PURPOSE: It is well established that the unusual actin arrangements known as cross-linked actin networks (CLANs) can be induced by dexamethasone (DEX) in trabecular meshwork (TM) cells. Recent work reporting their presence in elderly glaucomatous and nonglaucomatous tissue, however, has highlighted the presence of other inducers. In this study, the authors sought to identify CLAN induction agents that may be present within and around the outflow system. METHODS: Studies were conducted on confluent bovine TM (BTM) cells in culture, and actin was stained with Alexa-Fluor 488 phalloidin to identify CLANs in the target cells. The CLAN-inducing potential of aqueous humor was expanded and included investigation of transforming growth factor-beta 2 (TGF-β2). The effect of decorin and fetal calf serum (FCS) on BTM cell cytoskeleton was also investigated, and all were compared with DEX with an exposure period of up to 7 days. RESULTS: CLAN numbers were increased after 7 days of exposure to TGF-β2 (45%), aqueous humor (37%), and decorin (69%). Even FCS had some modest CLAN-inducing ability (reaching 12%) in BTM cells. Neutralization of TGF-β2 reduced CLAN incidence in aqueous humor conditions to baseline (12%) levels. Blocking TGF-β2 receptors reduced CLAN formation in TM cells by 25% to 30%, whereas the inhibition of Smad3 negated CLAN incidence. CONCLUSIONS: In this study the authors identified TGF-β2 as a CLAN-inducing component present in aqueous humor. Decorin was also implicated as another CLAN-inducing agent and it was confirmed that FCS has CLAN-inducing properties.
PURPOSE: It is well established that the unusual actin arrangements known as cross-linked actin networks (CLANs) can be induced by dexamethasone (DEX) in trabecular meshwork (TM) cells. Recent work reporting their presence in elderly glaucomatous and nonglaucomatous tissue, however, has highlighted the presence of other inducers. In this study, the authors sought to identify CLAN induction agents that may be present within and around the outflow system. METHODS: Studies were conducted on confluent bovine TM (BTM) cells in culture, and actin was stained with Alexa-Fluor 488 phalloidin to identify CLANs in the target cells. The CLAN-inducing potential of aqueous humor was expanded and included investigation of transforming growth factor-beta 2 (TGF-β2). The effect of decorin and fetal calf serum (FCS) on BTM cell cytoskeleton was also investigated, and all were compared with DEX with an exposure period of up to 7 days. RESULTS: CLAN numbers were increased after 7 days of exposure to TGF-β2 (45%), aqueous humor (37%), and decorin (69%). Even FCS had some modest CLAN-inducing ability (reaching 12%) in BTM cells. Neutralization of TGF-β2 reduced CLAN incidence in aqueous humor conditions to baseline (12%) levels. Blocking TGF-β2 receptors reduced CLAN formation in TM cells by 25% to 30%, whereas the inhibition of Smad3 negated CLAN incidence. CONCLUSIONS: In this study the authors identified TGF-β2 as a CLAN-inducing component present in aqueous humor. Decorin was also implicated as another CLAN-inducing agent and it was confirmed that FCS has CLAN-inducing properties.