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Objective: Chronic inflammation and oxidative stress mediate the pathological progression of diabetic complications, like diabetic retinopathy (DR), peripheral neuropathy (DPN) and impaired wound healing. Studies have shown that treatment with a stable form of arginase 1 that reduces l-arginine levels and increases ornithine and urea limits retinal injury and improves visual function in DR. We tested the therapeutic efficacy of PEGylated arginine deiminase (ADI-PEG20) that depletes l-arginine and elevates l-citrulline on diabetic complications in the db/db mouse model of type 2 diabetes (T2D)., Methods: Mice received intraperitoneal (IP), intramuscular (IM), or intravitreal (IVT) injections of ADI-PEG20 or PEG20 as control. Effects on body weight, fasting blood glucose levels, blood-retinal-barrier (BRB) function, visual acuity, contrast sensitivity, thermal sensitivity, and wound healing were determined. Studies using bone marrow-derived macrophages (BMDM) examined the underlying signaling pathway., Results: Systemic injections of ADI-PEG20 reduced body weight and blood glucose and decreased oxidative stress and inflammation in db/db retinas. These changes were associated with improved BRB and visual function along with thermal sensitivity and wound healing. IVT injections of either ADI-PEG20, anti-VEGF antibody or their combination also improved BRB and visual function. ADI-PEG20 treatment also prevented LPS/IFNℽ-induced activation of BMDM in vitro as did depletion of l-arginine and elevation of l-citrulline., Conclusions/interpretation: ADI-PEG20 treatment limited signs of DR and DPN and enhanced wound healing in db/db mice. Studies using BMDM suggest that the anti-inflammatory effects of ADI-PEG20 involve blockade of the JAK2-STAT1 signaling pathway via l-arginine depletion and l-citrulline production., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: R. B. Caldwell, M. Rojas, report financial support was provided by National Institutes of Health. Robert W. Caldwell reports a relationship with National Institutes of Health that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Angiogenesis plays a vital role for postnatal development and tissue repair following ischemia. Reactive oxygen species (ROS) generated by NADPH oxidases (NOXes) and mitochondria act as signaling molecules that promote angiogenesis in endothelial cells (ECs) which mainly relies on aerobic glycolysis for ATP production. However, the connections linking redox signaling with glycolysis are not well understood. The GTPase Drp1 is a member of the dynamin superfamily that moves from cytosol to mitochondria through posttranslational modifications to induce mitochondrial fission. The role of Drp1 in ROS-dependent VEGF signaling and angiogenesis in ECs has not been previously described. Here, we identify an unexpected function of endothelial Drp1 as a redox sensor, transmitting VEGF-induced H 2 O 2 signals to enhance glycolysis and angiogenesis. Loss of Drp1 expression in ECs inhibited VEGF-induced angiogenic responses. Mechanistically, VEGF rapidly induced the NOX4-dependent sulfenylation (CysOH) of Drp1 on Cys 644 , promoting disulfide bond formation with the metabolic kinase AMPK and subsequent sulfenylation of AMPK at Cys 299 / 304 via the mitochondrial fission-mitoROS axis. This cysteine oxidation of AMPK, in turn, enhanced glycolysis and angiogenesis. In vivo , mice with EC-specific Drp1 deficiency or CRISPR/Cas9-engineered "redox-dead" (Cys to Ala) Drp1 knock-in mutations exhibited impaired retinal angiogenesis and post-ischemic neovascularization. Our findings uncover a novel role for endothelial Drp1 in linking VEGF-induced mitochondrial redox signaling to glycolysis through a cysteine oxidation-mediated Drp1-AMPK redox relay, driving both developmental and reparative angiogenesis.

Anti-vascular endothelial growth factor therapy has had a substantial impact on the treatment of choroidal neovascularization (CNV) in patients with neovascular age-related macular degeneration (nAMD), the leading cause of vision loss in older adults. Despite treatment, many patients with nAMD still develop severe and irreversible visual impairment because of the development of subretinal fibrosis. We recently reported the anti-inflammatory and antiangiogenic effects of inhibiting the gene encoding adenosine receptor 2A ( Adora2a ), which has been implicated in cardiovascular disease. Here, using two mouse models of subretinal fibrosis (mice with laser injury-induced CNV or mice with a deficiency in the very low-density lipoprotein receptor), we found that deletion of Adora2a either globally or specifically in endothelial cells reduced subretinal fibrosis independently of angiogenesis. We showed that Adora2a -dependent endothelial-to-mesenchymal transition contributed to the development of subretinal fibrosis in mice with laser injury-induced CNV. Deficiency of Adora2a in cultured mouse and human choroidal endothelial cells suppressed induction of the endothelial-to-mesenchymal transition. A metabolomics analysis of cultured human choroidal endothelial cells showed that ADORA2A knockdown with an siRNA reversed the increase in succinate because of decreased succinate dehydrogenase B expression under fibrotic conditions. Pharmacological inhibition of ADORA2A with a small-molecule KW6002 in both mouse models recapitulated the reduction in subretinal fibrosis observed in mice with genetic deletion of Adora2a . ADORA2A inhibition may be a therapeutic approach to treat subretinal fibrosis associated with nAMD.

Compromised blood-retinal barrier (BRB) integrity is a significant factor in ocular diseases like uveitis and retinopathies, leading to pathological vascular permeability and retinal edema. Adherens and tight junction (AJ and TJ) dysregulation due to retinal inflammation plays a pivotal role in BRB disruption. We investigated the potential of ICG001, which inhibits β-catenin-mediated transcription, in stabilizing cell junctions and preventing BRB leakage. In vitro studies using human retinal endothelial cells (HRECs) showed that ICG001 treatment improved β-Catenin distribution within AJs post lipopolysaccharide (LPS) treatment and enhanced monolayer barrier resistance. The in vivo experiments involved a mouse model of LPS-induced ocular inflammation. LPS treatment resulted in increased albumin leakage from retinal vessels, elevated vascular endothelial growth factor (VEGF) and Plasmalemmal Vesicle-Associated Protein (PLVAP) expression, as well as microglia and macroglia activation. ICG001 treatment (i.p.) effectively mitigated albumin leakage, reduced VEGF and PLVAP expression, and reduced the number of activated microglia/macrophages. Furthermore, ICG001 treatment suppressed the surge in inflammatory cytokine synthesis induced by LPS. These findings highlight the potential of interventions targeting β-Catenin to enhance cell junction stability and improve compromised barrier integrity in various ocular inflammatory diseases, offering hope for better management and treatment options.

We previously found that global deletion of the mitochondrial enzyme arginase 2 (A2) limits optic nerve crush (ONC)-induced neuronal death. Herein, we examined the cell-specific role of A2 in this pathology by studies using wild type (WT), neuronal-specific calbindin 2 A2 KO (Calb2 cre/+ A2 f/f ), myeloid-specific A2 KO (LysM cre/+ A2 f/f ), endothelial-specific A2 KO (Cdh5 cre/+ A2 f/f ), and floxed controls. We also examined the impact of A2 overexpression on mitochondrial function in retinal neuronal R28 cells. Immunolabeling showed increased A2 expression in ganglion cell layer (GCL) neurons of WT mice within 6 h-post injury and inner retinal neurons after 7 days. Calb2 A2 KO mice showed improved neuronal survival, decreased TUNEL-positive neurons, and improved retinal function compared to floxed littermates. Neuronal loss was unchanged by A2 deletion in myeloid or endothelial cells. We also found increased expression of neurotrophins (BDNF, FGF2) and improved survival signaling (pAKT, pERK1/2) in Calb2 A2 KO retinas within 24-hour post-ONC along with suppression of inflammatory mediators (IL1β, TNFα, IL6, and iNOS) and apoptotic markers (cleavage of caspase3 and PARP). ONC increased GFAP and Iba1 immunostaining in floxed controls, and Calb2 A2 KO dampened this effect. Overexpression of A2 in R28 cells increased Drp1 expression, and decreased mitochondrial respiration, whereas ABH-induced inhibition of A2 decreased Drp1 expression and improved mitochondrial respiration. Finally, A2 overexpression or excitotoxic treatment with glutamate significantly impaired mitochondrial function in R28 cells as shown by significant reductions in basal respiration, maximal respiration, and ATP production. Further, glutamate treatment of A2 overexpressing cells did not induce further deterioration in their mitochondrial function, indicating that A2 overexpression or glutamate insult induce comparable alterations in mitochondrial function. Our data indicate that neuronal A2 expression is neurotoxic after injury, and A2 deletion in Calb2 expressing neurons limits ONC-induced retinal neurodegeneration and improves visual function., (© 2023. The Author(s).)

