Oral administration of a dual ETA/ETB receptor antagonist promotes neuroprotection in a rodent model of glaucoma.

Purpose: Glaucoma is a neurodegenerative disease associated with elevated intraocular pressure and characterized by optic nerve axonal degeneration, cupping of the optic disc, and loss of retinal ganglion cells (RGCs). The endothelin (ET) system of vasoactive peptides (ET-1, ET-2, ET-3) and their G-protein coupled receptors (ETA and ETB receptors) have been shown to contribute to the pathophysiology of glaucoma. The purpose of this study was to determine whether administration of the endothelin receptor antagonist macitentan was neuroprotective to RGCs and optic nerve axons when administered after the onset of intraocular pressure (IOP) elevation in ocular hypertensive rats. Methods: Male and female Brown Norway rats were subjected to the Morrison model of ocular hypertension by injection of hypertonic saline through the episcleral veins. Following IOP elevation, macitentan (5 mg/kg body wt) was administered orally 3 days per week, and rats with IOP elevation were maintained for 4 weeks. RGC function was determined by pattern electroretinography (PERG) at 2 and 4 weeks post-IOP elevation. Rats were euthanized by approved humane methods, and retinal flat mounts were generated and immunostained for the RGC-selective marker Brn3a. PPD-stained optic nerve sections were imaged by confocal microscopy. RGC and axon counts were conducted in a masked manner and compared between the treatment groups. Results: Significant protection against loss of RGCs and optic nerve axons was found following oral administration of macitentan in rats with elevated IOP. In addition, a protective trend for RGC function, as measured by pattern ERG analysis, was evident following macitentan treatment. Conclusions: Macitentan treatment had a neuroprotective effect on RGCs and their axons, independent of its IOP-lowering effect, suggesting that macitentan may complement existing treatments to prevent neurodegeneration during ocular hypertension. The findings presented have implications for the use of macitentan as an oral formulation to promote neuroprotection in glaucoma patients.

Neuroprotective Effects of Transferrin in Experimental Glaucoma Models.

Iron is essential for retinal metabolism, but an excess of ferrous iron causes oxidative stress. In glaucomatous eyes, retinal ganglion cell (RGC) death has been associated with dysregulation of iron homeostasis. Transferrin (TF) is an endogenous iron transporter that controls ocular iron levels. Intraocular administration of TF is neuroprotective in various models of retinal degeneration, preventing iron overload and reducing iron-induced oxidative stress. Herein, we assessed the protective effects of TF on RGC survival, using ex vivo rat retinal explants exposed to iron, NMDA-induced excitotoxicity, or CoCl2-induced hypoxia, and an in vivo rat model of ocular hypertension (OHT). TF significantly preserved RGCs against FeSO4-induced toxicity, NMDA-induced excitotoxicity, and CoCl2-induced hypoxia. TF protected RGCs from apoptosis, ferroptosis, and necrosis. In OHT rats, TF reduced RGC loss by about 70% compared to vehicle-treated animals and preserved about 47% of the axons. Finally, increased iron staining was shown in the retina of a glaucoma patient's eye as compared to non-glaucomatous eyes. These results indicate that TF can interfere with different cell-death mechanisms involved in glaucoma pathogenesis and demonstrate the ability of TF to protect RGCs exposed to elevated IOP. Altogether, these results suggest that TF is a promising treatment against glaucoma neuropathy.

Nanoencapsulated hybrid compound SA-2 with long-lasting intraocular pressure-lowering activity in rodent eyes.

