DISSERTATIONS | ELISA HYTÖNEN | JGENE THERAPY OF ATHEROSCLEROSIS AND FAMILIAL ... | No 519
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THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences
ISBN 978-952-61-3146-7 ISSN 1798-5706
Dissertations in Health Sciences
PUBLICATIONS OF
THE UNIVERSITY OF EASTERN FINLAND
ELISA HYTÖNEN
GENE THERAPY OF ATHEROSCLEROSIS AND FAMILIAL HYPERCHOLESTEROLEMIA
Efficacy and Safety of Liver Gene Therapy With Lentivirus and Adeno-associated Virus Vectors Familial hypercholesterolemia (FH) is a
monogenic disease resulting in high serum LDL levels regardless of conventional drug therapy and elevated LDL levels are a major
risk factor for atherosclerotic diseases. In this thesis the safety and efficacy of gene therapy for FH was evaluated in a preclinical
hyperlipidemic model. AAV and lentiviral vectors were compared, and liver-specific side
effects are also reported. In addition, gene therapy to reduce macrophage scavenger receptor function was studied as a means to
ameliorate atherosclerotic changes.
ELISA HYTÖNEN
GENE THERAPY OF ATHEROSCLEROSIS AND FAMILIAL HYPERCHOLESTEROLEMIA
EFFICACY AND SAFETY OF LIVER GENE THERAPY WITH LENTIVIRUS AND ADENO-ASSOCIATED VIRUS VECTORS
Elisa Hytönen (nèe Vähäkangas)
GENE THERAPY OF ATHEROSCLEROSIS AND FAMILIAL HYPERCHOLESTEROLEMIA
EFFICACY AND SAFETY OF LIVER GENE THERAPY WITH LENTIVIRUS AND ADENO-ASSOCIATED VIRUS VECTORS
Publications of the University of Eastern Finland Dissertations in Health Sciences
No 519
University of Eastern Finland Kuopio
2019
Series Editors
Professor Tomi Laitinen, M.D., Ph.D.
Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences
Associate professor (Tenure Track) Tarja Kvist, Ph.D.
Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D.
Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences
Associate Professor (Tenure Track) Tarja Malm, Ph.D.
A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences
Lecturer Veli-Pekka Ranta, Ph.D.
School of Pharmacy Faculty of Health Sciences
Distributor:
University of Eastern Finland Kuopio Campus Library
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www.uef.fi/kirjasto
Grano Jyväskylä 2019
ISBN: 978-952-61-3146-7 (print/nid.) ISBN: 978-952-61-3147-4 (PDF)
ISSNL: 1798-5706 ISSN: 1798-5706 ISSN: 1798-5714 (PDF)
Author’s address: A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland
KUOPIO FINLAND
Doctoral programme: Doctoral Program of Molecular Medicine Supervisors: Professor Seppo Ylä-Herttuala, M.D., Ph.D.
A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland
KUOPIO FINLAND
Assistant Professor Nihay Laham Karam, Ph.D.
A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland
KUOPIO FINLAND
Reviewers: Assistant Professor Pekka Taimen, M.D., Ph.D.
Institute of Biomedicine University of Turku TURKU
FINLAND
Docent Veijo Hukkanen, M.D., Ph.D.
Institute of Biomedicine University of Turku TURKU
FINLAND
Opponent: Professor Markku Savolainen, M.D., Ph.D.
Department of Medicine University of Oulu OULU
FINLAND
Hytönen (nèe Vähäkangas), Elisa
Gene Therapy of Atherosclerosis and Familial Hypercholesterolemia, Efficacy and Safety of Liver Gene Therapy with Lentivirus and Adeno-associated Virus Vectors Kuopio: University of Eastern Finland
Publications of the University of Eastern Finland Dissertations in Health Sciences 519. 2019, 85 p.
ISBN: 978-952-61-3146-7 (print) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3147-4 (PDF) ISSN: 1798-5714 (PDF)
ABSTRACT
Cardiovascular diseases (CVDs) continue to be a major cause of morbidity and mortality in the Western world. Atherosclerosis is the most common etiology for CVDs. While the disease group is multifactorial and can be treated with management of risk-factors such as hyperlipidemia, hypertension, smoking and diabetes, rare monogenic diseases such as familial hypercholesterolemia (FH) also contribute to the disease burden. FH is a disease caused by mutations in the high-affinity receptor for low-density lipoprotein (LDL) leading to increased levels of circulating LDL and premature coronary-artery disease (CAD).
The aim of this thesis was to evaluate the efficacy and safety of gene therapy approaches for the treatment of FH by vectors capable of expressing transgenes for long periods of time, up to years. We used an animal model of human FH, the Watanabe Heritable Hyperlipidemic Rabbit (WHHL) for intraportal gene transfers of lentiviral (LV) and adeno-associated virus (AAV) vectors carrying the rabbit low- density lipoprotein receptor (rLDLR). Since it is well known that foam cell formation is a crucial initial event in early atherogenesis, we also used AAV vectors carrying a secreted macrophage scavenger receptor (sMSR) for the prevention of foam cell formation in an attempt to reduce atherosclerosis in the same animal model.
In this thesis, gene transfer of rLDLR with LV led to a significant, long-lasting reduction in the serum total cholesterol levels with no significant side-effects.
Comparison of LV-rLDLR gene transfer to AAV2- and AAV9-rLDLR intraportal gene transfers highlighted the efficiency and good safety profile of LV-rLDLR compared to the AAV vectors. A surprising finding of a significant bile-duct proliferation one year after AAV2-rLDLR gene transfer was seen and was associated with increased expression of the matricellular protein Cyr61 in the liver. Intraportal gene transfer of sMSR did not reduce atherosclerotic lesion area in WHHL rabbit aorta. However, unlike in AAV2-rLDLR gene transfer no pathological findings in liver histology were seen in sMSR gene transfer, suggesting that the bile-duct
proliferation may have been induced by a specific combination of the vector and the transgene.
In conclusion, intraportal delivery of LV carrying rLDLR reduced total cholesterol levels more efficiently than AAV2 or AAV9 suggesting LV-LDLR as a feasible candidate for further clinical development for the treatment of homozygous human FH. The finding of bile-duct proliferation after intraportal AAV2 gene transfer of rLDL, but not sMSR, highlights the importance of long-term studies with gene transfer vectors to elucidate their efficacy, safety and possible long-term effects.
National Library of Medicine classification: WG 550, WD 200.5.H8, QU 560, QU 95, QY 58, WI 750, WX 185
Medical Subject Headings: Atherosclerosis; Hyperlipoproteinemia Type II; Cholesterol;
Genetic Therapy; Gene Transfer Techniques; Dependovirus; Lentivirus; Receptors, LDL;
Lipoproteins, LDL; Receptors, Scavenger; Transgenes; Rabbits; Models, Animal; Liver; Bile Ducts; Efficiency; Safety
Hytönen (nèe Vähäkangas), Elisa
Gene Therapy of Atherosclerosis and Familial Hypercholesterolemia, Efficacy and safety of liver gene therapy with Lentivirus and Adeno-associated virus vectors Kuopio: University of Eastern Finland
Publications of the University of Eastern Finland Dissertations in Health Sciences 519. 2019, 85 p.
ISBN: 978-952-61-3146-7 (print) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3147-4 (PDF) ISSN: 1798-5714 (PDF)
TIIVISTELMÄ
Sydän- ja verisuonisairaudet ovat yhä yksi suurimmista sairastavuuden ja kuolleisuuden aiheuttajista länsimaissa, jonka taustalla on tyypillisimmin valtimonkovettumatauti. Hoito perustuu hyvään valtimokovettaumatudin riskitekijöiden kuten korkean kolesterolin ja kohonneen verenpaineen hoitoon elämäntapamuutoksilla sekä lääkehoidoilla. Harvinaisissa tapauksissa valtimonkovettumataudin taustalla voi olla perinnöllinen sairaus kuten familiaalinen hyperkolesterolemia (FH). Sen aiheuttaa mutaatio maksan LDL- lipoproteiinia käsittelevässä reseptorissa. Tämä johtaa veren kolesterolipitoisuuksien kohoamiseen jo sikiöaikana, ja altistaa ennenaikaisen valtimonkovettumataudin sekä sen komplikaatioiden kuten sydäninfarktin kehittymiselle.
