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

Frontiers in ophthalmology · 14 authors, 11 centres

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POPULATIONRetired breeder male and female Brown Norway rats (8-11 months old)

INTERVENTIONOral administration of macitentan (5 mg/kg body weight/day) in dietary gel, starting 3 days prior to and continuing 7 days after intravitreal ET-1 injection

COMPARISONUntreated rats (dietary gel alone) receiving intravitreal ET-1 injection; vehicle-injected rats with or without macitentan

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TL;DR

In a rat model, intravitreal injection of endothelin-1 (ET-1) caused profound retinal vasoconstriction and a 40% loss of retinal ganglion cells (RGCs). Oral administration of the dual endothelin receptor antagonist macitentan (5 mg/kg) significantly attenuated vasoconstriction and reduced RGC loss to only 4%, but did not preserve RGC function or axonal integrity. These findings suggest macitentan may have potential as a neuroprotective adjunct therapy for glaucoma and other retinal ischemic disorders.

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**Background:** Glaucoma is a progressive optic neuropathy characterized by retinal ganglion cell (RGC) loss and optic nerve degeneration. While elevated intraocular pressure (IOP) is a major risk factor, vascular dysregulation and endothelin-1 (ET-1), a potent vasoconstrictor, are also implicated. ET-1 levels are elevated in glaucoma patients and animal models, and its receptors (ET_A and ET_B) contribute to neurodegeneration. Macitentan, a dual endothelin receptor antagonist approved for pulmonary hypertension, has shown neuroprotective effects in chronic ocular hypertension models. This study aimed to determine whether oral macitentan could protect against ET-1-induced vasoconstriction, RGC loss, and glial activation in an acute intravitreal ET-1 model in rats.

**Methods:** Retired breeder Brown Norway rats (8-11 months old) were used. Macitentan (5 mg/kg/day) was administered orally in DietGel® starting 3 days before and continuing 7 days after intravitreal injection of 2 nmol ET-1 or vehicle (4 µL of 500 µM ET-1 or vehicle). Retinal vasoconstriction was assessed by fundus fluorescein angiography and vessel diameter/density analysis using FIJI software at 0 and 10 minutes post-injection. IOP was measured with a TonoLab tonometer at multiple time points. RGC survival was quantified by Brn3a immunostaining of retinal flat mounts. RGC function was evaluated by pattern electroretinography (PERG) at baseline and 7 days. Optic nerve axon integrity was assessed by paraphenylenediamine (PPD) staining and axon counts. Glial fibrillary acidic protein (GFAP) expression was examined by immunohistochemistry in retinal sections and by qPCR in cultured rat optic nerve head astrocytes treated with 100 nM ET-1 ± 1 µM macitentan.

**Key Results:** - **Vasoconstriction:** ET-1 injection caused profound retinal vasoconstriction at 10 minutes, with mean vascular density decreasing from 38.7±1% (baseline) to 25.4±1% (p<0.05). Macitentan-treated rats showed significantly higher vascular density (37.7±2.8%) and vessel diameter at both central (E1) and peripheral (E2) eccentricities (p≤0.02 for E1 by unpaired t-test; p=0.02 for E2 by two-way ANOVA). - **IOP:** ET-1 caused a transient IOP elevation at 2 hours (p<0.05 vs vehicle), which was not attenuated by macitentan. IOP returned to baseline by 7 days. - **RGC survival:** ET-1 reduced RGC counts by 40% (2454±326 cells/mm² vs vehicle 4076±446; p<0.05). Macitentan treatment significantly reduced cell loss to only 4% (3928±356 cells/mm²), similar to controls. - **RGC function:** PERG amplitude declined significantly in ET-1-injected rats (from 7.9±2.2 µV at baseline to 3.3±1.8 µV at 7 days; p=0.015), and macitentan did not prevent this decline (8.0±2.6 µV to 2.8±1.7 µV; p=0.002). No significant latency changes were observed. - **Axonal integrity:** ET-1 caused significant optic nerve axon loss (p=0.002 vs vehicle), and macitentan did not protect axons (p=0.003 vs vehicle). - **Glial activation:** At 24 hours, ET-1 increased GFAP immunostaining in retina and optic nerve head in vivo, and in cultured astrocytes, ET-1 induced a 3-fold increase in GFAP mRNA and a 2-fold increase in fibronectin mRNA, both blocked by macitentan. At 7 days, macitentan-treated ET-1 rats showed significantly higher GFAP expression compared to other groups.

**Clinical Implications:** This study demonstrates that oral macitentan (5 mg/kg) attenuates ET-1-induced retinal vasoconstriction and promotes RGC soma survival in an acute rat model, but does not preserve RGC function or axonal integrity. The findings suggest that dual endothelin receptor blockade may be beneficial as an adjunct therapy for glaucoma and other retinal ischemic conditions (e.g., acute angle closure glaucoma, central retinal artery/vein occlusion) by reducing vascular dysfunction and promoting RGC survival. However, the lack of functional and axonal protection in this acute model highlights the need for further research in chronic models and the potential for combination therapies. Macitentan is FDA-approved for pulmonary hypertension with a favorable safety profile, supporting its repurposing for neuroprotection in ophthalmology.

PICO

PPOPULATION

Retired breeder male and female Brown Norway rats (8-11 months old)

IINTERVENTION

Oral administration of macitentan (5 mg/kg body weight/day) in dietary gel, starting 3 days prior to and continuing 7 days after intravitreal ET-1 injection

OOUTCOME

Retinal vessel diameter and density (vasoconstriction), RGC survival (Brn3a+ cell counts), RGC function (pattern ERG amplitude), optic nerve axon counts (PPD staining), GFAP expression (immunohistochemistry and qPCR)

STUDY TYPE

other

SPECIALTY

ophthalmology

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100% · A

ORIGINAL PAPER

PMC10921982

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

Frontiers in ophthalmology

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ABSTRACT

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, ET A and ET B , 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 ET A /ET B 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.

CITATION EXPORT

Bindu Kodati, Wei Zhang, Shaoqing He et al.. The endothelin receptor antagonist macitentan ameliorates endothelin-mediated vasoconstriction and promotes the survival of retinal ganglion cells in rats. Frontiers in ophthalmology. (2023). https://doi.org/10.3389/fopht.2023.1185755

AUTHORS

14 · 11 CENTRES

Bindu Kodati, Wei Zhang, Shaoqing He, Jennifer H. Pham, Kallen J. Beall, Zoe E. Swanger, Vignesh R. Krishnamoorthy, Payton E. Harris, Trent Hall, Ashley V. Tran, Renuka M. Chaphalkar, Sai H. Chavala, Dorota L. Stankowska, Raghu R. Krishnamoorthy

1Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, United States
2North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, TX, United States
3Department of Pathology, Children’s Health at Dallas, Dallas, TX, United States
4Department of General Surgery, Honor Health, Phoenix, AZ, United States
5Texas College of Osteopathic Medicine, University of North Texas Health Science Center, Fort Worth, TX, United States
6Department of Cell and Molecular Pharmacology, Loyola University, Maywood, IL, United States
7Department of Graduate Medical Education, Medical City, Fort Worth, TX, United States
8Williams College, Williamstown, MA, United States
9School of Natural Sciences and Mathematics, The University of Texas at Dallas, Richardson, TX, United States
10Department of Ophthalmology, School of Medicine, University of California, San Francisco, CA, United States
11Department of Surgery, Burnett School of Medicine at Texas Christian University (TCU), Fort Worth, TX, United States