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Angiogenesis in ocular neovascular diseases : from multitarget inhibition to gene therapy tools
No full textOcular neovascular diseases, such as neovascular age-related macular degeneration (nAMD) or proliferative retinopathies (PRs) are characterized by the growth of abnormal blood vessels in the eye, leading to vision impairment. Hypoxia-mediated responses trigger angiogenesis, the growth of new blood vessels from existing ones, a pivotal feature for the development of these diseases. Hypoxia-inducible factors (HIFs) and its target genes regulate various aspects of the angiogenic cascade. The vascular endothelial growth factor (VEGF) is one of the main initiators of the angiogenic process, and the urokinase plasminogen activator receptor (uPAR), is responsible for the extracellular matrix (ECM) remodeling, allowing endothelial cell migration. Due to angiogenesis complexity, current treatments for ocular neovascular diseases present various limitations, creating a need for novel therapies. In this thesis, I investigated novel approaches of treatments for ocular neovascular diseases by pursuing a multitarget inhibition strategy along with evaluating specific tools for the targeted delivery of gene therapy constructs in the eye.In Paper I we investigated the inhibition of hypoxia-induced angiogenesis by antagonizing the uPA/uPAR system with UPARANT using a novel ex vivo human iris organotypic cultures model. We discovered an unidentified endothelial-specific antagonistic effect of UPARANT on the uPA/uPAR system by disrupting the interaction between uPAR and the low-density lipoprotein receptor-related protein-1 (LRP-1). This led to the inhibition of _-catenin-mediated angiogenesis through the blockage of the uPA/uPAR system, rather than through VEGF inhibition. These findings expanded our understanding of the effects of uPA/uPAR system antagonism in the context of ocular angiogenesis.In Paper II we evaluated the efficacy of echinomycin, an inhibitor of HIF-1_ DNAbinding activity, in reducing hypoxia-driven angiogenesis for the first time in the eye. We examined the effects of echinomycin in vitro on retinal cells, and in vivo using a murine model of laser-induced choroidal neovascularization (CNV). The inhibition of HIF-1_ led to a decreased response of retinal cells to hypoxia, and a reduction of vascular area in CNV-induced mice. These data suggested that targeting HIF-1_ is pivotal for future implications in the treatment of patients with ocular neovascular diseases.In Paper III we aimed to establish a protocol for the redifferentiation of ARPE-19 cells into a more mature retinal pigment epithelium (RPE) phenotype, to improve the evaluation of RPE-specific gene therapy strategies. The combination of laminin (LN) 521 coating with nicotinamide (NAM) supplementation promoted the reorganization and expression of mature RPE signature proteins and genes, leading ARPE-19 closer to their in vivo phenotype. Redifferentiated ARPE-19 transduced adeno associated viral vectors (AAVs) at low multiplicity of infection, mimicking in vivo AAV tropism, essential to test new gene therapies for RPE-centered diseases.List of scientific papersI. Pesce NA*, Plastino F*, Reyes-Goya C, Bernd J, Pavone V, Kvanta A, Dal Monte M, Locri F#, AndrŽ H#. Mitigation of human iris angiogenesis through uPAR/LRP-1 interaction antagonism in an organotypic ex vivo model. FASEB J. 2024 Mar 15;38(5):e23533. *Co-first authors. #Co-last authors. https://doi.org/10.1096/fj.202301892RR II. Plastino F*, Santana-Garrido ç*, Pesce NA, Aronsson M, Lardner E, Mate A, Kvanta A, V‡zquez CM, AndrŽ H. Echinomycin mitigates ocular angiogenesis by transcriptional inhibition of the hypoxia-inducible factor-1. Exp Eye Res. 2021 May;206:108518. *Co-first authors. https://doi.org/10.1016/j.exer.2021.108518 III. Bernd J, Plastino F, Karayannis J J, Kvanta A, Filippo Locri#, Helder AndrŽ#. Accelerated maturation of ARPE-19 cells for the translational assessment of gene therapy. #Co-last authors. [Submitted]</p
On suicide and self-harm in university students and outcomes in persons with bipolar disorder and their offspring
No full textSuicide is one of the leading causes of death in young persons in Sweden and globally. The last decades have seen an increasing proportion of the youth population engaging in university level education. The university period usually coincides with other important life events such as moving away from home. Early adulthood is furthermore a period when severe mental disorders, such as bipolar disorder and schizophrenia, may become symptomatic.Bipolar disorder is highly heritable, with genetics accounting for approximately 60-80% of the risk. The disorder is often diagnosed with many years of delay, partly due to its episodic features. More than one third of all people with bipolar disorder have university level education and more than half hold an occupation. Suicide risk varies between people working in different occupations. Healthcare professionals, especially nurses, have a higher suicide risk, but there is a knowledge gap concerning whether there could be a selection of individuals with mental health vulnerability and vulnerability to suicide into these occupations. There is also a need for more knowledge concerning suicide risk and risk of other outcomes in people with bipolar disorder and in their children to better inform prevention strategies.In Study I, we explored the risk of suicide and other mortality during ongoing university studies compared with periods of not studying at university. The nonstudy time periods were stratified by attained educational level. We found that having ongoing university studies was associated with a twofold risk of suicide compared to periods of non-studies when having attained at least one semester of university education. Having attained the lowest level of education was, however, associated with