Arginase 2 Overexpression Aggravates Ischemic Injury in Retinal Vascular Endothelial Cells

Abstract

ObjectiveOur group has previously shown that degeneration of the retinal microvasculature after ischemia/reperfusion (I/R) injury is mediated through increased expression of the mitochondrial isoform of arginase, arginase 2 (A2). In contrast, increased expression of the cytosolic isoform, arginase 1 (A1) is involved in impairment of endothelial cell (EC)‐dependent vasorelaxation. We have shown that global deletion of A2 limits I/R‐induced vascular degeneration whereas EC‐specific deletion of A1 prevents diabetes‐induced EC dysfunction. However, the specific involvement of EC arginase expression in I/R‐induced vascular EC injury is as yet unknown. Now we have investigated the specific impact of increasing EC expression of A1 or A2 on EC survival in relation to mitochondrial function by studies using an in vitro model of I/R injury.MethodsStudies were performed using bovine retinal ECs transduced with or without adenoviral vectors to overexpress A1 or A2. I/R was modeled by subjecting the cultures to oxygen glucose deprivation (OGD) for 5 hours followed by reperfusion for 1 hour (OGD/R). Some cultures were treated with or without the specific but non‐selective inhibitor of arginase ABH (amino‐2‐borono‐6‐hexanoic acid). Cell extracts were collected for western blot. Media were collected to evaluate cell death using lactate dehydrogenase (LDH) release assay. Seahorse XF analyzer was used to evaluate mitochondrial function.ResultsStudies using normal ECs exposed to OGD/R in the presence or absence of ABH showed that inhibiting arginase protects against mitochondrial injury as shown by decreases in mitochondrial fragmentation and reduced expression of the mitochondrial fission marker Drp1 (dynamin related protein 1). Western blot analysis showed that OGD/R promoted a significant increase in A2 expression whereas A1 expression was unchanged. Seahorse analysis of the oxygen consumption rate (OCR), under normoxic conditions, showed that A2 overexpression significantly decreased basal respiration and ATP production indicating mitochondrial dysfunction. OGD/R insult in non‐transduced ECs caused mitochondrial dysfunction as demonstrated by decreased basal respiration, reduced ATP production and decreased spare respiratory capacity. A2 overexpression significantly aggravated the OGD/R‐induced mitochondrial dysfunction as shown by further decreases in basal respiration and ATP production. Overexpression of A1 had no effect on these parameters. A2 overexpression also increased phosphorylation of the stress kinase p38 mitogen‐activated protein kinase (p38‐MAPK) and increased release of LDH as compared to OGD/R alone indicating increases in cell stress and cell death. Overexpression of A1 had no effect on either measure.ConclusionsThe present study is the first to show that A2 could be playing a role in promoting OGD/R‐induced cell death through mediating mitochondrial dysfunction.Support or Funding InformationR01‐EY11766, VA grant BX001233, Culver Vision Discovery Institute at Augusta UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.