Publications by authors named "Joanna Kalucka"

37 Publications

Effects of the Novel PFKFB3 Inhibitor KAN0438757 on Colorectal Cancer Cells and Its Systemic Toxicity Evaluation In Vivo.

Cancers (Basel) 2021 Feb 28;13(5). Epub 2021 Feb 28.

Clinic of General, Visceral and Pediatric Surgery, University Medical Center Göttingen, Robert-Koch-Straβe 40, 37075 Göttingen, Germany.

Background: Despite substantial progress made in the last decades in colorectal cancer (CRC) research, new treatment approaches are still needed to improve patients' long-term survival. To date, the promising strategy to target tumor angiogenesis metabolically together with a sensitization of CRC to chemo- and/or radiotherapy by PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3) inhibition has never been tested. Therefore, initial evaluation and validation of newly developed compounds such as KAN0438757 and their effects on CRC cells are crucial steps preceding to in vivo preclinical studies, which in turn may consolidate new therapeutic targets.

Materials And Methods: The efficiency of KAN0438757 to block PFKFB3 expression and translation in human CRC cells was evaluated by immunoblotting and real-time PCR. Functional in vitro assays assessed the effects of KAN0438757 on cell viability, proliferation, survival, adhesion, migration and invasion. Additionally, we evaluated the effects of KAN0438757 on matched patient-derived normal and tumor organoids and its systemic toxicity in vivo in C57BL6/N mice.

Results: High PFKFB3 expression is correlated with a worse survival in CRC patients. KAN0438757 reduces PFKFB3 protein expression without affecting its transcriptional regulation. Additionally, a concentration-dependent anti-proliferative effect was observed. The migration and invasion capacity of cancer cells were significantly reduced, independent of the anti-proliferative effect. When treating colonic patient-derived organoids with KAN0438757 an impressive effect on tumor organoids growth was apparent, surprisingly sparing normal colonic organoids. No high-grade toxicity was observed in vivo.

Conclusion: The PFKFB3 inhibitor KAN0438757 significantly reduced CRC cell migration, invasion and survival. Moreover, on patient-derived cancer organoids KAN0438757 showed significant effects on growth, without being overly toxic in normal colon organoids and healthy mice. Our findings strongly encourage further translational studies to evaluate KAN0438757 in CRC therapy.
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http://dx.doi.org/10.3390/cancers13051011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957803PMC
February 2021

Angiogenesis in Adipose Tissue: The Interplay Between Adipose and Endothelial Cells.

Front Physiol 2020 9;11:624903. Epub 2021 Feb 9.

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

Obesity is a worldwide health problem, and as its prevalence increases, so does the burden of obesity-associated co-morbidities like type 2 diabetes or cardiovascular diseases (CVDs). Adipose tissue (AT) is an endocrine organ embedded in a dense vascular network. AT regulates the production of hormones, angiogenic factors, and cytokines. During the development of obesity, AT expands through the increase in fat cell size (hypertrophy) and/or fat cell number (hyperplasia). The plasticity and expansion of AT is related to its angiogenic capacities. Angiogenesis is a tightly orchestrated process, which involves endothelial cell (EC) proliferation, migration, invasion, and new tube formation. The expansion of AT is accelerated by hypoxia, inflammation, and structural remodeling of blood vessels. The paracrine signaling regulates the functional link between ECs and adipocytes. Adipocytes can secrete both pro-angiogenic molecules, e.g., tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), or vascular endothelial growth factor (VEGF), and anti-angiogenic factors, e.g., serpins. If the pro-angiogenic molecules dominate, the angiogenesis is dysregulated and the endothelium becomes dysfunctional. However, if anti-angiogenic molecules are overexpressed relative to the angiogenic regulators, the angiogenesis is repressed, and AT becomes hypoxic. Furthermore, in the presence of chronic nutritional excess, endothelium loses its primary function and contributes to the inflammation and fibrosis of AT, which increases the risk for CVDs. This review discusses the current understanding of ECs function in AT, the cross-talk between adipose and ECs, and how obesity can lead to its dysfunction. Understanding the interplay of angiogenesis with AT can be an approach to therapy obesity and obesity-related diseases such as CVDs.
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http://dx.doi.org/10.3389/fphys.2020.624903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900516PMC
February 2021

Role of the GLUT1 Glucose Transporter in Postnatal CNS Angiogenesis and Blood-Brain Barrier Integrity.

Circ Res 2020 07 14;127(4):466-482. Epub 2020 May 14.

