Novel markers for smooth muscle cell modulation in vascular injury and disease

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Department of Molecular Medicine and Surgery Karolinska Institutet, Stockholm, Sweden

Novel markers for smooth muscle cell modulation in vascular injury and disease

Urszula Rykaczewska

Stockholm 2022

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All previously published papers were reproduced with permission from the publisher.

Published by Karolinska Institutet.

Printed by Universitetsservice US-AB, 2022

Cover illustration: Triple staining of a brachiocephalic artery atherosclerotic plaque from a SMC lineage-tracing mouse (red – Tomato, blue – CD68, green – BCLAF1).

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NOVEL MARKERS FOR SMOOTH MUSCLE CELL

MODULATION IN VASCULAR INJURY AND DISEASE THESIS FOR DOCTORAL DEGREE (Ph.D.)

By

Urszula Rykaczewska

The thesis will be defended in public at BioClinicum, J3:11 Birger & Margareta Blombäck U220032100, Karolinska Universitetssjukhuset, Solnavägen 30, 26.08.2022 at 9:00.

Principal Supervisor:

Associate Professor Ljubica Matic Karolinska Institutet

Department of Molecular Medicine and Surgery Division of Vascular Surgery

Co-supervisor:

Professor Ulf Hedin Karolinska Institutet

Co-supervisor:

Associate Professor Joy Roy Karolinska Institutet

Co-supervisor:

Anton Razuvaev, MD, PhD Karolinska Institutet

Opponent:

Professor Joseph M. Miano Augusta University

Medical College of Georgia Examination Board:

Associate Professor Hans Grönlund Karolinska Institutet

Department of Clinical Neuroscience Professor Ulf Eriksson

Karolinska Institutet

Department of Medical Biochemistry and Biophysics

Professor Jan Boren

Gothenburg University, Sahlgrenska Academy

Department of Molecular and Clinical Medicine

Chair:

Assistant Professor Anton Gisterå Karolinska Institutet

Department of Medicine

Division of Cardiovascular Medicine

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Mojej ukochanej Rodzinie To my beloved Family

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It always seems impossible until it's done.

Nelson Mandela

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If you can meet with Triumph and Disaster and treat those two impostors just the same.

Inscription above the players’ entrance to the Centre Court at Wimbledon

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ABSTRACT

Smooth muscle cells (SMCs) are major constituents of the vascular wall, indispensable for basic physiological functions of a healthy vessel, such as regulating vascular tone and blood pressure, but also critical during disease development. With remarkable plasticity, SMCs act as early responders to vessel wall injury, where by activating molecular mechanisms, including phenotypic modulation and transdifferentiation, they counteract detrimental stimuli and aim to restore vascular homeostasis. The nature of SMC response to injury constitutes a major determinant of cardiovascular pathologies, including atherosclerosis, restenosis and aortic aneurysms, however, despite extensive progress in understanding the biology behind SMC phenotypic modulation, its many aspects remain elusive. With this perspective, the presented thesis aimed to identify and comprehensively characterize novel molecular signatures demarcating SMC phenotypic modulation, with a particular focus on transcriptional and cytoskeletal regulation of various SMC transitions.

Study I identified muscle contraction and actin cytoskeleton among the most downregulated pathways in atherosclerosis, while cytoskeleton-related leiomodin 1 (LMOD1), synaptopodin 2 (SYNPO2), PDZ And LIM Domain 7 (PDLIM7), phospholamban (PLN) and synemin (SYNM) emerged as the top molecular signatures repressed in atherosclerotic carotid plaques in comparison to control arteries. These genes positively correlated to classical contractility markers and showed abundant expression in SMCs in healthy arteries, but were largely absent from end-stage lesions. Subcellularly, the majority of the proteins localized to the SMC cytoskeleton and was significantly downregulated in response to atherosclerosis-relevant stimuli. Mechanistically, repression of PDLIM7 resulted in downregulation of SMC markers, and impaired cell spreading, but increased proliferation. Altogether, this study identifies a panel of novel sensitive SMC markers, which could serve as early indicators of SMC phenotypic modulation in vascular disease.

Study II investigated the role of proprotein convertase subtilisin/kexin 6 (PCSK6), previously identified as one of the top molecules upregulated in human atherosclerotic plaques. PCSK6 localized to fibrous cap and neovessels in carotid lesions as well as to injury- induced intimal hyperplasia, where it was expressed by proliferating smooth muscle alpha- actin (SMA) + cells and shown to colocalize and co-interact with matrix metalloproteinases (MMPs) 2 and 14. Pcsk6^-/-mice were characterized by the repression of SMC contractility markers and extracellular matrix (ECM) remodeling transcripts, displayed reduced intimal hyperplasia formation upon carotid ligation in vivo and impaired outgrowth of SMCs from aortic rings ex vivo, the latter two likely attributable to decreased MMP14 activity. In summary, this study establishes PCSK6 as a molecule of crucial importance for the SMC function in vascular remodeling.

Study III focused on key molecular signatures in carotid plaques stratified by ultrasound- assessed echogenicity. BCL2 Associated Transcription Factor 1 (BCLAF1) emerged as a top molecule downregulated in relation to plaque echolucency, abundantly expressed in SMA+

SMCs in the normal arteries, strongly repressed early during atheroprogression, however restored in cluster of differentiation 68 (CD68) + cells in advanced lesions, where it was also shown to co-interact with pro-survival B-Cell CLL/Lymphoma 2 (BCL2). Repression of BCLAF1 resulted in suppression of SMC contractility markers, decreased cell viability, as well as partially prevented oxLDL-induced SMC transdifferentiation into macrophage-like cells by preserving higher MYH11 expression and reducing levels of CD36 and CD68

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scavenger receptors. Overall, BCLAF1 emerged as a molecule indispensable for SMC survival and transdifferentiation into CD68+ macrophage-like cells.

Study IV aimed to identify key transcription factors (TFs) in the control of SMC phenotype and function in human atherosclerosis. Forkhead Box C1 (FOXC1) emerged as a master upstream regulator of genes differentially expressed in carotid plaques compared to control arteries and in relation to patient symptomatology, involved in the regulation of cell cycle, response to T3 hormone and cell adhesion. It was abundantly expressed in SMA+ cells in the control arteries and plaques, strongly downregulated in early phases of vascular wall healing, with its expression gradually restored concomitantly with SMCs regaining their contractile properties. Silencing of FOXC1 resulted in significant repression of SMC contractility markers, increased migration and proliferation, as well as partially abolished T3-induced SMC phenotypic modulation. Altogether, these results provide compelling evidence for FOXC1 being an important TF in the control of SMC quiescence vs. activation, especially in response to T3.

Collectively, by unraveling the intricacies of various aspects of SMC phenotypic modulation, this thesis contributes to a better understanding of molecular mechanisms underlying cardiovascular disease (CVD).

