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Abstract 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.

Retinitis pigmentosa (RP) is characterized by progressive loss of vision due to photoreceptor degeneration leading to secondary inflammation. The urokinase‐type plasminogen activator (uPA) system contributes to retinal inflammation, but its role in RP is unknown. In the rd10 mouse model of RP, we addressed this question with the use of the peptide UPARANT designed to interact with the uPA system. UPARANT was systemically administered from post‐natal day (PD) 10 to PD30 when its efficacy in RP rescue was investigated using electroretinographic recordings, Western blot and immunocytochemistry. Temporal profile of protein expression in the uPA system was also investigated. UPARANT reduced both Müller cell gliosis and up‐regulated levels of inflammatory markers and exerted major anti‐apoptotic effects without influencing the autophagy cascade. Rescue from retinal cell degeneration was accompanied by improved retinal function. No scotopic phototransduction was rescued in the UPARANT‐treated animals as determined by the kinetic analysis of rod‐mediated a‐waves and confirmed by rod photoreceptor markers. In contrast, the cone photopic b‐wave was recovered and its rescue was confirmed in the whole mounts using cone arrestin antibody. Investigation of the uPA system regulation over RP progression revealed extremely low levels of uPA and its receptor uPAR both of which were recovered by HIF‐1α stabilization indicating that HIF‐1 regulates the expression of the uPA/uPAR gene in the retina. Ameliorative effects of UPARANT were likely to occur through an inhibitory action on up‐regulated activity of the αvβ3 integrin/Rac1 pathway that was suggested as a novel target for the development of therapeutic approaches against RP.

Puncture-induced iris neovascularization (rubeosis iridis; RI) in mice is associated with upregulation of extracellular matrix (ECM) degradation and inflammatory factors. The anti-angiogenic and anti-inflammatory efficacy of UPARANT in reducing RI was determined by noninvasive, in vivo iris vascular densitometry, and confirmed in vitro by quantitative vascular-specific immunostaining. Intravitreal administration of UPARANT successfully and rapidly reduced RI to non-induced control levels. Molecular analysis revealed that UPARANT inhibits formyl peptide receptors through a predominantly anti-inflammatory response, accompanied with a significant reduction in ECM degradation and inflammation markers. Similar results were observed with UPARANT administered systemically by subcutaneous injection. These data suggest that the tetrapeptide UPARANT is an effective anti-angiogenic agent for the treatment of RI, both by local and systemic administrations. The effectiveness of UPARANT in reducing RI in a model independent of the canonical vascular endothelial growth factor (VEGF) proposes an alternative for patients that do not respond to anti-VEGF treatments, which could improve treatment in proliferative ocular diseases. Key messages UPARANT is effective in the treatment of rubeosis iridis, both by local and systemic administrations.UPARANT can reduce VEGF-independent neovascularization. Electronic supplementary material The online version of this article (10.1007/s00109-019-01794-w) contains supplementary material, which is available to authorized users.

Lisosan G (LG), a fermented powder obtained from whole grains, is a recognized antioxidant compound that improves the bioactivity and survival of different cell types. The purpose of this study was to investigate whether LG ameliorates both the neural and the vascular damage characterizing early stages of diabetic retinopathy (DR). The effects of LG were studied in cultured explants of mouse retinas challenged with oxidative stress (OS) or in retinas of streptozotocin (STZ)-treated rats. Apoptosis, vascular endothelial growth factor (VEGF) expression, OS markers, blood-retinal barrier (BRB) integrity, and inflammation were assessed, while retinal function was evaluated with electroretinogram (ERG). LG extensively inhibited apoptosis, VEGF expression, and OS both in retinal explants and in STZ rats. In addition, STZ rats treated with LG displayed an almost total BRB integrity, reduced levels of inflammatory markers and a partially restored visual function as evaluated with ERG. In summary, we demonstrated that LG exhibits antioxidant and anti-inflammatory effects that exert powerful protective actions against neural and vascular defects characteristic of DR. Therefore, LG-containing foods or supplements may be considered to implement DR treatments.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Optic neuritis is an acute inflammatory demyelinating disorder of the optic nerve (ON) and is an initial symptom of multiple sclerosis (MS). Optic neuritis is characterized by ON degeneration and retinal ganglion cell (RGC) loss that contributes to permanent visual disability and lacks a reliable treatment. Here, we used the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, a well-established model also for optic neuritis. In this model, C57BL6 mice, intraperitoneally injected with a fragment of the myelin oligodendrocyte glycoprotein (MOG), were found to develop inflammation, Müller cell gliosis, and infiltration of macrophages with increased production of oncomodulin (OCM), a calcium binding protein that acts as an atypical trophic factor for neurons enabling RGC axon regeneration. Immunolabeling of retinal whole mounts with a Brn3a antibody demonstrated drastic RGC loss. Dietary supplementation with Neuro-FAG (nFAG®), a balanced mixture of fatty acids (FAs), counteracted inflammatory and gliotic processes in the retina. In contrast, infiltration of macrophages and their production of OCM remained at elevated levels thus eventually preserving OCM trophic activity. In addition, the diet supplement with nFAG exerted a neuroprotective effect preventing MOG-induced RGC death. In conclusion, these data suggest that the balanced mixture of FAs may represent a useful form of diet supplementation to limit inflammatory events and death of RGCs associated to optic neuritis. This would occur without affecting macrophage infiltration and the release of OCM thus favoring the maintenance of OCM neuroprotective role.

