Abstract
Purpose :
Intraocular pressure (IOP) regulation, primarily through the trabecular meshwork (TM), is a key factor in glaucoma pathogenesis. Current therapies focus on IOP reduction, but addressing underlying TM degeneration is essential, in particular in steroid induced IOP elevation. Using a Dexamethasone treated TM cells as an experimental model, we explored the potential of Extracellular Vesicles (EVs) derived from corneal stromal stem cells (CSSCs) in modulating gene expression in TM cell. In particular, the MYOC gene, one of the first genes associated with glaucoma, and ANGPTL7, recently linked to glaucoma pathogenesis.
Methods :
Nine primary human TM cell lines were isolated and cultured from five donor corneoscleral rims. TM cells were harvested and expanded in gelatin-coated flasks containing DMEM-low glucose (1g/L), with GlutaMAX supplemented with 10% FBS and 1% Antibiotics/Antimycotics. Cells at passage 4 were utilized for this study. EVs were purified from CSSC conditioned media (CM) using size exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy, and ExoView technology. TM cells were treated with either 100nM dexamethasone (Dex) alone or 100nM Dex plus EVs for 5 days. The EV was replaced on day 3. After 5 days, total mRNA was isolated using the Qiagen RNeasy mini kit. Quantitative PCR was carried out using the Realplex 2 to quantify the mRNA level of MYOC and ANGPTL7.
Results :
EVs purified from CSSC conditioned media demonstrate an enrichment of particles with a mode size of 88.8nm. Transmission Electron Microscopy imaging demonstrated the presence of cup shaped structures with a lipid bilayer. Expression of tetraspannins CD9, CD81 and CD63 was confirmed by ExoView. Compared to the untreated TM cells (control), dexamethasone-treated TM cells, exhibited a 32 fold increase in the mRNA expression of MYOC and 290 fold increase in ANGPTL7 mRNA. When EVs were added to the Dex-treated TM cells, the expression of MYOC, was decreased by 44.4% and the ANGPTL7 was decreased in 88% of cultures.
Conclusions :
This preliminary study demonstrates the potential of CSSC-derived EVs in reducing MYOC and ANGPTL7 gene expression in TM cells induced by corticosteroids, Additional study is needed to further investigate the mechanism of EV in reversing the effect of corticosteroids in TM cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.