Research Aline Verstraeten

Abstract

Thoracic aortic aneurysm (TAA) entails a high risk for aortic dissection and rupture, which is a prominent cause of death in Western countries. Prophylactic surgery significantly reduces the mortality risk, but complications are relatively common. Moreover, in severe TAA conditions aneurysms often develop at other locations afterwards, exposing patients to repeated surgeries and, thus, threats. Current drug options only modestly slow down dilatation, without preventing dissections or ruptures. Clearly, a game changer in TAA patient management would be the availability of medical therapies capable of stopping or reversing aneurysm formation. Functional characterization of the known TAA genes, especially those that are linked to syndromic TAA, in relevant cell and/or mouse models has already delivered valuable insights into the disease mechanisms, prompting pre-clinical drug testing in mice. The mechanistic picture is incomplete though, encumbering the development of additional, and especially more effective, therapies. Another prevailing issue is the inefficient and/or unsuccessful translation of pharmacological mouse results to the clinic. Few compounds make it to clinical trials due to the high costs, lengthy time frames and difficulties as to patient recruitment. Additionally, while TAA mouse models allow us to study and therapeutically target disease in an in vivo setting, efficiency of ensuing compounds might not be recapitulated in humans. Building on intriguing preliminary data and the unique availability of mutant/control Fbn1 and Ipo8 mice and human induced pluripotent stem cells, this project aims to contribute to the resolution of these issues by further unravelling and therapeutically targeting the mechanisms underlying syndromic TAA. Additionally, BREAK-OUT will provide proof-of-concept that patient-derived aorta-on-a-chip models can be used for pre-clinical TAA research.

Researcher(s)

• Promoter: Verstraeten Aline

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Chondrodysplasias are skeletal disorders attributed to primary defects in hyaline cartilage. Disease severity differs considerably between subtypes, with some only inflicting joint abnormalities and others causing severe dwarfism or perinatal lethality. For many (severe) chondrodysplasias satisfactory therapies are lacking, prompting further research in the underlying disease mechanisms. Endoplasmatic reticulum (ER) stress, and the accompanying excess in chondrocyte apoptosis, have emerged as credible pathomechanisms in some chondrodysplasias, including COL2-pathies. In these conditions chaperone-oriented therapy represents an interesting pharmacological avenue. In this project, we will use iPSC-chondrocyte models to investigate whether ER stress and unfolded protein response (UPR) activation play a role in the etiology of BGN-related chondrodysplasia, which is a pathomechanistically unexplored form of serious dwarfism. IPSC-chondrocytes of patients suffering from severe but non-lethal COL2A1-related spondyloepiphyseal dysplasia congenita will be used as positive controls. Next, we will develop a novel iPSC-chondrocyte-based high-throughput microscopic high content assay, which will be used to pinpoint novel drug candidates (compound library screening) or existing drugs (repurposing) promoting protein folding in ER stress-related chondrodysplasias.

Researcher(s)

• Promoter: Verstraeten Aline
• Co-promoter: Peeters Silke

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Pathogenic variants in biglycan cause two divergent phenotypes: Meester-Loeys syndrome (MRLS) and X-linked spondyloepimetaphyseal dysplasia (SEMDX). The latter is characterized by a disproportionate short stature and caused by missense variants. MRLS, on the other hand, is a syndromic form of thoracic aortic aneurysm that is caused by loss-of-function variants. Intriguingly, MRLS patients with partial biglycan deletions present with a more severe skeletal phenotype. To date, discriminative pathomechanisms explaining why certain biglycan mutations cause MRLS and others SEMDX remain elusive. This PhD project aims to answer this research question using induced pluripotent stem cells (iPSCs) of both patient groups and their respective (isogenic) controls. IPSC-based disease modeling provides a unique opportunity for pathomechanistic investigation in a patient-, variant- and cell type-specific manner. After the creation of disease-relevant patient-derived iPSC-vascular smooth muscle cells and -chondrocytes, I will identify cell type-specific differences between MRLS and SEMDX using (1) functional assays tailored to existing pathomechanistic insights, and (2) hypothesis-free transcriptomic and proteomic approaches. Finally, I will investigate the mutational effect of partial biglycan deletions to establish a specific MRLS genotype-phenotype association.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Meester Josephina
• Co-promoter: Verstraeten Aline
• Fellow: Hebert Anne

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Thoracic aortic aneurysm (TAA) denotes a progressive enlargement of the thoracic aorta, entailing a significant risk for life-threatening aortic dissection and/or rupture. At present, mouse models are often used to investigate and therapeutically target the molecular defects underlying TAA, as native aortic samples of patients and, especially, control individuals are hard to collect. Yet, murine in vivo studies are often lengthy and drug testing results did previously not always recapitulate in patients. With the advent of induced pluripotent stem cells (iPSCs), the field is closing in on apt solutions to faithfully model patient and control aortas in a dish. The currently available vascular smooth muscle cell (VSMC) or endothelial cell (EC) monocultures are still overly simplified, as they fail to adequately replicate the complex multilayered and multicellular structure of the aorta. Taking advantage of available iPSCs from syndromic TAA patients (FBN1 & IPO8), my project aims to 1) develop and consolidate the validity of the first iPSC-derived TAA aorta-on-a-chip models, comprising the two VSMC subtypes populating the native ascending aorta along with a layer of arterial ECs, and 2) use the established model to further investigate the disease mechanisms underlying the relatively unexplored IPO8 syndrome. The anticipated outcomes will contribute to the replacement of mouse models (3R principle) and expedite pathophysiological TAA research and drug discovery.

