MicroRNAs in endothelial aging and atherosclerosis

(1)

DISSERTATIONS | SUVI KUOSMANEN | MICRORNAS IN ENDOTHELIAL AGING AND ATHEROSCLEROSIS | No 432

uef.fi

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences

ISBN 978-952-61-2374-5 ISSN 1798-5706

Dissertations in Health Sciences

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

SUVI KUOSMANEN

MICRORNAS IN ENDOTHELIAL AGING AND ATHEROSCLEROSIS

Atherosclerosis is a critical determinant of human lifespan and the main cause of morbidity and mortality worldwide. The hope of

successful future therapies lies in defining the specific molecular mechanisms that precede symptomatic disease onset. This thesis provides

important information about microRNA biology in aging and atherosclerosis.

Furthermore, it reveals an essential role for transcription factor NRF2 in the endothelial response to atherosclerotic oxidised lipids.

SUVI KUOSMANEN

(2)

(3)

MicroRNAs in Endothelial Aging and

Atherosclerosis

(4)

(5)

(6)

(7)

SUVI KUOSMANEN

MicroRNAs in Endothelial Aging and Atherosclerosis

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Snellmania Auditorium SN201, Kuopio,

on Friday, October 20^th 2017, at 12 noon

Publications of the University of Eastern Finland Dissertations in Health Sciences

Number 432

A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

University of Eastern Finland Kuopio

2017

(8)

Series Editors:

Professor Tomi Laitinen, M.D., Ph.D.

Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences

Professor Hannele Turunen, Ph.D.

Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D.

Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences

Associate Professor (Tenure Track) Tarja Malm, Ph.D.

A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

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

Faculty of Health Sciences Distributor:

University of Eastern Finland Kuopio Campus Library

P.O.Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto ISBN (print): 978-952-61-2374-5

ISBN (pdf): 978-952-61-2568-8 ISSN (print): 1798-5706

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

(9)

III

Author’s address: A. I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

University of Eastern Finland P.O. Box 1627

70211 KUOPIO FINLAND

Supervisors: Professor Anna-Liisa Levonen, M.D., Ph.D.

A. I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences

University of Eastern Finland KUOPIO

FINLAND

Adjunct Professor Sami Heikkinen, Ph.D.

Institute of Biomedicine, School of Medicine Faculty of Health Sciences

University of Eastern Finland KUOPIO

FINLAND

Adjunct Professor Merja Heinäniemi, Ph.D.

Institute of Biomedicine, School of Medicine University of Eastern Finland

KUOPIO FINLAND

Reviewers: Professor Vesa Olkkonen, Ph.D.

Minerva Institute for Medical Research Tukholmankatu 8

HELSINKI FINLAND

Associate Professor Noora Kotaja, Ph.D.

Institute of Biomedicine University of Turku TURKU

FINLAND

Opponent: Professor Reinier Boon, Ph.D.

Institute for Cardiovascular Regeneration University of Frankfurt

FRANKFURT GERMANY

(10)

(11)

V

Kuosmanen, Suvi

MicroRNAs in Endothelial Aging and Atherosclerosis University of Eastern Finland, Faculty of Health Sciences

Publications of the University of Eastern Finland. Dissertations in Health Sciences Number 432. 2017. 69 p.

ISBN (print): 978-952-61-2374-5 ISBN (pdf): 978-952-61-2568-8 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

ABSTRACT

Cardiovascular diseases (CVD) are the most common cause of morbidity and mortality worldwide. The most prevalent underlying disorder for CVD is atherosclerosis.

MicroRNAs (miRNAs) are potent regulators of cellular functions and are involved in all stages of atherogenesis. However, many aspects of their regulation and function are poorly described.

One of the earliest signs of atherosclerosis is endothelial dysfunction. Endothelium lines the inner surface of the heart and vasculature and its function is directly affected by the atherosclerotic risk factors, such as hyperlipidemia and aging. These factors expose endothelial cells to excess oxidative stress, which promotes endothelial dysfunction, barrier disruption and accumulation of lipids in the vascular wall. NRF2 is a transcription factor that orchestrates the antioxidant defence against oxidative species and is an important regulator of endothelial function. The aim of this thesis was to elucidate the function of miRNAs in atherosclerosis and endothelial aging with a special emphasis on NRF2- regulated miRNAs in human endothelial cells.

Pericardial fluid is the fluid within the pericardial space that lies between the fibrous and serous pericardial layers. In this study, it was shown for the first time that pericardial fluid contains miRNAs, especially those that are abundant in endothelial cells. Although cardiovascular disease- or stage-specific miRNAs could not be extracted from the profiling data obtained from heart failure patients, this data did contain several previously suggested markers for the diseases, which was concluded to result from an active and selective miRNA secretion from the cardiovascular cells.

In endothelial cells, the changes in miRNA expression between tissue-derived cells and cells that were cultured until senescence were characterized for the first time. In addition to alterations in the expression of mechano-sensitive and aging-related miRNAs, changes were observed in those associated with the transition of endothelial cells to mesenchymal phenotype suggesting a phenotype change in the aging endothelial cells.

For genome-wide NRF2 binding site mapping, a novel NRF2 binding model was created based on molecular modelling and protein-binding microarrays. Unique microarrays were designed and built to define the limits of the NRF2 binding site recognition, and the information was utilized to guide the binding prediction. The binding model was applied to finding novel atherosclerosis- and aging-associated NRF2-regulated miRNAs in endothelial cells and to further decipher their novel cellular functions.

Together, these studies provided important information about miRNA biology in aging endothelial cells and revealed an important role for NRF2 and NRF2-regulated miRNAs in endothelial response to atherosclerotic oxidised lipids.

National Library of Medicine Classification: QS 532.5.E7, QU 58.7, WG 505, WG 550

Medical Subject Headings: MicroRNAs; Atherosclerosis; Endothelium, Vascular; Endothelial Cells; Aging;

Pericardial Fluid; NF-E2-Related Factor 2; Oxidative Stress; Lipids; Phospholipids; Gene Expression; Binding Sites; Microarray Analysis

(12)

(13)

VII

Kuosmanen, Suvi

MicroRNAs in Endothelial Aging and Atherosclerosis Itä-Suomen yliopisto, terveystieteiden tiedekunta

Itä-Suomen yliopiston julkaisuja. Terveystieteiden tiedekunnan väitöskirjat Numero 432. 2017. 69 s.

