Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1225 The Role of Paladin in Endothelial Cell Signaling and Angiogenesis ANJA NITZSCHE ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-554-9578-7 UPPSALA urn:nbn:se:uu:diva-281708 2016 Dissertation presented at Uppsala University to be publicly examined in Fåhraeussalen, Rudbeck laboratory (C5), Dag Hammarskjölds väg 20, Uppsala, Thursday, 9 June 2016 at 09:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor Frank-Dietmar Böhmer (Institute of Molecular Cell Biology, Jena University Hospital, Jena, Germany). Abstract Nitzsche, A. 2016. The Role of Paladin in Endothelial Cell Signaling and Angiogenesis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1225. 47 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9578-7. Angiogenesis, the formation of new blood vessels from a pre-existing vasculature, is crucial during development and for many diseases including cancer. Despite tremendous progress in the understanding of the angiogenic process, many aspects are still not fully elucidated. Several attempts have been made to identify novel genes involved in endothelial cell biology and angiogenesis. Here we focused on Pald1, a recently identified, vascular-enriched gene encoding paladin. Our in vitro studies indicate that paladin is a lipid phosphatase catalyzing dephosphorylation of phosphatidylinositol phosphates, a process essential for endocytosis and intracellular vesicle trafficking. We confirmed paladin’s vascular expression pattern and revealed a shift from a broad endothelial cell expression during development to an arterial mural cell-restricted expression in several vascular beds in adult mice. Paladin expression in the lung, however, was not restricted to the vasculature, but was also observed in pneumocytes and myofibroblasts. Lungs of female, but not male, Pald1 null mice displayed an obstructive lung phenotype with increased alveolar air sacs that were already apparent early in the alveolarization process. Only endothelial cells, but not other main lung cell types, were affected by loss of paladin. Endothelial cell number was reduced in 4-week old mice, possibly due to increased endothelial turnover in Pald1 deficient lungs. Vascular defects were also found in the retina. Loss of paladin led to reduced retinal vascular outgrowth accompanied by a hyperdense and hypersprouting vascular front. Downstream signaling of the major angiogenic driver, vascular endothelial growth factor receptor 2 (VEGFR2) was sustained in Pald1 null mice, and VEGFR2 degradation was impaired. Furthermore, paladin inhibited endothelial cell junction stability and loss of paladin led to reduced vascular permeability. Whether the differences in VEGFR2 signaling and adherens junction stability are connected remains to be fully explored. The newly identified lipid phosphatase activity of paladin and its specific effects on VEGFR2 signaling and adherens junction stability indicate that paladin may be controlling the endocytic pathway. Keywords: Pald1, endothelium, lung, vascular permeability, phosphatase, angiogenesis Anja Nitzsche, Department of Immunology, Genetics and Pathology, Rudbecklaboratoriet, Uppsala University, SE-751 85 Uppsala, Sweden. © Anja Nitzsche 2016 ISSN 1651-6206 ISBN 978-91-554-9578-7 urn:nbn:se:uu:diva-281708 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-281708) To my family It always seems impossible until it’s done. Nelson Mandela Cover: Postnatal day 5 retina stained for endothelial cells (isolectin B4, green) and adherens junctions (VE-cadherin, red). List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Wallgard E, Nitzsche A, Larsson J, Guo X, Dieterich LC, Dimberg A, Olofsson T, Pontén FC, Mäkinen T, Kalen M, Hellström M (2012). Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development. Developmental Dy- namics, 241(4):770-786. II Egaña I, Nitzsche A, Kaito H, Becker L, Garrett L, Niaudet C, Liu W, Vanlandewijck M, Larsson J, Hrabe de Angelis M, Fuchs H, Gailus-Durner V, Vernaleken A, Klopstock T, Hölter SM, Wurst W, Rask-Andersen H, German Mouse Clinic Con- sortium, Yildirim AÖ, Hellström M: Female mice lacking Pald1 exhibit endothelial cell apoptosis and emphysema. Manuscript III Nitzsche A, Testini C, Ekvärn E, Larsson J, Bentley K, Philippides A, Roche FP, Egaña I, Smith R, Hellberg C, Ballmer-Hofer K, Hellström M: Paladin (Pald1) regulates en- dothelial sprouting, VE-cadherin junction stability and vascu- lar permeability. Manuscript Reprint of paper I was made with permission from publisher. Additional publication The author also contributed to the following paper not included in this thesis: Kalen M, Heikura T, Karvinen H, Nitzsche A, Weber H, Esser N, Yla- Herttuala S, Hellström M (2011). Gamma-secretase inhibitor treatment promotes VEGF-A-driven blood vessel growth and vascular leakage but disrupts neovascular perfusion. PLoS One 6:e18709. Contents Introduction ..................................................................................................... 