Researcher(s)

• Promoter: Verstraeten Aline
• Co-promoter: Loeys Bart
• Fellow: Fedoryshchenko Ivanna

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Thoracic aortic aneurysm (TAA) denotes a progressive enlargement of our body's largest artery, i.e. the aorta. It frequently remains unnoticed until aortic dissection and/or rupture occur, which are associated with high mortality rates. Although prophylactic aortic surgery can serve as a lifesaving event, it comes with important risks. Current drug therapies can only slow down TAA progression to some extent, but cannot fully prevent aortic dissections/ruptures. Better therapeutic options are clearly needed. TAA and the ensuing aortic complications are a hallmark of a rare connective tissue disorder, called Loeys-Dietz syndrome (LDS). While LDS-related TAA is relatively understudied as compared to other TAA conditions, it is an important study case considering its early onset and aggressive disease course. Taking advantage of the advent of the iPSC technology and our expertise in clinical and pathophysiological LDS research as well as iPSC-vascular smooth muscle cell and iPSC-endothelial cell disease modelling, we want to significantly expedite bench-to bedside translation of LDS research by developing and functionally validating iPSC-derived aorta-on-a-chip (AoC) models of TAA-presenting patients and isogenic controls. Upon demonstration of the known pathomechanisms and drug responses in SMAD3 mutant AoCs, we will investigate if our AoC models can also recapitulate between-patient variability in disease severity. In conclusion, we here take up the challenge to develop a novel pre-clinical tool allowing exploration and therapeutic targeting of LDS mechanisms in a human setting that more closely resembles the native aorta than ever before. The anticipated results will prove relevant for TAA in general, as the AoC expertise that will be acquired within the frame of this project can immediately be translated to other TAA conditions.

Researcher(s)

• Promoter: Verstraeten Aline
• Co-promoter: Loeys Bart
• Fellow: Bousbaa Amira

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Thoracic aortic aneurysm (TAA) stands for a pathological dilatation of the thoracic part of our body's largest artery, which entails a high risk for life-threatening aortic dissection and rupture. Prophylactic surgery significantly reduces the mortality risk, but complications are relatively common. Moreover, in severe TAA conditions aneurysms often develop at other locations afterwards, exposing patients to repeated surgeries and, thus, threats. Current drug options only modestly slow down dilatation, without preventing dissections or ruptures. Clearly, a game changer in TAA patient management would be the availability of medical therapies capable of stopping or reversing aneurysm formation. To develop such therapies, better pathomechanistic insights should be gained. To date, TAA insights have largely been acquired using single-gene functional genetic approaches. Disadvantages of such strategies include suboptimal exploration of less obvious disease culprits as well as the fact that the yet acquired insights may prove insufficient to develop a single efficient therapy for a genetically heterogeneous disease such as TAA. To find convergent TAA mechanisms, I used a hypothesis-free bulk transcriptomics approach on affected aortic root and ascending aorta samples from three distinct TAA mouse models and their respective wild-type littermates. A significant consistent dysregulation of several highly interesting candidate disease culprits (based on literature, other TAA transcriptomics datasets, phenotype of transgenic mice, etc.) was observed in all three models. Here, I aim to acquire preliminary evidence that neutralization of the top genes' expression levels in TAA-presenting Fbn1C1041G/+ mice can rescue the 3 TAA phenotype. To do so, crossbreeding of the TAA mouse with knockout or overexpression mice for the respective genes of interest will be done, after which the aorta of the different single- and double-transgenic offspring as well as their wildtype littermates will be phenotyped using echocardiography and histological stainings. Upon demonstration of phenotypic alleviation, additional offspring will be generated, whose aortic tissue samples will be subjected to standard molecular biology techniques to gain preliminary insights into the target genes' mode of action.

Researcher(s)

• Promoter: Verstraeten Aline

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Chondrodysplasias refer to a large and heterogeneous group of skeletal disorders caused by primary defects in hyaline cartilage. They have a combined prevalence of about 1/4000 births and differ considerably with respect to disease severity; with some only inflicting mild joint symptoms, and others coming with severe dwarfism or even perinatal lethality. Especially the complications that arise from major growth problems (e.g. respiratory difficulties, spinal cord compression, hydrocephaly) impact significantly on the patient's quality of life. For many chondrodysplasias no therapies are on the market yet. Over the past years, endoplasmatic reticulum (ER) stress and the resulting excess of apoptosis have emerged as convincing converging chondrodysplasia pathomechanisms. This project builds further on these findings and aims to significantly improve future chondrodysplasia patient management by 1) establishing the protocols to create and study iPSC-chondrocytes as well as to use them for high-throughput drug screening approaches, with a primary focus on COL2A1 and BGN-related dysplasias, 2) investigating whether ER stress and UPR activation play a role in the etiology of BGN-related chondrodysplasia (i.e. a pathomechanistically unexplored severe form of dwarfism), and 3) developing and applying a novel iPSC-chondrocyte-based high-throughput high content assay to discover putative drug candidates that promote protein folding in ER stress-related chondrodysplasias.

