Redefining the factors that determine tear film stability to develop novel therapeutics for evaporative dry eye disease

项目总结

Project Summary Dry eye disease (DED) is an ocular condition affecting about 6.8% of people in the United States, 87% of which have evaporative dry eye disease (EDED). In EDED, abnormalities in the tear film lipids cause tear film instability and increased evaporation. These abnormalities are typically caused by obstruction of the meibomian gland orifices or senescence of the acinar cells of the meibomian gland. Despite the high number of patients with EDED, current therapeutics only provide brief, palliative relief. To design therapeutics specifically for EDED, a better understanding of how the tear film is stabilized is required. To study tear film stability, the tear film of rabbits, a species with a naturally hyper-stable tear film, was compared to humans which identified four nonpolar lipids in rabbit tears that are absent from human tears. One nonpolar lipid (rNPL593) was formulated into a topical therapeutic and used as a treatment in rabbits with induced DED, which increased tear film stability and decreased damage to the cornea compared to untreated rabbits. To explore how rNPL593 increased tear film stability, we developed a novel in vitro experimental platform, which includes synthetic lipids that mimic the tear film lipid layer and the interactions of that layer with the environment and the rest of the tear film. With this model, we observed the spontaneous separation of the nonpolar lipids of the tear film. This spontaneous separation shares many characteristics with an intracellular phenomenon called liquid-liquid phase separation (LLPS). This finding conflicts with the current view of the tear film nonpolar lipid organization which consists of a lamellar arrangement of nonpolar lipids with weak, transient interactions. Instead, we propose that the different nonpolar species are mixed by blinking and that LLPS occurs during the interblink interval. This spontaneous separation destabilizes the tear film eventually leading to its breakup. Additionally, we propose that the alterations in the tear film lipids seen in patients with EDED promotes LLPS, causing their clinical signs. Exciting preliminary data suggests that the addition of rNPL593 to the in vitro model decreases the self-aggregation and phase separation of the nonpolar lipids. In this proposal, we will utilize fluorescence recovery after photobleaching and modulation of the ratios of the nonpolar lipids to further characterize the LLPS phenomenon and the addition of rNPL593 to further interrogate the effect of rNPL593. To determine the clinical efficacy of rNPL593, we will utilize a mouse model of EDED. These mice are deficient in acyl-CoA:wax alcohol acyltransferase 2 (Awat2), which causes alterations in meibomian gland secretions, decreased tear stability and secondary corneal damage, similar to moderate to severe EDED. Preliminary data suggests that Awat2 KO mice treated daily with rNPL593 ameliorated ocular surface disease and meibomian gland obstruction. This project aims to investigate how rNPL593 effects meibomian gland function in Awat2 knockout animals using transillumination meibography, transcriptomics and spatial lipidomics, laying the ground work for future clinical applications for patients with EDED.