Caldesmon transgene expression disrupts focal adhesions in HTM cells and increases outflow facility in organ-cultured human and monkey anterior segments.
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 (microl min-1 mmHg-1) for all anterior segments averaged (mean+/-sem): human, 0.19+/-0.03 (n=12); monkey, 0.36+/-0.02 (n=19). In human anterior segments, transduction with 10(7) plaque forming units of AdGFPCald increased outflow facility by 43+/-21% (p<or=0.11, n=6) 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 1.5x10(7) plaque forming units of AdGFPCald was increased by 35+/-18%, p<0.2, n=10 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 38+/-13%, p<0.02, n=16. Additional analysis of maximum responses in monkey anterior segments from 1 to 6 days after transduction showed outflow facility was increased by 66+/-18%, p<0.01, n=10. 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.
Gabelt
B'Ann True
BT
Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792, USA. btgabelt@wisc.edu
Hu
Yujie
Y
Vittitow
Jason L
JL
Rasmussen
Carol R
CR
Grosheva
Inna
I
Bershadsky
Alexander D
AD
Geiger
Benjamin
B
Borrás
Terete
T
Kaufman
Paul L
PL
D000199:Actins / Q000032:analysis; D000256:Adenoviridae; D000818:Animals; D000869:Anterior Eye Segment / Q000187:drug effects* / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000494:pharmacology; D002448:Cell Adhesion / Q000235:genetics; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects; D022001:Focal Adhesions / Q000187:drug effects / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007429:Intraocular Pressure / Q000187:drug effects; D008252:Macaca fascicularis; D008253:Macaca mulatta; D008856:Microscopy, Fluorescence / Q000379:methods; D009924:Organ Culture Techniques; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects; D019076:Transgenes / Q000235:genetics*; D016596:Vinculin / Q000032:analysis
D000199:Actins; D002148:Calmodulin-Binding Proteins; D016596:Vinculin; D049452:Green Fluorescent Proteins