The enzyme arginase 1 (A1) hydrolyzes the amino acid arginine to form L-ornithine and urea. Ornithine is further converted to polyamines by the ornithine decarboxylase (ODC) enzyme. We previously reported that deletion of myeloid A1 in mice exacerbates retinal damage after ischemia/reperfusion (IR) injury. Furthermore, treatment with A1 protects against retinal IR injury in wild-type mice. PEG-A1 also mitigates the exaggerated inflammatory response of A1 knockout (KO) macrophages in vitro. Here, we sought to identify the anti-inflammatory pathway that confers macrophage A1-mediated protection against retinal IR injury. Acute elevation of intraocular pressure was used to induce retinal IR injury in mice. A multiplex cytokine assay revealed a marked increase in the inflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the retina at day 5 after IR injury. In vitro, blocking the A1/ODC pathway augmented IL-1β and TNF-α production in stimulated macrophages. Furthermore, A1 treatment attenuated the stimulated macrophage metabolic switch to a pro-inflammatory glycolytic phenotype, whereas A1 deletion had the opposite effect. Screening for histone deacetylases (HDACs) which play a role in macrophage inflammatory response showed that A1 deletion or ODC inhibition increased the expression of HDAC3. We further showed the involvement of HDAC3 in the upregulation of TNF-α but not IL-1β in stimulated macrophages deficient in the A1/ODC pathway. Investigating HDAC3 KO macrophages showed a reduced inflammatory response and a less glycolytic phenotype upon stimulation. In vivo, HDAC3 co-localized with microglia/macrophages at day 2 after IR in WT retinas and was further increased in A1-deficient retinas. Collectively, our data provide initial evidence that A1 exerts its anti-inflammatory effect in macrophages via ODC-mediated suppression of HDAC3 and IL-1β. Collectively we propose that interventions that augment the A1/ODC pathway and inhibit HDAC3 may confer therapeutic benefits for the treatment of retinal ischemic diseases., (© 2023. The Author(s).)

Myeloid cells, specifically microglia and macrophages, are activated in retinal diseases and can improve or worsen retinopathy outcomes based on their inflammatory phenotype. However, assessing the myeloid cell response after retinal injury in mice remains challenging due to the small tissue size and the challenges of distinguishing microglia from infiltrating macrophages. In this protocol paper, we describe a flow cytometry-based protocol to assess retinal microglia/macrophage and their inflammatory phenotype after injury. The protocol is amenable to the incorporation of other markers of interest to other researchers. Key features This protocol describes a flow cytometry-based method to analyze the myeloid cell response in retinopathy mouse models. The protocol can distinguish between microglia- and monocyte-derived macrophages. It can be modified to incorporate markers of interest. We show representative results from three different retinopathy models, namely ischemia-reperfusion injury, endotoxin-induced uveitis, and oxygen-induced retinopathy., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)

Proliferative retinopathies are the leading cause of irreversible blindness in all ages, and there is a critical need to identify novel therapies. We investigated the impact of triciribine (TCBN), a tricyclic nucleoside analog and a weak Akt inhibitor, on retinal neurovascular injury, vascular permeability, and inflammation in oxygen-induced retinopathy (OIR). Post-natal day 7 (P7) mouse pups were subjected to OIR, and treated (i.p.) with TCBN or vehicle from P14-P16 and compared with age-matched, normoxic, vehicle or TCBN-treated controls. P17 retinas were processed for flat mounts, immunostaining, Western blotting, and qRT-PCR studies. Fluorescein angiography, electroretinography, and spectral domain optical coherence tomography were performed on days P21, P26, and P30, respectively. TCBN treatment significantly reduced pathological neovascularization, vaso-obliteration, and inflammation marked by reduced TNFα, IL6, MCP-1, Iba1, and F4/80 (macrophage/microglia markers) expression compared to the vehicle-treated OIR mouse retinas. Pathological expression of VEGF (vascular endothelial growth factor), and claudin-5 compromised the blood-retinal barrier integrity in the OIR retinas correlating with increased vascular permeability and neovascular tuft formation, which were blunted by TCBN treatment. Of note, there were no changes in the retinal architecture or retinal cell function in response to TCBN in the normoxia or OIR mice. We conclude that TCBN protects against pathological neovascularization, restores blood-retinal barrier homeostasis, and reduces retinal inflammation without adversely affecting the retinal structure and neuronal function in a mouse model of OIR. Our data suggest that TCBN may provide a novel therapeutic option for proliferative retinopathy., Competing Interests: Conflict of Interest Statement PRS is a scientific advisor of Ayma Therapeutics, NJ. All other authors declare that there are no financial or other conflicts of interest exist., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Advanced glycation end products (AGEs) contribute significantly to vascular dysfunction (VD) in diabetes. Decreased nitric oxide (NO) is a hallmark in VD. In endothelial cells, NO is produced by endothelial NO synthase (eNOS) from L-arginine. Arginase competes with NOS for L-arginine to produce urea and ornithine, limiting NO production. Arginase upregulation was reported in hyperglycemia; however, AGEs' role in arginase regulation is unknown. Here, we investigated the effects of methylglyoxal-modified albumin (MGA) on arginase activity and protein expression in mouse aortic endothelial cells (MAEC) and on vascular function in mice aortas. Exposure of MAEC to MGA increased arginase activity, which was abrogated by MEK/ERK1/2 inhibitor, p38 MAPK inhibitor, and ABH (arginase inhibitor). Immunodetection of arginase revealed MGA-induced protein expression for arginase I. In aortic rings, MGA pretreatment impaired acetylcholine (ACh)-induced vasorelaxation, which was reversed by ABH. Intracellular NO detection by DAF-2DA revealed blunted ACh-induced NO production with MGA treatment that was reversed by ABH. In conclusion, AGEs increase arginase activity probably through the ERK1/2/p38 MAPK pathway due to increased arginase I expression. Furthermore, AGEs impair vascular function that can be reversed by arginase inhibition. Therefore, AGEs may be pivotal in arginase deleterious effects in diabetic VD, providing a novel therapeutic target.

Background: We have investigated the efficacy of a new strategy to limit pathological retinal neovascularization (RNV) during ischemic retinopathy by targeting the cholesterol metabolizing enzyme acyl-coenzyme A: cholesterol transferase 1 (ACAT1). Dyslipidemia and cholesterol accumulation have been strongly implicated in promoting subretinal NV. However, little is known about the role of cholesterol metabolism in RNV. Here, we tested the effects of inhibiting ACAT1 on pathological RNV in the mouse model of oxygen-induced retinopathy (OIR)., Methods: In vivo studies used knockout mice that lack the receptor for LDL cholesterol (LDLR -/- ) and wild-type mice. The wild-type mice were treated with a specific inhibitor of ACAT1, K604 (10 mg/kg, i.p) or vehicle (PBS) during OIR. In vitro studies used human microglia exposed to oxygen-glucose deprivation (OGD) and treated with the ACAT1 inhibitor (1 μM) or PBS., Results: Analysis of OIR retinas showed that increased expression of inflammatory mediators and pathological RNV were associated with significant increases in expression of the LDLR, increased accumulation of neutral lipids, and formation of toxic levels of cholesterol ester (CE). Deletion of the LDLR completely blocked OIR-induced RNV and significantly reduced the AVA. The OIR-induced increase in CE formation was accompanied by significant increases in expression of ACAT1, VEGF and inflammatory factors (TREM1 and MCSF) (p < 0.05). ACAT1 was co-localized with TREM1, MCSF, and macrophage/microglia makers (F4/80 and Iba1) in areas of RNV. Treatment with K604 prevented retinal accumulation of neutral lipids and CE formation, inhibited RNV, and decreased the AVA as compared to controls (p < 0.05). The treatment also blocked upregulation of LDLR, ACAT1, TREM1, MCSF, and inflammatory cytokines but did not alter VEGF expression. K604 treatment of microglia cells also blocked the effects of OGD in increasing expression of ACAT1, TREM1, and MCSF without altering VEGF expression., Conclusions: OIR-induced RNV is closely associated with increases in lipid accumulation and CE formation along with increased expression of LDLR, ACAT1, TREM1, and MCSF. Inhibiting ACAT1 blocked these effects and limited RNV independently of alterations in VEGF expression. This pathway offers a novel strategy to limit vascular injury during ischemic retinopathy., (© 2023. The Author(s).)

