Molecular mechanisms of atherosclerotic disease

Molecular mechanisms of atherosclerotic disease

Studies on the role of the DNA glycosylase NEIL3 and the epitranscriptome in the development of atherosclerosis

Ana María Quiles-Jiménez

Thesis submitted for the degree of Philosophiae Doctor (PhD)

Research Institute of Internal Medicine Institute of Clinical Medicine

Oslo University Hospital Faculty of Medicine UNIVERSITY OF OSLO

2020

© Ana María Quiles-Jiménez, 2020

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-735-2

All rights reserved. No part of this publication may be

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

3 Table of Contents

ACKNOWLEDGMENTS ... 5

SELECTED ABBREVIATIONS ... 7

LIST OF SCIENTIFIC PAPERS ... 9

1. INTRODUCTION ... 11

1.1. Cardiovascular disease ... 11

1.1.1. Prevalence and costs ... 11

1.1.2. Risk factors ... 12

1.2. Atherosclerosis: a chronic inflammatory disease ... 13

1.2.1. Atherogenesis: fatty streak formation ... 14

1.2.2. Atherosclerotic plaque development ... 15

1.2.3. The vulnerable plaque and its complications ... 15

1.3. DNA damage response and repair during atherosclerosis ... 20

1.3.1. Base-excision repair pathway ... 21

1.3.2. DNA glycosylase NEIL3: a multipurpose enzyme ... 23

1.4. Epitranscriptomic regulation: an unexplored path in atherosclerosis ... 27

1.4.1. RNA editing ... 28

1.4.2. Covalent RNA modifications ... 28

1.4.3. Epitranscriptomic modifications in CVDs ... 29

2. AIMS OF THIS WORK ... 32

3. SUMMARY OF RESULTS ... 33

4. SELECTED METHODOLOGICAL CONSIDERATIONS ... 35

4.1. Patient and control samples: “bedside to bench – and back to bedside”... 35

4.2. Animal models of atherosclerosis... 36

4.2.1. Murine models of atherosclerosis ... 36

4.2.2. The Apoe^-/-/Neil3^-/- mouse model ... 39

4.3. Histology and immunohistochemistry ... 40

4.4. Experimental cell-based models ... 42

4.4.1. Cell-based models in atherosclerosis research ... 43

4.4.2. Generation of a NEIL3-knockdown cell-based model ... 44

4.5. Mass spectrometry for detection of N6-methyladenosine and regulatory proteins in atherosclerotic plaques... 45

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4.6. Gene expression detection and analysis ... 46

4.6.1. RNA and DNA isolation, and quality control ... 46

4.6.2. RNA sequencing and transcriptome analysis ... 48

4.7. Statistical considerations ... 49

4.8. Ethical considerations ... 50

5. GENERAL DISCUSSION ... 53

5.1. NEIL3 in atherosclerosis ... 53

5.1.1. Neil3 deficiency affects VSMC phenotype ... 53

5.1.2. Neil3/NEIL3 regulates VSMC proliferation ... 54

5.1.3. Neil3/NEIL3 deficiency impacts canonical VSMC marker expression ... 55

5.1.4. Neil3/NEIL3 deficiency influences VSMC function ... 56

5.1.5. How does Neil3/NEIL3 affect VSMC phenotypic modulation?... 57

5.1.6. A non-canonical mechanism of Neil3/NEIL3? ... 59

5.2. Epitranscriptomic regulation during atherosclerosis ... 60

5.2.1. rRNA-modifying proteins are regulated in human atherosclerosis ... 61

5.2.2. mRNA-modifying proteins are regulated in human atherosclerosis ... 63

5.2.3. What is the biological meaning of the RNA epitranscriptome regulation in atherosclerosis? ... 65

5.3. Clinical relevance of this work ... 68

6. CONCLUDING REMARKS AND FUTURE PERSPECTIVES... 72

7. REFERENCES ... 74

5 ACKNOWLEDGMENTS

This work would not have been possible without the contribution of many people.

First, I wish to express my deepest gratitude to my supervisors Filip Segers, Ida Gregersen and Bente Halvorsen, for believing in my work throughout these PhD years, supporting me when times were tough, sharing insightful knowledge, and offering invaluable advice. You have inspired me to improve, push harder, have faith despite the setbacks, and become a better researcher. This has been a very precious professional and personal experience for me!

I am grateful to have such wonderful group colleagues, the Halvorsen’s Dream Team. Thanks for your great team spirit, your interesting scientific and non- scientific discussions, patiently helping me out with both administrative and lab work, sharing lab protocols and other non-lab protocols (cake recipes), and for our team gatherings full of joy.

I would like to recognize the invaluable work of both clinical doctors and researchers, collaborators and co-authors of my work. A special thanks to Pål Aukrust and Magnar Bjørås for caring so much about my projects. You have been essential for the fulfilment of this work.

I want to thank all my colleagues at the Research Institute of Internal Medicine, for all the interesting science, and fun lunches, Christmas dinners, spring seminars, and more we have shared.

I wish to show my gratitude to Universitet i Oslo, Oslo universitetssykehus Rikshospitalet, Helse Sør-Øst RHF, and Norges forskningsråd, as well as the animal facility personnel, and the patients who kindly contributed to part of the studies, for making this research work possible.

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Thanks to my dear friends living in Oslo and other parts of the world for supporting me all this time. I look forward to catching up with you soon!

Finally, I wish to acknowledge the support and great care of my dear family, and especially my partner. Thanks for understanding me and keeping me going, showing me your unconditional love. This work would not have been possible without you.

7 SELECTED ABBREVIATIONS

ACS Acute coronary syndrome

ACTA2 Actin Alpha 2, Smooth Muscle

ADAR Adenosine deaminase acting on RNA

AGA Aspartylglucosaminidase

AON Antisense oligonucleotide

Apoe Apolipoprotein E

BER Base-excision repair

CAD Coronary artery disease

CD Cluster of differentiation

CTSS Cathepsin S

CVD Cardiovascular disease

DDR DNA damage response

DEGs Differentially expressed genes

dsDNA Double stranded DNA

ECM Extracellular matrix

eIF Eukaryotic translation initiation factor

ES Embryonic stem

ET Endothelin

FBRL Fibrillarin

Fpg Formamidopyrimidine

FTO Fat mass and obesity-associated

GapmeR Synthetic RNA-DNA hybrid AON

HFD High-fat diet

ICAM-1 Intercellular adhesion molecule 1

IHC Immunohistochemistry

IL Interleukin

IFN Interferon

LDL Low-density lipoprotein

LPs Lipoproteins

m⁵C 5-methylcytosine

m⁶A N6-methyladenosine

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M-CSF Macrophage colony-stimulating factor

MCP-1 Monocyte chemoattractant protein 1

METTL Methyltransferase-like

MI Myocardial infarction

MMP Matrix metalloproteinase

mRNA Messenger RNA

MS Mass spectrometry

NEIL3 Nei-like 3

NETs Neutrophil extracellular traps

NGS Next generation sequencing

NSUN NOL1/NOP2/SUN domain

OxLDL Oxidized LDL

ORO Oil Red O

P-DGF Platelet-derived growth factor

PCSK9 Pro-protein convertase subtilisin/kexin type 9

ROS Reactive oxygen species

ssRNA Single stranded RNA

VEGFC Vascular endothelial growth factor

VSMC Vascular smooth muscle cell

RIN RNA integrity number

RNA Ribonucleic acid

RNA-seq RNA sequencing

rRNA Ribosomal RNA

RT-qPCR Reverse transcription quantitative polymerase chain reaction

TGF Transforming growth factor

TNF Tumor necrosis factor

tRNA Transfer RNA

VCAM-1 Vascular cell adhesion molecule 1

VLDL Very low-density lipoprotein

WMM Complex of WTAP, METTL3, and METTL14

WTAP Wilms tumour 1-associated protein

YTHDF YTH domain family protein

9 LIST OF SCIENTIFIC PAPERS

This thesis is based on the following scientific papers:

