Inhibition of Tissue Factor by Ixolaris in Models of Age-Related Macular Degeneration

Abstract

Purpose: Age related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. Angiogenesis and inflammation are key factors mediating the pathogenesis of AMD. Activation of tissue factor, a cell surface protein, can lead to angiogenic and inflammatory stimulation. Increased expression of tissue factor has been linked to AMD in mouse models and patients. This study aimed to evaluate if inhibition of tissue factor activity via a small protein, Ixolaris, could reduce the pro-angiogenic and pro-inflammatory responses in human retinal pigmented epithelial (ARPE-19) cells and our Ccl2^-/-/Cx3cr1^-/- rd8 (DKO^rd8) mouse model.

Methods: A factor X cleavage assay was used to assess the inhibitory effects of Ixolaris on tissue factor in ARPE-19 cells. Cells were grown for 24 hours, washed, and incubated with 1 nM FVIIa and 100 nM FX with or without 10 nM Ixolaris for one hour. FX cleavage by tissue factor was measured using chromogenic substrate S2222. DKO^rd8 mice were injected intravitreally with 1.5 μg of Ixolaris in one eye and PBS control in the other. Fundus images were recorded and graded monthly post-injection. Functional analysis was performed using electroretinography (ERG) recordings in dark and light-adapted conditions with UV and green light stimulation. Two months later, eyes were enucleated and processed for histology, electron microscopy, molecular analysis, and measurement of A2E. Statistics were performed using GraphPad Prism 6.

Results: Ixolaris was a potent inhibitor of tissue factor in ARPE-19 cells, as evidenced by the lack of FXa generation in Ixolaris-treated cells. In the DKO^rd8 mice, Ixolaris treatment led a significant improvement in retinal lesions by fundoscopy when compared to the contralateral eye. Less RPE and photoreceptor degeneration was noted in treated eyes by electron microscopy, though histology and ERG analysis showed no statistical differences. A2E, which is elevated in AMD eyes, was significantly higher in the treated group (p = 0.005).

Conclusions: This study shows that in vitro inhibition of tissue factor by Ixolaris is effective, but that its effects on retinal lesion progression in our mouse model of AMD are limited. These findings do not preclude tissue factor from playing a role in the pathogenesis of AMD-like lesions; rather, further optimization of intravitreal administration of Ixolaris may be necessary to draw beneficial effects.