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Stephen N. Schwartz, Rachel M. Beverley, Michael C. Giovingo, Ryan D. McCarty, Jeffrey P. Mayer, Paul A. Knepper; Actin Reorganization (CLANs) Induced by sCD44-treatment in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4634.
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Cross-linked reorganization of F-actin networks (CLANs) occurin glaucomatous and steroid-treated trabecular meshwork (TM)cells and may reduce outflow facility. Since soluble CD44 (sCD44)reduces outflow facility in porcine organ perfusion, the purposeof this study was to determine if sCD44 effects CLAN formationin TM cells.
Primary cultures of TM cells were treated with 0.1 ng immunopurifiedsCD44 (n=15) and appropriate controls including heat-inactivatedsCD44 (n=13), stained for F-actin, and visualized with a 100xoil immersion objective on a confocal microscope. The relativeamount of F-actin exhibiting CLAN-like characteristics was quantifiedwithin individual cells. The vertices of the actin (the endsor hubs of F-actin bundles) were identified and counted as exhibitingeither regular formation or CLAN-like formation if at least3 vertices were connected with strongly fluorescent F-actinspokes. The images were partitioned into a grid, and the verticeswere counted in a regular pattern until at least 100 verticeswere counted.
An increase in the percent of F-actin arranged in CLANs wasobserved in cells treated with sCD44 (34.75% vertices) as opposedto control cells (11.47% vertices), and the difference was highlysignificant (P<0.00001).
Our results indicate that sCD44 induces the formation of CLANsin TM cells. Furthermore, this finding suggests that sCD44 mayalter the outflow resistance of aqueous through its effectson CLAN formation.
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