Paper I. DNA glycosylase Neil3 controls vascular smooth muscle cell biology during atherosclerosis development

Ana Quiles-Jiménez*; Ida Gregersen*; Filip M Segers*; Tonje Skarpengland;

Penelope Kroustallaki; Kuan Yang; Xiang Yi Kong; Knut H Lauritzen; Maria B Olsen; Tom Rune Karlsen; Tuula A Nyman; Ellen L Sagen; Vigdis Bjerkeli; Rajikala Suganthan; Ståle Nygård; Katja Scheffler; Jurriën Prins; Eric Van der Veer; Jonas DS Øgaard; Yngvar Fløisand; Helle F Jørgensen; Kirsten B Holven; Erik A Biessen;

Hilde Nilsen; Tuva B Dahl; Sverre Holm; Martin R Bennett; Pål Aukrust; Magnar Bjørås; Bente Halvorsen.

*Shared first authorship. Submitted on 14^th April 2020.

Paper II. N6-methyladenosine in RNA of atherosclerotic plaques: an epitranscriptomic signature of human carotid atherosclerosis

Ana Quiles-Jiménez; Ida Gregersen; Mirta Mittelstedt Leal de Sousa; Azhar Abbas;

Xiang Yi Kong; Ingrun Alseth; Sverre Holm; Tuva B Dahl; Karolina Skagen; Mona Skjelland; Pål Aukrust; Magnar Bjørås; Bente Halvorsen.

Submitted on 23^rd April 2020.

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11 1. INTRODUCTION

1.1. Cardiovascular disease

Cardiovascular disease (CVD) consists of a group of non-communicable disorders of the heart and the blood vessels which can lead to disability or death.¹ Clinical manifestations of CVDs are varied, including myocardial infarction (MI), stroke, aortic aneurysms, renal artery stenosis and gangrene.² Atherosclerosis is the main underlying cause of CVD, and therefore epidemiological data and biological mechanisms involved in atherosclerosis can be considered to be similar to those of CVD.³

1.1.1. Prevalence and costs

CVD is the leading cause of death and productivity loss worldwide. As of 2017, an estimate of 17.8 million people died from CVDs globally. These deaths represent around 31% of all global deaths, of which 85% are due to MI and stroke.^4-6 Moreover, the number of deaths caused by CVDs has increased during the last 25 years, with a sociodemographic transition from high-income countries to low- and middle-income countries. This is, while trends in CVD mortality remain the same for high-income countries, in low- and middle-income countries CVD cases have increased and account for at least 75% of total deaths.¹ Although Norway has successfully reduced the mortality due to CVDs in the last decade, CVDs still remain one of the main causes of death among Norwegians, and is expected to increase.^{7, 8} Additionally, CVDs are placing a tremendous burden on the economies of all regions in the world, with global costs of CVD predicted to rise to US$ 1.04 trillion, a 22% increase compared to 2010.⁹ These facts highlight the need for more research in order to improve prognosis and treatment for prospective or prevalent patients suffering from CVDs.

12 1.1.2. Risk factors

During several decades, doctors and scientists have been trying to identify CVD risk factors, with the Framingham Heart Study in 1948 being the first study aimed to unravel the causes of CVDs in the city of Framingham, Massachusetts (US).¹⁰ In 2004, the INTERHEART investigators published a large international case-control study to acknowledge coronary artery disease (CAD) risk factors in both developed and low- and middle-income countries in different ethnic

groups.¹¹ These studies have contributed to the knowledge that CVDs are influenced by both modifiable and non-modifiable risks factors (Figure 1). Risk factors that can be modified, which are often behavioural, consist of unhealthy diet; the presence of increased fat particles in blood (hyperlipidaemia); tobacco use; high blood pressure; diabetes; obesity; physical inactivity; and recurrent infections.^11-13 As seen by the INTERHEART study, the effect of modifiable risk factors is consistent in men and women, across different geographic regions, and ethnic groups.¹¹ Family history, ethnicity, gender and age comprise the non- modifiable risk factors. Indeed, age is a major factor in our rapidly-aging society, linked to a progressive decline in the health state of the vasculature.¹⁴

Noteworthy, women compose a larger proportion of the elderly population with CVDs, influenced by sex-specific risk factors such as hormonal dysfunction, hypertensive disease during pregnancy or gestational diabetes.^{15, 16} Other factors influencing the risk of CVDs are psychosocial, like chronic stress and

socioeconomic status,¹⁷ use of certain medications; and environmental factors, such as particulate matter air pollution.¹⁸ In addition, multiple studies have shown that inflammatory biomarkers, in particular C-reactive protein, are robust predictors of cardiovascular events.¹⁹

13 Unhealthy diet Hyperlipidaemia Diabetes

Obesity Smoking

Lack of exercise Infections

Modifiable Non-modifiable

Family history Gender

Age

Ethnic group

Other factors Psychosocial Environmental

CVDs

Figure 1. Risk factors for cardiovascular diseases. Modified from Tzoulak et al. ²⁰

1.2. Atherosclerosis: a chronic inflammatory disease

As previously mentioned, atherosclerosis is the main subjacent cause of CVDs and therefore the leading cause of death worldwide. It is a progressive

inflammatory disease of the blood vessels characterised by two important

trademarks: lipid accumulation and inflammation. The name atherosclerosis has its origin in the Ancient Greek word athḗra meaning “gruel” or “porridge”, and sclerosis meaning “hardening”, due to the soft-like and hardened consistency of the atherosclerotic plaques formed in the arteries. In the last century,

atherosclerosis was considered to be a condition mainly characterised by excessive lipid accumulation in the vessel wall. However, recent experimental studies and clinical trials have demonstrated that inflammation controls many aspects of plaque structure and stability, and is able to trigger the thrombotic complications of atherosclerosis.^21-23

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1.2.1. Atherogenesis: fatty streak formation

Atherosclerosis begins with the accumulation of apolipoprotein B-containing lipoproteins (LPs) under the endothelial layer of the blood vessels (Figure 2). This phenomenon happens especially in areas where the normal laminar blood flow is focally disturbed, like in blood vessel bends or branch points.²⁴ Once inside the vessel wall, LPs and in particular low-density lipoprotein (LDL) are frequently modified by reactive oxygen species (ROS), generating oxidized LDL (oxLDL). This oxidation occurs via transition metals or different cellular enzymatic systems such as nitric oxide synthases, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, myeloperoxidases, lipoxygenases, and xanthine oxidases.^{25, 26} The endothelial dysfunction caused by retention of modified LPs in the vessel wall will lead to activation of the endothelium in a “response-to-retention”

manner.²⁷ This will trigger the recruitment of immune cells, namely neutrophils, mast and dendritic cells, monocytes and T cells to the endothelial layer, through an increase in permeability (via nitric oxide, endothelin, angiotensin II) and expression of adhesion molecules, like selectins, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1); cytokines like monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-8); and growth factors, such as platelet-derived growth factor (P-DGF) and macrophage colony-

stimulating factor (M-CSF).^{22, 28} In this context, recruited monocytes differentiate and engulf modified LPs through scavenger receptors, including steroid receptor RNA activator 1 (SR-A1) and cluster of differentiation 36 (CD36), becoming lipid- laden macrophages or foam cells. Foam cells secrete pro-inflammatory cytokines, i.e. IL-1, IL-6, tumor necrosis factor (TNF), chemokines such as MCP-1, and ROS,²⁹ increasing the inflammatory and oxidative burden. These early lesions termed fatty streaks can already occur at a young age, a process accelerated by the presence of the aforementioned risk factors.^{30, 31}