ISBN (print): 978-952-61-2374-5 ISBN (pdf): 978-952-61-2568-8 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706

TIIVISTELMÄ

Sydän- ja verisuonitaudit ovat maailmanlaajuisesti yleisin kuolinsyy ja ateroskleroosi niiden yleisin taustasyy. Verisuonten sisäpintaa peittävän endoteelin vanhenemisella on suuri merkitys ateroskleroosin kehittymisessä. mikro-RNA:t (miRNA:t) taas ovat keskeisiä tekijöitä niin solujen vanhenemisessa kuin ateroskleroosissakin, mutta niiden toimintaa tunnetaan heikosti. Yksi ateroskleroosin varhaisimmista vaiheista on suonen endoteelin epänormaali toiminta, mikä lisää endoteelin läpäisevyyttä ja mahdollistaa rasvajuosteen kertymisen suonen seinämään. Ateroskleroosin riskitekijät, kuten ikääntyminen ja veren kohonnut rasva-ainepitoisuus, lisäävät hapen reaktiivisten metaboliittien määrää ja niiden endoteelille aiheuttamaa kuormitusta. Hapetusstressi aktivoi NRF2-säätelytekijää, joka säätelee solujen antioksidanttipuolustusta hapen reaktiivisia yhdisteitä vastaan ja on keskeinen tekijä verisuonten terveydelle. Tämän väitöskirjatutkimuksen tavoitteena oli tutkia ateroskleroosiin ja endoteelisolujen vanhenemiseen liittyvien miRNA:iden toimintaa erityisesti NRF2:n säätelemien miRNA:iden osalta.

Tässä väitöskirjatutkimuksessa sydäntä ympäröivän perikardiumnesteen osoitettiin ensimmäistä kertaa sisältävän miRNA:ita. Sydän- ja verisuonitautipotilaiden näytteistä mitattiin merkittäviä määriä endoteelisoluissa esiintyviä miRNA:ita, mutta myös mahdollisia sydän- ja verisuonitautien diagnostisia miRNA:ita. Varsinaisia tautien tai taudin etenemisasteiden välisiä eroja aineistosta ei pystytty havaitsemaan. Lisäksi tutkimuksessa selviteltiin ensimmäistä kertaa endoteelisolujen miRNA-muutoksia solujen vanhenemisen eri vaiheissa suoraan kudoksesta eristetyistä soluista kasvukaarensa loppupuolella oleviin soluihin saakka. Suurimmat muutokset havaittiin solujen sopeutuessa soluviljelyolosuhteisiin, mutta solujen miRNA-profiileissa havaitiin muutoksia myös solujen vanhetessa. Vanhenemisen havaittiin aiheuttavan soluissa muutoksia, jotka viittasivat solujen fenotyypin muutokseen.

NRF2:n genominlaajuisten säätelykohtien löytämiseksi säätelytekijälle rakennettiin sitoutumismalli, joka pohjautui molekyylimallitukseen ja erityisillä tutkimusta varten kehitetyillä proteiinimikrosiruilla kerättyyn dataan. Luodun sitoutumismallin avulla identifioitiin NRF2-säätyviä miRNA:ita, jotka liittyvät ateroskleroosiin ja endoteelisolujen vanhenemiseen, ja selvitettiin niiden toimintaa ihmisen endoteelisoluissa. Tutkimuksessa tuotettiin uutta mekanistista tietoa ateroskleroosia aiheuttavien hapettuneiden rasvojen vaikutuksista endoteelisoluissa ja todettiin NRF2:n toimivan kytkimenä, joka säätelee endoteelisolujen metabolista aktiivisuutta yhdessä säätelemiensä miRNA:iden kanssa.

Tutkimuksen tulokset osoittavat, että NRF2 on keskeinen tekijä endoteelisolujen toiminnan säätelyssä ja että sen säätelemien miRNA:iden toimintaa karakterisoimalla voidaan saada uutta tietoa ateroskleroosin ja ikääntymisen solukohtaisista mekanismeista.

Luokitus: QS 532.5.E7, QU 58.7, WG 505, WG 550

Yleinen Suomalainen asiasanasto: mikro-RNA; ateroskleroosi; verisuonet; endoteeli; endoteelisolut;

ikääntyminen; vanheneminen; transkriptiotekijät; oksidatiivinen stressi; hapettuminen; lipidit;

geeniekspressio

(14)

(15)

IX

Acknowledgements

The work presented in this thesis was carried out during the years 2012-2017 in the group of Anna-Liisa Levonen in the A.I. Virtanen Institute for Molecular Sciences, Faculty of Health Sciences at the University of Eastern Finland, Kuopio. I am sincerely thankful to all the people who have helped me along the way in so many ways. I could not have done this without you.

My three supervisors Professor Anna-Liisa Levonen, Adjunct Professor Sami Heikkinen and Adjunct Professor Merja Heinäniemi are warmly acknowledged for their valuable guidance and support during the thesis work. I am deeply grateful to my principal supervisor, Anna-Liisa, for giving me the opportunity to work in her excellent research group. I owe deep gratitude also to Sami and Merja whose enthusiastic and inspiring attitude towards scientific problem solving is not only admirable, but has also helped me considerably over the years.

I am sincerely thankful to the official reviewers of this thesis, Professor Vesa Olkkonen and Associate Professor Noora Kotaja, for making the process so uncomplicated. The kind comments I received were much appreciated. I would also like to express my sincere thanks to Nihay Laham Karam for kindly performing the linguistic revision of the thesis and to Professor Reinier Boon for agreeing to be the official opponent at the public defence.

My sincere thanks go also to the talented and skilful co-authors of the original articles and to the wonderful present and former members of the Levonen group. Special thanks go to Virve Sihvola, Emilia Kansanen and Anna-Kaisa Ruotsalainen for their support and understanding. I would also like to express my gratitude to our excellent technician Arja Korhonen. Sometimes, she just works miracles. Warm thanks also to the ever-efficient and helpful Helena Pernu and to Assistant Professor Aikseng Ooi for the possibility to visit his laboratory in the Department of Pharmacology and Toxicology, University of Arizona.

Combining family, friends and “normal life” to science is not easy, but thankfully I know people who make it possible. To my lovely friends in science, Sari Viitala, Sami Poutiainen, Sari Toropainen & Tiia Turunen, thank you for making the dark days brighter, it truly is a gift. To my dear childhood friends, Sanna, Saija, Laura, Outi and Riina, thank you for all the years of love, support and sympathy. The story continues every time we meet. I owe my deep, loving gratitude to my parents, Pirjo and Raimo, to my godmother and –father, Marja and Eino, and to my siblings, Jari-Matti and Sari, for raising me to be the person I am today.

Special thanks go to Emilia and Asser for the everyday mental support they provided during the critical phase of the thesis work. Finally and most importantly, my loving thanks go to Johannes and our two beautiful, amazing daughters, Tiitu and Liinu. I am truly grateful to have you in my life.

Lapinlahti, September 7^th 2017

Suvi Kuosmanen

This study was supported by the much appreciated grants from the Aarne and Aili Turunen Foundation, the Aarne Koskelo Foundation, the Aleksanteri Mikkonen Foundation, the Antti and Tyyne Soininen Foundation, the Doctoral Program in Molecular Medicine, the Emil Aaltonen Foundation, the Finnish Cultural Foundation, North Savo Regional Fund, the Finnish Foundation for Cardiovascular Research, the Ida Montin Foundation, the Instrufoundation, the Kuopio University Foundation, the Maud Kuistila Memorial Foundation, the Saara Kuusisto and Salme Pennanen Foundation, the Sigrid Juselius Foundation, and the Faculty of Health Sciences, University of Eastern Finland.

(16)

(17)

XI

List of the original publications

This dissertation is based on the following original publications:

I Kuosmanen SM, Hartikainen J, Hippeläinen M, Kokki H, Levonen AL, Tavi P.