the highest risk of suicide and other mortality.In Study II, we went on to investigate whether certain university program categories were associated with a higher risk of suicide and self-harm. We were particularly interested in whether the association previously found between suicide risk and working as a nurse or a physician could be mirrored among students aiming for these occupations. We found that being a nursing student, but not a medical student, was associated with a higher risk of suicide and of self-harm within the first three years after starting university studies.In Study III, we examined a broad range of consequences, including suicide, selfharm, and low school grades, from birth to age 18 in offspring of one or two parents with bipolar disorder. We found that having a parent with bipolar disorder was associated with a more than tenfold risk of a bipolar disorder diagnosis, and with a higher risk of other psychiatric conditions as well as of some somatic conditions, low school grades, accidents, criminal behavior, selfharm, and suicide compared with not having a parent with bipolar disorder.In Study IV, we explored associations between having bipolar disorder and somatic conditions, accidents, injuries, victimization, and self-harm. We found that bipolar disorder was associated with an increased risk of cardiovascular, neurological, and other somatic conditions, with a threefold increased risk for sleep disorders, dementias, fibromyalgia, and restless leg syndrome compared with the general population. Risk of self-harm was increased more than sevenfold and similarly elevated regardless of sex or bipolar disorder subtype.In conclusion, this thesis shows that ongoing university studies can be a risk period for suicide and that there may be a selection of individuals with increased vulnerability to suicide into nursing education programs. Moreover, we show that offspring of parents with bipolar disorder are vulnerable to multiple psychiatric conditions, and adverse social outcomes during childhood and adolescence. Lastly, in people with bipolar disorder there is a higher risk of several somatic disorders, accidents, injuries, and self-harm compared with in people without bipolar disorder. Taken together our findings suggest that targeted support for the subgroups identified here as vulnerable to suicide or to multiple difficulties during childhood and adolescence could help decrease their risk of adversity, including suicide. Also, persons with bipolar disorder who have medical comorbidities may benefit from increased collaborations between somatic and psychiatric specialties.List of scientific papersI. Lageborn CT, Ljung R, Vaez M, Dahlin M. Ongoing university studies and the risk of suicide: a register-based nationwide cohort study of 5 million young and middle-aged individuals in Sweden, 1993-2011. BMJ Open. 2017;7(3):e014264. https://doi.org/10.1136/bmjopen-2016-014264 II. Lageborn CT, Bjureberg J, Song J, Runeson B, Möller J, Ljung R, Dahlin M. Risk of suicide and self-harm in university students entering different university programs - a national register-based cohort study in Sweden. Soc Psychiatry Psychiatr Epidemiol. 2023 Aug;58(8):1139-1149. https://doi.org/10.1007/s00127-023-02484-2 III. Lageborn CT, Zhou M, Boman M, Sjölander A, Larsson H, D’Onofrio BM, Pettersson E, Lichtenstein P, Landén M. Childhood and adolescence outcomes in offspring to parents with bipolar disorder: The impact of parental comorbidity, parental sex, and bipolar subtype. [Submitted]IV. Lageborn CT, Virtanen S, Sjölander A, Chang Z, Brikell I, Kuja-Halkola R, D'Onofrio BM, Garcia-Argibay M, Larsson H, Pettersson E, Lichtenstein P, Landén M. Somatic morbidity, accidents, injuries, and self-harm in bipolar disorder. [Manuscript]</p
Clinical and radiological features of thoracic aortic disease
No full textBackground:The pathologies of the aorta constitute a group of severe diseases often managed by means of a single variable, the maximum aortic diameter. This variable is insufficient, and the aim of this work is to help prevent severe aortic events, to help develop surgical decisions and improve the results of surgery.Methods and Results:Study I - Survival and events in patients with moderately dilated proximal thoracic aorta. All patients referred to the Thoracic Aortic outpatient clinic at Karolinska University Hospital between 1992-2011, with a baseline proximal aortic diameter of 4.5-5.4 cm without other established primary indication for surgery and subject to CT follow-up (n=80) were included. Data on aortic growth, all-cause mortality, aortic mortality, aortic events and relative survival were obtained from medical records and questionnaires, CT imaging, The National Cause of Death Register and the Human Mortality Database, respectively. Overall, 35% of patients experienced an event. Relative survival compared to a matched normal population was 82% (95% CI 55- 98%) at 10 years. Increasing diameter of the descending aorta was an independent predictor of all-cause death (HR 1.39) and aortic death (HR 1.96).Study II - Growth, survival and events in patients with aortic arch pathology. All patients referred to the Thoracic Aortic outpatient clinic at Karolinska University Hospital between 1992-2011, that had an index diameter ≥4.5 cm or other pathology in the native aortic arch and were subjects to CT follow-up (n=186) were included. Data on aortic growth, all-cause mortality, aortic mortality and aortic events were obtained from patient charts and questionnaires, CT imaging and the National Cause of Death Register, respectively. Twenty-five percent of patients had an event of any kind and 32% percent of events, both local and remote, were an acute aortic syndrome, preceded by growth in the aortic arch. Five years estimated freedom from all events was 66% (95% CI 56-79%)and an increasing descending aortic diameter was an independent predictor of all cause death (HR 2.16), aortic death (HR 4.81) and local event in the aortic arch (HR 1.71).Study III - Association between inguinal hernia and thoracic aortic dilatation. Men with prior inguinal hernia repair and as controls men with a prior cholecystectomy performed in the Department of Surgery at Ersta Hospital between 2016 and 2019 (n=470), were included. Data on thoracic aortic diameters and thoracic aortic dilatation (>45 mm root or ascending diameter; >35 mm isthmic or descending diameter) were obtained from patient questionnaires, CT imaging and the National Cause of Death register. Thoracic aortic dilatation prevalence was in the inguinal hernia group significantly higher vs. controls: 9.7% vs. 2.4%, p=0.001 for proximal aorta, 13% vs. 8.3%, p=0.049 for distal aorta, and 23% vs. 11%, pStudy IV - Outcomes in patients with proximal aortic repair. All nonsyndromic, asymptomatic patients who underwent elective, first-time, prophylactic proximal aortic surgery in the Department of Cardiothoracic Surgery, Karolinska University Hospital, between 2014 and 2019 (n=262) were included. Data on all-cause mortality and major complications were obtained from patient charts and the Swedish Population Register. Overall, mortality was 0.76% and remained unchanged at 1-year postoperative follow-up. The presence of major complications was in the 0-1.5% range. Freedom from aortic death was estimated to 97% (95% CI 91-99%) at 5 years postoperatively.Conclusions:More aortic related deaths with worse overall survival, more remote events and with an increasing descending aortic diameter as an independent predictor of all-cause death and aortic death were noticed.Continuing growth in the aortic arch prior to the occurrence of an event can be expected as well as events in terms of deaths and acute aortic syndromes. An increasing descending aortic diameter served as an independent predictor of all-cause death, aortic death and local event.In men with inguinal hernia, higher prevalence of aortic dilatation than in controls, was demonstrated.The outcomes of asymptomatic, nonsyndromic patients undergoing elective, firsttime proximal aortic surgery were excellent.List of scientific papersI. Carlestål E, Franco-Cereceda A, Olsson C. Aortic events and relative survival in patients with moderately dilated proximal thoracic aorta. Scandinavian Cardiovascular Journal. 2024;58(1):2330345. https://doi.org/10.1080/14017431.2024.2330345 II. Carlestål E, Franco-Cereceda A, Olsson C. Growth, survival and events in patients with aortic arch pathology. [Submitted]III. Carlestål E, Thorell A, Bergstrand L, Wilamowski F, Franco-Cereceda A, Olsson C. High prevalence of thoracic aortic dilatation in men with previous inguinal hernia repair. Aorta. (Stamford) 2022;10(3):122-130. https://doi.org/10.1055/s-0042-1749172 IV. Carlestål E, Ezer MS, Franco-Cereceda A, Olsson C. Proximal aortic repair in asymptomatic patients. JTCVS Open. 2021;7:1-9. https://doi.org/10.1016/j.xjon.2021.05.001 </p
Pathophysiology of myocardial infarction with non-obstructive coronary arteries and Takotsubo syndrome
No full textIntroduction: The working diagnosis myocardial infarction with non-obstructive coronary arteries (MINOCA), with the subgroup myocardial infarction with normal coronary arteries (MINCA), encompasses conditions of diverse aetiological origin. One of them is Takotsubo syndrome (TTS), a reversible and often stress-induced condition of acute heart failure, predominantly affecting postmenopausal women. Both MINOCA and TTS are associated with substantial morbidity and mortality, yet evidence-based treatment is lacking. The overall aim of this thesis was to expand the knowledge on pathophysiological mechanisms of MINOCA and TTS, with a view to guide future clinical management. The study specific aims were I) to characterise MINCA patients regarding risk factors and markers for endothelial function and atherosclerosis, II) to improve the diagnostics of MINOCA, III) to study if coronary microvascular dysfunction (CMD) is a prominent feature of acute TTS, and whether CMD in TTS is associated with clinical parameters, and IV) to determine whether sympathetic activity and reactivity are enhanced in the early recovery phase of TTS.Methods and results: In study I, the first Stockholm Myocardial Infarction with Normal Coronaries (SMINC) study, we included 100 MINCA patients together with two age- and sexmatched control groups of MI patients with coronary heart disease, respectively healthy volunteers. Endothelial function was evaluated with peripheral arterial tonometry measuring reactive hyperaemia index. Atherosclerosis was evaluated with carotid ultrasound measuring intima-media thickness. MINCA was associated with many established cardiovascular risk factors and female sex. Measures of endothelial function and atherosclerosis did not differ between study groups. One out of four MINCA patients received a clinical diagnosis of TTS.In study II, the SMINC-2 study, 148 MINOCA patients were examined by comprehensive cardiovascular magnetic resonance (CMR) imaging at median 3 days after admission. 