Purpose: Glaucoma is a neurodegenerative disease of the eye with an estimated prevalence of more than 111.8 million patients worldwide by 2040, with at least 6 to 8 million projected to become bilaterally blind. Clinically, the current method of slowing glaucomatous vision loss is to reduce intraocular pressure (IOP). In this manuscript, we describe the in vitro cytoprotective and in vivo long lasting IOP-lowering activity of the poly D, L-lactic-co-glycolic acid (PLGA) nanoparticle-encapsulated hybrid compound SA-2, possessing nitric oxide (NO) donating and superoxide radical scavenging functionalities. Methods: Previously characterized primary human trabecular meshwork (hTM) cells were used for the study. hTM cells were treated with SA-2 (100 µM, 200 µM, and 1,000 µM), SA-2 PLGA-loaded nanosuspension (SA-2 NPs, 0.1%), or vehicle for 30 min. Cyclic guanosine monophosphate (cGMP) and super oxide dismutase (SOD) levels were analyzed using commercial kits. In another experiment, hTM cells were pretreated with tert-butyl hydrogen peroxide (TBHP, 300 µM) for 30 min followed by treatment with escalating doses of SA-2 for 24 h, and CellTiter 96 cell proliferation assay was performed. For the biodistribution study, the cornea, aqueous humor, vitreous humor, retina, choroid, and sclera were collected after 1 h of administration of a single eye drop (30 µl) of SA-2 NPs (1% w/v) formulated in PBS to rat (n = 6) eyes. Compound SA-2 was quantified using high performance liquid chromatography /mass spectrometry (HPLC/MS). For the IOP-lowering activity study, a single SA-2 NPs (1%) eye drop was instilled in normotensive rats eyes and in the IOP-elevated rat eyes (n = 3/group, in the Morrison model of glaucoma), or Ad5TGFß2-induced ocular hypertensive (OHT) mouse eyes (n = 5/group). IOP was measured at various time points up to 72 h, and the experiment was repeated in triplicate. Mouse aqueous humor outflow facility was determined with multiple flow-rate infusion and episcleral venous pressure estimated with manometry. Results: SA-2 upregulated cGMP levels (six- to ten-fold) with an half maximal effective concentration (EC50) of 20.3 µM in the hTM cells and simultaneously upregulated (40-fold) the SOD enzyme when compared with the vehicle-treated hTM cells. SA-2 also protected hTM cells from TBHP-induced decrease in cell survival with an EC50 of 0.38 µM. A single dose of slow-release SA-2 NPs (1% w/v) delivered as an eye drop significantly lowered IOP (by 30%) in normotensive and OHT rodent eyes after 3 h post-dose, with the effect lasting up to 72 h. A statistically significant increase in aqueous outflow facility and a decrease in episcleral venous pressure was observed in rodents at this dose at 54 h. Conclusions: Hybrid compound SA-2 upregulated cGMP in hTM cells, increased outflow facility and decreased IOP in rodent models of OHT. Compound SA-2 possessing an antioxidant moiety provided additive cytoprotective activity to oxidatively stressed hTM cells by scavenging reactive oxygen species (ROS) and increasing SOD enzyme activity. Additionally, the PLGA nanosuspension formulation (SA-2 NPs) provided longer duration of IOP-lowering activity (up to 3 days) in comparison with the free non-encapsulated SA-2 drug. The data have implications for developing novel, non-prostaglandin therapeutics for IOP-lowering and cytoprotective effects with the possibility of an eye drop dosing regimen of once every 3 days for patients with glaucoma.

Sodium 4-Phenylbutyrate Reduces Ocular Hypertension by Degrading Extracellular Matrix Deposition via Activation of MMP9.

Ocular hypertension (OHT) is a serious adverse effect of the widely prescribed glucocorticoid (GC) therapy and, if left undiagnosed, it can lead to glaucoma and complete blindness. Previously, we have shown that the small chemical chaperone, sodium-4-phenylbutyrate (PBA), rescues GC-induced OHT by reducing ocular endoplasmic reticulum (ER) stress. However, the exact mechanism of how PBA rescues GC-induced OHT is not completely understood. The trabecular meshwork (TM) is a filter-like specialized contractile tissue consisting of TM cells embedded within extracellular matrix (ECM) that controls intraocular pressure (IOP) by constantly regulating aqueous humor (AH) outflow. Induction of abnormal ECM deposition in TM is a hallmark of GC-induced OHT. Here, we investigated whether PBA reduces GC-induced OHT by degrading abnormal ECM deposition in TM using mouse model of GC-induced OHT, ex vivo cultured human TM tissues and primary human TM cells. We show that topical ocular eye drops of PBA (1%) significantly lowers elevated IOP in mouse model of GC-induced OHT. Importantly, PBA prevents synthesis and deposition of GC-induced ECM in TM. We report for the first time that PBA can degrade existing abnormal ECM in normal human TM cells/tissues by inducing matrix metalloproteinase (MMP)9 expression and activity. Furthermore, inhibition of MMPs activity by chemical-inhibitor (minocycline) abrogated PBA's effect on ECM reduction and its associated ER stress. Our study indicates a non-chaperone activity of PBA via activation of MMP9 that degrades abnormal ECM accumulation in TM.

