Development of pegylated arginase 1 as a therapy for retinal ischemic injury

Abstract

Purpose : Arginase 1 (A1) hydrolyzes arginine to form ornithine and urea. Ornithine is further converted to polyamines through ornithine decarboxylase (ODC). We have recently shown that deletion of myeloid A1 exacerbates retinal damage after ischemia/reperfusion (IR) injury whereas PEGylated arginase 1 (PEG-A1) intravitreal treatment is neuroprotective. Furthermore, A1 knockout (KO) macrophages (MΦ) exhibit an increased inflammatory response which is ameliorated with PEG-A1 treatment. This study aimed to examine the utility of systemic PEG-A1 treatment and elucidate the epigenetic mechanisms underlying A1 protection.

Methods : Peritoneal MΦ were isolated from myeloid A1 KO or floxed control mice and treated with lipopolysaccharide (LPS) ± PEG-A1, the ODC inhibitor difluoromethylornithine (DFMO), or the specific HDAC3 inhibitor RGFP966, for 24h. A cohort of WT mice were subjected to retinal IR injury and treated with intraperitoneal PEG-A1 or RGFP966. Retina explants from myeloid A1 KO and control mice were subjected to oxygen-glucose deprivation (OGD – 3h) followed by reoxygenation (R – 21h).

Results : Screening for HDACs and inflammation showed that A1 deletion or ODC inhibition increased HDAC3 expression and exacerbated the inflammatory response (increasing iNOS, NLRP3, and pro-IL-1β) in LPS-treated MΦ. PEG-A1 treatment dampened the upregulation of HDAC3 and reduced the inflammatory response. HDAC3 inhibition ameliorated the inflammatory response in A1 KO MΦ. In vivo, HDAC3 co-localized with microglia/MΦ at 48 h after IR in WT retinas and was further increased in A1-deficient retinas. HDAC3 inhibition improved neuronal cell survival (76±14% of sham vs vehicle 63±10%, n=7-8, mean±S.D, p=0.07) and prevented retinal thinning n=3. Systemic PEG-A1 treatment was neuroprotective (62.4±21% vs vehicle 39.2±12.9%, n=6-8, mean±S.D, p=0.03) and successfully crossed the blood-retina barrier in the injured eye after IR but not the sham eye (n=3). Unlike our previous in vivo data, retina explants from myeloid A1 KO mice showed neurodegeneration similar to the control retinas after OGD, suggesting an important role of A1 in infiltrating myeloid cells in vivo.

Conclusions : A1 mediates its anti-inflammatory effects via ODC-mediated suppression of HDAC3, which is a central player in the MΦ inflammatory response. Systemic PEG-A1 administration provides an excellent route to deliver the drug to the retina after acute ischemic injury.

This is a 2020 ARVO Annual Meeting abstract.