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A major player in the homeostatic response to hypoxia is the hypoxia-inducible factor (HIF)-1 that transactivates a number of genes involved in neovessel proliferation in response to low oxygen tension. In the retina, hypoxia overstimulates β-adrenoceptors (β-ARs) which play a key role in the formation of pathogenic blood vessels. Among β-ARs, β3-AR expression is increased in proliferating vessels in concomitance with increased levels of HIF-1α and vascular endothelial growth factor (VEGF). Whether, similarly to VEGF, hypoxia-induced β3-AR upregulation is driven by HIF-1 is still unknown. We used the mouse model of oxygen-induced retinopathy (OIR), an acknowledged model of retinal angiogenesis, to verify the hypothesis of β3-AR transcriptional regulation by HIF-1. Investigation of β3-AR regulation over OIR progression revealed that the expression profile of β3-AR depends on oxygen tension, similar to VEGF. The additional evidence that HIF-1α stabilization decouples β3-AR expression from oxygen levels further indicates that HIF-1 regulates the expression of the β3-AR gene in the retina. Bioinformatics predicted the presence of six HIF-1 binding sites (HBS #1-6) upstream and inside the mouse β3-AR gene. Among these, HBS #1 has been identified as the most suitable HBS for HIF-1 binding. Chromatin immunoprecipitation-qPCR demonstrated an effective binding of HIF-1 to HBS #1 indicating the existence of a physical interaction between HIF-1 and the β3-AR gene. The additional finding that β3-AR gene expression is concomitantly activated indicates the possibility that HIF-1 transactivates the β3-AR gene. Our results are indicative of β3-AR involvement in HIF-1-mediated response to hypoxia.

In retinopathy of prematurity (ROP), the abnormal retinal neovascularization is often accompanied by retinal neuronal dysfunction. Here, a rat model of oxygen-induced retinopathy (OIR), which mimics the ROP disease, was used to investigate changes in the expression of key mediators of autophagy and markers of cell death in the rat retina. In addition, rats were treated from birth to postnatal day 14 and 18 with 3-methyladenine (3-MA), an inhibitor of autophagy. Immunoblot and immunofluorescence analysis demonstrated that autophagic mechanisms are dysregulated in the retina of OIR rats and indicated a possible correlation between autophagy and necroptosis, but not apoptosis. We found that 3-MA acts predominantly by reducing autophagic and necroptotic markers in the OIR retinas, having no effects on apoptotic markers. However, 3-MA does not ameliorate retinal function, which results compromised in this model. Taken together, these results revealed the crucial role of autophagy in retinal cells of OIR rats. Thus, inhibiting autophagy may be viewed as a putative strategy to counteract ROP., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Proliferative retinopathies (PR) lead to an increase in neovascularization and inflammation factors, at times culminating in pathologic rubeosis iridis (RI). In mice, uveal puncture combined with injection of hypoxia-conditioned media mimics RI associated with proliferative retinopathies. Here, we investigated the effects of the urokinase plasminogen activator receptor (uPAR) antagonist-UPARANT-on the angiogenic and inflammatory processes that are dysregulated in this model. In addition, the effects of UPARANT were compared with those of anti-vascular endothelial growth factor (VEGF) therapies. Administration of UPARANT promptly decreased iris vasculature, while anti-VEGF effects were slower and less pronounced. Immunoblot and qPCR analysis suggested that UPARANT acts predominantly by reducing the upregulated inflammatory and extracellular matrix degradation responses. UPARANT appears to be more effective in comparison to anti-VEGF in the treatment of RI associated with PR in the murine model, by modulating multiple uPAR-associated signaling pathways. Furthermore, UPARANT effectiveness was maintained when systemically administered, which could open to novel improved therapies for proliferative ocular diseases, particularly those associated with PR. KEY MESSAGES: • Further evidence of UPARANT effectiveness in normalizing pathological iris neovascularization. • Both systemic and local administration of UPARANT reduce iris neovascularization in a model associated with proliferative retinopathies. • In the mouse models of rubeosis iridis associated with proliferative retinopathy, UPARANT displays stronger effects when compared with anti-vascular endothelial growth factor regimen.