15 1.2.2. Atherosclerotic plaque development

If the inflammatory response is not able to neutralize or remove the triggering agents, fatty streaks will grow into more advanced atherosclerotic lesions called atheromas. This unresolved inflammation will elicit migration, proliferation and activation of more immune cells and vascular smooth muscle cells (VSMCs) which blend in the lesion forming the so-called fibrous cap.^{32, 33} Once in the atheroma, VSMCs will secrete extracellular matrix (ECM) components, such as collagen and elastin, which gives elasticity to the lesion. Remodelling and neovascularization also takes place within the atheroma to compensate for the thickening of the vessel wall, creating an intricate network of microvessels named vasa vasorum³⁴. Later on, the atherosclerotic plaque can evolve into a more complex injury where foam cells die and release proteases which degrade the ECM, and lipids crystallize into cholesterol crystals, causing more

inflammation.^{35, 36} The defective clearance of necrotic and apoptotic cells, and debris, results in the formation of a lipid-rich necrotic core, a feature of advanced plaques.

1.2.3. The vulnerable plaque and its complications

Persistent and progressive inflammatory cell infiltration, necrosis, apoptosis and ECM degradation will lead to plaque deterioration. The so-called culprit or

vulnerable plaques are responsible for final cardiovascular events. In this stage, two types of vulnerable plaques can be distinguished: rupture-prone plaques and erosion-prone plaques (Table 1).³⁷ Rupture-prone plaques are the most common form of plaque destabilization, and represent two thirds of fatal MIs and sudden cardiac deaths. Typically, a rupture-prone plaque is composed of an enlarged necrotic core separated from the vessel lumen by a weakened and thin fibrous cap. Fibrous cap thinning is caused by an imbalance between matrix synthesis from VSMCs and degradation by matrix metalloproteinases (MMPs), which break

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down collagen fibres. Inflammatory cytokines, namely interferon γ (IFN-γ), TNF-α, and IL-1β, lipid mediators and oxidative stress produced by the immune cells present in the atheroma, are responsible for the inhibition of collagen and induction of VSMCs apoptosis. In addition, apoptotic VSMCs can cause

recruitment of macrophages via IL-1α, IL-8 and MCP-1, and create a pro-coagulant environment due to loss of membrane anticoagulant components.^{38, 39}

Interestingly, recent studies suggest that VSMCs can also undergo phenotypic switching into macrophage- and mesenchymal stem cell-like cells that promote lesion expansion and instability.^40-42 Such phenotypic transdifferentiation

comprises loss of VSMC markers (e.g., actin alpha 2 smooth muscle; ACTA2), cell migration and proliferation. These processes promote the rupture of vulnerable plaques wherein the content of the necrotic core interacts with blood coagulation factors and activated platelets. The coagulation cascade is then triggered, causing thrombus formation (i.e. atherothrombosis) and blockage of the artery or smaller arteries downstream. This leads to complications that will depend on which arteries are blocked, the most common being MI in the heart, and transient ischemic attack (TIA) or stroke in the brain.

Less is known about eroded plaques, which represent one quarter of all acute coronary syndrome events. Erosion-prone plaques lack features associated with rupture-prone plaques, presenting thicker caps, large numbers of VSMCs, smaller necrotic cores, and little inflammation. Their ECM is more elastic, since there is a shift from collagen I towards collagen III, and the proteoglycan subtype

hyaluronan. These changes in ECM properties promote endothelial dysfunction due to detachment and reduced endothelial cell adhesion. Moreover, an increased number of neutrophils have been found near eroded plaques, where they seem to induce endothelial apoptosis, degrade ECM proteins, and release neutrophil extracellular traps (NETs) leading to a prothrombotic state and complications.^{37, 43}

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In fact, increased levels of NETs in the circulation have been associated with disease severity in patients with stable CAD.⁴⁴

Rupture-prone plaques Erosion-prone plaques Other plaque vulnerability factors

 Active inflammation

 Thin cap-fibroatheroma

 Calcification

 Endothelial dysfunction

 Fissured plaque

 Intraplaque haemorrhage

 Positive remodelling

 Endothelial denudation with thrombogenic proteoglycan substrate with/without thrombus

 Endothelial dysfunction

 Neutrophil extracellular traps (NETs)

 Lumen stenosis over 90%

 Hemodynamics (shear stress)

Table 1. Main characteristics of vulnerable plaques. Modified from Stefanadis et al.³⁷

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Figure 2. Stages in the development of atherosclerosis. Modified from Libby et al.³²

Acknowledgement to SERVIER Medical Art for use of medical art kits.

a. A healthy artery is composed of three layers: the tunica intima formed by a monolayer of endothelial cells in contact with the bloodstream; the tunica media, containing resident VSMCs surrounded by a complex extracellular matrix; and the adventitia, containing mast cells, nerve endings and microvessels.

b. After lipid retention, immune cells adhere to the endothelial layer and migrate into the intima. Moreover, monocytes maturate into macrophages, engulfing lipids and becoming foam cells inside the atherosclerotic lesion.

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c. Plaque progression also involves migration and proliferation of VSMCs into the intima and synthesis of extracellular matrix proteins such as collagen, forming the fibrous cap. VSMCs can also undergo phenotypic switching, promoting disease development. Atherosclerotic lesions can also contain cholesterol crystals and new microvessels from the vasa vasorum.

d. In more advanced plaques, foam cells and VSMCs can die by apoptosis, accumulating and forming a lipid-rich necrotic core. A weakened fibrous cap in later stages of atherosclerosis can lead to plaque rupture and thrombosis, ultimately causing MI or ischemic stroke.

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1.3. DNA damage response and repair during atherosclerosis

Each cell in our body suffers approximately 10⁴ – 10⁵ DNA lesions per day, highlightning the importance of DNA repair mechanisms to maintain correct cellular functions. The sources of DNA damage are numerous and often

classified as environmental, including UV light, radiation, and toxic chemicals;

and endogenous, such as ROS generated by cellular respiration and

lipoperoxidation, errors in replication and recombination, and spontaneous hydrolysis of nucleotide residues.⁴⁵ DNA damage affects base-pairing properties and consequently DNA functions, such as transcription.⁴⁶ If left unrepaired, DNA damage can therefore lead to mutations and cell death.

It is well established that DNA damage is involved in diseases such as cancer, CVDs, metabolic syndrome, neurological disorders, immunodeficiencies, and premature aging.⁴⁷ However, it is only in the last decades that a causal link

between DNA damage and atherosclerosis has been postulated. Evidence support that VSMCs and inflammatory cells within atherosclerotic lesions in both human and animal models express DNA damage markers, triggering cell senescence and apoptosis, which contribute to plaque instability.⁴⁸ Genomic lesions occur in both nuclear and mitochondrial DNA in atherosclerosis, and animal studies have shown that mitochondrial dysfunction is enough to promote atherosclerosis. To guarantee the correct conservation of genetic information, all organisms have evolved complex and highly specialized means to repair this inescapable damage.