Astragaloside IV Attenuates Ocular Hypertension in a Mouse Model of TGFß2 Induced Primary Open Angle Glaucoma.

Elevated intraocular pressure (IOP) is a major risk factor in developing primary open angle glaucoma (POAG), which is the most common form of glaucoma. Transforming growth factor-beta 2 (TGFß2) is a pro-fibrotic cytokine that plays an important role in POAG pathogenesis. TGFß2 induced extracellular matrix (ECM) production, deposition and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) contribute to increased aqueous humor (AH) outflow resistance and IOP elevation. Drugs which alter the glaucomatous fibrotic changes and ER stress in the TM may be effective in reducing ocular hypertension. Astragaloside IV (AS.IV), a novel saponin isolated from the roots of Astragalus membranaceus, has demonstrated antifibrotic and ER stress lowering effects in various tissues during disease conditions. However, the effect of AS.IV on glaucomatous TM fibrosis, ER stress and ocular hypertension has not been studied. Primary human TM cells treated with AS.IV decreased TGFß2 induced ECM (FN, Col-I) deposition and ER stress (KDEL, ATF4 and CHOP). Moreover, AS.IV treatment reduced TGFß2 induced NF-κB activation and αSMA expression in TM cells. We found that AS.IV treatment significantly increased levels of matrix metalloproteases (MMP9 and MMP2) and MMP2 enzymatic activity, indicating that the antifibrotic effects of AS.IV are mediated via inhibition of NF-κB and activation of MMPs. AS.IV treatment also reduced ER stress in TM3 cells stably expressing mutant myocilin. Interestingly, the topical ocular AS.IV eye drops (1 mM) significantly decreased TGFß2 induced ocular hypertension in mice, and this was associated with a decrease in FN, Col-1 (ECM), KDEL (ER stress) and αSMA in mouse TM tissues. Taken together, the results suggest that AS.IV prevents TGFß2 induced ocular hypertension by modulating ECM deposition and ER stress in the TM.

Mechanisms contributing to inhibition of retinal ganglion cell death by cell permeable peptain-1 under glaucomatous stress.

This study assesses the neuroprotective potential of CPP-P1, a conjugate of an anti-apoptotic peptain-1 (P1) and a cell-penetrating peptide (CPP) in in vitro, in vivo, and ex vivo glaucoma models. Primary retinal ganglion cells (RGCs) were subjected to either neurotrophic factor (NF) deprivation for 48 h or endothelin-3 (ET-3) treatment for 24 h and received either CPP-P1 or vehicle. RGC survival was analyzed using a Live/Dead assay. Axotomized human retinal explants were treated with CPP-P1 or vehicle for seven days, stained with RGC marker RBPMS, and RGC survival was analyzed. Brown Norway (BN) rats with elevated intraocular pressure (IOP) received weekly intravitreal injections of CPP-P1 or vehicle for six weeks. RGC function was evaluated using a pattern electroretinogram (PERG). RGC and axonal damage were also assessed. RGCs from ocular hypertensive rats treated with CPP-P1 or vehicle for seven days were isolated for transcriptomic analysis. RGCs subjected to 48 h of NF deprivation were used for qPCR target confirmation. NF deprivation led to a significant loss of RGCs, which was markedly reduced by CPP-P1 treatment. CPP-P1 also decreased ET-3-mediated RGC death. In ex vivo human retinal explants, CPP-P1 decreased RGC loss. IOP elevation resulted in significant RGC loss in mid-peripheral and peripheral retinas compared to that in naive rats, which was significantly reduced by CPP-P1 treatment. PERG amplitude decline in IOP-elevated rats was mitigated by CPP-P1 treatment. Following IOP elevation in BN rats, the transcriptomic analysis showed over 6,000 differentially expressed genes in the CPP-P1 group compared to the vehicle-treated group. Upregulated pathways included CREB signaling and synaptogenesis. A significant increase in Creb1 mRNA and elevated phosphorylated Creb were observed in CPP-P1-treated RGCs. Our study showed that CPP-P1 is neuroprotective through CREB signaling enhancement in several settings that mimic glaucomatous conditions. The findings from this study are significant as they address the pressing need for the development of efficacious therapeutic strategies to maintain RGC viability and functionality associated with glaucoma.

