Thesis
Reference
S100A4 is a key player for smooth muscle cell phenotypic transition:
implications in atherosclerosis
SAKIC, Antonija
Abstract
Atherosclerosis is the leading cause of cardiovascular disease worldwide. In atherosclerosis, smooth muscle cells (SMCs) accumulate in the intima where they undergo a transition from a contractile to a synthetic phenotype. During the two last decades, synthetic SMCs have been considered as beneficial players in atherosclerotic plaque development by essentially contributing to fibrous cap formation that protects plaque from rupture. However, SMCs exhibit a remarkable plasticity, and recent studies have demonstrated that a high proportion of SMCs in atherosclerotic plaques are hardly detectable with the classical SMC markers and acquire proinflammatory macrophage-like phenotype. We have previously identified S100A4 as a marker of the synthetic phenotype, both in vitro and in vivo in pigs and humans. The main project of this thesis aimed at deciphering the role of extracellular S100A4 in phenotypic transition of SMCs and atherosclerotic plaque progression. Our results indicated that extracellular S100A4 is causally related to atherosclerotic plaque progression, putting it forward as a prospective therapeutic target for [...]
SAKIC, Antonija. S100A4 is a key player for smooth muscle cell phenotypic transition:
implications in atherosclerosis. Thèse de doctorat : Univ. Genève, 2020, no. Sc. Vie 45
DOI : 10.13097/archive-ouverte/unige:143048 URN : urn:nbn:ch:unige-1430480
Available at:
http://archive-ouverte.unige.ch/unige:143048
Disclaimer: layout of this document may differ from the published version.
Titre de la thèse [minuscules pour titre français, ou]
Title of the Dissertation [Capital Letters for English Title]
THÈSE
présentée aux Facultés de médecine et des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences en sciences de la vie,
mention Choose an item.
par
Thèse No nnnn
GENÈVE
S100A4 IS A KEY PLAYER FOR SMOOTH MUSCLE CELL PHENOTYPIC TRANSITION: IMPLICATIONS IN ATHEROSCLEROSIS
THÈSE
présentée aux Facultés de médecine et des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences en sciences de la vie,
mention Sciences biomédicales
par
Antonija ŠAKIĆ
de
Zagreb, Croatie
Thèse No 45
GENÈVE UNIVERSITÉ DE GENÈVE
Section de Médecine Fondamentale FACULTÉ DE MÉDECINE
Département de Pathologie et Immunologie Dr. Marie‐Luce Bochaton‐Piallat
Acknowledgment
Every PhD thesis is a long road that requires a hard work, patience, good will and deep commitment, both in professional and personal way. In the end of it, one person is officially awarded with the title, but actually, many more contributed to its realization and that is why they deserve special, deep and honest gratitude.
For that, I would like to start with one big thank you to my supervisor, Dr. Marie‐Luce Bochaton‐Piallat as without her the beginning, the middle or the end of this thesis would not be possible. She was a supervisor that gave me a lot of freedom and space to grow in a professional and personal way, but again, was always present and available for any advice, help or a talk. Thank you my boss, for being Lourdes, for caring, for incredible commitment and energy you give to all of us, for your optimism, your limitless help and the most‐ for being a big human!
To my committee members; Prof. Guido de Meyer, Prof. Thierry Soldati and Prof.
Michelangelo Foti, thank you for accepting to read evaluate my work.
Dear Chiraz and dear Matteo. I still remember crystal clear my interview and two of you who supported me and gave me great advices even before I started my PhD. Thank you for that and all the knowledge that we shared in our common work!
Dear Anita, thank you very much for all the help and your beautiful personality that made my days in our lab very pleasant. I am very happy that I shared all my time with you!
Dear Aman, even though not for all the time of my PhD, I enjoyed sharing work and stories with you! Thank you!
For a long time, I was the only “thesis” person in the laboratory, and that has dramatically changed when Luís Miguel Cardoso dos Santos joined us, as he brought energy accounting for minimum 2 additional people. That energy was expressed and prominent in all possible ways: when we shared our work, laughed, hanged out outside the lab as well as when we fight. All of that led to having not only an excellent working colleague, but also a (I hope) a life‐long friend. Thank you for everything my, well known, friend Miguel !
Ksenia, even though you have just arrived, thank you for bringing cheerfulness and fun in the laboratory!
I would also like to thank to all students that were contributing to the good work and atmosphere in the laboratory: Aurore for her enthusiasm, Tristan that was sharing with me the beginning steps of my project, Kaori for her scientific input and our sushi time, and Alexandre for bringing a new vibe in the lab. Specially, I would like to thank my Marieke who spend last months of my thesis in our lab. You lady, did much more for me than you think‐
both, in scientific and personal way. I am seriously proud of you and very honoured that you
“ended up” working with me!
Many thanks to all “PATIM people”; it was a pleasure to share the department with people like you. Thank you for your helpful scientific input and for always being willing to help! Moreover, I would like to thank bioimaging and genomic facility of the CMU for a big help and Sylvain, without whom, my RNA sequencing data would always stay a raw data Notably, I would like to thank to Prof. Brenda Kwak and her laboratory for their generous help and advices!
My dear Sanjice, CMU would not be the same without you and our “healing‐lunch‐
sessions”. I am thankful to the all circumstances that located us on the same place, in the same time. Thank you for everything! Together with Sanjica, naturally, appears Yves: thank you for being you and for sharing a decent amount of chocolates, as well as science, with me.
Now, my ladies, Mannekomba and Ronke, my companions from the first until the last day (and ongoing). If I could choose, I could not get better support, friends and people to have next to me during our PhD journey‐ thank you million times!!!!
Divore, “moja rak‐rano”, you have a very special role and place in all of this. Thank you for (not every) word and minute you spent discussing with me. For being my support rock, and person that was always unselfishly available: for sharing great, good, a bit less good, very bad….actually everything! Thank you!
Ksenija, Hana, Yacine, Duje, Matko, thank you my friends for all the support and care!
Ivana, Anita and Neven, we know each other for 25+ years and I have to thank you for not letting the distance set us apart. Thank you for all the virtual coffees and for not forgetting to take me on important events with your phones Thank you for being my friends! P.s‐ Jaki, you made our team complete, thank you for supporting us!
My dear Anka, you always made me feel like home, happy and peaceful. Our weekends will always stay only ours‐ thank you for that and for your never ending support!Thank you Ivica and Anka for going through the big swiss paper work for me‐ without you I would not have my small piece of home here. Ivice‐ thank you for not so often, but long, fun and interesting talks.
