Caldesmon transgene expression disrupts focal adhesions in HTM cells and increases outflow facility in organ-cultured human and monkey anterior segments B’Ann True Gabelt a, * , Yujie Hu a , Jason L. Vittitow b , Carol R. Rasmussen a , Inna Grosheva c , Alexander D. Bershadsky c , Benjamin Geiger c , Terete Borra ´s b , Paul L. Kaufman a a Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792, USA b Department of Ophthalmology, University of North Carolina, 6109 NeuroSci Res Bldg, Box 7041, Chapel Hill, NC 27599, USA c Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Received 29 August 2005; accepted in revised form 9 December 2005 Available online 26 January 2006 Abstract Cytoskeleton modulating compounds have been shown to lower intraocular pressure (IOP) and increase outflow facility. Caldesmon is one protein that is involved in the regulation of actin stress fiber formation. The effects of rat non-muscle caldesmon (Cald) gene over-expression on focal adhesions in human trabecular meshwork (HTM) cells and on outflow facility in organ-cultured human and monkey anterior segments were determined. Treatment of HTM cells with adenovirus-delivered caldesmon (AdCaldGFP) resulted in characteristic changes in the actin cytoskeleton and matrix adhesions within 24–48 hr post-transduction. Stress fibers gradually disappeared and novel actin structures were formed (see manuscript by Grosheva et al., this issue). In cells with disrupted stress fibers, vinculin-containing focal adhesions were also disrupted. In organ-cultured anterior segments, baseline outflow facility (ml min K1 mmHg K1 ) for all anterior segments averaged (meanGSEM): human, 0.19G 0.03 (nZ12); monkey, 0.36G0.02 (nZ19). In human anterior segments, transduction with 10 7 plaque forming units of AdGFPCald increased outflow facility by 43G21% (p%0.11, nZ6) at 66 hr compared to baseline and corrected for the changes in outflow facility of the contralateral vehicle treated segment. Using the same time point, i.e. 2–3 days after injection, outflow facility in monkey anterior segments, transduced with 1.5!10 7 plaque forming units of AdGFPCald was increased by 35G18%, p!0.2, nZ10 compared to baseline and corrected for the change in outflow facility in the contralateral AdGFP treated segment. Combining human (66 hr) and monkey (2–3 days) data, outflow facility was increased by 38G13%, p!0.02, nZ16. Additional analysis of maximum responses in monkey anterior segments from 1 to 6 days after transduction showed outflow facility was increased by 66G18%, p!0.01, nZ10. Caldesmon over-expression, which relaxes cultured HTM cells and disrupts their actin cytoskeleton and cell–matrix adhesions, also appears to increase outflow facility in organ-cultured human and monkey anterior segments. This suggests that over-expression of the caldesmon gene in the TM may be an effective approach for the gene therapy of glaucoma. q 2006 Elsevier Ltd. All rights reserved. Keywords: actin cytoskeleton; caldesmon; focal adhesions; gene therapy; outflow facility; organ-cultured anterior segments; trabecular meshwork; vinculin 1. Introduction Present pharmacotherapy of glaucoma involves lowering intraocular pressure (IOP). This is accomplished by frequent repetitive administration of exogenous molecules to enhance aqueous humour outflow or reduce its inflow (Ritch et al., 1996). Modern molecular genetic technology raises the possibility of bypassing the external agent and ‘setting’ the inflow or outflow tissues to a different ‘performance’ level genetically. This would have the great advantage of eliminating the need for repetitive drug application by the patient (Goldberg, 1999). Cytoskeleton modulating compounds have been shown to lower IOP and/or enhance outflow facility in vivo and/or in vitro (review in, Epstein et al., 1999; Kaufman et al., 2000). Experimental Eye Research 82 (2006) 935–944 www.elsevier.com/locate/yexer 0014-4835/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.12.002 * Corresponding author. B’Ann True Gabelt, Department of Ophthalmology and Visual Sciences, University of Wisconsin, 600 Highland Ave, F4/340 CSC, Madison, WI 53972, USA. E-mail addresses: btgabelt@wisc.edu (B.T. Gabelt), yujiehu@wisc.edu (Y. Hu), jvittitow@inspirepharm.com (J.L. Vittitow), crasmussen@wisc.edu (C.R. Rasmussen), inna.grosheva@weizmann.ac.il (I. Grosheva), alexander. bershadsky@weizmann.ac.il (A.D. Bershadsky), benny.geiger@weizmann.ac. il (B. Geiger), tborras@med.unc.edu (T. Borra ´s), kaufmanp@mhub.ophth. wisc.edu (P.L. Kaufman).