Targeting proliferative retinopathy: Arginase 1 limits vitreoretinal neovascularization and promotes angiogenic repair.

Current therapies for treatment of proliferative retinopathy focus on retinal neovascularization (RNV) during advanced disease and can trigger adverse side-effects. Here, we have tested a new strategy for limiting neurovascular injury and promoting repair during early-stage disease. We have recently shown that treatment with a stable, pegylated drug form of the ureohydrolase enzyme arginase 1 (A1) provides neuroprotection in acute models of ischemia/reperfusion injury, optic nerve crush, and ischemic stroke. Now, we have determined the effects of this treatment on RNV, vascular repair, and retinal function in the mouse oxygen-induced retinopathy (OIR) model of retinopathy of prematurity (ROP). Our studies in the OIR model show that treatment with pegylated A1 (PEG-A1), inhibits pathological RNV, promotes angiogenic repair, and improves retinal function by a mechanism involving decreased expression of TNF, iNOS, and VEGF and increased expression of FGF2 and A1. We further show that A1 is expressed in myeloid cells and areas of RNV in retinal sections from mice with OIR and human diabetic retinopathy (DR) patients and in blood samples from ROP patients. Moreover, studies using knockout mice with hemizygous deletion of A1 show worsened RNV and retinal injury, supporting the protective role of A1 in limiting the OIR-induced pathology. Collectively, A1 is critically involved in reparative angiogenesis and neuroprotection in OIR. Pegylated A1 may offer a novel therapy for limiting retinal injury and promoting repair during proliferative retinopathy.

Introduction

Ischemic retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP), are major causes of blindness in adults and neonates, respectively. Both conditions are characterized by vaso-obliteration and lack of adequate vascular repair. This leads to relative hypoxia, which induces upregulation of pro-inflammatory and pro-angiogenic growth factors and promotes pathological retinal neovascularization (RNV). Laser photocoagulation is usually effective for treatment of advanced retinopathy but can impair vision and in some patients, the pathology continues to progress. Intra-ocular injections with anti-vascular endothelial growth factor (VEGF) agents show promise in both DR [1] and severe late-stage ROP [2, 3]. However, neither treatment promotes tissue repair and there is a potential risk of adverse effects and disease recurrence with anti-VEGF therapy [4, 5]. Thus, there is a great need for new therapies to limit the neurovascular injury and promote repair.

Our previous studies have shown that expression of the ureohydrolase enzyme arginase is critically involved in retinal injury and repair in models of retinopathy [6-11]. Arginase has two isoforms, arginase 1 (A1), which is cytosolic, and arginase 2 (A2), which is mitochondrial [12]. The two isoforms have very similar mechanisms of action. Both metabolize arginine to produce urea and ornithine. However, they differ substantially in terms of their tissue distribution and involvement in disease and injury. Arginase 1 is highly expressed in the liver where it plays a critical role in the urea cycle. Mutations in A1 can result in hyperammonemia and A1 global knockout mice die soon after birth. A2 is highly expressed in the kidney but is also expressed in many other tissues. In contrast with A1 deficiency, mice globally deficient in A2 show no noticeable phenotype. Studies have shown that A1 plays a key role in the repair phase of wound healing, whereas A2 has been implicated in chronic inflammatory disease conditions [12].

Both isoforms have also been implicated in retinal injury and repair. Our studies in mouse models of oxygen-induced retinopathy (OIR), retinal ischemia/reperfusion injury, and optic nerve crush have demonstrated the involvement of the A2 isoform in neurovascular injury. We showed that deletion of the A2 gene significantly reduced both neuronal and vascular injury during OIR, while enhancing vascular repair and limiting pathological RNV [6-8]. Studies using the ischemia/reperfusion and optic nerve crush models showed retinal protection with A2 gene deletion [9, 10]. In contrast, A1 deletion in A2 deficient mice with OIR or A1 deletion in the ischemia/reperfusion mouse model amplified the signs of injury [8, 11]. Consistently, studies in acute models of ischemia/reperfusion, optic nerve crush, or ischemic stroke showed that treatment with a stable (pegylated) form of A1 limited neuronal injury [11, 13]. Here, we have characterized the specific effects of this A1 treatment on RNV, vascular repair, and neuronal function in the OIR model and examined the underlying mechanisms.