Dysregulation of vascular networks is characteristic of eye diseases associated with retinal cell degeneration and visual loss. Visual impairment is also the consequence of photoreceptor degeneration in inherited eye diseases with a major inflammatory component, but without angiogenic profile. Among the pathways with high impact on vascular/degenerative diseases of the eye, a central role is played by a system formed by the ligand urokinase-type plasminogen activator (uPA) and its receptor uPAR. The uPAR system, although extensively investigated in tumors, still remains a key issue in vascular diseases of the eye and even less studied in inherited retinal pathologies such as retinitis pigmantosa (RP). Its spectrum of action has been extended far beyond a classical pro-angiogenic function and has emerged as a central actor in inflammation. Preclinical studies in more prevalent eye diseases characterized by neovascular formation, as in retinopathy of prematurity, wet macular degeneration and rubeosis iridis or vasopermeability excess as in diabetic retinopathy, suggest a critical role of increased uPAR signaling indicating the potentiality of its modulation to counteract neovessel formation and microvascular dysfunction. The additional observation that the uPAR system plays a major role in RP by limiting the inflammatory cascade triggered by rod degeneration rises further questions about its role in the diseased eye.

Hypoxia-dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin-4 (AQP4) is involved in the hypoxia-dependent VEGF upregulation in the retina of a mouse model of oxygen-induced retinopathy (OIR). The expression levels of VEGF, the hypoxia-inducible factor-1α (HIF-1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF-1 binding site (HBS) in the VEGF gene promoter, the binding of HIF-1α to the HBS, the retinal vascularization and function have been determined in the retina of wild-type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF-1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF-1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF-1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF-1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF-induced retinal damage in response to hypoxia., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Aquaporin-4 (AQP4) is the Central Nervous System water channel highly expressed at the perivascular glial domain. In the retina, two types of AQP4 expressing glial cells take part in the blood-retinal barrier (BRB), astrocytes and Müller cells. The aim of the present study is to investigate the effect of AQP4 deletion on the retinal vasculature by looking at typical pathological hallmark such as BRB dysfunction and gliotic condition. AQP4 dependent BRB properties were evaluated by measuring the number of extravasations in WT and AQP4 KO retinas by Evans blue injection assay. AQP4 deletion did not affect the retinal vasculature, as assessed by Isolectin B4 staining, but caused BRB impairment to the deep plexus capillaries while the superficial and intermediate capillaries were not compromised. To investigate for gliotic responses caused by AQP4 deletion, Müller cells and astrocytes were analysed by immunofluorescence and western blot, using the Müller cell marker Glutamine Synthetase (GS) and the astrocyte marker GFAP. While GS expression was not altered in AQP4 KO retinas, a strong GFAP upregulation was found at the level of AQP4 KO astrocytes at the superficial plexus and not at Müller cells at the intermediate and deep plexi. These data, together with the upregulation of inflammatory markers (TNF-α, IL-6, IL-1β and ICAM-1) in AQP4 KO retinas indicated AQP4 deletion as responsible for a gliotic phenotype. Interestingly, no GFAP altered expression was found in AQP4 siRNA treated astrocyte primary cultures. All together these results indicate that AQP4 deletion is directly responsible for BRB dysfunction and gliotic condition in the mouse retina. The selective activation of glial cells at the primary plexus suggests that different regulatory elements control the reaction of astrocytes and Müller cells. Finally, GFAP upregulation is strictly linked to gliovascular crosstalk, as it is absent in astrocytes in culture. This study is useful to understand the role of AQP4 in the perivascular domain in the retina and its possible implications in the pathogenesis of retinal vascular diseases and of Neuromyelitis Optica, a human disease characterized by anti-AQP4 auto-antibodies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: Pharmacologic control of neovascularization is a promising approach for the treatment of retinal angiogenesis. UPARANT, an inhibitor of the urokinase-type plasminogen activator receptor (uPAR), inhibits VEGF-driven angiogenesis in vitro and in vivo. This study investigates for the first time the effectiveness of UPARANT in counteracting pathologic neovascularization in the retina., Methods: Murine retinal fragments and a mouse model of oxygen-induced retinopathy (OIR) were used. In mice with OIR, UPARANT-treated retinas were analyzed for avascular area and neovascular tuft formation. Levels of transcription and proangiogenic factors were determined. UPARANT effects on the blood-retinal barrier (BRB), visual function, retinal cytoarchitecture, and inflammatory markers were also assessed. Human umbilical vein endothelial cells (HUVECs) and chick embryo chorioallantoic membrane (CAM) in which angiogenesis was induced by the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) were also used., Results: UPARANT reduced VEGF-induced angiogenesis in retinal fragments. In mice with OIR, UPARANT decreased neovascular response, VEGF, and VEGF receptor-2 activity. Transcription factors regulating VEGF expression were also reduced. UPARANT restored BRB integrity, recovered visual loss, and reduced levels of inflammatory markers. Restored electroretinogram does not involve any rescue in the retinal cytoarchitecture. Finally, UPARANT blocked PDR vitreous fluid-induced angiogenesis in HUVEC and CAM assays., Conclusions: The finding that UPARANT is effective against neovascularization may help to establish uPAR as a target in the treatment of proliferative retinopathies. The potential application of UPARANT in retinal diseases is further supported by UPARANT capacity to counteract the angiogenic activity of PDR vitreous fluid.