MicroRNA profiling of pericardial fluid samples from patients with heart failure.

PLoS One 10: e0119646, 2015.

II Kuosmanen SM*, Kansanen E*, Sihvola V, Levonen AL. MicroRNA Profiling Reveals Distinct Profiles for Tissue-Derived and Cultured Endothelial Cells.

Scientific Reports 7(1): 10943, 2017.

III Kuosmanen SM, Viitala S, Laitinen T, Peräkylä M, Pölönen P, Kansanen E, Leinonen H, Raju S, Wienecke-Paldacchino A, Närvänen A, Poso A, Heinäniemi M, Heikkinen S, Levonen AL. The Effects of Sequence Variation on Genome-wide NRF2 Binding – New Target Genes and Regulatory SNPs. Nucleic Acids Research 44: 1760-1775, 2016.

IV Kuosmanen SM, Kansanen E, Sihvola V, Kaikkonen MU, Pulkkinen K, Jyrkkänen HK, Tuoresmäki P, Hartikainen J, Hippeläinen M, Kokki H, Tavi P, Heikkinen S, Levonen AL. NRF2 Regulates Endothelial Glycolysis and Proliferation with miR-93 and Mediates the Effects of Oxidised Phospholipids on Endothelial Activation. Submitted Manuscript.

*Authors with equal contribution

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

(18)

(19)

XIII

Abbreviations

AGO Argonaute AKT Protein kinase B ApoB Apolipoprotein B-100 ApoE KO Apolipoprotein E knockout ARE Antioxidant response element ATF4 Activating transcription

factor 4

BMPs Bone morphogenetic proteins bZIP Basic leucine zipper

CAD Coronary artery disease Ccm Cerebral cavernous

malformations

CD36 Cluster determinant 36 ChIP-seq Chromatin

immunoprecipitation combined with sequencing CNC cap’n’collar

COX Cyclooxygenase CUL3 Cullin 3

CVD Cardiovascular diseases DLL4 Delta-like 4

EGFL7 EGF-like domain 7

EMT Epithelial-to-mesenchymal transition

EndoMT Endothelial-to-Mesenchymal Transition

EP2 E-type prostaglandin receptor FGF Fibroblast growth factors FGFR1 FGF receptor 1

FOXO1 Forkhead box protein O1 GCLC Glutamate-Cysteine Ligase

Catalytic Subunit

GCLM Glutamate-Cysteine Ligase Modifier Subunit

GRO-seq Global run-on sequencing GSK-3β glycogen synthase kinase 3β Gsta2 glutathione S-transferase Ya

subunit

HDL High-density lipoprotein HIF-1α Hypoxia-inducible factor -1

alpha

HO-1 Heme oxygenase-1 HUVEC Human Umbilical Vein

Endothelial Cell

IGV Integrative Genomics Viewer iPSC Induced pluropotent stem cell

KEAP1 Kelch ECH-associating protein 1

KLF2 Krüppel-like factor 2 LDL Low-density lipoprotein LDLR KO LDL receptor knockout LOX Lipoxygenase

MAPK mitogen-activated protein kinase

MCP-1 Monocyte chemotactic protein 1

miRNAs MicroRNAs

mmLDL Minimally modified LDL MMP Matrix metalloproteinase MYC Myc proto-oncogene protein Neh NRF2-ECH homology NFE2L2 Nuclear Factor Erythroid-2-

Like 2

NOS Nitric oxide synthases NOTCH1 NOTCH homolog 1

NOX NADPH oxidase

NQO1 NADPH:quinone reductase NRF2 Nuclear factor E2-Related

Factor 2

NYHA New York Heart Association oxLDL Oxidised LDL

oxPAPC Oxidised PAPC

PAF Platelet activating factor PAPC 1-palmitoyl-2-arachidonoyl-

sn-glycero-3-phosphocholine PFKFB3 6-Phosphofructo-2-

kinase/fructose-2,6- bisphosphatase 3

PGPC 1-palmitoyl-2-glutaroyl-sn- glycero-3-phosphocholine PI3K Phosphoinositide 3-kinase POVPC 1-palmitoyl-2-(5-oxovaleroyl)-

sn-glycero-3-phosphocholine PTEN Phosphatase and tensin

homolog

qPCR Real-time quantitative polymerase chain reaction RBX1 Really interesting new gene-

box protein 1

RISC RNA-induced silencing complex

RNA Pol II RNA Polymerase II

(22)

ROS Reactive oxygen species SASP Senescence-associated

secretory phenotype siRNA Small interfering RNA SIRT1 Sirtuin 1

SNP single nucleotide polymorphism SQSTM1 Sequestesome-1 SRB1 Scavenger receptor B1 TGFβ transforming growth factor

beta

TGFβR transforming growth factor beta receptor

TLR2 Toll-like receptor 2

TMEM30a Transmembrane protein 30A TNFα Tumour Necrosis Factor

alpha

VEGF Vascular endothelial growth factor

VEGFR2 Vascular endothelial growth factor receptor 2

VSMC Vascular smooth muscle cell XPO5 Exportin 5

β-TrCP β-transducin repeat- containing protein

(23)

1 Introduction

Atherosclerosis is a critical determinant of human lifespan and is the main cause of morbidity and mortality worldwide, despite progression in its prevention and treatment. In addition, it is a major financial burden to the health care system. The disease progression is a slow and silent process during which plaques form along the inner walls of the arteries narrowing the lumen and reducing blood flow to the target tissue. The first symptoms appear in the late stages of the disease. Often it progresses completely unannounced until the incidence of myocardial infarction or stroke.

Vascular branching is required for the blood distribution throughout the body, but it is also the cause of flow disturbances, which predispose to atherosclerotic changes in the arterial wall. Combined with the classical risk factors of atherosclerosis, such as elevated low-density lipoprotein (LDL), hypertension and aging, flow disturbances cause dysfunction in the endothelium, which is the inner layer of blood vessels. Endothelial dysfunction increases vascular permeability promoting lipid accumulation into the vessel wall and increases local oxidative stress leading to the formation of oxidized phospholipids, such as oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), which is the main active component of minimally modified LDL (mmLDL).

Nuclear factor E2-Related Factor 2 (NRF2) is a transcription factor that controls the expression of various antioxidant genes, and thus orchestrates the cellular response to oxidative stress. OxPAPC activates the NRF2 pathway, thereby decreasing local oxidative stress and inflammation in the vessel wall and improving endothelial function in the early phases of atherosclerosis. However, oxPAPC also stimulates neovascularization in the plaques, which may in the later stages of the disease result in the formation of rupture- prone vulnerable plaques and cause sudden death due to thrombotic events.

MicroRNAs (miRNAs) are small, non-coding RNAs that control gene expression, but they can also be secreted from the cells and act as signalling molecules. Circulating miRNAs are considered as candidate biomarkers in molecular diagnostics as well as potential drugs and drug targets. In atherosclerosis, miRNAs have been associated with all stages of the disease from plaque initiation to plaque rupture, and their manipulation through silencing and mimic technologies has emerged as a promising therapeutic strategy for atherosclerosis. However, many aspects of miRNA biology are still unknown, and the context- and cell-specific effects are poorly described. Therefore, systemic miRNA targeting might have unpredictable long-term effects. Gaining more insight into the logic and dynamics of these miRNA networks is essential for the design of safe miRNA-based therapeutics.

