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

重新定义决定泪膜稳定性的因素，开发蒸发性干眼病的新疗法

• 批准号: 10678045
• 负责人: Erin Alissa Hisey
• 金额: $ 4.03万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678045
• 关键词: Acinar Cell; Acyl Coenzyme A; Acyltransferase; Affect; Air; Alcohols; Animal Model; Atrophic; Autopsy; Binding; Biological Models; Blinking; Cell Aging; Characteristics; Cholesterol Esters; Clinical; Complex; Confocal Microscopy; Cornea; Data; Disease; Dry Eye Syndromes; Duct (organ) structure; Environment; Equation; Esters; Film; Fluorescence Recovery After Photobleaching; Formulation; Future; Gene Expression; Gland; Health; High Prevalence; Histopathology; Human; In Vitro; Investigation; Kinetics; Knockout Mice; Label; Lead; Lipids; Liquid substance; Maps; Methods; Modeling; Mus; Obstruction; Oryctolagus cuniculus; Patients; Persons; Phase; Phenotype; Prevalence; Rest; Role; Temperature; Therapeutic; Time; Tissues; Transillumination; United States; Visual impairment; Waxes; Work; aqueous; clinical application; clinical efficacy; corneal epithelium; design; evaporation; eye dryness; fluidity; improved; in vitro Model; in vivo; irritation; knockout animal; lipidomics; mRNA Expression; meibomian gland; meibomian gland dysfunction; mouse model; novel; novel therapeutics; ocular surface; ocular surface disease; palliative; segregation; senescence; targeted treatment; therapeutically effective; time use; transcriptomics; treatment group

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.

项目摘要 干眼症（DED）是一种眼部疾病，影响了约6.8％的美国，其中87％ 患有蒸发干眼病（EDED）。在EDED中，催泪膜中的异常脂质引起撕裂膜 不稳定和蒸发增加。这些异常通常是由梅博米安的阻塞引起的 Meibomian腺的腺泡细胞的腺孔或衰老。尽管患者数量很高 借助EDED，当前的治疗剂仅提供简短的姑息治疗。专门设计用于EDED的治疗剂， 需要更好地了解如何稳定泪膜。 为了研究撕裂膜的稳定性，兔子的撕裂膜是一种天然超稳定的撕裂膜的物种，是 与人类在兔子眼泪中鉴定出四种非极性脂质的人相比，人眼泪不存在。一 非极性脂质（RNPL593）被配合到局部治疗中，并用作兔子的治疗 DED，与未经处理的兔子相比，DED提高了撕裂膜的稳定性并减少了对角膜的损害。 为了探索RNPL593如何提高泪膜的稳定性，我们开发了一个新型的体外实验平台， 其中包括模仿泪膜脂质层的合成脂质以及该层与该层的相互作用 环境和其余的泪液薄膜。使用此模型，我们观察到了自发分离 泪膜的非极性脂质。这种自发分离具有许多特征 现象称为液态液相分离（LLP）。这一发现与目前的眼泪相冲突 膜非极性脂质组织，由弱脂质的层状布置组成 互动。取而代 在间隔间隔期间。这种自发的分离破坏了撕裂膜的稳定，最终导致了它的 拆散。此外，我们建议在EDED患者中看到的泪膜脂质的改变促进 LLP，导致其临床体征。令人兴奋的初步数据表明，在体外添加RNPL593 模型减少了非极性脂质的自聚集和相分离。在此提案中，我们将利用 光漂白后的荧光恢复和非极性脂质比率的调节以进一步 表征LLP现象和添加RNPL593来进一步询问RNPL593的效果。 为了确定RNPL593的临床功效，我们将使用EDED的小鼠模型。这些老鼠是 缺乏酰基-COA：蜡醇酰基转移酶2（AWAT2），这会导致Meibomian腺发生变化 分泌物，减少撕裂稳定性和继发性角膜损伤，类似于中度至重度ED。 初步数据表明，每天用RNPL593治疗的AWAT2 KO小鼠改善眼表疾病 和梅博米亚腺体阻塞。该项目旨在调查RNPL593如何影响Meibomian腺 使用透明量化量量，转录组学和空间脂质组学在AWAT2敲除动物中的功能， 为EDED患者的未来临床应用奠定基础工作。