Tämän väitöskirjatyön tavoitteena oli selvittää pitkään siirtogeenia ilmentävien lentivirus (LV) sekä adeno-assosioitu virus (AAV) -geenikuljettimien turvallisuutta ja tehokkuutta maksaan kohdistuvassa FH:n geenihoidossa. Työssä WHHL-kaneille, joka on FH:n eläinmalli, siirrettiin toimiva LDL-reseptorigeeni maksaan porttilaskimon kautta käyttäen LV- tai AAV-geenikuljettimia. Lisäksi pyrittiin hidastamaan valtimonkovettumataudin kehittymistä samassa eläinmallissa siirtämällä maksaan liukoinen, makrofageissa eli syöjäsoluissa normaalisti ilmenevä scavenger-reseptori (SR). Tämän on osoitettu olevan tärkeä valtikonkovettumataudin kehityksen alkuvaiheissa edistämällä makrofagien muuttumista rasvatäyteisiksi vaahtosoluiksi. Liukoisen scavenger-reseptorin on aiemmin osoitettu voivan vähentää valtimonkovettumatautia hiirimallissa.
Tässä työssä LV-välitteinen LDLR:n geeninsiirto laski veren kokonaiskolesterolipitoisuuksia pitkäkestoisesti ilman merkittäviä haittavaikutuksia. Verrattuna AAV-geeninkuljettimiin, LV osoittautui tehokkaammaksi ja turvallisemmaksi maksaan kohdistuvassa geeninsiirrossa.
Yllättäen AAV2-LDLR -hoitoryhmässä todettiin maksassa sappitieproliferaatiota vuosi geeninsiirron jälkeen ja se liittyi lisääntyneeseen Cyr61- matrisellulaariproteiinin ilmentymiseen näiden eläinten maksoissa. Liukoisen SR:n
geeninsiirto AAV2-geeninkuljetinta käyttäen maksaan porttilaskimon kautta ei vähentänyt valtimonkovettumatautia WHHL-kaneissa. Maksassa ei myöskään todettu patologisia muutoksia, toisin kuin AAV2-LDLR:n geeninsiirron jälkeen.
Tämä viittaa siihen, että sappitieproliferaation syntyyn on voinut vaikuttaa spesifinen siirtogeenin ja geenikuljettimen yhdistelmä.
Yhteenvetona voidaan todeta, että LV-välitteinen LDLR:n geeninsiirto laskee veren kolesterolipitoisuutta tehokkaammin kuin AAV2- tai AAV9-välitteinen geeninsiirto. LV osoittautui tässä työssä myös turvalliseksi geenikuljettimeksi maksaan kohdistuvassa geeninsiirrossa. Sappitieprolifraatio, jota nähtiin AAV2- LDLR:n mutta ei AAV2-sMSR:n geeninsiirron jälkeen, korostaa pitkän seurannan tärkeyttä arvioitaessa eri geenikuljettimien tehokkuutta, turvallisuutta sekä pitkäaikaisvaikutuksia.
Luokitus: WG 550, WD 200.5.H8, QU 560, QU 95, QY 58, WI 750, WX 185
Yleinen suomalainen asiasanasto: valtimonkovettumistauti; ateroskleroosi;
hyperkolesterolemia; kolesteroli; geeniterapia; lipoproteiinit; lentivirukset; geenitekniikka;
koe-eläinmallit; reseptorit; maksa; sappitiet; tehokkuus; turvallisuus
”Not everything that can be counted counts, and not everything that counts can be counted.”
- William Bruce Cameron
ACKNOWLEDGEMENTS
This study was carried out in the A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, over many years. I am deeply grateful to all the people who I’ve had the privilege to work with and who have contributed to the research during these years.
First, I wish to thank my main supervisor Seppo Ylä-Herttuala. I am deeply appreciative for the opportunity to work in gene therapy research and challenge myself in the field of viral vectors, namely AAVs. I admire your optimistic attitude and scientific enthusiasm which are contagious. I also appreciate the patience and encouragement with the many challenges in some of the practical work, and especially getting the AAV-lab up and running.
I am forever grateful to my second supervisor Nihay Laham Karam. I am in awe of your wide expertise, helpful and friendly attitude towards all and the dedication to work. I am thankful for our friendship that will hopefully outlast our time together at AIVI. I also want to thank you for the linguistic revision of this thesis.
I am grateful to the official reviewers of this thesis, Assistant Professor Pekka Taimen and Docent Veijo Hukkanen, for their constructive criticism and helpful feedback which helped to improve this thesis.
I’m thankful to all my co-authors for their valuable contributions to this thesis. I wish to thank Katri Pajusola and Hansruedi Bueler for the opportunity to learn the production of AAV vectors during my visit to the University of Zurich.
During the years, I have had the privilege to work with dedicated, hard-working, expert, wonderful people. Hanna Kankkonen, Johanna Tietäväinen and Anniina Laurema: you were there in the beginning and helped to form the foundation for everything. Hanna, you took me fearlessly into your projects and are the reason I know how to function in a lab. Johanna, thank you for the possibility to work with scavenger receptors and giving the kickstart for future work with AAVs. Anniina, thank you for great times at the animal facilities and teaching me about animal surgery.
I am grateful to my past and present roommates Suvi Jauhiainen, Jenni Huusko, Markku Lähteenvuo, Mari Merentie and Eveliina Pasanen for the enjoyable working atmosphere, help, laughs and just general support whether the issue was scientific or not.
I’m very lucky to have been able to have my best friends working in same group.
Sanna-Kaisa and Jossu, it is absolutely impossible to put into words how much our friendship means to me, and how much fun it was also being able to work in the same place. I’m happy and grateful that we remain close.
My warmest thanks belong to Erika Gurzeler, Line Lottonen-Raikaslehto, Hanna Sallinen, Jonna Koponen, Anna-Kaisa Ruotsalainen, Emilia Kansanen, Suvi Heinonen, Henna Niemi, Henna-Kaisa Jyrkkänen, Annukka Kivelä, Hanna Stedt and
Marike Dijkstra for friendship, laughs, conversations and help both in the lab and otherwise.
I would like to thank Tuula Salonen, Anne Martikainen, Eila Korhonen, Jarmo Asikainen and Joonas Malinen for their valuable contribution in setting up and running the AAV-lab over the years. I also appreciate the technical assistance by Mervi Nieminen, Seija Sahrio, Tiina Koponen, Sari Järveläinen, Maarit Mähönen, Svetlana Laidinen, Aila Erkinheimo and Anneli Miettinen during the years in making this work possible. I am thankful for the always kind and patient secreterial help of Helena Pernu, Marja Poikolainen, Marjo-Riitta Salminkoski and Jatta Pitkänen.
My warmest thanks belong to my parents Kirsi and Jouko, whose endless love and support I have always been able to count on. You are also wonderful grandparents to Enni, and without all the practical help towards the end of this work, it may never have been finished. My mother-in-law Ritva, thank you for relaxing times spent together; I got very lucky in the mother-in-law department. My aunt Kati and her husband Jasu are thanked for providing a home away from home with good conversations lasting into the night, laughs and good food.
Finally, my loving thanks go to my husband Jarkko and daughter Enni. Jarkko, laughter makes for a long (and fun) life, thank you for always putting a smile on my face no matter what. Enni, I promise there will be much more time for parks and playing princess in the future. I love you both to bits!