Pathological angiogenesis is a major cause of irreversible blindness in individuals of all age groups with proliferative retinopathy (PR). Mononuclear phagocytes (MPs) within neovascular areas contribute to aberrant retinal angiogenesis. Due to their cellular heterogeneity, defining the roles of MP subsets in PR onset and progression has been challenging. Here, we aimed to investigate the heterogeneity of microglia associated with neovascularization and to characterize the transcriptional profiles and metabolic pathways of proangiogenic microglia in a mouse model of oxygen-induced PR (OIR). Using transcriptional single-cell sorting, we comprehensively mapped all microglia populations in retinas of room air (RA) and OIR mice. We have unveiled several unique types of PR-associated microglia (PRAM) and identified markers, signaling pathways, and regulons associated with these cells. Among these microglia subpopulations, we found a highly proliferative microglia subset with high self-renewal capacity and a hypermetabolic microglia subset that expresses high levels of activating microglia markers, glycolytic enzymes, and proangiogenic Igf1. IHC staining shows that these PRAM were spatially located within or around neovascular tufts. These unique types of microglia have the potential to promote retinal angiogenesis, which may have important implications for future treatment of PR and other pathological ocular angiogenesis-related diseases.

Background and Purpose: Pathological angiogenesis is a major cause of irreversible blindness in individuals with neovascular age-related macular degeneration (nAMD). Macrophages and microglia (MΦ) contribute to aberrant ocular angiogenesis. However, the role of glucose metabolism of MΦ in nAMD is still undefined. Here, we have investigated the involvement of glycolysis, driven by the kinase/phosphatase PFKFB3, in the development of choroidal neovascularization (CNV)., Experimental Approach: CNV was induced in mice with laser photocoagulation. Choroid/retinal pigment epithelium (RPE) complexes and MΦ were isolated for analysis by qRT-PCR, western blot, flow cytometry, immunostaining, metabolic measurements and angiogenesis assays., Key Results: MΦ accumulated within the CNV of murine nAMD models and expressed high levels of glycolysis-related enzymes and M1/M2 polarization markers. This phenotype of hyper-glycolytic and activated MΦ was replicated in bone marrow-derived macrophages stimulated by necrotic RPE in vitro. Myeloid cell-specific knockout of PFKFB3, a key glycolytic activator, attenuated pathological neovascularization in laser-induced CNV, which was associated with decreased expression of MΦ polarization markers and pro-angiogenic factors, along with decreased sprouting of vessels in choroid/RPE complexes. Mechanistically, necrotic RPE increased PFKFB3-driven glycolysis in macrophages, leading to activation of HIF-1α/HIF-2α and NF-κB, and subsequent induction of M1/M2 markers and pro-angiogenic cytokines, finally promoting macrophage reprogramming towards an angiogenic phenotype to facilitate development of CNV. The PFKFB3 inhibitor AZ67 also inhibited activation of HIF-1α/HIF-2α and NF-κB signalling and almost completely prevented laser-induced CNV in mice., Conclusions and Implications: Modulation of PFKFB3-mediated macrophage glycolysis and activation is a promising strategy for the treatment of nAMD., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Diabetic retinopathy (DR) is a serious complication of diabetes that results from sustained hyperglycemia, hyperlipidemia, and oxidative stress. Under these conditions, inducible nitric oxide synthase (iNOS) expression is upregulated in the macrophages (MΦ) and microglia, resulting in increased production of reactive oxygen species (ROS) and inflammatory cytokines, which contribute to disease progression. Arginase 1 (Arg1) is a ureohydrolase that competes with iNOS for their common substrate, L-arginine. We hypothesized that the administration of a stable form of Arg1 would deplete L-arginine's availability for iNOS, thus decreasing inflammation and oxidative stress in the retina. Using an obese Type 2 diabetic (T2DM) db/db mouse, this study characterized DR in this model and determined if systemic treatment with pegylated Arg1 (PEG-Arg1) altered the progression of DR. PEG-Arg1 treatment of db/db mice thrice weekly for two weeks improved visual function compared with untreated db/db controls. Retinal expression of inflammatory factors (iNOS, IL-1β, TNF-α, IL-6) was significantly increased in the untreated db/db mice compared with the lean littermate controls. The increased retinal inflammatory and oxidative stress markers in db/db mice were suppressed with PEG-Arg1 treatment. Additionally, PEG-Arg1 treatment restored the blood-retinal barrier (BRB) function, as evidenced by the decreased tissue albumin extravasation and an improved endothelial ZO-1 tight junction integrity compared with untreated db/db mice.

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., (© 2022. The Author(s).)

Diabetic retinopathy (DR) is the leading cause of vision loss in working age adults. Understanding the retinal metabolic response to circulating high glucose levels in diabetic patients is critical for development of new therapeutics to treat DR. Measuring retinal metabolic function using the Seahorse analyzer is a promising technique to investigate the effect of hyperglycemia on retinal glycolysis and mitochondrial respiration. Here, we analyzed the retinal metabolic function in young and old diabetic and control mice. We also compared the expression of key glycolytic enzymes between the two groups. The Seahorse XF analyzer was used to measure the metabolic function of retina explants from young and old type 1 diabetic Akita ( Ins2 Akita ) mice and their control littermates. Rate-limiting glycolytic enzymes were analyzed in retina lysates from the two age groups by Western blotting. Retinas from young adult Akita mice showed a decreased glycolytic response as compared to control littermates. However, this was not observed in the older mice. Western blotting analysis showed decreased expression of the glycolytic enzyme PFKFB3 in the young Akita mice retinas. Measurement of the oxygen consumption rate showed no difference in retinal mitochondrial respiration between Akita and WT littermates under normal glucose conditions ex vivo despite mitochondrial fragmentation in the Akita retinas as examined by electron microscopy. However, Akita mice retinas showed decreased mitochondrial respiration under glucose-free conditions. In conclusion, diabetic retinas display a decreased glycolytic response during the early course of diabetes which is accompanied by a reduction in PFKFB3. Diabetic retinas exhibit decreased mitochondrial respiration under glucose deprivation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shosha, Qin, Lemtalsi, Zaidi, Rojas, Xu, Caldwell, Caldwell and Fouda.)

Diabetic retinopathy (DR) and diabetic macular edema (DME) are retinal complications of diabetes that can lead to loss of vision and impaired quality of life. The current gold standard therapies for treatment of DR and DME focus on advanced disease, are invasive, expensive, and can trigger adverse side-effects, necessitating the development of more effective, affordable, and accessible therapies that can target early stage disease. The pathogenesis and pathophysiology of DR is complex and multifactorial, involving the interplay between the effects of hyperglycemia, hyperlipidemia, hypoxia, and production of reactive oxygen species (ROS) in the promotion of neurovascular dysfunction and immune cell polarization to a proinflammatory state. The pathophysiology of DR provides several therapeutic targets that have the potential to attenuate disease progression. Current novel DR and DME therapies under investigation include erythropoietin-derived peptides, inducers of antioxidant gene expression, activators of nitric oxide/cyclic GMP signaling pathways, and manipulation of arginase activity. This review aims to aid understanding of DR and DME pathophysiology and explore novel therapies that capitalize on our knowledge of these diabetic retinal complications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bunch, Abdelrahman, Caldwell and Caldwell.)