Role of Glucocorticoids and Glucocorticoid Receptors in Glaucoma Pathogenesis.

The glucocorticoid receptor (GR), including both alternative spliced isoforms (GRα and GRß), has been implicated in the development of primary open-angle glaucoma (POAG) and iatrogenic glucocorticoid-induced glaucoma (GIG). POAG is the most common form of glaucoma, which is the leading cause of irreversible vision loss and blindness in the world. Glucocorticoids (GCs) are commonly used therapeutically for ocular and numerous other diseases/conditions. One serious side effect of prolonged GC therapy is the development of iatrogenic secondary ocular hypertension (OHT) and OAG (i.e., GC-induced glaucoma (GIG)) that clinically and pathologically mimics POAG. GC-induced OHT is caused by pathogenic damage to the trabecular meshwork (TM), a tissue involved in regulating aqueous humor outflow and intraocular pressure. TM cells derived from POAG eyes (GTM cells) have a lower expression of GRß, a dominant negative regulator of GC activity, compared to TM cells from age-matched control eyes. Therefore, GTM cells have a greater pathogenic response to GCs. Almost all POAG patients develop GC-OHT when treated with GCs, in contrast to a GC responder rate of 40% in the normal population. An increased expression of GRß can block GC-induced pathogenic changes in TM cells and reverse GC-OHT in mice. The endogenous expression of GRß in the TM may relate to differences in the development of GC-OHT in the normal population. A number of studies have suggested increased levels of endogenous cortisol in POAG patients as well as differences in cortisol metabolism, suggesting that GCs may be involved in the development of POAG. Additional studies are warranted to better understand the molecular mechanisms involved in POAG and GIG in order to develop new disease-modifying therapies to better treat these two sight threatening forms of glaucoma. The purpose of this timely review is to highlight the pathological and clinical features of GC-OHT and GIG, mechanisms responsible for GC responsiveness, potential therapeutic options, as well as to compare the similar features of GIG with POAG.

Therapeutic Potential of Antioxidants and Hybrid TEMPOL Derivatives in Ocular Neurodegenerative Diseases: A Glimpse into the Future.

Reactive oxygen species play a significant role in the pathogenesis of various ocular neurodegenerative diseases especially glaucoma, age-related macular degeneration (AMD), and ocular ischemic stroke. Increased oxidative stress and the accumulation of ROS have been implicated in the progression of these diseases. As a result, there has been growing interest in exploring potential therapeutic and prophylactic strategies involving exogenous antioxidants. In recent years, there have been significant advancements in the development of synthetic therapeutic antioxidants for targeting reactive oxygen species (ROS) in neurodegenerative diseases. One area of focus has been the development of hybrid TEMPOL derivatives. In the context of ocular diseases, the application of next-generation hybrid TEMPOL antioxidants may offer new avenues for neuroprotection. By targeting ROS and reducing oxidative stress in the retina and optic nerve, these compounds have the potential to preserve retinal ganglion cells and trabecular meshwork and protect against optic nerve damage, mitigating irreversible blindness associated with these diseases. This review seeks to highlight the potential impact of hybrid TEMPOL antioxidants and their derivatives on ocular neurodegenerative disorders.

Role of mitophagy in ocular neurodegeneration.

