S. Priya Narayanan

Arginase: an old enzyme with new tricks

Arginase has roots in early life-forms. It converts L-arginine to urea and ornithine. The former provides protection against NH3; the latter serves to stimulate cell growth and other physiological functions. Excessive arginase activity in mammals has been associated with cardiovascular and nervous system dysfunction and disease. Two relevant aspects of this elevated activity may be involved in these disease states. First, excessive arginase activity reduces the supply of L-arginine needed by nitric oxide (NO) synthase to produce NO. Second, excessive production of ornithine leads to vascular structural problems and neural toxicity. Recent research has identified inflammatory agents and reactive oxygen species (ROS) as drivers of this pathologic elevation of arginase activity and expression. We review the involvement of arginase in cardiovascular and nervous system dysfunction, and discuss potential therapeutic interventions targeting excess arginase.

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.

Arginase as a mediator of diabetic retinopathy.

We have shown previously that diabetes causes increases in retinal arginase activity that are associated with impairment of endothelial cell (EC)-dependent vasodilation and increased formation of the peroxynitrite biomarker nitrotyrosine. Arginase blockade normalizes vasodilation responses and reduces nitrotyrosine formation, suggesting that overactive arginase contributes to diabetic retinopathy by reducing NO and increasing oxidative stress. We tested this hypothesis by studies in streptozotocin-induced diabetic mice and high glucose (HG) treated retinal ECs. Our results show that arginase activity is increased in both diabetic retinas and HG-treated retinal ECs as compared with the controls. Western blot shows that both arginase isoforms are present in retinal vessels and ECs and arginase I is increased in the diabetic vessels and HG-treated retinal ECs. Nitrate/nitrite levels are significantly increased in diabetic retinas, indicating an increase in total NO products. However, levels of nitrite, an indicator of bioavailable NO, are reduced by diabetes. Imaging analysis of NO formation in retinal sections confirmed decreases in NO formation in diabetic retinas. The decrease in NO is accompanied by increased O2.− formation and increased leukocyte attachment in retinal vessels. Studies in knockout mice show that arginase gene deletion enhances NO formation, reduces O2.− and prevents leukostasis in the diabetic retinas. HG treatment of retinal ECs also reduces NO release, increases oxidative stress, increases ICAM-1, and induces EC death. Arginase inhibitor treatment reverses these effects. In conclusion, diabetes- and HG-induced signs of retinal vascular activation and injury are associated with increased arginase activity and expression, decreased bioavailable NO, and increased O2.− formation. Blockade of the arginase pathway prevents these alterations, suggesting a primary role of arginase in the pathophysiological process.

Activation of the endothelin system mediates pathological angiogenesis during ischemic retinopathy.

Retinopathy of prematurity adversely affects premature infants because of oxygen-induced damage of the immature retinal vasculature, resulting in pathological neovascularization (NV). Our pilot studies using the mouse model of oxygen-induced retinopathy (OIR) showed marked increases in angiogenic mediators, including endothelins and endothelin receptor (EDNR) A. We hypothesized that activation of the endothelin system via EDNRA plays a causal role in pathological angiogenesis and up-regulation of angiogenic mediators, including vascular endothelial growth factor A (VEGFA) in OIR. Mice were exposed to 75% oxygen from post-natal day P7 to P12, treated with either vehicle or EDNRA antagonist BQ-123 or EDNRB antagonist BQ-788 on P12, and kept at room air from P12 to P17 (ischemic phase). RT-PCR analysis revealed increased levels of EDN2 and EDNRA mRNA, and Western blot analysis revealed increased EDN2 expression during the ischemic phase. EDNRA inhibition significantly increased vessel sprouting, resulting in enhanced physiological angiogenesis and decreased pathological NV, whereas EDNRB inhibition modestly improved vascular repair. OIR triggered significant increases in VEGFA protein and mRNA for delta-like ligand 4, apelin, angiopoietin-2, and monocyte chemoattractant protein-1. BQ-123 treatment significantly reduced these alterations. EDN2 expression was localized to retinal glia and pathological NV tufts of the OIR retinas. EDN2 also induced VEGFA protein expression in cultured astrocytes. In conclusion, inhibition of the EDNRA during OIR suppresses pathological NV and promotes physiological angiogenesis.

