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Jimmunol.1401364.Full.Pdf Loss of Mouse P2Y4 Nucleotide Receptor Protects against Myocardial Infarction through Endothelin-1 Downregulation This information is current as Michael Horckmans, Hrag Esfahani, Christophe Beauloye, of September 25, 2021. Sophie Clouet, Larissa di Pietrantonio, Bernard Robaye, Jean-Luc Balligand, Jean-Marie Boeynaems, Chantal Dessy and Didier Communi J Immunol published online 16 January 2015 http://www.jimmunol.org/content/early/2015/01/16/jimmun Downloaded from ol.1401364 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 25, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 16, 2015, doi:10.4049/jimmunol.1401364 The Journal of Immunology Loss of Mouse P2Y4 Nucleotide Receptor Protects against Myocardial Infarction through Endothelin-1 Downregulation Michael Horckmans,*,1 Hrag Esfahani,†,1 Christophe Beauloye,‡ Sophie Clouet,* Larissa di Pietrantonio,* Bernard Robaye,x Jean-Luc Balligand,† Jean-Marie Boeynaems,*,{ Chantal Dessy,†,2 and Didier Communi*,2 Nucleotides are released in the heart under pathological conditions, but little is known about their contribution to cardiac inflam- mation. The present study defines the P2Y4 nucleotide receptor, expressed on cardiac microvascular endothelial cells and involved in postnatal heart development, as an important regulator of the inflammatory response to cardiac ischemia. P2Y4-null mice displayed smaller infarcts in the left descending artery ligation model, as well as reduced neutrophil infiltration and fibrosis. Gene profiling identified inter alia endothelin-1 (ET-1) as one of the target genes of P2Y4 in ischemic heart. The reduced level of ET-1 was correlated with reduction of microvascular hyperpermeability, neutrophil infiltration, and endothelial adhesion molecule expression, Downloaded from and it could be explained by the decreased number of endothelial cells in P2Y4-null mice. Expression analysis of metalloproteinases and their tissue inhibitors in ischemic heart revealed reduced expression of matrix metalloproteinase (MMP)-9, reported to be potentially regulated by ET-1, and MMP-8, considered as neutrophil collagenase, as well as reduction of tissue inhibitor of MMP-1 and tissue inhibitor of MMP-4 in P2Y4-null mice. Reduction of cardiac permeability and neutrophil infiltration was also observed in P2Y4-null mice in LPS-induced inflammation model. Protection against infarction resulting from loss of P2Y4 brings new therapeutic perspectives for cardiac ischemia and remodeling. The Journal of Immunology, 2015, 194: 000–000. http://www.jimmunol.org/ ardiac injury is a great challenge owing to the limited the recruitment of leukocytes into the infarcted area. Neutrophils regenerative ability of the heart. Myocardial infarction and macrophages clear the wound from dead cells and matrix C (MI) is the most common source of cardiac injuries debris (2). However, MI is a multifactorial process that leads also leading to ischemic death of a large number of cardiomyocytes (1). in the first hours to a postischemic edema that develops as a result After this phase, dying cells trigger an inflammatory reaction with of increased microvascular permeability. Myocardial edema con- tributes to vessel collapse and impaired heart function, including reperfusion arrhythmias and stunning, and could affect ventricular by guest on September 25, 2021 *Institut de Recherche Interdisciplinaire en Biologie Humaine et Mole´culaire, Uni- remodeling by altering myocardial stiffness (3). Among the pro- † versite´ Libre de Bruxelles, 1070 Brussels, Belgium; Unite´ de Pharmacologie et de teins regulating the inflammatory response to cardiac ischemia, The´rapeutique, Universite´ Catholique de Louvain, 1200 Brussels, Belgium; ‡Poˆle de Recherche Cardiovasculaire, Institut de Recherche Expe´rimentale et Clin- endothelin-1 (ET-1) plays an important role. Indeed ET-1 is in- ique, Universite´ Catholique de Louvain, 1200 Brussels, Belgium; xInstitut de volved in neutrophil trafficking and increases cardiac microvas- Recherche Interdisciplinaire en Biologie Humaine et Mole´culaire, Universite´ Libre { cular permeability (4, 5). After the edema step, the healing process de Bruxelles, 6041 Gosselies, Belgium; and De´partement de Me´decine de Labora- toire, Hoˆpital Erasme, Universite´ Libre de Bruxelles, 1070 Brussels, Belgium proceeds to the formation of granulation tissue, which