LECs SH mAb WM aorta 127401 Biolegend I

PROX1 LECs Goat mAb WM heart AF2727 R&D Systems III

VEGFR2 VEGFR2 Rabbit mAb WB liver 55B11 Cell Signaling II

VEGFR3 VEGFR3 Rat mAb WB liver 14-5988-

82 Thermo Fischer

Scientific II

α-hFc Fc domain of IgG Goat pAb WB plasma I2136 Sigma-Aldrich I

α-sma SMCs Mouse mAb IHC heart C6198 Sigma-Aldrich III

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,


34

4.4 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 real-time 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.

35

Table 6. Antibodies used in immunohistochemical stainings and Western blot.

Antibody Specifity Type Method Tissue Product code Manufacturer Article

Akt Akt Rabbit pAb WB liver 9272 Cell Signaling II

ApoB Apolipoprotein B Rabbit pAb WB plasma ab20737 Abcam II

ApoC-III Apolipoprotein C-III WB plasma Ionis

Pharmaceuticals II

ApoE Apolipoprotein E Rabbit pAb WB plasma K23100R Meridian Life

Sciences II

CD138 Syndecan-1 Rat mAb IHC liver 281-2 BD Biosciences II

CD3 T-cells Rabbit mAb IHC liver ab16669 Abcam I

CD31 Endothelial cells Rat mAb WM aorta 553370 BD Biosciences I

CD45 Lymphocytes Rat mAb IHC heart 550539 BD Biosciences III

ERK1/2 ERK1/2 Rabbit mAb WB liver 137F5 Cell Signaling II

F4-80 Macrophages Rat mAb IHC heart MCA497 BioRad II, III

LYVE1 LECs Rabbit pAb

IHC, WM

skin, aortic root, heart 103-PA50 ReliaTech I, III

mMQ Macrophages Rabbit IHC aorta AIA31240 Accurate I

mTOR mTOR Rabbit mAb WB liver 2983 Cell Signaling II

p-Akt Phosporylated AKT


p-Erk1/2 Phosporylated ERK1/2

Rabbit mAb WB liver 20G11 Cell Signaling II

p-mTOR Phosphorylated mTOR


Podoplanin LECs SH mAb WM aorta 127401 Biolegend I

PROX1 LECs Goat mAb WM heart AF2727 R&D Systems III

VEGFR2 VEGFR2 Rabbit mAb WB liver 55B11 Cell Signaling II

VEGFR3 VEGFR3 Rat mAb WB liver 14-5988-

82 Thermo Fischer

Scientific II

α-hFc Fc domain of IgG Goat pAb WB plasma I2136 Sigma-Aldrich I

α-sma SMCs Mouse mAb IHC heart C6198 Sigma-Aldrich III

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,


36

4.7 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.

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-/-/Apob100/100mice in order to study the effects of lymphatic deficiency during hyperlipidemia and in the development of atherosclerosis. Ldlr-/-/Apob100/100

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.


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 [3H]-labeled triolein. (B) LDL turnover was analyzed by measuring radioactivity in blood after the i.v. injection of human [125I]-LDL. (C) In vivo RCT was measured by loading macrophages with [3H]-cholesterol. Macrophages were injected i.p. to the receiver mice and radioactivity was measured from plasma. Data is presented as mean ± SEM.

39

Atherosclerotic plaques were analyzed from mice aged 3-4 months (young cohort), 6-7 months (middle cohort) and 11-12 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.


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.

41

5.2 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-/-/Apob100/100 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-/-/Apob100/100

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<0.001.

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.


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 [3H]-labeled triolein by following its retention in plasma. The appearance of [3H]-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 [3H]-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 [3H]-labeled triolein. Lipid absorption was similar between VEGF-D KO mice and controls. (B) Extended study with [3H]-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.

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.


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, phospho-mTOR, 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.

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.


46

5.3 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-/-/Apob100/100 background. For these experiments, Ldlr-/-/Apob100/100 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.

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<0.001. 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 sheet-like morphology (Figure 16A and 16B).


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<0.001.

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.

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<0.001.

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


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.

51

6 Discussion

6.1 THE ROLE OF VEGFR3 MEDIATED SIGNALING IN LIPID METABOLISM AND ATHEROSCLEROSIS (I)

The lymphatic vasculature forms a blind-ended network of vessels that drain excess fluid, proteins and inflammatory cells from tissues in to the circulation. Lymphatic vessels also play an essential role in lipoprotein metabolism since they carry CMs from the intestine to the blood as well as transport extravasated lipoproteins. Recent data suggest that lymphatic vessels participate in the metabolism of HDL by transporting HDL from the interstitial space into the blood and liver for excretion. Thus, lymphatic vessels may not function only as a passive drainage system but they can actively regulate the metabolism of lipids. (Randolph, Miller, 2014) In the first study, we used two mouse strains with defective VEGFR3 signaling (sVEGFR3 and Chy), on a atherosclerotic Ldlr-/-/Apob100/100 background to evaluate the role of lymphatic vessels in the lipoprotein metabolism and atherosclerosis. sVEGFR3 and Chy mice displayed lymphedema in the paws and tails, thus verifying the downregulation of lymphangiogenic signaling. By surprise, we discovered that both mouse strains displayed severe hypercholesterolemia compared to controls, when fed both the normal chow and Western-type high fat diets. However, the levels of triglycerides were only mildly, but not significantly, increased. The lipoprotein profiling revealed that excess cholesterol was carried in large lipoprotein particles such as in CMs and VLDLs as well as in smaller LDL particles in sVEGFR3 mice.

Our first assumption was that the defective function of lymphatic vessels in the intestine would modify lipid absorption through lacteals (Nurmi et al., 2015, Kim, Sung & Koh, 2007). However, we did not discover any changes in the appearance of radiolabeled lipid in the blood after oral feeding indicating normal processing of the lipids in the intestine and their absorption into the blood circulation through lymphatic vessels. However, it is possible that CM particles are modified in the lymphatic system before they reach the blood. Since lymph contains large amounts of HDL particles (Nanjee et al., 2001), the exchange of apolipoproteins between HDL and CM particles may take place already in lymph and modifications in this process could affect the catabolism of CM particles later in plasma. For example, an abnormal composition of apolipoproteins in CM particles might influence lipolysis performed by LPL (Goldberg et al., 1990). However, the activity of LPL was normal in sVEGFR3 mice and they had similar triglyceride levels than control littermates indicating that the lipolysis was not altered. Alternatively, changes in the uptake of lipoproteins into the liver may also strongly influence plasma lipid levels. The retention of lipoprotein remnants or LDL would lead to an increase of plasma lipids, especially cholesterol. LDL turnover using the intravenous injection of radiolabeled LDL revealed a tendency to slower LDL uptake in the livers of sVEGFR3 mice indicating that the expression of sVEGFR3 might indeed decrease the LDL uptake into hepatocytes.

As lymphatic vessels have been shown to absorb and transport HDL (Lim et al., 2009), the accumulation of cholesterol-loaded HDL in the plasma in the lymphatic deficient mice could have also influenced the development of hypercholesterolemia. However, lipoprotein profiling revealed only minor levels of cholesterol in HDL fraction in sVEGFR3 mice and controls compared to other lipoproteins. Furthermore, we did not discover any differences in the RCT when macrophages were loaded with radiolabeled cholesterol and injected into the peritoneum. Therefore, defects in HDL metabolism most likely do not explain the accumulation of cholesterol in the plasma of sVEGFR3 and Chy mice.


52

Another important finding from this study was that atherogenesis was accelerated in sVEGFR3 and Chy mice during the high fat feeding. The primary factor for atherosclerosis in these mice is most likely changes in lipoprotein metabolism leading to hypercholesterolemia, the main culprit for atherogenesis. Interestingly, we discovered decreased number of lymphatic vessels in the atherosclerotic plaques of sVEGFR3 mice and Chy mice compared to controls and therefore, impaired lymphatic flow in aortic plaques could have also affected the plaque growth. The absence of lymphatic flow in the plaques could have altered the RCT from plaque residing macrophages and promoted the formation of foam cells (Martel et al., 2013). Additionally, adventitial lymphatic vessels might provide a route for transporting unretained cholesterol and lipoproteins from the vessel wall. (Xu et al., 2007). Furthermore, the lymphatic vasculature has been shown to regulate the inflammatory reaction. Although the number of macrophages in atherosclerosis lesions was not significantly changed, careful pathological examination revealed massive accumulations of inflammatory cells in the livers and adipose tissue of the neck area in older sVEGFR3 mice, which might indicate the activation and recruitment of specific types of inflammatory cells and possibly reduced clearance of tissue-retained immune cells in these mice.

In summary, this study showed for the first time that lymphatic insufficiency clearly aggravates plasma lipid profile and leads to accelerated atherogenesis. Delayed LDL turnover indicated that the hypercholesterolemia in sVEGFR3 mice most likely results from the reduced uptake of lipoproteins into the liver but the exact modifications in lipoprotein particles or hepatic receptors require further studies. It is intriguing to speculate the possibilities how the modulation of lymphatic network could be beneficial in the treatment of hyperlipidemia and atherosclerosis. The activation of lymphangiogenesis within atherosclerotic plaques could enhance cholesterol removal and inhibit atherogenesis. In contrast, the reduction of lymphatic flow in the intestine might prove useful in treating obesity. Further studies are needed to test both of these hypotheses.

6.2 THE FUNCTION OF VEGF-D IN LIPOPROTEIN METABOLISM (II)

While the importance of VEGF-C in regulating lymphagiogenesis has been well-established, the physiological role of VEGF-D, another VEGFR3 ligand has remainedundetermined. VEGF-D has shown great potential as a gene therapy agent when used to treatpatients with refractory angina (Hartikainen et al., 2017), but its role in lipoproteinmetabolism has been unknown. To evaluate the effects of VEGF-D on lipoproteinmetabolism, we crossed VEGF-D KO mice into the pro-atherosclerotic Ldlr-/-/Apob100/100

background. These mice do not have any visible phenotype and they breed normally.Additionally, they display normal lymphatic vasculature indicating that VEGF-D isdispensable for VEGFR3 mediated lymphangiogenic signaling during development (Baldwin et al., 2005). Unexpectedly, the deletion of VEGF-D in atherosclerotic background led to severe hypertriglyceridemia and hypercholesterolemia on Western-type high fat diet. Lipoprotein profiling revealed that excess triglycerides and choles-terol were mostly carried in large lipoprotein particles comprising of CMs, VLDLs and their remnants. In accordance with the lipoprotein profiles, retinol excursion study with radiolabeled retinol revealed an accumulation of radiolabeled lipid in the plasma 4 h after the lipid bolus and significantly reduced uptake in to the liver. The results from remnant clearance studies, as well as normal LPL activity, confirmed that the absorption and lipolysis of CMs were both efficient in VEGF-D mice, but the uptake of CM remnants in the liver was decreased.

In humans, patients suffering from type III hyperlipoproteinemia display hyperlipidemiaresulting from the increased levels of CM and VLDL remnants. This condition is caused bymutated ApoE protein that is unable to bind hepatic receptors leading to the retention oflipoproteins in the plasma. (Mahley, Huang & Rall, 1999). Therefore, findings fromlipoprotein uptake studies made us to hypothesize that the hepatic uptake of CM and VLDL

53

remnants through hepatic receptors is reduced due to the absence of VEGF-D. While the expression of Lrp1 was similar in VEGF-D KO mice and controls, Sdc1 was significantly downregulated. The amount of SDC1 protein analyzed with Western blotting was also decreased and immunohistochemical analysis revealed patchy SDC1 staining in the liver sinusoids of VEGF-D KO mice. Since CM and VLDL remnants are internalized to the liver by LRP1 and HSPGs during LDLR deficiency (Foley et al., 2013), the downregulation of SDC1 in VEGF-D KO mice has most likely resulted in the accumulation of lipoprotein remnants in plasma. The mechanism leading to the downregulation SDC1 by VEGF-D is still under investigation. Data from a rare genetic disease LAM could provide some clues for determining the molecular mechanisms. LAM is characterized by the accumulation of VEGF-D expressing SMCs in the lungs (Seyama et al., 2006). In cells derived from LAM patients, deletions in the gene of Tuberous sclerosis protein (TSC1 or TSC2) lead to the hyperactivation of Mammalian target of rapamycin complex (mTORC) 1 causing aberrant cell growth. Treatment of these patients with mTORC1 inhibitor Sirolimus leads to normalized VEGF-D concentration. (Taveira-DaSilva, Moss, 2015). Interestingly, Sirolimus treatment can also cause increased plasma triglyceride, cholesterol and FFA levels as a side effect (Morrisett et al., 2002). In mice, VEGF-D binds exclusively to VEGFR3, which mediates signaling pathways through PI3K, AKT and ERK (Koch et al., 2011). Western blot analysis revealed decreased phosphorylation of AKT in VEGF-D KO mice indicating downregulation of this important signaling pathway. However, the analysis of other pathways or mTORC levels did not show any major changes between VEGF-D KO mice and the controls.

In the 1970s, increased plasma triglyceride levels and thus the accumulation of triglyceride-rich lipoprotein particles were not considered as a risk factor for coronary artery disease (Boren et al., 2014) but more recent studies have shown that remnant lipoproteins are just as atherogenic as LDL (Varbo et al., 2013). Even though VEGF-D KO mice displayed severe hyperlipidemia, they did not show increased atherosclerosis after 12 weeks of high-fat feeding. The discrepancy between recent clinical studies and findings from VEGF-D KO mice could be explained by the study design of our work: 12 week follow-up time might have been too long to shown any differences in the size of atherosclerotic plaques as atherosclerotic lesions in mice on the Ldlr-/-/Apob100/100 background seem to reach a plateau after extended periods of high-fat diet. Furthermore, the size of a lipoprotein particle can determine their capability to transverse vascular endothelium. It is possible that the majority of the excess cholesterol in VEGF-D KO mice is carried in particles so large that they cannot cross the vascular endothelium (Proctor, Vine & Mamo, 2002, Boren et al., 2014). Furthermore, sulphated proteoglycans and HSPGs have been shown to be important in trapping lipoprotein within the subendothelial space of vascular wall (Gordts, Esko, 2018, Yla-Herttuala et al., 1987). Although the expression of HSPGs were not analyzed in the arteries, it is possible that the deletion of VEGF-D has globally decreased HSPG levels resulting in decreased lipoprotein retention in the vascular wall.

Here we showed for the first time that the modulation of VEGF-D levels affects lipoprotein metabolism, at least in the mice on the Ldlr-/-/Apob100/100 background. Although lipoprotein profile in Ldlr-/-/Apob100/100 mice resembles human lipoprotein distribution, some major differences remain (such as the absence of CETP in mice) and the role of VEGF-D in human lipoprotein metabolism needs to be verified. Changes in plasma VEGF-D levels could be measured in humans in order to evaluate the role of VEGF-D in hyperlipidemia. Intriguingly, recent reports have shown a correlation between plasma VEGF-D levels and incidence of HF (Borne et al., 2018), atrial fibrillation and stroke (Berntsson et al., 2018) suggesting the importance of VEGF-D in the development of CVDs in humans as well.


54

6.3 CARDIAC LYMPHATIC VESSELS IN MYOCARDIAL INFARCTION (III)

In the third study, our aim was to analyze the role of VEGFR3 in the healthy hearts and after MI. By using sVEGFR3 and Chy mice in the pro-atherosclerotic Ldlr-/-/Apob100/100

background, we discovered that the downregulation of VEGFR3 mediated signaling in mice led to modifications in cardiac lymphatic morphology: cardiac lymphatic vessels were significantly dilated in sVEGFR3 mice and the lymphatic network had completely lost its structure in Chy mice. As VEGF-C/VEGFR3 signaling is essential for the formation of the lymphatic vasculature (Kukk et al., 1996, Dumont et al., 1998) it is probable that the structural changes in the cardiac lymphatic vessel network have occurred already during the embryogenesis or during the first few postnatal weeks, when the maturation of cardiac lymphatic system still occurs (Klotz et al., 2015). Hearts from sVEGFR3, Chy and control mice were anatomically similar and cardiac function measured with echocardiography and MRI did not show any major changes between groups. Previous studies have shown a 3.5% fluid increase within the myocardium can already lead to a 30% decrease in cardiac output, at least in large animal models (Laine, Allen, 1991). Therefore, although the structure of the lymphatic network was altered, cardiac lymphatic flow appeared to be sufficient to maintain the fluid homeostasis in healthy animals.

Oxygen deprivation during MI causes massive cell death, which initiates inflammatory processes leading to the removal of dead cell debris and the replacement of necrotic areas with fibrotic tissue. The lymphatic system is involved in both of these processes. To evaluate the role of modified cardiac lymphatic system in sVEGFR3 mice in the healing after MI, we performed permanent LAD ligation, which is regularly used to model myocardial changes that occur after coronary artery occlusion in humans (Klocke et al., 2007). LAD ligation was performed with a novel method, in which heart is “popped” out from the thoracic cavity through 4th intercostal space (Gao et al., 2010). Even though this method is fast and causes less tissue damage and lower death rate during surgery than conventional open chest surgery methods, there is still a significant mortality during the operation. Mice display anatomical variation in their coronary artery tree, which might affect the location, severity and size of the infarction. (Chen et al., 2017). Furthermore, mice on Ldlr-/-/Apob100/100 background have excessive amounts of fat in their thoracic cavity, which can restrain the heart and cause tissue damage when the heart is exposed. If the animals recovered from anesthesia, the survival of the animals was generally good. Interestingly, sVEGFR3 mice displayed a 25% higher mortality compared to controls during 8-days follow up after LAD ligation. Although the average size of the infarcted areas was not significantly different between sVEGFR3 mice and their control littermates, sVEGFR3 mice showed a tendency to develop larger infarctions.

