Glaucoma, a complex neurodegenerative disorder affecting over 80 million people worldwide, results in the denervation of retinal ganglion cells and their axons on the optic nerve and can eventually lead to blindness. The endothelin system, well known for its role in regulating blood pressure, is also implicated in the development of various forms of glaucoma. Therefore targeting the endothelin system to prevent glaucoma has therapeutic potential; however, the cell types involved as well as the exact mechanisms of action remain elusive.
New research from scientists at The Jackson Laboratory led by Dr. Gareth Howell characterizes the endothelin system in DBA/2J mice (000671, Fig.1), which are commonly used as a model of hereditary glaucoma. They specifically examined cell types of the optical nerve head (ONH), a key site where the retinal ganglion cell axons are damaged by early and critical insult. They also demonstrated that early combinatorial targeting of both the endothelin and the complement systems prevents glaucoma development in the mouse (Howell, et al. 2014).
Howell’s team previously identified molecular stages of disease that precede conventional detectable damage in DBA/2J mice (Howell, et al. 2011a; 2012b). Seven temporally ordered stages of glaucoma that occur in the ONH were described in these studies using gene expression profiling combined with computational approaches. The first five stages (stages 1a, 1b, 1c, 2, and 3) were recognized as early stages resulting from gene expression changes preceding axon loss and other conventional signs of glaucoma such as gliosis.
The researchers examined the expression levels of three endothelin ligands (Edn1, Edn2, and Edn3) in all seven stages of glaucoma in the ONH during the development of glaucoma in DBA/2J and control mice. Edn2 was significantly up-regulated as compared to the control group (D2-Gpnmb+, Howell, et al. 2007b) first in stage 2, followed by an over 10-fold increase by stage 3—when eyes are about to experience damage and axon loss. This increased expression of Edn2 was maintained through stages 4 and 5, whereas expression of Edn1 and Edn3 was only modestly increased (1.5 fold) in only one or two stages. RNA in situ hybridization combined with immunofluorescence revealed that Edn2 was expressed in IBA1+ cells and GFAP+ cells; the first observation of Edn2 expression in astrocytes in DBA/2J glaucoma.
The researchers further investigated the effect of Edn2 up-regulation on the ONH in early stage glaucoma, and found that the expression levels for both receptors of Edn2, Ednra and Ednrb were also significantly up-regulated. Furthermore, Ednra was shown to localize to endothelial cells in the ONH, consistent with a role in mediating early vascular alterations previously reported by the same group (Howell, et al. 2011a).
Retinal ganglion cell loss can be reduced by inhibiting the endothelin system (Howell, et al. 2011a; Minton, et al. 2012). Therefore, the investigators then explored the potential for targeting the endothelin system for glaucoma by testing Macitentan, a derivative of the dual endothelin receptor antagonist Bosentan (Iglarz, et al. 2008; Bolli, et al. 2012). Macitentan was administered to 6-month old DBA/2J mice and physical examinations, intraocular pressure (IOP) levels, and retinal ganglion cell loss were assessed between 10.5 and 12 months of age. Macitentan significantly reduced retinal ganglion cell loss and at 10.5 months of age, approximately 80% of eyes from the Macitentan group showed no glaucoma compared to 50% in control group (p = 1.4 × 10−7). By 12 months of age, approximately 45% of eyes from the Macitentan group showed no glaucoma compared to 35% in the controls (p = 0.05).
It is evident that targeting the endothelin system alone is not effective to prevent glaucoma, particularly in older animals. The investigators then tested the effectiveness of inhibiting the complement cascade together with endothelin during early stages. The complement system is also one of the earliest processes up-regulated in glaucoma, however inhibiting this pathway is also not sufficient to prevent glaucoma (Stasi, et al. 2006; Steele, et al. 2006; Stevens, et al. 2007, Howell, et al. 2011a). Howell’s group found that combinatorial blocking of both the endothelin system and the classical complement cascade robustly protected animals at 12 months of age (80% of eyes had no detectable glaucoma), which was more effective than blocking either pathway alone.
Combinatorial treatments targeting multiple early pathways are likely to be the most effective in protecting against glaucoma development, considering the complex nature of the disease as well as the diverse cell types and pathways involved (Soto and Howell, 2014). This study opens a new avenue for neuroprotection against glaucoma.