Neurons in the central nervous system are among the most metabolically active cells in the body, characterized by high oxygen consumption utilizing glucose both aerobically and anaerobically. Neurons have an abundance of mitochondria which generate adequate ATP to keep up with the high metabolic demand. One consequence of the oxidative phosphorylation mechanism of ATP synthesis, is the generation of reactive oxygen species which produces cellular injury as well as damage to mitochondria. Mitochondria respond to injury by fusion which serves to ameliorate the damage through genetic complementation. Mitochondria also undergo fission to meet an increased energy demand. Loss of mitochondria is also compensated by increased biogenesis to generate new mitochondria. Damaged mitochondria are removed by mitophagy, an autophagic process, in which damaged mitochondria are surrounded by a membrane to form an autophagosome which ultimately fuses with the lysosome resulting in degradation of faulty mitochondria. Dysregulation of mitophagy has been reported in several central nervous system disorders, including, Alzheimer's disease and Parkinson's disease. Recent studies point to aberrant mitophagy in ocular neurodegenerative disorders which could be an important contributor to the disease etiology/pathology. This review article highlights some of the recent findings that point to dysregulation of mitophagy and it's underlying mechanisms in ocular neurodegenerative diseases, including, glaucoma, age-related macular degeneration and diabetic retinopathy.

Therapeutic applications of CRISPR/Cas9 gene editing technology for the treatment of ocular diseases.

Ocular diseases are a highly heterogeneous group of phenotypes, caused by a spectrum of genetic variants and environmental factors that exhibit diverse clinical symptoms. As a result of its anatomical location, structure and immune privilege, the eye is an ideal system to assess and validate novel genetic therapies. Advances in genome editing have revolutionized the field of biomedical science, enabling researchers to understand the biology behind disease mechanisms and allow the treatment of several health conditions, including ocular pathologies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing facilitates efficient and specific genetic modifications in the nucleic acid sequence, resulting in permanent changes at the genomic level. This approach has advantages over other treatment strategies and is promising for the treatment of various genetic and non-genetic ocular conditions. This review provides an overview of the CRISPR/CRISPR-associated protein 9 (Cas9) system and summarizes recent advances in the therapeutic application of CRISPR/Cas9 for the treatment of various ocular pathologies, as well as future challenges.

The endothelin receptor antagonist macitentan ameliorates endothelin-mediated vasoconstriction and promotes the survival of retinal ganglion cells in rats.

Glaucoma is a chronic and progressive eye disease, commonly associated with elevated intraocular pressure (IOP) and characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells (RGCs). The pathological changes in glaucoma are triggered by multiple mechanisms and both mechanical effects and vascular factors are thought to contribute to the etiology of glaucoma. Various studies have shown that endothelin-1 (ET-1), a vasoactive peptide, acting through its G protein coupled receptors, ETA and ETB, plays a pathophysiologic role in glaucoma. However, the mechanisms by which ET-1 contribute to neurodegeneration remain to be completely understood. Our laboratory and others demonstrated that macitentan (MAC), a pan endothelin receptor antagonist, has neuroprotective effects in rodent models of IOP elevation. The current study aimed to determine if oral administration of a dual endothelin antagonist, macitentan, could promote neuroprotection in an acute model of intravitreal administration of ET-1. We demonstrate that vasoconstriction following the intravitreal administration of ET-1 was attenuated by dietary administration of the ETA/ETB dual receptor antagonist, macitentan (5 mg/kg body weight) in retired breeder Brown Norway rats. ET-1 intravitreal injection produced a 40% loss of RGCs, which was significantly lower in macitentan-treated rats. We also evaluated the expression levels of glial fibrillary acidic protein (GFAP) at 24 h and 7 days post intravitreal administration of ET-1 in Brown Norway rats as well as following ET-1 treatment in cultured human optic nerve head astrocytes. We observed that at the 24 h time point the expression levels of GFAP was upregulated (indicative of glial activation) following intravitreal ET-1 administration in both retina and optic nerve head regions. However, following macitentan administration for 7 days after intravitreal ET-1 administration, we observed an upregulation of GFAP expression, compared to untreated rats injected intravitreally with ET-1 alone. Macitentan treatment in ET-1 administered rats showed protection of RGC somas but was not able to preserve axonal integrity and functionality. The endothelin receptor antagonist, macitentan, has neuroprotective effects in the retinas of Brown Norway rats acting through different mechanisms, including enhancement of RGC survival and reduction of ET-1 mediated vasoconstriction.

Early-Onset Glaucoma in egl1 Mice Homozygous for Pitx2 Mutation.