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LIST OF SCIENTIFIC PUBLICATIONS

I. Perisic L, Rykaczewska U, Razuvaev A, Sabater-Lleal M, Lengquist M, Miller CL, Ericsson I, Röhl S, Kronqvist M, Aldi S, Gonzalez Diez M, Roy J, Baldassarre D, Veglia F, Humphries SE, de Faire U, Tremoli E, on behalf of the IMPROVE study group, Odeberg J, Vukojević V, Paulsson-Berne G, Hansson GK, Lindeman JHN, Eriksson P, Quertermous T, Hamsten A, Hedin U. Phenotypic modulation of smooth muscle cells in atherosclerosis is associated with downregulation of LMOD1, SYNPO2, PDLIM7, PLN and SYNM. Arteriosclerosis, Thrombosis, and Vascular Surgery (ATVB). 2016 Sep;36(9):1947-61.

II. Rykaczewska U*, Suur BE*, Röhl S*, Razuvaev A, Lengquist M, Sabater-Lleal M, van der Laan SW, Miller CL, Wirka R, Kronqvist M, Gonzalez Diez M, Vesterlund M, Gillgren P, Odeberg J, Lindeman J, Veglia F, Humphries SE, de Faire U, Baldassarre D, Tremoli E, on behalf of the IMPROVE study group; Lehtiö J, Hansson GK, Paulsson- Berne G, Pasterkamp G, Quertermous T, Hamsten A, Eriksson P, Hedin U# and Matic L

#. PCSK6 is a key protease in the control of smooth muscle cell function in vascular remodeling. Circulation Research. 2020 Feb 28;126(5):571-585.

III. Rykaczewska U, Zhao Q, Saliba-Gustafsson P, Lengquist M, Kronqvist M, Bergman O, Huang Z, Lund K, Waden K, Pons Vila Z, Caidahl K, Skogsberg J, Vukojevic V, Lindeman JHN, Roy J, Hansson GK, Treuter E, Leeper NJ, Eriksson P, Ehrenborg E, Razuvaev A, Hedin U* and Matic L*. Plaque evaluation by ultrasound and transcriptomics reveals BCLAF1 as a regulator of smooth muscle cell lipid transdifferentiation in atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Surgery (ATVB). 2022 May;42(5):659-676.

IV. Rykaczewska U, Suur BE, Zhao Q, Narayanan S, Lengquist M, Kronqvist, Razuvaev A, Eriksson P, Quertermous T, Hedin U, Matic L. Transcription factor FOXC1 controls smooth muscle cell phenotype and function in atherosclerosis. Manuscript.

*, # authors contributed equally

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OTHER RELATED PUBLICATIONS

V. Röhl S*, Rykaczewska U*, Seime T*, Suur BE, Diez MG, Gådin JR, Gainullina A, Sergushichev AA, Wirka R, Lengquist M, Kronqvist M, Bergman O, Odeberg J, Lindeman JHN, Quertermous T, Hamsten A, Eriksson P, Hedin U, Razuvaev A, Matic LP. Transcriptomic profiling of experimental arterial injury reveals new mechanisms and temporal dynamics in vascular healing response. JVS-Vascular Science. 2020 Feb 7;1:13-27.

VI. Gallina AL, Rykaczewska U, Wirka RC, Caravaca AS, Shavva VS, Youness M, Karadimou G, Lengquist M, Razuvaev A, Paulsson-Berne G, Quertermous T, Gisterå A, Malin SG, Tarnawski L, Matic L, Olofsson PS. AMPA-Type Glutamate Receptors Associated With Vascular Smooth Muscle Cell Subpopulations in Atherosclerosis and Vascular Injury. Frontiers in Cardiovascular Medicine. 2021 Apr 20;8:655869.

* authors contributed equally

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LIST OF MOST FREQUENT ABBREVIATIONS

ACTA2 actin alpha 2

AFx Amaurosis Fugax

AHA American Heart Associacion ApoE Apolipoprotein E

BCL2 B-Cell CLL/Lymphoma 2

BCLAF1 BCL2 Associated Transcription Factor 1 BiKE Biobank of Karolinska Endarterectomies BIRCA Biobank of Rat Carotid Arteries

CASP3 caspase 3

CD68 cluster of differentiation 68

ChIP-seq chromatin immunoprecipitation-sequencing cIMT carotid intima/media thickness

CNN1 calponin 1

CT computed tomography

CVD cardiovascular disease ECM extracellular matrix ECs endothelial cells

eQTL Expression quantitative trait loci FGF fibroblast growth factor

FOXC1 Forkhead Box C1

GJA1 Gap Junction Protein Alpha 1

GLS Glutaminase

GLUL Glutamate-ammonia ligase

GRIA Glutamate Ionotropic Receptor AMPA Type Subunit GSM greyscale median

GWAS genome-wide association studies HDL high density lipoprotein

IFNg interferon-gamma IL interleukin

KLF4 Kruppel-like factor 4 LDL low density lipoproteins LGALS3 Galectin 3

LMOD1 Leiomodin 1

MCP1 Monocyte chemoattractant protein 1 MS minor/major stroke

MMPs matrix metalloproteinases MRI magnetic resonance imaging

mRNA Messenger RNA

MRTF-B myocardin-related transcription factor-B MYH11 myosin heavy chain 11

MYOCD myocardin

NASCET The North American Symptomatic Carotid Endarterectomy Trial OCT4 Octamer-binding transcription factor 4

oxLDL oxydized low-density lipoprotein PBMCs peripheral blood mononuclear cells PCSK proprotein convertase subtilisin/kexin PDGFB platelet-derived growth factor subunit B PDLIM7 PDZ And LIM Domain 7

PLA proximity ligation assay

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PLN phospholamban

ROS reactive oxygen species

RUNX2 Runt-related transcription factor 2 SASP senescent secretory phenotype SCA1 Stem cells antigen-1

scRNA-seq single cell RNA-sequencing SMA smooth muscle alpha-actin SMAD3 SMAD Family Member 3 SMCs smooth muscle cells SMTN smoothelin

SNPs single nucleotide polymorphisms SOX2 SRY-Box Transcription Factor 2 SRF serum response factor

STMN stathmin

SYNM synemin

SYNPO2 Synaptopodin 2 T3 triiodothyronine

TCF21 Transcription Factor 21 TF transcription factor

TGF-β transforming growth factor-beta TAGLN transgelin

THRB Thyroid Hormone Receptor Beta TIA transitory ischemic attack

TIMPs tissue inhibitors of MMPs TRLs triglyceride-rich lipoproteins

US ultrasound

vWF von Willebrand factor

WT wild type

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LIST OF ILLUSTRATIONS

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1 INTRODUCTION

1.1 CARDIOVASCULAR DISEASE

1.1.1 Epidemiology, prevalence and risk factors

Despite substantial progress in prevention, diagnosis and treatment, cardiovascular disease (CVD) is still considered to represent the leading cause of morbidity and mortality worldwide. According to the 2019 Global Burden of Disease study, CVD is responsible for 18.6 million deaths globally (corresponding to > 30% of all deaths), with significantly higher prevalence in low- and middle-income countries^{1, 2}. Only in Europe, 4 million people die every year from CVD, of which 1.4 million do not reach the age of 75³. In 2015 over 85 million Europeans were living with CVD, with the estimated cost for EU economies being placed at approximately €210 billion/year, of which €111 billion was related to health care expenditure and €99 billion to productivity loss and patient care combined⁴. CVD is a broad term and includes diseases related to the heart and/or blood vessels, such as coronary and peripheral artery disease, cerebrovascular disease, renal artery stenosis and aortic aneurysm.