Researcher(s)

• Promoter: Verstraeten Aline
• Co-promoter: Meester Josephina
• Fellow: De Kinderen Pauline

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Thoracic aortic aneurysm (TAA) is an abnormal widening of the aorta in the chest, caused by the weakening of the aortic wall. TAAs can lead to rupture or dissection, a devastating complication with a mortality rate of 50%. Despite considerable efforts to gain insights on the molecular mechanisms underlying TAAs, there is currently no therapy that effectively stops or reverses TAA development. Single-cell RNA sequencing (scRNA-seq) is emerging as a ground-breaking technology to investigate gene expression at single-cell level and is opening new avenues to discover yet unexplored disease pathways. In my project, I will apply this technique to investigate a novel TAA disorder caused by bi-allelic pathogenic variants in the IPO8 gene, recently discovered in our Cardiogenomics research group. I will search for differentially expressed genes (DEGs) within the different aortic cell populations from an Ipo8-/- mouse model that recapitulates the human aortic aneurysmal phenotype. I will also investigate shared DEGs between Ipo8-/- mice and additional TAAs mouse models to find convergent disease pathways in clinically related TAA disorders. Subsequently, I will validate the role of the identified candidate culprits in mouse TAA development in a human setting, by using CRISPR-inhibition or -activation in iPSCs derived vascular smooth muscle cells or endothelial cells. The predicted outcomes will potentially pinpoint novel TAA drivers and hence, unveil potential new therapeutic targets.

Researcher(s)

• Promoter: Verstraeten Aline
• Co-promoter: Loeys Bart
• Co-promoter: Meester Josephina
• Fellow: Valdivia Callejon Irene

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Thoracic aortic aneurysm (TAA) is an abnormal widening of the thoracic aorta caused by blood vessel wall weakness. TAAs entail a high risk for aortic rupture or dissection, commonly leading to sudden death. To date, genetic defects in >35 genes have been linked with TAA, providing a molecular cause for about 30% of patients. Their identification and functional characterization have been key in acquiring our current pathomechanistic aortopathy knowledge. Yet, the genetic and mechanistic picture for TAA is far from complete, hampering identification of predictive markers for aneurysm formation and development of therapies capable of stopping or reversing aneurysm formation. In search for novel TAA genes, our research group most recently identified recessive truncating IPO8 mutations as a novel cause of syndromic TAA. This project builds on this exciting finding, remarkable Ipo8-/- mouse background differences and the availability of IPO8 mutant iPSCs and isogenic controls. More specifically, we aim to significantly improve our current pathomechanistic insight in TAA caused by IPO8 deficiency based on 1) transcriptomics to unravel the involved biological pathways; and 2) identification of proteins and miRNAs with an abnormal cytosol/nucleus distribution upon IPO8 depletion. In the long term, this project's anticipated results will identify new targets for drug therapies, improving TAA patient management.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Meester Josephina
• Co-promoter: Verstraeten Aline
• Fellow: Buccioli Lucia

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

The Vevo LAZR-X is an imaging platform for preclinical applications capable of acquiring in vivo anatomical, functional and molecular data. It combines ultra high frequency ultrasound with photoacoustic imaging (a new biomedical imaging modality based on the use of lasergenerated ultrasound) for high resolution images as well as software for analysis and quantification. This equipment will be used in the context of the study of (cardio)vascular diseases, genetics of the heart, heart valves and aortic dissection, kidney diseases and their effects on the heart and blood vessels, and for cancer research.

Researcher(s)

• Promoter: De Meyer Guido
• Co-promoter: De Keulenaer Gilles
• Co-promoter: Heidbuchel Hein
• Co-promoter: Loeys Bart
• Co-promoter: Smits Evelien
• Co-promoter: Van Craenenbroeck Emeline
• Co-promoter: Verhulst Anja
• Co-promoter: Verstraeten Aline

Research team(s)

• Physiopharmacology (PHYSPHAR)

Project type(s)

• Research Project

Abstract

In this application, we request financing for three benchtop CERO 3D Cell Culture Bioreactor units for the culture of 3D cell cultures, including spheroids and organoids, that are increasingly being used in biomedical research. Currently, 3D organoids and spheroids are cultured in traditional cell culture plates under static or shaking (using orbital shaker) conditions in a standard CO2 cell culture incubator, which is suboptimal for long-term and large-scale culture of spheroids and organoids. A bioreactor system would take organoid and spheroid culture at the campus to a next level in terms of quality (improved viability, maturation and homogeneity) as well as quantity. Each CERO 3D Cell culture bioreactor unit can maintain four 50 mL organoid cultures, including monitoring and control of temperature, pH and carbon dioxide levels. In total, the envisaged bioreactor infrastructure will be able to accommodate twelve simultaneous organoid cultures under highly controlled conditions. The envisaged CERO 3D Bioreactor units will be applied for multiple research domains at the University of Antwerp, and more specifically for upscaled culture of stem cell-derived spheroids and organoids, tumoroids derived from primary tumour material of patients, stem cell-derived cardiomyocytes, stem cell-derived cartilage tissue and intestinal organoids. Furthermore, based on our own experience in upscaled organoid culture, the instalment of bioreactor units has become an urgent need to progress towards future valorisation activities.

Researcher(s)

• Promoter: Ponsaerts Peter
• Co-promoter: Alaerts Maaike
• Co-promoter: Lardon Filip
• Co-promoter: Verstraeten Aline

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Progressive dilatation of the aorta leads to the development of thoracic aortic aneurysms (TAAs), frequently resulting in aortic dissection or rupture. The latter events associate with an ultimate mortality rate of 50% and, hence, represent a prominent cause of morbidity and mortality in the Western population. Prophylactic surgery of TAA patients reduces the mortality rate down to about 5%, but comes with a relatively high risk of complications. Medical therapies capable of stopping or even reversing aneurysm formation are clearly highly needed, but are not available yet. Further deciphering of the mechanisms underlying TAA is essential to develop more efficient drugs. Owing to the advent of -omics technologies, it is now possible to dig into pan-TAA pathomechanisms in a hypothesis-free manner, opening new avenues to discover yet unexplored disease pathways and, hence, novel therapeutic targets. With this project, we want to be the first to take up the challenge of discovering convergent disease-linked pathways for TAA in a hypothesis-free manner using mouse and iPSC-derived cell models. The anticipated findings will advance the pathomechanistic TAA knowledge significantly beyond the current understanding and will greatly facilitate the development of novel therapeutic strategies.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Verstraeten Aline