In the experimental autoimmune encephalomyelitis (EAE) mouse model of optic neuritis, we recently demonstrated that diet supplementation with a balanced mixture of fatty acids (FAs), including omega 3 and omega 6, efficiently limited inflammatory events in the retina and prevented retinal ganglion cell (RGC) death, although mechanisms underlying the efficacy of FAs were to be elucidated. Whether FAs effectiveness was accompanied by efficient rescue of demyelinating events in the optic nerve was also unresolved. Finally, the possibility that RGC rescue might result in ameliorated visual performance remained to be investigated. Here, the EAE model of optic neuritis was used to investigate mechanisms underlying the anti-inflammatory effects of FAs, including their potential efficacy on macrophage polarization. In addition, we determined how FAs-induced rescue of RGC degeneration was related to optic nerve histopathology by performing ultrastructural morphometric analysis with transmission electron microscopy. Finally, RGC rescue was correlated with visual performance by recording photopic electroretinogram, an efficient methodology to unravel the role of RGCs in the generation of electroretinographic waves. We conclude that the ameliorative effects of FAs were dependent on a predominant anti-inflammatory action including a role on promoting the shift of macrophages from the inflammatory M1 phenotype towards the anti-inflammatory M2 phenotype. This would finally result in restored optic nerve histopathology and ameliorated visual performance. These findings can now offer new perspectives for implementing our knowledge on the effectiveness of diet supplementation in counteracting optic neuritis and suggest the importance of FAs as possible adjuvants in therapies against inflammatory diseases of the eye.

In a mouse model of oxygen induced retinopathy (OIR), beta adrenergic receptor (BAR) blockade has been shown to recover hypoxia-associated retinal damages. Although the adrenergic signaling is an important regulator of apoptotic and autophagic processes, the role of BARs in retinal cell death remains to be elucidated. The present study was aimed at investigating whether ameliorative effects of BAR blockers may occur through their coordinated action on apoptosis and autophagy. To this aim, retinas from control and OIR mice untreated or treated with propranolol, a non-selective BAR1/2 blocker, were characterized in terms of expression and localization of apoptosis and autophagy markers. The effects of propranolol on autophagy signaling were also evaluated and specific autophagy modulators were used to get functional information on the autophagic effects of BAR antagonism. Finally, propranolol effects on neurodegenerative processes were associated to an electrophysiological investigation of retinal function by recording electroretinogram (ERG). We found that retinas of OIR mice are characterized by increased apoptosis and decreased autophagy, while propranolol reduces apoptosis and stimulates autophagy. In particular, propranolol triggers autophagosome formation in bipolar, amacrine and ganglion cells that are committed to die by apoptosis in response to hypoxia. Also our data argue that propranolol, through the inhibition of the Akt-mammalian target of rapamycin pathway, activates autophagy which decreases retinal cell death. At the functional level, propranolol recovers dysfunctional ERG by recovering the amplitude of a- and b-waves, and oscillatory potentials, thus indicating an efficient restoring of retinal transduction. Overall, our results demonstrate that BAR1/2 are key regulators of retinal apoptosis/autophagy, and that BAR1/2 blockade leads to autophagy-mediated neuroprotection. Reinstating the balance between apoptotic and autophagic machines may therefore be viewed as a future goal in the treatment of retinopathies.