Although all present themselves with various clinical manifestations, population-based studies show that many share the same underlying pathology, atherosclerosis, as well as common risk factors⁵, such as genetic predisposition, age, smoking, alcohol, high-fat diet, lack of physical activity, obesity, diabetes and hypertension⁶ (Figure 1).

With a significantly increased life expectancy of the general population, age is indisputably the strongest risk factor for CVD^7-10. This is likely linked to profound changes in arterial homeostasis and regenerative potential, both impaired in aging vessels. Vasculature repair

Figure 1. Modifiable and non-modifiable risk factors for CVD.

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capacity is typically associated with two types of cells: smooth muscle cells (SMCs) and bone marrow-derived progenitors. As SMCs constitute the major portion of the vessel wall and are usually the most pronounced responders to the injury¹¹, it is of crucial importance that accumulation of senescent SMCs has been reported in the aging vascular wall¹². Characterized by irreversible growth arrest combined with the secretion of proteases and inflammatory cytokines, these senescent SMCs not only limit the reparative potential but also contribute to sustained chronic inflammation in the vessel milieu^{12, 13}. This, further amplified by age-related severe impairment in the reparative capacity of bone marrow-derived progenitors^{7, 10}, consequently alters the balance towards development and progression of CVD⁷.

Dyslipidemia defines a group of disorders with abnormally elevated or reduced lipid levels, including triglycerides, cholesterol and phospholipids, with monogenic and familial hypercholesterolemias being the most common¹⁴. Loss-of-function mutations in genes regulating lipid uptake (LDLR, APOB)^{15, 16} or metabolism (LPL, APOC2)^{17, 18} profoundly alter lipid profile in favor of significantly elevated circulating low-density lipoprotein (LDL) and triglyceride-rich lipoproteins (TRLs), including VLDL, both strong risk factors for cardiovascular disease^{19, 20}. While LDL accumulation within the vascular wall and its subsequent oxidation promote the recruitment of inflammatory cells and atheroprogression²¹, similar processes are attributable to TRLs, which induce the production of reactive oxygen species (ROS) and mediate macrophage-to-foam cell conversion²².

Smoking has been postulated to represent the major modifiable risk factor for CVD, with its importance emphasized by it being accountable for approximately 1 in 4 CVD-related deaths²³. Three major constituents of cigarette smoke were considered to be responsible for the observed effect: nicotine, carbon monoxide and, most importantly for the development of CVD, oxidant gases. Long-term exposure to nicotine has been shown to, via sustained stimulation of the sympathetic nervous system, accelerate heart rate²⁴, which combined with the likely contribution of nicotine to endothelial dysfunction, insulin resistance and altered lipid metabolism²⁵, strengthen its position as a strong contributor to CVD. Another major component of cigarette smoke, carbon monoxide (CO), has been reported to bind to hemoglobin and reduce its oxygen binding capacity, therefore aggravating ischemia²⁵. Acting in concert with the previous two, oxidizing chemicals, reduce the levels of antioxidants²⁶ and in turn increase the amount of ROS²⁷, therefore contributing to lipid peroxidation²⁸, as well as endothelial dysfunction and oxidation of LDL²⁹. Altogether, these components are potent factors destabilizing vessel wall homeostasis, significantly contributing to the risk of CVD.

Overall, the significance of cardiovascular disease prevention cannot be denied, as control of well-defined major modifiable risk factors has been proven to substantially (up to 90%) reduce the disease burden, as estimated by recent reports³⁰.

1.1.2 Vascular wall injury and healing

1.1.2.1 Physiological conditions

Irrespectively of their size and function, healthy arteries are composed of three separate layers: tunica intima, tunica media and tunica adventitia³¹ (Figure 2). Although the overall vessel wall architecture is preserved among different kinds of arterial beds, the structure and proportion of each layer may vary depending on the localization and function of the particular artery. Tunica intima, the innermost part of the artery, comprises a monolayer of endothelial cells (ECs), the adjacent basement membrane and subendothelial connective tissue, which anchors cells to the vessel wall. Due to its direct contact with the blood flow, intima is critical

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for maintaining vascular homeostasis, as it is involved in processes such as coagulation, regulation of blood pressure and inflammation³². Tunica media, separated with internal and external elastic laminae, is composed predominantly of contractile SMCs arranged in concentric circles, characterized by profound involvement of myofilaments constituting even 70-90% of cytoplasm³³ and high expression of cytoskeleton-related proteins, such as myosin heavy chain 11 (MYH11), actin alpha 2 (ACTA2), transgelin (TAGLN) or calponin 1 (CNN1)³⁴. Cellular composition, where contracting SMCs are intermixed with elastic fibers and embedded in extracellular matrix (ECM), determines the importance of media in the control of the vascular tone and blood pressure, as well as vessel wall healing³⁵. Tunica adventitia constitutes the outermost part of the artery that comprises collagen- and fibronectin-enriched tissue³⁶, dynamic microvasculature called vasa vasorum, perivascular nerves and various kinds of resident cells (fibroblasts, T-cells, B-cells, macrophages and both endothelial and mural progenitor cells)^37-40. It nourishes both media and intima, provides mechanical stabilization and participates in the growth and repair of the vessel wall⁴¹.

Intimal hyperplasia is a generic healing response to vascular wall injury that aims at replenishing defects in the tissue, to preserve its full functionality (Figure 2). Although various cell types are involved in this process, in healthy arteries central role is attributed to SMCs as major components of the arterial wall¹¹. Upon injury inflicted to the arterial wall, a healing response is initiated. Endothelial denudation results in exposure to underlying SMCs, which triggers thrombosis and inflammatory cell recruitment. Inhibition of NO-dependent anti-proliferative signaling combined with growth factors released from adherent platelets, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and transforming growth factor b (TGF-b) stimulate SMC phenotypic modulation^42-45. It is manifested in strong downregulation of typical contractility markers, the transition from spindle- to rhomboid-shaped cells, as well as protease-dependent degradation of basement lamina^{46, 47}. Synthetic SMCs are in direct contact with the interstitial matrix components, such as fibronectin and various proteoglycans, which have been shown to further promote their phenotypic modulation⁴⁸. Following the initial activation, migration of SMCs to the intimal layer is initiated. It is a complex process that requires cell adhesion alterations, activation of several matrix metalloproteinases (MMPs), as well as bioavailability of various mitogenic and chemotactic factors⁴⁹. First, proteinases such as MMP-2 and MMP-9 release SMCs from the constraints of the basal membrane, which results in the subsequent

Figure 2. Structure of the artery under physiological conditions. The healthy coronary artery is composed of a single layer of ECs, media with multiple layers of SMCs and adventitia. Intimal hyperplasia arises as a response to the vascular injury and constitutes a mechanism of vascular repair. (Figure adapted from Macabrey et al. Front Cardiovasc Med. 2022 Apr 11;9:876639.)

