Abstract
Purpose :
Matrix metalloproteinase (MMP) 1 and MMP14 are found co-localized with reactive astrocytes in the lamina cribrosa (LC) of human optic nerve heads in primary open-angle glaucoma (POAG) (Agapova et al. 2001). Increased intensity of MMP2 immunostaining in the LC (Yan et al. 2000) and increased concentration of MMP9 in the aqueous humor (Markiewicz et al. 2015) is also observed in POAG eyes when compared to normal eyes. These studies suggest that MMP activity may play an important role in the neurodegenerative processes in POAG. We have previously found that MMP2 production is significantly increased in normal LC cells following transforming growth factor beta 2 (TGFβ2) treatment, while in POAG LC cells MMP2 production remains unaltered. In this study, we monitor MMP activity using an MMP probe to assess the effect of TGFβ2 on porcine LC cells.
Methods :
Porcine LC cells characterized as alpha smooth muscle actin positive and glial fibrillary acidic protein negative were plated on a 96-well plate. After culture for 48 hours, 10 ng/mL of recombinant human TGFβ2 (R&D Systems) or 200 nM of MMPSense750FAST (PerkinElmer) were added to each well. The supernatant was removed at 24 hours to record absorbance at 750 nm and fluorescence at 775 nm with excitation at 750 nm. Fluorescence intensity of MMPSense in LC cells with or without TGFβ2 is shown as mean ± standard deviation (n=10) and unpaired t tests were performed to determine the significance level at p<0.05.
Results :
The max absorbance of MMPSense was detected at 680 nm with optical density 0.415. A two-tailed unpaired t test shows a significant increase (p=0.0132) in TGFβ2-treated group (525.6±48.7) when compared to the non-treated group (477.5±26.4) (Figure 1). No significant difference was detected between the non-treated group and the probe only (p=0.8217).
Conclusions :
TGFβ2 significantly increases MMP activity in LC cells. We are currently investigating which MMP protein activates MMPSense predominant in TGFβ2-treated group and whether the MMP activity reduces in response to tissue inhibitor of metalloproteinase or plasminogen activator inhibitor. We will also investigate if TGFβ2-induced MMP activity is substrate stiffness dependent or can be influenced by oxidative stress. Understanding the roles of these proteins will provide information for development of therapeutic targets in glaucoma.
This is a 2020 ARVO Annual Meeting abstract.