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Natalie M. Pollock, Steven O'Reilly, Luminita Paraoan, Abe Clark, Ian Grierson; Is TGF-β2 The Component In Aqueous Humor Responsible For Induction Of Cross-Linked Actin Networks (CLANs) In Trabecular Meshwork Cells?. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4660.
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We have shown previously that addition of aqueous humor to trabecular meshwork (TM) cells induces the formation of cross-linked actin networks (CLANs); however, it is not clear which component of the aqueous humor was responsible for this effect. TGF-β2 has been shown to be increased in glaucoma and is suspected to play a role in altered outflow. The purpose of this work was to identify whether TGF-β2 was the main CLAN inducing agent in aqueous humor.
Primary cultured bovine TM (BTM) served as our test cells. Bovine aqueous humor was added, (1:1) with media containing 1% serum, to confluent cultures of BTM cells. Recombinant human TGF-β2 (Invitrogen) was added at varying concentrations (1-8ng/ml) to cultures. We employed several methods to neutralize TGF-β2. Anti-TGF-β2 antibody (1.6µg/ml in media containing 1% serum) (R&D Systems) was added simultaneously with aqueous humor to cells. In separate experiments agents that block TGF-β RI and RIII were also added to cultures. Known inhibitors of TGF-β, decorin and mannose-6-phosphate, were also added to cultures at varying concentrations. In all cases media containing 1% and 10% serum were run as comparators. After 3 and 7 days cells were fixed in 10% neutral buffered formalin, stained with Alexa-fluor-488 phalloidin and propridium iodide (PI) (Invitrogen) and imaged using fluorescence and/or confocal microscopy.
Following 7 days treatment with aqueous humor CLAN incidence was 43%; these levels are similar to the incidence with 2ng/ml TGF-β2 (47%). Treatment with receptor blockers reduced CLAN incidence to 30-35% while addition of TGF-β2 neutralizing antibody to aqueous humor significantly reduced the incidence of CLANs to within baseline levels at 7day time point (11%). In all cultures grown with serum there is a baseline level of CLANs (6-12%) that was non-responsive to TGF-β2 neutralization. BTM cells treated with mannose-6-phosphate showed straight stress fiber arrangements with low CLAN incidence (<20%) while decorin was found to induce CLAN formation (70% at 25µg/ml) by mechanisms still to be determined.
This data shows that CLANs can be induced by factors present in aqueous humor, particularly TGF-β2, but there are inducers in serum that are not TGF-β2 related.
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