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Thoracic aortic aneurysm (TAA) is an abnormal widening of the thoracic aorta caused by blood vessel wall weakness. TAAs entail a high risk for aortic rupture or dissection, commonly leading to sudden death. This dramatic event may leave family members of the deceased terrified and oblivious. To date, genetic defects in >30 genes have been linked with TAA, providing a molecular cause for about 30% of patients. Their identification and functional characterization have been key in acquiring our current pathomechanistic aortopathy knowledge. Yet, the genetic and mechanistic picture for TAA is far from complete, hampering identification of predictive markers for aneurysm formation and development of therapies capable of stopping or reversing aneurysm formation. In search for novel TAA genes, we most recently identified recessive truncating mutations in IPO8 as a novel cause of syndromic TAA. This project builds on this exciting finding. More specifically, we aim to significantly improve our current TAA pathomechanistic insight and future TAA patient management by (1) aortic phenotyping and functional characterization of an Ipo8 null mouse line, (2) validation of the mouse findings in the human context using patient- and control-derived iPSC-VSMCs, and (3) identifying putative drug compounds for IPO8-related aortopathy using a cell-based matrix metalloproteinase inhibition assay.

Researcher(s)

• Promoter: Verstraeten Aline
• Co-promoter: Luyckx Ilse

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Loeys-Dietz syndrome (LDS) is a genetic disorder presenting with thoracic aortic aneurysm (TAA), causing abnormal widening of the aorta, which leads to aortic rupture or dissection, a life-threatening complication that occurs unexpectedly. LDS is caused by genetic defects in six different genes of the TGF? pathway (TGFBR1/2, SMAD2/3, TGFB2/3), which is vital in the proper development of the body's connective tissue. Despite the progress in unraveling its genetic basis, there is a lack of understanding of the wide range of severity of cardiovascular involvement. In my project, I will focus on patients within families, carrying pathogenic SMAD3 variants, which show either no or early-onset aortic aneurysmal disease. I hypothesize that genetic modifiers of the primary SMAD3 mutation are the main contributors to the striking aortopathy variability in LDS-SMAD3 families. In this project, an innovative strategy will be used to identify genetic modifiers. I will perform genome-wide single nucleotide polymorphism-based linkage analysis on two large SMAD3 families and whole-genome sequencing on selected individuals, combined with SMAD3 iPSC-VSMC (induced pluripotent stem cell-derived vascular smooth muscle cells) model creation and characterization and subsequent CRISPR/Cas9-based validation of the identified modifier(s). The predicted outcomes will advance the LDS and TAA knowledge, contributing to the discovery and development of novel therapeutic targets and personalized medicine.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Alaerts Maaike
• Co-promoter: Verstraeten Aline
• Fellow: Velchev Joe Davis

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Cardiovascular diseases are worldwide the leading cause of mortality (31% of all deaths, WHO) and disability. These diseases include heart failure, coronary artery disease, heart disease, hypertension, cerebrovascular and peripheral vascular diseases. Dysfunction of endothelial cells (ECs) lining the inner wall of the vasculature is a major initiator that fuels the progression of cardiovascular disease. Mutations in genes encoding different components of the bone morphogenetic protein (BMP) pathway cause various severe vascular diseases such as hereditary hemorrhagic telangiectasia, bicuspid aortic valve with thoracic aortic aneurysms and pulmonary arterial hypertension. BMPs are secreted factors that belong to the larger transforming growth factor (TGF)β family. Signaling by BMPs contributes to the morphological, functional and molecular differences ('heterogeneity') among ECs in different vessel types like arteries, veins, lymphatic vessels and in different organs. Understanding how BMP signaling co-regulates EC heterogeneity in homeostasis and how its deregulation can contribute to disease is key to obtain insights in the genesis of vessel-type restricted disorders and design improved disease-tailored therapies with reduced side effects.

Researcher(s)

• Promoter: Verstraeten Aline

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with pleiotropic ocular, skeletal and cardiovascular manifestations. Morbidity and mortality are mostly determined by aortic root aneurysm, dissection and rupture. Although mutations in FBN1, coding for fibrillin-1, are the sole genetic MFS cause, there is a poor correlation between the MFS phenotype and the nature or location of the FBN1 variant. Wide intra- and interfamilial phenotypic variability, ranging from completely asymptomatic to sudden death at young age, is observed. The precise mechanisms underlying this variability remain elusive. In this project, I have selected an innovative strategy to fully understand the functional effects of the FBN1 mutation and discover genetic modifiers of MFS aortopathy with the following objectives: (1) CRISPR/Cas9 correction of the recurrent FBN1 p.Ile2585Thr in patient-derived iPSC-VSMCs and functional comparison to FBN1 mutation and control iPSC-VSMCs. (2) Whole genome sequencing, and RNA-seq of patient iPSCVSMCs at the extreme ends of the phenotypical spectrum for genetic modifier identification. (3) CRISPR-modification for validation of their modifying capacities. The understanding of the functional effects of the FBN1 mutation and the identification of genetic modifiers will advance the knowledge on aortopathy-mechanisms beyond current understanding, it will allow to individualize treatment protocols and will offer new leads to novel therapeutic targets.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Verstraeten Aline
• Fellow: Van Den Heuvel Lotte