Kuopio, July 2019
Elisa Hytönen
This work was supported by grants from the Finnish Academy, ERC Advanced
LIST OF ORIGINAL PUBLICATIONS
This dissertation is based on the following original publications:
I Kankkonen HM, Vähäkangas E, Marr RA, Pakkanen T, Laurema A, Leppänen P, Jalkanen J, Verma IM, Ylä-Herttuala S. Long-term lowering of plasma cholesterol levels in LDL-receptor deficient WHHL rabbits by gene therapy.
Molecular Therapy 9(4):548-56, 2004.
II Hytönen E, Laurema A, Kankkonen H, Miyanohara A, Kärjä V, Hujo M, Laham-Karam N, Ylä-herttuala S. Bile-duct proliferation as an unexpected side-effect after AAV2-LDLR gene transfer to rabbit liver.
Scientific Reports 2019 May 6; 9(1):6934
III Hytönen E, Laurema A, Jalkanen J, Leppänen P, Pajusola K, Büeler H, Ylä- Herttuala S. AAV mediated gene transfer of secreted macrophage scavenger receptor to WHHL rabbit liver. Manuscript, 2019.
The publications were adapted with the permission of the copyright owners.
CONTENTS
ABSTRACT ... 7
TIIVISTELMÄ ... 9
ACKNOWLEDGEMENTS ... 13
1 INTRODUCTION ... 27
2 REVIEW OF THE LITERATURE ... 29
2.1 Cardiovascular diseases ... 29
2.1.1 Definitions, risk factors and treatment ... 29
2.1.2 Epidemiology ... 30
2.2 Atherosclerosis ... 30
2.2.1 Initial lesion, fatty streak and intermediate lesion ... 30
2.2.2 Atheroma, advanced lesions and complicated lesions ... 31
2.3 Familial hypercholesterolemia ... 33
2.3.1 Diagnosis and clinical manifestations ... 33
2.3.2 Genetics and prevalence ... 34
2.3.3 Current treatment of familial hypercholesterolemia ... 35
2.3.4 Animal models of familial hypercholesterolemia ... 37
2.3.5 Rabbit models ... 37
2.4 Lipoprotein metabolism ... 38
2.4.1 Lipoproteins and their functions ... 38
2.4.2 Exogenous pathway ... 39
2.4.3 Endogenous pathway ... 39
2.4.4 Low-density lipoprotein receptor ... 40
2.4.5 Metabolism of Low-density lipoproteins ... 41
2.4.6 Reverse cholesterol transport ... 43
2.5 Scavenger receptors ... 43
2.5.1 Scavenger receptor A ... 43
2.5.2 Other scavenger receptors ... 44
2.5.3 Scavenger receptors in atherosclerosis ... 44
2.5.4 Soluble receptors ... 45
2.6 Gene therapy ... 45
2.6.1 Gene transfer techniques and vectors ... 45
2.6.2 Lentiviral vectors ... 46
2.6.3 Adeno-associated virus vectors ... 47
2.6.4 Other vectors ... 48
2.6.5 LV- and AAV-vectors in the treatment of atherosclerosis and lipid- disorders ... 49
2.6.6 Future directions ... 50
2.6.7 Challenges in gene therapy ... 50
3 AIMS OF THE STUDY ... 53
4 MATERIALS AND METHODS ... 55
4.1 Production of viral vectors ... 55
4.2 Cell culture experiments ... 56 4.2.1 Degradation of I125-LDL, -oxLDL or -acLDL ... 56 4.2.2 Binding and uptake of fluorescent DiI-LDL ... 57 4.3 Animal experiments ... 57 4.3.1 In vivo gene transfers ... 57 4.3.2 Uptake of DiI-LDL in liver ... 58 4.4 Lipids and lipoprotein metabolism ... 58 4.4.1 Analysis of blood samples ... 58 4.4.2 Isolation and DiI-labeling of LDL ... 58 4.4.3 En face -lesion area ... 58 4.5 Tissue analyses ... 58 4.6 Molecular biology analyses ... 59 4.6.1 Extraction of nucleic acids ... 59 4.6.2 PCR and RT-PCR (I) ... 59 4.6.3 qPCR and RT-qPCR (II, III) ... 60 4.6.4 ELISA-assay (III) ... 60 4.7 Statistical analyses ... 60 5 RESULTS ... 61
5.1 intraportal gene transfer of LV-LDLR leads to a long-lasting reduction in serum total cholesterol in WHHL rabbits (I) ... 61 5.1.1 In vitro analysis of vectors ... 61 5.1.2 In vivo experiments ... 61 5.2 LV-rLDLR is more efficient than AAV2- or AAV9-rLDLR in the treatment of
FH (II) ... 61 5.2.1 Expression of transgenes and clinical chemistry ... 61 5.2.2 Histology and characterization of bile-duct proliferation ... 62 5.2.3 Atherosclerosis after AAV2- and AAV9 gene transfers (unpublished
findings) ... 64 5.2.4 Uptake of DiI-LDL in liver (unpublished findings) ... 64 5.3 sMSR does not reduce lesion area in WHHL rabbits (III) ... 65 5.3.1 Clinical chemistry ... 65 5.3.2 Atherosclerosis and inflammation ... 65 6 DISCUSSION ... 67 6.1 LV- rLDLR in the treatment of FH ... 67 6.2 Comparison of LV- and AAV-rLDLR for treatment of FH ... 68 6.2.1 Effect on hypercholesterolemia ... 68 6.2.2 Safety ... 69 6.3 sMSR gene transfer for reducing atherosclerosis ... 70 7 SUMMARY AND CONCLUSIONS ... 73 REFERENCES ... 75
ABBREVIATIONS
AAV Adeno-associated virus ABCA1 ATP-cassette binding
subfamily A member 1 ABCG1 ATP-cassette binding
subfamily G member 1 ACAT Lecithin:cholesterol
acyltransferase Ad Adenovirus
AcLDL Acetylated low-density lipoprotein
Alb Albumin
ALT Alanine aminotransferase ApoAI Apolipoprotein AI ApoB Apolipoprotein B ApoB48 Apolipoprotein B48 ApoE Apolipoprotein E APT Alkaline phosphatase AST Aspartate aminotransferase Bil Bilirubin
CABG Coronary artery bypass grafting
CAD Coronary artery disease
CAR Coxsackie-adenovirus receptor
CE Cholesterol ester
CETP Cholesteryl ester transfer protein
CHD Coronary heart disease CM Chylomicron
CN Copy number CRP C-reactive protein CVD Cardiovascular disease Cyr61 Cysteine-rich angiogenic
inducer 61
DALY Disability-adjusted life year DGAT Diacylglycerol
acyltransferase
DiI 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindocarbocyanine perchlorate
DNA Deoxyribonucleic acid EC Endothelial cell ECM Extracellular matrix FA Fatty acid
FATP Fatty acid transfer protein
FH Familial hypercholesterolemia HDL High-density lipoprotein heFH Heterozygous familial
hypercholesterolemia HIV Human immunodeficiency
virus
HL Hepatic lipase
HMG-CoA 3-hydroxy-3-methyl glutaryl-coenzyme–A
hoFH Homozygous familial hypercholesterolemia HSPG Heparan-sulfate
proteoglycan
ICAM-1 Intercellular adhesion molecule 1
LCAT Lecithin:cholesterol acyltransferase IDL Intermediate density
lipoprotein
IDOL E3 ubiquitin ligase inducible degrader of the LDL receptor LDL Low-density lipoprotein LDLR Low-density lipoprotein
receptor
LMO2 LIM-domain only 2
LRP1 Low-density lipoprotein related protein 1
LTR Long-terminal repeat LV Lentivirus
MCP1 Monocyte chemoattractant protein 1
M-CSF Macrophage colony- stimulating factor MGAT Monoacylglycerol
acytransferase
mRNA Messenger ribonucleic acid MTP Mitochondrial transfer
protein
NPC1L1 NPC1 like intracellular cholesterol transporter 1 ORF Open-reading frame oxLDL Oxidized low-density
lipoprotein
PAD Periferial arterial disease PCR Polymerase chain reaction PCSK9 Proprotein convertase
subtilisin/kexin type 9 PL Phospholipid
rER Rough endoplamic reticulum
SMC. Smooth muscle cell sMSR Secreted macrophage
scavenger receptor SNP Single-nucleotide
polymorphism SR Scavenger receptor TC Total cholesterol TG Triglyceride
VCAM Vascular cell adhesion molecule
VEGFR-1 Vascular endothelial growth factor receptor 1
VLDL Very-low density lipoprotein VLDLR Very-low density lipoprotein
receptor
VSVG Vesicular stomatitis virus protein G
WHHL Watanabe heritable hyperlipidemic rabbit X-SCID X-linked severe combined
immunodefiency
1 INTRODUCTION
Atherosclerosis, and its clinical manifestations of coronary artery disease (CAD), stroke and peripheral arterial disease (PAD), remain one of the most significant causes of mortality and morbidity in the Western world. Atherosclerosis is a multifactorial, inflammatory disease, causing narrowing of arteries due to the accumulation of lipids, inflammatory cells and necrotic debris into the vessel wall.