Ventricular arrhythmias caused by the disturbance of electric conductivity are the most common reason for death following MI. Among other factors, cardiac edema caused by the hyperpermeability of vasculature can lead to arrhythmias (Weis, Cheresh, 2005, Cui, 2010). To resolve the excessive accumulation of fluids within the myocardium, lymphangiogenesis is activated after MI (Sun, Wang & Guo, 2012). In this study, control animals exhibited strong increase in the number of lymphatic vessels during the healing after MI. However, the number of lymphatic vessels did not significantly increase in sVEGFR3 mice indicating unresponsiveness to fluid accumulation. Although we did not find significant changes in the edema formation with T2-weighed MRI, even small changes in water content can alter the function of the heart (Laine, Allen, 1991, Henri et al., 2016). Additionally, the leakiness of the vasculature can lead to hemorrhages especially after reperfusion (Ghugre et al., 2017). Interestingly, the infarcted areas of sVEGFR3 mice displayed higher amounts of red blood cells than control mice indicating the extravasation of erythrocytes from blood vessels. The role of hemorrhages is not completely understood but they have been associated with larger infarction scars and adverse remodeling of the heart (Betgem et al., 2015). We discovered that the amount of VEGFR2 was increased in the healthy hearts of sVEGFR3 mice, which could

55

lead to the increased activity of the VEGF-A/VEGFR2 pathway and promote leakiness of the blood vasculature (Heinolainen et al., 2017).

Furthermore, the lymphatic system controls the trafficking of immune cells and lymphangiogenesis is activated in several inflammatory conditions. For example, the accumulation of CD4-positive cells and macrophages has been associated with increased vascular leakage and tissue edema (Gousopoulos et al., 2016). Although we did not discover any differences in the amount of macrophages and lymphocytes in the hearts of sVEGFR3 mice and control mice, it is possible that the amount of certain types of inflammatory cells might have contributed to the more severe outcome after MI in sVEGFR3 mice. For example, prolonged activity of innate immune cells (Vieira et al., 2018) or changes in macrophage polarization (Karaman et al., 2014) could influence the healing after MI.

The other central hallmarks of MI are the activation of myofibroblasts and the formation of a fibrotic infarction scar. The development of a scar can be either beneficial or detrimental and correct timing is essential for the repair process. The development of the fibrotic scar prevents the rupture of the heart but can lead to the stiffness of the muscle and reduced pumping efficacy, which can eventually lead to HF. Interestingly, some sVEGFR3 mice displayed reduced staining of type I and III collagens in infarcted areas, indicating either slower or attenuated production of ECM components in these mice. To support this finding, a novel MRI method TRAFF4 relaxation time revealed a modified composition of infarcted area in sVEGFR3 mice compared to controls. However, this difference was not detected with more conventional MRI method T1ρ, which has generally been used for evaluation of fibrotic area in the infarcted heart. Therefore, it is possible that TRAFFn is able to detect more specific modifications in the infarcted area, which are yet to be revealed.

In summary, this study highlights the importance of VEGFR3 signaling in the development and maintenance of the cardiac lymphatic network and verifies its important role in regulating both lymphangiogenesis and vascular function after MI. Complex cellular responses take place after MI and cardiac lymphatic vessels most likely influence most of them. Therefore, the activation of lymphangiogenesis therapeutically after MI could be beneficial for restoring cardiac function by resolving edema, fibrosis and inflammatory reaction.


56

57

7 Conclusions

Based on this thesis, the following conclusions can be made:

I) Lymphatic insufficiency in atherosclerotic mice increased plasma cholesterol levels on both chow and on high-fat diet. Hypercholesterolemia and reduced number of lymphatic vessels in the atherosclerotic aortas led to accelerated atherogenesis.

II) The deletion of VEGF-D decreased the expression of hepatic Syndecan-1 leading to the retention of CM remnants and causing severe hyperlipidemia in atherosclerotic mice. However, the accumulation of large CM remnants in plasma did not enhance the development of atherosclerosis.

III) The downregulation of VEGFR3 signaling altered the organization of the cardiac lymphatic networks. Moreover, mice expressing soluble VEGFR3 had an increased mortality after MI, increased level of hemorrhages in the myocardium and modified structure of the infarction area, as measured by a novel MRI method, TRAFF4.

In summary, these studies highlighted the importance of VEGFR3 and its ligands inlipoprotein metabolism and in the development of CVDs. For the first time, thedownregulation of VEGFR3 signaling leading to lymphatic insufficiency was associatedwith hypercholesterolemia, atherogenesis and higher mortality after MI. Furthermore,VEGFR3 ligand VEGF-D was shown to play a major role in the hepatic uptake of largelipoproteins. As the burden of CVDs is still increasing globally and new treatment optionsare required, the activation of lymphangiogenesis might provide a useful method in order toenhance the removal cholesterol from atherosclerotic lesions as well as mediate the clearanceof inflammatory cells and excessive fluids after MI. Additionally, plasma VEGF-D levelsmight function as a biomarker to predict the development of hyperlipidemia as well as futurecardiovascular events.


58

59

8 References

Aalto-Setala, K., Fisher, E.A., Chen, X., Chajek-Shaul, T., Hayek, T., Zechner, R., Walsh, A., Ramakrishnan, R., Ginsberg, H.N. & Breslow, J.L. 1992, "Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles", The Journal of clinical investigation, vol. 90, no. 5, pp. 1889-1900.

Aase, K., von Euler, G., Li, X., Ponten, A., Thoren, P., Cao, R., Cao, Y., Olofsson, B., Gebre-Medhin, S., Pekny, M., Alitalo, K., Betsholtz, C. & Eriksson, U. 2001, "Vascular endothelial growth factor-B-deficient mice display an atrial conduction defect", Circulation, vol. 104, no. 3, pp. 358-364.

Achen, M.G., Jeltsch, M., Kukk, E., Makinen, T., Vitali, A., Wilks, A.F., Alitalo, K. & Stacker, S.A. 1998, "Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4)", Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 2, pp. 548-553.

Acton, S., Rigotti, A., Landschulz, K.T., Xu, S., Hobbs, H.H. & Krieger, M. 1996, "Identification of scavenger receptor SR-BI as a high density lipoprotein receptor", Science (New York, N.Y.), vol. 271, no. 5248, pp. 518-520.

Adorni, M.P., Zimetti, F., Billheimer, J.T., Wang, N., Rader, D.J., Phillips, M.C. & Rothblat, G.H. 2007, "The roles of different pathways in the release of cholesterol from macrophages", Journal of lipid research, vol. 48, no. 11, pp. 2453-2462.

Albuquerque, R.J., Hayashi, T., Cho, W.G., Kleinman, M.E., Dridi, S., Takeda, A., Baffi, J.Z., Yamada, K., Kaneko, H., Green, M.G., Chappell, J., Wilting, J., Weich, H.A., Yamagami, S., Amano, S., Mizuki, N., Alexander, J.S., Peterson, M.L., Brekken, R.A., Hirashima, M., Capoor, S., Usui, T., Ambati, B.K. & Ambati, J. 2009, "Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth", Nature medicine, vol. 15, no. 9, pp. 1023-1030.

Alitalo, K. 2011, "The lymphatic vasculature in disease", Nature medicine, vol. 17, no. 11, pp. 1371-1380. Andrassy, M., Volz, H.C., Igwe, J.C., Funke, B., Eichberger, S.N., Kaya, Z., Buss, S., Autschbach, F.,

Pleger, S.T., Lukic, I.K., Bea, F., Hardt, S.E., Humpert, P.M., Bianchi, M.E., Mairbaurl, H., Nawroth, P.P., Remppis, A., Katus, H.A. & Bierhaus, A. 2008, "High-mobility group box-1 in ischemia-reperfusion injury of the heart", Circulation, vol. 117, no. 25, pp. 3216-3226.

Anisimov, A., Alitalo, A., Korpisalo, P., Soronen, J., Kaijalainen, S., Leppanen, V.M., Jeltsch, M., Yla-Herttuala, S. & Alitalo, K. 2009, "Activated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle", Circulation research, vol. 104, no. 11, pp. 1302-1312.

Anisimov, A., Leppanen, V.M., Tvorogov, D., Zarkada, G., Jeltsch, M., Holopainen, T., Kaijalainen, S. & Alitalo, K. 2013, "The basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands", Science signaling, vol. 6, no. 282, pp. ra52.

Aprelikova, O., Pajusola, K., Partanen, J., Armstrong, E., Alitalo, R., Bailey, S.K., McMahon, J., Wasmuth, J., Huebner, K. & Alitalo, K. 1992, "FLT4, a novel class III receptor tyrosine kinase in chromosome 5q33-qter", Cancer research, vol. 52, no. 3, pp. 746-748.

Argraves, K.M., Battey, F.D., MacCalman, C.D., McCrae, K.R., Gafvels, M., Kozarsky, K.F., Chappell, D.A., Strauss, J.F.,3rd & Strickland, D.K. 1995, "The very low density lipoprotein receptor mediates the cellular catabolism of lipoprotein lipase and urokinase-plasminogen activator inhibitor type I complexes", The Journal of biological chemistry, vol. 270, no. 44, pp. 26550-26557.


60

Arslan, F., Smeets, M.B., O'Neill, L.A., Keogh, B., McGuirk, P., Timmers, L., Tersteeg, C., Hoefer, I.E., Doevendans, P.A., Pasterkamp, G. & de Kleijn, D.P. 2010, "Myocardial ischemia/reperfusion injury is mediated by leukocytic toll-like receptor-2 and reduced by systemic administration of a novel anti-toll-like receptor-2 antibody", Circulation, vol. 121, no. 1, pp. 80-90.

Aspelund, A., Robciuc, M.R., Karaman, S., Makinen, T. & Alitalo, K. 2016, "Lymphatic System in Cardiovascular Medicine", Circulation research, vol. 118, no. 3, pp. 515-530.

Baldwin, M.E., Catimel, B., Nice, E.C., Roufail, S., Hall, N.E., Stenvers, K.L., Karkkainen, M.J., Alitalo, K., Stacker, S.A. & Achen, M.G. 2001, "The specificity of receptor binding by vascular endothelial growth factor-d is different in mouse and man", The Journal of biological chemistry, vol. 276, no. 22, pp. 19166-19171.

Baldwin, M.E., Halford, M.M., Roufail, S., Williams, R.A., Hibbs, M.L., Grail, D., Kubo, H., Stacker, S.A. & Achen, M.G. 2005, "Vascular endothelial growth factor D is dispensable for development of the lymphatic system", Molecular and cellular biology, vol. 25, no. 6, pp. 2441-2449.

Ballmer-Hofer, K., Andersson, A.E., Ratcliffe, L.E. & Berger, P. 2011, "Neuropilin-1 promotes VEGFR-2 trafficking through Rab11 vesicles thereby specifying signal output", Blood, vol. 118, no. 3, pp. 816-826.

Banai, S., Shweiki, D., Pinson, A., Chandra, M., Lazarovici, G. & Keshet, E. 1994, "Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: implications for coronary angiogenesis", Cardiovascular research, vol. 28, no. 8, pp. 1176-1179.

Baumgartner, I., Pieczek, A., Manor, O., Blair, R., Kearney, M., Walsh, K. & Isner, J.M. 1998, "Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia", Circulation, vol. 97, no. 12, pp. 1114-1123.

Becker, P.M., Waltenberger, J., Yachechko, R., Mirzapoiazova, T., Sham, J.S., Lee, C.G., Elias, J.A. & Verin, A.D. 2005, "Neuropilin-1 regulates vascular endothelial growth factor-mediated endothelial permeability", Circulation research, vol. 96, no. 12, pp. 1257-1265.

Bellomo, D., Headrick, J.P., Silins, G.U., Paterson, C.A., Thomas, P.S., Gartside, M., Mould, A., Cahill, M.M., Tonks, I.D., Grimmond, S.M., Townson, S., Wells, C., Little, M., Cummings, M.C., Hayward, N.K. & Kay, G.F. 2000, "Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia", Circulation research, vol. 86, no. 2, pp. E29-35.

Benjamin, E.J., Blaha, M.J., Chiuve, S.E., Cushman, M., Das, S.R., Deo, R., de Ferranti, S.D., Floyd, J., Fornage, M., Gillespie, C., Isasi, C.R., Jimenez, M.C., Jordan, L.C., Judd, S.E., Lackland, D., et al. & American Heart Association Statistics Committee and Stroke Statistics Subcommittee 2017, "Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association", Circulation, vol. 135, no. 10, pp. e146-e603.

Bennett, M.R., Sinha, S. & Owens, G.K. 2016, "Vascular Smooth Muscle Cells in Atherosclerosis", Circulation research, vol. 118, no. 4, pp. 692-702.

Berbee, J.F., van der Hoogt, C.C., Sundararaman, D., Havekes, L.M. & Rensen, P.C. 2005, "Severe hypertriglyceridemia in human APOC1 transgenic mice is caused by apoC-I-induced inhibition of LPL", Journal of lipid research, vol. 46, no. 2, pp. 297-306.

Berliner, J.A., Territo, M.C., Sevanian, A., Ramin, S., Kim, J.A., Bamshad, B., Esterson, M. & Fogelman, A.M. 1990, "Minimally modified low density lipoprotein stimulates monocyte endothelial interactions", The Journal of clinical investigation, vol. 85, no. 4, pp. 1260-1266.

Bernatchez, P.N., Soker, S. & Sirois, M.G. 1999, "Vascular endothelial growth factor effect on endothelial cell proliferation, migration, and platelet-activating factor synthesis is Flk-1-dependent", The Journal of biological chemistry, vol. 274, no. 43, pp. 31047-31054.

Bernier-Latmani, J., Cisarovsky, C., Demir, C.S., Bruand, M., Jaquet, M., Davanture, S., Ragusa, S., Siegert, S., Dormond, O., Benedito, R., Radtke, F., Luther, S.A. & Petrova, T.V. 2015, "DLL4

61

promotes continuous adult intestinal lacteal regeneration and dietary fat transport", The Journal of clinical investigation, vol. 125, no. 12, pp. 4572-4586.

Berntsson, J., Smith, J.G., Johnson, L.S.B., Soderholm, M., Borne, Y., Melander, O., Orho-Melander, M., Nilsson, J. & Engstrom, G. 2018, "Increased vascular endothelial growth factor D is associated with atrial fibrillation and ischaemic stroke", Heart (British Cardiac Society), .

Betgem, R.P., de Waard, G.A., Nijveldt, R., Beek, A.M., Escaned, J. & van Royen, N. 2015, "Intramyocardial haemorrhage after acute myocardial infarction", Nature reviews.Cardiology, vol. 12, no. 3, pp. 156-167.

Beyers, R.J., Smith, R.S., Xu, Y., Piras, B.A., Salerno, M., Berr, S.S., Meyer, C.H., Kramer, C.M., French, B.A. & Epstein, F.H. 2012, "T(2) -weighted MRI of post-infarct myocardial edema in mice", Magnetic resonance in medicine, vol. 67, no. 1, pp. 201-209.

Bietrix, F., Yan, D., Nauze, M., Rolland, C., Bertrand-Michel, J., Comera, C., Schaak, S., Barbaras, R., Groen, A.K., Perret, B., Terce, F. & Collet, X. 2006, "Accelerated lipid absorption in mice overexpressing intestinal SR-BI", The Journal of biological chemistry, vol. 281, no. 11, pp. 7214-7219.

Bisgaier, C.L., Sachdev, O.P., Megna, L. & Glickman, R.M. 1985, "Distribution of apolipoprotein A-IV in human plasma", Journal of lipid research, vol. 26, no. 1, pp. 11-25.

Blancher, C., Moore, J.W., Talks, K.L., Houlbrook, S. & Harris, A.L. 2000, "Relationship of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression to vascular endothelial growth factor induction and hypoxia survival in human breast cancer cell lines", Cancer research, vol. 60, no. 24, pp. 7106-7113.

Blanco-Vaca, F., Escola-Gil, J.C., Martin-Campos, J.M. & Julve, J. 2001, "Role of apoA-II in lipid metabolism and atherosclerosis: advances in the study of an enigmatic protein", Journal of lipid research, vol. 42, no. 11, pp. 1727-1739.

Blann, A.D., Belgore, F.M., Constans, J., Conri, C. & Lip, G.Y. 2001, "Plasma vascular endothelial growth factor and its receptor Flt-1 in patients with hyperlipidemia and atherosclerosis and the effects of fluvastatin or fenofibrate", The American Journal of Cardiology, vol. 87, no. 10, pp. 1160-1163.

Blom, D.J., Hala, T., Bolognese, M., Lillestol, M.J., Toth, P.D., Burgess, L., Ceska, R., Roth, E., Koren, M.J., Ballantyne, C.M., Monsalvo, M.L., Tsirtsonis, K., Kim, J.B., Scott, R., Wasserman, S.M., Stein, E.A. & DESCARTES Investigators 2014, "A 52-week placebo-controlled trial of evolocumab in hyperlipidemia", The New England journal of medicine, vol. 370, no. 19, pp. 1809-1819.

