Increased Arginase Expression and Decreased Nitric Oxide in Pig Donor Lungs after Normothermic Ex Vivo Lung Perfusion.

Farshad Tavasoli,David R Grant,Mingyao Liu,Marcelo Cypel,Riccardo Bonato,Tiago Machuca,Shaf Keshavjee,Hartmut Grasemann

doi:10.3390/biom10020300

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.

Highlights

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 [1,2,3]
Our data show that NOx levels in lung tissue after 6 h CIT and 12 h ex vivo lung perfusion (EVLP) were significantly reduced when compared to normal control lung, and remained decreased after 1 hr of reperfusion
As NO and l-citrulline are both products of l-arginine metabolism from nitric oxide synthase (NOS), these data, suggest that EVLP preservation resulted in reduced NOS activity and reduced NO formation in lung

Summary

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 [1,2,3]. In a rabbit model of lung transplantation, administration of the NO-precursor l-arginine during reperfusion reduced I/R injury after cold ischemia by preventing vascular endothelial dysfunction [20]. L-arginine supplementation during warm reperfusion following 12 h of cold ischemia time in neonatal piglet lungs resulted in improved pulmonary function as measured by partial oxygen pressure and lung compliance [21]. Normothermic ex vivo lung perfusion (EVLP) renders physiological conditions to maintain normal cellular metabolism in the donor organ and thereby provides the opportunity to evaluate lung function, tissue recovery and therapeutic interventions prior to transplantation, without increasing the risk of post-transplant complications [22,23,24,25,26,27,28,29]. We studied the pulmonary l-arginine/NO metabolism in the immediate pre- and post-transplantation period utilizing an established pig model of normothermic EVLP and lung transplantation

Animals

Tissue Processing and Analyses

Statistics

Findings

Discussion