Arginase 1 (A1) is the enzyme that hydrolyzes the amino acid, L-arginine, to ornithine and urea. We have previously shown that A1 deletion worsens retinal ischemic injury, suggesting a protective role of A1. In this translational study, we aimed to study the utility of systemic pegylated A1 (PEG-A1, recombinant human arginase linked to polyethylene glycol) treatment in mouse models of acute retinal and brain injury. Cohorts of WT mice were subjected to retinal ischemia-reperfusion (IR) injury, traumatic optic neuropathy (TON) or brain cerebral ischemia via middle cerebral artery occlusion (MCAO) and treated with intraperitoneal injections of PEG-A1 or vehicle (PEG only). Drug penetration into retina and brain tissues was measured by western blotting and immunolabeling for PEG. Neuroprotection was measured in a blinded fashion by quantitation of NeuN (neuronal marker) immunolabeling of retina flat-mounts and brain infarct area using triphenyl tetrazolium chloride (TTC) staining. Furthermore, ex vivo retina explants and in vitro retina neuron cultures were subjected to oxygen-glucose deprivation (OGD) followed by reoxygenation (R) and treated with PEG-A1. PEG-A1 given systemically did not cross the intact blood-retina/brain barriers in sham controls but reached the retina and brain after injury. PEG-A1 provided neuroprotection after retinal IR injury, TON and cerebral ischemia. PEG-A1 treatment was also neuroprotective in retina explants subjected to OGD/R but did not improve survival in retinal neuronal cultures exposed to OGD/R. In summary, systemic PEG-A1 administration is neuroprotective and provides an excellent route to deliver the drug to the retina and the brain after acute injury., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor receptor type 2 (VEGFR2, also known as KDR and FLK1) signalling in endothelial cells (ECs) is essential for developmental and reparative angiogenesis. Reactive oxygen species and copper (Cu) are also involved in these processes. However, their inter-relationship is poorly understood. Evidence of the role of the endothelial Cu importer CTR1 (also known as SLC31A1) in VEGFR2 signalling and angiogenesis in vivo is lacking. Here, we show that CTR1 functions as a redox sensor to promote angiogenesis in ECs. CTR1-depleted ECs showed reduced VEGF-induced VEGFR2 signalling and angiogenic responses. Mechanistically, CTR1 was rapidly sulfenylated at Cys189 at its cytosolic C terminus after stimulation with VEGF, which induced CTR1-VEGFR2 disulfide bond formation and their co-internalization to early endosomes, driving sustained VEGFR2 signalling. In vivo, EC-specific Ctr1-deficient mice or CRISPR-Cas9-generated redox-dead Ctr1(C187A)-knockin mutant mice had impaired developmental and reparative angiogenesis. Thus, oxidation of CTR1 at Cys189 promotes VEGFR2 internalization and signalling to enhance angiogenesis. Our study uncovers an important mechanism for sensing reactive oxygen species through CTR1 to drive neovascularization., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: Retinal ischemic disease is a major cause of vision loss. Current treatment options are limited to late-stage diseases, and the molecular mechanisms of the initial insult are not fully understood. We have previously shown that the deletion of the mitochondrial arginase isoform, arginase 2 (A2), limits neurovascular injury in models of ischemic retinopathy. Here, we investigated the involvement of A2-mediated alterations in mitochondrial dynamics and function in the pathology., Methods: We used wild-type (WT), global A2 knockout (A2KO-) mice, cell-specific A2 knockout mice subjected to retinal ischemia/reperfusion (I/R), and bovine retinal endothelial cells (BRECs) subjected to an oxygen-glucose deprivation/reperfusion (OGD/R) insult. We used western blotting to measure levels of cell stress and death markers and the mitochondrial fragmentation protein, dynamin related protein 1 (Drp1). We also used live cell mitochondrial labeling and Seahorse XF analysis to evaluate mitochondrial fragmentation and function, respectively., Results: We found that the global deletion of A2 limited the I/R-induced disruption of retinal layers, fundus abnormalities, and albumin extravasation. The specific deletion of A2 in endothelial cells was protective against I/R-induced neurodegeneration. The OGD/R insult in BRECs increased A2 expression and induced cell stress and cell death, along with decreased mitochondrial respiration, increased Drp1 expression, and mitochondrial fragmentation. The overexpression of A2 in BREC also decreased mitochondrial respiration, promoted increases in the expression of Drp1, mitochondrial fragmentation, and cell stress and resulted in decreased cell survival. In contrast, the overexpression of the cytosolic isoform, arginase 1 (A1), did not affect these parameters., Conclusions: This study is the first to show that A2 in endothelial cells mediates retinal ischemic injury through a mechanism involving alterations in mitochondrial dynamics and function., (Published by Elsevier GmbH.)

Background: Doxorubicin (DOX) is a commonly used chemotherapeutic drug but is limited in clinical applications by its cardiotoxicity. Neiguan acupoint (PC6) is a well-recognized acupoint for the treatment of cardiothoracic disease. However, whether acupuncture at PC6 could be effective in preventing DOX-induced cardiotoxicity is still unknown., Methods: A set of experiments were performed with myocardial cells, wild type, inducible nitric oxide synthase knockout (iNOS-/-), and myocardial-specific ablation arginase 2 (Myh6-ARG 2-/-) mice. We investigated the protective effect and the underlying mechanisms for electroacupuncture (EA) against DOX-induced cardiotoxicity by echocardiography, immunostaining, biochemical analysis, and molecular biotechnology in vivo and in vitro analysis., Results: We found that DOX-mediated nitric oxide (NO) production was positively correlated with the iNOS level but has a negative correlation with the arginase 2 (ARG 2) level in both myocardial cells and tissues. Meanwhile, EA at PC6 alleviated cardiac dysfunction and cardiac hypertrophy in DOX-treated mice. EA at PC6 blocked the upregulation of NO production in accompanied with the downregulated iNOS and upregulated ARG 2 levels in myocardial tissue induced by DOX. Furthermore, knockout iNOS prevented cardiotoxicity and EA treatment did not cause the further improvement of cardiac function in iNOS-/- mice treated by DOX. In contrast, deficiency of myocardial ARG 2 aggravated DOX-induced cardiotoxicity and reduced EA protective effect., Conclusion: These results suggest that EA treatment at PC6 can prevent DOX-induced cardiotoxicity through modulating NO production by modulating the iNOS/ARG 2 balance in myocardial cells., Competing Interests: The author declares that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Jingya Wang et al.)

Vision impairment due to optic neuritis (ON) is one of the major clinical presentations in Multiple Sclerosis (MS) and is characterized by inflammation and degeneration of the optic nerve and retina. Currently available treatments are only partially effective and have a limited impact on the neuroinflammatory pathology of the disease. A recent study from our laboratory highlighted the beneficial effect of arginase 2 (A2) deletion in suppressing retinal neurodegeneration and inflammation in an experimental model of MS. Utilizing the same model, the present study investigated the impact of A2 deficiency on MS-induced optic neuritis. Experimental autoimmune encephalomyelitis (EAE) was induced in wild-type (WT) and A2 knockout (A2-/-) mice. EAE-induced cellular infiltration, as well as activation of microglia and macrophages, were reduced in A2-/- optic nerves. Axonal degeneration and demyelination seen in EAE optic nerves were observed to be reduced with A2 deletion. Further, the lack of A2 significantly ameliorated astrogliosis induced by EAE. In conclusion, our findings demonstrate a critical involvement of arginase 2 in mediating neuroinflammation in optic neuritis and suggest the potential of A2 blockade as a targeted therapy for MS-induced optic neuritis., Competing Interests: The authors have declared that no competing interests exist.