150 patients from the screening phase of the SMINC-1 study, examined by standard CMR imaging at median 12 days after admission, served as historical controls. The underlying diagnosis was identified in 77% of patients in the SMINC-2 study, compared to 47% of patients in SMINC-1. The improved diagnostic yield was mainly due to increased detection of myocarditis and TTS.In study III, the Sympathetic And vascular Function in Takotsubo syndrome (SAFT) study, we included 27 female TTS patients together with age- and sexmatched controls with ischemia and no obstructive coronary arteries (INOCA). CMD was assessed invasively with thermodilution technique during index coronary angiography. Cardiac function was determined by echocardiography and CMR imaging in TTS patients. CMD was more common in TTS patients than in INOCA controls (78% vs. 44%). CMD was related to left ventricular function in TTS and more pronounced in the apical than midventricular phenotype.In study IV, muscle sympathetic nerve activity (MSNA) was determined by microneurography of the peroneal nerve at rest and during stress in 18 TTS patients from the SAFT study together with 13 age- and sex-matched healthy controls. Cardiac specific sympathetic activity was depicted by 123Imetaiodobenzylguanidine scintigraphy in 10 TTS patients. Despite signs of increased cardiac sympathetic activity in TTS, MSNA at rest and in response to stress did not differ between TTS patients and controls.Conclusions: MINCA is associated with many cardiovascular risk factors, female sex, and TTS, but not with markers of endothelial function and atherosclerosis. Early and comprehensive CMR imaging significantly improves the diagnostic yield in MINOCA and is therefore strongly suggested as a standard diagnostic tool. CMD is highly prevalent in the acute phase of TTS, associated with left ventricular function, and thus proposed as a key mediator in TTS pathophysiology. An altered cardiac response to sympathetic activation, rather than an underlying sympathetic hyperactivity or hyperreactivity, is likely central for TTS development.List of scientific papersI. Daniel M*, Ekenbäck C*, Agewall S, Brolin EB, Caidahl K, Cederlund K, Collste O, Eurenius L, Frick M, Younis-Hassan S, Henareh L, Jernberg T, Malmqvist K, Spaak J, Sörensson P, Hofman-Bang C, Tornvall P. Risk Factors and Markers for Acute Myocardial Infarction With Angiographically Normal Coronary Arteries. Am J Cardiol. 2015 Sep 15;116(6):838-44. *Shared first authorship. https://doi.org/10.1016/j.amjcard.2015.06.011 II. Sörensson P*, Ekenbäck C*, Lundin M, Agewall S, Bacsovics Brolin E, Caidahl K, Cederlund K, Collste O, Daniel M, Jensen J, Y-Hassan S, Henareh L, Hofman-Bang C, Lyngå P, Maret E, Sarkar N, Spaak J, Winnberg O, Ugander M, Tornvall P. Early Comprehensive Cardiovascular Magnetic Resonance Imaging in Patients With Myocardial Infarction With Nonobstructive Coronary Arteries. JACC Cardiovasc Imaging. 2021 Sep;14(9):1774-1783. *Shared first authorship. https://doi.org/10.1016/j.jcmg.2021.02.021 III. Ekenbäck C, Nickander J, Jokhaji F, Tornvall P, Engblom H, Spaak J, Persson J. Coronary microvascular dysfunction in Takotsubo syndrome and associations with left ventricular function. ESC Heart Fail. 2023 Aug;10(4):2395-2405. https://doi.org/10.1002/ehf2.14394 IV. Ekenbäck C, Persson J, Tornvall P, Forsberg L, Spaak J. Sympathetic nerve activity and response to stress in Takotsubo syndrome. [Manuscript]</p
NK cells in renal cell carcinoma : toward biomarker discovery and improved immunotherapeutic strategies
No full textRenal cell carcinoma (RCC) represents a significant clinical challenge due to its resistance to conventional therapies and its complex tumor microenvironment (TME). RCC's immunogenic nature, characterized by the infiltration of immune cells, highlights the potential of immunotherapeutic strategies. Among these immune cells, natural killer (NK) cells play a crucial role in the body's defense against tumors, including RCC. However, the functionality of NK cells is often impaired within the RCC TME due to various suppressive factors, leading to decreased tumor infiltration and cytotoxic activity. This thesis delves into the intricate interactions between NK cells and the RCC TME. It aims to uncover biomarkers for disease progression and devise strategies to enhance the efficacy of NK cell-based therapies.In Paper I, we conduct a comprehensive immune profiling to dissect the immune landscape of RCC, focusing on the secretome and phenotypes of NK cells and other immune populations in both the blood and tumors of RCC patients. Our analysis reveals significant alterations in cellular and soluble factors, pointing to potential biomarkers for disease progression, including CXCL8 levels, which correlate with aggressive tumor behavior and patient relapse. Papers II through IV investigate specific suppressive factors within the RCC TME that hinder NK cell infiltration and function. Paper II focuses on the role of the tumor poliovirus receptor (PVR) and its interaction with the activating receptor DNAM-1 on NK cells. We demonstrate that PVR-positive tumors downregulate DNAM-1, impairing NK cell-mediated cytotoxicity and highlighting PVR/DNAM-1 axis as a target to enhance NK cell function in RCC. Paper III examines the impact of regulatory T cells (Tregs) on NK cells. Our findings suggest that IL-15 primed NK cells, expressing higher levels of CD25, can better withstand Treg-mediated suppression. This points to the potential of IL-15 stimulation and CD25 expression as strategies to empower NK cells within the TME. Lastly, Paper IV addresses the effect of hypoxia, a hallmark of RCC due to VHL mutation, on NK cell activity. We show that hypoxia-inducible factors (HIFs), stabilized in the absence of VHL, create an immunosuppressive environment that reduces NK cell infiltration and activity. Restoration of VHL in RCC models enhances NK cell infiltration and antitumor activity, proposing a novel approach to combat hypoxia-induced NK cell dysfunction.Together, these studies provide a comprehensive understanding of the factors that limit NK cell efficacy in RCC and propose novel biomarkers and therapeutic strategies to harness NK cells' potential in combating this challenging disease. This thesis lays the groundwork for future research into optimizing NK cell-based immunotherapies for RCC, aiming to improve patient outcomes through targeted modulation of TME.List of scientific papersI. Tong L*, Kremer V*, Neo SY, Seitz C, Tobin NP, Harmenberg U, Colón E, Plogell AHS, Liu LL, Lundqvist A. Immune profiling reveals cellular and soluble factors as putative biomarkers in the prognosis of renal cell carcinoma patients. *Equal contributions. [Manuscript]II. Tong L, Kremer V, Neo SY, Liu Y, Chen Y, Wagner AK, Yang Y, Chen Z, Seitz C, Tobin NP, Ligtenberg MA, Alici E, Chen X, Haglund F, Seliger B, Harmenberg U, Colón E, Plogell AHS, Liu LL, Lundqvist A. Renal cell carcinoma escapes NK cell-mediated immune surveillance through the downregulation of DNAM-1. Cancer Communications. 2023, 43(7), p.855. https://doi.org/10.1002/cac2.12446 III. Chen Z*, Tong L*, Neo SY, Li S, Gao J, Schlisio S, Lundqvist A#. CD25bright NK cells display superior function and metabolic activity under regulatory T cell-mediated suppression. OncoImmunology. 2023, 12:2175517. *Equal contributions. https://doi.org/10.1080/2162402X.2023.2175517 IV. Tong L*, Tay AHM*, Cui W, Liu Y, Su Y, Lyu J, Hoedemakers L, Haglund F, Ehnman M, Nordlund P, Holmgren L, Neo SY, Lundqvist A. VHL restoration in clear cell renal cell carcinoma improves NK cell infiltration and function. *Equal contributions. [Manuscript]</p
