Abstract
Purpose :
Glaucoma is one of the leading causes of visual disability throughout the world and increased intraocular pressure (IOP) is considered a risk factor. Disruption of aqueous humour (AH) drainage is thought to contribute to increased IOP and has been shown to be regulated by actomyosin contraction of trabecular meshwork cells (TM) following cytoskeletal reorganization and formation of stress fibers (CLANs). Here, we have investigated the role of myocardin related transcription factor (MRTF-A) in the induction of TM cell contraction and cytoskeletal reorganization.
Methods :
Primary human trabecular meshwork (HTM) cells were cultured using a standard protocol and incubated with serum free media for 24 hrs prior to any treatment. HTM cells were incubated with MRTF-A inhibitor, CCG-203971 (5µM) in the presence or absence of dexamethasone (DEX) (500 nM) for 72 hrs. Real-time deformability cytometry (RT-DC), western blot and immunofluorescence analysis was carried out and data was statistically analyzed using Graphpad Prism software.
Results :
Our RT-DC analysis revealed that in the presence of DEX, HTM cells show a ~20% decrease in deformation when compared to untreated HTM cells. However, incubation of TM cells with CCG-203971, prevented DEX-induced stiffness of HTM cells. Furthermore, we show that CCG-203971 was able to prevent DEX-induced nuclear translocation of MRTF-A as revealed by immunofluorescence analysis. Our western blot analysis further revealed a significant reduction (~2.4-fold) in DEX-induced expression of αSMA (p<0.0001; dex- vs dex+CCG-203971- treated HTM cells), a key contractile cytoskeletal protein and a marker of cellular transformation, in the presence of CCG-203971.
Conclusions :
The inability of DEX to induce stiffness in HTM cells in the presence of the MRTF-A inhibitor, CCG-203971, suggests a role of MRTF-A in the regulation of proteins responsible for glucocorticoid-induced cellular stiffness. Further, prevention of DEX-induced nuclear translocation of MRTF-A and the subsequent reduction of DEX-induced αSMA expression in the presence of CCG-203971 suggests a correlation between MRTF-A nuclear translocation and αSMA expression in HTM cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.