With time this results in restricted blood flow to the affected tissue causing ischemia and its clinical symptoms of angina, claudication and stroke-related symptoms.
Treatment consists of life-style modifications and medication to reduce the known risk factors of hypercholesterolemia, hypertension, diabetes and smoking.
A difficult to treat hypercholesterolemia can be familial hypercholesterolemia (FH) which is a dominantly inherited disease of lipid metabolism. The disease is caused by mutations in genes involved in the metabolism of low-density lipoprotein (LDL) in the liver, leading to persistently elevated levels of circulating LDL already from infancy, and subsequently premature atherosclerosis. While the heterozygous form of FH responds well to treatments with currently available cholesterol lowering drugs, apart from liver transfer, the more severe homozygous FH is often refractory to currently available treatments.
The earliest visible atherosclerotic changes in the vessel wall are fatty streaks composed mainly of macrophage foam cells. Scavenger receptors have been implicated in the pathogenesis of atherosclerosis as they are able to bind and internalize oxidized LDL leading to the formation of these lipid-laden foam cells.
Scavenger receptors comprise a large and diverse family of receptors involved in the recognition and clearance of non-self and altered-self antigens and ligands.
The aim of this thesis was to evaluate intraportal gene therapy options for the treatment of atherosclerosis and FH. Intraportal gene transfer of the rabbit LDLR receptor (rLDLR) into the liver of hypercholesterolemic Watanabe Heritable Hyperlipidemic (WHHL) rabbits with lentiviral vectors (LVs) and adeno-associated virus vectors (AAVs) were evaluated for long-term efficacy and safety. Additionally, the same approach of intraportal gene transfer was used to assess the potential of a secreted macrophage scavenger receptor (sMSR) to reduce atherosclerosis in WHHL rabbits.
2 REVIEW OF THE LITERATURE
2.1 CARDIOVASCULAR DISEASES 2.1.1 Definitions, risk factors and treatment
Cardiovascular diseases comprise a range of diseases of the cardiovascular system that is the heart and blood vessels. The most common diseases include CAD (that predisposes to angina or myocardial infarction), PAD and stroke. Other CVDs include heart failure, arrhythmias, cardiomyopathies and valvular heart diseases but these are more commonly referred to as heart diseases.
The common pathological process behind ischemic diseases is atherosclerosis (Glass and Witztum 2001; Lusis 2000). The clinical manifestations of atherosclerosis are due either to the narrowing of the vessel diameter, thus restricting blood flow to tissues, or to thrombus formation which in the worst case can lead to sudden death.
CAD, stroke and PAD are the most common sequelae of atherosclerosis. The symptoms arise from insufficient blood flow to the affected tissue, resulting in oxygen deprivation and ischemia. Depending on the tissue the symptoms caused by ischemia vary; ischemia of the cardiac tissue results in chest pain, either during exercise when the narrowing of the vessel, or stenosis, does not exceed 70% of the vessel diameter, or at rest when the stenosis reaches 90%. Ischemia in the periphery leads to claudication, while ischemia in the brain may cause a stroke. Risk-factors known to be closely associated with CVDs include modifiable risk factors such as high levels of circulating low-density lipoprotein (LDL), hypertension, diabetes, smoking and physical inactivity, as well as non-modifiable factors such as family history and male gender (Herrington et al. 2016; Lusis 2000).
Treatment of CVDs includes management of the modifiable risk factors and comprises life-style changes such as cessation of smoking and increased physical activity. Medication for high-blood pressure, hypercholesterolemia and diabetes are also needed (J.-G. Park and Oh 2019). Statins are used to lower elevated LDL levels via inhibition of the 3-hydroxy-3-methyl-glutaryl-coenzyme–A (HMG-CoA) reductase, a key enzyme in the cholesterol synthesis pathway. Besides cholesterol lowering, statins have beneficial pleiotropic effects including improved endothelial function through endothelial nitric oxide synthase activation, reduced oxidative stress and enhanced plaque stability (Oesterle, Laufs, and Liao 2017). It has also been shown that statins can affect foam cell formation in macrophages through disruption of pro-inflammatory pathways and dysregulated cholesterol handling in macrophages. In statin intolerant patients Ezetimibe, a drug inhibing cholesterol absorption in the small intestine, can be used. It can also be combined with statins in patients unable to reach their cholesterol targets with statins alone. When the disease progresses despite life-style changes and drug treatment, both advanced stable
coronary artery disease and acute myocardial infarction can be treated with balloon angioplasty and stenting or coronary artery bypass grafting (CABG).
2.1.2 Epidemiology
The prevalence of CVDs in Europe is high with 6.1 million new cases of CVD diagnosed (2015) and 1.8 million deaths yearly attributed to CVDs in the European Union. Although the absolute number of CVD cases has increased in the EU in the last 25 years, the age-standardized prevalence rate of CVD has fallen in most European countries. The disability-adjusted life years (DALYs) due to CVD have been decreasing in Europe over the last decade but still account for the loss of more than 26 million DALYs in the EU (Herrington et al. 2016; Wilkins et al. 2017). In Finland ischemic heart disease is still the major cause of death although the age- standardized mortality from diseases of the circulatory system has decreased by over 40 percent in the last twenty years (Official Statistics of Finland 2017).
2.2 ATHEROSCLEROSIS
2.2.1 Initial lesion, fatty streak and intermediate lesion
Atherosclerosis is a progressive, inflammatory disease of large and medium sized arteries where lipid, inflammatory cells and fibrous elements accumulate in the vessel wall causing narrowing of the arterial lumen (Figure 1) (Lusis 2000). The initiating event in atherosclerosis is endothelial dysfunction which can be caused by numerous factors including modified/oxidized LDL, shear stress or inflammation.
(Ylä-Herttuala et al. 1989). Predilect sites for the development of atherosclerosis include arterial branch points due to disturbed laminar flow and increased permeability to macromolecules like LDL (Gimbrone 1999). LDL accumulates in the subendothelial space and interactions of ApoB in LDL with matrix proteoglycans leads to retention of the LDL in the vessel wall, where it is susceptible to oxidative modifications by reactive oxygen species or by enzymes such as myeloperoxidase or lipoxygenases that can be released from inflammatory cells (Ylä-Herttuala et al.
1988).
Modified LDL stimulates overlying endothelial cells (ECs) to produce pro- inflammatory molecules including adhesion molecules (ICAM-1, VCAM), chemotactic proteins (MCP-1) and selectins (P-and E-selectin), leading to the recruitment of monocytes and T-cells to the vessel wall. There, monocytes differentiate to macrophages and proliferate due to the secreted macrophage colony- stimulating factor (M-CSF). (Glass and Witztum 2001; Weber and Noels 2011).
Macrophages are able to bind and internalize modified LDL such as oxidized (ox)-
as fatty streaks. (Lusis 2000; Stary et al. 1994). The type (III) lesion or ‘pre-atheroma’
differs from the fatty streak by microscopically visible extracellular lipid-droplets and particles. These early lesions are non-symptomatic.