Gjino, short and clear‐ you are the one knowing me the best and the one that is there for me when it is the hardest, but also the nicest. Thank you for being a wind at my back since a very long time and for encouraging me when it was the most important! I know that you are not a fan of words, but I have to say that in this PhD story, you are a big piece of puzzle, and thank you for that!
Ivane, thank you for being the best brother, and Anita, thank you for being the best wife he could have. Two of you made my grey weekends much brighter!
To the smallest ones‐ Mislav and Klara…Once you grow up, I want you to read this to know that you turned our lives upside‐down, by bringing us huge amounts of happiness and love. Mislave, the worst days would disappear instantly when you would call and thank you for that. I am honoured that I was both, your Pero and teta Tonka. Thank you for the purest, the most honest and valuable talks and love!
I am thankful that in different moments of my life I had people supporting me and sharing with me happy and a bit less happy moments. Nevertheless, throughout my whole life I had and I have two people that were constantly giving me limitless and unconditional support, care and love. Becoming a parent is a matter of biology, but being a parent is a matter of unselfish commitment that never ends. Mama i tata, thank you for always being next to me. Without you I would not be happy and accomplished person in any segment of my life.
Thank you for all the sacrifice you did for me and if I hope that I managed to make you feel proud. Words will never be enough to express my gratitude and without you, nothing would be possible. Voli vas vaša Tonka!
Abstract
Atherosclerosis is the leading cause of cardiovascular disease worldwide, including coronary artery disease, stroke, hypertension and peripheral artery occlusive disease. In atherosclerosis, smooth muscle cells (SMCs) accumulate in the intima where they undergo a transition from a contractile to a synthetic phenotype. This phenomenon includes a process of cell dedifferentiation characterized by altered expression of contractile proteins, as well as increased production of extracellular matrix components. During the two last decades, synthetic SMCs have been considered as beneficial players in atherosclerotic plaque development by essentially contributing to fibrous cap formation that protects plaque from rupture. However, SMCs exhibit a remarkable plasticity depending on environmental cues/signals, and recent studies have demonstrated that a high proportion of SMCs in atherosclerotic plaques are hardly detectable with the classical SMC markers and acquire pro‐
inflammatory macrophage‐like phenotype. Therefore, there is a need for identification of factors and mechanisms responsible for the detrimental SMC phenotypic changes, with the aim of developing appropriate tools to influence SMC phenotypic profile and promote plaque stabilization and/or regression. We have previously identified S100A4 as a marker of the synthetic phenotype, both in vitro and in vivo in pigs and humans. S100A4 exhibits intra‐ and extracellular functions and, in vitro, extracellular S100A4 is active once in oligomeric conformation. The main project of this thesis aimed at deciphering the role of extracellular S100A4 in phenotypic transition of SMCs.
Chapter I is dedicated to the pathogenesis of atherosclerosis, focusing on the role of SMCs. The main aims of my thesis are described in Chapter II. In the first part of Chapter III, we describe a human study, in which we have characterized SMC from atherosclerotic plaque and underlying media in coronary arteries from young sudden cardiac death victims, by using
typical SMC differentiation markers. We have demonstrated that SMCs exhibited highly differentiated phenotype, both in media and intima. Our results suggest that SMC contractility might contribute to transient coronary spasm leading to myocardial ischemia and sudden cardiac death. The main project of my thesis is described in the second part of Chapter III. By treating porcine coronary artery SMCs with platelet derived growth factor BB and oligomeric S100A4, we have demonstrated that, when in combination, they synergistically induced a complete phenotypic transition of SMCs toward a synthetic phenotype associated with pro‐inflammatory properties. In Apolipoprotein E knockout mice, which develop atherosclerotic plaques after being fed with high cholesterol diet, we have demonstrated that extracellular S100A4 neutralization, by using the specific neutralizing antibody (clone 6B12), decreased systemic inflammation as well as the area of atherosclerotic lesions in aortas.
Moreover, S100A4 neutralization promoted plaque stability as shown by increased number of differentiated intimal SMCs within the fibrous cap and decreased expression of makers typical of inflammatory cells. These results indicate that extracellular S100A4 is causally related to atherosclerotic plaque progression, putting it forward as a prospective therapeutic target for plaque stabilization and/or regression. In Chapter IV, findings of my thesis are discussed and future perspectives of the project are presented. Chapter V contains references, and Chapter VI consists of two additional publications, in which I partially contributed.
Résumé
L'athérosclérose est la principale cause des maladies cardiovasculaires dans le monde, y compris la coronaropathie, l'accident vasculaire cérébral, l'hypertension et la maladie occlusive des artères périphériques. Dans l'athérosclérose, les cellules musculaires lisses (CML) s'accumulent dans l'intima où elles subissent une transition d'un phénotype contractile à synthétique. Ce phénomène comprend un processus de dédifférenciation cellulaire caractérisé par une expression altérée des protéines contractiles, ainsi qu'une production accrue de composants de la matrice extracellulaire. Au cours des deux dernières décennies, les CML synthétiques ont été considérées comme des acteurs bénéfiques dans le développement de la plaque d'athérome en contribuant principalement à la formation d'une capsule fibreuse qui la protège de la rupture. Cependant, les CML présentent une plasticité remarquable en fonction de leur environnement. Ainsi des études récentes ont démontré qu'une proportion élevée de CML dans les plaques d'athérome sont difficilement détectables avec les marqueurs classiques des CML et acquièrent un phénotype pro‐inflammatoire typique des macrophages. Il est donc nécessaire d'identifier les facteurs et les mécanismes responsables des changements phénotypiques délétères des CML dans le but de développer des outils appropriés pour influencer le profil phénotypique des CML et promouvoir la stabilisation et/ou la régression de la plaque d’athérome. Notre laboratoire avait identifié la protéine S100A4 comme étant un marqueur du phénotype synthétique, à la fois in vitro et in vivo, chez le porc et l'homme. Celle‐ci présente des fonctions intra et extracellulaires; in vitro, la S100A4 extracellulaire est active dans sa conformation oligomérique. Le projet principal de cette thèse est dédié à l’étude du rôle du S100A4 extracellulaire dans la transition phénotypique des CML.
Le Chapitre I est consacré à la pathogenèse de l'athérosclérose, en mettant l'accent sur le rôle des CML. Les objectifs principaux de ma thèse sont décrits dans le Chapitre II. Dans la première partie du Chapitre III, nous décrivons une étude de spécimens humains dans lesquels nous avons caractérisé les CML de la lésion athérosclérotique et de la média sous‐
jacente d’artères coronaires collectées chez de jeunes victimes de mort subite d'origine cardiaque, en utilisant des marqueurs de différenciation typiques des CML. Nous avons démontré que les CML présentaient un phénotype très différencié, à la fois dans la média et la lésion. Nos résultats suggèrent que la contractilité des CML pourrait contribuer à un spasme coronarien transitoire entraînant une ischémie myocardique puis une mort cardiaque subite.
