title: Elevated pressure downregulates ZO-1 expression and disrupts cytoskeleton and focal adhesion in human trabecular meshwork cells.
abstract: OBJECTIVE
  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 (GTM(3)).
  
  
  METHODS
  iHTM and GTM(3) 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 GTM(3). However, in both iHTM and GTM(3), 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 GTM(3), the distribution of ZO-1 and vinculin in GTM(3) was not as regular as that in iHTM. After exposuring in elevated pressure, the changes in GTM(3) 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 GTM(3) 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).
authors: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
authors: Xuejiao
authors: X
authors: Yang
authors: Bingqian
authors: B
authors: Liu
authors: Yujing
authors: Y
authors: Bai
authors: Min
authors: M
authors: Chen
authors: Yiqing
authors: Y
authors: Li
authors: Mengfei
authors: M
authors: Chen
authors: Yantao
authors: Y
authors: Wei
authors: Jian
authors: J
authors: Ge
authors: Yehong
authors: Y
authors: Zhuo
mesh_terms: D008841:Actin Cytoskeleton / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002461:Cell Line, Transformed; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D022001:Focal Adhesions / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005902:Glaucoma, Open-Angle / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D006874:Hydrostatic Pressure / Q000009:adverse effects; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008853:Microscopy; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D016596:Vinculin / Q000235:genetics / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
chemical_list: D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D016596:Vinculin