is charac- 1M.H. and H.E. contributed equally to this work. terized by the presence of fibroblasts, macrophages, myofibro- 2C.D. and D.C. contributed equally to this work. blasts, new blood vessels, and extracellular matrix proteins (6, 7), ultimately resulting in scar formation, which is characterized by Received for publication May 28, 2014. Accepted for publication December 8, 2014. acellular and cross-linked collagen-rich regions (8–11). This work was supported by an Action de Recherche Concerte´e of the Communaute´ Franc¸aise de Belgique, an Interuniversity Attraction Pole grant from the Politique Cardiac endothelial cells and cardiomyocytes are an important Scientifique Fe´de´rale (Grant IAP-P6/30), Prime Minister’s Office, Federal Service for source of extracellular nucleotides within the heart, especially Science, Technology and Culture, by grants of the Fonds de la Recherche Scientifique under ischemia (12). Extracellular nucleotides are involved in the Me´dicale, the Fonds d’Encouragement a` la Recherche, the Fonds Emile Defay, the Fonds de la Recherche Scientifique Me´dicale of Belgium, the Walloon Region (Pro- protection of rat cardiomyocytes from hypoxic stress through gramme d’Excellence CIBLES), and by LifeSciHealth Program of the European activation of P2Y2 nucleotide receptors (13). UTP administration Community Grant LSHB-2003-503337. D.C. and C.D. are Senior Research Associ- ates of the Fonds National de la Recherche Scientifique. M.H. and S.C. are supported to rats reduced infarct size and enhanced myocardial function by the Fonds National de la Recherche Scientifique/Fonds pour la Recherche dans (14). The implication of P2Y2 receptor in the cardioprotection l’lndustrie et l’Agriculture, Belgium. The funders had no role in study design, data mediated in vivo by UTP has been recently demonstrated (15). collection and analysis, decision to publish, or preparation of the manuscript. We investigated in the present study the potential role of the Address correspondence and reprint requests to Dr. Didier Communi, Institut de Recherche Interdisciplinaire en Biologie Humaine et Mole´culaire, Universite´ Libre P2Y4 receptor in cardiac inflammation and myocardial infarction. de Bruxelles, Building C (5th Floor), Campus Erasme, 808 Route de Lennik, 1070 The human P2Y4 receptor is a UTP receptor coupled to the Brussels, Belgium. E-mail address: [email protected] phosphoinositide/Ca2+ pathway (16). Recombinant rat and mouse Abbreviations used in this article: EF, ejection fraction; ET-1, endothelin-1; HPRT, orthologs of human P2Y4 receptor are activated by UTP and hypoxanthine phosphoribosyltransferase; LAD, left anterior descending artery; LVED, left ventricular end-diastolic volume; LVES, left ventricular end-systolic ATP (17–19), as described for the ubiquitously expressed mouse volume; MI, myocardial infarction; MMP, matrix metalloproteinase; MPO, myelo- P2Y2 receptor (20). Interestingly, P2Y2 and P2Y4 receptors were peroxidase; TIMP, tissue inhibitor of matrix metalloproteinase; VEGF, vascular en- abundantly detected in the rat developing heart (21). P2Y -null dothelial growth factor. 4 mice have been generated in our laboratory (22) and we demon- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 strated P2Y4 expression in cardiac endothelial cells but not in www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401364 2 LOSS OF MOUSE P2Y4 PROTECTS AGAINST MYOCARDIAL INFARCTION cardiomyocytes (23). We showed recently that this receptor is in- Allylamine-induced heart fibrosis volved in postnatal heart development (23), as well as in exercise 0/+ 0/2 Male P2Y4 and P2Y4 mice were given 0.1% allylamine-HCl as tolerance and exercise-induced cardiac hypertrophy (24). drinking fluid ad libitum. These mice demonstrated a sustained decreased In this study, we demonstrated that loss of mouse P2Y4 receptor fluid consumption. For histopathological study, mice were given allyl- is associated with an unexpected protection against infarction and amine for 3, 7, 14, or 30 d and were killed by cervical dislocation. The a reduction of cardiac inflammation, permeability, and fibrosis. hearts were rapidly removed and frozen. Quantification of fibrosis in ischemic or allylamine-treated Materials and Methods mice +/+ 2/2 Ischemia experiments: LAD ligation of P2Y4 and P2Y4 Heart sections of ischemic or allylamine-treated mice were
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