Boren, J., Lee, I., Zhu, W., Arnold, K., Taylor, S. & Innerarity, T.L. 1998, "Identification of the low density lipoprotein receptor-binding site in apolipoprotein B100 and the modulation of its binding activity by the carboxyl terminus in familial defective apo-B100", The Journal of clinical investigation, vol. 101, no. 5, pp. 1084-1093.

Boren, J., Matikainen, N., Adiels, M. & Taskinen, M.R. 2014, "Postprandial hypertriglyceridemia as a coronary risk factor", Clinica chimica acta; international journal of clinical chemistry, vol. 431, pp. 131-142.

Borne, Y., Gransbo, K., Nilsson, J., Melander, O., Orho-Melander, M., Smith, J.G. & Engstrom, G. 2018, "Vascular Endothelial Growth Factor D, Pulmonary Congestion, and Incidence of Heart Failure", Journal of the American College of Cardiology, vol. 71, no. 5, pp. 580-582.

Boucher, P., Gotthardt, M., Li, W.P., Anderson, R.G. & Herz, J. 2003, "LRP: role in vascular wall integrity and protection from atherosclerosis", Science (New York, N.Y.), vol. 300, no. 5617, pp. 329-332.

Braun, A., Trigatti, B.L., Post, M.J., Sato, K., Simons, M., Edelberg, J.M., Rosenberg, R.D., Schrenzel, M. & Krieger, M. 2002, "Loss of SR-BI expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice", Circulation research, vol. 90, no. 3, pp. 270-276.

Braunwald, E. 2013, "Heart failure", JACC.Heart failure, vol. 1, no. 1, pp. 1-20.


62

Brown, A. & Sharpe, L. 2016, "Cholesterol Synthesis" in Biochemistry of Lipids, Lipoproteins and Membranes, eds. N. Ridgway & R. McLeod, 6th edn, Elsevier, , pp. 327.

Brown, M.S. & Goldstein, J.L. 1986, "A receptor-mediated pathway for cholesterol homeostasis", Science (New York, N.Y.), vol. 232, no. 4746, pp. 34-47.

Bui, A.L., Horwich, T.B. & Fonarow, G.C. 2011, "Epidemiology and risk profile of heart failure", Nature reviews.Cardiology, vol. 8, no. 1, pp. 30-41.

Bui, H.M., Enis, D., Robciuc, M.R., Nurmi, H.J., Cohen, J., Chen, M., Yang, Y., Dhillon, V., Johnson, K., Zhang, H., Kirkpatrick, R., Traxler, E., Anisimov, A., Alitalo, K. & Kahn, M.L. 2016, "Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD", The Journal of clinical investigation, vol. 126, no. 6, pp. 2167-2180.

Buttet, M., Traynard, V., Tran, T.T., Besnard, P., Poirier, H. & Niot, I. 2014, "From fatty-acid sensing to chylomicron synthesis: role of intestinal lipid-binding proteins", Biochimie, vol. 96, pp. 37-47.

Calvo, D. & Vega, M.A. 1993, "Identification, primary structure, and distribution of CLA-1, a novel member of the CD36/LIMPII gene family", The Journal of biological chemistry, vol. 268, no. 25, pp. 18929-18935.

Campbell, G.R. & Campbell, J.H. 1985, "Smooth muscle phenotypic changes in arterial wall homeostasis: implications for the pathogenesis of atherosclerosis", Experimental and molecular pathology, vol. 42, no. 2, pp. 139-162.

Cao, Y. 2013, "Angiogenesis and vascular functions in modulation of obesity, adipose metabolism, and insulin sensitivity", Cell metabolism, vol. 18, no. 4, pp. 478-489.

Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C., Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W. & Nagy, A. 1996, "Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele", Nature, vol. 380, no. 6573, pp. 435-439.

Carmeliet, P., Moons, L., Luttun, A., Vincenti, V., Compernolle, V., De Mol, M., Wu, Y., Bono, F., Devy, L., Beck, H., Scholz, D., Acker, T., DiPalma, T., Dewerchin, M., Noel, A., Stalmans, I., Barra, A., Blacher, S., VandenDriessche, T., Ponten, A., Eriksson, U., Plate, K.H., Foidart, J.M., Schaper, W., Charnock-Jones, D.S., Hicklin, D.J., Herbert, J.M., Collen, D. & Persico, M.G. 2001, "Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions", Nature medicine, vol. 7, no. 5, pp. 575-583.

Celletti, F.L., Hilfiker, P.R., Ghafouri, P. & Dake, M.D. 2001a, "Effect of human recombinant vascular endothelial growth factor165 on progression of atherosclerotic plaque", Journal of the American College of Cardiology, vol. 37, no. 8, pp. 2126-2130.

Celletti, F.L., Waugh, J.M., Amabile, P.G., Brendolan, A., Hilfiker, P.R. & Dake, M.D. 2001b, "Vascular endothelial growth factor enhances atherosclerotic plaque progression", Nature medicine, vol. 7, no. 4, pp. 425-429.

Chaudhary, R., Garg, J., Shah, N. & Sumner, A. 2017, "PCSK9 inhibitors: A new era of lipid lowering therapy", World journal of cardiology, vol. 9, no. 2, pp. 76-91.

Chen, H., Chedotal, A., He, Z., Goodman, C.S. & Tessier-Lavigne, M. 1997, "Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III", Neuron, vol. 19, no. 3, pp. 547-559.

Chen, J., Ceholski, D.K., Liang, L., Fish, K. & Hajjar, R.J. 2017, "Variability in coronary artery anatomy affects consistency of cardiac damage after myocardial infarction in mice", American journal of physiology.Heart and circulatory physiology, vol. 313, no. 2, pp. H275-H282.

Chen, S.H., Habib, G., Yang, C.Y., Gu, Z.W., Lee, B.R., Weng, S.A., Silberman, S.R., Cai, S.J., Deslypere, J.P. & Rosseneu, M. 1987, "Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon", Science (New York, N.Y.), vol. 238, no. 4825, pp. 363-366.

63

Chen, W.J., Goldstein, J.L. & Brown, M.S. 1990, "NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor", The Journal of biological chemistry, vol. 265, no. 6, pp. 3116-3123.

Cohen, R.D., Castellani, L.W., Qiao, J.H., Van Lenten, B.J., Lusis, A.J. & Reue, K. 1997, "Reduced aortic lesions and elevated high density lipoprotein levels in transgenic mice overexpressing mouse apolipoprotein A-IV", The Journal of clinical investigation, vol. 99, no. 8, pp. 1906-1916.

Collet, X., Tall, A.R., Serajuddin, H., Guendouzi, K., Royer, L., Oliveira, H., Barbaras, R., Jiang, X.C. & Francone, O.L. 1999, "Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I", Journal of lipid research, vol. 40, no. 7, pp. 1185-1193.

Colucci, W.S. 1997, "Molecular and cellular mechanisms of myocardial failure", The American Journal of Cardiology, vol. 80, no. 11A, pp. 15L-25L.

Corder, E.H., Saunders, A.M., Strittmatter, W.J., Schmechel, D.E., Gaskell, P.C., Small, G.W., Roses, A.D., Haines, J.L. & Pericak-Vance, M.A. 1993, "Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families", Science (New York, N.Y.), vol. 261, no. 5123, pp. 921-923.

Cui, Y. 2010, "The role of lymphatic vessels in the heart", Pathophysiology :the official journal of the International Society for Pathophysiology / ISP, vol. 17, no. 4, pp. 307-314.

Daniels, T.F., Killinger, K.M., Michal, J.J., Wright, R.W.,Jr & Jiang, Z. 2009, "Lipoproteins, cholesterol homeostasis and cardiac health", International journal of biological sciences, vol. 5, no. 5, pp. 474-488.

Dash, S., Xiao, C., Morgantini, C. & Lewis, G.F. 2015, "New Insights into the Regulation of Chylomicron Production", Annual Review of Nutrition, vol. 35, pp. 265-294.

Davidson, N.O. & Shelness, G.S. 2000, "APOLIPOPROTEIN B: mRNA editing, lipoprotein assembly, and presecretory degradation", Annual Review of Nutrition, vol. 20, pp. 169-193.

Davis, K.L., Laine, G.A., Geissler, H.J., Mehlhorn, U., Brennan, M. & Allen, S.J. 2000, "Effects of myocardial edema on the development of myocardial interstitial fibrosis", Microcirculation (New York, N.Y.: 1994), vol. 7, no. 4, pp. 269-280.

Davis, R.B., Kechele, D.O., Blakeney, E.S., Pawlak, J.B. & Caron, K.M. 2017, "Lymphatic deletion of calcitonin receptor-like receptor exacerbates intestinal inflammation", JCI insight, vol. 2, no. 6, pp. e92465.

de Beer, M.C., Durbin, D.M., Cai, L., Mirocha, N., Jonas, A., Webb, N.R., de Beer, F.C. & van Der Westhuyzen, D.R. 2001, "Apolipoprotein A-II modulates the binding and selective lipid uptake of reconstituted high density lipoprotein by scavenger receptor BI", The Journal of biological chemistry, vol. 276, no. 19, pp. 15832-15839.

de Silva, H.V., Lauer, S.J., Wang, J., Simonet, W.S., Weisgraber, K.H., Mahley, R.W. & Taylor, J.M. 1994, "Overexpression of human apolipoprotein C-III in transgenic mice results in an accumulation of apolipoprotein B48 remnants that is corrected by excess apolipoprotein E", The Journal of biological chemistry, vol. 269, no. 3, pp. 2324-2335.

de Vries, C., Escobedo, J.A., Ueno, H., Houck, K., Ferrara, N. & Williams, L.T. 1992, "The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor", Science (New York, N.Y.), vol. 255, no. 5047, pp. 989-991.

de Winther, M.P., Kanters, E., Kraal, G. & Hofker, M.H. 2005, "Nuclear factor kappaB signaling in atherogenesis", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 25, no. 5, pp. 904-914.

Deng, Y., Foley, E.M., Gonzales, J.C., Gordts, P.L., Li, Y. & Esko, J.D. 2012, "Shedding of syndecan-1 from human hepatocytes alters very low density lipoprotein clearance", Hepatology (Baltimore, Md.), vol. 55, no. 1, pp. 277-286.

Dieckmann, M., Dietrich, M.F. & Herz, J. 2010, "Lipoprotein receptors--an evolutionarily ancient multifunctional receptor family", Biological chemistry, vol. 391, no. 11, pp. 1341-1363.


64

Dijkstra, M.H., Pirinen, E., Huusko, J., Kivela, R., Schenkwein, D., Alitalo, K. & Yla-Herttuala, S. 2014, "Lack of cardiac and high-fat diet induced metabolic phenotypes in two independent strains of Vegf-b knockout mice", Scientific reports, vol. 4, pp. 6238.

Dixelius, J., Makinen, T., Wirzenius, M., Karkkainen, M.J., Wernstedt, C., Alitalo, K. & Claesson-Welsh, L. 2003, "Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites", The Journal of biological chemistry, vol. 278, no. 42, pp. 40973-40979.

Dixon, J.B. 2010, "Mechanisms of chylomicron uptake into lacteals", Annals of the New York Academy of Sciences, vol. 1207 Suppl 1, pp. E52-7.

Dominiczak, M.H. & Caslake, M.J. 2011, "Apolipoproteins: metabolic role and clinical biochemistry applications", Annals of Clinical Biochemistry, vol. 48, no. Pt 6, pp. 498-515.

Dumont, D.J., Jussila, L., Taipale, J., Lymboussaki, A., Mustonen, T., Pajusola, K., Breitman, M. & Alitalo, K. 1998, "Cardiovascular failure in mouse embryos deficient in VEGF receptor-3", Science (New York, N.Y.), vol. 282, no. 5390, pp. 946-949.

Elias, I., Franckhauser, S., Ferre, T., Vila, L., Tafuro, S., Munoz, S., Roca, C., Ramos, D., Pujol, A., Riu, E., Ruberte, J. & Bosch, F. 2012, "Adipose tissue overexpression of vascular endothelial growth factor protects against diet-induced obesity and insulin resistance", Diabetes, vol. 61, no. 7, pp. 1801-1813.

Enholm, B., Paavonen, K., Ristimaki, A., Kumar, V., Gunji, Y., Klefstrom, J., Kivinen, L., Laiho, M., Olofsson, B., Joukov, V., Eriksson, U. & Alitalo, K. 1997, "Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia", Oncogene, vol. 14, no. 20, pp. 2475-2483.

Escobedo, N. & Oliver, G. 2017, "The Lymphatic Vasculature: Its Role in Adipose Metabolism and Obesity", Cell metabolism, vol. 26, no. 4, pp. 598-609.

Escobedo, N., Proulx, S.T., Karaman, S., Dillard, M.E., Johnson, N., Detmar, M. & Oliver, G. 2016, "Restoration of lymphatic function rescues obesity in Prox1-haploinsufficient mice", JCI insight, vol. 1, no. 2, pp. 10.1172/jci.insight.85096. Epub 2016 Feb 25.

Evans, I.M., Yamaji, M., Britton, G., Pellet-Many, C., Lockie, C., Zachary, I.C. & Frankel, P. 2011, "Neuropilin-1 signaling through p130Cas tyrosine phosphorylation is essential for growth factor-dependent migration of glioma and endothelial cells", Molecular and cellular biology, vol. 31, no. 6, pp. 1174-1185.

Favier, B., Alam, A., Barron, P., Bonnin, J., Laboudie, P., Fons, P., Mandron, M., Herault, J.P., Neufeld, G., Savi, P., Herbert, J.M. & Bono, F. 2006, "Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration", Blood, vol. 108, no. 4, pp. 1243-1250.

Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., O'Shea, K.S., Powell-Braxton, L., Hillan, K.J. & Moore, M.W. 1996, "Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene", Nature, vol. 380, no. 6573, pp. 439-442.

Ferrara, N., Gerber, H.P. & LeCouter, J. 2003, "The biology of VEGF and its receptors", Nature medicine, vol. 9, no. 6, pp. 669-676.

Ferrara, N. & Henzel, W.J. 1989, "Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells", Biochemical and biophysical research communications, vol. 161, no. 2, pp. 851-858.

Flood, C., Gustafsson, M., Richardson, P.E., Harvey, S.C., Segrest, J.P. & Boren, J. 2002, "Identification of the proteoglycan binding site in apolipoprotein B48", The Journal of biological chemistry, vol. 277, no. 35, pp. 32228-32233.

Foley, E.M. & Esko, J.D. 2010, "Hepatic heparan sulfate proteoglycans and endocytic clearance of triglyceride-rich lipoproteins", Progress in molecular biology and translational science, vol. 93, pp. 213-233.

65

Foley, E.M., Gordts, P.L.S.M., Stanford, K.I., Gonzales, J.C., Lawrence, R., Stoddard, N. & Esko, J.D. 2013, "Hepatic remnant lipoprotein clearance by heparan sulfate proteoglycans and low-density lipoprotein receptors depend on dietary conditions in mice", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 33, no. 9, pp. 2065-2074.

Fong, G.H., Rossant, J., Gertsenstein, M. & Breitman, M.L. 1995, "Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium", Nature, vol. 376, no. 6535, pp. 66-70.

Fong, L.G., Young, S.G., Beigneux, A.P., Bensadoun, A., Oberer, M., Jiang, H. & Ploug, M. 2016, "GPIHBP1 and Plasma Triglyceride Metabolism", Trends in endocrinology and metabolism: TEM, vol. 27, no. 7, pp. 455-469.

Forsythe, J.A., Jiang, B.H., Iyer, N.V., Agani, F., Leung, S.W., Koos, R.D. & Semenza, G.L. 1996, "Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1", Molecular and cellular biology, vol. 16, no. 9, pp. 4604-4613.

Frangogiannis, N.G. 2014, "The inflammatory response in myocardial injury, repair, and remodelling", Nature reviews.Cardiology, vol. 11, no. 5, pp. 255-265.

Fraser, R., Dobbs, B.R. & Rogers, G.W. 1995, "Lipoproteins and the liver sieve: the role of the fenestrated sinusoidal endothelium in lipoprotein metabolism, atherosclerosis, and cirrhosis", Hepatology (Baltimore, Md.), vol. 21, no. 3, pp. 863-874.

Frykman, P.K., Brown, M.S., Yamamoto, T., Goldstein, J.L. & Herz, J. 1995, "Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor", Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 18, pp. 8453-8457.

Fuki, I.V., Kuhn, K.M., Lomazov, I.R., Rothman, V.L., Tuszynski, G.P., Iozzo, R.V., Swenson, T.L., Fisher, E.A. & Williams, K.J. 1997, "The syndecan family of proteoglycans. Novel receptors mediating internalization of atherogenic lipoproteins in vitro", The Journal of clinical investigation, vol. 100, no. 6, pp. 1611-1622.

Gajda, A.M. & Storch, J. 2015, "Enterocyte fatty acid-binding proteins (FABPs): different functions of liver and intestinal FABPs in the intestine", Prostaglandins, leukotrienes, and essential fatty acids, vol. 93, pp. 9-16.

Gao, E., Lei, Y.H., Shang, X., Huang, Z.M., Zuo, L., Boucher, M., Fan, Q., Chuprun, J.K., Ma, X.L. & Koch, W.J. 2010, "A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse", Circulation research, vol. 107, no. 12, pp. 1445-1453.

Gelissen, I.C., Harris, M., Rye, K.A., Quinn, C., Brown, A.J., Kockx, M., Cartland, S., Packianathan, M., Kritharides, L. & Jessup, W. 2006, "ABCA1 and ABCG1 synergize to mediate cholesterol export to apoA-I", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 26, no. 3, pp. 534-540.

