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S. O'Reilly, D. Brotchie, R. Williams, I. Greirson, A. Clark; Modulation of Contraction of Bovine Trabecular Meshwork Cells by Cross-Linked Actin Networks (clans). Invest. Ophthalmol. Vis. Sci. 2009;50(13):4878.
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CLANs are polygonal arrangements of actin. They have been found in human trabecular meshwork (TM) tissue in situ and are increased in glaucomatous TM whole tissue. We have demonstrated in bovine TM cells exposure to dexamethasone (DEX) increases the incidence of CLANs robustly. The actin cytoskeleton is essential in TM cell function, in particular, contraction. So we sought to demonstrate a functional effect of the presence of CLANs in bovine TM cells.
TM cells were cultured from bovine eyes. Cells were cultured within (3D) and upon (2D) a collagen matrix matrices (1.5mg/ml) in 24 well culture plates. The matrices were overlayed with either medium or medium plus DEX (10-7M), detached from the base of the wells and diameter measured after 1, 3, 5 and 7 days in culture. Matrices were then fixed in 10% formalin and stained for F-actin with phalloidin and nuclei with propridium iodide and examined using confocal microscopy. In addition TM cells were exposed to medium or medium plus DEX and at the same time points stained and examined for morphological signs of apoptosis.
Bovine TM cells contracted the collagen matrices in a time -and dose dependant manner in both 3D and 2D collagen matrices. The contraction in 2D collagen matrices was faster and more robust than 3D matrices. In 3D matrices the contraction was not inhibited by DEX; however, 2D matrices contraction was inhibited by DEX treatment compared to control (p=0.001). CLANs were demonstrated in DEX-treated collagen matrices by confocal laser microscopy at the time point when contraction was impaired. Stress fibers were the dominant actin pattern in medium only exposed matrices. No increase of apoptotic index was demonstrated in DEX-treated TM cells at any time point examined and was comparable to controls without DEX.
DEX inhibits contraction of collagen matrices when bovine TM cells are seeded onto the collagen rather than into the collagen. The inhibition is associated with the appearance of CLANs in the bovine TM cytoplasm. We think that excessive DEX-induced CLAN formation is a key step in contractility failure within the TM sheet of cells. Apoptotic cell death was not a feature of this model.
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