Retinal neovascular pathologies, such as diabetic retinopathy, retinopathy of prematurity (ROP) and age-related macular degeneration, may be treated with intravitreal injections of drugs targeting vascular endothelial growth factor (VEGF), the main inducer of neoangiogenesis; however further improvements and alternative strategies are needed. In the last few years, an intense research activity has focused on the β-adrenergic system. The results indicate that, in different experimental models, a decrease of the β-adrenergic function may result either in reduction or in exacerbation of the vascular changes, thus suggesting possible dual effects of β-adrenoreceptor (β-AR) modulation depending on the experimental setting. In in vivo models of proliferative retinopathies, most of the data point to a strong inhibitory role against vascular changes exerted by the blockade of specific β-ARs. In particular, the β2-AR seems to be the mostly involved in these responses, and the β1-/β2-AR blocker propranolol results highly effective in inhibiting both the increase of VEGF expression caused by a hypoxic insult and the consequent neovascular response. These observations have prompted clinical trials in preterm infants with ROP, where oral administrations of propranolol produced positive results in terms of efficacy, although safety problems were also reported. In addition, the possibility of using topical propranolol administrations in the form of eye drops opens new potential routes of drug administration in humans. A further point that should be considered is that there are data demonstrating significant antiapoptotic effects exerted by β-ARs, therefore if β-AR blockers were used to inhibit aberrant neovascularization, there may be a burden to pay in terms of impaired neuronal viability., (Copyright © 2014. Published by Elsevier Ltd.)

Purpose: To evaluate if coenzyme Q10 (CoQ10) can protect retinal ganglion cells (RGCs) from apoptosis and, when instilled as eye drops on the cornea, if it can reach the retina and exert its antiapoptotic activity in this area in a mouse model of kainate (KA)-induced retinal damage., Methods: Rat primary or cultured RGCs were subjected to glutamate (50 μM) or chemical hypoxia (Antimycin A, 200 μM) or serum withdrawal (FBS, 0.5%) in the presence or absence of CoQ10 (10 μM). Cell viability was evaluated by light microscopy and fluorescence-activated cell sorting analyses. Apoptosis was evaluated by caspase 3/7 activity and mitochondrion depolarization tetramethylrhodamine ethyl ester analysis. CoQ10 transfer to the retina following its instillation as eye drops on the cornea was quantified by HPLC. Retinal protection by CoQ10 (10 μM) eye drops instilled on the cornea was then evaluated in a mouse model of KA-induced excitotoxic retinal cell apoptosis by cleaved caspase 3 immunohistofluorescence, caspase 3/7 activity assays, and quantification of inhibition of RGC loss., Results: CoQ10 significantly increased viable cells by preventing RGC apoptosis. Furthermore, when topically applied as eye drops to the cornea, it reached the retina, thus substantially increasing local CoQ10 concentration and protecting retinal layers from apoptosis., Conclusions: The ability of CoQ10 eye drops to protect retinal cells from apoptosis in the mouse model of KA-induced retinal damage suggests that topical CoQ10 may be evaluated in designing therapies for treating apoptosis-driven retinopathies.