Le projet principal de ma thèse est décrit dans la deuxième partie du Chapitre 3. En traitant les CML, isolées de l'artère coronaire porcine, avec le facteur de croissance « platelet‐
derived growth factor‐BB » (PDGF‐BB) et la S100A4 oligomérique, nous avons démontré que leur combinaison induisait de façon synergique une transition phénotypique complète des CML vers un phénotype synthétique associé à des propriétés pro‐inflammatoires. Chez les souris ApoE‐/‐, qui développent des plaques d'athérosclérose après avoir été nourries avec un régime riche en cholestérol, nous avons montré que la neutralisation de la S100A4 extracellulaire par un anticorps neutralisant spécifique (clone 6B12) diminuait l'inflammation systémique et la surface des plaques d’athérome dans l’aorte. De plus, ce traitement favorisait la stabilité de la plaque, car il augmentait le nombre de CML différenciées dans la capsule fibreuse de la plaque et diminuait l'expression des marqueurs typiques des cellules inflammatoires. Ces résultats indiquent que la S100A4 extracellulaire et la progression de la plaque athérosclérotique ont un lien de cause à effet, ce qui fait de la S100A4 extracellulaire une cible thérapeutique potentielle pour la stabilisation et/ou la régression de la plaque.
Dans le Chapitre IV, je discute les résultats de ma thèse et présente les perspectives futures du projet. Le Chapitre V est dédié aux références et le Chapitre VI comprend deux autres publications dans lesquelles j'ai partiellement contribué.
Table of Contents
List of abbreviations ... 1
Chapter I: Introduction ... 4
1. Atherosclerosis ... 5
1.1 Cause of cardiovascular disease ... 5
1.2 Threat in all life stages ... 7
1.3 Atherosclerosis through history ... 8
1.4 Pathogenesis ... 10
1.5 Clinical classification ... 15
1.6 Experimental models ... 18
2. SMCs in atherosclerosis ... 20
2.1 Contractile vs synthetic phenotype of SMCs ... 20
2.2 Regulation of SMC differentiation levels ... 21
2.3 Clonality of SMCs ... 23
2.4 Characterization of SMC synthetic phenotype ... 24
2.5 S100 proteins ... 27
2.6 S100A4‐ a marker of synthetic SMCs ... 29
2.7 Role of SMCs in atherosclerosis inflammation ... 31
Chapter II: Aims of the thesis ... 36
1. Human study ... 37
2. Mechanisms involved in S100A4‐induced SMC phenotypic transition ... 37
3. Role of S100A4 in atherosclerosis in mouse model ... 38
Chapter III: Results ... 39
1. Sudden coronary death in the young: Evidence of contractile phenotype of smooth muscle cells in the culprit atherosclerotic plaque ... 40
2. Neutralization of S100A4 induces stabilization of atherosclerotic plaques: role of smooth muscle cells ... 52
Chapter IV: Discussion ... 111
1. Human study ... 113
2. S100A4 and PDGF‐BB: duo leading to high pro‐inflammatory profile of SMCs ... 114
3. Therapeutic achievements in the field. ... 116
4. S100A4, a new anti‐inflammatory therapeutic candidate. ... 118
5. General remarks ... 120
Chapter V: References ... 122
Chapter VI: Appendix ... 132
List of abbreviations
‐SMA, ‐smooth muscle actin
ABCA1, ATP‐binding cassette transporter ApoE, Apolipoprotein E
CCL1, Chemokine C‐C motif ligand 1 CCR2, C‐C chemokine receptor type 2 CD, cluster of differentiation
CRP, C‐reactive protein CVD, cardiovascular disease
DAMP, damage‐associated molecular pattern dS100A4, dimeric S100A4
EC, endothelial cell ECM, extracellular matrix
EGFR, epidermal growth factor receptor Egr‐1, Early growth response protein 1
ELK‐1, transcription activator ETS Like‐1 protein E‐SMC, epithelioid smooth muscle cell
GM‐CSF, granulocyte‐macrophage colony stimulating factor hCad, heavy caldesmon
HCD, high cholesterol diet HDL, high density lipoprotein IFN‐γ, interferon‐gamma IL, interleukin
KLF4, Kruppel‐like factor 4 LDL, low density lipoproteins
MAPK, mitogen‐activated protein kinase MCP‐1, monocyte chemotactic protein‐1 miRNA, microRNA
MMP, matrix‐metalloproteinase MSCs, mesenchymal stem cells MYOCD, myocardin
NF‐κB, nuclear factor‐κB NO, nitric oxide
oS100A4, oligomeric S100A4 oxLDL, oxidized LDL
PAMP, pathogen associated molecular pattern PCSK9, proprotein convertase subtilisin/kexin PDGF‐BB, platelet derived growth factor‐BB PLA, proximity ligation assay
PRR, pattern recognition receptor
RAGE, receptor for advanced glycation end products ROS, reactive oxygen species
R‐SMC, rhomboid‐shaped smooth muscle cell SAA, serum amyloid A
Sca1, stem cells antigen‐1 SCD, sudden cardiac death
SM22α, Smooth Muscle Protein 22‐Alpha SMC, smooth muscle cell
SMMHCs, smooth muscle myosin heavy chains Sp1, specificity protein 1
SRF, serum‐response factor
S‐SMC, spindle‐shaped smooth muscle cell TAGLN1, transgelin
TET2, ten‐eleven translocation‐2 TLR, toll‐like receptor
TNF‐α, tumor necrosis factor alpha VCAM‐1, vascular cell adhesion protein VLDL, very low‐density lipoproteins YFP, yellow fluorescent protein
Chapter I: Introduction
1. Atherosclerosis
1.1 Cause of cardiovascular disease
The last epidemiological studies performed by the European Society of Cardiology committee (2017) shows that cardiovascular disease (CVD) remains the first cause of death in developed countries with 3.9 million deaths in Europe (accounting for 45% of all deaths in Europe), out of which 1.8 million persist in countries of European Union (accounting for 37% of all deaths in European Union) (1). CVD comprises a plethora of diseases: coronary heart disease including myocardial infarction (commonly called heart attack) and angina pectoris (chest pain as a consequence of coronary heart disease), heart failure, cerebrovascular disease (including stroke), abdominal aortic aneurysm, peripheral arterial disease, rheumatic and congenital heart diseases and venous thromboembolism (2). Interestingly, statistics for the latest available year in Europe demonstrated the presence of sex/gender differences in CVD caused mortality: 49% of all deaths in woman and 40% in men are caused by CVD (1) . Risk factors for CVD are well described and they include the modifiable (i.e. smoking, obesity, high blood pressure, diabetes, diet rich in fat and lack of exercise) and non‐modifiable ones (i.e.
genetics, family history, hemodynamic factors, male gender and age). Even though much has been improved in the last 20 years, in terms of prevention (medicines reducing the cholesterol levels, hypertension and anti‐diabetic treatments) and clinical interventions (e.g.
implantation of stent, tube‐shaped device usually implanted in coronary arteries to open narrowed vessel and allow blood supply to the heart), as well as smoking decline and nutrition improvement, CVD still remains main global health problem. Nevertheless, the mortality rate after myocardial infarction in the last decades has dramatically dropped (3).
