Myeloid Arginase 1 Protects Against Retinal Ischemia‐Reperfusion Injury
Authors: FOUDA ABDELRAHMAN , Xu Zhimin , Shosha Esraa , Chen Jijun , Caldwell William , Narayanan Priya , Caldwell Ruth 
Published: June 2021

PurposeWe have recently shown that the mitochondrial, ureahydrolase enzyme arginase 2 (A2) mediates retinal neurovascular degeneration after ischemia‐reperfusion (IR) injury by increasing oxidative stress and inflammation. This project aimed to examine the role of the cytosolic enzyme isoform, arginase 1 (A1) in retinal IR injury. We specifically focused on endothelial and myeloid cells because A1 competes with the nitric oxide synthase (NOS) enzymes in these cells for the common substrate L‐arginine. L‐arginine depletion reduces NO formation. This promotes vascular dysfunction when endothelial NOS is involved but prevents inflammatory injury when inducible NOS is involved.MethodsWild type (WT) mice, global A1+/− knock out (KO), endothelial specific (Cdh5‐cre;A1f/f) A1 KO, and myeloid specific (LysM‐cre;A1f/f) A1 KO mice were subjected to retinal ischemia for 40 minutes through raising the intraocular pressure in the right eye followed by reperfusion to achieve IR‐injury. The left eye served as sham control. A cohort of WT mice were treated with intravitreal injection of pegylated (peg) A1 (1.7 ng) or PBS at 3 hours after IR. Retinas were collected at different time‐points for analysis. At 7 days, retinal thinning was measured using optical coherence tomography (OCT) in live mice and morphometric analysis of hematoxylin and eosin (H & E) stained tissue sections. Neurodegeneration and microglia/macrophage activation were analyzed by confocal imaging in retinal flat mounts using NeuN and Iba‐1 labeling of inner retinal neurons and microglia/macrophages, respectively. At 14 days, retina function was assessed in live mice using electroretinography (ERG) while vascular degeneration was assessed by retina trypsin digestion to quantify the loss of vascular endothelial cells and pericytes. In vitro experiments using primary peritoneal macrophages (MΦ) were conducted to examine the role of A1 in MΦ inflammatory response to lipopolysaccharide (LPS) stimulation.ResultsGlobal as well as myeloid specific A1 KO mice showed worsened IR‐induced neuronal loss and retinal thinning. Deletion of A1 in endothelial cells had no effect while treatment with peg A1 improved neuronal survival in WT mice (table). In addition to worsened neurodegeneration, A1+/− KO mice showed reduced retina function and worsened vascular injury manifested by a 50 % increase in endothelial cell/pericyte loss (p<0.05, n=4–6). Western blotting analysis of retinal tissue showed that the worsened IR outcome with A1 deletion was associated with tumor necrosis factor α (TNFα)/receptor interacting protein 3 kinase (RIP3)/dynamin‐related protein 1 (DRP1) mediated necroptosis.In vitro experiments showed that macrophage (MΦ) lacking A1 exhibit increased inflammatory response upon LPS stimulation, as demonstrated by increased iNOS expression, activation of the NLRP3 inflammasome, increased IL‐1β, TNFα and nitric oxide (NO) production. PegA1 treatment dampened this inflammatory response. Moreover, intravitreal injection of WT MΦ at 3 days after IR improved neuronal survival compared to A1 KO MΦ while systemic MΦ depletion with clodronate liposomes increased neuronal loss (table).ConclusionA1 reduces IR‐induced retinal neurovascular degeneration and necroptosis via dampening macrophage inflammatory responses. Increasing A1 offers a novel strategy for limiting neurovascular injury and promoting macrophage‐mediated repair in the early stages of ischemic retinopathy.Support or Funding InformationThis work was supported by grants from The National Institute of Health (NIH grant R01‐EY11766 (RBC)), the Department of Veterans Affairs, Veterans Health Administration (RBC), Office of Research and Development, Biomedical Laboratory Research and Development (BX001233), and the Culver Vision Discovery Institute at Augusta University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.