Current knowledge of the benefits of nutrition supplements for eye pathologies is based largely on the use of appropriate animal models, together with defined dietary supplementation. Here, C57BL6 mice were subretinally injected with polyethylene glycol (PEG)-400, an established model of retinal degeneration with a dry age-related macular degeneration (AMD)-like phenotype, an eye pathology that lacks treatment. In response to PEG-400, markers of the complement system, angiogenesis, inflammation, gliosis, and macrophage infiltration were upregulated in both retinas and retinal pigment epithelium (RPE)/choroids, whereas dietary supplementation with a mixture based on fatty acids counteracted their upregulation. Major effects include a reduction of inflammation, in both retinas and RPE/choroids, and an inhibition of macrophage infiltration in the choroid, yet not in the retina, suggesting a targeted action through the choroidal vasculature. Histological analysis revealed a thinning of the outer nuclear layer (ONL), together with dysregulation of the epithelium layer in response to PEG-400. In addition, immunohistofluorescence demonstrated Muller cell gliosis and macrophage infiltration into subretinal tissues supporting the molecular findings. Reduced ONL thickness, gliosis, and macrophage infiltration were counteracted by the diet supplement. The present data suggest that fatty acids may represent a useful form of diet supplementation to prevent or limit the progression of dry AMD.

Purpose The activation of the urokinase-type plasminogen activator and its receptor system is associated with retinal diseases. Among peptide inhibitors of this system, UPARANT acts by preventing the onset of pathologic signs of neovascular ocular diseases. We investigated whether systemic UPARANT may act in a therapeutic regimen by suppressing the retinal damage that characterizes diabetic retinopathy using a rat model of streptozotocin-induced diabetes. Methods In healthy rats, plasma, eye, and retina concentrations of UPARANT were evaluated by mass spectrometry. In rat models of streptozotocin-induced diabetes, the appearance of diabetic retinopathy was assessed by electroretinogram (ERG). UPARANT was then administered at different dosages and daily regimens. ERG recording, Evans blue perfusion, and real-time PCR were used to evaluate UPARANT efficacy. UPARANT safety was also determined. Results UPARANT was found in plasma, eye, and retina soon after its administration and remained detectable after 24 hours. Between the 4th and the 5th week after diabetes onset, UPARANT at 8 mg/kg (daily for 5 days) was effective in recovering dysfunctional ERG. Three-day treatments at 8 mg/kg or a half dose for 5 days were ineffective. ERG recovery lasted approximately 2 weeks. ERG recovery was accompanied by restored blood-retinal barrier integrity and inhibition of inflammatory and angiogenic responses. UPARANT showed a safety profile. Conclusions These data suggest that targeting the urokinase-type plasminogen activator and its receptor system by systemic UPARANT is a potential therapeutic approach for the treatment of early diabetic retinopathy, thus providing a potential alternative approach to delay disease progression in humans.

Purpose To assess iris neovascularization by uveal puncture of the mouse eye and determine the role of angiogenic factors during iris neovascularization. Methods Uveal punctures were performed on BalbC mouse eyes to induce iris angiogenesis. VEGF-blockage was used as an anti-angiogenic treatment, while normoxia- and hypoxia-conditioned media from retinal pigment epithelium (RPE) cells was used as an angiogenic-inducer in this model. Iris vasculature was determined in vivo by noninvasive methods. Iris blood vessels were stained for platelet endothelial cell adhesion molecule-1 and vascular sprouts were counted as markers of angiogenesis. Expression of angiogenic and inflammatory factors in the puncture-induced model were determined by qPCR and western blot. Results Punctures led to increased neovascularization and sprouting of the iris. qPCR and protein analysis showed an increase of angiogenic factors, particularly in the plasminogen-activating receptor and inflammatory systems. VEGF-blockage partly reduced iris neovascularization, and treatment with hypoxia-conditioned RPE medium led to a statistically significant increase in iris neovascularization. Conclusions This study presents the first evidence of a puncture-induced iris angiogenesis model in the mouse. In a broader context, this novel in vivo model of neovascularization has the potential for noninvasive evaluation of angiogenesis modulating substances.