The coordination of metabolic signals among different cellular components in pathological retinal angiogenesis is poorly understood. Here, we showed that in the pathological angiogenic vascular niche, retinal myeloid cells, particularly macrophages/microglia that are spatially adjacent to endothelial cells (ECs), are highly glycolytic. We refer to these macrophages/microglia that exhibit a unique angiogenic phenotype with increased expression of both M1 and M2 markers and enhanced production of both proinflammatory and proangiogenic cytokines as pathological retinal angiogenesis-associated glycolytic macrophages/microglia (PRAGMs). The phenotype of PRAGMs was recapitulated in bone marrow-derived macrophages or retinal microglia stimulated by lactate that was produced by hypoxic retinal ECs. Knockout of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase ( PFKFB3 ; Pfkfb3 for rodents), a glycolytic activator in myeloid cells, impaired the ability of macrophages/microglia to acquire an angiogenic phenotype, rendering them unable to promote EC proliferation and sprouting and pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Mechanistically, hyperglycolytic macrophages/microglia produced large amount of acetyl-coenzyme A, leading to histone acetylation and PRAGM-related gene induction, thus reprogramming macrophages/microglia into an angiogenic phenotype. These findings reveal a critical role of glycolytic metabolites as initiators of reciprocal activation of macrophages/microglia and ECs in the retinal angiogenic niche and suggest that strategies targeting the metabolic communication between these cell types may be efficacious in the treatment of pathological retinal angiogenesis., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Ischemic retinopathies represent a major cause of visual impairment and blindness. They include diabetic retinopathy (DR), acute glaucoma, retinopathy of prematurity (ROP), and central (or branch) retinal artery occlusion (CRAO). These conditions share in common a period of ischemia or reduced blood supply to the retinal tissue that eventually leads to neuronal degeneration. Similarly, acute brain injury from ischemia or trauma leads to neurodegeneration and can have devastating consequences in patients with stroke or traumatic brain injury (TBI). In all of these conditions, current treatment strategies are limited by their lack of effectiveness, adverse effects or short time window for administration. Therefore, there is a great need to identify new therapies for acute central nervous system (CNS) injury. In this brief review article, we focus on the pathway of the arginase enzyme as a novel therapeutic target for acute CNS injury. We review the recent work on the role of arginase enzyme and its downstream components in neuroprotection in both retina and brain acute injury models. Delineating the similarities and differences between the role of arginase in the retina and brain neurodegeneration will allow for better understanding of the role of arginase in CNS disorders. This will also facilitate repurposing the arginase pathway as a new therapeutic target in both retina and brain diseases., (Copyright © 2020 Fouda, Eldahshan, Narayanan, Caldwell and Caldwell.)

Purpose: The lysozyme 2 (Lyz2 or LysM) cre mouse is extensively used to achieve genetic manipulation in myeloid cells and it has been widely employed in retinal research. However, LysM has been recently described to be expressed in brain neurons and there is a debate on whether it is also expressed by resident microglia in addition to infiltrating macrophages., Methods: We examined LysM-cre recombination in retinal tissue using a LysM-cre/tdTomato reporter mouse together with immunolabeling for several retinal cell markers. We further compared LysM-cre tdTomato recombination with that of Cdh5-cre driver, which is expressed in both endothelial and hematopoietic cells., Results: LysM-cre was strongly expressed in most microglia/resident macrophages in neonatal retinas (P8) and to a lesser extent in microglia of adult retinas. In addition, there was some neuronal recombination (8 %) of LysM-cre specifically in adult retinal ganglion cells and amacrine cells. After retinal ischemia-reperfusion injury, LysM-cre was strongly expressed in microglia/infiltrating macrophages. Cdh5-cre was expressed in endothelial and myeloid cells of P8 pups retinas. Unexpectedly, Cdh5 showed additional expression in adult mouse retinal ganglion cells and brain neurons., Conclusions: LysM-cre is expressed in macrophages and a subset of microglia together with a small but significant recombination of LysM-cre in the retinal neurons of adult mice. Cdh5 also showed some neuronal expression in both retina and brain of adult mice. These findings should be taken into consideration when interpreting results from central nervous system research using LysM-cre and Cdh5-cre mice.

Diabetic retinopathy (DR) is the leading cause of blindness in working age Americans. Clinicians diagnose DR based on its characteristic vascular pathology, which is evident upon clinical exam. However, extensive research has shown that diabetes causes significant neurovascular dysfunction prior to the development of clinically apparent vascular damage. While laser photocoagulation and/or anti-vascular endothelial growth factor (VEGF) therapies are often effective for limiting the late-stage vascular pathology, we still do not have an effective treatment to limit the neurovascular dysfunction or promote repair during the early stages of DR. This review addresses the role of arginase as a mediator of retinal neurovascular injury and therapeutic target for early stage DR. Arginase is the ureohydrolase enzyme that catalyzes the production of L-ornithine and urea from L-arginine. Arginase upregulation has been associated with inflammation, oxidative stress, and peripheral vascular dysfunction in models of both types of diabetes. The arginase enzyme has been identified as a therapeutic target in cardiovascular disease and central nervous system disease including stroke and ischemic retinopathies. Here, we discuss and review the literature on arginase-induced retinal neurovascular dysfunction in models of DR. We also speculate on the therapeutic potential of arginase in DR and its related underlying mechanisms.

Diabetic retinopathy (DR) is a significant cause of blindness in working-age adults worldwide. Lack of effective strategies to prevent or reduce vision loss is a major problem. Since the degeneration of retinal neurons is an early event in the diabetic retina, studies to characterize the molecular mechanisms of diabetes-induced retinal neuronal damage and dysfunction are of high significance. We have demonstrated that spermine oxidase (SMOX), a mediator of polyamine oxidation is critically involved in causing neurovascular damage in the retina. The involvement of SMOX in diabetes-induced retinal neuronal damage is completely unknown. Utilizing the streptozotocin-induced mouse model of diabetes, the impact of the SMOX inhibitor, MDL 72527, on neuronal damage and dysfunction in the diabetic retina was investigated. Retinal function was assessed by electroretinography (ERG) and retinal architecture was evaluated using spectral domain-optical coherence tomography. Retinal cryosections were prepared for immunolabeling of inner retinal neurons and retinal lysates were used for Western blotting. We observed a marked decrease in retinal function in diabetic mice compared to the non-diabetic controls. Treatment with MDL 72527 significantly improved the ERG responses in diabetic retinas. Diabetes-induced retinal thinning was also inhibited by the MDL 72527 treatment. Our analysis further showed that diabetes-induced retinal ganglion cell damage and neurodegeneration were markedly attenuated by MDL 72527 treatment. These results strongly implicate SMOX in diabetes-induced retinal neurodegeneration and visual dysfunction., Competing Interests: The authors declare that they have no conflict of interest.

Western diet-induced obesity is linked to the development of metabolic dysfunctions, including type 2 diabetes and complications that include retinopathy, a leading cause of blindness. Aberrant activation of the inflammasome cascade leads to the progression of obesity-induced pathologies. Our lab showed the critical role of arginase 2 (A2), the mitochondrial isoform of this ureahydrolase, in obesity-induced metabolic dysfunction and inflammation. A2 deletion also has been shown to be protective against retinal inflammation in models of ischemic retinopathy and multiple sclerosis. We investigated the effect of A2 deletion on western diet-induced retinopathy. Wild-type mice fed a high-fat, high-sucrose western diet for 16 weeks exhibited elevated retinal expression of A2, markers of the inflammasome pathway, oxidative stress, and activation of microglia/macrophages. Western diet feeding induced exaggerated retinal light responses without affecting visual acuity or retinal morphology. These effects were reduced or absent in mice with global A2 deletion. Exposure of retinal endothelial cells to palmitate and high glucose, a mimic of the obese state, increased expression of A2 and inflammatory mediators and induced cell death. These effects, except for A2, were prevented by pretreatment with an arginase inhibitor. Collectively, our study demonstrated a substantial role of A2 in early manifestations of diabetic retinopathy., Competing Interests: The authors declare no conflicts of interest.