In this study, the objective was to characterise novel miRNAs that participate in the development or prevention of atherosclerosis, and as such could potentially be considered as biomarkers. To this end, pericardial fluid of cardiovascular patients was collected and profiled for miRNAs, and the diagnostic and prognostic potential of the detected miRNAs in cardiovascular disorders were estimated in the study population. In addition, the global changes in human primary endothelial cell miRNA profiles from young freshly isolated cells to old cultured cells were characterized. Then, a genome-wide binding profile for NRF2 was established utilising protein binding microarrays, which were developed in this study. This genome-wide NRF2 binding profile in combination with data from oxPAPC- treated endothelial cells was used to select a putative NRF2-regulated candidate for further studies. The selected candidate was also abundant in atherosclerosis patient samples and aging endothelial cells. Finally, the function of the candidate was elucidated in endothelial cells and detailed mechanisms were deciphered. The study established novel mechanisms through which oxPAPC can activate endothelial cells and potentially affect neovascularization.

(24)

(25)

3

2 Background

2.1 ATHEROSCLEROSIS

Atherosclerosis is the most prevalent underlying cause of myocardial infarction, stroke and sudden cardiac death. Coronary artery disease (CAD), that is atherosclerosis of the coronary arteries, is the leading cause of morbidity and mortality worldwide, resulting in millions of deaths annually (Libby, Bornfeldt and Tall, 2016). The initiation and progression of atherosclerosis is a silent process starting early in life and continuing over several decades during which plaques form along the inner walls of the arteries narrowing the lumen and reducing blood flow to target tissues and organs. Early atherosclerotic lesions develop during adolescence in everyone, and with aging and other contributing risk factors, such as elevated low-density lipoprotein (LDL) cholesterol, hypertension, diabetes, obesity and smoking, the lesions can progress to advanced stages and become symptomatic (Libby, Ridker and Hansson, 2011; Schober, Nazari-Jahantigh and Weber, 2015). The first symptoms commonly appear when the blood flow to the target tissue has already been compromised, the very first sign often being myocardial infarction or stroke.

2.1.1 Early Events in Atherosclerosis: Endothelial Dysfunction

Blood vessel branching is required for efficient blood distribution and normal organ function, but it is also the cause of blood flow disturbances (Schober, Nazari-Jahantigh and Weber, 2015). Atherosclerotic lesions develop site-specifically primarily at the branching points of large and medium arteries where the dynamic flow patterns are more complex than in the unbranched parts of the vessel. In the unbranched parts, blood flows in streamlined, parallel fashion known as the laminar flow, which causes high shear stress on the inner vessel wall, the endothelium. At the branching points, however, the flow becomes turbulent, which results in low shear stress.

The inner surface of all blood vessels is formed of a single-layer of endothelial cells, which represents a natural barrier between the blood and other tissues. Endothelial cells are specialized and have various roles depending on their location and microenvironment.

Arterial endothelial cells have adapted to pulsatile, high-pressure and high shear stress conditions, which inhibit proliferation and induce cell survival and quiescence. However, despite their plasticity, endothelial cells are unable to adjust to disturbed flow, which eventually leads to endothelial activation and subsequent coordinated cycles of endothelial cell death and proliferation. Consequently, enhanced endothelial turnover disrupts the normal barrier function of the endothelium increasing its permeability, inducing inflammatory signalling and leading to endothelial dysfunction (Schober, Nazari-Jahantigh and Weber, 2015).

2.1.2 Progression of Atherosclerosis: LDL and Oxidative Stress

Endothelial dysfunction results in the earliest detectable manifestation of atherosclerosis (Figure 1): the focal permeation, trapping and modification of circulating LDL particles in the subendothelial space, the intima (Gimbrone and García-Cardeña, 2016). Furthermore, endothelial activation increases reactive oxygen species (ROS) production leading to increased oxidative stress and oxidation of LDL particles (Panieri and Santoro, 2015). The formation of oxidised LDL (oxLDL) stimulates endothelial cells to express adhesion molecules on the cell surface, which attracts monocytes to the site and causes their infiltration into the intima. In the intima, monocytes differentiate into macrophages. They

(26)

internalize and degrade modified lipoproteins thereby removing cholesterol from the vessel wall through high-density lipoprotein (HDL) particles in a process called reverse cholesterol transport. Over time, however, the constant and increasing influx of lipoproteins overwhelms the removal capacity of the macrophages. This results in lipid accumulation in the macrophages and subsequently their transformation into foam cells.

The intimal accumulation of oxLDL products further increases oxidative stress and inflammatory signalling thus stimulating endothelial activation and sustaining chronic inflammation in the vessel wall (Gimbrone and García-Cardeña, 2016).

Figure 1. Atherosclerotic plaque formation from initiation to rupture. mmLDL = Minimally-modified LDL, MMPs = Matrix metalloproteinases, VSMC = Vascular smooth muscle cell. Adapted from original figures of Emilia Kansanen.

2.1.3 Advanced Stages of Atherosclerosis

As the lesions progress, foam cells form fatty streaks. Macrophages and endothelial cells produce more chemokines and growth factors, which induce vascular smooth muscle cell proliferation and their migration from the medial layer into the intima. In addition, intimal production of extracellular matrix components increases. This progressive structural remodelling eventually leads to formation of a fibrous cap overlaying a lipid-rich necrotic core of oxidized lipoproteins, cholesterol crystals, cellular debris, and varying degrees of remodelled matrix and calcification (Gimbrone and García-Cardeña, 2016). Furthermore, induction of angiogenic factors promotes angiogenesis and neovascularization to increase the local flow of oxygen and nutrients to enable plaque growth (Camaré et al., 2017).

2.1.4 Vulnerable Atherosclerosis

The repetitive and sustained damage caused by the lesion progression can cause thickening of the vessel wall, which leads to narrowing of the vascular lumen and causes ischemic symptoms. However, acute cardiovascular events, such as myocardial infarction, stroke and sudden cardiac death, are mainly caused by atherothrombosis resulting from erosion or rupture of unstable or vulnerable plaques (Libby, 2013; Gimbrone and García-Cardeña, 2016). Erosion-prone plaques exhibit increased superficial endothelial apoptosis, a low number of macrophages and accumulation of smooth muscle cells and proteoglycans.

Rupture-prone plaques, on the other hand, have been associated with an enlarged necrotic core, a thin fibrous cap, increased intraplaque angiogenesis and neovascularization, as well as decreased calcification (Badimon and Vilahur, 2014). The occurrence of rupture and

LDL

ROS oxLDL

mmLDL

Monocyte Adhesion molecule

oxLDL uptake

Macrophage

HDL

Foam cell

Fa=y streak Lipid

droplet Cholesterol eﬄux Endothelial dysfuncBon

ROS MMPs Cytokines Chemokines

Growth Factors

migraBon

Plaque ini0a0on Fa2y streak forma0on Stable plaque Unstable Plaque

Endothelium In0ma

Media

Leucocyte

Endothelial acBvaBon

Vascular smooth muscle cells Internal elasBc lamina

Erosion Rupture Thrombosis Fibrous cap

InﬂammaBon

NeovascularizaBon

Vessel wall Vascular lumen

Shear stress

VSMC proliferaBon SMC

(27)

5

erosion varies by syndrome, however plaque rupture has been suggested to be the more prevalent cause of atherothrombosis.

