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.

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.

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.

Neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma.

PURPOSE: Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP), leading to optic nerve head (ONH) cupping, axon loss, and apoptosis of retinal ganglion cells (RGCs), which could ultimately result in blindness. Brn3b is a class-4 POU domain transcription factor that plays a key role in RGC development, axon outgrowth, and pathfinding. Previous studies suggest that a decrease in Brn3b levels occurs in animal models of glaucoma. The goal of this study was to determine if adeno-associated virus (AAV)-directed overexpression of the Brn3b protein could have neuroprotective effects following elevated IOP-mediated neurodegeneration. METHODS: Intraocular pressure was elevated in one eye of Brown Norway rats (Rattus norvegicus), following which the IOP-elevated eyes were intravitreally injected with AAV constructs encoding either the GFP (rAAV-CMV-GFP and rAAV-hsyn-GFP) or Brn3b (rAAV-CMV-Brn3b and rAAV-hsyn-Brn3b). Retina sections through the ONH were stained for synaptic plasticity markers and neuroprotection was assessed by RGC counts and visual acuity tests. RESULTS: Adeno-associated virus-mediated expression of the Brn3b protein in IOP-elevated rat eyes promoted an upregulation of growth associated protein-43 (GAP-43), actin binding LIM protein (abLIM) and acetylated α-tubulin (ac-Tuba) both posterior to the ONH and in RGCs. The RGC survival as well as axon integrity score were significantly improved in IOP-elevated rAAV-hsyn-Brn3b-injected rats compared with those of the IOP-elevated rAAV-hsyn-GFP- injected rats. Additionally, intravitreal rAAV-hsyn-Brn3b administration significantly restored the visual optomotor response in IOP-elevated rat eyes. CONCLUSIONS: Adeno-associated virus-mediated Brn3b protein expression may be a suitable approach for promoting neuroprotection in animal models of glaucoma.

Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma.

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B)) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B) receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET(B) receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET(B) receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B) receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.