Gerber, H.P., Hillan, K.J., Ryan, A.M., Kowalski, J., Keller, G.A., Rangell, L., Wright, B.D., Radtke, F., Aguet, M. & Ferrara, N. 1999, "VEGF is required for growth and survival in neonatal mice", Development (Cambridge, England), vol. 126, no. 6, pp. 1149-1159.

Getz, G.S. & Reardon, C.A. 2016, "Do the Apoe-/- and Ldlr-/- Mice Yield the Same Insight on Atherogenesis?", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 36, no. 9, pp. 1734-1741.

Getz, G.S. & Reardon, C.A. 2012, "Animal models of atherosclerosis", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 32, no. 5, pp. 1104-1115.

Ghugre, N.R., Pop, M., Thomas, R., Newbigging, S., Qi, X., Barry, J., Strauss, B.H. & Wright, G.A. 2017, "Hemorrhage promotes inflammation and myocardial damage following acute myocardial infarction: insights from a novel preclinical model and cardiovascular magnetic resonance", Journal of cardiovascular magnetic resonance : official journal of the Society for Cardiovascular Magnetic Resonance, vol. 19, no. 1, pp. 50-017-0361-7.

Giacca, M. & Zacchigna, S. 2012, "VEGF gene therapy: therapeutic angiogenesis in the clinic and beyond", Gene therapy, vol. 19, no. 6, pp. 622-629.

Giger, R.J., Cloutier, J.F., Sahay, A., Prinjha, R.K., Levengood, D.V., Moore, S.E., Pickering, S., Simmons, D., Rastan, S., Walsh, F.S., Kolodkin, A.L., Ginty, D.D. & Geppert, M. 2000,


66

"Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins", Neuron, vol. 25, no. 1, pp. 29-41.

Gleissner, C.A. 2016, "Translational atherosclerosis research: From experimental models to coronary artery disease in humans", Atherosclerosis, vol. 248, pp. 110-116.

Gluzman-Poltorak, Z., Cohen, T., Herzog, Y. & Neufeld, G. 2000, "Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165 [corrected", The Journal of biological chemistry, vol. 275, no. 24, pp. 18040-18045.

Gluzman-Poltorak, Z., Cohen, T., Shibuya, M. & Neufeld, G. 2001, "Vascular endothelial growth factor receptor-1 and neuropilin-2 form complexes", The Journal of biological chemistry, vol. 276, no. 22, pp. 18688-18694.

Goldberg, I.J., Scheraldi, C.A., Yacoub, L.K., Saxena, U. & Bisgaier, C.L. 1990, "Lipoprotein ApoC-II activation of lipoprotein lipase. Modulation by apolipoprotein A-IV", The Journal of biological chemistry, vol. 265, no. 8, pp. 4266-4272.

Goldman, J., Le, T.X., Skobe, M. & Swartz, M.A. 2005, "Overexpression of VEGF-C causes transient lymphatic hyperplasia but not increased lymphangiogenesis in regenerating skin", Circulation research, vol. 96, no. 11, pp. 1193-1199.

Goldstein, J.L. & Brown, M.S. 2009, "The LDL receptor", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 29, no. 4, pp. 431-438.

Goldstein, J.L. & Brown, M.S. 1974, "Binding and degradation of low density lipoproteins by cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia", The Journal of biological chemistry, vol. 249, no. 16, pp. 5153-5162.

Goldstein, J.L., Dana, S.E., Faust, J.R., Beaudet, A.L. & Brown, M.S. 1975, "Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease", The Journal of biological chemistry, vol. 250, no. 21, pp. 8487-8495.

Gomez-Ambrosi, J., Catalan, V., Rodriguez, A., Ramirez, B., Silva, C., Gil, M.J., Salvador, J. & Fruhbeck, G. 2010, "Involvement of serum vascular endothelial growth factor family members in the development of obesity in mice and humans", The Journal of nutritional biochemistry, vol. 21, no. 8, pp. 774-780.

Gonias, S.L. & Campana, W.M. 2014, "LDL receptor-related protein-1: a regulator of inflammation in atherosclerosis, cancer, and injury to the nervous system", The American journal of pathology, vol. 184, no. 1, pp. 18-27.

Gordon, K., Schulte, D., Brice, G., Simpson, M.A., Roukens, M.G., van Impel, A., Connell, F., Kalidas, K., Jeffery, S., Mortimer, P.S., Mansour, S., Schulte-Merker, S. & Ostergaard, P. 2013, "Mutation in vascular endothelial growth factor-C, a ligand for vascular endothelial growth factor receptor-3, is associated with autosomal dominant milroy-like primary lymphedema", Circulation research, vol. 112, no. 6, pp. 956-960.

Gordts, P.L., Nock, R., Son, N.H., Ramms, B., Lew, I., Gonzales, J.C., Thacker, B.E., Basu, D., Lee, R.G., Mullick, A.E., Graham, M.J., Goldberg, I.J., Crooke, R.M., Witztum, J.L. & Esko, J.D. 2016, "ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors", The Journal of clinical investigation, vol. 126, no. 8, pp. 2855-2866.

Gordts, P.L.S.M. & Esko, J.D. 2018, "The heparan sulfate proteoglycan grip on hyperlipidemia and atherosclerosis", Matrix biology journal of the International Society for Matrix Biology, .

Gotink, K.J. & Verheul, H.M. 2010, "Anti-angiogenic tyrosine kinase inhibitors: what is their mechanism of action?", Angiogenesis, vol. 13, no. 1, pp. 1-14.

Goudriaan, J.R., Tacken, P.J., Dahlmans, V.E., Gijbels, M.J., van Dijk, K.W., Havekes, L.M. & Jong, M.C. 2001, "Protection from obesity in mice lacking the VLDL receptor", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 21, no. 9, pp. 1488-1493.

67

Goulbourne, C.N., Gin, P., Tatar, A., Nobumori, C., Hoenger, A., Jiang, H., Grovenor, C.R., Adeyo, O., Esko, J.D., Goldberg, I.J., Reue, K., Tontonoz, P., Bensadoun, A., Beigneux, A.P., Young, S.G. & Fong, L.G. 2014, "The GPIHBP1-LPL complex is responsible for the margination of triglyceride-rich lipoproteins in capillaries", Cell metabolism, vol. 19, no. 5, pp. 849-860.

Gousopoulos, E., Proulx, S.T., Scholl, J., Uecker, M. & Detmar, M. 2016, "Prominent Lymphatic Vessel Hyperplasia with Progressive Dysfunction and Distinct Immune Cell Infiltration in Lymphedema", The American journal of pathology, vol. 186, no. 8, pp. 2193-2203.

Gowdak, L.H., Poliakova, L., Wang, X., Kovesdi, I., Fishbein, K.W., Zacheo, A., Palumbo, R., Straino, S., Emanueli, C., Marrocco-Trischitta, M., Lakatta, E.G., Anversa, P., Spencer, R.G., Talan, M. & Capogrossi, M.C. 2000, "Adenovirus-mediated VEGF(121) gene transfer stimulates angiogenesis in normoperfused skeletal muscle and preserves tissue perfusion after induction of ischemia", Circulation, vol. 102, no. 5, pp. 565-571.

Greeve, J., Altkemper, I., Dieterich, J.H., Greten, H. & Windler, E. 1993, "Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins", Journal of lipid research, vol. 34, no. 8, pp. 1367-1383.

Grosskreutz, C.L., Anand-Apte, B., Duplaa, C., Quinn, T.P., Terman, B.I., Zetter, B. & D'Amore, P.A. 1999, "Vascular endothelial growth factor-induced migration of vascular smooth muscle cells in vitro", Microvascular research, vol. 58, no. 2, pp. 128-136.

Hagberg, C.E., Falkevall, A., Wang, X., Larsson, E., Huusko, J., Nilsson, I., van Meeteren, L.A., Samen, E., Lu, L., Vanwildemeersch, M., Klar, J., Genove, G., Pietras, K., Stone-Elander, S., Claesson-Welsh, L., Yla-Herttuala, S., Lindahl, P. & Eriksson, U. 2010, "Vascular endothelial growth factor B controls endothelial fatty acid uptake", Nature, vol. 464, no. 7290, pp. 917-921.

Hagberg, C.E., Mehlem, A., Falkevall, A., Muhl, L., Fam, B.C., Ortsater, H., Scotney, P., Nyqvist, D., Samen, E., Lu, L., Stone-Elander, S., Proietto, J., Andrikopoulos, S., Sjoholm, A., Nash, A. & Eriksson, U. 2012, "Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes", Nature, vol. 490, no. 7420, pp. 426-430.

Haiko, P., Makinen, T., Keskitalo, S., Taipale, J., Karkkainen, M.J., Baldwin, M.E., Stacker, S.A., Achen, M.G. & Alitalo, K. 2008, "Deletion of vascular endothelial growth factor C (VEGF-C) and VEGF-D is not equivalent to VEGF receptor 3 deletion in mouse embryos", Molecular and cellular biology, vol. 28, no. 15, pp. 4843-4850.

Harris, N.C., Paavonen, K., Davydova, N., Roufail, S., Sato, T., Zhang, Y.F., Karnezis, T., Stacker, S.A. & Achen, M.G. 2011, "Proteolytic processing of vascular endothelial growth factor-D is essential for its capacity to promote the growth and spread of cancer", The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, vol. 25, no. 8, pp. 2615-2625.

Hartikainen, J., Hassinen, I., Hedman, A., Kivela, A., Saraste, A., Knuuti, J., Husso, M., Mussalo, H., Hedman, M., Rissanen, T.T., Toivanen, P., Heikura, T., Witztum, J.L., Tsimikas, S. & Yla-Herttuala, S. 2017, "Adenoviral intramyocardial VEGF-DDeltaNDeltaC gene transfer increases myocardial perfusion reserve in refractory angina patients: a phase I/IIa study with 1-year follow-up", European heart journal, vol. 38, no. 33, pp. 2547-2555.

Harvey, N.L., Srinivasan, R.S., Dillard, M.E., Johnson, N.C., Witte, M.H., Boyd, K., Sleeman, M.W. & Oliver, G. 2005, "Lymphatic vascular defects promoted by Prox1 haploinsufficiency cause adult-onset obesity", Nature genetics, vol. 37, no. 10, pp. 1072-1081.

He, G., Gupta, S., Yi, M., Michaely, P., Hobbs, H.H. & Cohen, J.C. 2002, "ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and AP-2", The Journal of biological chemistry, vol. 277, no. 46, pp. 44044-44049.

He, Z. & Tessier-Lavigne, M. 1997, "Neuropilin is a receptor for the axonal chemorepellent Semaphorin III", Cell, vol. 90, no. 4, pp. 739-751.

Hedman, M., Muona, K., Hedman, A., Kivela, A., Syvanne, M., Eranen, J., Rantala, A., Stjernvall, J., Nieminen, M.S., Hartikainen, J. & Yla-Herttuala, S. 2009, "Eight-year safety follow-up of coronary


68

artery disease patients after local intracoronary VEGF gene transfer", Gene therapy, vol. 16, no. 5, pp. 629-634.

Hegele, R.A. 2009, "Plasma lipoproteins: genetic influences and clinical implications", Nature reviews.Genetics, vol. 10, no. 2, pp. 109-121.

Heinolainen, K., Karaman, S., D'Amico, G., Tammela, T., Sormunen, R., Eklund, L., Alitalo, K. & Zarkada, G. 2017, "VEGFR3 Modulates Vascular Permeability by Controlling VEGF/VEGFR2 Signaling", Circulation research, vol. 120, no. 9, pp. 1414-1425.

Heinonen, S.E., Kivela, A.M., Huusko, J., Dijkstra, M.H., Gurzeler, E., Makinen, P.I., Leppanen, P., Olkkonen, V.M., Eriksson, U., Jauhiainen, M. & Yla-Herttuala, S. 2013, "The effects of VEGF-A on atherosclerosis, lipoprotein profile, and lipoprotein lipase in hyperlipidaemic mouse models", Cardiovascular research, vol. 99, no. 4, pp. 716-723.

Henri, O., Pouehe, C., Houssari, M., Galas, L., Nicol, L., Edwards-Levy, F., Henry, J.P., Dumesnil, A., Boukhalfa, I., Banquet, S., Schapman, D., Thuillez, C., Richard, V., Mulder, P. & Brakenhielm, E. 2016, "Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction", Circulation, vol. 133, no. 15, pp. 1484-97; discussion 1497.

Henry, T.D., Annex, B.H., McKendall, G.R., Azrin, M.A., Lopez, J.J., Giordano, F.J., Shah, P.K., Willerson, J.T., Benza, R.L., Berman, D.S., Gibson, C.M., Bajamonde, A., Rundle, A.C., Fine, J., McCluskey, E.R. & VIVA Investigators 2003, "The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis", Circulation, vol. 107, no. 10, pp. 1359-1365.

Herz, J., Clouthier, D.E. & Hammer, R.E. 1992, "LDL receptor-related protein internalizes and degrades uPA-PAI-1 complexes and is essential for embryo implantation", Cell, vol. 71, no. 3, pp. 411-421.

Higgins, M.E., Davies, J.P., Chen, F.W. & Ioannou, Y.A. 1999, "Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network", Molecular genetics and metabolism, vol. 68, no. 1, pp. 1-13.

Hirano, K., Min, J., Funahashi, T. & Davidson, N.O. 1997, "Cloning and characterization of the rat apobec-1 gene: a comparative analysis of gene structure and promoter usage in rat and mouse", Journal of lipid research, vol. 38, no. 6, pp. 1103-1119.

Hiratsuka, S., Minowa, O., Kuno, J., Noda, T. & Shibuya, M. 1998, "Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice", Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 16, pp. 9349-9354.

Ho, V.C., Duan, L.J., Cronin, C., Liang, B.T. & Fong, G.H. 2012, "Elevated vascular endothelial growth factor receptor-2 abundance contributes to increased angiogenesis in vascular endothelial growth factor receptor-1-deficient mice", Circulation, vol. 126, no. 6, pp. 741-752.

Honkonen, K.M., Visuri, M.T., Tervala, T.V., Halonen, P.J., Koivisto, M., Lahteenvuo, M.T., Alitalo, K.K., Yla-Herttuala, S. & Saaristo, A.M. 2013, "Lymph node transfer and perinodal lymphatic growth factor treatment for lymphedema", Annals of Surgery, vol. 257, no. 5, pp. 961-967.

Horton, J.D., Cohen, J.C. & Hobbs, H.H. 2007, "Molecular biology of PCSK9: its role in LDL metabolism", Trends in biochemical sciences, vol. 32, no. 2, pp. 71-77.

Huang, K., Andersson, C., Roomans, G.M., Ito, N. & Claesson-Welsh, L. 2001, "Signaling properties of VEGF receptor-1 and -2 homo- and heterodimers", The international journal of biochemistry & cell biology, vol. 33, no. 4, pp. 315-324.

Huang, L.H., Lavine, K.J. & Randolph, G.J. 2017, "Cardiac Lymphatic Vessels, Transport, and Healing of the Infarcted Heart", JACC.Basic to translational science, vol. 2, no. 4, pp. 477-483.

Huang, Y. & Mahley, R.W. 2014, "Apolipoprotein E: structure and function in lipid metabolism, neurobiology, and Alzheimer's diseases", Neurobiology of disease, vol. 72 Pt A, pp. 3-12.

Hussain, M.M., Shi, J. & Dreizen, P. 2003, "Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly", Journal of lipid research, vol. 44, no. 1, pp. 22-32.

69

Huusko, J., Merentie, M., Dijkstra, M.H., Ryhanen, M.M., Karvinen, H., Rissanen, T.T., Vanwildemeersch, M., Hedman, M., Lipponen, J., Heinonen, S.E., Eriksson, U., Shibuya, M. & Yla-Herttuala, S. 2010, "The effects of VEGF-R1 and VEGF-R2 ligands on angiogenic responses and left ventricular function in mice", Cardiovascular research, vol. 86, no. 1, pp. 122-130.

Inazu, A., Brown, M.L., Hesler, C.B., Agellon, L.B., Koizumi, J., Takata, K., Maruhama, Y., Mabuchi, H. & Tall, A.R. 1990, "Increased high-density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation", The New England journal of medicine, vol. 323, no. 18, pp. 1234-1238.

Innerarity, T.L., Weisgraber, K.H., Arnold, K.S., Mahley, R.W., Krauss, R.M., Vega, G.L. & Grundy, S.M. 1987, "Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding", Proceedings of the National Academy of Sciences of the United States of America, vol. 84, no. 19, pp. 6919-6923.

Iqbal, J., Rudel, L.L. & Hussain, M.M. 2008, "Microsomal triglyceride transfer protein enhances cellular cholesteryl esterification by relieving product inhibition", The Journal of biological chemistry, vol. 283, no. 29, pp. 19967-19980.

Ishibashi, S., Brown, M.S., Goldstein, J.L., Gerard, R.D., Hammer, R.E. & Herz, J. 1993, "Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery", The Journal of clinical investigation, vol. 92, no. 2, pp. 883-893.

Istvan, E.S. & Deisenhofer, J. 2001, "Structural mechanism for statin inhibition of HMG-CoA reductase", Science (New York, N.Y.), vol. 292, no. 5519, pp. 1160-1164.

Ito, Y., Azrolan, N., O'Connell, A., Walsh, A. & Breslow, J.L. 1990, "Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice", Science (New York, N.Y.), vol. 249, no. 4970, pp. 790-793.