Socioeconomic determinants of quality of care and outcomes in multiple sclerosis
No full textBackground: Multiple sclerosis (MS) is a chronic, inflammatory disease affecting the central nervous system (CNS), leading to varying degrees of neurological disability. While disease-modifying treatment (DMT) can ameliorate the severity of certain subtypes of this heterogenous disease, recent research has increasingly focused on understanding other factors influencing MS outcomes. Two such factors are explored in this thesis: quality of MS care, and sociodemographic status.Aims and Hypotheses: This thesis aims to explore the relationship between social determinants of health, quality of care (QoC) and MS outcomes in a universal healthcare context. It hypothesises that: 1. Higher quality of clinical care, including earlier initiation of DMT, is associated with more favourable clinical and patient-reported outcomes; 2. Socioeconomic status influences the quality of MS care received, as well as MS outcomes; 3. Race is associated with disparities in treatment and disability severity among people living with MS.Materials and Methods: This research comprises a series of observational studies using data from the Swedish MS Registry, linked with national administrative and healthcare databases, and a cross-sectional observational study conducted in a tertiary hospital in the United Kingdom (UK).Study 1 and Study 2 used longitudinal data from the Swedish MS Registry to explore the relationship between QoC indicators and patient outcomes. Study 1 assessed the effectiveness of early treatment – a QoC indicator - in improving symptoms and quality of life of people living with relapsing-remitting MS, while study 2 explored whether clinics’ performance on quality indicators set by the Swedish MS society guidelines – including treatment timeliness, outpatient visit frequency, Magnetic Resonance Imaging (MRI) frequency, and completeness of data entry - correlated with clinical and patient-reported outcomes in people with relapsing and progressive subtypes of disease.Study 3 used data from the Swedish MS Registry linked to national administrative databases to explore whether sociodemographic factors prior to disease onset - such as educational attainment, income, and marital status – are associated with future severity of disability and symptoms of MS.Study 4 compared the quality of MS care received by individuals of differing socioeconomic statuses within Sweden's universal healthcare system. We measured patients’ time from disease onset to diagnosis, time from diagnosis to treatment initiation, frequency of neurology clinic visits, and the number of MRI scans conducted within the first four years post-diagnosis. These indicators were analysed in relation to premorbid educational attainment and income levels.Study 5 utilised cross-sectional data from a major tertiary hospital in London. It explored the relationship between race, disease severity and treatment intensity within a nominally equitable healthcare system. All studies included both descriptive and multivariable analyses, as well as causal methodologies when comparing binary treatment exposures.Results: Studies 1 and 2 revealed that QoC was associated with clinical and patientreported outcomes in relapse-onset but not progressive-onset MS. Early DMT initiation was significantly associated with lower clinical disability as well as lower physical and psychological symptom burden, though overall quality of life was unaffected. More frequent clinic visits and MRI scans also appeared to correlate to physical health outcomes in people with relapse-onset MS.Study 3 found that higher premorbid educational attainment and income were both associated with significantly more favourable disability scores and symptoms. Single people and married/partnered people experienced comparable disease severity, while people who underwent marital separation prior to disease onset experienced significantly worse outcomes.Study 4 found that people with higher premorbid income had a faster time from diagnosis to DMT start, while those with higher educational attainment had a higher visit and MRI frequency in the first four years from diagnosis.Study 5 found significant racial disparities in a contemporary cohort of people with MS in the UK, with Black and Asian people experiencing worse disability outcomes compared to White people. Black people faced longer delays in receiving their first DMT and spent a smaller proportion of their disease duration on treatment compared to White people.Conclusions: The findings from these studies collectively indicate that both healthcare and sociodemographic factors significantly influence outcomes of MS patients. Early intervention and sustained, high-quality healthcare are crucial for improving patient outcomes. However, MS outcomes are not only influenced by healthcare factors, but also by socioeconomic status, marital status and race. This highlights the need for strategies beyond affordable healthcare in order to achieve health equity, such as cultural competency, enhanced support and outreach for vulnerable patients, and future research to understand the mechanisms through which these disparities occur. Holistic MS management must consider not just clinical, but also social determinants of health.List of scientific papersI. He A, Spelman T, Manouchehrinia A, Ciccarelli O, Hillert J, McKay K. Association between early treatment of multiple sclerosis and patient-reported outcomes: a nationwide observational cohort study. J Neurol Neurosurg Psychiatry. 