EEL – external elastic lamina; IEL – internal elastic lamina

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downregulation of its constituents such as laminin, nidogen, collagen IV and perlecan^{50, 51} as well as a number of integrins, including a1b1 integrin, responsible for SMC-collagen IV contact⁵². Instead, it is replaced by a2b1, a5b1, aVb1 and aVb3, which all favor the interaction between the SMCs and other ECM components^{53, 54}. Further, membrane-bound MMPs, such as MMP-14, localized to the cells’ leading edge, proteolytically remodel the surrounding extracellular matrix (ECM), creating gaps necessary for cell repositioning⁵⁵. At the same time, synthetic SMCs, adherent platelets and inflammatory cells secrete various growth factors, including PDGF, which further stimulate chemotactic migration of SMCs^56,

57. Once SMCs migrate to the intima, they extensively proliferate, hence contributing to the increased intimal cellularity. With time, the proliferative index decreases, while excessive ECM deposition becomes evident. It has been shown that synthetic SMCs within the intima secrete even approximately 40 times more collagen than their contractile counterparts in the media⁵⁸. Successful re-endothelialization and associated restoration of NO signaling, cease SMC proliferation⁵⁹. At this point, intimal hyperplasia is fully developed, and most of the time will not progress further. SMC phenotypic switch is a reversible process, therefore when the vascular repair is completed, SMCs regain their contractile phenotype^{11, 60, 61}.

1.1.2.2 Excessive healing response – restenosis

Clinically relevant vascular wall healing occurs in response to surgical interventions when mechanical interference in the structure of the blood vessel triggers pronounced SMC activation and proliferation. While in the majority of cases such a healing response confers a survival advantage by virtue of thrombosis prevention, it may also contribute to severe (>50%) luminal narrowing, termed restenosis. Restenosis is one of the major adverse events after angioplasty, where initially bare-metal stents have been used in order to restore blood flow. Due to the high rate of post-stenting re-occlusion, they were replaced by drug-eluting ones^{62, 63}. Although the latter utilize anti-proliferative compounds such as paclitaxel or rapamycin, and hence significantly reduce intimal hyperplasia, they confer a greater risk of late stent thrombosis associated with impaired healing response, therefore highlighting the importance of balanced repair in maintaining vascular integrity^64-67.

1.1.3 Carotid stenosis: clinical presentation, diagnostics and treatment options Carotid stenosis is associated with progressive narrowing or occlusion of internal carotid arteries, two major vessels that supply the brain with oxygenated blood. Most often it is attributable to the developing atherosclerotic lesions, to which carotid arteries are predisposed, due to the presence of oscillatory and turbulent blood flow in their bifurcation

68. In the majority of cases the disease is asymptomatic, and for many years may remain undetected, even despite severe luminal stenosis. On the other hand, it has been shown that symptomatic carotid plaques account for 10-20% of ischemic strokes⁶⁹, which manifest by either transitory ischemic attack (TIA), minor/major stroke (MS) or retinal stroke (amaurosis fugax, AFx). These cerebrovascular events are a consequence of atherosclerotic lesion destabilization, associated with thinning of the fibrous cap and its subsequent rupture, which results in acute thrombosis. Thromboembolic material may detach from the side of the rupture, travel with the bloodstream and be a direct cause of occlusion in small cerebral vessels. To identify patients at risk and to detect the characteristics of unstable lesions, several imaging modalities have been employed. Ultrasound (US) is the single most commonly used technique for visualization of the lumen with its flow velocities, as well as the structure of the vessel wall, and therefore provides complex information regarding the disease status.

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Carotid Intima-Media Thickness (cIMT) measurements by US are considered to be indicative of the extent of carotid atherosclerosis. While cIMT for a healthy middle-aged individual usually varies between 0.5 - 0.8 mm⁷⁰, thickness values above 1 mm may signify a high risk of CVD⁷¹.

Interestingly, as single cIMT measurements are commonly accepted as surrogates of subclinical atherosclerosis^72-74, cIMT progression over time has been linked to increased cardiovascular risk⁷⁵. Assessment of the plaque morphology based on greyscale median (GSM), divides lesions according to the pixel value intensity into echolucent and echodense ones (Figure 3)⁷⁶. Interestingly, previous integrations of ultrasound with plaque histology revealed that echolucent plaques share distinct lipid-rich morphology with the presence of large lipid-rich necrotic cores, inflammatory cell infiltrates and possibly also intraplaque hemorrhage, features which are considered to be indicative of rupture vulnerability⁷⁷. Echodense lesions, on the other hand, were shown to contain more fibrous tissue⁷⁸, calcifications⁷⁹ and SMC-rich areas, which collectively provide stability and durability to the whole structure. Since the examination is fast, inexpensive and safe, US has become the most commonly used imaging modality in the visualization of vascular stenosis, with limitations that encompass acoustic shadows (associated with among others calcifications), operator dependency and variability of measurements depending on the anatomical features⁸⁰. Most of these issues can be overcome by magnetic resonance imaging (MRI), a multiparametric technique that allows for detailed visualization of lesion components. As it can successfully detect features associated with lesion vulnerability, such as thin fibrous cap, large necrotic core, neovessels and thrombus, it is frequently used in prospective clinical studies^81-83. Despite numerous advantages over the conventional US, the use of MRI for diagnostic purposes is severely limited by its availability, high cost and metal sensitivity. Computed tomography (CT) is an X-ray-based diagnostic method with various medical applications.

Since it is considered to be a relatively simple detection technique with wide availability, high spatial resolution and specificity towards calcium deposits, it has been utilized for the characterization of atherosclerotic lesions⁸⁴. Although components such as calcifications, lipids and fibrous tissue can be detected with CT aided by additional image processing software, its weakness is the use of contrast and the fact that patients are exposed to radiation^85, ⁸⁶. To summarize, each of the aforementioned imaging modalities confers both pros and cons, which result in neither of them being routinely used to characterize plaque composition.

Due to the lack of highly effective methods for identifying ‘vulnerable’ rupture-prone lesions, available treatment is usually limited to pharmacological management of the already present advanced disease, in a fraction of cases combined with surgical removal of atherosclerotic plaques.