Jakobsson, L., Kreuger, J., Holmborn, K., Lundin, L., Eriksson, I., Kjellen, L. & Claesson-Welsh, L. 2006, "Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis", Developmental cell, vol. 10, no. 5, pp. 625-634.

Jarvelainen, H., Sainio, A., Koulu, M., Wight, T.N. & Penttinen, R. 2009, "Extracellular matrix molecules: potential targets in pharmacotherapy", Pharmacological reviews, vol. 61, no. 2, pp. 198-223.

Jeltsch, M., Jha, S.K., Tvorogov, D., Anisimov, A., Leppanen, V.M., Holopainen, T., Kivela, R., Ortega, S., Karpanen, T. & Alitalo, K. 2014, "CCBE1 enhances lymphangiogenesis via A disintegrin and metalloprotease with thrombospondin motifs-3-mediated vascular endothelial growth factor-C activation", Circulation, vol. 129, no. 19, pp. 1962-1971.

Jeltsch, M., Kaipainen, A., Joukov, V., Meng, X., Lakso, M., Rauvala, H., Swartz, M., Fukumura, D., Jain, R.K. & Alitalo, K. 1997, "Hyperplasia of lymphatic vessels in VEGF-C transgenic mice", Science (New York, N.Y.), vol. 276, no. 5317, pp. 1423-1425.

Jin, F., Hagemann, N., Brockmeier, U., Schafer, S.T., Zechariah, A. & Hermann, D.M. 2013, "LDL attenuates VEGF-induced angiogenesis via mechanisms involving VEGFR2 internalization and degradation following endosome-trans-Golgi network trafficking", Angiogenesis, vol. 16, no. 3, pp. 625-637.

Jonas, A. 2000, "Lecithin cholesterol acyltransferase", Biochimica et biophysica acta, vol. 1529, no. 1-3, pp. 245-256.

Jong, M.C., Dahlmans, V.E., van Gorp, P.J., van Dijk, K.W., Breuer, M.L., Hofker, M.H. & Havekes, L.M. 1996, "In the absence of the low density lipoprotein receptor, human apolipoprotein C1 overexpression in transgenic mice inhibits the hepatic uptake of very low density lipoproteins via a receptor-associated protein-sensitive pathway", The Journal of clinical investigation, vol. 98, no. 10, pp. 2259-2267.


70

Jong, M.C., Hofker, M.H. & Havekes, L.M. 1999, "Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 19, no. 3, pp. 472-484.

Joseph, P., Leong, D., McKee, M., Anand, S.S., Schwalm, J.D., Teo, K., Mente, A. & Yusuf, S. 2017, "Reducing the Global Burden of Cardiovascular Disease, Part 1: The Epidemiology and Risk Factors", Circulation research, vol. 121, no. 6, pp. 677-694.

Joukov, V., Pajusola, K., Kaipainen, A., Chilov, D., Lahtinen, I., Kukk, E., Saksela, O., Kalkkinen, N. & Alitalo, K. 1996, "A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases", The EMBO journal, vol. 15, no. 7, pp. 1751.

Joukov, V., Sorsa, T., Kumar, V., Jeltsch, M., Claesson-Welsh, L., Cao, Y., Saksela, O., Kalkkinen, N. & Alitalo, K. 1997, "Proteolytic processing regulates receptor specificity and activity of VEGF-C", The EMBO journal, vol. 16, no. 13, pp. 3898-3911.

Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, V.W., Fang, G.H., Dumont, D., Breitman, M. & Alitalo, K. 1995, "Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development", Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 8, pp. 3566-3570.

Karaman, S., Hollmen, M., Robciuc, M.R., Alitalo, A., Nurmi, H., Morf, B., Buschle, D., Alkan, H.F., Ochsenbein, A.M., Alitalo, K., Wolfrum, C. & Detmar, M. 2014, "Blockade of VEGF-C and VEGF-D modulates adipose tissue inflammation and improves metabolic parameters under high-fat diet", Molecular metabolism, vol. 4, no. 2, pp. 93-105.

Karaman, S., Hollmen, M., Yoon, S.Y., Alkan, H.F., Alitalo, K., Wolfrum, C. & Detmar, M. 2016, "Transgenic overexpression of VEGF-C induces weight gain and insulin resistance in mice", Scientific reports, vol. 6, pp. 31566.

Karkkainen, M.J., Ferrell, R.E., Lawrence, E.C., Kimak, M.A., Levinson, K.L., McTigue, M.A., Alitalo, K. & Finegold, D.N. 2000, "Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema", Nature genetics, vol. 25, no. 2, pp. 153-159.

Karkkainen, M.J., Haiko, P., Sainio, K., Partanen, J., Taipale, J., Petrova, T.V., Jeltsch, M., Jackson, D.G., Talikka, M., Rauvala, H., Betsholtz, C. & Alitalo, K. 2004, "Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins", Nature immunology, vol. 5, no. 1, pp. 74-80.

Karkkainen, M.J., Saaristo, A., Jussila, L., Karila, K.A., Lawrence, E.C., Pajusola, K., Bueler, H., Eichmann, A., Kauppinen, R., Kettunen, M.I., Yla-Herttuala, S., Finegold, D.N., Ferrell, R.E. & Alitalo, K. 2001, "A model for gene therapy of human hereditary lymphedema", Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 22, pp. 12677-12682.

Karpanen, T., Bry, M., Ollila, H.M., Seppanen-Laakso, T., Liimatta, E., Leskinen, H., Kivela, R., Helkamaa, T., Merentie, M., Jeltsch, M., Paavonen, K., Andersson, L.C., Mervaala, E., Hassinen, I.E., Yla-Herttuala, S., Oresic, M. & Alitalo, K. 2008, "Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy", Circulation research, vol. 103, no. 9, pp. 1018-1026.

Karpanen, T., Heckman, C.A., Keskitalo, S., Jeltsch, M., Ollila, H., Neufeld, G., Tamagnone, L. & Alitalo, K. 2006a, "Functional interaction of VEGF-C and VEGF-D with neuropilin receptors", The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, vol. 20, no. 9, pp. 1462-1472.

Karpanen, T., Wirzenius, M., Makinen, T., Veikkola, T., Haisma, H.J., Achen, M.G., Stacker, S.A., Pytowski, B., Yla-Herttuala, S. & Alitalo, K. 2006b, "Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation", The American journal of pathology, vol. 169, no. 2, pp. 708-718.

Kawamura, H., Li, X., Goishi, K., van Meeteren, L.A., Jakobsson, L., Cebe-Suarez, S., Shimizu, A., Edholm, D., Ballmer-Hofer, K., Kjellen, L., Klagsbrun, M. & Claesson-Welsh, L. 2008,

71

"Neuropilin-1 in regulation of VEGF-induced activation of p38MAPK and endothelial cell organization", Blood, vol. 112, no. 9, pp. 3638-3649.

Kawasaki, T., Kitsukawa, T., Bekku, Y., Matsuda, Y., Sanbo, M., Yagi, T. & Fujisawa, H. 1999, "A requirement for neuropilin-1 in embryonic vessel formation", Development (Cambridge, England), vol. 126, no. 21, pp. 4895-4902.

Kemp, C.D. & Conte, J.V. 2012, "The pathophysiology of heart failure", Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology, vol. 21, no. 5, pp. 365-371.

Kersten, S. 2014, "Physiological regulation of lipoprotein lipase", Biochimica et biophysica acta, vol. 1841, no. 7, pp. 919-933.

Kholova, I., Dragneva, G., Cermakova, P., Laidinen, S., Kaskenpaa, N., Hazes, T., Cermakova, E., Steiner, I. & Yla-Herttuala, S. 2011, "Lymphatic vasculature is increased in heart valves, ischaemic and inflamed hearts and in cholesterol-rich and calcified atherosclerotic lesions", European journal of clinical investigation, vol. 41, no. 5, pp. 487-497.

Kim, K.E., Sung, H.K. & Koh, G.Y. 2007, "Lymphatic development in mouse small intestine", Developmental dynamics : an official publication of the American Association of Anatomists, vol. 236, no. 7, pp. 2020-2025.

Kinnunen, P.K., Jackson, R.L., Smith, L.C., Gotto, A.M.,Jr & Sparrow, J.T. 1977, "Activation of lipoprotein lipase by native and synthetic fragments of human plasma apolipoprotein C-II", Proceedings of the National Academy of Sciences of the United States of America, vol. 74, no. 11, pp. 4848-4851.

Kitsukawa, T., Shimizu, M., Sanbo, M., Hirata, T., Taniguchi, M., Bekku, Y., Yagi, T. & Fujisawa, H. 1997, "Neuropilin-semaphorin III/D-mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice", Neuron, vol. 19, no. 5, pp. 995-1005.

Klocke, R., Tian, W., Kuhlmann, M.T. & Nikol, S. 2007, "Surgical animal models of heart failure related to coronary heart disease", Cardiovascular research, vol. 74, no. 1, pp. 29-38.

Klotz, L., Norman, S., Vieira, J.M., Masters, M., Rohling, M., Dube, K.N., Bollini, S., Matsuzaki, F., Carr, C.A. & Riley, P.R. 2015, "Cardiac lymphatics are heterogeneous in origin and respond to injury", Nature, vol. 522, no. 7554, pp. 62-67.

Koch, S., Tugues, S., Li, X., Gualandi, L. & Claesson-Welsh, L. 2011, "Signal transduction by vascular endothelial growth factor receptors", The Biochemical journal, vol. 437, no. 2, pp. 169-183.

Kohan, A.B., Wang, F., Li, X., Bradshaw, S., Yang, Q., Caldwell, J.L., Bullock, T.M. & Tso, P. 2012, "Apolipoprotein A-IV regulates chylomicron metabolism-mechanism and function", American journal of physiology.Gastrointestinal and liver physiology, vol. 302, no. 6, pp. G628-36.

Kohan, A.B., Wang, F., Lo, C.M., Liu, M. & Tso, P. 2015, "ApoA-IV: current and emerging roles in intestinal lipid metabolism, glucose homeostasis, and satiety", American journal of physiology.Gastrointestinal and liver physiology, vol. 308, no. 6, pp. G472-81.

Kukk, E., Lymboussaki, A., Taira, S., Kaipainen, A., Jeltsch, M., Joukov, V. & Alitalo, K. 1996, "VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development", Development (Cambridge, England), vol. 122, no. 12, pp. 3829-3837.

Lahteenvuo, J.E., Lahteenvuo, M.T., Kivela, A., Rosenlew, C., Falkevall, A., Klar, J., Heikura, T., Rissanen, T.T., Vahakangas, E., Korpisalo, P., Enholm, B., Carmeliet, P., Alitalo, K., Eriksson, U. & Yla-Herttuala, S. 2009, "Vascular endothelial growth factor-B induces myocardium-specific angiogenesis and arteriogenesis via vascular endothelial growth factor receptor-1- and neuropilin receptor-1-dependent mechanisms", Circulation, vol. 119, no. 6, pp. 845-856.

Lahteenvuo, M., Honkonen, K., Tervala, T., Tammela, T., Suominen, E., Lahteenvuo, J., Kholova, I., Alitalo, K., Yla-Herttuala, S. & Saaristo, A. 2011, "Growth factor therapy and autologous lymph node transfer in lymphedema", Circulation, vol. 123, no. 6, pp. 613-620.

Laine, G.A. & Allen, S.J. 1991, "Left ventricular myocardial edema. Lymph flow, interstitial fibrosis, and cardiac function", Circulation research, vol. 68, no. 6, pp. 1713-1721.


72

Lanahan, A., Zhang, X., Fantin, A., Zhuang, Z., Rivera-Molina, F., Speichinger, K., Prahst, C., Zhang, J., Wang, Y., Davis, G., Toomre, D., Ruhrberg, C. & Simons, M. 2013, "The neuropilin 1 cytoplasmic domain is required for VEGF-A-dependent arteriogenesis", Developmental cell, vol. 25, no. 2, pp. 156-168.

LaRosa, J.C., He, J. & Vupputuri, S. 1999, "Effect of statins on risk of coronary disease: a meta-analysis of randomized controlled trials", Jama, vol. 282, no. 24, pp. 2340-2346.

Larsson, M., Allan, C.M., Jung, R.S., Heizer, P.J., Beigneux, A.P., Young, S.G. & Fong, L.G. 2017, "Apolipoprotein C-III inhibits triglyceride hydrolysis by GPIHBP1-bound LPL", Journal of lipid research, vol. 58, no. 9, pp. 1893-1902.

Le Bras, B., Barallobre, M.J., Homman-Ludiye, J., Ny, A., Wyns, S., Tammela, T., Haiko, P., Karkkainen, M.J., Yuan, L., Muriel, M.P., Chatzopoulou, E., Breant, C., Zalc, B., Carmeliet, P., Alitalo, K., Eichmann, A. & Thomas, J.L. 2006, "VEGF-C is a trophic factor for neural progenitors in the vertebrate embryonic brain", Nature neuroscience, vol. 9, no. 3, pp. 340-348.

Lee, S.H., Wolf, P.L., Escudero, R., Deutsch, R., Jamieson, S.W. & Thistlethwaite, P.A. 2000, "Early expression of angiogenesis factors in acute myocardial ischemia and infarction", The New England journal of medicine, vol. 342, no. 9, pp. 626-633.

Lehner, R. & Quiroga, A. 2016, "Fatty Acid Handling in Mammalian Cells" in Biochemistry of Lipids, Lipoproteins and Membranes, eds. N. Ridgway & R. McLeod, 6th edn, Elsevier, , pp. 149-184.

Leppanen, P., Koota, S., Kholova, I., Koponen, J., Fieber, C., Eriksson, U., Alitalo, K. & Yla-Herttuala, S. 2005, "Gene transfers of vascular endothelial growth factor-A, vascular endothelial growth factor-B, vascular endothelial growth factor-C, and vascular endothelial growth factor-D have no effects on atherosclerosis in hypercholesterolemic low-density lipoprotein-receptor/apolipoprotein B48-deficient mice", Circulation, vol. 112, no. 9, pp. 1347-1352.

Lewis, G.F. & Rader, D.J. 2005, "New insights into the regulation of HDL metabolism and reverse cholesterol transport", Circulation research, vol. 96, no. 12, pp. 1221-1232.

Li, Y., He, P.P., Zhang, D.W., Zheng, X.L., Cayabyab, F.S., Yin, W.D. & Tang, C.K. 2014, "Lipoprotein lipase: from gene to atherosclerosis", Atherosclerosis, vol. 237, no. 2, pp. 597-608.

Liao, J.K. & Laufs, U. 2005, "Pleiotropic effects of statins", Annual Review of Pharmacology and Toxicology, vol. 45, pp. 89-118.

Lijnen, H.R., Christiaens, V., Scroyen, I., Voros, G., Tjwa, M., Carmeliet, P. & Collen, D. 2006, "Impaired adipose tissue development in mice with inactivation of placental growth factor function", Diabetes, vol. 55, no. 10, pp. 2698-2704.

Lim, H.Y., Rutkowski, J.M., Helft, J., Reddy, S.T., Swartz, M.A., Randolph, G.J. & Angeli, V. 2009, "Hypercholesterolemic mice exhibit lymphatic vessel dysfunction and degeneration", The American journal of pathology, vol. 175, no. 3, pp. 1328-1337.

Lim, H.Y., Thiam, C.H., Yeo, K.P., Bisoendial, R., Hii, C.S., McGrath, K.C., Tan, K.W., Heather, A., Alexander, J.S. & Angeli, V. 2013, "Lymphatic Vessels Are Essential for the Removal of Cholesterol from Peripheral Tissues by SR-BI-Mediated Transport of HDL", Cell metabolism, vol. 17, no. 5, pp. 671-684.

Lookene, A., Beckstead, J.A., Nilsson, S., Olivecrona, G. & Ryan, R.O. 2005, "Apolipoprotein A-V-heparin interactions: implications for plasma lipoprotein metabolism", The Journal of biological chemistry, vol. 280, no. 27, pp. 25383-25387.

Lopez, J.J., Laham, R.J., Stamler, A., Pearlman, J.D., Bunting, S., Kaplan, A., Carrozza, J.P., Sellke, F.W. & Simons, M. 1998, "VEGF administration in chronic myocardial ischemia in pigs", Cardiovascular research, vol. 40, no. 2, pp. 272-281.

Lucerna, M., Zernecke, A., de Nooijer, R., de Jager, S.C., Bot, I., van der Lans, C., Kholova, I., Liehn, E.A., van Berkel, T.J., Yla-Herttuala, S., Weber, C. & Biessen, E.A. 2007, "Vascular endothelial growth factor-A induces plaque expansion in ApoE knock-out mice by promoting de novo leukocyte recruitment", Blood, vol. 109, no. 1, pp. 122-129.

Lusis, A.J. 2000, "Atherosclerosis", Nature, vol. 407, no. 6801, pp. 233-241.

73

Luttun, A., Brusselmans, K., Fukao, H., Tjwa, M., Ueshima, S., Herbert, J.M., Matsuo, O., Collen, D., Carmeliet, P. & Moons, L. 2002, "Loss of placental growth factor protects mice against vascular permeability in pathological conditions", Biochemical and biophysical research communications, vol. 295, no. 2, pp. 428-434.

Lyttle, D.J., Fraser, K.M., Fleming, S.B., Mercer, A.A. & Robinson, A.J. 1994, "Homologs of vascular endothelial growth factor are encoded by the poxvirus orf virus", Journal of virology, vol. 68, no. 1, pp. 84-92.

