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

项目摘要 干眼症（DED）是一种眼部疾病，影响美国约6.8%的人，87%的人， 患有蒸发性干眼病（EDED）。在EDED中，泪膜脂质的异常导致泪膜 不稳定和蒸发增加。这些异常通常是由睑板肌阻塞引起的 睑板腺腺泡细胞的衰老。尽管有大量的病人 对于EDED，目前的治疗方法只能提供短暂的缓解。为了设计专门针对EDED的治疗方法， 需要更好地理解泪膜是如何稳定的。 为了研究泪膜的稳定性，对具有天然超稳定泪膜的兔的泪膜进行了研究。 与人类相比，在兔子眼泪中发现了四种人类眼泪中不存在的非极性脂质。一 将非极性脂质（rNPL 593）配制成局部治疗剂，并用作诱导的 DED，与未经治疗的兔子相比，它增加了泪膜的稳定性，减少了对角膜的损伤。 为了探索rNPL 593如何增加泪膜稳定性，我们开发了一种新的体外实验平台， 其包括模拟泪膜脂质层的合成脂质以及该层与泪液的相互作用。 环境和泪膜的其余部分。在这个模型中，我们观察到了 泪膜的非极性脂质。这种自发的分离与细胞内的 这种现象称为液-液相分离（LLPS）。这一发现与目前对撕裂的看法相冲突， 膜非极性脂质组织，由非极性脂质的层状排列组成，具有弱的、短暂的 交互.相反，我们提出，不同的非极性物种混合闪烁和LLPS发生 在眨眼间隔期间。这种自发的分离使泪膜不稳定，最终导致其 分手此外，我们认为EDED患者泪膜脂质的改变促进了 LLPS，导致他们的临床症状。令人兴奋的初步数据表明，将rNPL 593添加到体外 模型降低了非极性脂质的自聚集和相分离。在本提案中，我们将利用 光漂白后的荧光恢复和非极性脂质与另外的脂质的比率的调制 表征LLPS现象并加入rNPL 593以进一步研究rNPL 593的作用。 为了确定rNPL 593的临床功效，我们将利用EDED的小鼠模型。这些小鼠 缺乏酰基辅酶A：蜡醇酰基转移酶2（Awat 2），导致睑板腺改变 分泌物，泪液稳定性下降和继发性角膜损伤，类似于中度至重度EDED。 初步数据表明，每天用rNPL 593治疗的Awat 2 KO小鼠改善了眼表疾病 和睑板腺阻塞。本项目旨在研究rNPL 593如何影响睑板腺 使用透照睑板照相术、转录组学和空间脂质组学在Awat 2敲除动物中的功能， 为将来临床应用于EDED患者奠定基础。

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.