Catalase therapy corrects oxidative stress-induced pathophysiology in incipient diabetic retinopathy.

Accumulating evidence supports diabetes-evoked rod-dominated retinal oxidative stress as a pathogenic factor in experimental diabetic retinopathy.1–4 However, targeting such stress using conventional antioxidant treatment is complicated by the fact that reactive oxygen species (ROS) have a central role in a number of normal cellular activities, including endomembrane signaling cascades, communication networks, and metabolic regulatory complexes.5 Here, we examine the effects of a peroxisome-targeted protein biologic on specific biomarkers associated with diabetic retinopathy. The peroxisome is a critical, but understudied subcellular constituent that is involved in a number of important redox-based metabolic processes in the cell.6,7 These peroxisomal reactions are significant contributors to the oxidative load in aged cells and in several degenerative diseases, including diabetes.7,8 Peroxisomal ROS accumulates for a number of reasons including, perhaps most importantly, mislocalization or impaired activity of catalase, its major resident antioxidant enzyme.7,9 Diabetes reduces whole retinal catalase levels, although a specific contribution of peroxisomes per se in diabetic retinopathy is unclear.10–14 The goal of this study was to begin to determine if targeting cells with catalase supplementation could confer antioxidant protection in the context of diabetic retinopathy. To this end, we examined oxidative stress in retinal cells exposed to high glucose conditions, and determined if an in vivo biomarker sensitive to antioxidant treatment in diabetic models also was positively impacted by targeted catalase treatment. In this feasibility study, hydrogen peroxide (H2O2) levels were first evaluated in retinal (Muller and RPE) cell lines chronically exposed to high glucose levels and treated with CAT-SKL, a cell penetrating catalase derivative designed to traffic to peroxisomes (described in a previous study8 and references therein). Polyamines, which include spermine, spermidine, and putrescine, are prevalent in the retina.15 These molecules have been reported to possess, among other activities, antioxidiant/radical scavenger action. Interestingly, polyamines also have been suggested to contribute to diabetes-induced oxidative stress in endothelial cells.16 The latter effect may be mediated by increased activity of the H2O2-generating peroxisomal enzyme, polyamine oxidase. Indeed, in a clinical study, diabetes increased ocular polyamines raising the possibility of enhanced polyamine oxidase activity.17 Thus, we examined the effects of the polyamine synthesis inhibitor, difluoromethylornithine (DFMO), which demonstrates antioxidant benefits in certain contexts, but whose impact on diabetic retinopathy have yet to be described. Our study represents a first step in parsing the contributions of these different activities in diabetic retinopathy. We also examined whether CAT-SKL or DFMO might be useful in vivo using manganese-enhanced magnetic resonance imaging (MEMRI), a noninvasive measurement of intraretinal uptake of manganese ion (Mn2+, a strong MRI contrast agent) as an analytical biomarker of intraretinal calcium channel activity in the retina encoded while animals are awake and freely moving.18–20 MEMRI recently has emerged as the imaging modality of choice when performing studies of retinal ion activity.21 In diabetic rats and mice, reduced intraretinal uptake of manganese precedes the appearance of frank retinal histopathology.22,23 Importantly, antioxidant therapies that correct these early MEMRI impairments also correct later diabetic retinopathy.22,23 For these reasons, we used MEMRI to begin an analysis of CAT-SKL and DMFO's in vivo efficacies. Here, we find that CAT-SKL reduced diabetes-evoked H2O2 production in retinal cells and partly improved the subnormal retinal uptake of manganese in vivo. On the other hand, DFMO showed no such effects on retinal cells or in our in vivo analyses. These results suggest the use of a targeted antioxidant, CAT-SKL, as a potential useful treatment against the oxidative stress associated with diabetic retinopathy.