MacArthur, J.M., Bishop, J.R., Stanford, K.I., Wang, L., Bensadoun, A., Witztum, J.L. & Esko, J.D. 2007, "Liver heparan sulfate proteoglycans mediate clearance of triglyceride-rich lipoproteins independently of LDL receptor family members", The Journal of clinical investigation, vol. 117, no. 1, pp. 153-164.

Maeda, N., Li, H., Lee, D., Oliver, P., Quarfordt, S.H. & Osada, J. 1994, "Targeted disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridemia and protection from postprandial hypertriglyceridemia", The Journal of biological chemistry, vol. 269, no. 38, pp. 23610-23616.

Maglione, D., Guerriero, V., Viglietto, G., Delli-Bovi, P. & Persico, M.G. 1991, "Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor", Proceedings of the National Academy of Sciences of the United States of America, vol. 88, no. 20, pp. 9267-9271.

Mahley, R.W. 1988, "Apolipoprotein E: cholesterol transport protein with expanding role in cell biology", Science (New York, N.Y.), vol. 240, no. 4852, pp. 622-630.

Mahley, R.W. & Huang, Y. 2007, "Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing", The Journal of clinical investigation, vol. 117, no. 1, pp. 94-98.

Mahley, R.W., Huang, Y. & Rall, S.C.,Jr 1999, "Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia). Questions, quandaries, and paradoxes", Journal of lipid research, vol. 40, no. 11, pp. 1933-1949.

Mahley, R.W., Huang, Y. & Weisgraber, K.H. 2006, "Putting cholesterol in its place: apoE and reverse cholesterol transport", The Journal of clinical investigation, vol. 116, no. 5, pp. 1226-1229.

Mahley, R.W. & Ji, Z.S. 1999, "Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E", Journal of lipid research, vol. 40, no. 1, pp. 1-16.

Mahley, R.W., Weisgraber, K.H. & Huang, Y. 2009, "Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer's disease to AIDS", Journal of lipid research, vol. 50 Suppl, pp. S183-8.

Makinen, T., Jussila, L., Veikkola, T., Karpanen, T., Kettunen, M.I., Pulkkanen, K.J., Kauppinen, R., Jackson, D.G., Kubo, H., Nishikawa, S., Yla-Herttuala, S. & Alitalo, K. 2001, "Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3", Nature medicine, vol. 7, no. 2, pp. 199-205.

Makinen, T., Olofsson, B., Karpanen, T., Hellman, U., Soker, S., Klagsbrun, M., Eriksson, U. & Alitalo, K. 1999, "Differential binding of vascular endothelial growth factor B splice and proteolytic isoforms to neuropilin-1", The Journal of biological chemistry, vol. 274, no. 30, pp. 21217-21222.

Mann, D.L. 2011, "The emerging role of innate immunity in the heart and vascular system: for whom the cell tolls", Circulation research, vol. 108, no. 9, pp. 1133-1145.

Mansbach, C.M.,2nd & Siddiqi, S. 2016, "Control of chylomicron export from the intestine", American journal of physiology.Gastrointestinal and liver physiology, vol. 310, no. 9, pp. G659-68.

Marks, D., Thorogood, M., Neil, H.A. & Humphries, S.E. 2003, "A review on the diagnosis, natural history, and treatment of familial hypercholesterolaemia", Atherosclerosis, vol. 168, no. 1, pp. 1-14.

Martel, C., Li, W., Fulp, B., Platt, A.M., Gautier, E.L., Westerterp, M., Bittman, R., Tall, A.R., Chen, S.H., Thomas, M.J., Kreisel, D., Swartz, M.A., Sorci-Thomas, M.G. & Randolph, G.J. 2013,


74

"Lymphatic vasculature mediates macrophage reverse cholesterol transport in mice", The Journal of clinical investigation, vol. 123, no. 4, pp. 1571-1579.

Marzal-Casacuberta, A., Blanco-Vaca, F., Ishida, B.Y., Julve-Gil, J., Shen, J., Calvet-Marquez, S., Gonzalez-Sastre, F. & Chan, L. 1996, "Functional lecithin:cholesterol acyltransferase deficiency and high density lipoprotein deficiency in transgenic mice overexpressing human apolipoprotein A-II", The Journal of biological chemistry, vol. 271, no. 12, pp. 6720-6728.

Matsunaga, Y., Yamazaki, Y., Suzuki, H. & Morita, T. 2009, "VEGF-A and VEGF-F evoke distinct changes in vascular ultrastructure", Biochemical and biophysical research communications, vol. 379, no. 4, pp. 872-875.

Matthews, W., Jordan, C.T., Gavin, M., Jenkins, N.A., Copeland, N.G. & Lemischka, I.R. 1991, "A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit", Proceedings of the National Academy of Sciences of the United States of America, vol. 88, no. 20, pp. 9026-9030.

McLaughlin, S.A., Wright, M.J., Morris, K.T., Giron, G.L., Sampson, M.R., Brockway, J.P., Hurley, K.E., Riedel, E.R. & Van Zee, K.J. 2008, "Prevalence of lymphedema in women with breast cancer 5 years after sentinel lymph node biopsy or axillary dissection: objective measurements", Journal of clinical oncology : official journal of the American Society of Clinical Oncology, vol. 26, no. 32, pp. 5213-5219.

McPherson, R. & Tybjaerg-Hansen, A. 2016, "Genetics of Coronary Artery Disease", Circulation research, vol. 118, no. 4, pp. 564-578.

Mehlem, A., Palombo, I., Wang, X., Hagberg, C.E., Eriksson, U. & Falkevall, A. 2016, "PGC-1alpha Coordinates Mitochondrial Respiratory Capacity and Muscular Fatty Acid Uptake via Regulation of VEGF-B", Diabetes, vol. 65, no. 4, pp. 861-873.

Meyer, M., Clauss, M., Lepple-Wienhues, A., Waltenberger, J., Augustin, H.G., Ziche, M., Lanz, C., Buttner, M., Rziha, H.J. & Dehio, C. 1999, "A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases", The EMBO journal, vol. 18, no. 2, pp. 363-374.

Migdal, M., Huppertz, B., Tessler, S., Comforti, A., Shibuya, M., Reich, R., Baumann, H. & Neufeld, G. 1998, "Neuropilin-1 is a placenta growth factor-2 receptor", The Journal of biological chemistry, vol. 273, no. 35, pp. 22272-22278.

Moore, K.J. & Freeman, M.W. 2006, "Scavenger receptors in atherosclerosis: beyond lipid uptake", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 26, no. 8, pp. 1702-1711.

Morrisett, J.D., Abdel-Fattah, G., Hoogeveen, R., Mitchell, E., Ballantyne, C.M., Pownall, H.J., Opekun, A.R., Jaffe, J.S., Oppermann, S. & Kahan, B.D. 2002, "Effects of sirolimus on plasma lipids, lipoprotein levels, and fatty acid metabolism in renal transplant patients", Journal of lipid research, vol. 43, no. 8, pp. 1170-1180.

Murakami, M., Zheng, Y., Hirashima, M., Suda, T., Morita, Y., Ooehara, J., Ema, H., Fong, G.H. & Shibuya, M. 2008, "VEGFR1 tyrosine kinase signaling promotes lymphangiogenesis as well as angiogenesis indirectly via macrophage recruitment", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 28, no. 4, pp. 658-664.

Musthafa, H.S., Dragneva, G., Lottonen, L., Merentie, M., Petrov, L., Heikura, T., Yla-Herttuala, E., Yla-Herttuala, S., Grohn, O. & Liimatainen, T. 2013, "Longitudinal rotating frame relaxation time measurements in infarcted mouse myocardium in vivo", Magnetic resonance in medicine, vol. 69, no. 5, pp. 1389-1395.

Nanjee, M.N., Cooke, C.J., Olszewski, W.L. & Miller, N.E. 2000, "Lipid and apolipoprotein concentrations in prenodal leg lymph of fasted humans. Associations with plasma concentrations in normal subjects, lipoprotein lipase deficiency, and LCAT deficiency", Journal of lipid research, vol. 41, no. 8, pp. 1317-1327.

Nanjee, M.N., Cooke, C.J., Wong, J.S., Hamilton, R.L., Olszewski, W.L. & Miller, N.E. 2001, "Composition and ultrastructure of size subclasses of normal human peripheral lymph

75

lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids", Journal of lipid research, vol. 42, no. 4, pp. 639-648.

Nauli, A.M., Nassir, F., Zheng, S., Yang, Q., Lo, C.M., Vonlehmden, S.B., Lee, D., Jandacek, R.J., Abumrad, N.A. & Tso, P. 2006, "CD36 is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine", Gastroenterology, vol. 131, no. 4, pp. 1197-1207.

Nguyen, T.M., Sawyer, J.K., Kelley, K.L., Davis, M.A. & Rudel, L.L. 2012, "Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation", Journal of lipid research, vol. 53, no. 1, pp. 95-104.

Nilsson, J.C., Nielsen, G., Groenning, B.A., Fritz-Hansen, T., Sondergaard, L., Jensen, G.B. & Larsson, H.B. 2001, "Sustained postinfarction myocardial oedema in humans visualised by magnetic resonance imaging", Heart (British Cardiac Society), vol. 85, no. 6, pp. 639-642.

Nomura, M., Yamagishi, S., Harada, S., Hayashi, Y., Yamashima, T., Yamashita, J. & Yamamoto, H. 1995, "Possible participation of autocrine and paracrine vascular endothelial growth factors in hypoxia-induced proliferation of endothelial cells and pericytes", The Journal of biological chemistry, vol. 270, no. 47, pp. 28316-28324.

Norman, S. & Riley, P.R. 2016, "Anatomy and development of the cardiac lymphatic vasculature: Its role in injury and disease", Clinical anatomy (New York, N.Y.), vol. 29, no. 3, pp. 305-315.

Nurmi, H., Saharinen, P., Zarkada, G., Zheng, W., Robciuc, M.R. & Alitalo, K. 2015, "VEGF-C is required for intestinal lymphatic vessel maintenance and lipid absorption", EMBO molecular medicine, vol. 7, no. 11, pp. 1418-1425.

Nurro, J., Halonen, P.J., Kuivanen, A., Tarkia, M., Saraste, A., Honkonen, K., Lahteenvuo, J., Rissanen, T.T., Knuuti, J. & Yla-Herttuala, S. 2016, "AdVEGF-B186 and AdVEGF-DDeltaNDeltaC induce angiogenesis and increase perfusion in porcine myocardium", Heart (British Cardiac Society), vol. 102, no. 21, pp. 1716-1720.

Ogawa, S., Oku, A., Sawano, A., Yamaguchi, S., Yazaki, Y. & Shibuya, M. 1998, "A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain", The Journal of biological chemistry, vol. 273, no. 47, pp. 31273-31282.

Olofsson, B., Korpelainen, E., Pepper, M.S., Mandriota, S.J., Aase, K., Kumar, V., Gunji, Y., Jeltsch, M.M., Shibuya, M., Alitalo, K. & Eriksson, U. 1998, "Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells", Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 20, pp. 11709-11714.

Olofsson, B., Pajusola, K., Kaipainen, A., von Euler, G., Joukov, V., Saksela, O., Orpana, A., Pettersson, R.F., Alitalo, K. & Eriksson, U. 1996, "Vascular endothelial growth factor B, a novel growth factor for endothelial cells", Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 6, pp. 2576-2581.

Olsson, A.K., Dimberg, A., Kreuger, J. & Claesson-Welsh, L. 2006, "VEGF receptor signalling - in control of vascular function", Nature reviews.Molecular cell biology, vol. 7, no. 5, pp. 359-371.

Oram, J.F. 2003, "HDL apolipoproteins and ABCA1: partners in the removal of excess cellular cholesterol", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 23, no. 5, pp. 720-727.

Orlandini, M., Marconcini, L., Ferruzzi, R. & Oliviero, S. 1996, "Identification of a c-fos-induced gene that is related to the platelet-derived growth factor/vascular endothelial growth factor family", Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 21, pp. 11675-11680.

Orlandini, M., Spreafico, A., Bardelli, M., Rocchigiani, M., Salameh, A., Nucciotti, S., Capperucci, C., Frediani, B. & Oliviero, S. 2006, "Vascular endothelial growth factor-D activates VEGFR-3 expressed in osteoblasts inducing their differentiation", The Journal of biological chemistry, vol. 281, no. 26, pp. 17961-17967.


76

Overton, C.D., Yancey, P.G., Major, A.S., Linton, M.F. & Fazio, S. 2007, "Deletion of macrophage LDL receptor-related protein increases atherogenesis in the mouse", Circulation research, vol. 100, no. 5, pp. 670-677.

Park, J.E., Chen, H.H., Winer, J., Houck, K.A. & Ferrara, N. 1994, "Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR", The Journal of biological chemistry, vol. 269, no. 41, pp. 25646-25654.

Pellet-Many, C., Frankel, P., Jia, H. & Zachary, I. 2008, "Neuropilins: structure, function and role in disease", The Biochemical journal, vol. 411, no. 2, pp. 211-226.

Pervanidou, P., Chouliaras, G., Akalestos, A., Bastaki, D., Apostolakou, F., Papassotiriou, I. & Chrousos, G.P. 2014, "Increased placental growth factor (PlGF) concentrations in children and adolescents with obesity and the metabolic syndrome", Hormones (Athens, Greece), vol. 13, no. 3, pp. 369-374.

Peterfy, M., Ben-Zeev, O., Mao, H.Z., Weissglas-Volkov, D., Aouizerat, B.E., Pullinger, C.R., Frost, P.H., Kane, J.P., Malloy, M.J., Reue, K., Pajukanta, P. & Doolittle, M.H. 2007, "Mutations in LMF1 cause combined lipase deficiency and severe hypertriglyceridemia", Nature genetics, vol. 39, no. 12, pp. 1483-1487.

Phan, B.A., Dayspring, T.D. & Toth, P.P. 2012, "Ezetimibe therapy: mechanism of action and clinical update", Vascular health and risk management, vol. 8, pp. 415-427.

Phillips, M.C. 2014a, "Apolipoprotein E isoforms and lipoprotein metabolism", IUBMB life, vol. 66, no. 9, pp. 616-623.

Phillips, M.C. 2014b, "Molecular mechanisms of cellular cholesterol efflux", The Journal of biological chemistry, vol. 289, no. 35, pp. 24020-24029.

Piepoli, M.F., Hoes, A.W., Agewall, S., Albus, C., Brotons, C., Catapano, A.L., Cooney, M.T., Corra, U., Cosyns, B., Deaton, C., Graham, I., Hall, M.S., Hobbs, F.D.R., Lochen, M.L., Lollgen, H., Marques-Vidal, P., Perk, J., Prescott, E., Redon, J., Richter, D.J., Sattar, N., Smulders, Y., Tiberi, M., van der Worp, H.B., van Dis, I., Verschuren, W.M.M., Binno, S. & ESC Scientific Document Group 2016, "2016 European Guidelines on cardiovascular disease prevention in clinical practice: The Sixth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of 10 societies and by invited experts)Developed with the special contribution of the European Association for Cardiovascular Prevention & Rehabilitation (EACPR)", European heart journal, vol. 37, no. 29, pp. 2315-2381.

Plump, A.S., Smith, J.D., Hayek, T., Aalto-Setala, K., Walsh, A., Verstuyft, J.G., Rubin, E.M. & Breslow, J.L. 1992, "Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells", Cell, vol. 71, no. 2, pp. 343-353.

Powell, L.M., Wallis, S.C., Pease, R.J., Edwards, Y.H., Knott, T.J. & Scott, J. 1987, "A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine", Cell, vol. 50, no. 6, pp. 831-840.

Pownall, H.J., Gillard, B.K. & Gotto, A.M.,Jr 2013, "Setting the course for apoAII: a port in sight?", Clinical lipidology, vol. 8, no. 5, pp. 551-560.

Proctor, S.D., Vine, D.F. & Mamo, J.C. 2002, "Arterial retention of apolipoprotein B(48)- and B(100)-containing lipoproteins in atherogenesis", Current opinion in lipidology, vol. 13, no. 5, pp. 461-470.

Randolph, G.J., Ivanov, S., Zinselmeyer, B.H. & Scallan, J.P. 2017, "The Lymphatic System: Integral Roles in Immunity", Annual Review of Immunology, vol. 35, pp. 31-52.

Randolph, G.J. & Miller, N.E. 2014, "Lymphatic transport of high-density lipoproteins and chylomicrons", The Journal of clinical investigation, vol. 124, no. 3, pp. 929-935.

Rhoades, R. & Pflanzer, R. 2003, Human Physiology, 4th edn, Thomson Learning Inc., Pacific Grove, The United States Of America.

Ribatti, D. 2008, "The discovery of the placental growth factor and its role in angiogenesis: a historical review", Angiogenesis, vol. 11, no. 3, pp. 215-221.

77

Ridgway, N. 2016, "Phospholipid Synthesis in Mammalian Cells" in Biochemistry of Lipids, Lipoproteins and Membranes, eds. N. Ridgway & R. McLeod, 6th edn, Elsevier Science, , pp. 209-236.

Rigotti, A., Trigatti, B.L., Penman, M., Rayburn, H., Herz, J. & Krieger, M. 1997, "A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism", Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 23, pp. 12610-12615.