2.2 VASCULAR ENDOTHELIUM

The vascular endothelium forms the inner lining of the heart and blood vessels. It consists of a single-layer of endothelial cells and functions as a selective barrier between the vessel lumen and the surrounding tissue allowing the exchange of nutrients and gases between the blood and tissues. However, vascular endothelium is not just a passive barrier, it actively secretes signals that modify its own and neighbouring cell and tissue function. For example, endothelium controls vascular tone through secretion of vasoactive substances, which influence smooth muscle cell function, and participates in chemokine and growth factor signalling by producing both growth-inducing and -inhibiting substances to regulate normal vascular growth and angiogenesis. Endothelium is also an active modulator of thrombotic events through secretion of anti- and procoagulant agents and fibrinolytic substances. Moreover, it protects the surrounding tissues from exogenous pathogens by evoking inflammatory responses and expressing adhesion molecules, which attract immune cells to the site of injury or infection. Not surprisingly, endothelial dysfunction has serious consequences and it plays an important role in the pathogenesis of major vascular diseases, such as atherosclerosis. Understanding the causes and mechanisms of vascular dysfunction in chronic diseases is crucial for countering their detrimental consequences.

Figure 2. Structure of the vascular wall

2.2.1 Vascular Structure

Blood vasculature consists of three principal types of blood vessels that circulate blood throughout the body, namely arteries, veins and their interconnecting capillaries. It forms a closed system that transports gases, nutrients, metabolites, cells and various signalling molecules to surrounding tissues (Potente and Mäkinen, 2017). The systemic circulation is maintained by the heart, which pumps oxygen-rich blood from arteries to capillaries where the oxygen and nutrients diffuse to the tissues, and carbon dioxide diffuses back to the capillary blood. From the capillaries, the oxygen-deprived, carbon dioxide rich blood gathers to veins, which return it back to the heart. Finally, the heart circulates the oxygen- depleted blood to the lungs for re-oxygenation before the start of a new cycle. The local blood flow is also maintained by the arterial function (contraction and dilation), which ensures sufficient oxygen and nutrient supply to active tissues, whereas veins function as blood reservoirs storing large quantities of blood, which can be sent to the arterial side and used to restore blood pressure.

Mammalian vessel walls comprise of three histological layers, namely tunicas intima, media and adventitia (Figure 2). The innermost layer, the intima, faces the blood flow, and consists of the endothelial monolayer and the underlying connective tissue on top of the

Tunica in(ma Endothelium Internal elas1c lamina

Tunica Media Smooth muscle cells External elas1c lamina Tunica Adven((a Fibroblasts

Loose connec1ve 1ssue Vasa vasorum

Blood ﬂow

Connec1ve 1ssue Vasa vasorum External lamina Smooth muscle cells Internal lamina Endothelium

Fibroblast

(28)

internal elastic lamina. Endothelial cells are connected to each other through tight junctions and gap junctions, which are involved in intercellular cohesion and electrochemical coupling, respectively. The thin subendothelial layer of connective tissue, the extracellular matrix, serves as a transducer of physical and chemical microenvironmental signals, a reservoir of growth factors and an adhesive scaffold required for anchorage-dependent survival of endothelial cells. In normal arteries the subendothelial space is thin, whereas in atherosclerosis intimal thickening is one of the earliest signs of the disease. The intermediate layer below intima, tunica media, is separated from the intima by the internal elastic lamina and from the adventitia by the external elastic lamina. The main constituents of media are smooth muscle cells and extracellular matrix components, such as collagen, elastin and proteoglycans. The outer layer of the vessel is tunica adventitia. The main ingredients of adventitia are fibroblasts and loose connective tissue, which contains a network of small blood vessels, the vasa vasorum, that supply the walls of the large vessels with oxygen and nutrients. In healthy vessels, the intima and the inner part of media are nourished by diffusion from the vascular lumen, but in an atherosclerotic plaque, the vasa vasorum penetrates the media and extends to feed the pathological, thickened intima (Camaré et al., 2017).

Figure 3. Vasculogenesis and angiogenesis. Modified from (Potente and Mäkinen, 2017)

2.2.2 Vascular Development

Vasculature is among the first organ systems to develop during embryogenesis and is essential for the growth, survival and function of all other organ systems (Dejana, Hirschi and Simons, 2017). The two main processes, which determine the generation of blood vessels both during embryonic development and later in life, are vasculogenesis and angiogenesis (Figure 3). Vasculogenesis refers to the formation of new blood vessels from endothelial progenitor cells, and is therefore more common during embryogenesis. In contrast, angiogenesis refers to the formation of blood vessels from pre-existing vasculature by capillary sprouting, and is utilised in tissue regeneration such as wound healing or during the female reproductive cycle (Potente and Mäkinen, 2017). Pathological growth may utilise both vasculogenesis and angiogenesis, but also collateral growth of the vessels.

During vasculogenesis, multipotent mesodermal cells first differentiate to endothelial precursors known as angioblasts in response to growth factor signals, such as fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs) and vascular endothelial growth factors (VEGFs), which derive from the adjacent visceral endoderm. These endothelial progenitor cells then migrate to discrete locations, undergo rapid expansion

(29)

7

and coalesce to form lumenised tubes in a tree-like network of blood vessels, which are further remodelled into the arterial-venous networks capable of sustaining systemic circulation. The remodelling and maturation of the new blood vessels requires coordinated migration, cell cycle control, endothelial subtype specialisation and smooth muscle cell recruitment. Later on, a subset of venous endothelial cells continues to specialise into the lymphatic subtype to form the lymphatic vasculature (Goligorsky and Hirschi, 2016;

Potente and Mäkinen, 2017).

For angiogenesis, the main physiological stimulator is hypoxia. In hypoxic cells, low oxygen partial pressure activates a transcription factor called hypoxia-inducible factor-1 alpha (HIF-1α), which upregulates proangiogenic factors, such as VEGFs and nitric oxide synthases (NOS) that regulate vascular tone. Hence, the initial steps in angiogenesis involve vasodilatation and increased vascular permeability, disruption of endothelial junctions, as well as proteolysis of the basement membrane and the surrounding extracellular matrix.

These changes enable the activated endothelial cells to migrate and proliferate, which results in the formation of an angiogenic bud formed by tip and stalk cells. Tip cells are migratory and invasive cells that guide the new sprouts towards a VEGF gradient, and secrete proteolytic enzymes that degrade the surrounding extracellular matrix to facilitate bud expansion. The dynamic behaviour of actin filaments and the associated myosin enable the movement of the cells towards the VEGF stimulus. Tip cells are followed by proliferating stalk cells, which support the sprout elongation (Figure 3). When two elongating sprouts meet, they merge to generate a new network, and form a vascular lumen. The new vessel is stabilised with pericytes, smooth muscle cells, and shear stress- induced synthesis of extracellular matrix and basal lamina, whereas the nonperfused segments regress (Camaré et al., 2017; Potente and Mäkinen, 2017).