Atherosclerosis is the underlying leading cause of CVD, such as heart attack, strokes and peripheral artery disease. The pathology of atherosclerosis is characterized by the narrowing and occlusion of the arterial lumen, caused by the accumulation and dynamic interplay of many cell types and a plethora of factors in the subendothelial layer of the vessel.
The healthy artery wall consists of 3 concentric layers: intima, media and adventitia (Figure 1). Intima, the innermost layer, is composed of a monolayer of endothelial cells (ECs), which are in contact with blood, and subendothelial layer (also called basal lamina) consisting mostly of connective tissue. In contrast to animal, human subendothelial space contains a few resident smooth muscle cells (SMCs). ECs provide an anti‐thrombogenic surface (e.g heparin sulfates present on the surface of ECs that serve as cofactor for antithrombin activation inhibiting several factors in the coagulation process) and mediate vascular tone. The intima is separated from the media by the internal elastic membrane. The media consists entirely of SMCs embedded in extracellular matrix (ECM), containing mostly elastin and collagen. In elastic arteries such as the aorta, the media contains SMCs and many elastic fibers that allow the elastic recoil necessary to distribute blood through the peripheral vascular system, while in muscular arteries such as carotid and coronary arteries, SMCs are responsible for the vasoconstriction and vasodilatation. The media is separated from the outermost layer of the vessel, the adventitia, by the external elastic membrane. The adventitia mostly consists of fibroblasts surrounded by proteoglycans and collagen and contains vasa vasorum (nutrition vessels) and nerves. It is an additional support to the vessel, helps in setting the vessel in surrounding tissue and protects the vessel from overexpansion (4, 5).
Figure 1. Cross section representation of normal human artery (5).
1.2 Threat in all life stages
Most of the atherosclerosis‐induced CVD victims are of an old age and it is well accepted that atherosclerosis generally develops and progresses over decades. Nevertheless, numerous studies have demonstrated that atherosclerosis, mostly induced by standard risk factors (6), also develops in young and results in fatal CVD outcomes.
Autopsies of US army victims died in Korean (1950‐1953) and Vietnam (1954‐1975) wars demonstrated a high prevalence of coronary atherosclerosis (77% Korean (7) and 45%
in the Vietnam War (8)) in second and third decades of life. No clinical appearance for ischemic heart disease was present in victims. The follow‐up study, based on autopsies of US army victims died in Operations Enduring Freedom or Iraqi Freedom/New Dawn between 2001 and 2011 demonstrated a dramatic drop in prevalence of coronary atherosclerosis
should be taken with high caution as this drop could also be a consequence of methodology discrepancies, analysis and selection biases (9). In a study performed in 1990 in Italy, through the characterization of the coronary arterial trees of 100 young people (1‐20 years old), in which CVD was not a cause of death, authors observed the presence of diverse degrees of intimal thickening in 95.3% of samples that correlated with a high degree of sudden cardiac death (SCD) cases in youth from the same geographic origin (10). Recently, in a study performed by our group, in collaboration with clinical pathologists (group of Prof. Basso, Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Padua, Italy), we have characterized atherosclerotic coronary artery specimens of young SCD victims, of which the youngest was 17 years old (11), again demonstrating the presence of atherosclerosis with fatal outcomes in youth. Furthermore, juvenile idiopathic arthritis, an inflammatory disease affecting the population of infants to teenagers, has direct correlation with subclinical atherosclerosis in the studied group (12‐14). Besides the inflammatory nature of this disease, authors have proposed that lower physical activity could be an additional trigger for atherosclerosis development in patients with juvenile idiopathic arthritis. Studies focused on fetal and infant atherosclerotic lesions have suggested an association of maternal smoking and early intimal alterations in the prenatal and infancy period as well as association of maternal cholesterol level during pregnancy and epigenetic signature in offspring (15, 16).
1.3 Atherosclerosis through history
The first clue of atherosclerosis, being a cause of heart attack, was found on "Sudden Death" tomb relief, in which the sudden death of the noble Egyptian is depicted (17).
Furthermore, a study conducted on 137 mummies, up to 4000 old, from different origins (ancient Egypt, ancient Peru, the Ancestral Puebloans of southwest America, and the
Unangan of the Aleutian Islands), showed that atherosclerosis of similar features found nowadays, was present in all of the studied populations. This suggests that atherosclerosis is not exclusively a disease of the modern human, dependent on certain geographic origin, feeding habits or lifestyle (18). In 1799, during an autopsy, a British physician Caleb Hillier Parry, found a hard gritty matter in coronary arteries. Taking this notion in consideration when examining his patients, he correctly suggested the correlation between vessel blockage, angina pectoris and coronary heart disease (19, 20). Although today we know that this correlation was correct, his statement was underestimated for a century. Almost 50 years after Parry`s observation, in 1844, autopsy after a sudden heart attack of the famous artist Bertel Thorvaldsen in the Royal Theatre in Copenhagen, showed that the plaque rupture in the left coronary artery led to his death. This was the first report about plaque rupture (21).
Further on, two main branches of understanding atherosclerosis have been postulated: the first one was based on “humoral” theory of Carl von Rokitansky, who has seen the process of inflammation as a consequence of blood components and fibrin modifications, leading to accumulation of cholesterol and fat globules in the inner layer of the vessel wall. In sharp contrast, Rudolf Virchow has seen atherosclerosis as a “cellular disease”, in which, as a response to local vessel wall injury, inflammatory cells play a pivotal role in pathogenesis of the disease (21, 22). These findings gave inspiration for the work of Alexander I. Ignatowski, followed by the work of Anitschkow and Chalatov in early 1900s when they introduced the
“lipid model of atherosclerosis”. They have shown in different animal models that the feeding with cholesterol‐rich diets results in the development of atherosclerosis (23). Furthermore, Anitschow has demonstrated the existence of cholesterol loaded cells within the lesions, and correctly suggested the influence of hemodynamics on the particular localization of lesions within vessels (lesions are preferentially occurring at arterial branch points where the shear
stress is low, turbulente and/or oscillatory) (24). Lipid driven atherosclerosis continued to be the principal dogma of atherosclerosis for future decades. Noteworthy, in 1973, Russell Ross and John Glomset stated that the proliferation of SMCs is a key player in atherosclerosis development, raising the importance of this cell type in the pathogenesis of a disease (25).