Rissanen, T.T., Markkanen, J.E., Gruchala, M., Heikura, T., Puranen, A., Kettunen, M.I., Kholova, I., Kauppinen, R.A., Achen, M.G., Stacker, S.A., Alitalo, K. & Yla-Herttuala, S. 2003, "VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses", Circulation research, vol. 92, no. 10, pp. 1098-1106.

Robciuc, M.R., Kivela, R., Williams, I.M., de Boer, J.F., van Dijk, T.H., Elamaa, H., Tigistu-Sahle, F., Molotkov, D., Leppanen, V.M., Kakela, R., Eklund, L., Wasserman, D.H., Groen, A.K. & Alitalo, K. 2016, "VEGFB/VEGFR1-Induced Expansion of Adipose Vasculature Counteracts Obesity and Related Metabolic Complications", Cell metabolism, vol. 23, no. 4, pp. 712-724.

Robinson, C.J. & Stringer, S.E. 2001, "The splice variants of vascular endothelial growth factor (VEGF) and their receptors", Journal of cell science, vol. 114, no. Pt 5, pp. 853-865.

Robinson, J.G., Farnier, M., Krempf, M., Bergeron, J., Luc, G., Averna, M., Stroes, E.S., Langslet, G., Raal, F.J., El Shahawy, M., Koren, M.J., Lepor, N.E., Lorenzato, C., Pordy, R., Chaudhari, U., Kastelein, J.J. & ODYSSEY LONG TERM Investigators 2015, "Efficacy and safety of alirocumab in reducing lipids and cardiovascular events", The New England journal of medicine, vol. 372, no. 16, pp. 1489-1499.

Rodewell, V., Bender, D., Botham, K., Kennelly, P. & Weil, P. 2018, Harper' Illustrated Biochemistry, 31st edn, McGraw-Hill Education.

Rohlmann, A., Gotthardt, M., Hammer, R.E. & Herz, J. 1998, "Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants", The Journal of clinical investigation, vol. 101, no. 3, pp. 689-695.

Ross, R. 1999, "Atherosclerosis--an inflammatory disease", The New England journal of medicine, vol. 340, no. 2, pp. 115-126.

Roth, G.A., Johnson, C., Abajobir, A., Abd-Allah, F., Abera, S.F., Abyu, G., Ahmed, M., Aksut, B., Alam, T., Alam, K., Alla, F., Alvis-Guzman, N., Amrock, S., Ansari, H., Arnlov, J., et al. 2017, "Global, Regional, and National Burden of Cardiovascular Diseases for 10 Causes, 1990 to 2015", Journal of the American College of Cardiology, vol. 70, no. 1, pp. 1-25.

Rudenko, G., Henry, L., Henderson, K., Ichtchenko, K., Brown, M.S., Goldstein, J.L. & Deisenhofer, J. 2002, "Structure of the LDL receptor extracellular domain at endosomal pH", Science (New York, N.Y.), vol. 298, no. 5602, pp. 2353-2358.

Rutanen, J., Rissanen, T.T., Markkanen, J.E., Gruchala, M., Silvennoinen, P., Kivela, A., Hedman, A., Hedman, M., Heikura, T., Orden, M.R., Stacker, S.A., Achen, M.G., Hartikainen, J. & Yla-Herttuala, S. 2004, "Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart", Circulation, vol. 109, no. 8, pp. 1029-1035.

Rutanen, J., Turunen, A.M., Teittinen, M., Rissanen, T.T., Heikura, T., Koponen, J.K., Gruchala, M., Inkala, M., Jauhiainen, S., Hiltunen, M.O., Turunen, M.P., Stacker, S.A., Achen, M.G. & Yla-Herttuala, S. 2005, "Gene transfer using the mature form of VEGF-D reduces neointimal thickening through nitric oxide-dependent mechanism", Gene therapy, vol. 12, no. 12, pp. 980-987.

Rutkowski, J.M., Markhus, C.E., Gyenge, C.C., Alitalo, K., Wiig, H. & Swartz, M.A. 2010, "Dermal collagen and lipid deposition correlate with tissue swelling and hydraulic conductivity in murine primary lymphedema", The American journal of pathology, vol. 176, no. 3, pp. 1122-1129.

Saito, H., Dhanasekaran, P., Nguyen, D., Baldwin, F., Weisgraber, K.H., Wehrli, S., Phillips, M.C. & Lund-Katz, S. 2003, "Characterization of the heparin binding sites in human apolipoprotein E", The Journal of biological chemistry, vol. 278, no. 17, pp. 14782-14787.


78

Sakai, J., Hoshino, A., Takahashi, S., Miura, Y., Ishii, H., Suzuki, H., Kawarabayasi, Y. & Yamamoto, T. 1994, "Structure, chromosome location, and expression of the human very low density lipoprotein receptor gene", The Journal of biological chemistry, vol. 269, no. 3, pp. 2173-2182.

Sakurai, Y., Ohgimoto, K., Kataoka, Y., Yoshida, N. & Shibuya, M. 2005, "Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis in mice", Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 4, pp. 1076-1081.

Sane, A.T., Sinnett, D., Delvin, E., Bendayan, M., Marcil, V., Menard, D., Beaulieu, J.F. & Levy, E. 2006, "Localization and role of NPC1L1 in cholesterol absorption in human intestine", Journal of lipid research, vol. 47, no. 10, pp. 2112-2120.

Sarrazin, S., Lamanna, W.C. & Esko, J.D. 2011, "Heparan sulfate proteoglycans", Cold Spring Harbor perspectives in biology, vol. 3, no. 7, pp. 10.1101/cshperspect.a004952.

Saunders, A.M., Strittmatter, W.J., Schmechel, D., George-Hyslop, P.H., Pericak-Vance, M.A., Joo, S.H., Rosi, B.L., Gusella, J.F., Crapper-MacLachlan, D.R. & Alberts, M.J. 1993, "Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer's disease", Neurology, vol. 43, no. 8, pp. 1467-1472.

Sawano, A., Iwai, S., Sakurai, Y., Ito, M., Shitara, K., Nakahata, T. & Shibuya, M. 2001, "Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans", Blood, vol. 97, no. 3, pp. 785-791.

Saxena, U., Klein, M.G. & Goldberg, I.J. 1991, "Identification and characterization of the endothelial cell surface lipoprotein lipase receptor", The Journal of biological chemistry, vol. 266, no. 26, pp. 17516-17521.

Schmeisser, A., Christoph, M., Augstein, A., Marquetant, R., Kasper, M., Braun-Dullaeus, R.C. & Strasser, R.H. 2006, "Apoptosis of human macrophages by Flt-4 signaling: implications for atherosclerotic plaque pathology", Cardiovascular research, vol. 71, no. 4, pp. 774-784.

Schwarz, E.R., Speakman, M.T., Patterson, M., Hale, S.S., Isner, J.M., Kedes, L.H. & Kloner, R.A. 2000, "Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial infarction model in the rat--angiogenesis and angioma formation", Journal of the American College of Cardiology, vol. 35, no. 5, pp. 1323-1330.

Segrest, J.P., Jones, M.K., De Loof, H. & Dashti, N. 2001, "Structure of apolipoprotein B-100 in low density lipoproteins", Journal of lipid research, vol. 42, no. 9, pp. 1346-1367.

Senger, D.R., Galli, S.J., Dvorak, A.M., Perruzzi, C.A., Harvey, V.S. & Dvorak, H.F. 1983, "Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid", Science (New York, N.Y.), vol. 219, no. 4587, pp. 983-985.

Seyama, K., Kumasaka, T., Souma, S., Sato, T., Kurihara, M., Mitani, K., Tominaga, S. & Fukuchi, Y. 2006, "Vascular endothelial growth factor-D is increased in serum of patients with lymphangioleiomyomatosis", Lymphatic research and biology, vol. 4, no. 3, pp. 143-152.

Shachter, N.S., Ebara, T., Ramakrishnan, R., Steiner, G., Breslow, J.L., Ginsberg, H.N. & Smith, J.D. 1996, "Combined hyperlipidemia in transgenic mice overexpressing human apolipoprotein Cl", The Journal of clinical investigation, vol. 98, no. 3, pp. 846-855.

Shachter, N.S., Hayek, T., Leff, T., Smith, J.D., Rosenberg, D.W., Walsh, A., Ramakrishnan, R., Goldberg, I.J., Ginsberg, H.N. & Breslow, J.L. 1994, "Overexpression of apolipoprotein CII causes hypertriglyceridemia in transgenic mice", The Journal of clinical investigation, vol. 93, no. 4, pp. 1683-1690.

Shalaby, F., Rossant, J., Yamaguchi, T.P., Gertsenstein, M., Wu, X.F., Breitman, M.L. & Schuh, A.C. 1995, "Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice", Nature, vol. 376, no. 6535, pp. 62-66.

Shelness, G.S., Ingram, M.F., Huang, X.F. & DeLozier, J.A. 1999, "Apolipoprotein B in the rough endoplasmic reticulum: translation, translocation and the initiation of lipoprotein assembly", The Journal of nutrition, vol. 129, no. 2S Suppl, pp. 456S-462S.

79

Shiau, Y.F. 1990, "Mechanism of intestinal fatty acid uptake in the rat: the role of an acidic microclimate", The Journal of physiology, vol. 421, pp. 463-474.

Shibuya, M. 2009, "Unique signal transduction of the VEGF family members VEGF-A and VEGF-E", Biochemical Society transactions, vol. 37, no. Pt 6, pp. 1161-1166.

Shibuya, M., Yamaguchi, S., Yamane, A., Ikeda, T., Tojo, A., Matsushime, H. & Sato, M. 1990, "Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family", Oncogene, vol. 5, no. 4, pp. 519-524.

Silha, J.V., Krsek, M., Sucharda, P. & Murphy, L.J. 2005, "Angiogenic factors are elevated in overweight and obese individuals", International journal of obesity (2005), vol. 29, no. 11, pp. 1308-1314.

Skobe, M., Hawighorst, T., Jackson, D.G., Prevo, R., Janes, L., Velasco, P., Riccardi, L., Alitalo, K., Claffey, K. & Detmar, M. 2001, "Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis", Nature medicine, vol. 7, no. 2, pp. 192-198.

Soker, S., Miao, H.Q., Nomi, M., Takashima, S. & Klagsbrun, M. 2002, "VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding", Journal of cellular biochemistry, vol. 85, no. 2, pp. 357-368.

Soker, S., Takashima, S., Miao, H.Q., Neufeld, G. & Klagsbrun, M. 1998, "Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor", Cell, vol. 92, no. 6, pp. 735-745.

Stacker, S.A., Stenvers, K., Caesar, C., Vitali, A., Domagala, T., Nice, E., Roufail, S., Simpson, R.J., Moritz, R., Karpanen, T., Alitalo, K. & Achen, M.G. 1999, "Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers", The Journal of biological chemistry, vol. 274, no. 45, pp. 32127-32136.

Stahl, A., Hirsch, D.J., Gimeno, R.E., Punreddy, S., Ge, P., Watson, N., Patel, S., Kotler, M., Raimondi, A., Tartaglia, L.A. & Lodish, H.F. 1999, "Identification of the major intestinal fatty acid transport protein", Molecular cell, vol. 4, no. 3, pp. 299-308.

Stanford, K.I., Bishop, J.R., Foley, E.M., Gonzales, J.C., Niesman, I.R., Witztum, J.L. & Esko, J.D. 2009, "Syndecan-1 is the primary heparan sulfate proteoglycan mediating hepatic clearance of triglyceride-rich lipoproteins in mice", The Journal of clinical investigation, vol. 119, no. 11, pp. 3236-3245.

Stanton, A.W., Modi, S., Mellor, R.H., Levick, J.R. & Mortimer, P.S. 2009, "Recent advances in breast cancer-related lymphedema of the arm: lymphatic pump failure and predisposing factors", Lymphatic research and biology, vol. 7, no. 1, pp. 29-45.

Stary, H.C., Chandler, A.B., Dinsmore, R.E., Fuster, V., Glagov, S., Insull, W.,Jr, Rosenfeld, M.E., Schwartz, C.J., Wagner, W.D. & Wissler, R.W. 1995, "A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association", Circulation, vol. 92, no. 5, pp. 1355-1374.

Stathopoulou, M.G., Bonnefond, A., Ndiaye, N.C., Azimi-Nezhad, M., El Shamieh, S., Saleh, A., Rancier, M., Siest, G., Lamont, J., Fitzgerald, P. & Visvikis-Siest, S. 2013, "A common variant highly associated with plasma VEGFA levels also contributes to the variation of both LDL-C and HDL-C", Journal of lipid research, vol. 54, no. 2, pp. 535-541.

Steinberg, D., Parthasarathy, S., Carew, T.E., Khoo, J.C. & Witztum, J.L. 1989, "Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity", The New England journal of medicine, vol. 320, no. 14, pp. 915-924.

Steinmetz, A., Barbaras, R., Ghalim, N., Clavey, V., Fruchart, J.C. & Ailhaud, G. 1990, "Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells", The Journal of biological chemistry, vol. 265, no. 14, pp. 7859-7863.

Steinmetz, A. & Utermann, G. 1985, "Activation of lecithin: cholesterol acyltransferase by human apolipoprotein A-IV", The Journal of biological chemistry, vol. 260, no. 4, pp. 2258-2264.


80

Stolt, P.C. & Bock, H.H. 2006, "Modulation of lipoprotein receptor functions by intracellular adaptor proteins", Cellular signalling, vol. 18, no. 10, pp. 1560-1571.

Su, H., Lu, R. & Kan, Y.W. 2000, "Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart", Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 25, pp. 13801-13806.

Sukonina, V., Lookene, A., Olivecrona, T. & Olivecrona, G. 2006, "Angiopoietin-like protein 4 converts lipoprotein lipase to inactive monomers and modulates lipase activity in adipose tissue", Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 46, pp. 17450-17455.

Sun, C.Y., Lee, C.C., Hsieh, M.F., Chen, C.H. & Chou, K.M. 2014, "Clinical association of circulating VEGF-B levels with hyperlipidemia and target organ damage in type 2 diabetic patients", Journal of Biological Regulators and Homeostatic Agents, vol. 28, no. 2, pp. 225-236.

Sun, K., Wernstedt Asterholm, I., Kusminski, C.M., Bueno, A.C., Wang, Z.V., Pollard, J.W., Brekken, R.A. & Scherer, P.E. 2012, "Dichotomous effects of VEGF-A on adipose tissue dysfunction", Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 15, pp. 5874-5879.

Sun, Q.N., Wang, Y.F. & Guo, Z.K. 2012, "Reconstitution of myocardial lymphatic vessels after acute infarction of rat heart", Lymphology, vol. 45, no. 2, pp. 80-86.

Sundaram, M. & Yao, Z. 2010, "Recent progress in understanding protein and lipid factors affecting hepatic VLDL assembly and secretion", Nutrition & metabolism, vol. 7, pp. 35-7075-7-35.

Sung, H.K., Doh, K.O., Son, J.E., Park, J.G., Bae, Y., Choi, S., Nelson, S.M., Cowling, R., Nagy, K., Michael, I.P., Koh, G.Y., Adamson, S.L., Pawson, T. & Nagy, A. 2013, "Adipose vascular endothelial growth factor regulates metabolic homeostasis through angiogenesis", Cell metabolism, vol. 17, no. 1, pp. 61-72.

Swartz, M.A. 2001, "The physiology of the lymphatic system", Advanced Drug Delivery Reviews, vol. 50, no. 1-2, pp. 3-20.

Szekanecz, Z. & Koch, A.E. 2007, "Mechanisms of Disease: angiogenesis in inflammatory diseases", Nature clinical practice.Rheumatology, vol. 3, no. 11, pp. 635-643.

Tabas, I., Williams, K.J. & Boren, J. 2007, "Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications", Circulation, vol. 116, no. 16, pp. 1832-1844.

Takahashi, S., Kawarabayasi, Y., Nakai, T., Sakai, J. & Yamamoto, T. 1992, "Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity", Proceedings of the National Academy of Sciences of the United States of America, vol. 89, no. 19, pp. 9252-9256.

Takahashi, S., Sakai, J., Fujino, T., Hattori, H., Zenimaru, Y., Suzuki, J., Miyamori, I. & Yamamoto, T.T. 2004, "The very low-density lipoprotein (VLDL) receptor: characterization and functions as a peripheral lipoprotein receptor", Journal of Atherosclerosis and Thrombosis, vol. 11, no. 4, pp. 200-208.

Talman, V. & Ruskoaho, H. 2016, "Cardiac fibrosis in myocardial infarction-from repair and remodeling to regeneration", Cell and tissue research, vol. 365, no. 3, pp. 563-581.

Tammela, T., Zarkada, G., Wallgard, E., Murtomaki, A., Suchting, S., Wirzenius, M., Waltari, M., Hellstrom, M., Schomber, T., Peltonen, R., Freitas, C., Duarte, A., Isoniemi, H., Laakkonen, P., Christofori, G., Yla-Herttuala, S., Shibuya, M., Pytowski, B., Eichmann, A., Betsholtz, C. & Alitalo, K. 2008, "Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation", Nature, .

Tan, J.T., Ng, M.K. & Bursill, C.A. 2015, "The role of high-density lipoproteins in the regulation of angiogenesis", Cardiovascular research, vol. 106, no. 2, pp. 184-193.

Taveira-DaSilva, A.M. & Moss, J. 2015, "Clinical features, epidemiology, and therapy of lymphangioleiomyomatosis", Clinical epidemiology, vol. 7, pp. 249-257.

