title: Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger.
abstract: Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.
authors.affiliation: From Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary's University of London, London EC1M 6BQ, United Kingdom, p.andrikopoulos@qmul.ac.uk.
authors.forename: Petros
authors.initials: P
authors.lastname: Andrikopoulos
authors.affiliation: From Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary's University of London, London EC1M 6BQ, United Kingdom.
authors.forename: Julius
authors.initials: J
authors.lastname: Kieswich
authors.affiliation: From Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary's University of London, London EC1M 6BQ, United Kingdom.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Harwood
authors.affiliation: the Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe-shi, Hyogo 650-8530, Japan.
authors.forename: Akemichi
authors.initials: A
authors.lastname: Baba
authors.affiliation: the Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan, and.
authors.forename: Toshio
authors.initials: T
authors.lastname: Matsuda
authors.affiliation: the Division of Cancer Therapeutics, Institute of Cancer Research, London SW7 3RP, United Kingdom.
authors.forename: Olivier
authors.initials: O
authors.lastname: Barbeau
authors.affiliation: the Division of Cancer Therapeutics, Institute of Cancer Research, London SW7 3RP, United Kingdom.
authors.forename: Keith
authors.initials: K
authors.lastname: Jones
authors.affiliation: the Division of Cancer Therapeutics, Institute of Cancer Research, London SW7 3RP, United Kingdom.
authors.forename: Suzanne A
authors.initials: SA
authors.lastname: Eccles
authors.affiliation: From Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary's University of London, London EC1M 6BQ, United Kingdom.
authors.forename: Muhammad M
authors.initials: MM
authors.lastname: Yaqoob
mesh_terms: D000814:Aniline Compounds / Q000008:administration & dosage; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004727:Endothelium / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000235:genetics*; D010539:Permeability / Q000187:drug effects; D010647:Phenyl Ethers / Q000008:administration & dosage; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D013917:Thrombin / Q000008:administration & dosage
chemical_list: D000814:Aniline Compounds; D008562:Membrane Glycoproteins; C499849:NCX1 protein, mouse; D010647:Phenyl Ethers; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D044463:Receptor, PAR-1; C430918:SEA 0400; D019831:Sodium-Calcium Exchanger; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D013917:Thrombin; D002118:Calcium
keywords: NADPH oxidase; angiogenesis; calcium; endothelium; extracellular-signal-regulated kinase (ERK); permeability; sodium-calcium exchange; thrombin