This extensive work led to the well‐known “response‐to‐injury” theory, postulated by Russel Ross, in which he stated that atherosclerosis is initiated when endothelium gets dysfunctional due to different stimuli known as cardiovascular risk factors. Endothelial dysfunction results in the infiltration of inflammatory cells in the subendothelial layer and upon uptake of lipids these cells differentiated towards the foam cells. Additionally, this accumulation provokes the insult of SMCs located in arterial wall. The interplay between these cells in a highly dynamic manner, results in the formation of atherosclerotic lesion (26). Current global perception of atherosclerosis is based on the definition given by Ross in 1999, when he described atherosclerosis as an inflammatory disease, which comprises different cell types, each having its particular role in the pathogenesis of the disease (27).
1.4 Pathogenesis
Atherosclerosis is a disease that develops over decades and its evolution is characterized by different steps and cellular processes (Figure 2). Atherosclerotic lesions appear in areas where ECs are intact, thus showing that dysfunctional and not mechanically injured endothelium is the onset of most atherosclerosis (28). Under physiological laminar shear stress, nitric oxide (NO) is produced by ECs, maintaining the atheroprotective (anti‐inflammatory, anti‐
thrombotic and anti‐proliferative) environment within the vascular space (29, 30). When laminar shear stress is changed due to anatomical (bifurcations and curvatures), lesion/intervention related (changed vessel geometry due to presence of atherosclerotic
plaque or stent implantation), or blood viscosity related cues (diabetic and hypercholesterolemic patients) (31, 32), ECs increase NO production, which induces vasodilation of the vessel, probably as a counter mechanism for maintaining the constant shear stress in the vessel (29, 33). However, the hemodynamic perturbations (e.g on arterial bifurcations) or hyperlipidemia can also disturb NO signaling and increase reactive oxygen species (ROS) production, leading to activation/dysfunction of ECs (30, 34, 35).
Figure 2. Pathogenesis of atherosclerosis (28).
Such dysfunctional endothelium becomes conductive for lipid, monocyte and T‐cell adherence. The most pro‐atherogenic lipids are low density lipoproteins (LDL; essential transporter of lipids in human body), and small very low‐density lipoproteins (VLDL) (36).
Once accumulated to subendothelial space, lipids become modified (mostly oxidized) and monocytes differentiate towards macrophages. The latter and T cells are the most abundant inflammatory cells within the atherosclerotic lesions (37, 38). Different chemoattractans and
cytokines released by activated endothelium are needed for the migration of cells towards subendothelial compartment. Vascular cell adhesion protein (VCAM)‐1 and intercellular adhesion molecule (ICAM)‐1 are upregulated by oxidized LDL (oxLDL). These adhesion molecules allow lipid, monocyte and T cell adherence to the ECs and consequently migration in the subendothelial compartment (39, 40). Furthermore, disturbed laminar flow induces the upregulation of monocyte chemotactic protein‐1 (MCP‐1), which facilitates adhesion and migration of monocytes in the subendothelial space (41). Finally, various interleukins (IL) affect the endothelial permeability by different mechanisms (e.g IL‐8 modifies the expression of tight junctions), thus allowing cell migration (42, 43).
Once in the subendothelial compartment, macrophages can uptake oxLDL through its scavenger receptors. This uptake leads to the formation of foam cells. Loosely regulated uptake of oxLDL eventually leads to apoptosis/necrosis of macrophages (44). The other destiny of this cell is a “reverse cholesterol transport” where macrophages/foam cells may efflux cellular cholesterol to extracellular high density lipoprotein (HDL) (45). As oxLDL is recognized as damage‐associated molecular pattern (DAMP) molecule, its binding to macrophage scavenger receptors (e.g. scavenger receptors, receptor for oxidized low‐density lipoprotein [LOX‐1], cluster of differentiation 36 [CD36], and toll‐like receptor [TLR]‐4), triggers the pro‐inflammatory response characterized by production of cytokines (e.g. IL‐1, IL‐
6, IL‐8, granulocyte‐macrophage colony stimulating factor [GM‐CSF], MCP‐1 and others) (28, 37, 46), and foam cell formation (47). Moreover, macrophages produce matrix‐
metalloproteinases (MMPs) and growth factors (e.g. platelet derived growth factor [PDGF‐
BB]). Additionally, accumulated T cells release high levels of interferon‐gamma (IFN‐γ) and tumor necrosis factor alpha (TNF‐α) (48, 49). Endothelium dysfunction also triggers platelet
activation and adherence to endothelial surface, resulting in platelet releasing inflammatory mediators and growth factors which will further activate ECs and attract monocytes (50, 51).
The interplay of these cells definitely and irreversibly promote pro‐inflammatory environment and define atherosclerosis as a chronic inflammatory disease.
OxLDL and growth factors released by inflammatory cells attract and stimulate the proliferation of SMCs, originally located in the media, towards the intima. SMCs are not terminally differentiated and therefore possess high degree of plasticity. During the migration towards the intima, SMCs change their phenotype from a differentiated/contractile towards a dedifferentiated/synthetic phenotype. The contractile phenotype is typical of SMCs in the media of healthy vessel. These SMCs contain many microfilament bundles. The synthetic phenotype is typical of pathological vessels and is characterized by cytoplasm with a predominance of rough endoplasmic reticulum and a well‐developed Golgi apparatus. The SMC phenotypic transition is characterized by altered expression of contractile proteins, increased proliferative and migratory activities and ability of SMCs to synthetize ECM components and different proteases (52). Contribution of SMCs to pathogenesis of atherosclerosis was previously seen as beneficial, as SMCs form the fibrous cap where they produce collagen and proteoglycans in the fibrous matrix, which protects plaque from rupture. Plaques containing thick fibrous cap, rich in SMCs and with low content of leukocytes and lipids are considered as stable plaques. In contrast, higher content of leukocytes and lipids and thinner fibrous cap are attributed to unstable/vulnerable plaque that has higher risk to rupture and trigger the fatal outcomes (48) (Figure 3). Nowadays the increasing number of studies are continuously demonstrating that SMCs can acquire characteristics conventionally attributed to macrophages such as expression of inflammatory markers and formation of
foam cells, revealing the potential role of SMCs not only related to plaque stability, but also to plaque vulnerability/rupture (53‐55). SMCs and their role in atherosclerosis is the main interest in this thesis and will be elaborated extensively in Chapter 2.