2.2.3 Endothelial Metabolism and Angiogenesis

Recently, endothelial cell metabolism has emerged as an important mediator of endothelial function and angiogenesis (Teuwen et al., 2017). The role of endothelial metabolism as a major determinant of tip and stalk cell behaviour is not surprising considering that angiogenesis is metabolically very demanding. Migrating tip cells are required to move toward hypoxic regions, whereas stalk cells proliferate and elongate the emerging vessel sprout. The major source of energy in endothelial cells is glycolysis, which contributes to 85% of the endothelial ATP production, and upon endothelial activation the rate of glycolysis further doubles (De Bock et al., 2013; Schoors et al., 2014). Boosted glycolysis provides a competitive advantage for tip cell phenotype through ATP-driven endothelial cell rearrangement. Blockade of the most potent glycolytic activator, 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), and the resulting reduction of glycolysis compromises vascular expansion. Another critical metabolic process required for vessel sprouting is fatty acid β-oxidation, which provides carbons for nucleotide synthesis in DNA replication during endothelial proliferation (Potente and Mäkinen, 2017).

2.2.4 Endothelial Quiescence

After establishing new vascular networks to relieve local nutrient and oxygen deprivation, endothelial cells resume quiescence, the so called ‘phalanx’ phenotype. Quiescence is a reversible state characterised by a non-dividing and non-migratory phenotype, which is common in most adult blood vessels (Potente and Mäkinen, 2017). Two previously identified critical drivers of quiescence are phosphatase and tensin homolog (PTEN) and forkhead box protein O1 (FOXO1) (Serra et al., 2015; Wilhelm et al., 2016). PTEN is a target of delta-like 4-notch homolog 1 (DLL4-NOTCH1) signalling and functions to arrest cell cycle progression in stalk cells through inhibition of phosphoinositide 3-kinase/Protein Kinase B (PI3K-AKT) signalling (Serra et al., 2015). Inhibition of PI3K-AKT in turn prevents AKT-mediated phosphorylation of FOXO1 and promotes its activation. FOXO1 activation

(30)

inhibits Myc proto-oncogene protein (MYC), which is a potent activator of endothelial cell proliferation, growth and metabolism (Wilhelm et al., 2016). Thus, endothelial quiescence is maintained through the regulation of both the metabolic and cell cycle machinery.

Similarly, physiological laminar shear stress maintains endothelial quiescence through the transcription factor Krüppel-like factor 2 (KLF2), which suppresses the expression of several glycolytic and angiogenic genes, such as PFKFB3 and VEGF receptor 2 (VEGFR2), thus promoting quiescence (Doddaballapur et al., 2015; Schober, Nazari-Jahantigh and Weber, 2015).

2.2.5 Neovascularisation in the Atherosclerotic Plaques

A major contributor to atherosclerotic plaque instability and the ensuing thromboembolic events is the neovascularisation of atherosclerotic plaques. Angiogenic inhibitors have proven efficient in hindering athero-progression in experimental animal models. However, the currently available antiangiogenic agents, which have been tested in human clinical trials for cancer therapy, increase the risk of cardiovascular events in atherosclerotic patients, and are therefore unsuitable for atherosclerosis treatment (de Vries and Quax, 2016; Camaré et al., 2017).

The main process for atherosclerotic plaque vascularization is sprouting angiogenesis from the pre-existing vasa vasorum, whereas postnatal vasculogenesis seems to play only a minor role (Camaré et al., 2017). Successful angiogenesis requires a dynamically regulated process where a delicate balance is maintained between a complex array of angiogenic and angiostatic factors, which increase, stabilize and reduce the vascular network (Xu, Lu and Shi, 2015; de Vries and Quax, 2016; Camaré et al., 2017; Parma et al., 2017). In atherosclerotic areas, angiogenesis involves the same classical angiogenic mechanisms as in physiological conditions, but in addition is affected by specific atherosclerosis-related factors. These factors can for instance sustain angiogenic signalling even after oxygen supply has already been restored, and inhibit stabilization of neovessels. However, the exact mechanisms leading to plaque destabilizing neovascularization in human atherosclerosis remain unclear.

In early atherosclerotic lesions, moderate inflammation combined with oxidative stress and relative hypoxia in the intima promotes angiogenic neovascularization from the adventitial vasa vasorum thereby enabling further plaque growth. In the advanced stages, oxidised lipids, proteases and chronic inflammation may further promote angiogenesis, but the resulting neovessels are malformed and leaky as well as highly susceptible to injury by cytotoxic agents that are generated within the plaque. These leaky vessels contribute to intraplaque haemorrhages that release blood cells, coagulation factors and proteases within the plaque. The inflowing blood cells (activated platelets, leukocytes and erythrocytes) release free cholesterol from their cholesterol-rich membranes thereby contributing to cholesterol crystal formation. Cholesterol crystals, in turn, can trigger inflammatory responses, erode neovessels and the fibrous cap, disrupt biological membranes, and protrude into the vessel lumen, where they may cause thromboembolic events.

Haemoglobin, heme and iron resulting from small intraplaque haemorrhages promote free- radical and reactive oxygen species generation, which inactivates vasorelaxant nitric oxide, promotes lipid peroxidation, and sustains the inflammatory burden. Furthermore, activation of various proteases degrades the fibrous plaque thereby destabilizing the plaque and predisposing to its rupture, which is often associated with atherothrombotic events and may cause sudden death (Xu, Lu and Shi, 2015; de Vries and Quax, 2016; Camaré et al., 2017; Parma et al., 2017).

2.2.6 Endothelial to Mesenchymal Transition

Endothelial cells are constantly exposed to an environment full of various signalling molecules, nutrients, metabolites, oxygen and mechanical stimuli that further shape their

(31)

9

phenotype. Thus, maintenance of endothelial phenotype is an active process that requires energy and support from both intrinsic and environmental sources. These maintenance pathways are poorly understood, but recent studies have linked maintenance of endothelial barrier function, suppression of apoptosis and transcriptional regulation of key proteins, such as VEGFR2 and FGF receptor 1 (FGFR1), with regulation of endothelial fate drift (Dejana, Hirschi and Simons, 2017). For example, temporary inhibition of FGF signalling leads to progressive loss of endothelial cell-cell interactions, efficient barrier function and eventually, vascular integrity, whereas more prolonged secession leads to endothelial apoptosis and vascular rarefaction, as well as loss of the vasa vasorum. Moreover, meddling with FGF signalling jeopardises maintenance of endothelial phenotype, and leads to the induction of Endothelial-to-Mesenchymal Transition (EndoMT) (Piera-Velazquez, Mendoza and Jimenez, 2016; Xiao and Dudley, 2017).

