Elevated pressure downregulates ZO-1 expression and disrupts cytoskeleton and focal adhesion in human trabecular meshwork cells

Purpose: To investigate the effect of elevated hydrostatic pressure on the expression and distribution of zonula occludens-1 (ZO-1), and its effect on cytoskeleton and focal adhesion in immortal human trabecular meshwork cells (iHTM) and glaucomatous human trabecular meshwork cells (GTM3). Methods: iHTM and GTM3 were exposed to 60 mmHg hydrostatic pressure for 6, 12, and 24 h. As a control, the cells were incubated simultaneously in a conventional incubator. Morphology changes were observed with an inverted microscope. The expression of ZO-1was examined with western blot, and the distribution of ZO-1 was assessed by immunofluorescence. Actin cytoskeleton and focal adhesion (vinculin) were also assessed by immunofluorescence. Data were analyzed with commercial data analysis software and a p<0.05 was considered to be statistically significant. Results: There was no evident morphology change after 24 h culture in 60 mmHg pressure in iHTM and GTM3. However, in both iHTM and GTM3, elevated pressure attenuated the expression of ZO-1 at 12 h and 24 h, detected by western blot. Meanwhile, high pressure disrupted the organization of ZO-1, actin cytoskeleton, and vinculin, assessed by immunofluorescence. When comparing iHTM with GTM3, the distribution of ZO-1 and vinculin in GTM3 was not as regular as that in iHTM. After exposuring in elevated pressure, the changes in GTM3 were more obvious than that in iHTM. Conclusions: Sustained pressure elevation may directly damage trabecular meshwork cells by injuring ZO-1, cytoskeleton, and foal adhesions. And GTM3 was more susceptible to damage than iHTM. We suggest that elevated pressure seems to be not only the results of damaged TM, but also an important factor for the injury of TM cells, stop or reverse the process may help developing new target for the treatment of primary open angle glaucoma (POAG).