Mechanisms of Retinal Ischemia/Reperfusion Injury: Arginase and the Mitochondria

Abstract

ObjectiveOur group has previously shown that the ureahydrolyase mitochondrial isoenzyme, arginase 2 (A2) is involved in ischemia reperfusion (I/R)‐induced retinal neurovascular degeneration. We found that A2 homozygous deletion significantly protected against necroptosis, neurovascular degeneration, retinal thinning, and impairment of retinal function. Interestingly, our group has also shown a link between hypoxia‐induced increases in A2 and impaired angiogenesis in endothelial cells. The cytosolic isoform arginase 1 (A1) was not affected which suggests distinct roles of A1 versus A2 under hypoxic conditions. However, the exact mechanisms of arginase‐induced neurovascular damage in retinal I/R are not yet known. In the current study, we investigated the involvement of arginase in retinal I/R injury in vivo and in vitro in relation to mitochondrial function.MethodsI/R insult was conducted on the right eyes of wild type (WT) and A2 homozygous knockout (A2−/−) mice. The left eyes were used as sham controls. Bovine retinal endothelial cells (BRECs) subjected to oxygen glucose deprivation (OGD/R) were used as an in vitro model. Cells were treated with the arginase inhibitor, Amino‐2‐Borono‐6‐Hexanoic Acid (ABH, 100μM), recombinant Pegylated A1 (PegA1, 1μg/ml), or vehicle. Western blot was used to evaluate different protein levels. Mitochondria structure and function were evaluated using Rhodamine 123 labeling and Seahorse XF analyzer respectively.ResultsWe found that the mitochondrial fission protein, dynamin related protein (Drp1) was significantly increased in WT I/R retinas compared to sham controls at 3 and 6 hours. With A2 deletion, the Drp1 levels were also elevated after I/R but were significantly reduced to sham control levels at 6 hours. In cell culture studies, A2 protein levels were significantly increased after 6h OGD/ 6h R. The cleavage of poly ADP‐ribose polymerase (PARP) and phosphorylation of p38‐MAPK were significantly increased after OGD/R. Arginase inhibition significantly reduced the phosphorylation of p38 and slightly reduced PARP cleavage. Western blot analysis showed increased Drp1 expression and mitochondria labeling showed more fragmented mitochondria after OGD/R. With arginase inhibition, the mitochondria were restored and resumed a more normal morphology with significantly less Drp1 expression. Seahorse XF analysis of the oxygen consumption rate (OCR) showed a significant decrease in maximal respiration and spare respiratory capacity after OGD/R indicating impaired mitochondrial function. Treatment with PegA1 significantly prevented the OGD/R‐induced mitochondrial dysfunction as reflected by increased spare respiratory capacity and maximal respiration. However, treatment with the non‐selective arginase inhibitor (ABH) did not improve mitochondrial function under OGD/R conditions.ConclusionsThis study is the first to show that A1 and A2 could be playing different roles under ischemic conditions by affecting the mitochondrial function in distinct ways. Furthermore, PegA1 therapy may offer a new strategy for limiting hypoxia‐induced impairment of angiogenic function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.