Glaucoma is a multifactorial blinding disease with a major inflammatory component ultimately leading to apoptotic retinal ganglion cell (RGC) death. Pharmacological treatments lowering intraocular pressure can help slow or prevent vision loss although the damage caused by glaucoma cannot be reversed. Recently, nutritional approaches have been evaluated for their efficacy in preventing degenerative events in the retina although mechanisms underlying their effectiveness remain to be elucidated. Here, we evaluated the efficacy of a diet supplement consisting of forskolin, homotaurine, spearmint extract, and vitamins of the B group in counteracting retinal dysfunction in a mouse model of optic nerve crush (ONC) used as an in vivo model of glaucoma. After demonstrating that ONC did not affect retinal vasculature by fluorescein angiography, we determined the effect of the diet supplement on the photopic negative response (PhNR) whose amplitude is strictly related to RGC integrity and is therefore drastically reduced in concomitance with RGC death. We found that the diet supplementation prevents the reduction of PhNR amplitude (p &lt, 0.001) and concomitantly counteracts RGC death, as in supplemented mice, RGC number assessed immunohistochemically is significantly higher than that in non-supplemented animals (p &lt, 0.01). Major determinants of the protective efficacy of the compound are due to a reduction of ONC-associated cytokine secretion leading to decreased levels of apoptotic markers that in supplemented mice are significantly lower than in non-supplemented animals (p &lt, 0.001), ultimately causing RGC survival and ameliorated visual dysfunction. Overall, our data suggest that the above association of compounds plays a neuroprotective role in this mouse model of glaucoma thus offering a new perspective in inflammation-associated neurodegenerative diseases of the inner retina.

The spontaneously diabetic Torii (SDT) rat is of increasing preclinical interest because of its similarities to human type 2 diabetic retinopathy (DR). The system formed by urokinase-type plasminogen activator (uPA) and its receptor (uPAR) is a player in blood-retinal barrier (BRB) breakdown in DR. Here, we investigated whether in SDT rats, preventive administration of UPARANT, an inhibitor of the uPAR pathway, counteracts the retinal impairment in response to chronic hyperglycemia. Electroretinogram (ERG) monitoring was followed over time. Fluorescein-dextran microscopy, CD31 immunohistochemistry, quantitative PCR, ELISA, Evans blue perfusion, and Western blot were also used. UPARANT prevented ERG dysfunction, upregulation of vascular endothelial growth factor and fibroblast growth factor-2, BRB leakage, gliosis, and retinal cell death. The mechanisms underlying UPARANT benefits were studied comparing them with the acute streptozotocin (STZ) model in which UPARANT is known to inhibit DR signs. In SDT rats, but not in the STZ model, UPARANT downregulated the expression of uPAR and its membrane partners. In both models, UPARANT reduced the levels of transcription factors coupled to inflammation or inflammatory factors themselves. These findings may help to establish the uPAR system as putative target for the development of novel drugs that may prevent type 2 DR.

PURPOSE A mouse model of age-related macular degeneration (AMD) was used to investigate the anti-angiogenic and anti-inflammatory role of UPARANT in laser-induced choroidal neovascularization (CNV). METHODS Choroidal neovascularization was induced by laser photocoagulation, and UPARANT was intravitreally injected. Some experiments were also performed after either intravitreal injection of anti-VEGF drugs or systemic administration of UPARANT. Immunohistochemistry using CD31 antibodies was used to evaluate the area of CNV. Evans blue dye extravasation was quantitatively assessed. Transcripts of markers of outer blood retinal barrier were measured by quantitative RT-PCR, also used to evaluate angiogenesis and inflammation markers. Western blot was used to determine levels of transcription factors encoding genes involved in angiogenesis and inflammation. Levels of urokinase-type plasminogen activator (uPA), its receptor (uPAR), and formyl peptide receptors (FPRs) were determined at the transcript and the protein level. RESULTS Intravitreal UPARANT reduced the CNV area and the leakage from the choroid. The uPA/uPAR/FPR system was upregulated in CNV, but was not influenced by UPARANT. UPARANT recovered laser-induced upregulation of transcription factors encoding angiogenic and inflammatory markers. Accordingly, angiogenic and inflammatory factors were also reduced. UPARANT as compared to anti-VEGF drugs displayed similar effects on CNV area. CONCLUSIONS UPARANT mitigates laser-induced CNV by inhibiting angiogenesis and inflammation through an action on transcription factors encoding angiogenesis and inflammatory genes. The finding that UPARANT is effective against CNV may help to establish uPAR and its membrane partners as putative targets in the treatment of AMD.

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.