biomolecules Article Increased Arginase Expression and Decreased Nitric Oxide in Pig Donor Lungs after Normothermic Ex Vivo Lung Perfusion Farshad Tavasoli 1,2 , Mingyao Liu 2,3,4 , Tiago Machuca 3,4 , Riccardo Bonato 3,4 , David R. Grant 5 , Marcelo Cypel 2,3,4 , Shaf Keshavjee 2,3,4 and Hartmut Grasemann 1,2,6, * 1 Translational Medicine, SickKids Research Institute, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; f_tavasoli@yahoo.ca 2 Institute of Medical Science, University of Toronto, Toronto, ON M5S 1A8, Canada; Mingyao.Liu@utoronto.ca (M.L.); Marcelo.Cypel@uhn.ca (M.C.); Shaf.Keshavjee@uhn.ca (S.K.) 3 Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute, Toronto, ON M5G 2C4, Canada; Tiago.machuca@surgery.ufl.edu (T.M.); riccardo.bonato@uhn.ca (R.B.) 4 Toronto Lung Transplant Program, University Health Network, Toronto, ON M5G 2C4, Canada 5 Department of Surgery, Toronto General Hospital, Toronto, ON M5G 2C4, Canada; david.grant@uhn.ca 6 Division of Respiratory Medicine, Department of Paediatrics, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada * Correspondence: hartmut.grasemann@sickkids.ca; Tel.: +416-813-7654 (ext. 6346); Fax: +416-813-6246 Received: 27 September 2019; Accepted: 11 February 2020; Published: 14 February 2020   Abstract: An established pig lung transplantation model was used to study the effects of cold ischemia time, normothermic acellular ex vivo lung perfusion (EVLP) and reperfusion after lung transplantation on l-arginine/NO metabolism in lung tissue. Lung tissue homogenates were analyzed for NO metabolite (NOx) concentrations by chemiluminescent NO-analyzer technique, and l-arginine, l-ornithine, l-citrulline and asymmetric dimethylarginine (ADMA) quantified using liquid chromatography-mass spectrometry (LC-MS/MS). The expression of arginase and nitric oxide synthase (NOS) isoforms in lung was measured by real-time polymerase chain reaction. EVLP preservation resulted in a significant decrease in concentrations of NOx and l-citrulline, both products of NOS, at the end of EVLP and after reperfusion following transplantation, compared to control, respectively. The ratio of l-ornithine over l-citrulline, a marker of the balance between l-arginine metabolizing enzymes, was increased in the EVLP group prior to reperfusion. The expression of both arginase isoforms was increased from baseline 1 h post reperfusion in EVLP but not in the no-EVLP group. These data suggest that EVLP results in a shift of the l-arginine balance towards arginase, leading to NO deficiency in the lung. The arginase/NOS balance may, therefore, represent a therapeutic target to improve lung quality during EVLP and, subsequently, transplant outcomes. Keywords: lung transplantation; ex vivo lung perfusion; nitric oxide; arginase 1. Introduction Endogenous nitric oxide (NO) is important in the regulation of various physiological and patho-physiological conditions, including airway smooth muscle tone, vascular resistance and immune responses [1–3]. NO is produced by nitric oxide synthases (NOSs) that catalyze the reaction of l-arginine to l-citrulline. The constitutive NOS isoenzymes, neuronal NOS (nNOS) and endothelial NOS (eNOS) can be distinguished from inducible NOS (iNOS). The expression of iNOS is increased in response to inflammatory stimuli [2–4]. NOS activity depends on the presence of substrate l-arginine, and low l-arginine availability can cause NOS uncoupling and consequent formation of reactive Biomolecules 2020, 10, 300; doi:10.3390/biom10020300 www.mdpi.com/journal/biomolecules