Figure 3. Stable vs unstable atherosclerotic plaques. Adapted from (48)
Numerous studies have demonstrated an important role of TLR receptors in the development of atherosclerosis. TLRs and especially TLR4 are the best characterized pattern recognition receptors (PRRs) and thus, play a major role in innate immune system. PRRs are recognizing pathogen associated molecular patterns (PAMPs) as well DAMPs, and are expressed by most immune cells (e.g macrophages, dendritic cells) (56) as well as by ECs (57, 58), and SMCs (59). In the context of atherosclerosis, it has been demonstrated that the lack of Myd88 (adaptor protein necessary for TLR signalling), TLR4 or TLR2 results in beneficial effects in atherosclerosis progression (e.g decrease in lesion size and macrophage content) (60).
TLR4 is expressed in human coronary atherosclerotic plaques (61), involved in coronary artery disease development (62), and associated with heart failure after acute myocardial infarction (63). In TLR4 dependent manner, oxLDL induces differentiation of
Unstable Plaques in Atherosclerosis Stable Plaques in Atherosclerosis
Additionally, TLR4 may be implicated in macrophage apoptosis, thus favouring the formation of the necrotic core and processes of plaque vulnerability (66). In initial steps of atherosclerosis development, TLR4 is a regulator of the foam cell formation (67), while in the advanced lesions it mediates mostly oxLDL‐induced release of proinflammatory cytokines and MMP‐9 production (47). Cellular fibronectin containing extra domain A is abundant in the arteries of patients with atherosclerosis. It is produced after tissue injury and its prothrombotic activity is promoted by interaction with TLR4 expressed on platelets, leading to accelerated arterial thrombosis (68). In Apolipoprotein E knockout (ApoE‐/‐) mice, fibronectin containing extra domain A, exacerbates atherosclerosis in TLR4‐dependent manner and induces MMP‐9 expression by macrophages, thus promoting plaque instability (69).
1.5 Clinical classification
Extensive work has been done in order to define a correlation between clinical presence and histological analysis of lesions. A major contribution to the classification was given by Stary et al. in a series of reports, where authors gave definitions and descriptions of different phenotypes and biological composition of different atherosclerotic plaque development stages (70, 71). Later, Virmani et al. have modified this classification by adding additional categories (72). All stages with corresponding description are presented in Table 1.
The initial, first type of atherosclerosis (xanthoma or fatty streak) is characterized by luminal accumulation of foam cells and these lesions do not require clinical or pharmacological intervention as they are usually regressive. The second type of lesion is intimal thickening in which SMCs themselves form a mass, but without a presence of foam
cells. Both types are non‐atherosclerotic intimal lesions. Furthermore, there are several types of lesions within the category of progressive atherosclerotic lesions. Presence of SMCs and lipids, but absence of necrosis in the intima characterizes the least advanced lesion. The lesion progresses as necrotic core and fibrous cap develop. In this stage, plaque erosion (thrombus is formed over a plaque without endothelium but not ruptured) might take place eliciting 20‐
40% of sudden coronary artery death. Furthermore, infiltration of macrophages into the fibrous cap, growing necrotic core, processes of inflammation, calcification, cell death and proteolysis lead to vulnerable phenotype of the plaque and eventually to plaque rupture responsible for 70‐ 80% of sudden coronary artery deaths.
Description Thrombosis
Non‐atherosclerotic intimal lesions
Intimal xanthoma or "fatty streak" Luminal accumulation of foam cells without a necrotic core or fibrous cap.
Based on animal and human data, such lesions usually regress
Thrombus absent
Intimal thickening The normal accumulation of SMCs in the intima in the absence of lipid or macrophage foam cells
Thrombus absent
Progressive atherosclerotic lesions
Pathological intimal thickening SMCs in a proteoglycan‐rich matrix with areas of extracellular lipid accumulation without necrosis
Thrombus absent
Fibrous cap atheroma Well‐formed necrotic core with an overlying fibrous cap
Thrombus absent
Erosion
Luminal thrombosis; plaque same as above; no communication of thrombus with necrotic core
Thrombus Infrequently occlusive
Thin fibrous cap atheroma
A thin fibrous cap infiltrated by macrophages and lymphocytes with rare SMCs and an underlying necrotic core
Absent; may contain intraplaque hemorrhage/fibrin
Plaque rupture Fibroatheroma with cap disruption;
luminal thrombus communicates with the underlying necrotic core
Thrombus usually occlusive
Calcified nodule Eruptive nodular calcification with underlying fibrocalcific plaque
Thrombus usually nonocclusive
Fibrocalcific plaque Collagen‐rich plaque with significant stenosis usually contains large areas of calcification with few inflammatory cells; a necrotic core may be present.
Thrombus absent
Table 1. Classification of atherosclerosis. Adapted from (70‐72).
1.6 Experimental models
Although there are many difficulties to resemble human vascular anatomy and pathophysiology, several animal models are used to study atherosclerosis such as mouse and rabbit and less commonly, pigs and non‐human primates (73). Of those, the foremost model used is the mouse model because of the ease of genetic modification, handling and reproduction, and well established ethical concerns. Among all mouse strains, C57Bl/6 strain is the most appropriate to study atherosclerosis because of the low HDL levels and presence of T cells that are of the pro‐atherogenic profile (e.g producing IFN‐γ). Capability of this strain to develop atherosclerosis relatively fast increases once the mice are fed with the high fat diet (74).