Optic neuritis is a major clinical feature of multiple sclerosis (MS) and can lead to temporary or permanent vision loss. Previous studies from our laboratory have demonstrated the critical involvement of arginase 2 (A2) in retinal neurodegeneration in models of ischemic retinopathy. The current study was undertaken to investigate the role of A2 in MS-mediated retinal neuronal damage and degeneration. Experimental autoimmune encephalomyelitis (EAE) was induced in wild-type (WT) and A2 knockout (A2 -/- ) mice. EAE-induced motor deficits, loss of retinal ganglion cells, retinal thinning, inflammatory signaling, and glial activation were studied in EAE-treated WT and A2 -/- mice and their respective controls. Increased expression of A2 was observed in WT retinas in response to EAE induction. EAE-induced motor deficits were markedly reduced in A2 -/- mice compared with WT controls. Retinal flat mount studies demonstrated a significant reduction in the number of RGCs in WT EAE retinas in comparison with normal control mice. A significant improvement in neuronal survival was evident in retinas of EAE-induced A2 -/- mice compared with WT. RNA levels of the proinflammatory molecules CCL2, COX2, IL-1α, and IL-12α were significantly reduced in the A2 -/- EAE retinas compared with WT EAE. EAE-induced activation of glia (microglia and Müller cells) was markedly reduced in A2 -/- retinas compared with WT. Western blot analyses showed increased levels of phospho-ERK1/2 and reduced levels of phospho-BAD in the WT EAE retina, while these changes were prevented in A2 -/- mice. In conclusion, our studies establish EAE as an excellent model to study MS-mediated retinal neuronal damage and suggest the potential value of targeting A2 as a therapy to prevent MS-mediated retinal neuronal injury.

Chronic stress-induced anxiety disorder is a highly-prevalent, modern social disease in which oxidative stress plays an important role. It is necessary to determine the underlying mechanisms governing this disorder to establish an effective treatment target for anxiety disorders. In this study, we examined the behavioral changes in mice subjected to chronic mild stress (CMS). We found that CMS exposure leads to anxiety-like phenotypes and increased levels of oxidative stress in the ventral hippocampus of mice. Furthermore, CMS increased the excitatory synaptic transmission of pyramidal cells in the ventral CA1 (vCA1). Administration of 4-hydroxy-3-methoxy-acetophenone (apocynin), an inhibitor of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, clearly ameliorated the changes induced by CMS exposure. In addition, our results of behavioral tests and analyses of reactive oxygen species (ROS) using NOX2-deficient mice indicate that CMS-induced enhanced oxidative stress level is primarily caused by the increased expression of NOX2. NOX2-derived oxidative stress can serve as a target for anxiety therapy led by chronic stress., (Copyright © 2019. Published by Elsevier Ltd.)

Visceral adipose tissue (VAT) inflammation and metabolic dysregulation are key components of obesity-induced metabolic disease. Upregulated arginase, a ureahydrolase enzyme with two isoforms (A1-cytosolic and A2-mitochondrial), is implicated in pathologies associated with obesity and diabetes. This study examined A2 involvement in obesity-associated metabolic and vascular disorders. WT and globally deleted A2( -/- ) or A1( +/- ) mice were fed either a high fat/high sucrose (HFHS) diet or normal diet (ND) for 16 weeks. Increases in body and VAT weight of HFHS-fed WT mice were abrogated in A2 -/- , but not A1 +/- , mice. Additionally, A2 -/- HFHS-fed mice exhibited higher energy expenditure, lower blood glucose, and insulin levels compared to WT HFHS mice. VAT and adipocytes from WT HFHS fed mice showed greater A2 expression and adipocyte size and reduced expression of PGC-1α, PPAR-γ, and adiponectin. A2 deletion blunted these effects, increased levels of active AMPK-α , and upregulated genes involved in fatty acid metabolism. A2 deletion prevented HFHS-induced VAT collagen deposition and inflammation, which are involved in adipocyte metabolic dysfunction. Endothelium-dependent vasorelaxation, impaired by HFHS diet, was significantly preserved in A2 -/- mice, but more prominently maintained in A1 +/- mice. In summary, A2 is critically involved in HFHS-induced VAT inflammation and metabolic dysfunction.

Obesity has reached epidemic proportions and its prevalence is climbing. Obesity is characterized by hypertrophied adipocytes with a dysregulated adipokine secretion profile, increased recruitment of inflammatory cells, and impaired metabolic homeostasis that eventually results in the development of systemic insulin resistance, a phenotype of type 2 diabetes. Nitric oxide synthase (NOS) is an enzyme that converts L-arginine to nitric oxide (NO), which functions to maintain vascular and adipocyte homeostasis. Arginase is a ureohydrolase enzyme that competes with NOS for L-arginine. Arginase activity/expression is upregulated in obesity, which results in diminished bioavailability of NO, impairing both adipocyte and vascular endothelial cell function. Given the emerging role of NO in the regulation of adipocyte physiology and metabolic capacity, this review explores the interplay between arginase and NO, and their effect on the development of metabolic disorders, cardiovascular diseases, and mitochondrial dysfunction in obesity. A comprehensive understanding of the mechanisms involved in the development of obesity-induced metabolic and vascular dysfunction is necessary for the identification of more effective and tailored therapeutic avenues for their prevention and treatment.

Diabetes-induced vascular endothelial dysfunction has been reported to involve hyperglycemia-induced increases in arginase activity. However, upstream mediators of this effect are not clear. Here, we have tested involvement of Rho kinase, ERK1/2 and p38 MAPK pathways in this process. Studies were performed with aortas isolated from wild type or hemizygous arginase 1 knockout (Arg1 +/- ) mice and bovine aortic endothelial cells exposed to high glucose (HG, 25 mmol/l) or normal glucose (NG, 5.5 mmol/l) conditions for different times. Effects of inhibitors of arginase, p38 MAPK, ERK1/2 or ROCK and ex vivo adenoviral delivery of active Arg1 and inactive (D128-Arg1) cDNA were also determined. Exposure in wild type aorta or endothelial cells to HG significantly increased arginase activity and Arg1 expression and impaired aortic relaxation. Transduction of wild type aorta with active Arg1 cDNA impaired vascular relaxation, whereas inactive Arg1 had no effect. The HG-induced vascular endothelial dysfunction was associated with increased phosphorylation (activation) of ERK1/2 and p38 MAPK. Pretreatment with inhibitors of ERK1/2, p38 MAPK, ROCK or arginase blocked HG-induced elevation of arginase activity and Arg1 expression and prevented the vascular dysfunction. Inhibition of ROCK blunted the HG-induced activation of ERK1/2 and p38 MAPK. In summary, activated ROCK and subsequent activation of ERK1/2 or p38 MAPK elevates arginase activity and Arg1 expression in hyperglycemic states. Targeting this pathway may provide an effective means for preventing diabetes/hyperglycemia-induced vascular endothelial dysfunction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Dysfunction of retinal neurons is a major cause of vision impairment in blinding diseases that affect children and adults worldwide. Cellular damage resulting from polyamine catabolism has been demonstrated to be a major player in many neurodegenerative conditions. We have previously shown that inhibition of polyamine oxidase (PAO) using MDL 72527 significantly reduced retinal neurodegeneration and cell death signaling pathways in hyperoxia-mediated retinopathy. In the present study, we investigated the impact of PAO inhibition in limiting retinal neurodegeneration in a model of NMDA ( N-Methyl-D-aspartate )-induced excitotoxicity. Adult mice (8-10 weeks old) were given intravitreal injections (20 nmoles) of NMDA or NMLA ( N-Methyl-L-aspartate , control). Intraperitoneal injection of MDL 72527 (40 mg/kg body weight/day) or vehicle (normal saline) was given 24 h before NMDA or NMLA treatment and continued until the animals were sacrificed (varied from 1 to 7 days). Analyses of retinal ganglion cell (RGC) layer cell survival was performed on retinal flatmounts. Retinal cryostat sections were prepared for immunostaining, TUNEL assay and retinal thickness measurements. Fresh frozen retinal samples were used for Western blotting analysis. A marked decrease in the neuronal survival in the RGC layer was observed in NMDA treated retinas compared to their NMLA treated controls, as studied by NeuN immunostaining of retinal flatmounts. Treatment with MDL 72527 significantly improved survival of NeuN positive cells in the NMDA treated retinas. Excitotoxicity induced neurodegeneration was also demonstrated by reduced levels of synaptophysin and degeneration of inner retinal neurons in NMDA treated retinas compared to controls. TUNEL labeling studies showed increased cell death in the NMDA treated retinas. However, treatment with MDL 72527 markedly reduced these changes. Analysis of signaling pathways during excitotoxic injury revealed the downregulation of pro-survival signaling molecules p-ERK and p-Akt, and the upregulation of a pro-apoptotic molecule BID, which were normalized with PAO inhibition. Our data demonstrate that inhibition of polyamine oxidase blocks NMDA-induced retinal neurodegeneration and promotes cell survival, thus offering a new therapeutic target for retinal neurodegenerative disease conditions.

