Title Participants Abstract "Simply the B(r)east: endothelial cell heterogeneity in health and disease. Single cell analysis of transcriptomes in endothelial cells." "Vincent Geldhof" "Traditional anti-angiogenesis strategies attempt to prune tumor vessels. However, their success is restricted by toxicity and resistance. There is thus an unmet need for new anti-angiogenic strategies with fundamentally distinct mechanisms. An alternative therapeutic paradigm is tumor vessel normalization (TVN). By healing disorganized vessels, TVN improves perfusion and oxygenation, tightens the leaky vascular wall, thus reducing metastasis and enhancing chemotherapy delivery and responses. The host laboratory showed that haplodeficiency of the glycolytic activator PFKFB3 in endothelial cells or treatment with the PFKFB3 inhibitor 3PO induces TVN, reduces metastasis and improves the delivery of and response to cisplatin. Here, I propose to broaden this concept to anti-cancer immunotherapy. Given the emerging importance of immunotherapy, I will investigate whether TVN induced by targeting EC glycolysis also improves intratumoral delivery and activation of immune cells, and therefore has an increased efficacy. Moreover, I will evaluate whether TVN affects the metabolism of cancer cells and whether targeting these metabolic drivers enhances the vulnerability of cancer cells to immunotherapy. This SB PhD proposal promises to yield entirely novel insights in the therapeutic potential of new metabolic targets which may improve immune checkpoint blockade. My PhD application will be crucial for my future as physician-scientist and to develop as an independent scientist." "Primary Human Derived Blood Outgrowth Endothelial Cells: An Appropriate In Vitro Model to Study Shiga Toxin Mediated Damage of Endothelial Cells" "Bert van den Heuvel" "Hemolytic uremic syndrome (HUS) is a rare disease primarily characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Endothelial damage is the hallmark of the pathogenesis of HUS with an infection with the Shiga toxin (Stx) producing Escherichia coli (STEC-HUS) as the main underlying cause in childhood. In this study, blood outgrowth endothelial cells (BOECs) were isolated from healthy donors serving as controls and patients recovered from STEC-HUS. We hypothesized that Stx is more cytotoxic for STEC-HUS BOECs compared to healthy donor control BOECs explained via a higher amount of Stx bound to the cell surface. Binding of Shiga toxin-2a (Stx2a) was investigated and the effect on cytotoxicity, protein synthesis, wound healing, and cell proliferation was studied in static conditions. Results show that BOECs are highly susceptible for Stx2a. Stx2a is able to bind to the cell surface of BOECs with cytotoxicity in a dose-dependent manner as a result. Pre-treatment with tumor necrosis factor alpha (TNF-α) results in enhanced Stx binding with 20-30% increased lactate dehydrogenase (LDH) release. Endothelial wound healing is delayed in a Stx2a-rich environment; however, this is not caused by an effect on the proliferation rate of BOECs. No significant differences were found between control BOECs and BOECs from recovered STEC-HUS patients in terms of Stx2a binding and inhibition of protein synthesis." "Cytokines trigger disruption of endothelium barrier function and p38 MAP kinase activation in BMPR2-silenced human lung microvascular endothelial cells" "Birger Tielemans, Rik Gijsbers, Catharina Belge, Marion Delcroix, Rozenn Quarck" "The bone morphogenetic protein receptor II (BMPRII) signaling pathway is impaired in pulmonary arterial hypertension and mutations in the BMPR2 gene have been observed in both heritable and idiopathic pulmonary arterial hypertension. However, all BMPR2 mutation carriers do not develop pulmonary arterial hypertension, and inflammation could trigger the development of the disease in BMPR2 mutation carriers. Circulating levels and/or lung tissue expression of cytokines such as tumor necrosis factor-α or interleukin-18 are elevated in patients with pulmonary arterial hypertension and could be involved in the pathogenesis of pulmonary arterial hypertension. We consequently hypothesized that cytokines could trigger endothelial dysfunction in addition to impaired BMPRII signaling. Our aim was to determine whether impairment of BMPRII signaling might affect endothelium barrier function and adhesiveness to monocytes, in response to cytokines. BMPR2 was silenced in human lung microvascular endothelial cells (HLMVECs) using lentiviral vectors encoding microRNA-based hairpins. Effects of tumor necrosis factor-α and interleukin-18 on HLMVEC adhesiveness to the human monocyte cell line THP-1, adhesion molecule expression, endothelial barrier function and activation of P38MAPK were investigated in vitro. Stable BMPR2 silencing in HLMVECs resulted in impaired endothelial barrier function and constitutive activation of P38MAPK. Adhesiveness of BMPR2-silenced HLMVECs to THP-1 cells was enhanced by tumor necrosis factor-α and interleukin-18 through ICAM-1 adhesion molecule. Interestingly, tumor necrosis factor-α induced activation of P38MAPK and disrupted endothelial barrier function in BMPR2-silenced HLMVECs. Altogether, our findings showed that stable BMPR2 silencing resulted in impaired endothelial barrier function and activation of P38MAPK in HLMVECs. In BMPR2-silenced HLMVECs, cytokines enhanced adhesiveness capacities, activation of P38MAPK and impaired endothelial barrier function suggesting that cytokines could trigger the development of pulmonary arterial hypertension in a context of impaired BMPRII signaling pathway." "Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche." "Johnny Bonnardel, Wouter T'Jonck, Djoere Gaublomme, Robin Browaeys, Charlotte Scott, Kremer Anneke, Van Hamme Evelien, Pieter De Bleser, Alain Beschin, Lambrecht Bart Norbert, Saskia Lippens, Dirk Elewaut, Yvan Saeys, Martin Guilliams" "Functional Gene Analysis Reveals Cell Cycle Changes and Inflammation in Endothelial Cells Irradiated with a Single X-ray Dose" "Bjorn Baselet, Niels