Materials and methods

Owing to word limit, only main experiments are described here and detailed methods for the rest of experiments together with full (uncropped) Western blots are included in the supplementary file.

OIR mouse model

OIR was induced in newborn mice according to the protocol of Smith, et al. with some adjustment [14, 15]. On P7, pups of both sexes were placed along with their dams in a hyperoxia (75% oxygen) chamber for up to 5 days, after which they were transferred back to room air (RA, 21% oxygen) on P12. The OIR model is characterized by vaso-obliteration of the central retinal vessels (P7-P12) followed by vascular regrowth (P12-P17) and pathological retinal neovascularization (RNV, P14-P17) [15]. Controls were maintained in RA. Mice were sacrificed during the periods of vaso-obliteration, regrowth, and RNV (Fig. S1).

Mice were compared to their littermate controls and therefore no specific randomization scheme was used. Data were pooled together to minimize variability between litters due to differences in litter size/weight [16]. Both sexes were combined given the lack of differences between sexes. Experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the institutional animal care and use committee (Animal Welfare Assurance no. A3307–01).

PEG-A1 treatment in OIR

A pharmaceutical grade of PEG-A1 was a kind gift from Bio-Cancer Treatment International Limited (BCT, Hong Kong). PEG-A1 is a recombinant human arginase (rhArg) covalently attached to methoxy polyethylene glycol (mPEG-SPA; MW 5000) to increase its stability and half-life in vivo, which has been reported to be 3 days vs. a few minutes for the native enzyme [17]. PEG-A1 was prepared from a 3.4 mg/mL stock by dilution in PBS (1:250 ratio) to achieve final concentration of 13.6 ng/μL. PBS was used as vehicle control. Intravitreal injections were performed on anesthetized pups using a 36-gauge NanoFil needle mounted to a 10-μL Hamilton syringe (World Precision Instruments). Two treatment strategies were employed. To examine vaso-obliteration, wildtype (WT, C57BL6J) pups received single intravitreal injection of PEG-A1 (6.8 ng in 0.5 μL—based on a preliminary dose/response study) at P7 then subjected to hyperoxia (75% oxygen) for 2 days and sacrificed at P9. The P9 time point was selected based on the fact that vaso-obliteration occurs within the first 48 h of hyperoxia treatment [15, 18, 19]. Another cohort of WT pups was placed in hyperoxia (75% oxygen) on P7, switched to RA on P12, immediately given single intravitreal PEG-A1 injections (6.8 ng in 0.5 μL), and sacrificed on P17 (Fig. S1A).

A1 deletion in OIR

To assess the specific role of A1 expression in OIR, we used A1 knock out mice and WT littermates (Fig. S1B) [20-22]. As deletion of both copies of A1 is lethal due to hyperammonemia, we used heterozygous mice lacking 1 copy of A1 (A1+/− or A1 KO), which is sufficient to dampen its activity [20-22]. These mice develop normally. A 70% oxygen concentration was used for experiments involving A1 KO mice based on preliminary experiments showing intolerance of the A1 KO mice to hyperoxia treatment.

Myeloid and endothelial cell-specific A1 KO mice were used in this study under LysM cre and Cdh5 cre respectively as described and characterized before [11].

Statistical analysis

Sample size was determined based on our previous experience with the OIR model. Outliers were determined and excluded based on GraphPad Prism outliers calculator. Data were analyzed by investigators blinded to the group identity. Statistical analysis was conducted using GraphPad Prism 9 software. Differences between two groups were determined by student’s t-test. Differences between multiple groups were analyzed by ANOVA with Tukey’s post hoc test. P-values < 0.05 were considered statistically significant. Graphs were prepared using GraphPad Prism 9 software and data were presented as mean ± standard deviation (SD).