2023 Apr;94(4):284-289. Epub 2022 Dec 7. PMCID: PMC10086460. https://doi.org/10.1136/jnnp-2022-330169 II. He AH, Manouchehrinia A, Glaser A, Ciccarelli O, Butzkueven H, Hillert J, McKay KA. Association between clinic-level quality of care and patient-level outcomes in multiple sclerosis. Mult Scler. 2023 Aug;29(9):1126-1135. Epub 2023 Jun 30. PMCID: PMC10413789. https://doi.org/10.1177/13524585231181578 III. He A, Manouchehrinia A, Glaser A, Ciccarelli O, Butzkueven H, Hillert J, McKay KA. Premorbid Sociodemographic Status and Multiple Sclerosis Outcomes in a Universal Health Care Context. JAMA Netw Open. 2023 Sep 5;6(9):e2334675. PMCID: PMC10523174. https://doi.org/10.1001/jamanetworkopen.2023.34675 IV. He A, Manouchehrinia A, Glaser A, Ciccarelli O, Butzkueven H, Hillert J, McKay KA. Socioeconomic status is associated with quality of multiple sclerosis care in Sweden. MSARD 2024. [Submitted]V. He A, Mohamud S, Benjamin L, Abdel-Mannan O, Hacohen Y, Ciccarelli O. Racial disparities in multiple sclerosis treatment and disability outcomes in a universal healthcare context. [Manuscript]</p
Microglia : guardians across the lifespan and disease spectrum
No full textMicroglia, the resident immune cells of the central nervous system, are crucial for maintaining homeostasis and responding to injury and disease. In this thesis, I aim to elucidate the molecular diversity and regulatory mechanisms governing microglia phenotype acquisition. The studies highlight microglia's roles in development, aging, and brain tumour context, underscoring the potential for therapeutic interventions targeting microglia-specific pathways.Paper I: ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain. In the first project, we describe a newly identified subtype of microglia expressing the metabolic enzyme arginase-1 (ARG1), predominantly located in the basal forebrain and ventral striatum during early postnatal development. These ARG1+ microglia are integral to neurodevelopment, as their knockdown leads to impaired cholinergic innervation and dendritic spine maturation, culminating in cognitive deficits. This discovery enhances our understanding of microglial diversity and provides a foundation for future research into microglia subtype-specific functions.Paper II: Age-associated microglial transcriptome leads to diminished immunogenicity and dysregulation of MCT4 and P2RY12/P2RY13 related functions. The second project examines the aging process in microglia, focusing on their phenotype acquisition. Long-term cultivation of BV2 microglia revealed that aged microglia exhibit a distinct gene expression profile and a downregulated response to pro-inflammatory stimuli. Comparative analysis with datasets from aged mice and humans identified a conserved aging signature, Specifically the upregulation of the lactate pump MCT4 and downregulation of the homeostatic marker P2RY12 that work as sensing protein. This study underscores the importance of these molecular determinants in microglial aging, providing insights for potential interventions to reprogram aged microglia and help treat age-related neurological disorders.Paper III: ID2-ETS2 axis regulates the transcriptional acquisition of protumoral microglia phenotype in glioma. The third project explores the mechanisms through which glioma cells reprogram microglia into a tumour-supportive state. Our research identifies a molecular axis in microglia that involves the co-regulator of transcription factors, inhibitor of DNA binding 2 (ID2), and the transcription factor ETS proto-oncogene 2 (ETS2). This axis, activated in microglia exposed to glioma cells, appears to drive the reprogramming process. Notably, this molecular pathway was also active in microglia from human glioblastoma biopsies, underscoring its potential as a therapeutic target for modulating microglial functions to inhibit glioma progression.Paper IV: Glioma-induced DNMT3A-dependent reduction of DNA methylation in microglia promotes a transient anti-tumoral phenotype. The fourth project hypothesizes that microglia, upon stimulation by glioma cells, may transit through a reactive state characterized by increased inflammatory and immunogenic properties before acquiring a tumour-supportive phenotype. This state is marked by reduced DNMT3A chromatin occupancy and DNA demethylation, promoting pro-inflammatory gene expression. This intermediate state could offer a novel therapeutic angle for glioma treatment by leveraging the microglial response before it becomes tumour-supportive.List of scientific papersI. ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain. Vassilis Stratoulias, Roc’o Ruiz, Shigeaki Kanatani, Ahmed M. Osman, Lily Keane, Jose A. Armengol, Antonio Rodr’guez-Moreno, Adriana-Natalia Murgoci, Irene Garc’a-Dom’nguez, Isabel Alonso-Bellido, Fernando Gonz‡lez Ib‡–ez, Katherine Picard, Guillermo V‡zquez-Cabrera, Mercedes Posada-PŽrez, Nathalie Vernoux, Dario Tejera, Kathleen Grabert, Mathilde Cheray, Patricia Gonz‡lez-Rodr’guez, Eva M. PŽrez-Villegas, Irene Mart’nez-Gallego, Alejandro Lastra-Romero, David Brodin, Javier AvilaCari–o, Yang Cao, Mikko Airavaara, Per UhlŽn, Michael T. Heneka, Marie-éve Tremblay, Klas Blomgren, Jose L. Venero & Bertrand Joseph. Nat Neurosci. 