Among the medications commonly used to slow down the progression of atherosclerosis, statins are the gold standard. By blocking 3-hydroxy-3-methylglutaryl-coenzyme A (HMG- CoA) reductase, they decrease cholesterol production in the liver, therefore significantly reducing circulating cholesterol. Statins are used both in primary prevention in individuals at high risk of developing CVD, as well as in secondary prevention in patients with already established disease, but at increased risk of adverse events. Next to statins, a new class of lipid-lowering drugs has been recently introduced, namely proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors. By binding and inactivation of PCSK9 protein,

<50% echolucent >70% echolucent

Figure 3. Ultrasound images of echodense and echolucent lesions. Adapted from Study III, Figure 1.

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PCSK9 inhibitors dramatically elevate the number of LDL receptors in liver cells, therefore contributing to its effective clearance and reduced LDL levels in the blood⁸⁷. While not recapitulating the effectiveness of statins in reducing all-cause and cardiovascular deaths, PCSK9 inhibitors were shown to have the highest efficacy in improving lipid levels, but simultaneously not associating with statin-related side effects⁸⁸. Apart from the lipid- lowering drugs, other classes of medications are being used in order to minimize the risk of adverse events related to vascular stenosis. Platelet aggregation inhibitors, such as aspirin, reduce the possibility of excessive blood clotting in the narrowed arteries, while antihypertensive drugs lower blood pressure, by which they help to reduce the risk of disease- related complications, such as heart attack.

Clinical guidelines for surgical intervention were pre-defined by two multi-center randomized control trials carried out in 1991-1998, European Carotid Surgery Trial (ECST)⁸⁹ and North American Symptomatic Carotid Endarterectomy Trial Collaborators (NASCET)^90,

91. The aim of both studies was to investigate the benefit of carotid endarterectomy in patients with documented recent cerebrovascular adverse events in reducing future risk for stroke or death compared to the pharmacological disease management alone. In NASCET, surgical intervention has been recognized as highly beneficial for patients with severe (>70%) stenosis, as the absolute risk reduction in major or fatal ipsilateral stroke within the next 2 years has been estimated around approximately 10.6% compared to the control group on medications only. Among symptomatic patients with 50-69% stenosis degree, endarterectomy yielded only moderate advantage, as it has been shown that in order to prevent 1 stroke within 5 years 15 patients have to be operated. Therefore, the general recommendation for this patient group was to ground the decision-making not only on the stenosis degree but also on other variables, such as existing risk factors or the practitioner’s surgical skills. Interestingly, no benefit from surgical intervention was observed for patients with stenosis <50%. ECST, on the other hand, motivates surgical intervention only in the case of patients presenting >80% stenosis. The apparent discrepancy in stenosis degree between these two trials is attributable to differing methodologies and in fact, when angiographic conversions are applied, >80% stenosis as measured by ECST corresponds to that of >60% in NASCET, which puts both studies in broad agreement⁹¹. Current international surgical guidelines from Society for Vascular Surgery (SVS) and the European Society for Vascular Surgery (ESVS) are largely based on solid foundations laid by these studies.

Altogether, as atherosclerosis is a multifactorial disease usually affecting different vascular beds and frequently coexisting with other comorbidities, patient-oriented molecular biology- based personalized medicine may be the most attractive future approach.

1.2 ATHEROSCLEROSIS

1.2.1 Brief history of atherosclerosis research

During decades of atherosclerosis research, several hypotheses postulated to explain its underlying causes. One of the first was the response-to-injury hypothesis, proposed by Ross et al⁹². It was a continuation of the idea presented by Rudolph Virchow in 1856 and was based on the assumption that atherosclerotic lesion arises from the injury inflicted to the vessel wall. Chronic elevation of plasma lipids sustained inflammation or altered fluid shear stress have been proposed to be a direct source of injury, leading to the disruption of the endothelial layer, with subsequent exposure of underlying collagen and activation of the coagulation cascade. The release of platelet-derived factors into the arterial wall was

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suggested to trigger SMC activation, migration and proliferation in order to form intimal hyperplasia. The Discovery of adhesion molecules on the endothelial surface gave rise to another theory called the response-to-inflammation hypothesis. Once expressed, VCAM- 1 and ICAM-1 have been shown to attract monocytes and T-cells, and enable their migration into the vascular wall⁹³. As inflammatory cells have been shown to adhere to the intact endothelium⁹⁴, Ross’s theory regarding injury as a prerequisite for disease initiation was questioned. Once resident within the intimal layer, monocytes differentiate into macrophages, engulf lipids and become apoptotic, hereby contributing to the growing necrotic core. In 1995 Kevin J. Williams together with Ira Tabas came up with the response- to-retention hypothesis, which assumed that lipoprotein retention within the arterial wall is a key pathologic event triggering atherosclerosis initiation⁹⁵. Their reasoning was based on the observation that although all of the processes mentioned in the previous hypotheses substantially contributed to disease development, neither of them was necessary nor sufficient to provoke lesion formation. Neither except the lipid retention, since it has been shown that plaques do not develop in the presence of low plasma lipoprotein levels, even when the other major risk factors exist^{96, 97}. Interestingly, with recent advances in the field of atherosclerosis, a new SMC-centered theory proposing that these cells are causally linked to atherosclerosis initiation is gaining in importance⁹⁸.

1.2.2 Atheroinitiation

Despite decades of extensive research, a true consensus has not yet been reached in regard to the sequence of events triggering atherosclerosis initiation. Several pathophysiological mechanisms were proposed to be directly implicated in the onset of the disease, however it is rather unlikely that one of them solely could be accounted for driving the process. With respect to the multifactorial nature of atherosclerosis, it is believed that in order to commence lesion development, a well-tuned interplay among several factors is required.