2.2.2 Atheroma, advanced lesions and complicated lesions
Advanced atherosclerotic lesions are characterized by an increasing amount of extracellular lipid as well as accumulation of smooth muscle cells (SMCs) and the extracellular matrix (ECM) that they secrete. Migration and proliferation of SMCs are stimulated by cytokines and growth factors secreted by macrophages and T-cells.
(Weber and Noels 2011). SMCs, which normally exhibit a contractile, non- proliferative phenotype, once activated, proliferate and produce large amounts of ECM proteins to form a fibrous cap for the lesion.
Atheroma or type (IV) lesion has a well-defined area of extracellular lipid in the intima, a lipid core, disrupting the intimal SMCs and the intercellular matrix. The layer between the lipid core and the ECs, however, still resembles the normal intima.
Type (IV) lesions are located at the same sites as adaptive intimal thickening, and often do not narrow the lumen. They may, however, be clinically significant due to fissures and rupture from the periphery of the lesion, or the shoulder area, which contains abundant macrophage foam cells. (Lusis 2000; Stary et al. 1995).
The distinction between a type (IV) and type (V) lesion is in the structure of the intima above the lipid core. In type (IV) lesions, it is largely ‘normal’ intima, while in type (V) lesions an increase in fibrous tissue (collagen) of this area takes place. These lesions are largely indistinguishable from each other by regular microscopy and are both often referred to as fibrous plaques.
Type (VI) lesions bear the morphology of type (IV) or (V) lesions with additional features of calcification, hemorrhage or lack of a lipid core (Moore and Tabas 2011;
Stary et al. 1995). As with type IV lesions clinical significance comes from the occurrence of fissures, haematoma and thrombus formation leading to acute clinical syndromes like myocardial infarction. Although currently there is no consensus about the characteristics of the so-called vulnerable plaques some common features have been suggested. A thin fibrous cap might predispose to rupture as well as an increased number of inflammatory cells which produce factors affecting both matrix production and degradation. Vulnerable plaques show decreased numbers of SMCs and macrophages have been shown to trigger apoptosis in SMCs via activation of their Fas apoptotic pathway. (Moore and Tabas 2011). The stability of the lesion may also be influenced by neovascularization and calcification.
Figure 1. Summary of the pathogenesis of atherosclerosis. Picture modified from Atherosclerosis in McMaster Pathophysiology Review
(http://www.pathophys.org/atherosclerosis/). Histology pictures from Pathology Education Informational Resource (PEIR) Digital Library
(http://peir.path.uab.edu/library/index.php?/search/9590). LDL (low-density lipoprotein); EC (endothelial cell); SMC (smooth muscle cell); SR (scavenger receptor); ACS (acute coronary syndrome).
2.3 FAMILIAL HYPERCHOLESTEROLEMIA 2.3.1 Diagnosis and clinical manifestations
FH is an inherited disease of lipid metabolism characterized by persistently elevated levels of LDL-C and premature CVD (Goldstein, Hobbs, and Brown 2001).
Clinical diagnosis is most often based on Dutch Lipid Clinic Network (DLCN) criteria (Table 1), less frequently on Simon Broone or MEDPED criteria. (Austin et al.
2004; Haase and Goldberg 2012; Nordestgaard et al. 2013; Williams et al. 1993).
A score of over 8 points based on the DLCN criteria is diagnostic for FH. If the score is between 6 to 8 points FH is probable and with 3 to 5 points possible. Definitive diagnosis is achieved by identification of a pathogenic variant in one of the three genes (APOB, LDLR and PCSK9) known to be associated with FH.
Clinical manifestations of FH vary widely depending on the mutations. Typical clinical signs include a varying extent of hypercholesterolemia (especially elevated LDL-C), cholesterol deposits in tendons called xanthomas, cholesterol deposits around the eye called xantelasmas and arcus cornea which is a deposition of lipids in the cornea of the eye. The life-long and persistent elevation of LDL-C, if untreated, leads to premature and accelerated atherosclerotic cardiovascular disease. The risk of cardiovascular disease is 2.5 to 10-fold increased in FH but when diagnosed and treated early in life, is reduced by up to 80%. This highlights the importance of early diagnosis and initiation of cholesterol lowering treatment for prevention of future cardiovascular complications.
Cascade screening is a systematic process of identifying individuals at risk for FH based on the identification of a person with the disease and/or a pathogenic variant, and then testing the at-risk biological relatives of this person. As new cases or pathogenic variants are identified more rounds of screening are carried out. Cascade screening has proven effective in identifying new cases when a monogenic background has been verified for the index case. As FH is still vastly underdiagnosed, some countries like the Netherlands have adopted active screening programs to improve the discovery of new FH cases. (Louter, Defesche, and Roeters van Lennep 2017; Umans-Eckenhausen et al. 2001).
While on the one hand there is a definite need for more intensive screening to identify FH patients for treatment to reduce future CVD risk, on the other hand in 25-30% of clinically diagnosed FH patients no pathogenic variants can be identified (Taylor et al. 2010). In these cases, a likely polygenic background explains the hypercholesterolemia and cascade screening might not be cost-effective.
Table 1. Dutch Lipid Clinic Network diagnostic criteria for familial hypercholesterolemia
Criteria Points
Family History
First-degree relative with known premature* coronary and vascular disease OR First-
degree relative with known LDL-C level over the 95th percentile 1 First-degree relative with tendon xanthomata and/or arcus cornealis OR
Children aged less than 18 with LDL-C level over the 95th percentile 2 Clinical History
Patient with premature* coronary artery disease 2
Patient with premature* cerebral or peripherial vascular disease 1 Physical Examination
Tendinous xanthomata 6
Arcus cornealis prior to age 45 years 4
Cholesterol levels (mmol/l)
LDL-C > 8.5 8
LDL-C 6.5-8.4 5
LDL-C 5.0-6.4 3
LDL-C 4.0-4.9 1
DNA Analysis
Functional mutation in the APOB, LDLR or PSCK9 gene 8
Diagnosis (based on the total number of points obtained)
Definitive Familial Hypercholesterolemia >8
Probable Familial Hypercholesterolemia 6-8
Possible Familial Hypercholesterolemia 3-5
Unlikely Familial Hypercholesterolemia <3
*premature = <55 years in men; <60 years in women.
2.3.2 Genetics and prevalence
Defects in three genes, LDLR, APOB or PCSK9, are known to lead to familial hypercholesterolemia (Marks et al. 2003; Nordestgaard et al. 2013). Most of the mutations (60-80%) causing the FH phenotype arise from mutations in the LDLR gene. Overall, an excess of over 2000 disease-causing variants for familial hypercholesterolemia have been identified to date (Leigh et al. 2017). These include exonic substitutions, exonic small rearrengements, large rearrangements, promoter variants, intronic variants and one variant in the 3´untranslated sequence. Out of the identified variants 81% have been classified as pathogenic, 12% as non-pathogenic and 7% as variants of unknown significance (Leigh et al. 2017). Continued effort to find new mutations behind FH have been encouraged by the fact that with every
FH according to the DLCN criteria, a mutation can only be found in about 25-30% of cases. It has been suggested that most of these cases are actually polygenic, where patients have inherited a greater-than-average number of cholesterol-raising variants. An LDL-C SNP-score has been developed and tested and indeed these SNPs have been estimated to account for 80% of the cases where a clinical diagnosis of possible FH has been made. (Futema et al. 2015; Talmud et al. 2013). Since the phenotype of FH is similar irrespective of genetic background, is there benefit in the knowledge of a monogenic or polygenic background? It has been shown that the prevalence of CHD is higher in monogenic compared to polygenic hypercholesterolemia (Humphries et al. 2006; Khera et al. 2016); more severe intimal thickening in the carotid artery of monogenic vs. polygenic FH patients has been demonstrated despite similar cholesterol levels, and higher calcium scores of lesions from monogenic patients has been demonstrated. (Sharifi et al. 2017).