Apolipoprotein E is a transporter of lipids, fat‐soluble vitamins, and cholesterol to the lymph system and then to the blood which interacts with LDL receptor. ApoE‐/‐ and LDL receptor knockout mice are the most widely used mouse models for studying the initiation and progression of atherosclerosis as well as the underlying mechanisms. Both strains exhibit higher plasma cholesterol levels and finally, atherosclerosis. The development of atherosclerosis is accelerated when mice are fed with the high cholesterol diet (HCD) for ≥6 weeks. An ApoE/LDL receptor double‐knockout mouse develops more severe hyperlipidemia and atherosclerosis, and does not require a high fat diet for reaching this phenotype. The ApoE3‐Leiden (variant of apolipoprotein E that is defective in binding to the LDL receptor) transgenic mouse model is similar to ApoE‐/‐ and LDL receptor knockout mice, except that the basal lipid levels are lower. Nevertheless, these mice are highly responsive to food rich in fat, sugar and cholesterol and can reach high lipid profiles. Proprotein convertase subtilisin/kexin (PCSK) 9 is involved in degradation of LDL receptors. A model based on recombinant adeno‐
associated virus vector induced PCSK9 gain‐of function has been developed. High
hyperlipidemia and advanced stage of atherosclerosis in this model are amplified once crossed with ApoE‐/‐ mice. A disadvantage of all described experimental models is the absence of plaque rupture, thrombus formation, and/or haemorrhage, events frequently observed in human situations. With the aim of mimicking spontaneous plaque rupture event, different surgical intervention techniques have been developed (e.g ligation of the arteries) but none of them fully resembled human situations: spontaneous rupture was very rarely achieved.
Moreover, common clinical outcomes of plaque rupture in human such as heart attack or stroke are nearly never seen in these models. ApoE‐deficient Fibrillin‐1 mutant (ApoE‐/‐
Fbn1C1039G+/‐) model is the only mouse model that, under the high fat diet, reaches the stage of plaque rupture. Fibrilin‐1, as a major component of microfibrils, provides structural support to elastin core. Heterozygous mutation of fibrilin‐1 gene leads to disruption of elastic fibers (Marfan syndrome), and when combined with ApoE‐/‐ background and high fat diet, results in advanced unstable plaques with large necrotic cores and thin fibrous cap. This model resembles well the late‐stage of human atherosclerosis (vulnerable plaque leading to rupture) and these mice develop heart attack or die of a sudden death, thus being a good model to study mechanisms of plaque rupture and potential therapeutic targets for its prevention.
Despite certain limitations, mouse models are valuable for deciphering the mechanisms and developing new tools to influence the evolution of atherosclerotic lesions with the final goal of reducing adverse outcome of atherosclerosis in clinical events. In line with that, Baylis et al. (75) pointed out the importance of intervention studies (applying of potential therapies once atherosclerotic plaques in mice are established) over the prevention ones as there is a high need for therapies that could resolve critical factors inducing plaque vulnerability/rupture.
2. SMCs in atherosclerosis
Classically, SMCs are considered as beneficial players in atherosclerotic plaque development by essentially contributing to fibrous cap formation that protects plaque from rupture. However, our understanding of the role of SMCs in atherosclerosis has considerably changed. Plasticity is the feature that makes SMCs unique and distinct from other muscle cells that are terminally differentiated. The ability of SMCs to become dedifferentiated, during their transition from contractile towards a synthetic phenotype, is one of the hallmarks of atherosclerosis.
2.1 Contractile vs synthetic phenotype of SMCs
The contractile phenotype is typical of medial SMCs in healthy vessel and is characterized by a high degree of differentiation. In contrast, dedifferentiated synthetic phenotype is typical of intimal SMCs. Contractile SMCs contain many microfilament bundles, while the synthetic SMCs contain cytoplasm with a predominance of rough endoplasmic reticulum and a well‐developed Golgi apparatus (52, 76). Synthetic phenotype is defined by a decrease or loss of SMC‐specific cytoskeletal proteins such as ‐smooth muscle actin (‐SMA, ACTA‐2 gene), smooth muscle myosin heavy chains (SMMHCs, MYH11 gene), transgelin (or SM22α, TAGLN gene), calponin (CNN1 gene), heavy caldesmon (hCad), smoothelin (55, 76) or leiomodin‐1 (77, 78). To characterize differences between contractile and synthetic phenotypes, several in vitro models were established. In 1986, Walker et al. (79) for the first time isolated two different phenotypes of SMCs from rat carotid arteries. From media of the vessel, they digested spindle‐shaped (S‐) SMCs that grew in hill and valley pattern and represented the contractile phenotype, while the epithelioid (E‐) SMCs digested from mechanical‐injury‐induced intimal thickening grew in a monolayer and exhibited a
cobblestone morphology. Later, E‐SMC clones were obtained from normal media and intimal thickening (80), thus implicating that this particular population of E‐ SMCs originally located in the media is prone to accumulate into the intimal thickening. Many other studies have confirmed the heterogeneity of SMCs in different models (81‐83), and in humans (84). Based on in vivo observation that in mature bovine pulmonary arteries 4 different SMC phenotypes could be found, Frid et al. (85) isolated 4 different subtypes of SMCs from the inner, middle, and outer compartments of the pulmonary artery and aortic arch arterial media. Among them, two types represented typical S‐SMCs phenotype, while the other two displayed characteristics of E‐SMC, or were of a cobblestone morphology, but elongated, thus being named rhomboid (R‐) SMCs. In our laboratory, two distinct populations of SMCs (S‐ and R‐
SMCs) were isolated from porcine coronary artery (86) as well as two morphologically different SMC populations, large (exhibiting the features of S‐SMCs) and small SMCs (exhibiting the features of E‐ or R‐SMCs) from the media of human carotid artery collected with endarectomy. Small SMCs were recovered only after a coculture with CD68‐positive macrophage foam cells isolated from the plaque, suggesting that foam cells promote the dedifferentiation and recruitment of a particular medial SMC population (87).
2.2 Regulation of SMC differentiation levels
There are two regulation pathways of SMC differentiation levels. The first one is based on transcriptional and the second one on the epigenetic regulation of cytoskeletal proteins expressed by SMCs (Figure 4). The mechanism of transcriptional regulation is controlled by myocardin (MYOCD)/ serum‐response factor (SRF) complex that binds to CArG box and stimulates the expression of SMC differentiation markers. Binding of Kruppel‐like factor 4 (KLF4; transcription factor expressed in synthetic SMCs) and transcription activator ETS Like‐
1 protein (ELK‐1) to G/C repressor element results in inhibition of MYOCD/SRF complex, thus decreased expression of SMC differentiation markers. Moreover, in contractile phenotype DNA is acetylated and allows binding of SRF to CArG box, thereby inducing SMC differentiation marker expression. Histone deacetylase 2 (recruited to CArG box by e.g oxLDL and KLF‐4) induces DNA metylation and closing of chromatin, thereby repressing SMC differentiation marker expression. DNA demethylation by ten‐eleven translocation‐2 (TET2; downregulated in synthetic phenotype) DNA demethylase increases DNA accessibility to transcription factors resulting in increased expression of SMC differentiation markers. Additionally, microRNA (miR) and long non‐coding RNA (88), play important roles in SMC phenotypic transition.
