Ophthalmic Extracellular Vesicles Research Lab

PURPOSE. To elucidate the role of phosphatases in the eye drainage system by overexpressing the receptor tyrosine phosphatase sigma (RPTP-σ) in a human normal trabecular meshwork (NTM) cell line. METHODS. The efficacy, expression, and location of RPTP- σ were evaluated following its transfection in NTM cells (NTM^T) and in NTM control cells. The cells were also analyzed for viability, matrix metalloproteinase (MMP) activity, and phosphatase activity following oxidative stress conditions. Assays were conducted in the presence or absence of a specific RPTP-σ inhibitor. RESULTS. Transfection efficacy measurements revealed that RPTP-σ expression measured via GFP fluorescence was significantly higher (×3.8) in NTM^T cells than in control cells. Western blot analyses showed that RPTP- σ expression was significantly higher (×2.25) in NTM^T cells than in control cells. No significant differences were observed in cell viability between NTM^T and control cells after oxidative stress. We found that pro-MMP-2 and pro-MMP-9 showed a significantly higher activity (×2.18 and ×1.9; respectively) in NTM^T cells than in control cells. Serine/threonine phosphatase activity in NTM^T cells was significantly increased following oxidative stress. The specific phosphatase inhibitor PTP-IV inhibited 15% of the RPTP-σ expression in NTM cells and 31% in NTM^T cells. The activity of pro-MMP-9, pro- MMP-2, and MMP-9 was significantly inhibited (48%, 35%, and 78% respectively). CONCLUSIONS. The findings indicate that RPTP-σ is expressed constituently in NTM cells and that oxidative stress changes the general phosphatase balance in NTM cells. In addition, the results show that expression levels of RPTP-σ affect the activity of various forms of MMP.