1.2.2.1 Genetic predisposition

For many years it has been postulated that genetic predisposition constitutes one of the major risk factors for CVD, accounting for even 30-60% of interindividual variation in susceptibility to the disease⁹⁹. Current knowledge regarding genetic variants influencing the risk of CVD comes mainly from genome-wide association studies (GWAS), which are based on the comparison of DNA from individuals with varying phenotypes in order to find associations between common single nucleotide polymorphisms (SNPs) and particular traits or diseases. So far, over 160 DNA variants have been associated with CVD^100-102 and while the majority of these susceptibility loci were linked to inflammation and lipids, several have been found also to be associated with SMCs. Lead SMC-related SNPs were shown to encode changes in genes determining their contractile phenotype (i.e. REST¹⁰³, NOS3¹⁰⁰, LMOD1¹⁰⁴), as well as in those regulating their phenotypic activation, migration and proliferation (i.e. Transcription Factor 21 (TCF21)¹⁰⁵, SMAD Family Member 3 (SMAD3)¹⁰⁶, SWAP70¹⁰⁷, ABHD2¹⁰⁸), indicating that SMCs may be causally linked to CVD susceptibility. Among the so far best described SMC-related genes, likely causally linked to CVD, are CDKN2B, TCF21, SMAD3 and LMOD1. CAD risk locus identified at chromosome 9p21.3 was associated with repressed SMC expression of CDKN2A and CDKN2B, where downregulation of the latter has been previously correlated with increased SMC proliferation and investment into atherosclerotic lesions¹⁰⁹. Two other CAD-associated SNPs, rs12190287 and rs12524865, were shown to regulate TCF21 expression in coronary SMCs¹¹⁰. As a transcription factor, TCF21 has been implicated in governing SMC phenotypic modulation,

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since its target genes were involved predominantly in the repression of SMC contractility markers, as well as induction of migration and proliferation of these cells, with a significant role in the stabilization of the fibrous cap¹¹¹. Contrarywise, both SMAD3 and LMOD1, with CAD SNPs localized to chromosomes 15q22.33 and 1q32.1104, 112, 113, respectively, have been shown to act as positive regulators of SMC differentiation104, 112-114. Loss of SMAD3 in SMCs resulted in increased cell invasiveness¹¹⁴, while Lmod1 global knockout was characterized by reduced levels of filamentous actin and impaired SMC contraction¹¹⁵. In addition, while LMOD1 was reported to be abundantly expressed in normal arteries, it was significantly downregulated in atherosclerotic lesions and co-localized with classical SMC markers such as MYH11, ACTA2, myocardin (MYOCD) and CNN1⁶¹. Altogether, there is a growing body of evidence supporting the causal implication of SMCs in CVD, highlighting the central role of these cells in both vessel wall homeostasis and various vascular pathologies.

1.2.2.2 Shear stress

Atherosclerosis is a multifocal disease, which affects reproducible regions along the vascular wall. Segments characterized by altered fluid mechanics, such as carotid bifurcation, coronary and iliofemoral arteries, located in close proximity to inner curvatures or branch points, are particularly susceptible to disease development¹¹⁶. The role of shear stress in designating predilection sites for atherosclerosis has been widely recognized and the mechanism through which it happens involves disruption of endothelial homeostasis and subsequent activation of resident SMCs. In healthy vessels friction between the blood and the most inner part of the vascular wall generates shear stress parallel to the surface and therefore affects predominantly endothelial cells (ECs)¹¹⁷, which use flow-responsive ion channels, integrins and glycocalyx as mechanosensors responsive to even the slightest changes in the blood flow¹¹⁸. Under steady laminar conditions, these cells secrete factors that inhibit excessive SMC proliferation, inflammatory cell recruitment as well as coagulation, and therefore are considered to be atheroprotective^{119, 120}. On the contrary, in the regions characterized by disturbed flow, where low/oscillatory shear stress is perpendicular to the vessel inner layer, all cells within the vascular wall are affected^{121, 122}. As a result of flow- induced local hypoxia, ECs display an inflammatory phenotype defined by aggravated turnover and poor alignment¹²³, which combined with the secretion of various growth factors (PDGF, TGF-β) transduce the signal to the medial SMCs. In response, by interaction with ECM, SMCs are capable of modulating conformation of their membrane proteins, including ion channels, receptors and transmembrane molecules, which ultimately results in activation of downstream signaling cascades and force-dependent changes in gene expression¹²⁴. Of particular importance for the process are integrins, well known not only for their role in interconnecting cells, ECM and cytoskeleton but also in converting mechanical force into chemical signal¹²⁵. The so far best-documented integrin role in transmitting the perception of mechanical stress changes in SMCs is attributable to a5b1 and aVb3, both upregulated in stretched vascular cells¹²⁶. Integrin a5b1 has been reported to be one of the physiological regulators of blood pressure, as by triggering Ca(2+) bursts in response to increased mechanical force it promotes SMC contraction and vasoconstriction¹²⁷. Contrarywise, in a mechanism likely activated under increased circumferential stress conditions, a5b1 was shown to affect the expression of focal adhesion kinase (FAK) and therefore promote proliferation and migration of SMCs¹²⁸. Similar effect on activation of SMCs has been documented for aVb3, where induction of aVb3/PI3-K/Akt pathway, as well as aVb3- mediated FAK phosphorylation were both linked to actin cytoskeleton reorganization and the associated increase in SMC proliferation and migration126, 129, 130.

Altogether, sheer stress-induced increased endothelial layer permeability, accelerated transport of various macromolecules into the vessel wall (including LDL), as well as

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associated profound changes in SMC signaling result in excessive activation of the latter^131,

132. Adaptive intimal thickening is then formed, which may provide soil for atherosclerotic plaque development (Figure 4A).

1.2.2.3 Influence of lipids

Plasma lipoprotein particles can be divided into ApoA- (HDL) and ApoB-containing proteins (chylomicrons, VLDL, IDL, LDL). HDL is high-density lipoprotein, which was previously reported to exert atheroprotective effects, attributable mainly to its anti-oxidative properties, as well as capacity of cholesterol clearance from the vessel wall¹³³. Indeed, many studies so far provided evidence for the negative correlation between HDL plasma levels and risk for CVD, demonstrating that low HDL concentration is a strong predictor of future adverse events ^{134, 135}. On the contrary, cholesterol-rich LDL is considered to be the principal driver of atherosclerosis initiation^{136, 137}. Constant exposure to increased LDL concentrations is associated with decreased NO bioavailability and subsequent endothelial dysfunction, both preceding the entry of LDL into the vessel wall¹³⁸. The importance of sustained high LDL plasma concentrations for its retention in the subendothelial layer has been confirmed in studies on familial hyperlipidemia, where it has been shown that patients with a genetically conditioned tendency to maintain very high blood cholesterol concentrations develop atherosclerosis prematurely at the age of 30 to 40¹³⁹.

1.2.3 Atheroprogression

Regardless of the initial atherosclerotic catalyst, the overall sequence of events leading to the formation of advanced atherosclerotic plaques remains similar. LDL infiltrating the vessel wall binds to intimal proteoglycans, such as perlecan via its heparan sulfate chains¹⁴⁰, and is subjected to various modifications, including oxidation and aggregation^{141, 142}. Oxidized lipid fractions induce ECs and SMCs to express adhesion molecules (VCAM1, ICAM1), chemoattractants (MCP1) and growth factors (M-CSF) that attract leukocytes and monocytes, and guide their homing, migration, as well as in case of the latter also transition into macrophages¹⁴³. Macrophages recruited to the site of lipoprotein retention engulf lipids in an attempt to clear the vessel wall, and become foam cells¹⁴⁴. Initially, these lipid-laden cells accumulate within the intimal proteoglycan layer and form so-called fatty streaks or xanthomas (Figure 4B).