BELMANS, Emma Coninx, Donna Lowe, Ann Janssen, Arlette Michaux, Kevin Tabury, Kenneth Raj, Roel Quintens, Mohammed A Benotmane, Sarah Baatout, Pierre Sonveaux, An Aerts" "Background and Purpose: Epidemiological data suggests an excess risk of cardiovascular disease (CVD) at low doses (0.05 and 0.1 Gy) of ionizing radiation (IR). Furthermore, the underlying biological and molecular mechanisms of radiation-induced CVD are still unclear. Because damage to the endothelium could be critical in IR-related CVD, this study aimed to identify the effects of radiation on immortalized endothelial cells in the context of atherosclerosis. Material and Methods: Microarrays and RT-qPCR were used to compare the response of endothelial cells irradiated with a single X-ray dose (0.05, 0.1, 0.5, 2 Gy) measured after various post-irradiation (repair) times (1 day, 7 days, 14 days). To consolidate and mechanistically support the endothelial cell response to X-ray exposure identified via microarray analysis, DNA repair signaling (γH2AX/TP53BP1-foci quantification), cell cycle progression (BrdU/7AAD flow cytometric analysis), cellular senescence (β-galactosidase assay with CPRG and IGFBP7 quantification) and pro-inflammatory status (IL6 and CCL2) was assessed. Results: Microarray results indicated persistent changes in cell cycle progression and inflammation. Cells underwent G1 arrest in a dose-dependent manner after high doses (0.5 and 2 Gy), which was compensated by increased proliferation after 1 week and almost normalized after 2 weeks. However, at this point irradiated cells showed an increased β-Gal activity and IGFBP7 secretion, indicative of premature senescence. The production of pro-inflammatory cytokines IL6 and CCL2 was increased at early time points. Conclusions: IR induces pro-atherosclerotic processes in endothelial cells in a dose-dependent manner. These findings give an incentive for further research on the shape of the dose-response curve, as we show that even low doses of IR can induce premature endothelial senescence at later time points. Furthermore, our findings on the time- and dose-dependent response regarding differentially expressed genes, cell cycle progression, inflammation and senescence bring novel insights into the underlying molecular mechanisms of the endothelial response to X-ray radiation. This may in turn lead to the development of risk-reducing strategies to prevent IR-induced CVD, such as the use of cell cycle modulators and anti-inflammatory drugs as radioprotectors and/or radiation mitigators." "Endothelial cells and cancer cells: metabolic partners in crime?" "Annalisa Zecchin, Gitte Borgers, Peter Carmeliet" "Endothelial cells line the blood vessel lumen and are critical for blood flow homeostasis. Excessive and deregulated vessel overgrowth is a hallmark of pathological (tumor) angiogenesis. The purpose of this review is to describe the metabolic features of endothelial cells, in comparison with those of the cancer cells, and to discuss novel antiangiogenesis approaches based on targeting endothelial cell metabolism." "Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting" "Holger Gerhardt" "Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting." "Mechanical stimulation-induced calcium wave propagation in cell monolayers: the example of bovine corneal endothelial cells" "Catheleyne D'hondt, Bernard Himpens, Geert Bultynck" "Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca(2+)-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca(2+) that initiate the propagation of the Ca(2+)-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca(2+)-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca(2+)-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca(2+)-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca(2+)-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research." "Single cell atlas identifies lipid-processing and immunomodulatory endothelial cells in healthy and malignant breast." "Vincent Geldhof, de Rooij Laura P M, Liliana Sokol, Jacob Amersfoort, Katerina Rohlenova, Federico Taverna, Shawez Khan, Truong Anh-Co Khanh, Laure-Anne Teuwen, Lucas Treps, Stefan Vinckier, Luc Schoonjans, Joanna Kalucka, Mazzone Max, Mieke Dewerchin, Guy Eelen, Peter Carmeliet" "Cell Biological Responses after Shiga Toxin-1 Exposure to Primary Human Glomerular Microvascular Endothelial Cells from Pediatric and Adult Origin" "Bert van den Heuvel" "Hemolytic uremic syndrome (HUS) is characterized by a triad of symptoms consisting of hemolytic anemia, thrombocytopenia and acute renal failure. The most common form of HUS is caused by an infection with Shiga toxin (Stx) producing Escherichia coli bacteria (STEC-HUS), and the kidneys are the major organs affected. The development of HUS after an infection with Stx occurs most frequently in children under the age of 5 years. However, the cause for the higher incidence of STEC-HUS in children compared to adults is still not well understood. Human glomerular microvascular endothelial cells (HGMVECs) isolated and cultured from pediatric and adult kidney tissue were investigated with respect to Stx binding and different cellular responses. Shiga toxin-1 (Stx-1) inhibited protein synthesis in both pediatric and adult HGMVECs in a dose-dependent manner at basal conditions. The preincubation of pediatric and adult HGMVECs for 24 hrs with TNFα resulted in increased Stx binding to the cell surface and a 20-40% increase in protein synthesis inhibition in both age groups. A decreased proliferation of cells was found when a bromodeoxyuridine (BrdU) assay was performed. A trend towards a delay in endothelial wound closure was visible when pediatric and adult HGMVECs were incubated with Stx-1. Although minor differences between pediatric HGMVECs and adult HGMVECs were found in the assays applied in this study, no significant differences were observed. In conclusion, we have demonstrated that in vitro primary HGMVECs isolated from pediatric and adult kidneys do not significantly differ in their cell biological responses to Stx-1."