Our previous studies have implicated expression of the mitochondrial isoform of the arginase enzyme arginase 2 (A2) in neurovascular injury during ischemic retinopathies. The aim of this study was to characterize the specific involvement of A2 in retinal injury following optic nerve crush (ONC). To accomplish this, wild-type (WT) or A2 knockout (A2 -/- ) mice were subjected to ONC injury. The contralateral eye served as sham control. Quantitative RT-PCR and western blot were used to evaluate mRNA and protein expression. Retinal ganglion cell (RGC) survival was assessed in retinal whole mounts. Axonal sprouting was determined by anterograde transport of Cholera Toxin B (CTB). These analyses showed increased A2 expression following ONC. Numbers of NeuN-positive neurons as well as Brn3a- and RBPMS-positive RGC were decreased in the WT retinas at 14 days after ONC as compared to the sham controls. This ONC-induced neuronal loss was diminished in the A2 -/- retinas. Similarly, axonal degeneration was ameliorated by A2 deletion whereas axon sprouting was enhanced. Significant retinal thinning was also seen in WT retinas at 21 days after ONC, and this was blocked in A2 -/- mice. Cell death studies showed an increase in TUNEL positive cells in the RGC layer at 5 days after ONC in the WT retinas, and this was attenuated by A2 deletion. ONC increased glial cell activation in WT retinas, and this was significantly reduced by A2 deletion. Western blotting showed a marked increase in the neurotrophin, brain derived neurotrophic factor (BDNF) and its downstream signaling in A2 -/- retinas vs. WT after ONC. This was associated with increases in the axonal regeneration marker GAP-43 in A2 -/- retinas. Furthermore, A2 -/- retinas showed decreased NLRP3 inflammasome activation and lower interleukin (IL-) 1β/IL-18 levels as compared to WT retinas subjected to ONC. Collectively, our results show that deletion of A2 limits ONC-induced neurodegeneration and glial activation, and enhances axonal sprouting by a mechanism involving increases in BDNF and decreases in retinal inflammation. These data demonstrate that A2 plays an important role in ONC-induced retinal damage. Blockade of A2 activity may offer a therapeutic strategy for preventing vision loss induced by traumatic retinal injury.

Activation-induced cytidine deaminase (AID) is required for the immunoglobulin diversification processes of somatic hypermutation, gene conversion and class-switch recombination. The targeting of AID's deamination activity is thought to be a combination of cis- and trans-acting elements, but has not been fully elucidated. Deletion analysis of putative proximal cis-regulatory motifs, while helpful, fails to identify additive versus cumulative effects, redundancy, and may create new motifs where none previously existed. In contrast, gain-of-function analysis can be more insightful with fewer of the same drawbacks and the output is a positive result. Here, we show five defined DNA regions of the avian Igλ locus that are sufficient to confer events of hypermutation to a target gene. In our analysis, the essential cis-targeting elements fully reconstituted diversification of a transgene under heterologous promotion in the avian B-cell line DT40. Furthermore, to the best of our knowledge two of the five regions we report on here have not previously been described as individually having an influence on somatic hypermutation., (© 2018 The Authors Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australasian Society for Immunology Inc.)

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.

In pathological retinal neovascularization (RNV) disorders, the retina is infiltrated by activated leukocytes and macrophages. Triggering receptor expressed on myeloid cells 1 (TREM-1), an inflammation amplifier, activates monocytes and macrophages and plays an important role in cancer, autoimmune and other inflammation-associated disorders. Hypoxia-inducible TREM-1 is involved in cancer angiogenesis but its role in RNV remains unclear. Here, to close this gap, we evaluated the role of TREM-1 in RNV using a mouse model of oxygen-induced retinopathy (OIR). We found that hypoxia induced overexpression of TREM-1 in the OIR retinas compared to that of the room air group. TREM-1 was observed specifically in areas of pathological RNV, largely colocalizing with macrophage colony-stimulating factor (M-CSF) and CD45- and Iba-1-positive cells. TREM-1 blockade using systemically administered first-in-class ligand-independent TREM-1 inhibitory peptides rationally designed using the signaling chain homooligomerization (SCHOOL) strategy significantly (up to 95%) reduced vitreoretinal neovascularization. The peptides were well-tolerated when formulated into lipopeptide complexes for peptide half-life extension and targeted delivery. TREM-1 inhibition substantially downregulated retinal protein levels of TREM-1 and M-CSF suggesting that TREM-1-dependent suppression of pathological angiogenesis involves M-CSF. Targeting TREM-1 using TREM-1-specific SCHOOL peptide inhibitors represents a novel strategy to treat retinal diseases that are accompanied by neovascularization including retinopathy of prematurity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: Neurofibromatosis type 1 (NF1) is the result of inherited mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. Eye manifestations are common in NF1 with recent reports describing a vascular dysplasia in the retina and choroid. Common features of NF1 retinopathy include tortuous and dilated feeder vessels that terminate in capillary tufts, increased endothelial permeability, and neovascularization. Given the retinal vascular phenotype observed in persons with NF1, we hypothesize that preserving neurofibromin may be a novel strategy to control pathologic retinal neovascularization., Methods: Nf1 expression in human endothelial cells (EC) was reduced using small hairpin (sh) RNA and EC proliferation, migration, and capacity to form vessel-like networks were assessed in response to VEGF and hypoxia. Wild-type (WT), Nf1 heterozygous (Nf1+/-), and Nf1flox/+;Tie2cre pups were subjected to hyperoxia/hypoxia using the oxygen-induced retinopathy model. Retinas were analyzed quantitatively for extent of retinal vessel dropout, neovascularization, and capillary branching., Results: Neurofibromin expression was suppressed in response to VEGF, which corresponded with activation of Mek-Erk and PI3-K-Akt signaling. Neurofibromin-deficient EC exhibited enhanced proliferation and network formation in response to VEGF and hypoxia via an Akt-dependent mechanism. In response to hyperoxia/hypoxia, Nf1+/- retinas exhibited increased vessel dropout and neovascularization when compared with WT retinas. Neovascularization was similar between Nf1+/- and Nf1flox/+;Tie2cre retinas, but capillary drop out in Nf1flox/+;Tie2cre retinas was significantly reduced when compared with Nf1+/- retinas., Conclusions: These data suggest that neurofibromin expression is essential for controlling endothelial cell proliferation and retinal neovascularization and therapies targeting neurofibromin-deficient EC may be beneficial.

