Publications

Two Faces Of Adult Blood Vessel Formation: Vasculogenesis And Angiogenesis

During development our blood vascular system successively goes through a vasculogenic and angiogenic growth phase during which a primitive and mature vascular network, respectively, is laid down to provide all tissues with oxygen and nutrients. Rather than being a network of rigid tubes, the established blood vasculature turns out to be a very dynamic system that also after birth can be reshaped to meet the perfusional needs of our organs or to respond to pathological triggers such as cancer. Initially, it was thought that this reshaping only occurred through angiogenic mechanisms1Associate Professor, Molecular and Vascular Biology Research Unit, Endothelial Cell Biology Unit, Department of Cardiovascular Sciences, Katholieke Universiteit Leuven, Campus Gasthuisberg, Onderwijs & Navorsing 1, Herestraat 49, B-3000 Leuven, Belgium. Email: aernout.luttun@med.kuleuven.be 2Professor, Vesalius Research Center, Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, VIB – Katholieke Universiteit Leuven, Campus Gasthuisberg, Onderwijs & Navorsing 4, Herestraat 49, B-3000 Leuven, Belgium. Email: peter.carmeliet@vib-kuleuven.be *Corresponding authorList of abbreviations after the text.

Synergizing bioprinting and 3D cell culture to enhance tissue formation in printed synthetic constructs

Abstract Bioprinting is currently the most promising method to biofabricate complex tissues in vitro with the potential to transform the future of organ transplantation and drug discovery. Efforts to create such tissues are, however, almost exclusively based on animal-derived materials, such as gelatin methacryloyl, which have demonstrated efficacy in bioprinting of complex tissues. While these materials are already used in clinical applications, uncertainty about their safety still remains due to their animal origin. Alternatively, synthetic bioinks have been developed that match the printability of natural bioinks but lack their biological complexity, and thereby often fail to support cell growth and facilitate tissue formation. Additionally, most synthetic materials do not meet the mechanical demands of bioprint stable constructs while providing a suitable environment for cells to grow, limiting the number of available bioinks. To bridge this gap and synergize bioprinting and 3D cell culture, we developed a polyethylene glycol-based bioink system to promote the growth and spreading of cell spheroids that consist of human primary endothelial cells and fibroblasts. The 3D bioprinted centimeter-scale constructs have a high shape fidelity and accelerated softening to provide sufficient space for cells to grow. Adjusting the rate of degradability, induced by the integration of ester-functionalized crosslinkers in addition to protease cleavable crosslinkers into the hydrogel network, improves the growth of spheroids in larger printed hydrogel constructs containing an interconnected channel structure. The perfusable constructs enable extensive spheroid sprouting and the formation of a cellular network upon fusion of sprouts as initial steps toward tissue formation with the potential for clinical translation.

THU-03 Angiogenesis inhibition in cancer

PHD1 and PHD3 are expressed in bone cells but are not essential for bone homeostasis

HIF–1 induced VEGF augments, but is not essential for normal retinal vascular development

Placental growth factor plays a role in the mesenteric neo-angiogenesis of portal hypertensive mice

Molecular Mechanisms of Angiogenesis

Blood vessels provide essential nutrients to almost every organ system. It followsthat abnormal blood vessel growth is implicated in many clinically relevantdisease processes. Excess vessel growth is a feature of cancer, arthritis, psoriasis,diabetic retinopathy, infections, obesity, allergy, atherosclerosis, nasal polyps,and endometriosis, to name but a few. Inadequate vessel growth or excess vesselregression is most frequently linked to tissue ischemia, such as occurs during theevolution of a myocardial infarct, stroke, or limb ischemia. However, abnormalvessel regression also contributes to many other disorders including, for example,pre-eclampsia, age-dependent skin changes, nephropathies, bone loss, diabetes,macular degeneration, and such neurodegenerative disorders as amyotrophiclateral sclerosis.

Tissue-Type Plasminogen Activator Deficiency Exacerbates Arthritis

Abstract Fibrin deposition, cell migration, and tissue remodeling are key components in the lesions of inflammatory joint diseases, such as rheumatoid arthritis. The plasminogen activators (PAs), namely, tissue-type PA (t-PA) and urokinase PA, are implicated in these aspects of an inflammatory response, although their precise roles are yet to be defined. We therefore used gene-deficient mice to explore their role in a two-stage arthritis model involving intraarticular methylated BSA injection, followed by systemic IL-1 treatment. We report in this study that both t-PA and urokinase PA are protective for the mild arthritis induced by intraarticular methylated BSA injection alone, since absence of either of them exacerbates the response; following s.c. IL-1 injection, t-PA−/− mice had particularly severe disease. Fibrin deposition appeared to parallel disease severity under the various conditions, suggesting that PA-mediated fibrinolysis may be normally playing a protective role in inflammatory joint disease.

Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility

Protein C inhibitor (PCI) is a nonspecific, heparin-binding serpin (serine protease inhibitor) that inactivates many plasmatic and extravascular serine proteases by forming stable 1:1 complexes. Proteases inhibited by PCI include the anticoagulant activated protein C, the plasminogen activator urokinase, and the sperm protease acrosin. In humans PCI circulates as a plasma protein but is also present at high concentrations in organs of the male reproductive tract. The biological role of PCI has not been defined so far. However, the colocalization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. We generated mice lacking PCI by homologous recombination. Here we show that PCI(-/-) mice are apparently healthy but that males of this genotype are infertile. Infertility was apparently caused by abnormal spermatogenesis due to destruction of the Sertoli cell barrier, perhaps due to unopposed proteolytic activity. The resulting sperm are malformed and are morphologically similar to abnormal sperm seen in some cases of human male infertility. This animal model might therefore be useful for analyzing the molecular bases of these human conditions.

PlGF: A Multitasking Cytokine with Disease-Restricted Activity

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family that also comprises VEGF-A (VEGF), VEGF-B, VEGF-C, and VEGF-D. Unlike VEGF, PlGF is dispensable for development and health but has diverse nonredundant roles in tissue ischemia, malignancy, inflammation, and multiple other diseases. Genetic and pharmacological gain-of-function and loss-of-function studies have identified molecular mechanisms of this multitasking cytokine and characterized the therapeutic potential of delivering or blocking PlGF for various disorders.

Abstracts: Basic Science in arrhythmias: mechanisms

DNA Methylation Repels Binding of HIF Transcription Factors to Maintain Tumour Immunotolerance

A bstract Background Hypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia. Results Here, we report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modelling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid-down by the differential expression and binding of other transcription factors under normoxia control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumours with high immune checkpoint expression, but not in tumours with low immune checkpoint expression, where they would compromise tumour immunotolerance. In a low-immunogenic tumour model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumour growth. Conclusions Our data elucidate the mechanism underlying cell-type specific responses to hypoxia, and suggest DNA methylation and hypoxia to underlie tumour immunotolerance.

Improved metabolite identification with MIDAS and MAGMa through MS/MS spectral dataset-driven parameter optimization

VEGF-B Improves Metabolic Health through Vascular Pruning of Fat

Activation of the UNC5B receptor by Netrin-1 inhibits neovascularization

Heterozygous deficiency of the oxygen sensor PHD2 prevents metastasis by inducing vessel normalization

Abstract 202: Thrombospondin-2 Protects Against Uncontrolled Cardiac Inflammation and Oxidative Stress During Doxorubicin-Induced Myocarditis

Background Doxorubicin is a live-saving chemotherapeutic agent in young patients affected by cancer. However, its use is limited by its cardiotoxicity caused by increased oxidative stress and exagg...