Cells rely on the DNA damage response (DDR), an orchestrated cascade of sensors, transducers and effectors aimed to repair DNA lesions. The DDR network is composed of several distinctive repair pathways that are activated depending on the damaging agent, type of damage and position in the cell cycle where the DNA insult occurred.⁴⁵ Specifically in atherosclerosis, oxidative stress due to ROS generation is the major source of DNA damage. This type of lesion

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can be repaired by the highly conserved base-excision repair (BER) pathway, which repairs oxidation-damaged DNA bases.

1.3.1. Base-excision repair pathway

The BER pathway can be initiated by one of 11 DNA glycosylases.⁴⁹ Mammalian DNA glycosylases are subdivided into four superfamilies based on their structure:

uracil DNA glycosylases (UDGs); helix-hairpin-helix (HhH) glycosylases; 3-methyl- purine glycosylases (MPG); and endonuclease VIII-like (NEIL) glycosylases. During repair, DNA glycosylases flip the damaged base out of the DNA helix by cleavage of the N-glycosylic bond between the base and the deoxyribose moieties of the nucleotide residue. This creates an apurinic/apyrimidinic (AP)-site which is removed by an AP-endonuclease or an AP-lyase which cleaves the DNA strand.

Later, a phosphodiesterase excises the remaining phosphate residue from the deoxyribose, generating a gap which is filled with the new base by DNA polymerase, and finally sealed by DNA ligase (Figure 3).⁵⁰

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Figure 3. Subpathways in base excision repair (BER). BER comprises either short-patch repair or long-patch repair which use different proteins downstream of the base excision. The repair process occurs in four core steps: (1) excision of the base, (2) incision, (3) end processing, and (4) repair synthesis, including gap filling and ligation. Reprinted by permission from CSH Perspectives.⁵⁰

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1.3.2. DNA glycosylase NEIL3: a multipurpose enzyme

DNA glycosylases are key enzymes in the context of diseases such as cancer, metabolic syndrome, and neurodegeneration.⁵¹ One of them, Nei-like 3 (NEIL3), has been shown to be upregulated in both human asymptomatic and

symptomatic atherosclerotic plaques.⁵² Nevertheless, the role of NEIL3 in

atherosclerosis development is not yet fully elucidated, and this will be the main focus of the present thesis.

NEIL3 gene and protein structures

NEIL3 as well as its other counterparts NEIL1 and NEIL2, is only expressed in vertebrates.⁵³ NEIL proteins are homologous to the E. coli DNA glycosylases Formamidopyrimidine (Fpg) and Endonuclease VIII (Nei) proteins, sharing structural similarities and partly overlapping substrate affinity,⁵⁴ hence highly conserved during evolution. Human NEIL proteins were firstly identified by several research groups more than 15 years ago.^55-58 While NEIL1 and NEIL2 have been thoroughly characterised,⁵³ the biological role of NEIL3 needs to be better understood.

A great effort has led to the characterization of both human NEIL3 and mouse Neil3 gene and protein in the last years (Table 2).^59-61 The human NEIL3 gene is located on Chromosome 4q34.3 and encoded by the plus strand. On the minus strand, NEIL3 locus is flanked by two genes, the Aspartylglucosaminidase (AGA) gene and the Vascular endothelial growth factor c (VEGFC) gene. NEIL3 is about 53.25 kb consisting of 10 exons which result in a full-length protein with 605 amino-acids, almost twice the size of its bacterial counterparts,⁵⁴ and a molecular weight of 68 kDa. On the other hand, the murine Neil3 gene is located on

Chromosome 8 B1.3 and encoded on the minus strand, also flanked by Aga and Vegfc. Neil3 contains 11 exons which full-length protein comprises 606 amino-

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acids and has a molecular weight of 67.41 kDa.⁶² Full-length human NEIL3 and murine Neil3 proteins share at least 74% sequence identity. The promoter region of NEIL3 displays similar characteristics to the cell cycle-regulated genes (GC-rich, but TATA-less), and can activate NEIL3 transcription upon oxidative and

inflammatory stress in a epigenetic-like manner.⁶³ Several transcription factor binding sites have been predicted in close proximity to NEIL3 transcription initiation site, including cell cycle dependent element/cell cycle gene homology region cis-regulatory elements. NEIL3 expression patterns are shown to be upregulated in early S phase, with the highest levels in G2 phase, in several human cell lines, supporting the role of NEIL3 in replication associated DNA repair in proliferating cells.⁶⁴

NEIL3 has distinct characteristics from other Fpg/Nei homologs. The N-terminal half of the Neil3 proteins are homologous to the bacterial Fpg/Nei proteins, with the signature H2TH motif and the zinc finger motif for DNA binding. However, unlike most of the Fpg/Nei family members, Neil3 proteins contain a unique catalytic valine in the N-terminus and additional zinc finger motifs in the C- terminus. Expression of Neil3 transcripts seem to be tissue-specific and is highly expressed in mouse hematopoietic tissues such as spleen, bone marrow, thymus, B cell lines, and brain, where it was detected in regions harbouring progenitor cells especially during mouse embryogenesis, as well as in human testis and primary malignant melanomas associated with metastasis.^{53, 65} At the subcellular level, NEIL3 activity has only been detected in the nucleus, and not in

mitochondria like its other family members, NEIL1 and NEIL2.

NEIL3 functions

Although bifunctional, Neil3 mainly acts as a monofunctional DNA glycosylase with a very weak lyase activity, requiring the action of an AP endonuclease (APE1)

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to cleave the AP site.⁶⁶ Neil3 can excise both oxidized-damaged pyrimidines and purines, and has a broad substrate recognition spectrum in different DNA structures.⁵⁴ Regions of ssDNA such as looped structures and structures similar to replication forks are preferred by Neil3, where it removes

spiroiminodihytantoin (Sp), guanodinohydantoin (Gh) and thymine glycol (Tg), as well as the ring-saturated pyrimidines, dihydrothymine (DHT) and dihydrouracil (DHU), and the oxidized pyrimidines, 5-hydroxycytosine (5-OHC) and 5-

hydroxyuracil (5-OHU). Neil3 can also remove Sp and Gh in dsDNA, as well as FapyG and FapyA in γ-irradiated DNA. Interestingly, Neil3 can also recognize and excise Tg in G4 structures as well as in dsDNA and ssDNA telomeric sequences, essential structures in gene regulation.⁶⁷ Neil3 is also involved in the control of gene expression as it has been shown to work as a dynamic epigenetic reader of 5-methylcytosine (m⁵C), a methylation marker of oxidized derivatives.⁶⁸

NEIL3 in disease development

Considering the unique characteristics of NEIL3, it has been hypothesized that its biological functions could go beyond traditional DNA repair. Indeed, recent studies have demonstrated a role of NEIL3 in autoimmunity,⁶⁹ neurogenesis,⁷⁰ MI,⁷¹ and cancer.⁷² We have previously shown that atherosclerotic carotid plaques have increased levels of NEIL3 compared to controls,⁵² and that the NEIL3

rs12645561 SNP TT genotype was associated with increased risk of MI in a nested case-control study.⁷³ More recently, we have demonstrated that Neil3 deficiency in mice on a high-fat diet (HFD) promotes atherosclerosis, with Neil3 balancing lipid metabolism and macrophage function.⁵² Based on these findings, we believe that the biological role of NEIL3 surpasses that of a DNA glycosylase in traditional DNA repair, possibly playing an important role in gene expression regulation during atherosclerosis development.