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Connective tissue disease (CTD) refers to a large and diverse group of disorders affecting the protein-rich tissues that support our body's organ systems. Patients most typically present with skin, spinal cord, eye, heart, blood vessel and/or skeletal manifestations. CTDs can either be inherited or provoked by environmental factors. With respect to hereditary CTD, an increasing number of genetic causes has been discovered over the past ten years owing to the advent of high-throughput DNA sequencing technologies. In-depth investigation of these defects' molecular mode of action at the cellular and tissue level is now increasingly needed in order to complete the mechanistic CTD puzzles and to facilitate the development of novel drug therapies. I will establish a research group that aims to address these CTD needs, with a primary focus on thoracic aortic aneurysm and dissection (TAAD) and skeletal dysplasia. TAAD denotes an abnormal widening and/or rupture of the largest human artery, i.e. the aorta, and entails a high risk for sudden death due to severe internal bleeding. It is estimated to account for 1-2% of all deaths in the Western population. Skeletal dysplasias are a group of more than 200 disorders that affect bone and cartilage growth, resulting in abnormal skeleton shape and size. At first glance, these two conditions might seem oddly dissimilar. From a molecular point of view they have quite a lot in common though. Different defects in a set of genes have been shown to cause both TAAD and skeletal dysplasia. Moreover, significant overlap exists with regard to the yet described dysregulated subcellular processes. By comprehensively studying the entire disease continuum, I will contribute synergistically to a better understanding of the disease mechanisms and, hence, the treatment of both separate clinical entities. Three major strategic research pillars have been defined on which I desire to concentrate: (1) identification of the DNA variants that explain why some subjects carrying a certain disease-causing genetic defect are more severely affected than others with that identical defect (i.e. modifier variants); (2) elucidation of the molecular mode of action of disease-causing and disease-modifying genetic variants; and (3) discovery of novel disease-remedying drug compounds as well as the genetic determinants that explain variation in drug response between patients. The experimental set-up will be determined in a project-by-project manner, but will typically involve high-throughput DNA, RNA and/or protein analyses (-omics) as well as classical molecular biology strategies in relevant mouse and induced pluripotent stem cell (iPSC)-derived models. IPSCs are somatic cells (e.g. from skin or blood) that have been reprogrammed to pluripotent cells, and can be differentiated into virtually any cell type of interest. Patient- and control-derived iPSC-vascular smooth muscle cells and iPSC-chondrocytes will be used (i.e. relevant TAAD and skeletal dysplasia cell types, respectively) because of limited access to their native counterparts.

Researcher(s)

• Promoter: Verstraeten Aline
• Fellow: Verstraeten Aline

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

A progressive widening of the aorta (called aortic aneurysm) affecting our body's main blood vessel in the chest (thorax) can lead to a catastrophic event with a tear or rupture of the aortic wall (dissection or rupture). Unfortunately, a thoracic aortic aneurysm most often remains asymptomatic until catastrophic dissections or ruptures happen. These latter complications are a major cause of sudden cardiac death in the western world. Currently, aortic aneurysms are mostly detected incidentally upon imaging studies for other medical indications. Although not perfect, follow-up of the diameter of the aorta by imaging studies is considered the best predictor of the aortic dissection risk. At present, there are no markers in the blood (called biomarkers) that can predict the presence of a thoracic aortic aneurysm or the occurrence of a thoracic aortic dissection. Identification of such a biomarker would be of great help in the faster and easier diagnosis and follow-up of thoracic aortic aneurysm and dissection. Upon investigation of aneurysmal aortic gene expression profiles of three different symptomatic mouse models of Marfan and Loeys-Dietz syndrome, we observed high expression of a novel gene in all three models. This gene encodes for a growth factor that has not previously been associated with the pathogenesis of thoracic aortic aneurysm. Although the molecule has been linked to metabolism and cancer, its highest expression is in the aortic wall. In this study we would like to investigate if serum levels of this growth factor in Marfan and Loeys-Dietz syndrome mice correspond to their thoracic aortic aneurysm severity and progression. We will validate the findings in serum samples of patients with thoracic aortic aneurysm and dissection. If successful, the latter model can be used in future projects to test therapeutic compounds with the "simple" measurement of the levels of this growth factor as the outcome parameter. In summary, if successful our project will provide the strong foundations of future research that will further explore whether serum levels of this growth factor can detect asymptomatic thoracic aortic aneurysm, monitor disease progression and predict aortic dissection.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Verstraeten Aline

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Infection with SARS-CoV-2 mostly leads to a mild self-limiting respiratory tract illness, however, some patients develop severe progressive pneumonia, multiorgan failure, and death. This project aims to determine factors that dictate the course of COVID19 beyond cytokines. We have prior data that specific aberrantly expressed mucins, triggered by SARS-CoV-2, regulate ACE2 expression and affect lung barrier integrity. Such mucin alterations are clinically relevant as excessive mucin production is seen in severe COVID-19 illness obstructing the respiratory tract and complicating recovery. Here, we will first identify differentially expressed mucin isoforms in COVID-19 patients exhibiting the entire spectrum of disease severity. Thereafter, therapeutics currently used for COVID-19 will be screened for their ability to reduce mucin abundance. As mucin expression is also a critical factor in microbiome homeostasis and dysbiosis might modulate COVID-19 severity, this project secondly aims to map the microbiome associated with different degrees of disease severity. Unravelling mucin isoform-microbiome interactions that shape the course of SARS-CoV-2 infection will lead to the future identification of those patients who are in danger of progressing to severe disease. This project will also improve the choice for an appropriate treatment as well as the time frame of treatment options once infection occurs.