81

Teng, B., Burant, C.F. & Davidson, N.O. 1993, "Molecular cloning of an apolipoprotein B messenger RNA editing protein", Science (New York, N.Y.), vol. 260, no. 5115, pp. 1816-1819.

Terman, B.I., Carrion, M.E., Kovacs, E., Rasmussen, B.A., Eddy, R.L. & Shows, T.B. 1991, "Identification of a new endothelial cell growth factor receptor tyrosine kinase", Oncogene, vol. 6, no. 9, pp. 1677-1683.

Terman, B.I., Dougher-Vermazen, M., Carrion, M.E., Dimitrov, D., Armellino, D.C., Gospodarowicz, D. & Bohlen, P. 1992, "Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor", Biochemical and biophysical research communications, vol. 187, no. 3, pp. 1579-1586.

Thompson, G. 1989, A Handbook of Hyperlipidaemia, Current Science Ltd, London. Thygesen, K., Alpert, J.S., Jaffe, A.S., Simoons, M.L., Chaitman, B.R., White, H.D., Writing Group on

the Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction & ESC Committee for Practice Guidelines (CPG) 2012, "Third universal definition of myocardial infarction", European heart journal, vol. 33, no. 20, pp. 2551-2567.

Tiwari, S. & Siddiqi, S.A. 2012, "Intracellular trafficking and secretion of VLDL", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 32, no. 5, pp. 1079-1086.

Toivanen, P.I., Nieminen, T., Laakkonen, J.P., Heikura, T., Kaikkonen, M.U. & Yla-Herttuala, S. 2017, "Snake venom VEGF Vammin induces a highly efficient angiogenic response in skeletal muscle via VEGFR-2/NRP specific signaling", Scientific reports, vol. 7, no. 1, pp. 5525-017-05876-y.

Townsend, N., Wilson, L., Bhatnagar, P., Wickramasinghe, K., Rayner, M. & Nichols, M. 2016, "Cardiovascular disease in Europe: epidemiological update 2016", European heart journal, vol. 37, no. 42, pp. 3232-3245.

Trape, J., Morales, C., Molina, R., Filella, X., Marcos, J.M., Salinas, R. & Franquesa, J. 2006, "Vascular endothelial growth factor serum concentrations in hypercholesterolemic patients", Scandinavian Journal of Clinical and Laboratory Investigation, vol. 66, no. 3, pp. 261-267.

Van Dyck, F., Braem, C.V., Chen, Z., Declercq, J., Deckers, R., Kim, B.M., Ito, S., Wu, M.K., Cohen, D.E., Dewerchin, M., Derua, R., Waelkens, E., Fiette, L., Roebroek, A., Schuit, F., Van de Ven, W.J. & Shivdasani, R.A. 2007, "Loss of the PlagL2 transcription factor affects lacteal uptake of chylomicrons", Cell metabolism, vol. 6, no. 5, pp. 406-413.

Van Eck, M., Hoekstra, M., Out, R., Bos, I.S., Kruijt, J.K., Hildebrand, R.B. & Van Berkel, T.J. 2008, "Scavenger receptor BI facilitates the metabolism of VLDL lipoproteins in vivo", Journal of lipid research, vol. 49, no. 1, pp. 136-146.

Van Eck, M., Twisk, J., Hoekstra, M., Van Rij, B.T., Van der Lans, C.A., Bos, I.S., Kruijt, J.K., Kuipers, F. & Van Berkel, T.J. 2003, "Differential effects of scavenger receptor BI deficiency on lipid metabolism in cells of the arterial wall and in the liver", The Journal of biological chemistry, vol. 278, no. 26, pp. 23699-23705.

Varbo, A., Benn, M., Tybjaerg-Hansen, A., Jorgensen, A.B., Frikke-Schmidt, R. & Nordestgaard, B.G. 2013, "Remnant cholesterol as a causal risk factor for ischemic heart disease", Journal of the American College of Cardiology, vol. 61, no. 4, pp. 427-436.

Vasudev, N.S. & Reynolds, A.R. 2014, "Anti-angiogenic therapy for cancer: current progress, unresolved questions and future directions", Angiogenesis, vol. 17, no. 3, pp. 471-494.

Velagapudi, S., Yalcinkaya, M., Piemontese, A., Meier, R., Norrelykke, S.F., Perisa, D., Rzepiela, A., Stebler, M., Stoma, S., Zanoni, P., Rohrer, L. & von Eckardstein, A. 2017, "VEGF-A Regulates Cellular Localization of SR-BI as Well as Transendothelial Transport of HDL but Not LDL", Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 37, no. 5, pp. 794-803.

Veniant, M.M., Zlot, C.H., Walzem, R.L., Pierotti, V., Driscoll, R., Dichek, D., Herz, J. & Young, S.G. 1998, "Lipoprotein clearance mechanisms in LDL receptor-deficient "Apo-B48-only" and "Apo-B100-only" mice", The Journal of clinical investigation, vol. 102, no. 8, pp. 1559-1568.

Vergeer, M., Korporaal, S.J., Franssen, R., Meurs, I., Out, R., Hovingh, G.K., Hoekstra, M., Sierts, J.A., Dallinga-Thie, G.M., Motazacker, M.M., Holleboom, A.G., Van Berkel, T.J., Kastelein, J.J., Van


82

Eck, M. & Kuivenhoven, J.A. 2011, "Genetic variant of the scavenger receptor BI in humans", The New England journal of medicine, vol. 364, no. 2, pp. 136-145.

Vieira, J.M., Norman, S., Villa Del Campo, C., Cahill, T.J., Barnette, D.N., Gunadasa-Rohling, M., Johnson, L.A., Greaves, D.R., Carr, C.A., Jackson, D.G. & Riley, P.R. 2018, "The cardiac lymphatic system stimulates resolution of inflammation following myocardial infarction", The Journal of clinical investigation, vol. 128, no. 8, pp. 3402-3412.

Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M. & Heldin, C.H. 1994, "Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor", The Journal of biological chemistry, vol. 269, no. 43, pp. 26988-26995.

Wang, N., Lan, D., Chen, W., Matsuura, F. & Tall, A.R. 2004, "ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins", Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 26, pp. 9774-9779.

Weis, S.M. & Cheresh, D.A. 2005, "Pathophysiological consequences of VEGF-induced vascular permeability", Nature, vol. 437, no. 7058, pp. 497-504.

Weisgraber, K.H. & Rall, S.C.,Jr 1987, "Human apolipoprotein B-100 heparin-binding sites", The Journal of biological chemistry, vol. 262, no. 23, pp. 11097-11103.

Weitman, E.S., Aschen, S.Z., Farias-Eisner, G., Albano, N., Cuzzone, D.A., Ghanta, S., Zampell, J.C., Thorek, D. & Mehrara, B.J. 2013, "Obesity impairs lymphatic fluid transport and dendritic cell migration to lymph nodes", PloS one, vol. 8, no. 8, pp. e70703.

Wild, J.R., Staton, C.A., Chapple, K. & Corfe, B.M. 2012, "Neuropilins: expression and roles in the epithelium", International journal of experimental pathology, vol. 93, no. 2, pp. 81-103.

Willnow, T.E., Sheng, Z., Ishibashi, S. & Herz, J. 1994, "Inhibition of hepatic chylomicron remnant uptake by gene transfer of a receptor antagonist", Science (New York, N.Y.), vol. 264, no. 5164, pp. 1471-1474.

Xu, S., Laccotripe, M., Huang, X., Rigotti, A., Zannis, V.I. & Krieger, M. 1997, "Apolipoproteins of HDL can directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediates selective lipid uptake", Journal of lipid research, vol. 38, no. 7, pp. 1289-1298.

Xu, X., Lin, H., Lv, H., Zhang, M. & Zhang, Y. 2007, "Adventitial lymphatic vessels -- an important role in atherosclerosis", Medical hypotheses, vol. 69, no. 6, pp. 1238-1241.

Yamada, Y., Nezu, J., Shimane, M. & Hirata, Y. 1997, "Molecular cloning of a novel vascular endothelial growth factor, VEGF-D", Genomics, vol. 42, no. 3, pp. 483-488.

Yamamoto, T., Davis, C.G., Brown, M.S., Schneider, W.J., Casey, M.L., Goldstein, J.L. & Russell, D.W. 1984, "The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA", Cell, vol. 39, no. 1, pp. 27-38.

Yamamoto, T., Takahashi, S., Sakai, J. & Kawarabayasi, Y. 1993, "The very low density lipoprotein receptor A second lipoprotein receptor that may mediate uptake of fatty acids into muscle and fat cells", Trends in cardiovascular medicine, vol. 3, no. 4, pp. 144-148.

Yamazaki, Y., Matsunaga, Y., Tokunaga, Y., Obayashi, S., Saito, M. & Morita, T. 2009, "Snake venom Vascular Endothelial Growth Factors (VEGF-Fs) exclusively vary their structures and functions among species", The Journal of biological chemistry, vol. 284, no. 15, pp. 9885-9891.

Yamazaki, Y., Takani, K., Atoda, H. & Morita, T. 2003, "Snake venom vascular endothelial growth factors (VEGFs) exhibit potent activity through their specific recognition of KDR (VEGF receptor 2)", The Journal of biological chemistry, vol. 278, no. 52, pp. 51985-51988.

Yancey, P.G., de la Llera-Moya, M., Swarnakar, S., Monzo, P., Klein, S.M., Connelly, M.A., Johnson, W.J., Williams, D.L. & Rothblat, G.H. 2000, "High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI", The Journal of biological chemistry, vol. 275, no. 47, pp. 36596-36604.

83

Yancey, P.G., Jerome, W.G., Yu, H., Griffin, E.E., Cox, B.E., Babaev, V.R., Fazio, S. & Linton, M.F. 2007, "Severely altered cholesterol homeostasis in macrophages lacking apoE and SR-BI", Journal of lipid research, vol. 48, no. 5, pp. 1140-1149.

Yla-Herttuala, E., Laidinen, S., Laakso, H. & Liimatainen, T. 2018, "Quantification of myocardial infarct area based on TRAFFn relaxation time maps - comparison with cardiovascular magnetic resonance late gadolinium enhancement, T1rho and T2 in vivo", Journal of cardiovascular magnetic resonance : official journal of the Society for Cardiovascular Magnetic Resonance, vol. 20, no. 1, pp. 34-018-0463-x.

Yla-Herttuala, S., Bridges, C., Katz, M.G. & Korpisalo, P. 2017, "Angiogenic gene therapy in cardiovascular diseases: dream or vision?", European heart journal, vol. 38, no. 18, pp. 1365-1371.

Yla-Herttuala, S., Nikkari, T., Hirvonen, J., Laaksonen, H., Mottonen, M., Pesonen, E., Raekallio, J. & Akerblom, H.K. 1986, "Biochemical composition of coronary arteries in Finnish children", Arteriosclerosis (Dallas, Tex.), vol. 6, no. 2, pp. 230-236.

Yla-Herttuala, S., Palinski, W., Rosenfeld, M.E., Parthasarathy, S., Carew, T.E., Butler, S., Witztum, J.L. & Steinberg, D. 1989, "Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man", The Journal of clinical investigation, vol. 84, no. 4, pp. 1086-1095.

Yla-Herttuala, S., Rissanen, T.T., Vajanto, I. & Hartikainen, J. 2007, "Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine", Journal of the American College of Cardiology, vol. 49, no. 10, pp. 1015-1026.

Yla-Herttuala, S., Solakivi, T., Hirvonen, J., Laaksonen, H., Mottonen, M., Pesonen, E., Raekallio, J., Akerblom, H.K. & Nikkari, T. 1987, "Glycosaminoglycans and apolipoproteins B and A-I in human aortas. Chemical and immunological analysis of lesion-free aortas from children and adults", Arteriosclerosis (Dallas, Tex.), vol. 7, no. 4, pp. 333-340.

Young, S.G. 1990, "Recent progress in understanding apolipoprotein B", Circulation, vol. 82, no. 5, pp. 1574-1594.

Yuan, L., Moyon, D., Pardanaud, L., Breant, C., Karkkainen, M.J., Alitalo, K. & Eichmann, A. 2002, "Abnormal lymphatic vessel development in neuropilin 2 mutant mice", Development (Cambridge, England), vol. 129, no. 20, pp. 4797-4806.

Zachary, I. & Morgan, R.D. 2011, "Therapeutic angiogenesis for cardiovascular disease: biological context, challenges, prospects", Heart (British Cardiac Society), vol. 97, no. 3, pp. 181-189.

Zafar, M.I., Zheng, J., Kong, W., Ye, X., Gou, L., Regmi, A. & Chen, L.L. 2017, "The role of vascular endothelial growth factor-B in metabolic homoeostasis: current evidence", Bioscience reports, vol. 37, no. 4, pp. 10.1042/BSR20171089. Print 2017 Aug 31.

Zanoni, P., Khetarpal, S.A., Larach, D.B., Hancock-Cerutti, W.F., Millar, J.S., Cuchel, M., DerOhannessian, S., Kontush, A., Surendran, P., Saleheen, D., Trompet, S., Jukema, J.W., De Craen, A., Deloukas, P., Sattar, N., et al., CHD Exome+ Consortium, CARDIoGRAM Exome Consortium & Global Lipids Genetics Consortium 2016, "Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease", Science (New York, N.Y.), vol. 351, no. 6278, pp. 1166-1171.

Zelcer, N., Hong, C., Boyadjian, R. & Tontonoz, P. 2009, "LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor", Science (New York, N.Y.), vol. 325, no. 5936, pp. 100-104.

Zhang, F., Tang, Z., Hou, X., Lennartsson, J., Li, Y., Koch, A.W., Scotney, P., Lee, C., Arjunan, P., Dong, L., Kumar, A., Rissanen, T.T., Wang, B., Nagai, N., Fons, P., Fariss, R., Zhang, Y., Wawrousek, E., Tansey, G., Raber, J., Fong, G.H., Ding, H., Greenberg, D.A., Becker, K.G., Herbert, J.M., Nash, A., Yla-Herttuala, S., Cao, Y., Watts, R.J. & Li, X. 2009, "VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis", Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 15, pp. 6152-6157.


84

Zhang, F., Zarkada, G., Han, J., Li, J., Dubrac, A., Ola, R., Genet, G., Boye, K., Michon, P., Kunzel, S.E., Camporez, J.P., Singh, A.K., Fong, G.H., Simons, M., Tso, P., Fernandez-Hernando, C., Shulman, G.I., Sessa, W.C. & Eichmann, A. 2018, "Lacteal junction zippering protects against diet-induced obesity", Science (New York, N.Y.), vol. 361, no. 6402, pp. 599-603.

Zhang, K., Zhang, L. & Weinreb, R.N. 2012, "Ophthalmic drug discovery: novel targets and mechanisms for retinal diseases and glaucoma", Nature reviews.Drug discovery, vol. 11, no. 7, pp. 541-559.

Zhang, L., Zhou, F., Han, W., Shen, B., Luo, J., Shibuya, M. & He, Y. 2010, "VEGFR-3 ligand-binding and kinase activity are required for lymphangiogenesis but not for angiogenesis", Cell research, vol. 20, no. 12, pp. 1319-1331.

Zhang, S.H., Reddick, R.L., Burkey, B. & Maeda, N. 1994, "Diet-induced atherosclerosis in mice heterozygous and homozygous for apolipoprotein E gene disruption", The Journal of clinical investigation, vol. 94, no. 3, pp. 937-945.

Zhang, S.H., Reddick, R.L., Piedrahita, J.A. & Maeda, N. 1992, "Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E", Science (New York, N.Y.), vol. 258, no. 5081, pp. 468-471.

Zhou, M., Fisher, E.A. & Ginsberg, H.N. 1998, "Regulated Co-translational ubiquitination of apolipoprotein B100. A new paradigm for proteasomal degradation of a secretory protein", The Journal of biological chemistry, vol. 273, no. 38, pp. 24649-24653.

Ziche, M., Maglione, D., Ribatti, D., Morbidelli, L., Lago, C.T., Battisti, M., Paoletti, I., Barra, A., Tucci, M., Parise, G., Vincenti, V., Granger, H.J., Viglietto, G. & Persico, M.G. 1997, "Placenta growth factor-1 is chemotactic, mitogenic, and angiogenic", Laboratory investigation; a journal of technical methods and pathology, vol. 76, no. 4, pp. 517-531.

APPENDIX

ORIGINAL PUBLICATIONS (I-III)


DIS

SE

RT

AT

ION

S | T

AIN

A V

UO

RIO

| VA

SC

UL

AR

EN

DO

TH

EL

IAL

GR

OW

TH

FA

CT

OR

RE

CE

PT

OR

3 AN

D IT

S... | N

o 497

uef.fi



ISBN 978-952-61-2999-0ISSN 1798-5706



TAINA VUORIO

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

As the burden of cardiovascular diseases

remains substantially high, novel regulators of lipid metabolism and disease progression are anticipated. Lymphatic system regulates

tissue fluid homeostasis, lipid absorption and immune reactions, thereby modulating

many processes involved in the development of cardiovascular diseases. In this thesis, the function of lymphatic growth factor receptor VEGFR3 in lipoprotein metabolism as well as in atherosclerosis and myocardial infarction

was elucidated.

TAINA VUORIO

30984656_UEF_Vaitoskirja_NO_497_Taina_Vuorio_Terveystiede_kansi_19_01_02.indd 1 2.1.2019 9.03.40

dissertations in health sciences - uefmodified structure of the infarcted area in svegfr3 mice,...

Documents