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Table 2. Main structural and biological features of DNA glycosylase NEIL3/Neil3 gene and protein. Modified from Liu et al.⁵³

Feature Human NEIL3 Mouse Neil3

Chromosomic location 4q34.3 8 B1.3

Gene size, number of exons 53.25 kb, 10 exons 52.2 kb, 11 exons Protein size and weight 605 aa, 68 kDa 606 aa, 67.41 kDa

Protein structure N-terminus with signature H2TH motif, zinc finger motif for DNA binding, and unique catalytic valine; C-terminus with additional zinc finger motifs

Subcellular location Nucleus

Tissue expression Highly expressed in hematopoietic tissues e.g., spleen, bone marrow, thymus, B cell lines, brain (mouse), as well as testis and metastasis-associated primary malignant melanomas (human)

Preferred lesions Sp, Gh, FapyG, FapyA, MeFapyG *

Other lesions recognized DHU, DHT, 5-OHU, 5-OHC, 5-OHMH, Tg, 8-oxoA, AP **

Preferred DNA structures Large bubble and single-stranded DNA, small bubble, fork DNA, duplex DNA, G4 structures, dsDNA and ssDNA telomeric sequences

DNA glycosylase

functionality Bifunctional, but acts mainly as monofunctional (requires APE1)

Other functions described m⁵C epigenetic reader

Other possible roles in Autoimmunity, neurogenesis, neurological disorders, MI, cancer, atherosclerosis

* Sp, Spiroiminodihydantoin; Gh, Guanidinohydantoin; FapyG, 2,6-diamino-4-hydroxy-5- formamidopyrimidine; FapyA, 4,6-diamino-5-formamidopyrimidine; MeFapyG, 2,6- diamino-4-hydroxy-5-N-methylformamidopyrimidine.

** DHU, 5,6-dihydrouracil; DHT, 5,6-dihydrothymine; 5-OHU, 5-hydroxyruacil; 5-OHC, 5- hydroxycytosine; 5-OHMH, 5-hydroxy-5-methylhydantion; Tg, thymine glycol; 8-oxoA, 7,8- dihydro-8-oxoadenine; AP, apurinic or apyrimidinic site.

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1.4. Epitranscriptomic regulation: an unexplored path in atherosclerosis

According to the Central Dogma of molecular biology formulated by F. Crick in 1965, DNA carries the genetic information transferred to messenger ribonucleic acid (mRNA) molecules which are the template for protein production.⁷⁴ By this principle it is assumed that protein levels reflect mRNA levels. Nevertheless, studies have shown that around 30–40% of the variance in protein abundance is not explained by mRNA quantity.⁷⁵ Moreover, it is known that DNA and its correspondant mRNAs show extensive sequence differences in the human transcriptome.⁷⁶ This highlights the existence of a complex post-transcriptional regulation which is crucial in the fine-tuning of gene expression, and therefore in disease.

Epitranscriptomics, the field studying the set of post-transcriptional RNA modifications and its regulation, is a rapidly developing-research field studying modifications that affect RNA structure, localization, and function. Projects tailoring mRNA sequencing have demonstrated that almost every transcript has RNA modifications and editing sites, and these can be dynamically regulated throughout an organism’s life.⁷⁷ Their presence across species has been postulated to be a sign of evolutionarily conserved molecular toolboxes

modulating gene expression in response to environmental cues.⁷⁸ More than 170 different RNA modifications have been described to date.⁷⁹ This list is ever- increasing and has been shown to affect all types of RNA, including coding RNA (mRNA) and non-coding RNA, i.e., transfer RNA (tRNA), ribosomal RNA (rRNA), and long and small noncoding RNA (lncRNA and sncRNA, respectively).⁸⁰

Methylation, isomerization, thiolation or even addition of complex groups like aminoacids are some of the chemical changes that RNA bases can contain.

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Despite RNA modifications being described decades ago,⁸¹ we have just begun to understand them. In the last years, RNAmodifications have been increasingly mapped thanks to improved methodology, specially next-generation sequencing and mass spectrometry.⁷⁸ They can be classified into RNA editing and covalent RNA modifications.

1.4.1. RNA editing

RNA editing is a process in which RNA nucleotides are enzymatically modified in the co-transcriptional and post-transcriptional stages of gene expression. The most abundant types of RNA editing in mammals are the deamination of adenosine to generate inosine (A-to-I editing) catalyzed by the protein family adenosine deaminases acting on RNA (ADARs); and the deamination of cytidine into uridine (C-to-U editing), catalyzed by the cytidine deaminases

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBECs).

RNA editing can affect several stages of gene expression, such as splicing, RNA stability, localization, microRNA function, and translation;⁷⁷ and cellular

processes like host cell defense, antiviral defense, immunity, or recoding of transcripts.^{82, 83}

1.4.2. Covalent RNA modifications

Covalent RNA modifications have been found inside different types of RNA, containing modifications in all basic RNA bases. For example, known covalent RNA modifications are the 5’7-methylguanosine (m⁷G) cap and the poly(A) tail in the 3’ end which is added during mRNA processing, and involved in transcript stability, splicing, polyadenylation, nuclear export, and translation initiation.

Eukaryotic tRNA molecules contain the largest number of epitranscriptomic modifications with the widest chemical diversity, including an average 13

modifications.⁸⁴ Modifications in tRNA influence processes such as efficiency and

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fidelity of decoding, folding, cellular stability, and localization. Ribosomal RNA comprises more than 220 modification sites in humans, mainly accumulated in functional regions of the ribosome such as the peptidil transfer center,

fundamental for decoding, peptidyl transfer, and translocation.⁸⁵ Moreover, it has been seen that rRNA biogenesis can be blocked in the absence of certain RNA modifications, e.g., pseudouridylation.⁸⁴ Alhtough the number of studies on the RNA epitranscriptome is rapidly growing, the exact function of many

epitranscriptomic modifications and their regulators is still elusive.

1.4.3. Epitranscriptomic modifications in CVDs

Various research groups have shown RNA changes to be linked to human

diseases.⁸⁶ Aberrant RNA editing has been associated with severe human diseases, such as cancer and neurological disorders.⁸⁷ Interestingly, a recent study has shown that RNA editing is also involved in atherosclerotic disease.⁸⁸ In this study, the authors demonstrated that A-to-I editing controls gene expression of

cathepsin S (CTSS), an enzyme with cysteine protease activity associated with angiogenesis and atherosclerosis, highly edited in human endothelial cells. The 3’

untranslated region (3’UTR) of the CTSS transcript contains two Alu inverted repeats which ADAR1 binds to. This editing recruits the RNA-binding protein Human antigen R (HuR) to the 3’UTR of the CTSS transcript, thus controlling CTSS expression. In addition, ADAR1 levels and CTSS RNA editing have been associated with changes in cathepsin S in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, CAD, aortic aneurysms and advanced carotid atherosclerosis. Moreover, a new study in mice and human samples showed A-to-I editing of microRNA-487b, a novel, proangiogenic and highly edited microRNA involved in postischemic neovascularization.⁸⁹

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The most common and best studied covalent RNA modification is the

methylation of the N⁶ position of adenosine (m⁶A).90 In 2012, two pivotal studies by Dominissini and Meyer showed for the first time the human m⁶A

epitranscriptomic landscape, revealing m⁶A distribution in the coding and 3’

unstranslated regions with a significant enrichment upstream of the stop codon of mRNA.^{91, 92} These studies have also identified the key players mediating m⁶A levels in mRNA. Methyl groups are added by the WMM complex, formed by methyltransferase-like (METTL) 3, METTL14, Wilms tumour 1-associated protein (WTAP), and other RNA-binding motif (RBM) proteins. m⁶A can be demethylated by the AlkB family members Fat mass and obesity-associated (FTO) protein, and AlkB homologue 5 (ALKBH5). In addition, some proteins, such as YTH domain family (YTHDF) proteins, can bind to m⁶A and affect mRNA metabolism, e.g., alternative splicing, translation, and decay. Importantly, m⁶A has been involved in several diseases regulating, e.g., pluripotency transcription factors during the onset of several types of cancer, circadian clock gene expression during diabetes, and the normal hypertrophic response of cardiomyocytes during heart failure.^93-

95 However, literature is scarce or non-existing on the role of the

epitranscriptome and its regulatory enzymes in atherosclerosis. In this work, we investigate a possible role of the modification m⁶A and regulatory enzymes involved in RNA modifications, which will be discussed in the coming sections of this thesis.

