Impaired TRPV4-eNOS signaling in glaucomatous trabecular meshwork contributes to elevation of intraocular pressure

Abstract

Purpose : Nitric oxide (NO) is known to reduce intraocular pressure (IOP) by relaxation of the trabecular meshwork (TM) and distal vessels of the conventional outflow pathway. However, the intrinsic mechanisms by which outflow pathway tissues regulate NO production is yet to be elucidated. Here, we examined whether transient receptor potential vanilloid 4 (TRPV4) channels regulate IOP via endothelial nitic oxide synthase (eNOS) signaling. We also assess whether TRPV4-eNOS signaling is impaired in glaucoma.

Methods : In WT and glucocorticoid (GC)-induced ocular hypertensive (OHT) C57BL/6J mice, the effect of the TRPV4 agonist GSK1016790A (GSK101) on IOP and outflow facility was determined using rebound tonometry and a constant-flow infusion method, respectively. The effect of GSK101 on eNOS activation and NO production was determined using Western blot and a fluorometric DAF-FM assay in normal and glaucomatous primary human TM cells and ex vivocultured human TM donor tissues. We further examined whether TRPV4 channel function is impaired in glaucoma using high speed Ca²⁺ imaging, which records elementary Ca²⁺ influx events through TRPV4 channels (TRPV4 sparklets) in human primary TM cells from normal and glaucoma donor eyes (n=3 each).

Results : Topical administration of GSK101 (1 μM) significantly improved outflow facility (P=0.02) and reduced IOP (P<0.0001) in a mouse model of GC-induced OHT. TRPV4 channel activation by GSK101 (20 nM) resulted in increased eNOS phosphorylation in primary human TM cells and ex vivo cultured human TM donor tissues. Activation of TRPV4 channels in primary TM cells and ex vivo cultured human TM donor tissues resulted in increased DAF-FM fluorescence, which signifies an increase in TRPV4 channel-induced NO production. Genetic deletion of eNOS abrogated the IOP lowering effect of GSK101 in mice. Importantly, human glaucomatous primary TM cells exhibited significantly reduced TRPV4 sparklet activity, eNOS phosphorylation, and NO production in response to GSK101 when compared to human primary TM cells from normal donor eyes, suggesting impaired TRPV4 channel activity in glaucomatous TM cells.

Conclusions : The TRPV4 channel agonist GSK101 lowers IOP and improves outflow facility by regulating NO at the TM. Functional impairment of these channels may contribute to glaucomatous pathophysiology.

This is a 2020 ARVO Annual Meeting abstract.