Traditionally, the functional consequences of LDLR mutations have been classified into five categories: class I null, class II transport defective, class III binding defective, class IV internalization defective and class V recycling defective (Hobbs, Brown, and Goldstein 1992). However, better understanding of the different mechanisms that may affect normal LDLR function suggest that additional classes may be necessary (Koivisto, Hubbard, and Mellman 2001; Strøm et al. 2014; Strøm, Leren, and Laerdahl 2015).
The prevalence of heterozygous FH (heFH) has previously been cited as 1:500 but emerging data from white/European populations suggest that the prevalence of heFH may be as high as 1:200-1:250 (Nordestgaard et al. 2013). Homozygous FH (HoFH) is rare with a prevalence of 1:1000000, although based on the new studies estimates of 1:160000-300000 have been suggested (Cuchel et al. 2013). FH is more common in selected populations, such as the Finnish population, due to founder effects. (Lahtinen et al. 2015; Vuorio et al. 2001). In Finland eight founder mutations (FH-Helsinki, FH-Pohjois-Karjala, FH-Turku, FH-Pori, FH-Pogosta, FH-Keuruu, FH- Espoo and FH-Pro84Ser) account for approximately 80% of the FH cases (Vuorio et al. 2001).
2.3.3 Current treatment of familial hypercholesterolemia
Treatment focuses on lowering LDL-C via diet, exercise and cholesterol lowering drugs. Current guidelines for LDL-C targets for FH patients are <2.3 mmol/l for adults, <1.8mmol/l for adults with CHD or diabetes, and <3.5mmol/l for children.
These targets are achievable in most FH patients with statins, ezetimibe or the new PCSK9 inhibitors. In the most severe cases lomitapide or mipomersen alone or in combination with apheresis is required.
Statins are the cornerstone of treatment. They function via inhibiting the HMG- CoA reductase in the liver affecting cholesterol biosynthesis. Because the product of the HMG-CoA reductase reaction, mevalonate, is a precursor to many non-steroidal isoprenic compounds in addition to cholesterol, benefits beyond cholesterol lowering have been discovered. (Bellosta et al. 2000; Oesterle, Laufs, and Liao 2017). In heFH
statins reduce LDL levels and CHD mortality but in homozygous patients, especially patients who are null-mutants, the efficacy of statins is modest due to the lack of functioning LDL receptors.
A major addition to the treatment of elevated LDL cholesterol has been the introduction of PCSK9 inhibitors, in form of PCSK9-directed antibodies (He et al.
2017). Currently there are two commercially available anti-PCSK9 monoclonal antibodies Alirocumab and Evolocumab (Navarese et al. 2015). In clinical trials responses have been good in patients with receptor defective mutations but those with one receptor negative allele had lower reductions in cholesterol and one patient with two receptor negative alleles had no response to therapy highlighting that current therapies are still not sufficient for all patients. (Bergeron et al. 2015; Stein et al. 2012). Monoclonal antibodies reduce also lipoprotein (a) in addition to apoliprotein-B, non-HDL cholesterol and triglycerides (TGs). Levels of HDL and apolipoprotein A1 are increased as with statin therapy.
Other FH treatments include mipomersen which is an anti-sense oligonucleotide that binds the coding region of human ApoB mRNA and triggers its degradation, resulting in reduction of all ApoB containing atherogenic lipoproteins. An added benefit for hoFH is that the mipomersen-mediated reductions in LDL-C are not dependent on LDLR expression. In clinical studies elevations in liver transaminases and liver steatosis without inflammation or fibrosis were seen but were resolved after discontinuation of the drug. Mipomersen was also shown to reduce major cardiac events by 85%. (McGowan et al. 2012; Raal et al. 2010).
HoFH can also be treated with lomitapide which is a microsomal triglyseride transfer protein (MTP) inhibitor (Rader and Kastelein 2014). MTP is localized in the endoplasmic reticulum of hepatocytes and enterocytes playing a key role in the assembly and secretion of ApoB-containing lipoproteins. Loss-of-function mutations lead to hypocholesterolemia and reduced levels of ApoB containing lipoproteins. The reductions in LDL-C by lomitapide, similarly to mipomersen, are independent of LDLR expression.
Both lomitapide and mipomersen have been shown to cause accumulation of fat in the liver, although in recent trials no inflammation or fibrosis have been seen.
However, the possibility of the development of cirrhosis as a result of continuous, long-term fat accumulation in the liver can not be ruled out. An observational registry to monitor patients receiving lomitapide (LOWER: Lomitapide Observational Worldwide Evaluation Registry) has been set up and will follow lomitapide-treated FH patients for up to 10 years.
LDL apheresis is the physical removal of lipoproteins from the blood. It is indicated in FH patients who are unresponsive or have an inadequate response to cholesterol lowering drugs. It is mostly used in hoFH patients but occasionally is necessary also in severe heFH patients unresponsive to statin treatment. Reductions
The only curative treatment for hoFH is liver transplantation by which normal LDLR function can be restored in the liver (Bilheimer et al. 1984; Starzl et al. 1984).
However, this does not reverse existing cardiovascular complications, and for maximal benefit would need to be performed prior to the development of these. In addition, due to the poor availability of transplants, technical difficulty of the procedure and the life-long immunosuppression required, it is an option only for the most severe cases of hoFH.
2.3.4 Animal models of familial hypercholesterolemia
Several animal models have been available to study hypercholesterolemia and atherosclerosis in vivo including mouse, hamster and rabbit. Mice and rabbits with mutations in the LDLR gene recapitulate the disease and are the most appropriate for use in FH research. While the lipid metabolism of mice differs from humans significantly (Getz and Reardon, 2012), the development of human chimeric mouse models has improved congruity with human FH (Bissig-Choisat et al., 2015).
However, due to the small size of mice and differences in the development and progression of atherosclerosis, the WHHL rabbit can be considered to be the most useful in modeling FH in vivo. (Emini Veseli et al., 2017).
2.3.5 Rabbit models
Rabbits are naturally resistant to the development of atherosclerosis, even on a high- fat diet. Therefore the discovery of a rabbit with significant, spontaneous hypercholesterolemia in a colony of Japanse white rabbits in the early 70’s led to the generation of the WHHL rabbit by selective in-breeding of this mutant (Watanabe, 1980). Later it was shown that the cause of the hypercholesterolemia was deficiency of LDL receptors in the liver and adrenal gland (Kita et al. 1981; Yamamoto et al.
1986). The rabbits show increased levels of total cholesterol, LDL-C and TG at various ages. They develop spontaneous aortic atherosclerosis that progresses in extent and severity with age, and xanthoma of digital joints, both also features of human FH.
Cultured fibroblasts from homozygous animals express less than 5% of the expected number of LDLR.
In WHHL rabbits, the first detectable lesions are seen in the aortic arch at about 2 months of age. Intimal lesions containing CE in SMCs and macrophage foam cells predominate. Raised lesions in all parts of the aorta are visible by age of 6 months, and advanced atherosclerotic plaques, resembling those seen in humans, are present by the age of 10-12 months. Advanced lesions have a necrotic cholesterol-filled core, calcification and a fibrous cap. (Buja et al., 1990). Despite recapitulating the human disease extremely well, differences still exist. In the WHHL rabbit early lesions develop in the thoracic aorta, followed by the abdominal aorta. This relates most likely to the hemodynamic differences between four-legged animals and humans.
Also, aortic stenosis typically present in hoFH is not seen in the WHHL rabbits.
Finally, while both humans and rabbits display elevated plasma cholesterol levels, only WHHL rabbits show marked elevations in TG levels.
A recent publication by Lu and co-workers describes the generation of LDLR KO- rabbits using CRISPR/Cas9 to induce biallelic mutations to exon 7 of the LDLR, with subsequent development of spontaneous hypercholesterolemia and atherosclerosis on a normal chow-diet. While there was great variation in lesion area in this model, it may provide more opportunities to study the heterogenous molecular defects underlying human FH in the future (Lu et al. 2018).