We have recently found that diabetes-induced premature senescence of retinal endothelial cells is accompanied by NOX2-NADPH oxidase-induced increases in the ureohydrolase enzyme arginase 1 (A1). Here, we used genetic strategies to determine the specific involvement of A1 in diabetes-induced endothelial cell senescence. We used A1 knockout mice and wild type mice that were rendered diabetic with streptozotocin and retinal endothelial cells (ECs) exposed to high glucose or transduced with adenovirus to overexpress A1 for these experiments. ABH [2(S)-Amino-6-boronohexanoic acid] was used to inhibit arginase activity. We used Western blotting, immunolabeling, quantitative PCR, and senescence associated &beta;-galactosidase (SA &beta;-Gal) activity to evaluate senescence. Analyses of retinal tissue extracts from diabetic mice showed significant increases in mRNA expression of the senescence-related proteins p16 INK4a , p21, and p53 when compared with non-diabetic mice. SA &beta;-Gal activity and p16 INK4a immunoreactivity were also increased in retinal vessels from diabetic mice. A1 gene deletion or pharmacological inhibition protected against the induction of premature senescence. A1 overexpression or high glucose treatment increased SA &beta;-Gal activity in cultured ECs. These results demonstrate that A1 is critically involved in diabetes-induced senescence of retinal ECs. Inhibition of arginase activity may therefore be an effective therapeutic strategy to alleviate diabetic retinopathy by preventing premature senescence.

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.

Obesity-induced vascular dysfunction involves pathological remodeling of the visceral adipose tissue (VAT) and increased inflammation. Our previous studies showed that arginase 1 (A1) in endothelial cells (ECs) is critically involved in obesity-induced vascular dysfunction. We tested the hypothesis that EC-A1 activity also drives obesity-related VAT remodeling and inflammation. Our studies utilized wild-type and EC-A1 knockout (KO) mice made obese by high-fat/high-sucrose (HFHS) diet. HFHS diet induced increases in body weight, fasting blood glucose, and VAT expansion. This was accompanied by increased arginase activity and A1 expression in vascular ECs and increased expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in both VAT and ECs. HFHS also markedly increased circulating inflammatory monocytes and VAT infiltration by inflammatory macrophages, while reducing reparative macrophages. Additionally, adipocyte size and fibrosis increased and capillary density decreased in VAT. These effects of HFHS, except for weight gain and hyperglycemia, were prevented or reduced in mice lacking EC-A1 or treated with the arginase inhibitor 2-( S )-amino-6-boronohexanoic acid (ABH). In mouse aortic ECs, exposure to high glucose (25 mM) and Na palmitate (200 μM) reduced nitric oxide production and increased A1, TNF-α, VCAM-1, ICAM-1, and MCP-1 mRNA, and monocyte adhesion. Knockout of EC-A1 or ABH prevented these effects. HFHS diet-induced VAT inflammation is mediated by EC-A1 expression/activity. Limiting arginase activity is a possible therapeutic means of controlling obesity-induced vascular and VAT inflammation.

Aims: Elevation of arginase activity has been linked to vascular dysfunction in diabetes and hypertension by a mechanism involving decreased nitric oxide (NO) bioavailability due to L-arginine depletion. Excessive arginase activity also can drive L-arginine metabolism towards the production of ornithine, polyamines, and proline, promoting proliferation of vascular smooth muscle cells and collagen formation, leading to perivascular fibrosis. We hypothesized that there is a specific involvement of arginase 1 expression within the vascular endothelial cells in this pathology., Methods and Results: To test this proposition, we used models of type 2 diabetes and metabolic syndrome. Studies were performed using wild type (WT), endothelial-specific arginase 1 knockout (EC-A1-/-) and littermate controls(A1con) mice fed high fat-high sucrose (HFHS) or normal diet (ND) for 6 months and isolated vessels exposed to palmitate-high glucose (PA/HG) media. Some WT mice or isolated vessels were treated with an arginase inhibitor, ABH [2-(S)-amino-6-boronohexanoic acid. In WT mice, the HFHS diet promoted increases in body weight, fasting blood glucose, and post-prandial insulin levels along with arterial stiffening and fibrosis, elevated blood pressure, decreased plasma levels of L-arginine, and elevated L-ornithine. The HFHS diet or PA/HG treatment also induced increases in vascular arginase activity along with oxidative stress, reduced vascular NO levels, and impaired endothelial-dependent vasorelaxation. All of these effects except obesity and hypercholesterolemia were prevented or significantly reduced by endothelial-specific deletion of arginase 1 or ABH treatment., Conclusion: Vascular dysfunctions in diet-induced obesity are prevented by deletion of arginase 1 in vascular endothelial cells or arginase inhibition. These findings indicate that upregulation of arginase 1 expression/activity in vascular endothelial cells has an integral role in diet-induced cardiovascular dysfunction and metabolic syndrome., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Background: Netrin-1, a secreted laminin-like protein identified as an axon guidance molecule, has been shown to be of critical importance in the cardiovascular system. Recent studies have revealed pro-angiogenic, anti-apoptotic and anti-inflammatory properties of netrin-1 as well as cardioprotective actions against myocardial injury in diabetic mice., Aim: To examine the role of netrin-1 in diabetes-and high glucose (HG)-induced vascular endothelial dysfunction (VED) using netrin-1 transgenic mice (Tg3) and cultured bovine aortic endothelial cells (BAEC)., Main Outcome: Overexpression of netrin-1 prevented diabetes-induced VED in aorta from diabetic mice and netrin-1 treatment attenuated HG-induced impairment of nitric oxide synthase (NOS) function in BAECs., Methods and Results: Experiments were performed in Tg3 and littermate control (WT) mice rendered diabetic with streptozotocin (STZ) and in BAECs treated with HG (25 mmol/L). Levels of netrin-1 and its receptor DCC, markers of inflammation and apoptosis and vascular function were assessed in aortas from diabetic and non-diabetic Tg3 and WT mice. Vascular netrin-1 in WT mice was reduced under diabetic conditions. Aortas from non-diabetic Tg3 and WT mice showed similar maximum endothelium-dependent relaxation (MEDR) (83% and 87%, respectively). MEDR was markedly impaired in aorta from diabetic WT mice (51%). This effect was significantly blunted in Tg3 diabetic aortas (70%). Improved vascular relaxation in Tg3 diabetic mice was associated with increased levels of phospho-ERK1/2 and reduced levels of oxidant stress, NFκB, COX-2, p16INK4A, cleaved caspase-3 and p16 and p53 mRNA. Netrin-1 treatment prevented the HG-induced decrease in NO production and elevation of oxidative stress and apoptosis in BAECs., Conclusions: Diabetes decreases aortic levels of netrin-1. However, overexpression of netrin-1 attenuates diabetes-induced VED and limits the reduction of NO levels, while increasing expression of p-ERK1/2, and suppressing oxidative stress and inflammatory and apoptotic processes. Enhancement of netrin-1 function may be a useful therapeutic means for preventing vascular dysfunction in diabetes.

[This corrects the article DOI: 10.1371/journal.pone.0071868.].

Adenosine/adenosine receptor-mediated signaling has been implicated in the development of various ischemic diseases, including ischemic retinopathies. Here, we show that the adenosine A2a receptor (ADORA2A) promotes hypoxia-inducible transcription factor-1 (HIF-1)-dependent endothelial cell glycolysis, which is crucial for pathological angiogenesis in proliferative retinopathies. Adora2a expression is markedly increased in the retina of mice with oxygen-induced retinopathy (OIR). Endothelial cell-specific, but not macrophage-specific Adora2a deletion decreases key glycolytic enzymes and reduces pathological neovascularization in the OIR mice. In human primary retinal microvascular endothelial cells, hypoxia induces the expression of ADORA2A by activating HIF-2α. ADORA2A knockdown decreases hypoxia-induced glycolytic enzyme expression, glycolytic flux, and endothelial cell proliferation, sprouting and tubule formation. Mechanistically, ADORA2A activation promotes the transcriptional induction of glycolytic enzymes via ERK- and Akt-dependent translational activation of HIF-1α protein. Taken together, these findings advance translation of ADORA2A as a therapeutic target in the treatment of proliferative retinopathies and other diseases dependent on pathological angiogenesis.Pathological angiogenesis in the retina is a major cause of blindness. Here the authors show that adenosine receptor A2A drives pathological angiogenesis in the oxygen-induced retinopathy mouse model by promoting glycolysis in endothelial cells via the ERK/Akt/HIF-1α pathway, thereby suggesting new therapeutic targets for disease treatment.