Purpose: To determine whether the somatostatin receptor 2 (sst(2)) influences angiogenesis and its associated factors in a model of hypoxia-induced retinal neovascularization., Methods: sst(1)-knockout (KO) mice, in which sst(2) is overexpressed and overfunctional, and sst(2)-KO mice were used. Angiogenesis was evaluated in fluorescein-perfused retinas. Angiogenesis-associated factors were determined by RT-PCR and immunohistochemistry., Results: Retinal neovascularization was increased in sst(2)-KO mice, but remained unchanged in sst(1)-KO compared with wild-type (WT) mice. Retinal levels of sst(2) mRNA were not affected by hypoxia. Normoxic levels of angiogenesis regulators were similar in WT and KO retinas except for mRNA levels of IGF-1, Ang-2, and its receptor Tie-2. In WT, hypoxia induced an increase in mRNA levels of (1) VEGF and its receptors, (2) IGF-1R, and (3) Ang-2 and Tie-2. The increase in VEGF and IGF-1R mRNAs was more pronounced after sst(2) loss, but was less pronounced when sst(2) was overexpressed. In addition, in hypoxic retinas, sst(2) loss increased IGF-1 mRNA, whereas it decreased Ang-1, Tie-1, and Tie-2 mRNA levels. Moreover, Tie-1 mRNA increased when sst(2) was overexpressed. Immunohistochemistry confirmed the results in hypoxic retinas on increased expression of VEGF, IGF-1, and their receptors after sst(2) loss. It also allowed the localization of these factors to specific retinal cells. In this respect, VEGFR-2, IGF-1, and IGF-1R were localized to Müller cells., Conclusions: These results suggest that sst(2) may be protective against angiogenesis. The immediate clinical importance lies in the establishment of a potential pharmacological target based on sst(2) pharmacology.

The somatostatinergic system of the retina has been investigated in a variety of studies. A considerable amount of experimental evidence is available concerning the patterns of expression of somatostatin (SRIF) and its receptors in vertebrate retinas. However the functional roles of this peptidergic system in retinal physiology are far from being elucidated. Nonetheless, data have been provided concerning the regulatory action of SRIF on the excitability of different retinal cell types and on the modulation of ion channels in different vertebrate retinas. The present review is focused on recent and unpublished investigations of the mouse retina relative to the involvement of specific SRIF receptors in the regulation of ion channels and transmitter release, the transduction pathways coupled to SRIF receptors, and the mechanisms regulating the expression of SRIF and its receptors as derived from studies in transgenic animal models. In these models, altered expression levels of SRIF or of specific SRIF receptors have also been found to affect the morphology of retinal cell types (namely the rod bipolar cells) and to result in functional alterations at the level of both ion channel regulation and transmitter release. These new pieces of evidence constitute an important step forward in the understanding of the functional actions of the retinal somatostatinergic system, although our current knowledge is far from being exhaustive. The ultimate goal of understanding SRIF functional actions in the retina is concerned with the possibility of using SRIF or its analogs as therapeutic agents to cure retinal diseases. Indeed, encouraging results are being obtained in clinical investigations focused on the use of SRIF analogs to treat diabetic retinopathy, a retinal disease with high social impact and originating as a complication of diabetes. The closing part of the present paper examines the evidence supporting SRIF as a promising therapeutic agent in this disease.

Purpose In the retina, hypoxia is considered one of the key factors to trigger angiogenesis and to promote apoptosis by reducing photoreceptors and other retinal neurons, each of them contributing to vision loss. We recently demonstrated that the beta‐adrenergic system interferes with angiogenesis‐dependent diseases in the retina by regulating endogenous VEGF. Aim of the present study was to investigate the role of distinct beta‐adrenergic receptors (BARs)in neurodegenerative processes in response to hypoxia. Methods Ex vivo mouse retinal explants and in vivo models were used. BARs were blocked with antagonists or silenced with siRNAs. VEGFR2 was blocked with SU1498, whereas iNOs and eNOS with AG, L‐NIO or L‐NAME. mRNA levels were evaluated with quantitative RT‐PCR. BARs, iNOS, eNOS and apoptotic signals were determined with Western blot. Immunohistochemistry was used to localize BARs and to assess neovascularization. VEGF release and NO production were measured with colorimetric assays. Hoechst and TUNEL were used to evaluate cell loss. Results Among the BARs, BAR3 is localized to blood vessels, is upregulated by hypoxia and acts through iNOS/NO to control VEGF levels and degenerative responses to the hypoxic insult. In particular, BAR3 blockade is accompanied by increased levels of apoptotic signals and cell loss. Pharmacological interaction with the VEGF signalling demonstrates that BAR3 exerts a protective action on retinal cells through a modulation of endogenous VEGF. Conclusion Our findings demonstrate that modulation of BAR3 activity during ischemia may be a powerful means to achieve neuroprotection. VEGF's role in retinal cell survival has important implications for treatments with VEGF blockade within the context of ocular vascular diseases. [ABSTRACT FROM AUTHOR]