Lastly, coding and non-coding RNA can be affected by either editing or covalent modifications in their stability, localization, and functions. This highlights the importance of epitranscriptomic regulatory processes as a novel checkpoint in gene expression during health and disease development. Exploring the RNA epitranscriptome could offer a new helpful insight into mechanistical processes

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involved in human gene regulation during atherogenesis. Ultimately, discoveries in the epitranscriptomics field could serve as diagnosis, stratification, and therapeutic tools for different diseases, including CVDs.

32 2. AIMS OF THIS WORK

The overall aim of this work was to study the underlying cause of atherogenesis at a molecular level. We investigated the role of the DNA glycosylase NEIL3 as a mediator in the onset and development of atherosclerosis. Moreover, we

investigated clinical atherosclerosis using a novel approach, looking at the RNA epitranscriptome and its regulation in human atherosclerotic plaques. Specific aims of each study:

Study I. To examine the role of NEIL3 deficiency and its implication in VSMC phenotype and function in an atherosclerosis-prone mouse model as well as in NEIL3-abrogated human primary aortic VSMCs.

Study II. To explore the role of the RNA modification N6-methyladenosine (m⁶A) and regulatory enzymes involved in post-transcriptional RNA modifications during the development of human atherosclerosis.

33 3. SUMMARY OF RESULTS

Paper I. DNA glycosylase Neil3 controls vascular smooth muscle cell biology during atherosclerosis development

The DNA repair enzyme NEIL3 has been previously suggested to have a role in the development of atherosclerosis, but the mechanisms are far from clear. In this study, we assess the role of Neil3/NEIL3 in atherogenesis by combining different experimental approaches, including studies on Neil3-deficient

atherosclerosis-prone mice and in vitro experiments in NEIL3-deficient primary human aortic VSMCs. Our main findings are:

 Increased atherosclerosis in Apoe^-/-/Neil3^-/- mice as compared to Apoe^-/- mice with no differences in circulating lipids, cytokines or blood pressure

 Increased medial VSMC area and layer disorganization in Apoe^-/-/Neil3^-/- mice

 Increased aortic VSMC proliferation in Apoe^-/-/Neil3^-/- mice and NEIL3- abrogated human primary aortic VSMCs, with no alterations in genomic DNA integrity

 Neil3/NEIL3 deficiency increases aortic VSMC lipid uptake and phenotypic modulation towards a more secretory macrophage-like cell profile

 Neil3/NEIL3 deficiency-dependent VSMC proliferation involves activation of the Akt signalling pathway

Our results suggest that NEIL3/Neil3 is a novel player in the regulation of VSMC biology, controlling cell proliferation and transdifferentiation. Lack of Neil3 in atherosclerosis-prone mice promotes a pro-atherogenic macrophage-like VSMC phenotype through non-canonical mechanisms that might involve the Akt signalling pathway.

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Paper II. N6-methyladenosine in RNA of atherosclerotic plaques: an epitranscriptomic signature of human carotid atherosclerosis

More than 170 post-transcriptional RNA modifications have been shown to affect the localization, processing and function of cellular RNAs, and the dysregulation of RNA modifications has been linked to human diseases, such as cancer and MI.

In this study, we examined a variety of RNA-modifying enzymes and the levels of the epitranscriptomic modification N6-methyladenosine (m⁶A) in human

atherosclerotic samples and donor controls. Our main findings are:

 Ribosomal RNA methyltransferase levels are altered in human atherosclerotic lesions

 Dysregulated m⁶A-mRNA modulators in human atherosclerotic lesions

 Decreased m⁶A RNA levels in human atherosclerotic lesions

We demonstrate that the levels of the RNA modification m⁶A as well as some RNA-modifying enzymes are regulated in atherosclerosis development, findings which could help creating new prognosis, stratification, and treatment strategies.

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4. SELECTED METHODOLOGICAL CONSIDERATIONS

4.1. Patient and control samples: “bedside to bench – and back to bedside”

Translational medicine is an interdisciplinary branch of the biomedical field supported by three main pillars: bench-side, bedside and community. Its goal is to combine disciplines, resources, expertise, and techniques within these pillars to promote innovative developments in prevention, diagnosis, and therapies to improve the global healthcare system.⁹⁶ Therefore, collaboration between clinical and basic researchers is essential to accomplish this goal.

In our studies, clinical material was collected from symptomatic patients with carotid atherosclerosis (presenting ischemic stroke, transient ischemic attack, or amaurosis fugax). These patients underwent carotid endarterectomy surgery at Østfold Hospital Trust in Fredrikstad (Norway), where atherosclerotic plaques were excised and snap-frozen, and analysed in Paper II. Thereafter, each plaque mechanically split in two parts: a central part representing highly developed atherosclerosis (i.e., ‘advanced’), and the distal part of the same plaque, representing early signs of atherosclerosis (i.e., ‘early’). This was done on the basis of known morphological differences between different sections of an atherosclerotic plaque,⁹⁷ as well as macroscopical (eyesight) and microscopical (immunostaning) differences when handling and analysing the samples. Due to ethical considerations, we could not acquire non-atherosclerotic carotid artery tissue as a control. We obtained then common iliac arteries of young deceased organ donors without known CVDs, provided by the Oslo Biobank. The common iliac arteries originate from the bifurcation of the aorta, which runs from the heart downwards the body. The smaller geometry of these arteries compared to the ascending aorta greatly influences blood flow and therefore the

predisposition for atherogenesis.⁹⁸ Moreover, given the younger age of the

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donors, one would imagine that stiffness and other structural qualities of these blood vessels to be different from those of older patients with atherosclerosis.

Nevertheless, common iliac arteries are a more accessible control tissue group.

Another limitation of this method is a possible ‘mechanical’ bias when splitting the sample into ‘advanced’ and ‘early’ atherosclerotic plaques. However,

considering the general difficulties in biobanking, this collection of clinical samples is very valuable and unique to study human atherosclerosis, since it represents very closely the in vivo situation.

4.2. Animal models of atherosclerosis

Several animal models have been used in atherosclerosis research, such as mice, rats, rabbits, pigs, and non-human primates. Murine models are the most used animal model in atherosclerosis studies.⁹⁹

4.2.1. Murine models of atherosclerosis

Mice are an attractive animal model for several reasons: (i) they can be genetically engineered to identify genetic links for atherosclerosis susceptibility, (ii) their small size and low cost of housing compared to other laboratory animals, allowing for bigger experimental numbers to ensure statistical significance, (iii) mice have a short gestation time, and (iv) in studies involving the testing of certain drugs where cost or availability is an issue, mice would require smaller amounts of the drug than the same studies conducted in larger mammals.¹⁰⁰ However, mice present limitations such as differences in lesion distribution, and small quantity of blood that can be withdrawn, and small lesion samples for characterization of both protein and RNA analysis. In addition, mice do not develop atherosclerosis naturally. Nevertheless, this can be overcome by using fat-rich diets (i.e., western diet) or by creating genetically modified mice deficient in genes involved in the clearance of cholesterol and triglyceride-rich lipoprotein

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particles from the blood, such as apolipoprotein E (Apoe) or LDL receptor (Ldlr)- deficient mice. Nowadays, both Apoe and Ldlr-deficient mice are the most used murine models of atherosclerosis (Table 3).