Figure 4. Regulation of SMC differentiation levels. (SRF, serum‐response factor; MYOCD, Myocardin; KLF 4, Krüppel‐like factor 4; ELK1, ETS domain‐containing protein‐1; CArG, CArG
box; G/C rep, G/C repressor elements; HDAC2, histone deacetylase 2; TET2, ten‐eleven translocation‐2; H, histones; Ac, acetyl group; Me, methyl group) (89).
2.3 Clonality of SMCs
In contrast to the “response‐to‐injury” theory postulated by Russel Ross, implying that any cell from the media could accumulate in the intima, the possibility that a pre‐existing SMC subpopulation in the media is prone to accumulate in the intima has recently gained a renewed interest. Besides isolation of different SMC phenotypes from media and intimal thickening, described by many groups (Chapter 2.1), other studies demonstrated the existence of monoclonality/oligoclonality of SMCs. In 1970s, it has been demonstrated that SMCs of human atheromatous plaque, in particular SMCs of the fibrous cap, exhibit features of monoclonal or oligoclonal lesions (90). Later, Murry al. (91) have shown that SMCs of the fibrous cap are monoclonal. Their observation was based on microdissection of different portions of human plaques followed by polymerase chain reaction amplification of the DNA of an X‐inactivated gene. Chappell and co‐workers (92) have generated ApoE‐/‐ mice, in which multicolor SMCs could be lineage‐traced (mice expressed tamoxifen‐inducible Cre recombinase under the SMC specific MYH11 (SMMHC) promoter), and demonstrated that the media exhibits multicolored SMCs with a stochastic distribution whereas SMCs in the intima were distributed in large monochromatic areas. Combination of this lineage tracing model and single‐cell transcriptomics pointed out the importance of stem cells antigen‐1 (Sca1) found in small SMC population within normal media. Authors have shown that Sca1 upregulation is a marker of SMCs undergoing a phenotypic transition and that equivalent population of Sca1 positive SMCs were found in atherosclerotic plaques (93). Moreover, SMCs in the fibrous cap and macrophage‐like SMCs in the necrotic core derive from clonal SMCs, a
notion supported by other studies (94, 95). Recently, it has been demonstrated that the clonal SMCs in the fibrous cap subsequently undergo a transition toward an inflammatory phenotype through integrin β3‐mediated pathway, characterized by increased expression of TLR4, the scavenger receptor CD36 and ox‐LDL uptake (96). These findings, together with a notion that E‐ or R‐ SMCs isolated in small proportion from media share the characteristics with SMCs isolated from intimal thickening, are important to design strategies aimed at promoting the expansion of SMC that populate the fibrous cap and stabilize the plaque.
2.4 Characterization of SMC synthetic phenotype
Characterization of SMCs is based on reduced expression, or the absence, of cytoskeletal markers of mature or differentiated phenotype (Table 2). It is hence of a great importance to find additional markers specific for the synthetic phenotype and several genes and/or proteins typical of the synthetic phenotype were reported (e.g osteopontin, cytokeratin 8 and 18, zonula occludens‐2‐protein, cingulin, connexion 43, calmodulin and others, Table 2). By using the proteomic approach, our laboratory has identified S100A4 being a marker of R‐SMCs in vitro and of intimal SMCs, in both pig and man (87, 97) as well as of dedifferentiated SMCs in human saccular intracranial aneurysms (98). Moreover, we have demonstrated that the extracellular form of S100A4 is essential for the establishment of the R‐ phenotype. Blockade of extracellular S100A4 in R‐SMCs with S100A4 neutralizing antibody induced a transition from R‐ to S‐phenotype, decreased proliferative activity and upregulation of SMC differentiation markers. Conversely, extracellular S100A4‐rich conditioned medium collected from S100A4‐transfected S‐SMCs induced nuclear factor‐κB (NF‐κB) activation, S‐ to R‐phenotypic transition and changes in proteolytic enzymes and inhibitors expression (99).
Recently, others have used S100A4 to characterize the phenotype of synthetic/dedifferentiated SMCs in various vascular disease and species (100‐104).
Table 2. Expression of typical proteins found in contractile and synthetic SMCs. Adapted from (89).
Protein expression
Contractile Synthetic
α‐smooth muscle actin S100A4
Smooth muscle myosin heavy chains Cytokeratin 8 and 18
Transgelin Zonula occludens‐2 protein
Desmin Cingulin
Metavinculin Connexin 43
Calponin Calmodulin
Heavy caldesmon Osteopontin
Smoothelin
Leiomodin‐1
SM α‐tropomyosin
2.5 S100 proteins
The S100 protein family is expressed exclusively in vertebrates and consists of 24 members, each having conserved EF hand domains for Ca2+ binding and diverse C‐terminal and hinge regions. Each S100 EF‐hand has a helix loop‐helix motif and binds Ca2+ via main‐
chain oxygen atoms and carboxylate side chains. S100 proteins can form non‐covalent oligomers. Both, homo‐ and heterodimers are characterized by a stable four‐helix domain with a hydrophobic core, which serves as a functional binding site (105). According to their function, they are divided in three main groups: S100 proteins with intracellular, extracellular, or both intra‐ and extracellular functions (106‐108). Intracellular role of S100 proteins is mainly associated with the processes of proliferation, differentiation, apoptosis, Ca2+ homeostasis, energy metabolism, inflammation and migration/invasion. Extracellulary, S100 proteins bind surface receptors, G‐protein‐coupled receptors, scavenger receptors, or heparan sulfate proteoglycans and N‐glycans, thus playing a role in regulation of cell proliferation, differentiation, survival and migration and inflammation and tissue repair.
Some of S100 proteins are used as disease markers as they are found in serum and other fluids of patients with pathological conditions (108). S100 proteins are well studied in fields of cancer and stress and inflammation induced responses and they major roles in these processes are depicted in Figure 5.
Figure 5: S100 proteins in stress and inflammation‐mediated responses. As a response to
cellular stress or inflammation, S100 proteins get released in extracellular space where they bind cell surface receptors and initiate multiple cellular processes such as cell differentiation, migration, apoptosis, proliferation, and inflammation. AP1, activator protein 1; ERK, extracellular signal‐regulated protein kinase; GPCR, G‐protein‐coupled receptor; IL‐1, interleukin 1; IL‐7, interleukin 7; IκBα, nuclear factor of kappa light polypeptide gene enhancer in B‐cells inhibitor alpha; JNK, c‐Jun N‐terminal kinase; P38, p38 mitogen‐activated protein kinase; RAGE, receptor for advanced glycation end products; TLR4, toll‐like receptor 4; Traf2, TNF receptor‐associated factor 2 (109).
