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M.-J. Hoare, R. Job, N. C. Wade, D. Brotchie, I. Grierson, A. F. Clark; Effects of Bovine Aqueous Humor (BAH) and TGFβ-2 on Cross Linked Actin Networks (CLANs) in Cultured Bovine Trabecular Meshwork (BTM) Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4876.
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The BTM cell cytoskeleton is thought to have an important functional role in maintaining the eye’s outflow system. CLANs are changes to the cell cytoskeleton that have been induced in culture using the steroid Dexamethasone (DEX) (10-7M). In this study we are testing if the application of BAH and TGFβ-2 has an effect on CLANs in cultured BTM cells.
BTM cells were cultured for 2 weeks in varying combinations of DEX, BAH (heat and acid denatured and ultra filtered - 30,000 Dalton cut off and 5,000 Dalton cut off) and TGFβ-2. Cells were fixed in 10% NBF, stained with phalloidin-Alexa 488 to show actin and the nuclear stain, Propidium Iodide (PI). Images were taken with confocal microscopy. The number of cells containing CLANs and CLAN size were recorded.
BAH exposure for 3 and 7 days had no obvious degenerative effect on cell morphology and there was little evidence of apoptosis based on PI nuclear staining. CLANs were present in all cells but only common in cells exposed to 100% BAH.3 day BTM cells in 100% BAH showed CLAN incidence over 3 times that in control wells (13 to 16% of cells in each exposed well had CLANs, P<0.01). Heat denaturing and acid (pH 3) treating of the 100% BAH appeared to removed CLAN forming properties from the fluid. Alkali exposure (pH 11) reduced CLAN incidence to half that found in positive controls (Students t test P<0.05). Ultra filtration of BAH (30,000 Da cut off) did not reduce CLAN incidence, but a 5,000 Da cut off filter reduced CLAN incidence to background levels. Cells treated with TGFβ-2 had fewer CLANs than those treated with a mix of TGFβ-2 and DEX. TGFβ-2 did not lead to an increase apoptosis.
We have shown that BTM cells have an elevated number of CLANs when incubated in 100% BAH and 50% BAH in DMEM when compared to DMEM alone. CLAN numbers were greatest in 100% BAH and were only slightly less than those produced by 10-7M DEX. Our analysis indicated that CLAN inducing action is probably associated with a factor that can be denatured and is between 50,000 and 5,000 Da in size. We have shown that an unknown component of BAH is a potent CLAN inducer in BTM cells. This may well be due in large part to the TGFβ-2 that was found to be a potent CLAN forming agent.
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