At this stage, lesions are still harmless and removal of the causative factor may potentially fully reverse the process of plaque formation¹⁴⁵. Although a majority of the xanthomas fail to progress further, some advance to form proper atherosclerotic lesions. Continuous accumulation of lipoproteins in combination with the sustained influx of inflammatory cells profoundly changes vessel wall milieu. In response to increased amounts of oxidized low- density lipoprotein (oxLDL), various cytokines, growth factors and other proatherogenic molecules, more and more vascular SMCs lose their contractile features and become activated^146-148. Phenotypically altered SMCs migrate to the luminal part of the lesion, where they extensively proliferate and secrete ECM, thus directly contributing to the plaque enlargement. Interestingly, SMCs were also shown to be capable of lipid uptake and formation of cluster of differentiation 68 (CD68) + foam-like cells, thereby contributing to the growing lipid accumulation within the plaque^{149, 150}. Although in the beginning both macrophages and to a smaller extent also SMCs cooperate to efficiently clear the lipids from the vessel wall, continued increase in intracellular cholesterol concentration leads to the formation of acellular lipid pools underneath the layer of foam cells. This kind of lesion, where the proper structure of the intima is still preserved, is called pathological intimal

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thickening (Figure 4C). At this point cells are already overloaded with lipids and bombarded with various pro-inflammatory/apoptotic factors, therefore caspase 3 (CASP3)-dependent apoptosis, as well as secondary necrosis, become more frequent in macrophages and SMCs^151-153. This is especially pronounced in the latter, as SMCs, despite their acquisition of macrophage-like phenotype, show impaired abilities to uptake and cope with lipids compared to classical macrophages^{149, 150}. Impaired clearance of apoptotic debris due to increased expression of anti-phagocytic molecules such as CD47, drives further deposition of lipid-rich cargo within the tissue and is responsible for the expansion of isolated lipid pools into a continuous necrotic core within the lesion¹⁵⁴. A lesion with the intima irreversibly disrupted by a necrotic core is termed fibroatheroma (Figure 4D).

1.2.4 Vascular Remodeling in Atherosclerosis

It is a well-known phenomenon that human arteries enlarge in compensation for growing atherosclerotic lesions. Therefore, it is not before a plaque occupies 40% of the internal elastic lamina that the functionally crucial lumen stenosis occurs¹⁵⁵. This compensation mechanism is called outward vascular remodeling and is associated with an increase in vessel wall thickness and stiffness. Underlying molecular mechanisms involve altered shear stress with EC activation, aggravation of inflammation and SMC phenotypic modulation, followed by MMP activation, alterations in ECM production and degradation as well as enhanced collagen deposition¹⁵⁶. In response to chronically increased blood flow, ECs alter their NO production, which results in sustained vasorelaxation and an increase in circumferential stress¹⁵⁷. Stretch-induced production of FGF and MMPs (MMP-1, MMP-2 and MMP-9)

Figure 4. Progression of atherosclerotic lesions. A, Adaptive intimal thickening arising from SMC activation, migration and proliferation within the intima. B, Xanthoma is characterized by the accumulation of macrophage and SMC-derived foam cells in the intimal layer. C, Pathological intimal thickening with extracellular lipid pools. D, Late-stage ‘stable’ atherosclerotic lesion with a SMC-rich fibrous cap, large necrotic core and macrocalcification. E, Advanced ruptured atherosclerotic lesion with pronounced inflammation, apoptosis and neovessel formation.

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results in increased SMC proliferation in combination with degradation of collagen in the periphery of the media, which results in expansion of the vessel wall^{158, 159}. Interestingly, recently also PCSK6 has been linked to the remodeling of the vessel wall, as its loss was associated with the increase in outward remodeling in mice¹⁶⁰. The second type of arterial remodeling associated with atherosclerosis is called inward remodeling, and its prevalence is estimated for 25% of all lesions¹⁶¹. Instead of increasing its circumference with the growth of the plaque, the vessel shrinks, thereby further exacerbating stenosis¹⁶². It has been shown that this kind of vessel remodeling often occurs in relation to calcified plaques and smoking¹⁶³. In conclusion, the type and extent of vessel wall remodeling may be at least equally relevant as the lesion size and should be considered in determining the severity of stenosis.

1.2.5 End-stage disease

1.2.5.1 Characteristics of advanced plaques

Human atherosclerotic plaques are considered to be advanced when the accumulation of cells, lipoproteins and various ECM components is linked to serious intimal thickening and tissue structural disorganization, which results in irreversible deformation of the vascular wall. Although every lesion is different and may have distinct cellular composition, the majority of the advanced plaques share the same structural features.

One of the most prominent components of late-stage atherosclerotic lesions is a large necrotic core, constituting a significant portion of the plaque. Its development is inevitably associated with pronounced cell apoptosis and secondary necrosis, followed by defective efferocytosis151, 152, 164. In healthy arteries apoptosis is a well-orchestrated type of cell death, and as estimated, around 50-70 billion cells in the human body undergo self-destruction each day, to ensure tissue homeostasis. This balance between cell survival and apoptosis is severely disturbed in atherosclerosis, when overloaded with lipids and stimulated with various kinds of inflammatory factors (TNFa, IFN-g and IL-1) SMCs and foam cells repress anti-apoptotic Bcl-2, but induce pro-apoptotic proteins such as Bax, Bok, or Bad instead, and therefore more eagerly undergo Caspase 3-dependent programmed cell death^165-167. B-Cell CLL/Lymphoma 2 (Bcl2) is, next to Bcl-xl, one of the main anti-apoptotic proteins maintaining the equilibrium between cell survival and apoptosis. It has been shown to be capable of rescuing cells from a critical phase in commitment to apoptosis, by constraining activation of pro-apoptotic members of its family^{168, 169}. Interestingly, within atherosclerotic lesions, it has been shown to play a protective role against macrophage apoptosis and the associated increase in the size of necrotic core¹⁷⁰ Experimental data indicate that, at least in the initial phases, efferocytosis should efficiently clear cell remnants¹⁷¹. Interestingly, it has been shown that while in the majority of other tissues apoptotic cells are identified and targeted for clearance within minutes, in atherosclerotic plaques efferocytosis capacity is reduced almost 20-fold¹⁷². Therefore, even though overwhelming apoptosis admittedly contributes to the necrotic core formation, it is the failure to remove apoptotic cells from the plaques that seems to be crucial for this process. As a result, instead of being cleared, cell debris is stored within the lesion, where it is subjected to secondary necrosis, and therefore contributes to the formation of a rigid necrotic core that influences vessel wall stiffness, plaque expansion and subsequent lumen narrowing¹⁷³.