The Apoe knockout (Apoe^-/-) mouse is one the most commonly used mouse models of atherosclerosis since it was created by targeted gene inactivation in 1992, and was used in Paper I. APOE is a plasma glycoprotein on the surface of most lipoproteins. An advantage of this mouse model is the spontaneous development of hypercholesterolemia due to increased chylomicron remnant, very low-density lipoprotein (VLDL) and LDL cholesterol levels in blood. Thus, Apoe^-/- mice can develop severe atherosclerosis rapidly, and although the rate of atherosclerotic lesions development varies between facilities, macrophage foam cells can be present in the aortic root within a month, growing into more

complex lesions overtime. A saturated fat-enriched diet, e.g., Western diet, is used to accelerate atherosclerosis, although it can alter or mask the outcomes of the disease due to the induction of increased levels of cholesterol that overstress the system. Furthermore, Apoe can influence macrophage biology, immune functions, and adipose tissue biology,¹⁰¹ which should be carefully considered when interpreting results.

The LDL receptor-deficient (Ldlr^-/-) mouse is the other most frequently used atherosclerotic model. LDLR is a cell surface receptor in liver cells that binds to APOE to clear lipoprotein particles from blood, where Ldlr deficiency affects lipoprotein uptake and clearance.⁹⁹ This model is more suitable to study whether a particular gene influences bone marrow-derived cells or non-hematopoietic vascular cells, since the extracellular macrophage Apoe from Apoe bone marrow- donors can rescue the atherosclerotic phenotype of the Apoe^-/- hosts.¹⁰²

38 Phenotype Apoe^-/- mice

B6.129P2-Apoe^tm1Unc/J Ldlr^-/- mice

B6.129S7-Ldlr^tm1Her/J Hypercholesterolemia ~400 mg/dl; 5X higher than

controls

~200-275 mg/dl; 2-3X higher than controls

VLDL Greatly increased Modestly increased

IDL/LDL Modestly increased Greatly increased

HDL Decreased Modestly increased

Spontaneous lesions At 6 months None

Diet induced lesions Fast, proportional to dietary cholesterol intake

Fatty streaks on the aorta Spontaneous plaques At 3 months: fatty streaks in

proximal aorta; at 8 months:

aortic plaques

None (needs Western diet feeding)

Diet induced plaques Abundant and large (by 14

weeks after atherogenic diet) Medium (after 12 weeks on a high cholesterol diet) Other Abnormal spatial learning Metabolic syndrome on a

high cholesterol diet

Table 3. Phenotypes of the two most popular Apoe and Ldlr-deficient strains used for atherosclerotic research. Both are backcrossed to the atherosclerosis-susceptible C57BL/6J

genetic background. Modified from Yeadon, J.¹⁰³ Here, ‘lesions’ refer to any atherosclerotic lesion or insult, and ‘plaques’ refer to atheromas, protruding atheromas, atherosclerotic debris, and plaque. VLDL, very low-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low- density lipoprotein; HDL, high-density lipoprotein.

In spite of technical constrains, genetically modified mice that develop

atherosclerosis are without doubt an essential experimental tool in translational atherosclerosis research. Indeed, a recent review of more than 9000 publications in the mouse literature indicates that atherosclerosis mouse models are of great value, especially for revealing essential molecular mechanisms involved in

atherogenesis¹⁰⁴. Using genome-wide association studies (GWAS), the authors of the aforementioned study showed that both mouse models of atherosclerosis and human patients share many disease features, including key genes and

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pathways related to cholesterol metabolism, inflammation, blood pressure, coagulation and vascular functions. Likewise, mouse models have been useful for elucidating the effect of individual genes in atherosclerosis, with following

confirmation of human relevance in many instances. For example, mouse studies provided the first evidence that liver pro-protein convertase subtilisin/kexin type 9 (PCSK9) increases plasma LDL cholesterol by reducing expression of LDL

receptors. As a result, neutralizing PCSK9 antibodies have been developed and preliminary data from recent randomized controlled trials suggest that PCSK9 antibodies are associated with a significant reduction in atherosclerotic events.¹⁰⁵ It is worth mentioning that recent guidelines are encouraging the inclusion of both male and female mice in preclinical studies, as sex differences affect the outcome of both human and murine atherosclerosis (due to e.g., sex hormones, and lipid metabolism).^{99, 106} We did not investigated, however, the effects of gender differences in our studies mainly as a result of logistic limitations.

Nevertheless, this variable should be considered in future studies aiming for a better understanding of the disease in both genders.

4.2.2. The Apoe^-/-/Neil3^-/- mouse model

As mentioned in the introductory chapter of this thesis, our group has previously shown that NEIL3 is involved in clinical atherosclerosis⁵². To further investigate the role of the DNA glycosylase NEIL3, a genetically modified constitutive Neil3 knockout (Neil3^-/-) mouse model was created. The Neil3-deficient mouse model was generated by targeted disruption of the Neil3 locus by replacing exons 3-5, harbouring the sequence encoding the conserved DNA-binding domain, with a positive selection cassette for neomycin resistance. Neil3-deficient mice were generated by injection of transfected 129-derived embryonic stem (ES) cells into blastocysts derived from C57BL/6 inbred mice, which were transferred to

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pseudopregnant foster mothers. The chimeric offspring carrying germ cells containing the transfected ES cell will transfer not only the induced null mutation, but also the 129-derived genes flanking the modified region to its progeny. To overcome possible genetic variations, the resulting heterozygous 129/BL6 is backcrossed into the BL6 strain for several generations. There is a consensus that more than 10 backcrosses should be enough to remove remaining recombination-dependent genetic variants occurred during homozygous

recombination between the DNA from ES cells and its foreign transfected DNA.

These Neil3^-/- mice have been reported to be viable, fertile and healthy during their lifespan.¹⁰⁷ In collaboration with Prof. Bjørås’ group, we have created a double knockout constitutive mouse model deficient in DNA glycosylase Neil3 with an atherosclerosis-prone Apoe-deficient background. In Paper I, we have used the Apoe^-/-/Neil3^-/- mouse model to help us elucidate the functions of DNA glycosylase Neil3 in the onset and progression of atherosclerotic plaque

formation and atherosclerosis.

All animal experiments performed and described in Paper I were approved by the Norwegian National Research Authority, in agreement with the European

Directive 2010/63/EU for the use of animals for scientific purposes,¹⁰⁸ and the Guide for the Care and Use of Laboratory Animals.¹⁰⁹

4.3. Histology and immunohistochemistry

Standardized methodology for the quantification of murine atherosclerosis is essential for data reproducibility and interpretation of results across studies. In Paper I, quantification of atherosclerosis was done in a similar manner as we have previously reported.⁵² Aortic root is the most common region for

atherosclerosis quantification,⁹⁹ since it is the first place of the aorta developing atherosclerosis. In spite of their small size, murine atherosclerotic lesions can be

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easily identified using several types of staining, which can be further quantified by image analysis software.

In our studies, we chose to use Oil Red-O (ORO) in addition to haematoxylin staining for quantification of murine atherosclerosis burden in the aortic root.

