Abstract
Purpose :
Dry eye disease presents a significant challenge in ophthalmology due to its prevalence and multifaceted pathophysiology, with Meibomian gland dysfunction (MGD) being a recognized contributor to its complexity. This study aims to elucidate the potential role of sphingolipid metabolism in modulating the health and functionality of Meibomian gland epithelial cells.
Methods :
We developed an in vitro model of cultured Human Meibomian Gland Epithelial Cells (HMGEC) and subjected them to osmolar stress to simulate dry eye disease. Immortal HMGEC were obtained from the Schepens Eye Research Institute and cultured in two different conditions- 1) keratinocyte serum-free media containing 5 ng/ml epidermal growth factor and 50 ug/ml bovine pituitary extract (BPE) to maintain their undifferentiated states and 2) with Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum that allows them to differentiate. Both Undifferentiated and Differentiated HMGECs were grown to 70 – 80 % confluence and subjected to osmolar stress for 24 hours. Harvested cell pellets were subjected to assay for sphingomyelinase (SMase) activity for both neutral and acidic SMases and lipidomic analysis using LC-MS/MS to determine sphingolipids composition.
Results :
Under osmolar stress, the Undifferentiated cells did not show changes in neutral and acidic SMase activity, while Differentiated cells demonstrated significantly increased activities. Mass spectrometry analysis of the major classes of sphingolipids showed significant differences in their composition between the Undifferentiated and Differentiated cells. Under osmolar stress, the level and composition of many sphingolipid species across all three classes were found to be altered in Differentiated cells, whereas only a few species were affected in Undifferentiated cells.
Conclusions :
We found that HMGECs that are allowed to differentiate were more sensitive to osmolar stress and had significantly different profiles of sphingolipids compared to the Undifferentiated cells. Salt stress increased SMase activity in Differentiated cells, corresponding to a significant alteration of the sphingolipid profile in these cells. These findings suggest a potential link between sphingolipids and the observed cellular responses, prompting our hypothesis that sphingolipids may play a pivotal role in the inflammation and cell death processes within Meibomian gland cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.