Laboratory of Exercise and Health, Department of Health Sciences and Technology, Swiss Federal Institute of Technology (ETHZ) Zurich (Z.F., M.G., T.G., P.G., R.A., K.D.B.).

Rationale: Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood.

Objective: We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB.

Methods And Results: Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets.

Conclusions: GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.316463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386868PMC
July 2020

Single-Cell RNA Sequencing Maps Endothelial Metabolic Plasticity in Pathological Angiogenesis.

Cell Metab 2020 04;31(4):862-877.e14

Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, VIB, Department of Oncology, Leuven Cancer Institute, KU Leuven, Leuven 3000, Belgium.

Endothelial cell (EC) metabolism is an emerging target for anti-angiogenic therapy in tumor angiogenesis and choroidal neovascularization (CNV), but little is known about individual EC metabolic transcriptomes. By single-cell RNA sequencing 28,337 murine choroidal ECs (CECs) and sprouting CNV-ECs, we constructed a taxonomy to characterize their heterogeneity. Comparison with murine lung tumor ECs (TECs) revealed congruent marker gene expression by distinct EC phenotypes across tissues and diseases, suggesting similar angiogenic mechanisms. Trajectory inference predicted that differentiation of venous to angiogenic ECs was accompanied by metabolic transcriptome plasticity. ECs displayed metabolic transcriptome heterogeneity during cell-cycle progression and in quiescence. Hypothesizing that conserved genes are important, we used an integrated analysis, based on congruent transcriptome analysis, CEC-tailored genome-scale metabolic modeling, and gene expression meta-analysis in cross-species datasets, followed by in vitro and in vivo validation, to identify SQLE and ALDH18A1 as previously unknown metabolic angiogenic targets.
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http://dx.doi.org/10.1016/j.cmet.2020.03.009DOI Listing
April 2020

Single-Cell Transcriptome Atlas of Murine Endothelial Cells.

Cell 2020 02 13;180(4):764-779.e20. Epub 2020 Feb 13.

Laboratory for Functional Epigenetics, Department of Human Genetics, KU Leuven, 3000 Leuven, Belgium.

The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7 intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.
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http://dx.doi.org/10.1016/j.cell.2020.01.015DOI Listing
February 2020

An Integrated Gene Expression Landscape Profiling Approach to Identify Lung Tumor Endothelial Cell Heterogeneity and Angiogenic Candidates.

Cancer Cell 2020 01;37(1):21-36.e13

Laboratory for Precision Cancer Medicine, Translational Cell & Tissue Research, Department of Imaging & Pathology, KU Leuven, Leuven 3000, Belgium.

Heterogeneity of lung tumor endothelial cell (TEC) phenotypes across patients, species (human/mouse), and models (in vivo/in vitro) remains poorly inventoried at the single-cell level. We single-cell RNA (scRNA)-sequenced 56,771 endothelial cells from human/mouse (peri)-tumoral lung and cultured human lung TECs, and detected 17 known and 16 previously unrecognized phenotypes, including TECs putatively regulating immune surveillance. We resolved the canonical tip TECs into a known migratory tip and a putative basement-membrane remodeling breach phenotype. Tip TEC signatures correlated with patient survival, and tip/breach TECs were most sensitive to vascular endothelial growth factor blockade. Only tip TECs were congruent across species/models and shared conserved markers. Integrated analysis of the scRNA-sequenced data with orthogonal multi-omics and meta-analysis data across different human tumors, validated by functional analysis, identified collagen modification as a candidate angiogenic pathway.
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http://dx.doi.org/10.1016/j.ccell.2019.12.001DOI Listing
January 2020

Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation.

J Am Soc Nephrol 2020 01 9;31(1):118-138. Epub 2019 Dec 9.

Department of Oncology, Laboratory of Angiogenesis and Vascular Metabolism, Katholieke Universiteit Leuven (KU Leuven), Leuven, Belgium;

Background: Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown.

Methods: We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model and in dehydrated mice .

Results: We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and-surprisingly-oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration.

Conclusions: This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.
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http://dx.doi.org/10.1681/ASN.2019080832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935008PMC
January 2020

Mononuclear phagocytes orchestrate prolyl hydroxylase inhibition-mediated renoprotection in chronic tubulointerstitial nephritis.

Kidney Int 2019 08 5;96(2):378-396. Epub 2019 Mar 5.

Department of Internal Medicine 4-Nephrology and Hypertension, Friedrich-Alexander-University Erlangen-Nürnberg (FAU) and University Hospital Erlangen, Erlangen, Germany. Electronic address:

Prolyl hydroxylase domain enzyme inhibitors (PHDIs) stabilize hypoxia-inducible factors (HIFs), and are protective in models of acute ischemic and inflammatory kidney disease. Whether PHDIs also confer protection in chronic inflammatory kidney disease models remains unknown. Here we investigated long-term effects of PHDI treatment in adenine-induced nephropathy as a model for chronic tubulointerstitial nephritis. After three weeks, renal dysfunction and tubulointerstitial damage, including proximal and distal tubular injury, tubular dilation and renal crystal deposition were significantly attenuated in PHDI-treated (the isoquinoline derivative ICA and Roxadustat) compared to vehicle-treated mice with adenine-induced nephropathy. Crystal-induced renal fibrosis was only partially diminished by treatment with ICA. Renoprotective effects of ICA treatment could not be attributed to changes in adenine metabolism or urinary excretion of the metabolite 2,8-dihydroxyadenine. ICA treatment reduced inflammatory infiltrates of F4/80+ mononuclear phagocytes in the kidneys and supported a regulatory, anti-inflammatory immune response. Furthermore, interstitial deposition of complement C1q was decreased in ICA-treated mice fed an adenine-enriched diet. Tubular cell-specific HIF-1α and myeloid cell-specific HIF-1α and HIF-2α expression were not required for the renoprotective effects of ICA. In contrast, depletion of mononuclear phagocytes with clodronate largely abolished the nephroprotective effects of PHD inhibition. Thus, our findings indicate novel and potent systemic anti-inflammatory properties of PHDIs that confer preservation of kidney function and structure in chronic tubulointerstitial inflammation and might counteract kidney disease progression.
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http://dx.doi.org/10.1016/j.kint.2019.02.016DOI Listing
August 2019

Analysis of Endothelial Fatty Acid Metabolism Using Tracer Metabolomics.

Methods Mol Biol 2019 ;1978:259-268

Laboratory of Angiogenesis and Vascular Metabolism, VIB Center for Cancer Biology (CCB), VIB, Leuven, Belgium.

Blood vessels are lined by a streamlined monolayer of quiescent endothelial cells (ECs). Although these cells can remain quiescent for years, different stimuli (ischemia, inflammation) and growth factors can activate them and drive a process of new vessel formation (angiogenesis). Emerging evidence reveals that cellular metabolism is a key determinant of the EC subtype specification. The use of stable isotope tracing and mass spectrometry analysis has been essential for the discovery that fatty acid metabolism contributes to EC proliferation and lymphatic EC differentiation. This chapter describes the methodology for setting up palmitate-based tracer metabolomics and the subsequent liquid chromatography-mass spectrometry (LC-MS)-based analysis. As such, tracer metabolomics can be used: (1) to identify the different metabolic pathways relying on carbons provided by fatty acid oxidation and (2) to quantify the relative contributions of palmitate-derived carbons. We begin by providing a background and general principles regarding the use of stable isotopes to study fatty acid metabolism. We then proceed with detailed procedures for the labeling conditions, sample preparation, and subsequent LC-MS analysis.
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http://dx.doi.org/10.1007/978-1-4939-9236-2_16DOI Listing
November 2019

EndoDB: a database of endothelial cell transcriptomics data.

Nucleic Acids Res 2019 01;47(D1):D736-D744

Department of Oncology and Leuven Cancer Institute (LKI), Laboratory of Angiogenesis and Vascular Metabolism, KU Leuven, 3000 Leuven, Belgium.

Endothelial cells (ECs) line blood vessels, regulate homeostatic processes (blood flow, immune cell trafficking), but are also involved in many prevalent diseases. The increasing use of high-throughput technologies such as gene expression microarrays and (single cell) RNA sequencing generated a wealth of data on the molecular basis of EC (dys-)function. Extracting biological insight from these datasets is challenging for scientists who are not proficient in bioinformatics. To facilitate the re-use of publicly available EC transcriptomics data, we developed the endothelial database EndoDB, a web-accessible collection of expert curated, quality assured and pre-analyzed data collected from 360 datasets comprising a total of 4741 bulk and 5847 single cell endothelial transcriptomes from six different organisms. Unlike other added-value databases, EndoDB allows to easily retrieve and explore data of specific studies, determine under which conditions genes and pathways of interest are deregulated and assess reprogramming of metabolism via principal component analysis, differential gene expression analysis, gene set enrichment analysis, heatmaps and metabolic and transcription factor analysis, while single cell data are visualized as gene expression color-coded t-SNE plots. Plots and tables in EndoDB are customizable, downloadable and interactive. EndoDB is freely available at https://vibcancer.be/software-tools/endodb, and will be updated to include new studies.
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http://dx.doi.org/10.1093/nar/gky997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324065PMC
January 2019

Role of glutamine synthetase in angiogenesis beyond glutamine synthesis.

Nature 2018 09 29;561(7721):63-69. Epub 2018 Aug 29.

Molecular Imaging and Photonics, University of Leuven, Leuven, Belgium.

Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.
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http://dx.doi.org/10.1038/s41586-018-0466-7DOI Listing
September 2018

Quiescent Endothelial Cells Upregulate Fatty Acid β-Oxidation for Vasculoprotection via Redox Homeostasis.

Cell Metab 2018 12 23;28(6):881-894.e13. Epub 2018 Aug 23.

Translational Cell & Tissue Research, Department of Imaging & Pathology, KU Leuven, 3000 Leuven, Belgium.

Little is known about the metabolism of quiescent endothelial cells (QECs). Nonetheless, when dysfunctional, QECs contribute to multiple diseases. Previously, we demonstrated that proliferating endothelial cells (PECs) use fatty acid β-oxidation (FAO) for de novo dNTP synthesis. We report now that QECs are not hypometabolic, but upregulate FAO >3-fold higher than PECs, not to support biomass or energy production but to sustain the tricarboxylic acid cycle for redox homeostasis through NADPH regeneration. Hence, endothelial loss of FAO-controlling CPT1A in CPT1A mice promotes EC dysfunction (leukocyte infiltration, barrier disruption) by increasing endothelial oxidative stress, rendering CPT1A mice more susceptible to LPS and inflammatory bowel disease. Mechanistically, Notch1 orchestrates the use of FAO for redox balance in QECs. Supplementation of acetate (metabolized to acetyl-coenzyme A) restores endothelial quiescence and counters oxidative stress-mediated EC dysfunction in CPT1A mice, offering therapeutic opportunities. Thus, QECs use FAO for vasculoprotection against oxidative stress-prone exposure.
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http://dx.doi.org/10.1016/j.cmet.2018.07.016DOI Listing
December 2018

Impairment of Angiogenesis by Fatty Acid Synthase Inhibition Involves mTOR Malonylation.

Cell Metab 2018 12 23;28(6):866-880.e15. Epub 2018 Aug 23.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, Guangdong, P.R. China. Electronic address:

The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASN) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASN elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASN ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade.
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http://dx.doi.org/10.1016/j.cmet.2018.07.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8057116PMC
December 2018

Serine Synthesis via PHGDH Is Essential for Heme Production in Endothelial Cells.

Cell Metab 2018 10 12;28(4):573-587.e13. Epub 2018 Jul 12.

Laboratory of Angiogenesis and Vascular Metabolism, VIB Center for Cancer Biology, VIB, Leuven 3000, Belgium; State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, Guangdong, P.R. China; Laboratory of Angiogenesis and Vascular Metabolism, VIB-KU Leuven Center for Cancer Biology, Department of Oncology, KU Leuven, Leuven 3000, Belgium. Electronic address:

The role of phosphoglycerate dehydrogenase (PHGDH), a key enzyme of the serine synthesis pathway (SSP), in endothelial cells (ECs) remains poorly characterized. We report that mouse neonates with EC-specific PHGDH deficiency suffer lethal vascular defects within days of gene inactivation, due to reduced EC proliferation and survival. In addition to nucleotide synthesis impairment, PHGDH knockdown (PHGDH) caused oxidative stress, due not only to decreased glutathione and NADPH synthesis but also to mitochondrial dysfunction. Electron transport chain (ETC) enzyme activities were compromised upon PHGDH because of insufficient heme production due to cellular serine depletion, not observed in other cell types. As a result of heme depletion, elevated reactive oxygen species levels caused EC demise. Supplementation of hemin in PHGDH ECs restored ETC function and rescued the apoptosis and angiogenesis defects. These data argue that ECs die upon PHGDH inhibition, even without external serine deprivation, illustrating an unusual importance of serine synthesis for ECs.
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http://dx.doi.org/10.1016/j.cmet.2018.06.009DOI Listing
October 2018

Consensus guidelines for the use and interpretation of angiogenesis assays.

Angiogenesis 2018 08;21(3):425-532

Laboratory of Cellular Metabolism and Metabolic Regulation, VIB Center for Cancer Biology, Leuven, Belgium.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.
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http://dx.doi.org/10.1007/s10456-018-9613-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237663PMC
August 2018

How Endothelial Cells Adapt Their Metabolism to Form Vessels in Tumors.

Front Immunol 2017 11;8:1750. Epub 2017 Dec 11.

Laboratory of Angiogenesis and Vascular Metabolism, Vesalius Research Center, VIB, Leuven, Belgium.

Endothelial cells (ECs) line blood vessels, i.e., vital conduits for oxygen and nutrient delivery to distant tissues. While mostly present as quiescent "phalanx" cells throughout adult life, ECs can rapidly switch to a migratory "tip" cell and a proliferative "stalk" cell, and sprout into avascular tissue to form new blood vessels. The angiogenic switch has long been considered to be primarily orchestrated by the activity of angiogenic molecules. However, recent evidence illustrates an instrumental role of cellular metabolism in vessel sprouting, whereby ECs require specific metabolic adaptations to grow. Here, we overview the emerging picture that tip, stalk, and phalanx cells have distinct metabolic signatures and discuss how these signatures can become deregulated in pathological conditions, such as in cancer.
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http://dx.doi.org/10.3389/fimmu.2017.01750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732229PMC
December 2017

Tumor vessel disintegration by maximum tolerable PFKFB3 blockade.

Angiogenesis 2017 Nov 5;20(4):599-613. Epub 2017 Sep 5.

Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, KU Leuven, 3000, Leuven, Belgium.

Blockade of the glycolytic activator PFKFB3 in cancer cells (using a maximum tolerable dose of 70 mg/kg of the PFKFB3 blocker 3PO) inhibits tumor growth in preclinical models and is currently being tested as a novel anticancer treatment in phase I clinical trials. However, a detailed preclinical analysis of the effects of such maximum tolerable dose of a PFKFB3 blocker on the tumor vasculature is lacking, even though tumor endothelial cells are hyper-glycolytic. We report here that a high dose of 3PO (70 mg/kg), which inhibits cancer cell proliferation and reduces primary tumor growth, causes tumor vessel disintegration, suppresses endothelial cell growth for protracted periods, (model-dependently) aggravates tumor hypoxia, and compromises vascular barrier integrity, thereby rendering tumor vessels more leaky and facilitating cancer cell intravasation and dissemination. These findings contrast to the effects of a low dose of 3PO (25 mg/kg), which induces tumor vessel normalization, characterized by vascular barrier tightening and maturation, but reduces cancer cell intravasation and metastasis. Our findings highlight the importance of adequately dosing a glycolytic inhibitor for anticancer treatment.
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http://dx.doi.org/10.1007/s10456-017-9573-6DOI Listing
November 2017

Role of glutamine and interlinked asparagine metabolism in vessel formation.

EMBO J 2017 08 28;36(16):2334-2352. Epub 2017 Jun 28.

Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, KU Leuven, Leuven, Belgium

Endothelial cell (EC) metabolism is emerging as a regulator of angiogenesis, but the precise role of glutamine metabolism in ECs is unknown. Here, we show that depriving ECs of glutamine or inhibiting glutaminase 1 (GLS1) caused vessel sprouting defects due to impaired proliferation and migration, and reduced pathological ocular angiogenesis. Inhibition of glutamine metabolism in ECs did not cause energy distress, but impaired tricarboxylic acid (TCA) cycle anaplerosis, macromolecule production, and redox homeostasis. Only the combination of TCA cycle replenishment plus asparagine supplementation restored the metabolic aberrations and proliferation defect caused by glutamine deprivation. Mechanistically, glutamine provided nitrogen for asparagine synthesis to sustain cellular homeostasis. While ECs can take up asparagine, silencing asparagine synthetase (ASNS, which converts glutamine-derived nitrogen and aspartate to asparagine) impaired EC sprouting even in the presence of glutamine and asparagine. Asparagine further proved crucial in glutamine-deprived ECs to restore protein synthesis, suppress ER stress, and reactivate mTOR signaling. These findings reveal a novel link between endothelial glutamine and asparagine metabolism in vessel sprouting.
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http://dx.doi.org/10.15252/embj.201695518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556263PMC
August 2017

How to Cross the Lymphatic Fence: Lessons From Solute Transport.

Circ Res 2017 04;120(9):1376-1378

From the Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, Leuven Cancer Institute (LKI), KU Leuven, Belgium (J.K., L.-A.T., V.G., P.C.); Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, Vesalius Research Center, Leuven, Belgium (J.K., L.-A.T., V.G., P.C.); GZA Hospitals Sint-Augustinus, Wilrijk, Belgium (L.-A.T.); and Center for Oncological Research, University of Antwerp, Belgium (L.-A.T.).

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http://dx.doi.org/10.1161/CIRCRESAHA.117.310916DOI Listing
April 2017

Interaction of endothelial cells with macrophages-linking molecular and metabolic signaling.

Pflugers Arch 2017 04 24;469(3-4):473-483. Epub 2017 Feb 24.

Laboratory of Angiogenesis and Vascular Metabolism, Vesalius Research Centre, VIB, Campus Gasthuisberg O&N4, Herestraat 49-912, Leuven, 3000, Belgium.

Angiogenesis and inflammation go hand in hand in various (patho-)physiological conditions. Several studies have highlighted the interconnection between endothelial cells (ECs) and macrophages in these conditions at the level of growth factor and cytokine signaling, yet the importance of metabolism and metabolic signaling has been largely overlooked. Modulating macrophage and/or endothelial functions by interfering with metabolic pathways offers new perspectives for therapeutic strategies. In this review, we highlight the complexity of the interrelationship between the inflammatory response and angiogenesis. More in particular, the interaction between macrophages and ECs will be discussed with a special focus on how their metabolism can contribute to (patho-)physiological conditions.
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http://dx.doi.org/10.1007/s00424-017-1946-6DOI Listing
April 2017

Vessel pruning or healing: endothelial metabolism as a novel target?

Expert Opin Ther Targets 2017 Mar 27;21(3):239-247. Epub 2017 Jan 27.

a Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology , KU Leuven , Leuven , Belgium.

Introduction: Antiangiogenic drugs were originally designed to starve tumors by cutting off their vascular supply. Unfortunately, when these agents are used as monotherapy or in combination with chemotherapy, they provide only modest survival benefits in the order of weeks to months in most cancer patients. Strategies normalizing the disorganized tumor vasculature offer the potential to increase tumor perfusion and oxygenation, and to improve the efficacy of radio-, chemo- and immunotherapy, while reducing metastasis. Areas covered: This review discusses tumor vascular normalization (TVN) as an alternative strategy for anti-angiogenic cancer treatment. We summarize (pre)-clinical strategies that have been developed to normalize tumor vessels as well as their potential to enhance standard therapy. Notably, we describe how targeting endothelial cell metabolism offers new possibilities for antiangiogenic therapy through evoking TVN. Expert opinion: Several drugs targeting VEGF signaling are now clinically used for antiangiogenic cancer treatment. However, excessive blood vessel pruning impedes perfusion and causes tumor hypoxia, known to promote cancer cell dissemination and impair radio-, chemo- and immunotherapy. Normalized vessels lessen tumor hypoxia, impair cancer cell intravasation and enhance anticancer treatment. New data indicate that targeting endothelial cell metabolism is an alternative strategy of antiangiogenic cancer treatment via promotion of TVN.
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http://dx.doi.org/10.1080/14728222.2017.1282465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526136PMC
March 2017

The role of fatty acid β-oxidation in lymphangiogenesis.

Nature 2017 02 26;542(7639):49-54. Epub 2016 Dec 26.

Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, KU Leuven, Leuven B-3000, Belgium.

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid β-oxidation, impairs lymphatic development. LECs use fatty acid β-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid β-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.
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http://dx.doi.org/10.1038/nature21028DOI Listing
February 2017

Inhibition of the Glycolytic Activator PFKFB3 in Endothelium Induces Tumor Vessel Normalization, Impairs Metastasis, and Improves Chemotherapy.

Cancer Cell 2016 Dec 17;30(6):968-985. Epub 2016 Nov 17.

Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA; Section of Cardiology, Department of Medicine, University of Illinois College of Medicine, Chicago, IL 60612, USA.

Abnormal tumor vessels promote metastasis and impair chemotherapy. Hence, tumor vessel normalization (TVN) is emerging as an anti-cancer treatment. Here, we show that tumor endothelial cells (ECs) have a hyper-glycolytic metabolism, shunting intermediates to nucleotide synthesis. EC haplo-deficiency or blockade of the glycolytic activator PFKFB3 did not affect tumor growth, but reduced cancer cell invasion, intravasation, and metastasis by normalizing tumor vessels, which improved vessel maturation and perfusion. Mechanistically, PFKFB3 inhibition tightened the vascular barrier by reducing VE-cadherin endocytosis in ECs, and rendering pericytes more quiescent and adhesive (via upregulation of N-cadherin) through glycolysis reduction; it also lowered the expression of cancer cell adhesion molecules in ECs by decreasing NF-κB signaling. PFKFB3-blockade treatment also improved chemotherapy of primary and metastatic tumors.
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http://dx.doi.org/10.1016/j.ccell.2016.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675554PMC
December 2016

Meta-analysis of clinical metabolic profiling studies in cancer: challenges and opportunities.

EMBO Mol Med 2016 10 4;8(10):1134-1142. Epub 2016 Oct 4.

Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, KU Leuven, Leuven, Belgium Laboratory of Angiogenesis and Vascular Metabolism, Vesalius Research Center, VIB, Leuven, Belgium

Cancer cell metabolism has received increasing attention. Despite a boost in the application of clinical metabolic profiling (CMP) in cancer patients, a meta-analysis has not been performed. The primary goal of this study was to assess whether public accessibility of metabolomics data and identification and reporting of metabolites were sufficient to assess which metabolites were consistently altered in cancer patients. We therefore retrospectively curated data from CMP studies in cancer patients published during 5 recent years and used an established vote-counting method to perform a semiquantitative meta-analysis of metabolites in tumor tissue and blood. This analysis confirmed well-known increases in glycolytic metabolites, but also unveiled unprecedented changes in other metabolites such as ketone bodies and amino acids (histidine, tryptophan). However, this study also highlighted that insufficient public accessibility of metabolomics data, and inadequate metabolite identification and reporting hamper the discovery potential of meta-analyses of CMP studies, calling for improved standardization of metabolomics studies.
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http://dx.doi.org/10.15252/emmm.201606798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5048364PMC
October 2016

Ferritin-Mediated Iron Sequestration Stabilizes Hypoxia-Inducible Factor-1α upon LPS Activation in the Presence of Ample Oxygen.

Cell Rep 2015 Dec 25;13(10):2048-55. Epub 2015 Nov 25.

Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen and Friedrich-Alexander Universität (FAU) Erlangen-Nürnberg, 91054 Erlangen, Germany; Institute of Clinical Microbiology and Hygiene, University Hospital of Regensburg and University of Regensburg, 93053 Regensburg, Germany. Electronic address:

Both hypoxic and inflammatory conditions activate transcription factors such as hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB, which play a crucial role in adaptive responses to these challenges. In dendritic cells (DC), lipopolysaccharide (LPS)-induced HIF1α accumulation requires NF-κB signaling and promotes inflammatory DC function. The mechanisms that drive LPS-induced HIF1α accumulation under normoxia are unclear. Here, we demonstrate that LPS inhibits prolyl hydroxylase domain enzyme (PHD) activity and thereby blocks HIF1α degradation. Of note, LPS-induced PHD inhibition was neither due to cosubstrate depletion (oxygen or α-ketoglutarate) nor due to increased levels of reactive oxygen species, fumarate, and succinate. Instead, LPS inhibited PHD activity through NF-κB-mediated induction of the iron storage protein ferritin and subsequent decrease of intracellular available iron, a critical cofactor of PHD. Thus, hypoxia and LPS both induce HIF1α accumulation via PHD inhibition but deploy distinct molecular mechanisms (lack of cosubstrate oxygen versus deprivation of co-factor iron).
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http://dx.doi.org/10.1016/j.celrep.2015.11.005DOI Listing
December 2015

Targeting fatty acid metabolism in cancer and endothelial cells.

Crit Rev Oncol Hematol 2016 Jan 1;97:15-21. Epub 2015 Nov 1.

Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, Department of Oncology, University of Leuven, Leuven B-3000, Belgium; Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven B-3000, Belgium. Electronic address:

Tumour angiogenesis has long been recognised as a target for anti-cancer therapy. The current approach of inhibiting the VEGF pathway has shown benefit in the clinic, though less than anticipated. We recently documented that glycolytic metabolism in endothelial cells (ECs) fuels angiogenesis, rendering it a possible target for inhibiting vascular growth in pathological conditions. More recently, we reported that the oxidation of fatty acids (FA) is irreplaceable for EC proliferation by providing carbons for de novo nucleotide synthesis. Furthermore, ECs are rather unique in this respect, creating novel therapeutic opportunities. Here, we review and compare the current understanding of FA utilisation in ECs and tumour cells (TCs).
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http://dx.doi.org/10.1016/j.critrevonc.2015.10.011DOI Listing
January 2016

Metabolic control of the cell cycle.

Cell Cycle 2015 ;14(21):3379-88

a Laboratory of Angiogenesis and Neurovascular link; Department of Oncology; KU Leuven ; Leuven , Belgium.

Cell division is a metabolically demanding process, requiring the production of large amounts of energy and biomass. Not surprisingly therefore, a cell's decision to initiate division is co-determined by its metabolic status and the availability of nutrients. Emerging evidence reveals that metabolism is not only undergoing substantial changes during the cell cycle, but it is becoming equally clear that metabolism regulates cell cycle progression. Here, we overview the emerging role of those metabolic pathways that have been best characterized to change during or influence cell cycle progression. We then studied how Notch signaling, a key angiogenic pathway that inhibits endothelial cell (EC) proliferation, controls EC metabolism (glycolysis) during the cell cycle.
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http://dx.doi.org/10.1080/15384101.2015.1090068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825590PMC
August 2016

Formalin evokes calcium transients from the endoplasmatic reticulum.

PLoS One 2015 15;10(4):e0123762. Epub 2015 Apr 15.

Institute of Physiology and Pathophysiology, University of Erlangen-Nürnberg, Erlangen, Germany.

The formalin test is the most widely used behavioral screening test for analgesic compounds. The cellular mechanism of action of formaldehyde, inducing a typically biphasic pain-related behavior in rodents is addressed in this study. The chemoreceptor channel TRPA1 was suggested as primary transducer, but the high concentrations used in the formalin test elicit a similar response in TRPA1 wildtype and knockout animals. Here we show that formaldehyde evokes a dose-dependent calcium release from intracellular stores in mouse sensory neurons and primary keratinocytes as well as in non-neuronal cell lines, and independent of TRPA1. The source of calcium is the endoplasmatic reticulum and inhibition of the sarco/endoplasmic reticulum calcium-ATPase has a major contribution. This TRPA1-independent mechanism may underlie formaldehyde-induced pan-neuronal excitation and subsequent inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123762PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398422PMC
January 2016

Kidney injury is independent of endothelial HIF-1α.

J Mol Med (Berl) 2015 Aug 11;93(8):891-904. Epub 2015 Mar 11.

Department of Nephrology and Hypertension, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Ulmenweg 18, 91054, Erlangen, Germany.

Unlabelled: Hypoxia-inducible transcription factors (HIFs) control cellular adaptation to low oxygen. In the kidney, activation of HIF is beneficial during injury; however, the specific contribution of HIF-1α in renal endothelial cells (EC) remains elusive. Since EC display tissue-specific heterogeneity, we investigated how HIF-1α affects key functions of glomerular EC in vitro and its contribution to renal development and pathophysiological adaptation to acute or chronic renal injury in vivo. Loss of HIF-1α in glomerular EC induces hypoxic cell death and reduces hypoxic adhesion of macrophages in vitro. In vivo, HIF-1α expression in EC in mouse kidneys is detectable but limited. Accordingly, EC-specific ablation of HIF-1α does not lead to developmental or phenotypical abnormalities in the kidney. Renal function and expression of adhesion molecules during acute ischemic kidney injury is independent of HIF-1α in EC. Likewise, inflammation and development of fibrosis after unilateral ureteric obstruction is not influenced by endothelial HIF-1α. Taken together, although HIF-1α exerts effects on glomerular EC in vitro, endothelial HIF-1α does not influence renal development and pathophysiological adaptation to kidney injury in vivo. This implies a profound difference of the hypoxic response of the renal vascular bed compared to other organs, such as the heart. This has implications for the development of pharmacological strategies targeting the endothelial hypoxic response pathways.

Key Message: HIF-1α controls hypoxic survival and adhesion on endothelial cells (EC) in vitro. In vivo, HIF-1α expression in renal EC is low. Deletion of HIF-1α in EC does not affect kidney development and function in mice. Renal function after acute and chronic kidney injury is independent of HIF-1α in EC. Data suggest organ-specific regulation of HIF-1α function in EC.
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http://dx.doi.org/10.1007/s00109-015-1264-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592817PMC
August 2015