Researcher(s)

• Promoter: De Winter Benedicte
• Co-promoter: Malhotra Surbhi
• Co-promoter: Smet Annemieke
• Co-promoter: Verstraeten Aline

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Infection with SARS-CoV-2 mostly leads to a mild self-limiting respiratory tract illness, however, some patients progress to develop severe progressive pneumonia, multiorgan failure, and death. The project aims to determine factors that dictate the severity of COVID-19. Firstly, guided by our prior data of interaction of certain mucins with the ACE2 receptor and the clinical evidence of excessive mucin production in severe COVID-19 illness, we intend to characterize different mucins for their role in both the initiation and progression of COVID-19. Secondly, based on a severe degree of edematous interstitial lung tissue pathology observed in COVID-19 autopsies and its hypothesized link to abnormally low PaO2 observed clinically, the project intends to characterize aquaporin (AQP) water channels that are responsible for fluid transport across cells. This has important therapeutic relevance for COVID-19 as specific AQP inhibitors have been shown to attenuate inflammation and lung injury and to block mucin hypersecretion. Lastly, mucin expression is also a critical factor in microbiome homeostasis and based on, so far, scarce data that co-infection with other respiratory pathogens and other microbial interactions might modulate COVID-19 severity, the project aims to characterize the microbiome associated with different degrees of disease severity. Identifying factors that shape the course of SARS-CoV-2 infection will lead to identification of plausible targets to treat COVID-19.

Researcher(s)

• Promoter: De Winter Benedicte
• Co-promoter: Jorens Philippe
• Co-promoter: Kumar-Singh Samir
• Co-promoter: Malhotra Surbhi
• Co-promoter: Smet Annemieke
• Co-promoter: Verstraeten Aline

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Loeys-Dietz syndrome (LDS) is a genetic disorder presenting with thoracic aortic aneurysm (TAA), causing abnormal widening of the aorta, which leads to aortic rupture or dissection, a life-threatening complication that occurs unexpectedly. LDS is caused by genetic defects in six different genes of the TGF? pathway (TGFBR1/2, SMAD2/3, TGFB2/3), which is vital in the proper development of the body's connective tissue. Despite the progress in unravelling its genetic basis, there is a lack of understanding of the wide range of severity of cardiovascular involvement. In my project, I will focus on patients within families, carrying pathogenic SMAD3 variants, which show either no or early onset aortic aneurysmal disease. I hypothesize that genetic modifiers of the primary SMAD3 mutation are the main contributors to the striking aortopathy variability in LDS-SMAD3 families. In this project, an innovative strategy will be used to identify genetic modifiers. I will perform genome-wide single nucleotide polymorphism-based linkage analysis on two large SMAD3 families and whole genome sequencing on selected individuals, combined with SMAD3 iPSC-VSMC (induced pluripotent stem cell-derived vascular smooth muscle cells) model creation and characterization and subsequent CRISPR/Cas9-based validation of the identified modifier(s). The predicted outcomes will advance the LDS and TAA knowledge, contributing to the discovery and development of novel therapeutic targets and personalized medicine.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Verstraeten Aline
• Fellow: Velchev Joe Davis

Research team(s)

Project type(s)

• Research Project

Abstract

Heterozygous missense mutations in the collagen type II-encoding gene COL2A1 explain about 95% and 70% of the hypochondrogenesis and spondyloepiphyseal dysplasia congenita patients, respectively, as well as a smaller fraction of patients with closely related phenotypes. Prior functional characterization of iPSC-derived and transdifferentiated chondrocytes of carriers of COL2A1 missense mutations revealed increased expression of endoplasmatic reticulum (ER) stress and apoptosis markers in addition to reduced levels of cartilage matrix proteins. Abnormal procollagen folding is considered a key pathogenic skeletal dysplasia mechanism, rendering chaperone-oriented therapy an interesting pharmacological avenue. Subjecting iPSC-chondrocytes of a COL2A1 glycine substitution carrier to a drug library comprising roughly 2,400 chaperone agonists and antagonists, we aim to identify a highly potent novel drug for skeletal dysplasias attributed to COL2A1 missense mutations. To evaluate the compounds' efficiency in restoring the cellular phenotype, integrated high content quantification of collagen type II as well as apoptosis and ER stress markers will be done. The most interesting compounds will be tested in knock-in COL2A1 mice to establish in vivo performance.

Researcher(s)

• Promoter: Verstraeten Aline
• Fellow: De Kinderen Pauline

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Thoracic aortic aneurysm (TAA) entails a high risk for aortic dissection and rupture. The latter events are a prominent cause of morbidity and mortality in the Western population. Over the past 25 years, extensive gene discovery efforts have identified over 30 genes in which variants impinge on TAA risk. Although collectively explaining less than 30% of all familial patients, their identification and functional characterization have been key in acquiring the vast majority of our current knowledge on aortopathy pathomechanisms. Yet, the genetic and mechanistic picture for TAA is far from complete, hampering further identification of predictive markers for aneurysm development and progression as well as novel therapies capable of stopping or even reversing the disease process. In search for novel entry points in the etiology of TAA, I most recently identified recessive truncating mutations in IPO8 as a novel cause of a TAA syndrome clinically resembling TGF-β-related syndromes such as Marfan syndrome, Loeys-Dietz syndrome and Shprintzen-Goldberg syndrome. More precisely, patients present with childhood-onset aneurysms at the level of the aortic root and/or aorta ascendens, global developmental delay, facial dysmorphism, joint laxity, neonatal hypotonia, pectus deformity and hernia. Not much is known about IPO8, except that it encodes a protein involved in cytosol-to-nucleus cargo shuttling (including pSMAD2, pSMAD3 and SMAD4) as well as miRNA processing. My project proposal builds further on this novel genetic finding. More specifically, I aim (1) to unravel the IPO8-related pathomechanisms using patient- and control-derived skin fibroblasts and induced pluripotent stem cell-derived vascular smooth muscle cells (iPSC-VSMCs), and (2) to discover FDA-approved drug compounds for aortopathy using a matrix metalloproteinase inhibition assay in iPSC-VSMCs of an IPO8 mutation-positive patient. The project's anticipated outcomes will advance TAA knowledge significantly beyond the current understanding and improve patient management.

Researcher(s)

• Promoter: Verstraeten Aline
• Co-promoter: Loeys Bart

Research team(s)

Project type(s)

• Research Project

Abstract

Thoracic aortic aneurysm (TAA) is an abnormal widening of the thoracic aorta caused by blood vessel wall weakness. TAAs entail a high risk for aortic rupture or dissection, commonly leading to sudden death. This dramatic event may leave family members of the deceased terrified and oblivious. To date, genetic defects in >30 genes have been linked with TAA, providing a molecular cause for about 30% of patients. Their identification and functional characterization have been key in acquiring our current aortopathy knowledge. Yet, the genetic and mechanistic picture for TAA is far from complete, hampering identification of predictive markers for aneurysm formation and development of therapies capable of stopping or reversing aneurysm formation. In search for novel TAA genes, the Antwerp Cardiogenetics research group most recently identified recessive truncating mutations in IPO8 as a novel cause of syndromic TAA. My PhD project builds on this exciting finding. More specifically, I aim to significantly improve our current TAA pathomechanistic insight and future TAA patient management by (1) investigating IPO8 as a novel syndromic TAA gene through characterization of an Ipo8 null mouse line, (2) elucidating the IPO8-related pathomechanisms using patient- and control-derived cell lines, and (3) identifying putative drug compounds for IPO8-related aortopathy using a cell-based matrix metalloproteinase inhibition assay.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Verstraeten Aline
• Fellow: Van Gucht Ilse

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Bicuspid aortic valve (BAV) is characterized by an aortic valve with only two valve leaflets instead of the normal three. With an estimated prevalence of about 1-2% in the general population, BAV represents the most common human congenital heart malformation. Although most BAV individuals remain asymptomatic, up to 30% of patients develop severe cardiovascular complications throughout their life, including thoracic aortic aneurysms (TAA) and dissections. The latter manifestations associate with significant morbidity and mortality. In view of the prevalent nature of BAV and the current lack of efficient pharmacological therapies for (BAV-related) TAA, BAV/TAA puts a substantial burden on our health care system. In search of new genetic entry points in the etiology of BAV/TAA, we most recently observed an enrichment of damaging SMAD6 variants in BAV/TAA patients compared to the general population. SMAD6 is highly expressed in the cardiovascular system and encodes an inhibitory SMAD protein that negatively regulates BMP and TGF-β signalling, which both have been linked to aortic valve development and aneurysm formation before. This project proposal builds on our genetic SMAD6 data and aims at advancing the knowledge on the pathomechanisms underlying BAV/TAA significantly beyond the current understanding by extensively investigating the key molecular elements and processes that relate SMAD6 deficiency to aortic valve and wall abnormalities. For this purpose, we will use aortic valve and wall tissue dissected from the Madh6-/- mouse model. The project's outcomes are expected to instigate the discovery of novel therapeutic targets for BAV-related TAA and prompt drug compound testing in Madh6-/- mice.

Researcher(s)

• Promoter: Verstraeten Aline

Research team(s)

Project type(s)

• Research Project

Abstract

Thoracic aortic aneurysms (TAAs) result from progressive dilatation of the aorta and entail a high risk for aortic dissection and rupture. The latter events associate with a mortality rate of 50%, representing a prominent cause of morbidity and sudden death in the Western population. Over the past 25 years, extensive gene identification efforts have pinpointed more than 25 genes associated with familial TAA risk, explaining about 30% of all familial TAA cases. Functional characterization of these genes has revealed perturbed extracellular matrix homeostasis, transforming growth factor‑β signaling, and vascular smooth muscle cell contractility as important TAA processes. To expedite the development of novel therapeutic strategies, acquisition of even more extensive insights into the genetic and mechanistic TAA picture is mandatory. Owing to the recent advent and fast evolution of next-generation sequencing technologies, we anticipate that the identification of additional genetic TAA causes will remain quite straightforward in the upcoming years. Given that TAA is characterized by greatly reduced penetrance and variable expressivity, modifier studies now represent a challenging, yet important, new avenue in the field of TAA genetics. In this project, we pursue the genetic modifiers that determine phenotypical variability in selected families with an autosomal dominantly inherited syndromic TAA form, namely Loeys-Dietz syndrome. State-of-the-art technologies, such as genome sequencing and creation of induced pluripotent stem cells, will be used. The anticipated outcomes will advance TAA knowledge significantly beyond the current understanding, aid genetic counseling, and offer unprecedented opportunities to find leads to novel therapeutic strategies.

Researcher(s)

• Promoter: Van Laer Lut
• Co-promoter: Verstraeten Aline
• Fellow: Perik Melanie

Research team(s)

Project type(s)

• Research Project

Abstract

Expansion of a weakened region of the thoracic aorta (aneurysm; TAA) entails a high risk for aortic dissection/rupture. The latter events associate with severe internal bleedings, often resulting in sudden death. Over time, defects in more than 20 genes have been found to influence TAA predisposition. Yet, the disease's genetic and mechanistic picture is still far from complete, hampering development of amended diagnostic tools and therapies. We will further resolve the TAA puzzle by identifying novel protective and risk-inferring variants/genes and by examining their mode of action. Multiple lines of evidence suggest that some to be identified TAA genes locate to the X-chromosome. Recently, we indeed discovered TAA-causing defects in an X-linked gene, biglycan (BGN). Interestingly, in certain mouse strains protection from BGN-related TAA has been documented. We aim at mapping the protective factor and at translating its protective effect to men. A second genetic approach builds on the observation that in Turner syndrome (TS) girls lacking either the short X-arm (Xp) or the entire X-chromosome, TAA is strikingly frequent. The known X-linked TAA genes, however, locate to the long X-arm. Hence, we also aim at identifying novel Xp-located TAA genes in TS girls. Finally, to further delineate existing disease pathways or to discover novel ones, we will functionally characterize the identified protective and risk-inferring defects in patient samples and transgenic model systems.

Researcher(s)

• Promoter: Loeys Bart
• Co-promoter: Van Laer Lut
• Co-promoter: Verstraeten Aline

Research team(s)

Project type(s)

• Research Project

Abstract

The aorta serves as the responsible artery for blood distribution from the heart towards the distal parts of the human body. Expansion of weakened regions of the thoracic aorta (aneuysms; TAA) entails a high risk for aortic dissection/rupture. The latter events associate with severe internal bleedings, often resulting in sudden death. As relatives of TAA patients are at high risk, genetic defects are certainly involved in disease development. Multiple lines of evidence suggest that at least one TAA-causing gene locates to a sex (i.e. the X-) chromosome: firstly, TAA is much more frequent in males and secondly, Turner syndrome (TS) girls, in whom one X-chromosome is partially or completely deleted, strikingly often present with TAA. Most recently, our research group identified genetic defects in the biglycan (BGN) gene, which is located on the long arm of the X-chromosome (Xq), causing syndromic TAA. This project partially builds on this finding. We aim at (1) elucidating the role of BGN promoter defects in non-syndromic TAA, (2) determining the downstream consequences of BGN loss and (3) identifying a genetic factor influencing manifestation of BGN-related TAA in mice. As TS patients missing the short X-arm (Xp) are more frequently affected with TAA, it is likely that an Xp-located TAA gene remains to be identified. Therefore, goal (4) encompasses identification of an Xp aortopathy gene using X-chromosome sequencing in TS patients.

Researcher(s)

• Promoter: Loeys Bart
• Fellow: Verstraeten Aline

Research team(s)

Project type(s)

• Research Project

Abstract

Bicuspid aortic valve (BAV) is the most common congenital heart disorder. Although BAV is intrinsically asymptomatic, it associates with thoracic aortic aneurysms (TAA) and ruptures that are highly mortal. BAV and TAA are strikingly frequent in Turner syndrome (TS), which affects approximately 1 in 2,500 live-born females and is caused by either partial or complete absence of one X-chromosome. One possible mechanistic hypothesis (a 2-hit hypothesis) states that increased BAV/TAA prevalence in TS is caused by mutations in a "cardiovascular" gene on the residual X-chromosome. To identify such X-linked aortopathy genes, all protein coding sequences of the X-chromosome will be sequenced in 22 TS patients with BAV as well as in 10 tricuspid TS patients. Supporting genetic evidence for the identified aortopathy candidate genes will be acquired by sequence analysis of their coding regions in additional TS/BAV samples as well as in a small non-syndromic male BAV/TAA discovery cohort. The expected experimental findings will prove beneficial for molecular diagnostic applications, genetic counselling or clinical follow-up of BAV/TAA families and, through acquisition of novel pathomechanical insights, development of preventive and more personalized therapies.

Researcher(s)

• Promoter: Verstraeten Aline

Research team(s)

Project type(s)

• Research Project

Abstract

Lewy body disorders (LBD) represent a heterogenic group of neurodegenerative brain diseases (NBD) characterized by the presence of intraneuronal α-synuclein containing inclusions, called Lewy bodies. The Lewy body variant of Alzheimer's disease marks one end of the spectrum, Parkinson's disease (PD) the other and Lewy body dementia and Parkinson with dementia are both located in-between. PD is the second most common NBD, affecting ~2 % of the population older than 65 years and causing major disability and reduction of the quality of life. Genetic linkage studies in rare families with autosomal dominant or recessive inheritance of PD identified at least five genes in which mutations lead to PD. The study of the corresponding gene products was the primary source of the current knowledge on PD pathogenesis. However, the contribution of these genes to the genetic etiology of PD in the Belgian population is relatively limited. The main aim of this PhD project is to contribute to the identification of novel causal PD genes using whole genome sequencing in Belgian families segregating PD. Furthermore, we aim at characterizing the pathogenic effect of the identified disease associated genetic variants using 'state of the art' functional genomics/genetics approaches. The expected results are thought to further elucidate the pathomechanisms underlying PD and related LBD and will pave the way for the development of more accurate diagnostic tools and effective drugs/therapies.

Researcher(s)

• Promoter: Van Broeckhoven Christine
• Co-promoter: Theuns Jessie
• Fellow: Verstraeten Aline

Research team(s)

Project type(s)

• Research Project