Abstract
Purpose :
The cell-signaling mechanisms inducing damage and eventual retinal ganglion cell (RGC) death during glaucoma disease progression are still unknown. Recently, we identified DAMP (damage associated molecular pattern) activated toll-like receptor 4 (TLR4) signaling as a novel pathway involved in extracellular (ECM) regulation in the ONH. Here, we examined whether TLR4 and TGFβ2 signaling crosstalk is essential for increased extracellular matrix (ECM) deposition by primary ONH astrocytes. We also examined whether increased activation of TLR4 signaling by the DAMP, fibronectin extra domain A (FN-EDA), leads to a severe glaucoma phenotype in mice.
Methods :
Human primary ONH astrocytes (N=3 strains) were isolated and exposed to exogenous TGFβ2 with or without the selective TLR4 inhibitor TAK-242 to quantify changes in ECM protein production by western blot and immunocytochemistry. We used our novel mouse model of glaucoma, B6.EDA+/+, which constitutively expressed the extra domain A (EDA) isoform of fibronectin. Intraocular pressure was measured using a rebound tonometer (N=10-12). RGC loss was measured via immunohistochemistry for RBPMS at one year (N=6-7) and two years (N=6-7) of age. Cells were counted in a masked manner 1400μm and 700μm from the ONH in all four quadrants of the retina.
Results :
TGFβ2 induces fibronectin protein expression (p<0.05), but concurrent treatment with the selective TLR4 inhibitor TAK-242 blocks this effect (p>0.05) in human ONH primary astrocytes. IOP was significantly elevated in B6.EDA+/+ mice at both 1 and 2 years of age (p<0.001). At one year of age, total RGC counts are significantly decreased in B6.EDA+/+ mice (p<0.01) compared to age-matched controls. Interestingly, RGC loss was only significant in the peripheral sections of the retina (p<0.01) and not in the central sections (p>0.05). However, at two years of age, total RGC counts in B6.EDA+/+ mice are significantly decreased (p<0.05) and both central and peripheral areas each show significant decreases (p<0.05, p<0.05).
Conclusions :
Here we show that TLR4 signaling is necessary for TGFβ2-dependent increases in ECM expression in ONH astrocytes. We also show constitutive activation of TLR4 by the DAMP FN-EDA generates a progressive glaucoma phenotype in our mouse model system. These results implicate TLR4 as an important regulator of glaucoma damage, providing a novel target pathway for new therapeutics.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.