In the absence of an active input that maintains their phenotype, endothelial cells either activate the apoptotic signalling cascade or undergo EndoMT. During embryogenesis, EndoMT of endocardial cells, that is endothelial cells that line the heart, gives rise to the mesenchymal cells that are necessary for proper heart development. Specifically, EndoMT contributes to valvular progenitor cell, cardiac fibroblast and smooth muscle cell generation and endocardial cushion formation. However, later in life endothelial cells that undergo EndoMT acquire characteristics of fibroblasts, smooth muscle cells, chondrocytes, osteocytes or adipocytes, and as a result loose proper barrier function capabilities and become more proliferative and migratory. In addition, the cells start to secrete and express various new components, such as the extracellular matrix proteins fibronectin and collagen, or leucocyte adhesion molecules. The loss of endothelial characteristics eventually results in a number of pathological consequences in diseases like tissue fibrosis, atherosclerosis and cancer (Dejana, Hirschi and Simons, 2017).

EndoMT has been implicated in the pathology of atherosclerosis as well as in transplant atherosclerosis, where data from explanted rejected hearts showed that 80% of the luminal and neointimal endothelial cells of the coronary arteries were undergoing transition (Chen et al., 2012, 2014). More recently, studies on atherosclerotic vessels have confirmed the frequent occurrence of EndoMT in atherosclerosis of coronary and carotid arteries (Chen et al., 2015; Moonen et al., 2015; Evrard et al., 2016). Moreover, the correlation between disease severity and the extent of EndoMT in luminal endothelial cells is high (Chen et al., 2015;

Evrard et al., 2016). The incidence of EndoMT has been shown to be higher in endothelial cells that overlie atherosclerotic plaque than in nearby cells, which are not in direct contact with the plaque (Chen et al., 2015).

Mechanistically, a number of triggers have been proposed. However, the two best characterised involve the activation of BMP signalling due to the loss of cerebral cavernous malformation (Ccm) 1 and Ccm2 gene expression, and the activation of transforming growth factor beta (TGFβ) receptor (TGFβR) in response to absent FGFR1 signalling and a decline in let-7 expression (Dejana, Hirschi and Simons, 2017). In human coronary arteries, the extent of EndoMT has been shown to correlated with the magnitude of endothelial TGFβ activation (Chen et al., 2015), which is one of the hallmarks of the transition. TGFβ activation is controlled by FGF signalling through FGF receptor, which in endothelial cells is predominantly FGFR1. FGFR1 deletion leads to severe downregulation of the let-7 miRNA family and dramatic upregulation of both TGFβ and TGFβR, which activates EndoMT. Accordingly, FGFR1 expression is low in atherosclerosis-prone areas in the arteries, whereas atherosclerosis-resistant areas express high levels of it. Other pathways and regulators that have been suggested to control EndoMT include Wnt/β-catenin signalling, which promotes EndoMT, miR-20a, which targets TGFBR1 and TGFBR2, and miR-21, which is induced by TGFβ and promotes EndoMT (Piera-Velazquez, Mendoza and Jimenez, 2016; Dejana, Hirschi and Simons, 2017).

(32)

2.2.7 Endothelial Redox Biology

In normal cell physiology, reactive oxygen species (ROS) are constantly produced in response to various exogenous and endogenous stimuli and transformed and consumed by tissue metabolism. To maintain redox homeostasis, cells control the balance between the ROS-generating pro-oxidants and their elimination with antioxidants. Thus, owing to their reserve antioxidant capacity, cells in normal vessel wall can tolerate a certain level of exogenous ROS stress. In endothelial cells, a moderate increase in ROS may even promote cell proliferation and survival. Moreover, appropriate levels of ROS are indispensable mediators of physiological processes and redox-regulated signalling pathways. However, prolonged exposure to increased ROS leads to vascular dysfunction and predisposes to atherosclerosis and aging due to DNA, protein and lipid damage, furthermore ROS levels that reach toxic threshold trigger cell death (Panieri and Santoro, 2015).

Oxidative stress describes a situation where the production of ROS exceeds the ability of the antioxidant system to detoxify these substances. The excess ROS can damage all components of the cells (nucleic acids, proteins and lipids), but also contribute to the initiating steps of diseases, such as atherosclerosis, where LDL particles in the vessel wall become oxidised by ROS function. ROS comprises free radicals that contain one or more unpaired electron, such as nitric oxide (NO), superoxide (O2-), and hydroxyl radical (OH), and nonradical oxidants, such as hydrogen peroxide (H2O2) and peroxynitrite (ONOO^-). The reactions between two radical species are kinetically fast and can trigger a chain reaction, which causes cellular damage. A good example that is relevant to vessel wall function is the reaction between NO and O2- in pathological conditions, which decreases the bioavailability of NO thereby predisposing arteries to remodelling, development of atherosclerotic lesions and thrombus formation. The product of the reaction is a very reactive oxidant, ONOO^-, which can further react with enzymes and other proteins, cause lipid peroxidation and promote endothelial dysfunction. In the vascular wall, several systems contribute to ROS production, particularly mitochondrial respiratory chain, cyclooxygenases (COXs), cytochrome P450, endothelial nitric oxide synthase (eNOS), lipoxygenases (LOXs), NADPH oxidases (NOXs), and xanthine oxidase (Chiu and Chien, 2011; Panieri and Santoro, 2015; Camaré et al., 2017).

The constant presence of cellular ROS has led to the development of protective mechanisms in cells to ensure the maintenance of cellular redox balance. The cellular defence is based on antioxidants, which can neutralise and degrade ROS through electron acceptance or donation. Antioxidants include enzymes (catalase, glutathione peroxidases, superoxide dismutases, peroxiredoxins, thioredoxins), as well as small antioxidants and ROS-scavengers from endogenous (glutathione, uric acid, bilirubin, coenzyme Q) and dietary origin (tocopherols, ascorbic acid, carotenoids, polyphenols). Many of the endogenous antioxidants are regulated by a transcription factor called NRF2, which is induced by oxidative stress (Hayes and Dinkova-Kostova, 2014).

2.3 OXIDISED PHOSPHOLIPIDS

Elevated LDL concentration in plasma is a hallmark of hyperlipidemia and atherosclerosis.

Physiologically, lipoproteins act as lipid carriers in blood. They transport lipids from the liver and intestines to the peripheral target tissues and from tissues back to the liver for storage or degradation. This transport function requires them to pass through the endothelial barrier and the subendothelial space, the intima, to reach their target sites.

Although the liver functions to remove excess lipoproteins from the bloodstream, its clearance capacity can be exceeded resulting in pathological levels of blood lipoproteins and lipid accumulation in the vessel walls. However, lipoproteins are not the only source of

(33)

11

oxidised lipids in pathological states. Oxidised lipids are also formed in cells both intracellularly and on the cell surface particularly in stressful conditions and during apoptosis.