Mutations in PITX2 cause Axenfeld-Rieger syndrome, with congenital glaucoma as an ocular feature. The egl1 mouse strain carries a chemically induced Pitx2 mutation and develops early-onset glaucoma. In this study, we characterized the glaucomatous features in egl1 mice. The eyes of egl1 and C57BL/6J control mice were assessed by slit lamp examination, total aqueous humor outflow facility, intraocular pressure (IOP) measurement, pattern electroretinography (PERG) recording, and histologic and immunohistochemistry assessment beginning at 3 weeks and up to 12 months of age. The egl1 mice developed elevated IOP as early as 4 weeks old. The IOP elevation was variable and asymmetric within and between the animals. The aqueous humor outflow facility was significantly reduced in 12-month-old animals. PERG detected a decreased response at 2 weeks after the development of IOP elevation. Retinal ganglion cell (RGC) loss was detected after 8 weeks of IOP elevation. Slit lamp and histologic evaluation revealed corneal opacity, iridocorneal adhesions (anterior synechiae), and ciliary body atrophy in egl1 mice. Immunohistochemistry assessment demonstrated glial cell activation and RGC axonal injury in response to IOP elevation. These results show that the eyes of egl1 mice exhibit anterior segment dysgenesis and early-onset glaucoma. The egl1 mouse strain may represent a useful model for the study of congenital glaucoma.

Involvement of c-Jun N-terminal kinase 2 (JNK2) in Endothelin-1 (ET-1) Mediated Neurodegeneration of Retinal Ganglion Cells.

Purpose: The goal of this study was to determine whether JNK2 played a causative role in endothelin-mediated loss of RGCs in mice. Methods: JNK2-/- and wild type (C57BL/6) mice were intravitreally injected in one eye with 1 nmole of ET-1, whereas the contralateral eye was injected with the vehicle. At two time points (two hours and 24 hours) after the intravitreal injections, mice were euthanized, and phosphorylated c-Jun was assessed in retinal sections. In a separate set of experiments, JNK2-/- and wild type mice were intravitreally injected with either 1 nmole of ET-1 or its vehicle and euthanized seven days after injection. Retinal flat mounts were stained with antibodies to the RGC marker, Brn3a, and surviving RGCs were quantified. Axonal degeneration was assessed in paraphenylenediamine stained optic nerve sections. Results: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice, which was appreciably lower in the JNK2 -/- mice. A significant (P < 0.05) 26% loss of RGCs was found in wild type mice, seven days after injection with ET-1. JNK2-/- mice showed a significant protection from RGC loss following ET-1 administration, compared to wild type mice injected with ET-1. A significant decrease in axonal counts and an increase in the collapsed axons was found in ET-1 injected wild type mice eyes. Conclusions: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice. Neuroprotective effects in JNK2-/- mice following ET-1 administration occur mainly in the soma and not in the axons of RGCs.

Endothelin-1 Mediated Decrease in Mitochondrial Gene Expression and Bioenergetics Contribute to Neurodegeneration of Retinal Ganglion Cells.

Endothelin-1 (ET-1) is a vasoactive peptide that is elevated in aqueous humor as well as circulation of primary open angle glaucoma (POAG) patients. ET-1 has been shown to promote degeneration of optic nerve axons and apoptosis of retinal ganglion cells (RGCs), however, the precise mechanisms are still largely unknown. In this study, RNA-seq analysis was used to assess changes in ET-1 mediated gene expression in primary RGCs, which revealed that 23 out of 156 differentially expressed genes (DEGs) had known or predicted mitochondrial function, of which oxidative phosphorylation emerged as the top-most enriched pathway. ET-1 treatment significantly decreased protein expression of key mitochondrial genes including cytochrome C oxidase copper chaperone (COX17) and ATP Synthase, H+ transporting, Mitochondrial Fo Complex (ATP5H) in primary RGCs and in vivo following intravitreal ET-1 injection in rats. A Seahorse ATP rate assay revealed a significant decrease in the rate of mitochondrial ATP production following ET-1 treatment. IOP elevation in Brown Norway rats showed a trend towards decreased expression of ATP5H. Our results demonstrate that ET-1 produced a decrease in expression of vital components of mitochondrial electron transport chain, which compromise bioenergetics and suggest a mechanism by which ET-1 promotes neurodegeneration of RGCs in glaucoma.