Elevated pressure downregulates ZO-1 expression and disrupts cytoskeleton and focal adhesion in human trabecular meshwork cells
Elevated pressure downregulates ZO-1 expression and disrupts cytoskeleton and focal adhesion in human trabecular meshwork cells
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升高的压力下调 ZO-1 表达并破坏人小梁网细胞的细胞骨架和粘着斑
作者：
卓业鸿
目的探讨高静水压对人小梁细胞（iHTM）和青光眼小梁细胞（GTM 3）中闭锁小带1（ZO 1）表达和分布的影响，以及对细胞骨架和粘着斑的影响。方法将iHTM和GTM 3暴露于60 mmHg静水压下6、12和24 h。作为对照，在常规培养箱中同时孵育细胞。倒置显微镜下观察形态学变化。Western blot检测ZO-1的表达，免疫荧光法检测ZO-1的分布。肌动蛋白细胞骨架和粘着斑（黏着斑蛋白）也进行了评估免疫荧光。用商业数据分析软件分析数据，认为p<0.05具有统计学显著性。结果iHTM和GTM 3在60 mmHg压力下培养24 h后，细胞形态无明显变化。然而，在iHTM和GTM 3中，升高的压力在12小时和24小时减弱ZO-1的表达，通过蛋白质印迹检测。同时，高压破坏了ZO-1，肌动蛋白细胞骨架和黏着斑蛋白的组织，通过免疫荧光评估。比较iHTM和GTM 3，ZO-1和纽蛋白在GTM 3中的分布不如iHTM中那样规则。高压灭菌后，GTM 3的变化比iHTM更明显。结论持续性压力升高可通过损伤ZO-1、细胞骨架和粘连直接损伤小梁细胞。GTM 3比iHTM更容易受到损伤。提示眼压升高不仅是TM受损的结果，而且是TM细胞损伤的重要因素，阻止或逆转这一过程可能有助于开发治疗原发性开角型青光眼（primary open angle glaucoma，POAG）的新靶点。
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).
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