2.4 LIPOPROTEIN METABOLISM 2.4.1 Lipoproteins and their functions
Lipoproteins are used by cells to transfer hydrophobic lipid molecules such as cholesterol esters and TG through the bloodstream. Their classificiation is based on the associated apolipoproteins and the content of cholesterol, TGs and phospholipids. The combined protein and lipid content define the buoyant density of the particle and separate the lipoproteins into chylomicrons (CM), CM remnants, very low-density lipoproteins (VLDL), intermediate density lipoproteins (IDL), LDL and HDL. Lipoprotein particles consist of a hydrophobic lipid core consisting of cholesteryl esters and TGs, surrounded by a hydrophilic membrane of phospholipids, free cholesterol and apolipoproteins. (Hegele 2009).
CMs are formed in enterocytes in the small intestine. They transport dietary TGs and cholesterol to peripheral tissues. CM remnants are formed after lipoprotein lipase (LPL)-mediated lipolysis and are taken up by the liver via the LDLR and low- density lipoprotein related protein 1 (LRP-1). (Hussain 2014). VLDL particles are synthesized by the liver and consist mainly of TGs and cholesterol and contain a single ApoB100 molecule. Similiar to CMs their TGs are hydrolyzed by LPL in peripheral tissues to release fatty acids (FAs). This release of TGs leads to the formation of VLDL remnants or IDL. These can be removed from the circulation via binding of ApoE to hepatic LDLR or LRP1 but while clearance of CM remnants is efficient, most of the TGs in IDL are further hydrolyzed by hepatic lipase (HL). Also, the exchangeable apolipoproteins of IDL are transferred from the IDL to other lipoproteins leading to the formation of cholesterol ester (CE)-rich LDL particles.
(Nakajima et al. 2011)
HDL is formed via multiple steps after synthesis of ApoAI by the liver and intestine. It then accumulates cholesterol and phospholipids (PLs) mediated by ABCA1 to eventually form mature HDL. Cholesterol in HDL particles are esterified to CEs by lecithin:cholesterol acyltransferase (LCAT). (Dominiczak and Caslake
Three main pathways are responsible for the production and transport of lipids within the body; the exogenous pathway, the endogenous pathway, and the pathway of reverse cholesterol transport.
2.4.2 Exogenous pathway
The exogenous pathway mediates the efficient transfer of dietary FAs to muscle and adipose tissue for use in energy metabolism and storage. Dietary TGs are emulsified by bile and hydrolyzed by pancreatic lipase. Released FAs are transported from the intestinal lumen to enterocytes where they are re-esterified by monoacylgycerol acyltransferase (MGAT) and diacylglycerolacyltransferase (DGAT) to form TGs.
Nascent CMs are assembled from TGs, cholesteryl esters and ApoB48 with the help of microsomal triglyceride transfer protein (MTP). They aquire Apo-AIV in the lipidation step and Apo-A1 and Apo-AII when processed in the Golgi. After being transported into the lacteals which are small lymphatic vessels, they enter the circulation from the thoracic duct where they further aquire Apo-CII, Apo-CIII and Apo-E from other lipoproteins such as HDL. (Hussain 2014).
LPL expressed from muscle and adipose tissue, and transported to the luminal surface of capillaries, then hydrolyzes the TGs carried in the CMs to form FAs which are then taken up by the muscle cells and adipocytes with the help of fatty-acid transport proteins (FATPs) and CD36 (Olivecrona 2016). The metabolism of CMs leads to a decrease in their size due to the loss of TGs, and formation of CM remnants.
Subsequently, these are efficiently cleared by hepatic LDLR, through receptor binding of the ApoE on the surface of the CM remnants. CM remnants are also taken up by VLDLR and LRP-1 which are able to bind both ApoB and ApoE. The size and composition of the CM particle is determined by the amount and type of fat ingested and absorbed by the intestine. Larger particles are produced when more fat is absorbed.
2.4.3 Endogenous pathway
The endogenous pathway refers to the synthesis and subsequent metabolism of VLDL to ultimately form LDL particles which are taken up by the liver via LDLR.
TGs and CEs in the liver are transferred by MTP to newly synthesized ApoB-100 to form VLDL. The rate-limiting step in this process is the availability of TGs which determines whether the synthesized ApoB is secreted or degraded. (Cohen and Fisher 2013). VLDL particles are transported to the peripheral tissues where, similar to CM, LPL hydrolyzes the TGs to release FAs to be taken up by the peripheral tissues. The formed IDL particle (VLDL remnant) is enriched in CE and acquires ApoE from HDL particles. Once the particle is small enough, it passess through fenestrae in the liver endothelium to the space of Disse where it is further modified before taken up by hepatic LDLR, LRP-1 and heparin sulfate proteoglycans (HSPG).
Even though ApoE is recognized by hepatic LDLR, the uptake of IDL is not as efficient as uptake of CM remnants, and only about 50 % of IDL is cleared. The
remaining TGs in IDL are hydrolysed by hepatic lipase (HL) and exchangeable apolipoproteins are transferred from IDL to other lipoproteins, leading to the formation of LDL. (Cohen and Fisher 2013).
Figure 2. The exogenous and endogenous pathways of lipoprotein metabolism. CMs are formed from dietary lipids, transported via the thoracic duct to the circulation and peripheral tissues where lipolysis by LPL hydrolyzes the TGs to release FFAs to be taken up by muscle and adipose tissue. Remnants are cleared via hepatic LDLR and LRP-1. VLDL is secreted by the liver to peripheral tissues where lipolysis by LPL similiar to CMs happens. The formed IDL is either taken up by the LDLR in the liver or further hydrolysed by HL to form LDL. Picture modified from (Lusis, Fogelman, and Fonarow 2004).
2.4.4 Low-density lipoprotein receptor
LDLR is a transmembrane protein and belongs to the low-density lipoprotein receptor gene family. It is encoded by the ldlr gene on chromosome 19p13.1-13.3 (Francke, Brown, and Goldstein 1984). Mutations in the ldlr gene lead to familial hypercholesterolemia as discussed in chapter 2.3. LDLR is expressed ubiquitously
homology region, a region containing O-linked sugars, a transmembrane domain and a C-terminal cytosolic domain (Südhof et al. 1985). The ligand binding domain consists of seven cysteine-rich repeats called LDLR class A (LA) repeats. This domain is crucial for binding of both LDL via ApoB and VLDL via ApoE. The EGF-precursor homology domain consists of three EGF-like repeats. Deletion of this domain does not affect VLDL binding but instead prevents the acid-dependent dissociation of ligand in endosomes. The function of the O-linked glycan region does not affect ligand binding, endocytosis or degradation, and its clear role remains to be elucidated. The hydrophobic 24 amino acid transmembrane domain anchors the LDLR in the lipid membrane while the cytosolic domain is responsible for recruitment to clathrin coated pits, endocytosis and intracellular transport of the ligand-receptor complex.
The LDLR pathway is shown in Figure 3b. Synthesis of the LDLR polypeptide from mRNA takes place on the ribosomes of the rough endoplasmic reticulum where also initial glycosylation of the polypeptide takes place (Goldstein, Hobbs, and Brown 2001). It is then transported to the Golgi complex where glycosylation is completed. The receptor is then transported to the cell surface where the receptors accumulate in clathrin-coated pits (Anderson, Brown, and Goldstein 1977; Anderson, Goldstein, and Brown 1976). LDLR binds LDL in the circulation, after which the ligand-receptor complex is internalized in coated endocytic vesicles. In the cell, uncoating of the vesicle takes place and the ligand-receptor complex is transported to the endosomes where the acidic environment facilitates the release of the ligand.
The receptor is rapidly recycled back to the cell surface, and the LDL is degraded in lysosomes by acid hydrolases and proteases to release unesterified cholesterol which is used in the production of steroid-homones, cell-membranes and bile-acids (Brown, Anderson, and Goldstein 1983).
