Abstract
Purpose :
Dry eye disease (DED) is a spectrum of ocular surface disease that affects 5-30% of people over 50 and an estimated 300M people in the world suffer from DED. Meibomian glands (MG), which are modified holocrine glands, secrete meibomian lipids or meibum for the tear film. DED is often driven by meibomian gland dysfunction (MGD). The Mechanism of MGD is not well understood. The purpose of this study was to investigate the health and functionality of meibomian gland cells in an in vitro model of osmolar stress.
Methods :
We developed an in vitro model of cultured Human Meibomian Gland Epithelial Cells (HMGEC) and subjected them to osmolar stress to simulate DED. Immortal HMGEC were gifted by Dr. David Sullivan (Schepens Eye Research Institute) and grown in two different conditions) keratinocyte serum-free media (KSFM) with 5ng/ml epidermal growth factor and 50ug/ml bovine pituitary extract to maintain their undifferentiated states and 2) with DMEM with 10% FBS that allows them to differentiate. HMGECs were grown to 70–80% confluence and subjected to osmolar stress with an increased dose of NaCl from 50–150mM for 24h. The dose-response of NaCl on cell viability and survival was determined by MTT and LDH assays. Expression of inflammatory, cell adhesion, and sphingolipid pathways genes were determined by qRT-PCR. The release of inflammatory mediators was assayed using a multiplex assay.
Results :
50–150mM NaCl increased the osmolarity of the media from 396 to 777mOsm/kg, the osmolarity of no salt was 297mOsm/kg. In KSFM, NaCl reduced cell viability by 7% (50), 43% (100), & 60% (150mM NaCl). Cell death was induced by 15% (100) & 29% (150mM NaCl). For differentiated cells, NaCl reduced cell viability by 2% (50), 12% (100), & 62% (150mM NaCl) and induced 59% cell death in150 mM NaCl. Only differentiated cells showed elevated expression of inflammatory genes (IL-1b and IL-18) under osmolar stress. On the other hand, undifferentiated cells released increased levels of IL-1b, MMP-3, TNF alpha, & CXCL11, while the levels of secreted MMP-2, MMP-9 & TIMP-1 reduced.
Conclusions :
This study underscores the adverse effects of increased osmolarity on meibomian gland cells and revealed distinct responses between states of the cells. The findings emphasize the vulnerability of these cells to osmotic stress, indicating potential pathways and mechanisms involved in dry eye pathogenesis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.