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K.K. Wentz-Hunter, R. Kubota, X. Shen, B.Y. Yue; Effects of Extracellular Myocilin on Cultured Human Trabecular Meshwork Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1154.
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Purpose:To study the influence of extracellular recombinant myocilin on the adhesion, morphology, and migratory and phagocytic activities of cultured human trabecular meshwork (TM) cells. Methods: Recombinant myocilin was expressed and purified from bacterial cultures. For adhesion assays, TM cells were plated onto wells coated with fibronectin, the purified myocilin, or fibronectin/myocilin mixtures. Morphology was recorded by phase contrast microscopy. Oregon Green phalloidin-488 was used to reveal the actin architecture. Cells were also stained with anti-paxillin to assess focal contact formation. To examine the effects of extracellular myocilin on migration, TM cells were plated on fibronectin/myocilin or fibronectin alone, wounded, and monitored 6 hours later. For phagocytic activity, TM cells plated on fibronectin with or without myocilin were challenged with rhodamine-labeled latex beads for 18 hours. Parallel experiments were performed on human corneal fibrobalsts. Results: TM cells, while adhering readily to fibronectin, failed to attach in myocilin coated wells. Cell adhesion on fibronectin was also compromised by myocilin in a dose dependent manner. Myocilin, in addition, triggered TM cells to assume a stellate appearance with broad cell bodies and microspikes. The integrity of actin structure was affected and the number of focal contacts was decreased. Migration of TM cells plated on fibronectin/myocilin was found to be retarded compared to those on fibronectin controls. Myocilin, on the other hand, had little impact on phagocytic activities of TM cells. Corneal fibroblasts, used as a control cell type, attached and migrated equally well on myocilin or any fibronectin/myocilin mixtures. Conclusions: The present results demonstrate the anti-adhesive and counter-migratory effects of myocilin on TM cells. An optimal level of myocilin in the extracellular matrix of TM tissues may be essential to allow both the flexibility and resilience required for TM cells to withstand the aqueous flow and intraocular pressure fluctuations.
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