2.4.5 Metabolism of Low-density lipoproteins
Plasma LDL concentration is determined by the rate of production and clearance of LDL which is partly determined by the number of LDL receptors in the liver. The production of LDL from IDL is partially dependent on LDLR activity. The liver accounts for about 70% of the clearance of LDL with the rest being taken up by extra- hepatic tissues (Goldstein, Hobbs, and Brown 2001). The most important regulator of LDLR expression in the liver is the cholesterol content of hepatocytes via sterol regulatory element binding proteins (SREPBs) (Horton, Goldstein, and Brown 2002).
These are transcription factors that mediate the expression of LDLR and other key genes involved in cholesterol and fatty acid metabolism. When there is a sufficient amount of cholesterol in the cell, SREBPs reside in the endoplasmic reticulum as inactive forms. They are activated by a decrease in intracellular cholesterol, whereby they are transported from the ER to the golgi where proteases cleave SREBPs into active transcription factors which then translocate to the nucleus to stimulate the transcription of LDLR and other genes. (Horton, Goldstein, and Brown 2002). In addition, LDLR transcription is induced by oxidized sterols which activate LXR, a
nuclear hormone receptor that is also a transcription factor. (Zhang et al. 2012).
Furthermore, LDLR activity is regulated by PCSK9 which targets the receptor for degradation. Loss-of-function mutations in PCSK9 lead to increased LDLR activity and decreased LDL levels. This has led to the development a novel group of drugs, PSCK9 inhibitors, to treat severe hypercholesterolemia. (Bergeron et al. 2015).
LDLR-independent pathway is the sole means of clearance of LDL from the plasma of hoFH patients with null-alleles. LDL can be taken up by a non-specific mechanism resembling phagocytosis. However, this is inefficient and only accounts for a minor part of the clearance of LDL from the plasma.
Figure 3. Structure and domains of LDL receptor (a) and metabolism of LDL and functional consequences of LDLR mutations (b). LDLR is synthesized at the endoplasmic reticulum (ER), transported to the Golgi where it is further processed. Mature LDLR is transported to the plasma membrane where it binds LDL particles via ApoB. The LDLR/LDL complex is endocytosed from clathrin-coated pits. In the cell, the LDL is targeted to lysosomal degradation whereas the receptor is recycled to the cell surface for multiple rounds of receptor-mediated endocytosis. LDLR mutations are classified into five functional classes based on the phenotypic behaviour of the mutant protein: Class I mutations affect the synthesis of the receptor (null-alleles) (1); class II: defective transport to Golgi or plasma membrane (2); class III binding-deficient (3); class IV impaired endocytosis (4) and class V recycling deficient (5).
Modified from (Benito-Vicente et al. 2018)
2.4.6 Reverse cholesterol transport
Reverse-cholesterol transport (RCT) is the process by which cholesterol is transported via HDL, from the periphery and lesions to the liver for excretion. It involves the efflux of free cholesterol (FC) from macrophages by ABCA1 to lipid- poor Apo-A1 secreted from the liver and intestinal enterocytes to form discoidal nascent HDL (nHDL) which contains FC, PL and ApoAI. Then the enzyme LCAT catalyzes the conversion of FC to CE to eventually form the spherical, mature HDL particles. (Cohen and Fisher 2013). The liver takes up FC and CE from the mature HDL particle via SR-BI leaving behind a remnant HDL and lipid-free ApoAI that returns for another cycle of RCT (Cohen and Fisher 2013). However, based on current evidence this traditional model of RCT has been suggested to be a minor mechanism and a ‘revised’ model has been proposed where LCAT plays a minor role in RCT.
This newer model suggests that most HDL-FC and nHDL-FC transfer to the liver rapidly independent of LCAT activity. (Gillard et al. 2018).
2.5 SCAVENGER RECEPTORS
Atherosclerosis is a chronic inflammatory disease where macrophages play an important role in mediating uptake of ox-LDL and thus foam cells formation in the early stages of atherosclerosis. This uptake is mediated through binding and internalization of these modified lipids to scavenger receptors expressed by macrophages.
2.5.1 Scavenger receptor A
Scavenger receptors (SRs) are a group of integral membrane proteins and soluble secreted extracellular domain isoforms which are able to bind a diverse array of ligands. These include modified lipoproteins, apoptotic cells, proteoglycans, carbohydrates, cholesterol ester, phospholipids and ferritin, subsequently SRs participate in a wide range of biological functions. The proteins are classified, according to their structure and biological function, into ten classes termed A-J.
(PrabhuDas et al. 2017).
Class A proteins are distinguished by a unique collagen-like domain which determines the collagen binding activity of SR-A homotrimers at the cell surface.
Members of this class include SR-A1, -A3, -A4, -A5 and -A6. SR-A1 is abundantly expressed on macrophages but can also be found on ECs and VSMCs. It binds a wide array of ligands including oxidized LDL (ox-LDL) (Kodama et al. 1990), β-amyloid (Frenkel et al. 2013), heat shock proteins (Berwin et al. 2003), as well as gram-positive (Dunne et al. 1994) and gram–negative (Hampton et al. 1991) bacteria. SR-A also has a role in monocyte migration and adhesion. Due to its ability to bind modified LDL and its role in monocyte migration and adhesion it was postulated to play a significant role in atherogenesis, and expression of SR-A1 has been shown in human
atherosclerotic lesions (Ylä-Herttuala et al. 1991). This is further discussed in chapter 2.5.3. Other members of the class A SRs are unable to bind oxidized LDL and play roles in innate immunity and immune reactions.
Class A SRs can be internalized from the plasma membrane either by clathrin- dependent or independent endocytosis or by phagocytosis by a lipid raft–dependent mechanism (Zani et al. 2015).
2.5.2 Other scavenger receptors
From the other scavenger receptors, SR-B1 and SR-B2 (previously CD36), SR-D1 (previously CD68) and SR-E1 (previously LOX-1) have also been implicated to play roles in atherogenesis. SR-B2 is expressed on Kupffer cells, liver sinusoidal ECs, monocyte/macrophages and hepatocytes among others. It binds HDL, oxLDL, long- chain FAs, thrombospondin and apoptotic cells, and is also able to mediate adhesion.
It is able to bind and internalize minimally modified LDL, unlike SR-A1, and has been detected in foam cells of human atherosclerotic lesions. ApoE-/-/CD36-/- double knock-out mice display a reduction in atherosclerotic lesion area compared to ApoE single knock-out mice on Western type diet. However, a prospective study of a Danish population no correlation with elevated plasma SR-B2 (CD36) levels and higher CHD risk in the general population was found (Y. Wang et al. 2018).
2.5.3 Scavenger receptors in atherosclerosis
Due to their ability to bind and take up modified LDL it has been postulated that scavenger receptors may play a significant role in atherosclerosis via foam cell formation and by mediating migration and adhesion of monocytes. Indeed, the expression of SR-A has been shown in human atherosclerotic lesions (Luoma et al.
1994; Ylä-Herttuala et al. 1991) however controversial evidence from animal studies has left the exact role of SR-A in atherosclerosis still unclear.
Originally it was shown that macrophages from SR-A deficient mice display an 80% reduction in the uptake of I125-labelled acetylated LDL (ac-LDL) but its clearance was unaffected (Suzuki et al. 1997). This was likely due to receptor redundancy as new members of the SR family were later cloned and characterized. Crossbreeding of the SR-A deficient mice into an ApoE-deficient background showed dramatic reductions in lesion area. However, in subsequent studies deletion of SR-A or SR-B2 (CD36) alone in ApoE-deficient mice caused an increase in aortic sinus lesion size, and in a separate study a clear decrease in atherosclerotic lesion size was seen with SR-B2 (CD36) deficiency in ApoE-/- background whereas in a triple knock-out of ApoE-/-, SR-A-/- and CD36-/-, the additional knock-out of the SR-A did not affect lesion area further. (Kuchibhotla et al. 2008; Moore et al. 2005) On the other hand,
