Jutamas Suwanpradid

Arginase 2 deletion reduces neuro-glial injury and improves retinal function in a model of retinopathy of prematurity.

Background Retinopathy of prematurity (ROP) is a major cause of vision impairment in low birth weight infants. While previous work has focused on defining the mechanisms of vascular injury leading to retinal neovascularization, recent studies show that neurons are also affected. This study was undertaken to determine the role of the mitochondrial arginine/ornithine regulating enzyme arginase 2 (A2) in retinal neuro-glial cell injury in the mouse model of ROP. Methods and Findings Studies were performed using wild type (WT) and A2 knockout (A2−/−) mice exposed to Oxygen Induced Retinopathy (OIR). Neuronal injury and apoptosis were assessed using immunohistochemistry, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end) labeling and Western blotting. Electroretinography (ERG) was used to assess retinal function. Neuro-glial injury in WT ROP mice was evident by TUNEL labeling, retinal thinning, decreases in number of rod bipolar cells and glial cell activation as compared with room air controls. Significant reduction in numbers of TUNEL positive cells, inhibition of retinal thinning, preservation of the rod bipolar cells and prevention of glial activation were observed in the A2−/− retinas. Retinal function was markedly impaired in the WT OIR mice as shown by decreases in amplitude of the b-wave of the ERG. This defect was significantly reduced in A2−/− mice. Levels of the pro-apoptotic proteins p53, cleaved caspase 9, cytochrome C and the mitochondrial protein Bim were markedly increased in WT OIR retinas compared to controls, whereas the pro-survival mitrochondrial protein BCL-xl was reduced. These alterations were largely blocked in the A2−/− OIR retina. Conclusions Our data implicate A2 in neurodegeneration during ROP. Deletion of A2 significantly improves neuronal survival and function, possibly through the regulation of mitochondrial membrane permeability mediated apoptosis during retinal ischemia. These molecular events are associated with decreased activation of glial cells, suggesting a rescue effect on macroglia as well.

Arginase in Retinopathy

Ischemic retinopathies, such as diabetic retinopathy (DR), retinopathy of prematurity and retinal vein occlusion are a major cause of blindness in developed nations worldwide. Each of these conditions is associated with early neurovascular dysfunction. However, conventional therapies target clinically significant macula edema or neovascularization, which occur much later. Intra-ocular injections of anti-VEGF show promise in reducing retinal edema, but the effects are usually transient and the need for repeated injections increases the risk of intraocular infection. Laser photocoagulation can control pathological neovascularization, but may impair vision and in some patients the retinopathy continues to progress. Moreover, neither treatment targets early stage disease or promotes repair. This review examines the potential role of the ureahydrolase enzyme arginase as a therapeutic target for the treatment of ischemic retinopathy. Arginase metabolizes l-arginine to form proline, polyamines and glutamate. Excessive arginase activity reduces the l-arginine supply for nitric oxide synthase (NOS), causing it to become uncoupled and produce superoxide and less NO. Superoxide and NO react and form the toxic oxidant peroxynitrite. The catabolic products of polyamine oxidation and glutamate can induce more oxidative stress and DNA damage, both of which can cause cellular injury. Studies indicate that neurovascular injury during retinopathy is associated with increased arginase expression/activity, decreased NO, polyamine oxidation, formation of superoxide and peroxynitrite and dysfunction and injury of both vascular and neural cells. Furthermore, data indicate that the cytosolic isoform arginase I (AI) is involved in hyperglycemia-induced dysfunction and injury of vascular endothelial cells whereas the mitochondrial isoform arginase II (AII) is involved in neurovascular dysfunction and death following hyperoxia exposure. Thus, we postulate that activation of the arginase pathway causes neurovascular injury by uncoupling NOS and inducing polyamine oxidation and glutamate formation, thereby reducing NO and increasing oxidative stress, all of which contribute to the retinopathic process.

Anti-angiogenic actions of the mangosteen polyphenolic xanthone derivative α-mangostin☆

Retinal neovascularization is a major cause of vision loss in diseases characterized by retinal ischemia and is characterized by the pathological growth of abnormal vessels. Vascular endothelial growth factor (VEGF) is known to play an important role in this process. Oxidative stress has been strongly implicated in up-regulation of VEGF associated with neovascularization in various tissues. Hence, compounds with anti-oxidant actions can prevent neovascularization. α-Mangostin, a component of mangosteen (Garcinia mangostana Linn), has been shown to have an anti-oxidant property in pathological conditions involving angiogenesis such as cancer. However, the effect of α-mangostin on ROS formation and angiogenic function in microvascular endothelial cells has not been studied. Hence, this study demonstrated the anti-angiogenic effects of α-mangostin in relation to ROS formation in bovine retinal endothelial cells (REC). α-Mangostin significantly and dose-dependently reduced formation of ROS in hypoxia-treated REC. α-Mangostin also significantly and dose-dependently suppressed VEGF-induced increases in permeability, proliferation, migration and tube formation in REC and blocked angiogenic sprouting in the ex vivo aortic ring assay. In addition, α-mangostin inhibited VEGF-induced phosphorylation of VEGFR2 and ERK1/2-MAPK. According to our results, α-mangostin reduces oxidative stress and limits VEGF-induced angiogenesis through a process involving abrogation of VEGFR2 and ERK1/2-MAPK activation.

Arginase 2 deficiency prevents oxidative stress and limits hyperoxia-induced retinal vascular degeneration.

Background Hyperoxia exposure of premature infants causes obliteration of the immature retinal microvessels, leading to a condition of proliferative vitreoretinal neovascularization termed retinopathy of prematurity (ROP). Previous work has demonstrated that the hyperoxia-induced vascular injury is mediated by dysfunction of endothelial nitric oxide synthase resulting in peroxynitrite formation. This study was undertaken to determine the involvement of the ureahydrolase enzyme arginase in this pathology. Methods and Findings Studies were performed using hyperoxia-treated bovine retinal endothelial cells (BRE) and mice with oxygen-induced retinopathy (OIR) as experimental models of ROP. Treatment with the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) prevented hyperoxia-induced apoptosis of BRE cells and reduced vaso-obliteration in the OIR model. Furthermore, deletion of the arginase 2 gene protected against hyperoxia-induced vaso-obliteration, enhanced physiological vascular repair, and reduced retinal neovascularization in the OIR model. Additional deletion of one copy of arginase 1 did not improve the vascular pathology. Analyses of peroxynitrite by quantitation of its biomarker nitrotyrosine, superoxide by dihydroethidium imaging and NO formation by diaminofluoroscein imaging showed that the protective actions of arginase 2 deletion were associated with blockade of superoxide and peroxynitrite formation and normalization of NOS activity. Conclusions Our data demonstrate the involvement of arginase activity and arginase 2 expression in hyperoxia-induced vascular injury. Arginase 2 deletion prevents hyperoxia-induced retinal vascular injury by preventing NOS uncoupling resulting in decreased reactive oxygen species formation and increased nitric oxide bioavailability.