2023 Jun;26(6):1008-1020. https://doi.org/10.1038/s41593-023-01326-3 II. Age-associated microglial transcriptome leads to diminished immunogenicity and dysregulation of MCT4 and P2RY12/P2RY13 related functions. Martin Skandik, Lara Friess, Guillermo V‡zquez-Cabrera, Lily Keane, Kathleen Grabert, Mireia Cruz De los Santos, Mercedes Posada Perez, Austeja Baleviciute, Mathilde Cheray and Bertrand Joseph. [Manuscript]III. ID2-ETS2 axis regulates the transcriptional acquisition of protumoral microglia phenotype in glioma. Guillermo V‡zquez-Cabrera, Martin _kand’k, NoŽmie Roncier, Farah Real Oualit, Mireia Cruz De Los Santos, Austeja Baleviciute, Mathilde Cheray and Bertrand Joseph. Cell Death Dis. 2024 Jul ;15(7):512. https://doi.org/10.1038/s41419-024-06903-3 IV. Glioma-induced DNMT3A-dependent reduction of DNA methylation in microglia promotes a transient anti-tumoral phenotype. Mathilde Cheray, Adriana-Natalia Murgoci, Adamantia Fragkopoulou, Carlos F.D. Rodrigues, Ahmed M. Osman, Christine Hong, Guillermo V‡zquez-Cabrera, Lara Friess, Lena-Maria Carlson, Shigeaki Kanatani, Yue Li, Anastasius Damdimopoulos, Per UhlŽn, Fredrik Kamme, Klas Blomgren, and Bertrand Joseph. [Manuscript]</p
Unveiling mechanisms of translational control in disease
No full textmRNA translation is a core cellular function that enables rapid alterations in proteome as a response to intra- or extracellular stimuli, with or without the corresponding changes in the transcriptome. Considering its prominent role in regulating numerous other cellular functions such as proliferation, metabolism, development, or stress response, its dysregulation contributes to the pathogenesis of multiple diseases.Tuberous sclerosis complex (TSC) is an inherited multi-system disorder caused by loss-of-function mutations in TSC1 or TSC2 genes characterized by hyperactive mTOR signaling. In Study I, we characterized the alterations of gene expression programs caused by TSC1 loss-of-function mutations in neural progenitor cells (NPCs) that are also recapitulated in postmortem brains from autism spectrum disorder (ASD) donors. While rapamycin is the only approved therapy for TSC, we showed that it could not reverse the alterations caused by loss of TSC1 and rescue phenotypes such as neurite overgrowth. On the other hand, the bisteric mTORC1 inhibitor, RMC-6272, reversed the alterations of gene expression programs and rescued phenotypes associated with TSC1 loss. Overall, we provided a detailed overview of TSC1-dependent gene expression programs in NPCs that are recapitulated in postmortem ASD brains. In addition, we highlighted the potential of bi-steric mTORC1 inhibitors in treating TSC and associated phenotypes, offering a promising therapeutic avenue for TSC and related neurodevelopmental disorders.The integrated stress response (ISR) enables the translation machinery to adjust to various stressors. In Study II, we present evidence of an osmoadaptation mechanism independent of the integrated stress response (ISR). This process involves reprogramming mRNA translation through the coordinated yet independent activities of the mTOR signaling and the plasma membrane amino acid transporter SNAT2. We revealed that the biphasic response to mild hyperosmotic stress involves a decrease in overall protein synthesis and mTOR signaling, followed by the translation of SNAT2. The induction of SNAT2 subsequently stimulates the intake of amino acids and restoration of mTOR activity and, thus, overall protein synthesis. This is accompanied by a partial reversal of the translation programs first affected by hyperosmotic stress during the early phase. Our results suggest that SNAT2 is a prominent mediator of osmoadaptation to mild hyperosmotic stress, serving as a molecular switch that controls the transition from inhibiting protein synthesis to establishing a biphasic osmoadaptive translation program.Breast cancer, the most prevalent type of cancer in women, is a disease with a wide range of underlying molecular mechanisms. Tumor classification based on these molecular mechanisms is a crucial step that estimates disease progression and allows patients to benefit from currently available treatment methods. To date, efforts have been made to categorize tumors based on histological characteristics or gene expression programs; however, these methods do not consider mRNA translation as a means of tumor classification. In Study III, we used a data set of transcriptome-wide patterns of mRNA translation generated using patient tumors to investigate how mRNA translation is regulated in breast cancer patients. Moreover, our efforts helped us to identify mechanisms that, when combined, explain a large proportion of variation in mRNA translation in individual patients. Future studies will assess if identifying and targeting these translation mechanisms could serve as an opportunity for personalized cancer treatment.List of scientific papersI. Translatome analysis of tuberous sclerosis complex 1 patient-derived neural progenitor cells reveals rapamycin-dependent and independent alterations. Aksoylu IS, Martin P, Robert F, Szkop KJ, Redmond NE, Bhattacharyya S, Wang J, Chen S, Beauchamp RL, Nobeli I, Pelletier J, Larsson O, Ramesh V. Mol Autism. 2023 Oct 25;14(1):39. https://doi.org/10.1186/s13229-023-00572-3II. Stress-induced perturbations in intracellular amino acids reprogram mRNA translation in osmoadaptation independently of the ISR. Krokowski D, Jobava R, Szkop KJ, Chen CW, Fu X, Venus S, Guan BJ, Wu J, Gao Z, Banaszuk W, Tchorzewski M, Mu T, Ropelewski P, Merrick WC, Mao Y, Aksoylu IS, Miranda H, Qian SB, Manifava M, Ktistakis NT, Vourekas A, Jankowsky E, Topisirovic I, Larsson O, Hatzoglou M. Cell Rep. 2022 Jul 19;40(3):111092.https://doi.org/10.1016/j.celrep.2022.11109III. Transcriptome-wide alterations in mRNA translation define breast cancer subtypes. Aksoylu IS, Bellato HM, Liang S, Lupinacci FCS, Masvidal-Sanz L, Oertlin C, Hajj GNM, Larsson O. [Manuscript]</p
The rRNA epitranscriptome and myonuclear SNORD landscape in skeletal muscle fibers contributes to ribosome heterogeneity and is altered by a hypertrophic stimulus.