A fibrous cap is another hallmark of advanced lesions. It arises from the migration and proliferation of activated SMCs in response to a plethora of atherogenic stimuli (oxLDL, cytokines, growth factors). The formation of a proper fibrous cap is associated with the merging of isolated lipid pools into a continuous necrotic core¹⁷⁴ and while SMCs are one of

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its major constituents, macrophages and T-lymphocytes can also be found within this region¹⁷⁵. Although coexisting in one lesional compartment, these cells exert completely opposing effects. Once present in the fibrous cap, SMCs proliferate and secrete matrix constituents such as collagen and elastin, and by this strengthen this protective layer¹⁷⁶. Inflammatory cells, on the other hand, contribute to increasing concentrations of cytokines and proteases (MMPs) that may negatively affect cap stability, and therefore they are believed to promote lesion destabilization¹⁷⁷.

Atherosclerotic plaque calcification is an active process that resembles bone formation and can start to occur already in the second decade of life^{178, 179}. With lesion progression, intraplaque calcium amounts increase, since apoptotic cell debris and ECM act as a nidus for calcium deposits. Although the mechanism directly responsible for the initiation of calcification is not known, it is presumed that SMC apoptosis is required for the formation of apoptotic bodies, which constitute attractive sites for microcalcification^{180, 181}.

1.2.5.2 The ‘vulnerable’ plaque

Clinical symptoms due to atherosclerosis are often caused by severe luminal narrowing or thrombus precipitation at the site of plaque rupture. The concept of a ‘vulnerable’ lesion has been raised in relation to the plaques that convey high risk of thrombotic events and associated future adverse events (MI, TIA, stroke). Although as much as 58% of the plaque ruptures may be silent¹⁸², the healing response can lead to the gradual narrowing of arterial lumen and severe obstruction of the blood flow, therefore many efforts have been directed towards identification of the main determinants of plaque instability¹⁸³ (Figure 5). Plaque rupture is associated with disruption of a fibrous cap and subsequent exposure of a highly thrombogenic necrotic core. It often occurs within the parts where the fibrous cap is very thin and infiltrated with inflammatory cells¹⁸⁴. As assessed by autopsy studies, the average thickness of the disrupted fibrous caps was 23 µm, while 95% didn’t exceed 65 µm¹⁸⁵.

Figure 5. Features of ’stable’ and ’vulnerable’ lesions. So-called ‘stable plaques’ are characterized by thick, SMC-rich fibrous cap and low inflammatory content. Contrarywise, ‘rupture-prone’ lesions consist of the thin, highly inflamed fibrous cap, neovessels and intraplaque hemorrhage.

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Several mechanisms have been so far implicated in fibrous cap thinning. One would be a gradual decrease in the number of SMCs. Whilst in healthy arteries SMC-related apoptotic process is tightly regulated and allows for the controlled ‘suicide’ of defective cells and their replacement to ensure tissue integrity, in diseased atherosclerotic tissue SMCs reside in a hostile milieu containing a mixture of pro-and anti-apoptotic factors, where pronounced inflammatory influx and oxidized lipids shift the balance towards increased apoptosis^{165, 166,}

186. It has been shown that disrupted fibrous cap contains fewer SMCs and therefore also less collagen and glycosaminoglycans, which makes them less resistant to any forces acting on the vessel wall¹⁸⁷. Simultaneously, macrophages residing in the fibrous cap secrete proteolytic enzymes (cathepsins, MMPs) that degrade the surrounding matrix. This, in combination with defective collagen synthesis by intimal SMCs, creates gaps in the tissue, further contributing to the decrease in the fibrous cap extensibility^{188, 189}.

However, no fibrous cap is present in lesions lacking the necrotic core, therefore it is reasonable to state that growing necrotic core confers a higher risk of plaque rupture and its subsequent clinical manifestation¹⁹⁰. This observed effect is likely attributable to: 1) mechanistic properties of the necrotic core, where its rigid structure exerts great tensile strength on the overlying cap and therefore reduces its thickness by eroding it from below;

2) the underlying molecular biology with pronounced secondary necrosis and subsequently augmented inflammation, the former previously associated with increased incidence of ischemia in humans¹⁹¹.

Presence of neovessels, particularly in the close proximity to the necrotic core and in the lesion shoulder regions, is another feature of plaques with decreased stability. The initial stimulus for neovessel formation comes from resident hypoxic macrophages and is dictated by insufficient oxygenation of the tissue¹⁹². In the majority derived from vasa vasorum, neovessels differ from mature vessels in that they mostly lack supporting mural cells and therefore are leaky and fragile, which enables extravasation of the blood¹⁹³. Intraplaque hemorrhages are common in advanced lesions, where they constitute an alternative entry route for inflammatory cells^194-196, of which CD4+ T cells have been shown to be of particular importance for lesion destabilization. Apart from releasing pro-inflammatory cytokines interferon-gamma (IFNg) and TNFa¹⁹⁷, these cells also interact with macrophages, which in turn activate a cascade of proatherogenic events, including increased secretion of ECM- degrading proteases, chemokines, cytokines (IL-1, IL-6) and reactive oxygen species (ROS)^{143, 198}. How important CD4+ T-cells are for atherosclerosis development and progression, was determined when immunodeficient Apolipoprotein E (ApoE) ^-/-mice, at baseline showing >70% reduction in the plaque size, displayed instead >160% increased atheromas, when obtaining a transfer of CD4+ T-cells¹⁹⁹.

Overall, while determinants of unstable lesions have been recognized, the key events initiating the transition from stable to vulnerable plaque phenotype remain largely unknown.

1.2.6 SMCs in vascular disease and atherosclerosis

Studies so far have identified various cell types to be involved in plaque formation, including ECs, monocytes, lymphocytes and SMCs^{200, 201}. While most consider ECs and inflammation to be crucial for the disease initiation, SMCs are discussed mainly in the context of atheroprogression when their migration, proliferation and production of ECM contribute to further development, but also stabilization of the lesion34, 176, 202. Interestingly, the role of SMCs in lesion initiation was suggested already in 1953, when histological evaluation of coronary arteries from 300 United States soldiers killed in Korea provided evidence that regions prone to atherosclerosis contain thick SMC-rich intima²⁰³. This was further extended

Novel markers for smooth muscle cell modulation in vascular injury and disease

Department of Molecular Medicine and Surgery Karolinska Institutet, Stockholm, Sweden

Novel markers for smooth muscle cell modulation in vascular injury and disease

NOVEL MARKERS FOR SMOOTH MUSCLE CELL

MODULATION IN VASCULAR INJURY AND DISEASE THESIS FOR DOCTORAL DEGREE (Ph.D.)

Urszula Rykaczewska

ABSTRACT

LIST OF SCIENTIFIC PUBLICATIONS

OTHER RELATED PUBLICATIONS

TABLE OF CONTENTS

LIST OF MOST FREQUENT ABBREVIATIONS

LIST OF ILLUSTRATIONS

1 INTRODUCTION

1.1 CARDIOVASCULAR DISEASE

1.2 ATHEROSCLEROSIS