These techniques are widely chosen due to their ease of use, reliability, and the large amount of information they provide.^{110, 111} Moreover, this combination of techniques is very useful to acquire information about the area of the aorta affected by atheroma, the area of each individual lesion, and the aortic

intima/media ratio. ORO is a fat-soluble bright red diazo dye which effectively stains the most hydrophobic and neutral lipids in cells, such as cholesterol esters, triglycerides and diacylglycerols, but cannot stain the polar lipids (phospholipids, sphingolipids, and ceramides) present in the cellular membranes. Therefore, ORO is useful to detect pathological deposits of fat present, for example, in the

atheromas. Haematoxylin is used as a counterstain, for detection of cell nuclei and proteoglycans and can provide useful information on both cell type and cellular density of the plaque. ORO staining can be performed on fresh, frozen, or formalin-fixed tissue samples, but not with paraffin-embedded samples since the chemicals used for deparaffinization will wash out most of the fats in the samples. In our studies, snap-frozen paraformaldehyde (PFA) fixed samples were used to quantify murine atherosclerosis. Murine hearts were snap-frozen in OCT compound to preserve integrity of chemical and structural components of the artery. The sample sectioning was performed under low temperature (-21°C to - 22°C) in a cryostat. The cryostat allows for reorientation of the specimen holder during sectioning, thus simplifying correct sectioning perpendicular to the aortic valve.

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Immunohistochemistry (IHC) is used to identify the distribution of specific proteins within individual cells from a fixed cell culture or tissue.¹¹² Therefore, we used IHC in Paper I to detect the presence of certain proteins and cell types involved in atherosclerosis. The selection of primary antibodies is a critical step for specific detection of the antigen of interest. Antigen detection is done

directly i.e., the primary antibody is linked to a label, or indirectly i.e., the label is linked to a secondary antibody which is directed against the invariant portion of the primary antibody. The antigen-antibody complexes are visualized under the microscope by means of either immunofluorescence, using fluorophore-

conjugated antibodies, or chemiluminiscence, using antibodies coupled to horse- radish peroxidase (HRP). Antigen detection is greatly improved by antigen

retrieval methods, which act by removing protein cross-links formed by fixation agents (e.g., PFA) that mask antigen sites. This step is not necessary if the sample has been snap-frozen in OCT or liquid nitrogen. In this context, the fixation agent depends on the nature of the tissue and the antigen detection method available. One of the main concerns in IHC is to overcome background signal and non-specific staining. For instance, this is improved by optimizing fixation

methods, antibody dilutions, time intervals, and use of blocking agents and wash buffers. Additionally, the incorporation of positive and negative controls in

staining experiment is essential to determine the specificity of the antibody used.

4.4. Experimental cell-based models

The body consists of an intricate set of interactions between cells and molecules, which are difficult to study in many instances. Experimental cell-based disease models are less complex systems to explore specific molecule interactions. They are easy to manipulate, reproduce, and interpret, and help us generate and test hypotheses. Therefore, they provide us with valuable mechanistic insights into

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disease onset and progression in atherosclerosis research. Cell-based models can be categorized into in vitro cell-based models and ex vivo cell-based models.

While in vitro models use single cells either in a monoculture to study cell mechanisms, or co-cultures (a combination of different cell types) to study cell interactions, ex vivo models use cells derived from explanted tissues of an organism. In addition, these two types of cell-based models can be combined to exploit their advantages. Moreover, as cell models lack cell to cell interactions and physiological homeostasis, cell-based experiments should be ideally

complemented by in vivo experiments in laboratory animal models, which is of particular interest due to the multifactorial nature of atherosclerosis. Finally, cell-based models do not only offer a better understanding of complex disease mechanisms, but also could provide us with a tool for personalized

atherosclerosis treatment.

4.4.1. Cell-based models in atherosclerosis research

In Paper I, we used cell-based models to investigate the role of NEIL3 in

atherosclerosis, including commercially available human primary aortic VSMCs isolated from plaque-free regions of the human aorta. Primary cells offer advantages over the use of transformed, immortalized cell lines. Though they have a finite lifespan and are more sensitive to work with, primary cells provide us with more reflective data of the in vivo situation. In contrast, most cell lines have been in culture for years and accumulate mutations, thus differing from their tissue of origin.¹¹³ Ex vivo models preserve the original tissue architecture and cell types from the in vivo setting. Therefore, they are a useful tool to study biological mechanisms under controlled conditions and serve as a bridge

between the in vivo and the in vitro cell-based systems. Also in Paper I, we

performed an ex vivo assay with aortic tissue from Apoe^-/-/Neil3^-/- and Apoe^-/- mice

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to investigate the proliferation profile of VSMCs in murine atherosclerosis, and therefore establish a link between our observations from in vivo and in vitro experiments.

4.4.2. Generation of a NEIL3-knockdown cell-based model

Generation of cells with partial or total abolition in a gene’s expression is an effective way to investigate the function of a given gene product. There are several strategies to generate such cells such as plasmid-mediated transfection of RNA-guided systems, e.g., CRISPR/Cas9. However, this technology is still challenging and very time-consuming.¹¹⁴ Antisense-mediated exon skipping is a useful, cheap and easy tool to study gene function as well as being a promising therapeutic application for multiple diseases, including atherosclerosis.¹¹⁵ The basis of this technology is antisense oligonucleotides (AONs). AONs can induce targeted degradation of mRNA or modulate splicing by hiding specific sites essential for exon inclusion from the splicing machinery.

In Paper I, we designed and used synthetic RNA-DNA hybrid AONs (also called GapmeRs) for targeted silencing of NEIL3 mRNA in vitro. NEIL3 GapmeRs were designed following specific criteria,¹¹⁶ including addition of sequence motives essential for exon inclusion into the mRNA, as well as resistance against endo- and exonucleases, ribonuclease (RNase)-H induced cleavage and appropriate thermodynamic properties. It is important here to target exons, as introns will be eliminated later on in mRNA maturation steps. GapmeRs have a hybrid ssRNA- dsDNA-ssRNA structure (Figure 4). While GapmeRs will hybridize to their target mRNA sequence due to sequence complementarity, a gap will be created in the non-complementary dsDNA-mRNA region. This will induce the cleavage of the hybrid by RNase H, therefore abolishing target mRNA expression.

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Figure 4. Antisense-mediated exon skipping mode of action.

When using GapmeRs we must remember that these have a non-lasting effect, as they are not integrative molecules, remaining episomal and being degraded or diluted over time. Therefore, considerable and reiterative amounts of GapmeRs are needed to prevent target mRNA expression. Moreover, some gene products will remain intact at the time of treatment, since GapmeRs only target mRNA and not protein.

4.5. Mass spectrometry for detection of N6-methyladenosine and regulatory proteins in atherosclerotic plaques

Mass spectrometry (MS) is a powerful analytical method in which a sample preparation is fragmented into ions which are sorted based on their mass-to- charge ratio. After the ions have been detected, it is possible to reconstruct the sequence of a molecule of interest (e.g., peptides or oligonucleotides) by

extrapolating information from the fragmentation spectra with general

knowledge on how a particular class of biomolecules fragments.¹¹⁷ In paper II, we used MS coupled with high-performance liquid chromatography (HPLC) for

detection of the RNA modification N6-methyladenosine (m⁶A), and targeted proteomics to identify previously-reported RNA-modifying enzymes, in atherosclerotic plaques. We used different mass spectrometers for analysing nucleic acids and proteins. Although new methods have are being developed to detect RNA modifications, MS is known to be one of the most reliable biophysical tools to detect and validate RNA modification sites.¹¹⁸ For detection of m⁶A levels