9 The vascular system ................................................................................... 9 Blood vessel formation ........................................................................ 10 In vivo models of developmental angiogenesis ................................... 11 Vascular endothelial growth factors and their receptors ...................... 11 Molecular mechanisms of sprouting angiogenesis .............................. 13 Endothelial cell adherens junctions and vascular permeability ........... 15 Endosomal trafficking ......................................................................... 18 Lung physiology and disease ................................................................... 20 Lung development ............................................................................... 20 Alveolar cell types ............................................................................... 21 Chronic obstructive pulmonary disease and emphysema .................... 22 Phosphatases............................................................................................. 23 Protein Tyrosine Phosphatases ............................................................ 23 Pseudophosphatases ............................................................................. 26 Paladin ...................................................................................................... 27 Present investigations .................................................................................... 29 Paper I ...................................................................................................... 29 Paper II ..................................................................................................... 30 Paper III .................................................................................................... 32 Concluding remarks and future perspectives ................................................ 34 Acknowledgments......................................................................................... 36 References ..................................................................................................... 38 Abbreviations Arf ADP-ribosylation factor BASC Bronchioalveolar stem cell COPD Chronic obstructive pulmonary disease Csk C-terminal Src tyrosine protein kinase DASC Distal airway stem cell DEP1 density enhanced phosphatase-1 Dll4 Delta-like ligand 4 DUSP Dual-specificity phosphatase E# Embryonic day ECM Extracellular matrix EEA1 Early endosome antigen 1 Erk Extracellular regulated kinase FAK Focal adhesion kinase GAP GTPase-activating protein GEF Guanine nucleotide exchange factor HIF Hypoxia-inducible factor LMW-PTP Low molecular weight protein tyrosine phosphatase MMP Matrix metalloproteinase MTM myotubularin N-cadherin Neuronal cadherin P# Postnatal day PAK p21-activated kinase PIP phosphatidylinositol phosphate PLC Phospholipase C PTEN phosphatase and tensin homolog deleted on chromo- some 10 PTP Protein tyrosine phosphatase ROS Reactive oxygen species RTK Receptor tyrosine kinase TNFα tumor necrosis factor TSAd T-cell specific adaptor VE-cadherin Vascular endothelial cadherin VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor VE-PTP Vascular endothelial protein tyrosine phosphatase Introduction The vascular system The complex vertebrate body depends on a functional, highly branched vas- cular system, comprising both blood and lymphatic vasculatures. The blood vessel network ensures supply of oxygen and nutrients to the tissues and removal of waste products1. Oxygenated blood from the lung is transported via the heart and, through arteries and arterioles, to the periphery where gas exchange is facilitated by thin capillaries (diameter < 10 µm). Through post- capillary venules and veins deoxygenated blood is transported back to the heart and enters the pulmonary circulation to be replenished with oxygen (Figure 1)2. The lymphatic vascular system ensures the drainage of excess interstitial fluids back into the systemic circulation1. Figure 1. The vascular system. Blood vessels are composed of endothelial cells. Pericytes sparsely cover capillaries, whereas arteries and vein are surrounded