The effect of C3 transgene expression on actin and cellular adhesions in cultured human trabecular meshwork cells and on outflow facility in organ cultured monkey eyes.
OBJECTIVE
To determine the effects of adenovirus-delivered exoenzyme C3 transferase (C3) gene expression on cultured human trabecular meshwork (HTM) cells and on outflow facility in organ cultured monkey anterior segments.


METHODS
An adenoviral (Ad) vector expressing both C3 and green fluorescent protein (GFP) was used to transduce cultured HTM cells. Changes in cell morphology and the organization of actin, vinculin, and beta-catenin were assessed using immunofluorescence. Cultured monkey eye anterior segments were used to test the effects of AdC3GFP on outflow facility.


RESULTS
Treatment of HTM cells with AdC3GFP resulted in dose-dependent morphological changes 3 or 4 days post-transduction. The AdC3GFP-transduced cells were either partially retracted, rounded, or very elongated compared to non-transduced cells. Compared to AdGFP-transduced cells, AdC3GFP-transduced cells demonstrated disrupted actin cytoskeleton, reduced vinculin-positive focal adhesions, and loss of beta-catenin staining. Cells transduced with AdGFP did not round up or retract. In organ culture studies, outflow facility was increased by 90+/-21% (n=15, p<0.001) in AdC3GFP-transduced eyes compared to baseline and corrected for AdGFP-transduced control eye washout on days 3-6 after transduction.


CONCLUSIONS
C3 transduction is effective in disrupting actin filaments, cytoskeleton, and cellular adhesions in HTM cells and in increasing outflow facility in organ cultured monkey anterior segments, suggesting that expressing the C3 gene in the trabecular meshwork may be an effective approach for glaucoma therapy.
Liu
Xuyang
X
Department of Ophthalmology & Visual Sciences, University of Wisconsin Medical School, Madison, WI, USA.
Hu
Yujie
Y
Filla
Mark S
MS
Gabelt
B'Ann T
BT
Peters
Donna M
DM
Brandt
Curtis R
CR
Kaufman
Paul L
PL
D036002:ADP Ribose Transferases / Q000235:genetics* / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000869:Anterior Eye Segment / Q000166:cytology / Q000378:metabolism; D001082:Aqueous Humor / Q000378:metabolism*; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000235:genetics* / Q000378:metabolism; D002448:Cell Adhesion; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008252:Macaca fascicularis; D008253:Macaca mulatta; D008856:Microscopy, Fluorescence; D009924:Organ Culture Techniques; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D014162:Transfection; D019076:Transgenes / Q000502:physiology; D016596:Vinculin / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
D000199:Actins; D011993:Recombinant Fusion Proteins; D051176:beta Catenin; D016596:Vinculin; D049452:Green Fluorescent Proteins; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins