While Study IV establishes the role of FOXC1 as a transcription factor in regulating SMC quiescence vs. activation, it also identifies a strong link between atherosclerosis, SMCs and thyroid hormone regulation, a connection previously unappreciated in CVD. In silico analyses from human plaques and rat intimal hyperplasia suggested a strong positive association between FOXC1 and Thyroid Hormone Receptor Beta (THRB), the latter being a nuclear receptor for triiodothyronine (T3) mediating biological effects. Previously, T3 has been reported to alter cell metabolic status by enhancing oxygen and energy consumption434, processes linked to the cell activation435. With relevance to the function of the cardiovascular system, T3 has been shown to potentiate NO-induced vasorelaxation and suppress SMC calcification, both likely mediated via PI3K/Akt signalling436, 437. Despite such a profound impact on the vasculature, the role of thyroid hormone in the regulation of SMC phenotype and function remains unknown. In this study, we for the first time show that plaques from patients with higher T3 levels in plasma are characterized by lower FOXC1 expression, while conversely, lesions from patients with lower amounts of circulating T3 display higher FOXC1 levels. Although this is just an indication of the likely functional association between T3 and FOXC1, it nicely complements our previous findings, as it should be of no surprise that T3, considered to induce cell activation, may also repress transcription factors controlling cell differentiation, such as FOXC1. Of note, no such effect was observed for thyroxine (T4), an inactive form of T3. The impact of T3 on SMC phenotype was elucidated in vitro, where T3 stimulation of cultured early-passage cells resulted in a downregulation of THRB, as well as classical contractility markers, likely due to the T3-dependent increase in activity of SMCs. Intriguingly, expression of FOXC1 was not affected, which can be explained in two ways: 1) downregulation of FOXC1 in response to T3 may be dependent on the presence of other cell types/factors in the plaque, an effect absent in the isolated monolayer SMC culture, and/or 2) the T3-associated downregulation of classical SMCs is not linked to the actual activation of SMCs, in the usual sense of that expression, with connection to increased cell migratory and proliferative capacity. The latter seems possible, as it has been shown that SMC phenotypic modulation characterized by altered expression of contractility markers can be independent of the functional consequences linked to cell migration and proliferation242. The relevance of FOXC1 for mediation of T3-related alterations in SMCs was confirmed, when silencing of FOXC1 prior to T3 stimulation led to partially abolished repression of ACTA2 and CNN1. We hypothesize this effect in plaques may be attributable to Gap Junction Protein Alpha 1 (GJA1), vascular SMC gap junction protein, identified as FOXC1 downstream target and upregulated in FOXC1-silenced T3-stimulated SMCs. While in normal arteries and with low circulating T3 FOXC1 likely represses GJA1 and prevents the activation of SMCs, this effect is abolished in plaques, where patients display high T3 plasma levels. Then, two scenarios are possible. One, where FOXC1 is not downregulated, GJA1 remains repressed and SMCs contribute to plaque stabilization and the other, where downregulation of FOXC1 leads to subsequent upregulation of GJA1 and partial restoration of contractile phenotype, feature when present in advanced lesions likely contributing to the
impaired investment of SMCs and ultimately lesion destabilization (Figure 22 Visual abstract). Interestingly, while upregulation of GJA1 in atherosclerosis has been previously reported438, its SMC-specific knockout displayed a 50% reduction in the lesion size with increased features of plaque stability, including thicker fibrous cap, less inflammation, but the increased investment of SMCs439, which combined with studies linking GJA1 also with SMC transitions440 and role of these cells in vascular wall healing441, 442, establish GJA1 as important for SMC phenotype and function. In the light of recent findings that thyroid hormone levels may constitute one of the important risk factors for CVD443, the T3-FOXC1-GJA1 axis may be of key importance in the regulation of SMC response to T3 in atherosclerosis. Further studies are required to comprehensively investigate the molecular mechanisms underlying thyroid hormone-related effects in atherosclerotic disease and/or response to vascular injury.
Considering the indispensability of thyroid hormone for the proper functioning of virtually every single cell and organ in the human body, identification of its direct connection with FOXC1 transcription factor involved in the regulation of SMC quiescence is a relevant novel discovery in this study, one that could provide a new angle for future studies and help understand intricacies behind plaque destabilization. Besides the data already discussed, immunostainings performed in >40 atherosclerotic lesions led to an intriguing discovery that approximately 50% of end-stage lesions completely lacked FOXC1 protein signal.
Hypothetically, based on our previous findings, this could be indicative of higher plasma T3 levels in atherosclerosis patients, but the relevant question is: Could such strong repression of FOXC1 protein and increased circulating T3 levels be somehow linked to the more active growth of the plaque and/or its destabilization? Cellular dormancy is a well-known phenomenon in cancer and relates to malignant cells residing temporarily in a quiescent state, with minimal proliferation and death rates444. It can be induced by various environmental cues, including among others ECM-specific alterations445, hypoxic microenvironment446, or endoplasmic reticulum stress447. It is a general feature of various types of cancers, meaning
Figure 22. Graphical abstract of the proposed FOXC1-mediated response to T3 in plaques. Adapted from Study IV, Figure 7.
it is not restricted to any particular cell lineage and, most importantly, can be reversed.
Considering the existence of profound similarities between cancer and atherosclerosis448, would it be so difficult to imagine similar processes could be of importance during 30-40 years of plaque progression? In this hypothesis, depending on the temporal changes in the state of the cells within (active vs. quiescent), atherosclerotic lesions would be expected to undergo rounds of latency periods with overall stabilization/quiescence and those of rapid growth, associated with, among others, extensive remodeling and healing, where SMCs would be of crucial importance for both. This concept is particularly intriguing given that dormant cancer cells are considered to be resistant to conventional anti-cancer therapy, which usually targets actively proliferating cells444. Altogether, although still hypothetical, the concept of the existence of interdependencies between thyroid hormone, FOXC1 and active growth/quiescence phases of lesion progression, could provide some insight into the still unresolved fundamental questions of atherosclerosis pathology.
5 CONCLUSIONS
Despite advancements in the understanding of molecular biology behind CVD, available diagnostic and treatment options remain limited, suggesting there may still be relevant gaps in our perception of the disease. As highlighted in the extensive 2014 Astra Zeneca review373, gaining in-depth knowledge about the likely future therapeutic targets before they reach clinical trials is of crucial importance, therefore basic science and its efforts to unravel the intricacies of cardiovascular pathologies cannot be overestimated.
With this in mind, all studies presented within this thesis aimed to identify and functionally characterize novel signatures demarcating SMC phenotypic modulation, with a particular focus on the key transcription factors in the regulation of processes related to plaque instability. Initial identification of candidate molecules in BiKE was extended by in vivo, ex vivo, in situ and in vitro studies providing a comprehensive pipeline assuring multilevel investigation and validations, as well as translatability of the research findings.
The major conclusions of this Ph.D. thesis are the following:
I. Downregulation of transcription factors FOXC1 and BCLAF1, as well as cytoskeleton-related molecules LMOD1, SYNPO2, PDLIM7, PLN and SYNM is indicative of early alterations in the phenotype of SMCs and could serve as sensitive markers of their dedifferentiation. (Study I, III and IV)
II. PCSK6 protease is a key modulator of SMC invasiveness in vascular remodeling and atherosclerosis through activation of the MMP14-MMP2 axis. (Study II)
III. Maintenance of BCLAF1 expression is critical for SMC survival in an atherosclerotic plaque environment via transdifferentiation into macrophage-like cells. (Study III) IV. FOXC1 is the key transcriptional regulator governing SMC activation vs. quiescence,
likely via mediating effects of thyroid hormone. (Study IV)
Together, these findings establish a panel of novel markers of crucial importance for the function of SMCs, both in physiological conditions and in vascular pathologies. Importantly, with assured relevance for human disease, all of the investigated molecules constitute attractive potential therapeutic targets for CVD.
6 SOCIETAL IMPACT AND TRANSLATIONAL POTENTIAL
Documented research on cardiovascular disorders dates at least from as far as the 1700s449 and has been systematically growing until a boom at the beginning of the 21st century, facilitated mainly by technological advances on an unprecedented scale. However, contrary to expectations and despite extensive progress in understanding CVD with estimated hundreds of scientific papers being published globally each week, we are faced with surprisingly low progress in developing new successful treatment options, likely due to the immense disease molecular complexity and population heterogeneity. In the light of WHO estimations that by 2030 23 million people will die from CVD each year worldwide, we are in great need of novel research strategies, which could facilitate advances in both prevention, as well as diagnostics and treatment of cardiovascular disorders.
Precision medicine, often referred to as personalized medicine, presents a new integrative approach in which an individual’s molecular profile, including genomics, transcriptomics and/or epigenomics, is used for decision-making regarding medical care, including surgical and pharmaceutical disease management450. Although still in its infancy, in some cases it is already being successfully used. For instance, pharmacological management of several types of hypercholesterolemia varies depending on the patient’s omics profile, where in case certain mutations in the genome are found indicative of potentially altered response to standard treatment, alternative therapeutic options are considered 451, 452, 453. With the recognition of the immense potential and likely significantly increased success rates of patient-tailored care, great efforts are directed into broadening its application, where medical decisions could be made not only based on the patient’s individual genetic background but also other variables, such as targeted diagnostic imaging or available therapeutic targets.
Molecular ultrasound imaging is a relatively new concept combining classical ultrasound with the targeting of disease-specific molecular markers. Although currently used mainly in cancer research and animal models of CVD454, it holds great potential also for human plaque diagnostics, as it could allow the identification of processes with key relevance for atherosclerotic disease, instead of general unspecific detection of plaque echogenicity.
Building on this idea, the presented thesis facilitated the identification of novel molecular signatures, previously not or only scarcely investigated in CVD, which could potentially serve as imaging targets and therefore facilitate patient-specific molecular characterization of atherosclerotic lesions with respect to underlying processes, such as SMC phenotypic modulation (FOXC1, LMOD1, SYNPO2, PDLIM7, PLN and SYNM), migration/proliferation with ECM remodeling (PCSK6), as well as lipid-induced SMC transdifferentiation (BCLAF1). Equipped with a better understanding of molecular mechanisms underlying the disease in particular individuals, physicians could in an informed way decide on the most appropriate therapeutic strategy, where the best treatment options could be offered to the ‘right patient’ at the ‘right time’.
Apart from the potential diagnostic value, several of the identified molecular mechanisms constitute potentially attractive therapeutic targets. Dedifferentiation with downregulation of markers related to SMC contractility is a hallmark of these cells’ activation. While targeting molecules involved in the maintenance of contractile phenotype seems attractive, caution must be applied, as SMC activation is not only essential for the disease progression but also for lesion stabilization. With this in mind, it is very likely that for this particular group of potential therapeutics equally significant as the efficacy may be the timing of the treatment administration. Early identification of patients with high risk for CVD would be of crucial importance, where treatment would facilitate the preservation of SMC contractile phenotype and as such hopefully provide desirable increased vessel wall resistance to the
atherogenic stimuli. While SMC migration and proliferation are crucial for both vascular wall healing and establishment of the fibrous cap in advanced atherosclerotic lesions, when excessive, both processes are considered detrimental to the vessel wall, as they may ultimately lead to disease progression and severe stenosis of the lumen. Preserving the proliferation of SMCs within the fibrous cap, but instead inhibiting their contribution to the necrotic core could potentially impact disease progression and prevent lesion destabilization.
Two other potentially attractive treatment options rely on targeting SMC survival and transdifferentiation concepts, however timing of the treatment once more is of the essence and determines the character of the therapeutic approach. Early restoration of physiological high expression of anti-apoptotic molecules, such as BCLAF1, in SMCs exposed to proatherogenic cues could result in the increased resistance towards these factors and delay or prevent associated SMC activation, as well as hopefully also the initiation of disease.
Contrarywise, manipulation of SMC conversion into macrophage-like cells in late-stage lesions could harbor the potential to prevent excessive cell death and further progression of the disease.
Importantly, while no evidence has been provided for either of the identified here molecular signatures to be able to serve as atherosclerosis plasma biomarkers, studies emerge showing ECM molecules shedded into the blood as a result of SMC phenotypic modulation can serve such a function335, 455, therefore further studies are needed to investigate this angle.
Altogether, this thesis identified and characterized novel molecular mechanisms underlying SMC phenotypic modulation, where some of the identified molecules could likely serve as surrogate markers of processes such as SMC dedifferentiation, proliferation/migration and lipid transdifferentiation, as well as constitute attractive therapeutic options for CVD treatment. Further comprehensive proof-of-concept studies are required to evaluate these possibilities in vivo.
7 ACKNOWLEDGEMENTS
‘When your dream is bigger than you are, you only have two choices: give up or get help.’
John C. Maxwell
This is probably the most important part of my Thesis book and despite my sincere efforts to keep it decent length, it just cannot be short, as I am fortunate enough to have so many amazing people in my life. You all made me the person I am today and for that, I will be forever grateful.
Time spent as a Ph.D. student is truly one of a kind and one prepares for it long before it starts. It represents the beauty of the scientific journey with all of the failed experiments, rejected manuscripts, repeated cell culture contaminations and (multiple) lab movings, none of which really matters in the light of the later smallest experimental success. More importantly, it is a life journey, where we mature and meet wonderful people with the same passion and hunger for knowledge that we recognize in ourselves. And before we realize, they become our family. My time in the Vascular Surgery Group was the most wonderful, challenging, difficult at times, but also extremely fun period of my life. It was my ‘dream comes true’ experience, one to which I will come back my whole life. None of this would be ever possible without many of you, to whom I finally have the opportunity to say: ‘Thank you!’.
With this I would like to express my sincere gratitude to the following:
First and foremost, my main supervisor Ljubica Matic, who is a true role model of a successful woman scientist. Ljubica, there are no words to express how grateful I am for everything you have done for me. You believed in me from the very beginning and gave me the chance to be your Ph.D.
student despite my lack of experience in basic molecular biology (now it really sounds unbelievable!).
You are a brilliant researcher, with an unmatched ability to connect dots that many of us cannot even see at first glance. Always striving for perfection and demanding nothing less from others. During those years I spent at Vascular Surgery Group, you continuously cared for my development by creating possibilities, pushing me and encouraging me to leave my comfort zone. You gave me all the tools and opportunities I could dream of, which resulted in 6 (!!) papers/manuscripts, 2 fantastic internships (including a dream-come-true Stanford University) and countless international/national presentations with several awards, just to mention a few. I still cannot believe I was a part of this fantastic experience. You taught me the basics of molecular biology and shaped me into the scientist I am today, but most importantly you helped me become the person I always wanted to be. Both you and Ulf changed my life forever when you accepted me as your Ph.D. student and despite many scientific challenges along the way, I would not change a single thing. Thank you for being my teacher and mentor. You will always stay very close to my heart. My co- supervisor, Ulf Hedin. From day one I was absolutely enchanted by the way you talk about science, your passion for it and your knowledge. Over the years you infected me with the same enthusiasm, love for research and endless curiosity. You have a clear vision and in, what appears like an effortless way, you make other people believe in it too. This is truly inspiring. Throughout my Ph.D. I always enjoyed our meetings, as every time I was sure I will leave with tons of new ideas, recharged batteries and much higher motivation to continue, even despite many bumps on the way. Importantly, I could always rely on you and, in case I really needed your advice or help, you always made yourself available. You were never judging, never impatient and you were leaving me space to realize my own mistakes and learn from them. You taught me to always keep an open mind, which rule I try to have in the back of my head also in my private life. And most of all, you always made me feel my work and opinion were important. I am (and forever will be) extremely grateful I could be part of your team and learn from you. Thank you Ulf so much for everything! My co-supervisors Anton Razuvaev and Joy Roy, some of the most knowledgeable, but also kindest and most sincere people I know. Anton, you are a true animal surgery genius and having the possibility to observe you and get guidance from you was just a ‘once in a
lifetime’ opportunity. Joy, I am extremely grateful for all of your insightful questions and comments that made me look at my research from another perspective and for always being available when I needed your help. You are both extraordinary supervisors and it was a privilege to be able to work with you and have you as my teachers. Nicholas Leeper, my external mentor, for all the invaluable support over the years. I am deeply thankful for your great collaborative spirit and the time you have taken from your busy schedule to discuss science with me. I will always be impressed with your scientific knowledge, positive attitude and incredible kindness.
Being far from home and loved ones is at times extremely difficult and brings challenges impossible to predict in advance. Although it was not always easy, I never felt alone and I am pretty sure it was because of my Swedish family and all of those amazing people from the Vascular Surgery Group that every day made Sweden feel like my second home. Mariette Lengquist and Malin Kronqvist, my Swedish moms and my go-to persons in the moments of biggest crises. I would never be able to thank you enough for everything you have done for me, both when it comes to work, but also privately. I hope you know how much you mean to me and that you were one of the reasons, why I could survive these almost 7 years and come out stronger from all of this. Just the knowledge you were around was enough for me to feel everything will be ok. Mette, in the toughest, most difficult moments you were there for me and I can truly call you my friend. You are an amazing person, so kind, warm and honest.
You gave me a sense of security and were invaluable support during my whole Ph.D. Professionally, I think you are extremely good at your job and, considering your impressive knowledge about immunostainings, I am lucky I could learn from you. Malin, you were never afraid to tell me when I did something wrong and I greatly appreciated that. I usually don’t take criticism lightly, but coming from you it never sounded bad and I immediately wanted to do better. You are one of the best people I know. Your good heart, kindness and strong sense of what is right and wrong are inspiring and when I add it to your unmatched expertise in RNA isolation, I am really grateful you were my teacher and my friend. My rock and guide in KI’s administrative labirynth, Siw Frebelius. Siw, you are a true genius in solving ‘unsolvable’ issues and I am sure I would never be able to get to this point without you and your support. Whenever I had doubts or troubles, you always helped me and responded to my desperate emails, literally any time of day and night (even after 11 pm and/or during the weekends). You are an incredibly positive person, always nice and very easy to talk to. I am happy we met, because I really learned a lot from you. Thank you! Suvi Liinamo, although we did not spend so much time working together, you managed to make a huge impression on me. You are an absolutely gorgeous person, so kind, good and smart. Thank you for all the life-related conversations we shared and for being a huge support, both when it comes to science and private life. And thanks for the brilliant advice to learn swedish from criminal series books (which I love!).
Bianca Suur, my lab sister, best friend and a Ph.D. life companion. We understand one another without words, hence I hope you know, how much I love you, even though I almost never express it.
You are a beautiful person, inside and out, and one of the best scientists I have ever met. I am every day impressed by your knowledge, skills, integrity and ability to do millions of things at the same time and do all of them well (and way better than me). I am sure you have a great future ahead of you and I will always be one of your biggest fans and supporters, I promise! I was extremely lucky to meet you all those years ago in CMM and now I cannot even imagine how I could have completed 2 years of my studies without you in the picture. I learned so much from you and in the darkest of times could always count on you and your advice. There are no words to say, how grateful I am for all of that. I will deeply miss sitting next to you, with all of the laughter, tears and complaining we shared.
But of course, our friendship does not end here, so rather than being sad, I am excited for all the time we still have to share in the future! Till Seime, my 8 days younger (yes, I know how important it is for you) lab brother with an unbelievably sharp sense of humor and true scientific passion. Till, it was a true pleasure to work with you and be able to discuss with you both science and life. You are incredibly smart and funny, which is a killer combination, and you brought a lot of laughter and fun into my Ph.D. life. Sometimes one joke was enough to take all of the stress away and you cannot even imagine, how grateful I am for this. Thanks for being such a great friend and for all our fantastic conversations. Although I may not be as fast as you in coming up with decent ‘come back’ lines, you have to admit I had my (very few, indeed) moments, which I am still proud of J I miss you greatly, but can’t wait until I hear all about your new adventures! Melody Chemaly, an absolutely fantastic person and one of the purest souls I know. Mel, I love talking to you, not only because you are
extremely smart and you have answers for almost all my questions, but also because we always have something to talk about and you give me this kind of a special feeling of ‘inner peace’. It does not matter how stressed, sad or angry I am, when I talk to you, life somehow gets better. Thank you Mel for everything you said or did for me (including teaching me confocal microscopy and sharing your booked microscope time with me!), I will never forget it. We of course stay in touch and I wish you all the best in your biggest life challenge – motherhood! Antti Siika, for our fantastic trip to California and all the laughter we had together. This is when I got to know you a bit better and you truly amazed me with how smart, funny and kind you are. It is never boring with you and you can make even the most trivial things funny and enjoyable, which is exactly what I sometimes needed. Thanks Antti for just being there for me! Glykeria Karadimou, for our project and teaching collaborations and for your always friendly attitude. I am so happy I could meet you and share those many motherhood-related discussions. You have everything a successful woman and a scientist should have, so I am keeping my fingers crossed for you and I am sure you have a great future ahead! Nikolaos Taxiarchis-Skenteris, you are one of the nicest and kindest people I met at KI, with impressive knowledge, skills and always positive energy. I really enjoyed our conversations about science and life in general, you always managed to say something I could not stop thinking about. I am extremely grateful for all the help, advice and instructions, both regarding my research projects but also thesis application, writing and printing! Sampath Narayanan, for all our cell culture discussions and for sharing with me your scientific expertise. It was great to have you on board for the FOXC1 project!
Samuel Röhl, for all the support you have given me, nice words and kindness. For your calmness and positive attitude towards life, which every time impress me just as much, as they did the first time we met. Thank you for training me in animal surgery (you are absolutely amazing!) and for all our fantastic discussions. Hong Jin, thank you for sharing with me your animal surgery expertise and for all the kind words and support you have given me from the very beginning. I really enjoyed our friendly chats, during which you usually managed to make me feel better about myself, even when I didn’t think it was possible. Thank you also for all of the fantastic collaborations and for the confidence you had in me. It really means the world to me! Katarina Wadén, Moritz Lindquist Liljeqvist, Eva Karlöf and Marko Bogdanovic, for all the fun, more or less serious conversations we shared and simply for making my time as a Ph.D. student so much more enjoyable. I am truly amazed by your ability to successfully combine clinic duties with science, especially that for most of the time I struggle with ’just’ the science part J Thank you for all your valuable input and comments regarding my projects and for making the ’translational science’ concept so much more clear. Andrew Buckler, for being such a computational genius and bringing new quality to the Vascular Surgery Group. Although I am sure I will never be able to fully understand the complexity behind your imaging software and your projects, I am truly grateful for all the questions and input from you. It made me put my projects into perspective and see the bigger picture. Linnea Eriksson, for being friendly and supportive from the moment we met. You helped me immensely with my transition to the Vascular Surgery Group and even though you officially were not my mentor, it kind of felt this way. It was a great pleasure to share the office with you and be able to discuss various aspects of science and life. Thank you Linnea, you are a huge inspiration to me. Xiang Zhang and Otto Bergman, for all the help with bioinformatics and complex molecular biology analyses. Without you making sense of all of the data would be so much more difficult! Olga Nilsson and Alicia Garcia Lantz, for your positive energy, smile and always kind, supportive attitude. I am so grateful I could meet you! Mona Ahmed, Björn Gustafsson, Silvia Aldi and Robert Saxelin, for all of our exciting conversations, the feedback I received from you and your incredible kindness, which made everything so much easier for me in the beginning. Sofija Vuckovic, Colin Willems and Xinran (Aurora) Dong, for all the fresh air, new ideas and energy you brought to the lab. Science needs more people like you and I am happy I could witness such brilliant young scientists growing up J
Vascular Surgery Clinic, for all the fantastic work you do caring about patients and for greatly contributing to the basic research of the Vascular Surgery Group. Especially, to Rebecka Hultgren, a true example of an outstanding woman doctor and scientist, for the fantastic courses on sex differences and all the kindness and understanding you showed me when I was pregnant. Thank you so much!
A special thank you to Ann-Britt Wikström for your invaluable support and help with the administrative issues. You were always one step ahead, keeping track of all of the required