Arginase 2 promotes neurovascular degeneration during ischemia/reperfusion injury

Retinal ischemia is a major cause of visual impairment and blindness and is involved in various disorders including diabetic retinopathy, glaucoma, optic neuropathies and retinopathy of prematurity. Neurovascular degeneration is a common feature of these pathologies. Our lab has previously reported that the ureahydrolase arginase 2 (A2) is involved in ischemic retinopathies. Here, we are introducing A2 as a therapeutic target to prevent neurovascular injury after retinal ischemia/reperfusion (I/R) insult. Studies were performed with mice lacking both copies of A2 (A2−/−) and wild-type (WT) controls (C57BL6J). I/R insult was conducted on the right eye and the left eye was used as control. Retinas were collected for analysis at different times (3 h–4 week after injury). Neuronal and microvascular degeneration were evaluated using NeuN staining and vascular digests, respectively. Glial activation was evaluated by glial fibrillary acidic protein expression. Necrotic cell death was studied by propidium iodide labeling and western blot for RIP-3. Arginase expression was determined by western blot and quantitative RT-PCR. Retinal function was determined by electroretinography (ERG). A2 mRNA and protein levels were increased in WT I/R. A2 deletion significantly reduced ganglion cell loss and microvascular degeneration and preserved retinal morphology after I/R. Glial activation, reactive oxygen species formation and cell death by necroptosis were significantly reduced by A2 deletion. ERG showed improved positive scotopic threshold response with A2 deletion. This study shows for the first time that neurovascular injury after retinal I/R is mediated through increased expression of A2. Deletion of A2 was found to be beneficial in reducing neurovascular degeneration after I/R.

Arginase: A multifaceted enzyme important in health and disease

The arginase enzyme developed in early life forms and was maintained during evolution. As the last step in the urea cycle, arginase cleaves l-arginine to form urea and l-ornithine. The urea cycle provides protection against excess ammonia, while l-ornithine is needed for cell proliferation, collagen formation, and other physiological functions. In mammals, increases in arginase activity have been linked to dysfunction and pathologies of the cardiovascular system, kidney, and central nervous system and also to dysfunction of the immune system and cancer. Two important aspects of the excessive activity of arginase may be involved in diseases. First, overly active arginase can reduce the supply of l-arginine needed for the production of nitric oxide (NO) by NO synthase. Second, too much l-ornithine can lead to structural problems in the vasculature, neuronal toxicity, and abnormal growth of tumor cells. Seminal studies have demonstrated that increased formation of reactive oxygen species and key inflammatory mediators promote this pathological elevation of arginase activity. Here, we review the involvement of arginase in diseases affecting the cardiovascular, renal, and central nervous system and cancer and discuss the value of therapies targeting the elevated activity of arginase.

Arginase 1 promotes retinal neurovascular protection from ischemia through suppression of macrophage inflammatory responses

The lack of effective therapies to limit neurovascular injury in ischemic retinopathy is a major clinical problem. This study aimed to examine the role of ureohydrolase enzyme, arginase 1 (A1), in retinal ischemia-reperfusion (IR) injury. A1 competes with nitric oxide synthase (NOS) for their common substrate l-arginine. A1-mediated l-arginine depletion reduces nitric oxide (NO) formation by NOS leading to vascular dysfunction when endothelial NOS is involved but prevents inflammatory injury when inducible NOS is involved. Studies were performed using wild-type (WT) mice, global A1+/− knockout (KO), endothelial-specific A1 KO, and myeloid-specific A1 KO mice subjected to retinal IR injury. Global as well as myeloid-specific A1 KO mice showed worsened IR-induced neuronal loss and retinal thinning. Deletion of A1 in endothelial cells had no effect, while treatment with PEGylated (PEG) A1 improved neuronal survival in WT mice. In addition, A1+/− KO mice showed worsened vascular injury manifested by increased acellular capillaries. Western blotting analysis of retinal tissue showed increased inflammatory and necroptotic markers with A1 deletion. In vitro experiments showed that macrophages lacking A1 exhibit increased inflammatory response upon LPS stimulation. PEG-A1 treatment dampened this inflammatory response and decreased the LPS-induced metabolic reprogramming. Moreover, intravitreal injection of A1 KO macrophages or systemic macrophage depletion with clodronate liposomes increased neuronal loss after IR injury. These results demonstrate that A1 reduces IR injury-induced retinal neurovascular degeneration via dampening macrophage inflammatory responses. Increasing A1 offers a novel strategy for limiting neurovascular injury and promoting macrophage-mediated repair.