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，在美国约有6.8%的人受到影响，87%的人 有蒸发性干眼病(EED)。在eed中，泪膜脂质的异常会导致泪膜。 不稳定和蒸发量增加。这些异常通常是由额肌受阻引起的。 眉板腺的腺孔或腺泡细胞衰老。尽管病人的数量很高 对于ED，目前的治疗方法只能提供短暂的姑息缓解。专门为艾德设计治疗方法， 需要更好地了解泪膜是如何稳定的。 为了研究泪膜的稳定性，兔的泪膜是一种自然具有超稳定泪膜的物种。 与人类相比，人类在兔子眼泪中发现了四种人类眼泪中没有的非极性脂类。一 将非极性脂类(RNPL593)制成局部治疗药物，用于治疗兔实验性脑缺血模型。 DED，与未经治疗的兔子相比，它增加了泪膜的稳定性，减少了对角膜的损害。 为了探索rNPL593如何增加泪膜的稳定性，我们开发了一种新型的体外实验平台， 它包括模拟泪膜脂层的合成脂类，以及该脂层与 环境和泪膜的其余部分。利用这个模型，我们观察到了细胞的自发分离 泪膜中的非极性脂类。这种自发的分离与细胞内的 这种现象称为液-液相分离(LLP)。这一发现与目前对撕裂的看法相冲突 薄膜非极性脂组织，由片状排列的非极性脂组成，具有弱的、瞬时的 互动。相反，我们认为不同的非极性物种通过眨眼混合在一起，出现了LLP 在眨眼间隔期间。这种自发的分离破坏了泪膜的稳定，最终导致了它 分手。此外，我们认为eED患者泪膜脂质的改变促进了 LLP，导致他们的临床症状。令人兴奋的初步数据表明，将rNPL593添加到体外 模型减少了非极性脂类的自聚集和相分离。在这个提案中，我们将利用 光漂白后的荧光恢复和非极性脂类比的调节 表征LLP现象和rNPL593的添加，以进一步询问rNPL593的作用。 为了确定rNPL593的临床疗效，我们将利用EED的小鼠模型。这些老鼠是 缺乏酰辅酶A：蜡醇酰基转移酶2(AWAT2)，导致眉毛腺改变 分泌物、泪液稳定性下降和继发性角膜损伤，类似于中到重度EDD。 初步数据表明，每天使用rNPL593治疗的Awat2KO小鼠可以改善眼表疾病 和眉毛腺阻塞。本项目旨在研究rNPL593对眉板腺的影响。 用透射式微管造影术、转录组学和空间脂组学研究Awat2基因敲除动物的功能， 为今后ED患者的临床应用奠定了基础。