No full textIn cell biology, ribosomal RNA (rRNA) 2'O-methyl (2'-O-Me) is the most prevalent posttranscriptional chemical modification contributing to ribosome heterogeneity. The modification involves a family of small nucleolar RNAs (snoRNAs) and is specified by box C/D snoRNAs (SNORDs). Given the importance of ribosome biogenesis for skeletal muscle growth, we asked if rRNA 2'-O-Me in nascent ribosomes synthesized in response to a growth stimulus is an unrecognized mode of ribosome heterogeneity in muscle. To determine the pattern and dynamics of 2'-O-Me rRNA, we used a sequencing-based profiling method called RiboMeth-seq (RMS). We applied this method to tissue-derived rRNA of skeletal muscle and rRNA specifically from the muscle fiber using an inducible myofiber-specific RiboTag mouse in sedentary and mechanically overloaded conditions. These analyses were complemented by myonuclear-specific small RNA sequencing to profile SNORDs and link the rRNA epitranscriptome to known regulatory elements generated within the muscle fiber. We demonstrate for the first time that mechanical overload of skeletal muscle 1) induces decreased 2'-O-Me at a subset of skeletal muscle rRNA and 2) alters the SNORD profile in isolated myonuclei. These findings point to a transient diversification of the ribosome pool via 2'-O-Me during growth and adaptation in skeletal muscle. These findings suggest changes in ribosome heterogeneity at the 2'-O-Me level during muscle hypertrophy and lay the foundation for studies investigating the functional implications of these newly identified "growth-induced" ribosomes.NEW & NOTEWORTHY Ribosomal RNAs (rRNAs) are posttranscriptionally modified by 2'O-methyl (2'-O-Me). This study applied RiboMeth-seq (RMS) to detect changes in 2'-O-Me levels during skeletal muscle hypertrophy, uncovering transient diversification of the ribosome pool in skeletal muscle fibers. This work implies a role for ribosome heterogeneity in skeletal muscle growth and adaptation.</p
Development and validation of novel deep learning-based models for cancer histopathology image analysis
No full textHistopathological assessment of resected tumour specimens remains the foundation of breast cancer diagnosis and plays a critical role in guiding clinical decision-making. Advances in computational pathology have shown the potential to improve routine diagnostics and enable more precise treatment planning for breast cancer patients. Deep learning, at the forefront of these advancements, not only replicates traditional pathological assessments but also introduces novel avenues beyond routine settings like improved risk- stratification, prognostic and response-to-treatment predictive models. Such avenues of deep learning tasks are referred to as AI-based precision pathology. In this thesis, we developed and validated deep learning-based models for AI- based precision pathology tasks to improve breast cancer diagnosis using routinely stained resected tumour specimens.In study I, we developed and prognostically validated a deep learning-based three-grade classification model (predGrade) using Haematoxylin and Eosin (H&E) stained whole slide images (WSIs), that mimics the conventional histomorphology based prognostic marker Nottingham Histological Grading (NHG) for invasive breast cancer patients. predGrade showed similar prognostic performance to the conventional NHG with the unadjusted hazard ratio (HR) for clinical NHG 2 versus 1 associated with recurrence-free survival (RFS) estimated to be 2.59 (p-value = 0.004) and clinical NHG 3 versus 1 estimated to be 3.58 (p- value In study II, we prognostically validated the CE-IVD-approved AI-based solution called Stratipath Breast, for prognostic risk-stratification of breast cancer patients using H&E WSIs in two independent hospital sites in Sweden (N=2719). In this retrospective validation study, we observed the HR associated with progression-free survival (PFS) to be 2.20 (95% CI: 1.22-3.98, p-value = 0.009) between Stratipath breast low- and high-risk groups, in the clinically relevant oestrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative/NHG 2 patient subgroup. Assignment of adjuvant chemotherapy to intermediate-risk/ER+/HER2- patients is ambiguous. Improved risk-stratification of this patient subgroup using an H&E WSI-based AI solution can potentially improve the under- and over-treatment of such patients. Further, it provides a fast and cost-effective solution over existing molecular profiling- based methods.In study III, we proposed a methodology to spatially interpret the deep learning- based weakly supervised models. In many of the precision pathology tasks like histological grading classification or prognostic risk score modelling, the label is only available at the slide-level and not the tile or pixel level. In such scenarios, there is a need to understand the association of local regions in WSI with the slide-level prediction task. Here, we introduce the Wsi rEgion sElection approach (WEEP), which provides the selection of tiles that are directly associated with the classification of the WSI. We observed the application of WEEP in a binary classification task (low vs high histological grade). Further, the methodology provides visual interpretability of the regions that are driving the classification of the WSI. It is a straightforward and simple methodology that has applications in both research and diagnostic applications.In study IV, we developed a deep learning-based multi-stain prognostic risk score prediction model for breast cancer patients. H&E and immunochemistry (IHC) stains are central for routine biomarker assessments in breast cancer pathology. Instead of discrete and categorical protein-expression values, the spatial combination of histomorphological features across different stains can potentially include more prognostic information. In this study, we utilised the WSI registration method to provide local and spatial alignment from different stains. We combined the tile-level features extracted from different stains using the foundation models UNI and CONCH. Further, we observed the improvement in prognostic risk prediction after the addition of multiple stains in comparison to individual stains (C-index: 0.72 [95% CI: 0.65 - 0.79]) in an aggregated 5-fold CV test set. The results show the potential of a multi-stain model to improve breast cancer patient risk-stratification over the single-stain modality.List of scientific papersIn this thesis, we have included the following list of scientific papers, that includes two published articles in peer-reviewed journals and two completed manuscripts available as pre-prints.1. Sharma A, Weitz P, Wang Y, Liu B, Vallon-Christersson J, Hartman J, et al. Development and prognostic validation of a three-level NHG-like deep learning-based model for histological grading of breast cancer. Breast Cancer Res. 2024 Jan 29;26(1):17. https://doi.org/10.1186/s13058-024-01770-42. Sharma A, Lövgren SK, Eriksson KL, Wang Y, Robertson S, Hartman J, et al. Validation of an AI-based solution for breast cancer risk stratification using routine digital histopathology images. Breast Cancer Res. 2024 Aug 14;26(1):123. https://doi.org/10.1186/s13058-024-01879-63. Sharma A, Liu B, Rantalainen M. WEEP: A method for spatial interpretation of weakly supervised CNN models in computational pathology [Internet]. arXiv [eess.IV]. 2024 [cited 2024 Sep 23]. Available from: http://arxiv.org/abs/2403.15238 [Manuscript Preprint]4. Sharma A, Gustafsson FK, Hartman J, Rantalainen M. Multi-stain modelling of histopathology slides for breast cancer prognosis prediction [Internet]. medRxiv. 2024 [cited 2024 Nov 20]. p. 2024.11.10.24317066. Available from: https://www.medrxiv.org/content/10.1101/2024.11.10.24317066v1.abstract [Manuscript Preprint]</p