2.3.1 Sources of Oxidised Phospholipids

LDL consists of approximately 1600 cholesteryl ester, 700 phospholipid, 600 unesterified cholesterol and 185 triglyceride molecules, as well as one molecule of apolipoprotein B-100 (Miller and Shyy, 2017). The hydrophobic core of LDL comprises cholesteryl esters and triglycerides, whereas phospholipids, unesterified cholesterol and apolipoprotein B-100 from a hydrophilic layer over the core allowing LDL transport in the bloodstream and entry into tissues. Cholesteryl esters, phospholipids and triglycerides incorporate one, two or three fatty acyl chains, respectively. Among the most common polyunsaturated fatty acyl groups incorporated into these compounds are linoleic (18:2), arachidonic (20:4) and docosahexaenoic (22:6) acids. Enzymatic or free radical oxidation of LDL results in hundreds of oxidative products, including oxidised phospholipids. These oxidised lipids can covalently modify proteins, be hydrolysed to produce oxidised free fatty acids, and fragment into highly reactive molecules, which in turn can covalently modify proteins and other phospholipids. In addition, phospholipids are the main components of lipid bilayers in cell membranes (Ashrav and Srivastava, 2012).

Glycerophospholipids and sphingolipids form two major classes of phospholipids.

Glycerophospholipids consist of a glycerol backbone, a polar phosphate head group, and two fatty acid chains (sn-1 and sn-2). They are further classified into phosphatidylglycerols, -cholines, -ethanolamines, -inositols and -serines depending on the type of the polar head group. Polyunsaturated fatty acid residues, which are prone to oxidation, are most commonly found at the sn-2 position. Phospholipid oxidation can be initiated by enzymatic (12/15-LOX) and non-enzymatic (ROS) free radical and radical-free processes, which commonly produce identical primary oxidation products, i.e. peroxyl radicals and hydroperoxides. The subsequent phospholipid oxidation produces a wide spectrum of oxidised phospholipids through oxidation of polyunsaturated fatty acid residues, cyclization of peroxyl radicals, or oxidative fragmentation of esterified polyunsaturated fatty acids (Bochkov et al., 2010; Ashrav and Srivastava, 2012).

2.3.2 Oxidised Phospholipids in Atherosclerotic Lesions

The role of oxidised phospholipids in the pathophysiology of disease was first recognised in atherosclerosis (Witztum and Steinberg, 1991; Berliner and Watson, 2005). In physiological states, low-level phospholipid oxidation occurs as a result of basal cellular metabolism, and the accumulation of oxidised phospholipids is prevented through rapid turnover and removal of the products. In atherosclerotic lesions, however, the capacity of the removal is exceeded leading to accumulation of the species, which further contributes to the initiation and development of the disease. During atherogenesis, high concentrations of oxidised phospholipids are continuously generated and degraded in the lesions.

Furthermore, concentrations of active oxidised phospholipids in atherosclerotic vessels reach local levels that are comparable to those inducing biological effects on cultured cells.

Moreover, atherosclerotic vessels express high amounts of proteins that are induced by oxidised phospholipids supporting their mechanistic role in atherogenesis (Bochkov et al., 2010).

2.3.3 OxPAPC

1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) is a naturally occurring phospholipid present in cell membranes, and one of the main phospholipids of lipoproteins (Bochkov et al., 2010). Due to its surface location, PAPC is prone to oxidation, which affects the polyunsaturated arachidonic acid chain generating a vast array of oxidation products,

(34)

such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1- palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC). The oxidation products are collectively referred to as oxPAPC (Figure 4).

The effects of OxPAPC are mediated by several cell surface receptors, such as cluster determinant 36 (CD36), E-type prostaglandin receptor (EP2), platelet activating factor (PAF) receptor, scavenger receptor B1 (SRB1), Toll-like receptor 2 (TLR2) and transmembrane protein 30A (TMEM30a) (Figure 5). In addition, oxPAPC also signals through covalent binding to other cell surface proteins and signalling factors, and through modifications on cellular membranes (Miller and Shyy, 2017). In physiological states, individual oxPAPC species are present at very low concentrations, but in pathological sates their concentrations are significantly higher and can reach the levels of tens of micromoles per litre (Bochkov et al., 2010).

OxPAPC is a strong stimulator of endothelial function. After exposure, the primary responses in second messengers occur as early as in 2 minutes, and secondary signals may be sustained for days (Lee et al., 2012). The affected pathways include but are not limited to sterol synthesis, unfolded protein response, redox signalling, inflammation, angiogenesis, cell division, endocytosis, filament formation, ecto-protease regulation and thrombosis (Gargalovic et al., 2006; Romanoski et al., 2011). The effects are both atheroprotective and atherogenic, but the net effect on vascular wall is thought to be proatherogenic (Lee et al., 2012).

Figure 4. PAPC oxidation leads to a variety of oxidation products as demonstrated by the mass spectroscopy image from (Spickett, Reis and Pitt, 2011). The structures for two of the products are shown.

2.3.4 OxPAPC in Atherosclerosis

In early atherosclerotic lesions, LDL entrance to the vessel wall leads to its oxidation by reactive oxygen species or 12/15-LOX and formation of mmLDL, which contains high amounts of oxPAPC (Bochkov et al., 2010; Lee et al., 2012). Low doses of oxPAPC decrease endothelial permeability by formation of adherens junctions. High levels of oxPAPC, on the other hand, increase the permeability through junction breakdown and stress fibre formation increasing LDL entrance into the vessel wall. OxPAPC action on cell membrane receptors leads to the production of cell surface receptors, growth factors and chemokines,

(35)

13

which attract monocytes and facilitate their entry into the vessel wall. OxPAPC action on monocyte receptors causes their differentiation into macrophage subtypes and dendritic cells and further increases chemokine and cytokine synthesis. Oxidised phosphatidylcholines of oxLDL also induce foam cell formation (Figure 5A).

In advanced lesions, increased chemokine and direct oxPAPC action on vascular smooth muscle cells cause muscle cell proliferation and migration and stimulate their collagen and fibronectin production (Lee et al., 2012). The migrating cells form a cap over the accumulating foam cells. OxPAPC interaction with macrophages induces their apoptosis.

Apoptotic cells and cell debris are taken up by the lipid-activated macrophages, which in early lesions can prevent atherosclerosis. In later stages, however, it contributes to the formation of necrotic core and thus aggravates the condition. Apoptotic fragments can also stimulate endothelial cells to produce angiogenic cytokines, which together with oxPAPC- induce angiogenic factors, such as VEGFA, cause angiogenesis and neovascularization from the adventitial vasa vasorum towards the media and intima. In addition, oxPAPC stimulates metalloproteinase production in macrophages, which weakens the plaque and predisposes to plaque rupture. OxPAPC increases platelet activation, as well as production of thrombotic factors in endothelial cells and macrophages (Figure 5B).

Figure 5. Oxidised phospholipid function in early (A) and late (B) atherosclerotic lesions. Modified from (Lee et al., 2012).

MicroRNAs in endothelial aging and atherosclerosis

uef.fi

Dissertations in Health Sciences

SUVI KUOSMANEN

MICRORNAS IN ENDOTHELIAL AGING AND ATHEROSCLEROSIS

SUVI KUOSMANEN

MicroRNAs in Endothelial Aging and

Atherosclerosis

MicroRNAs in Endothelial Aging and Atherosclerosis

Acknowledgements

List of the original publications

Contents

Abbreviations

1 Introduction

2 Background