Viral Vector Effects on Exoenzyme C3 Transferase-Mediated Actin Disruption and on Outflow Facility

Abstract

Purpose: Purified Clostridium botulinum exoenzyme C3 transferase (C3) effects on the actin cytoskeleton in human trabecular meshwork cells (HTM) and on the outflow facility response in monkey organ-cultured anterior segments (MOCAS) were determined in the presence or absence of viral vectors.

Methods: Human adenovirus type 5 (AdV) and feline immunodeficiency virus (FIV) vectors were produced using kits. Cell soluble purified C3 (C3cs) was purchased commercially. Recombinant C3 (C3rec) cDNA was overexpressed in Escherichia coli and purified. The HTM cells were incubated with up to 10 μg/mL C3cs or with 5 μg of C3rec and/or viral vector (multiplicity of infection [MOI] = 25). Cells then were fixed and stained for actin. Outflow facility in MOCAS was measured at baseline, 4 hours, 24 hours, and 3 to 4 days following bolus injection of AdV (1.6 × 107 transducing units) and/or 2.5 μg C3rec.

Results: The HTM cells treated for 4 hours with C3cs (all doses) or for 24 hours with C3rec developed a rounded morphology and lost stress fibers. Cells transduced with vectors alone showed no changes at any time point. Cells exposed to C3rec and cotransduced with either viral vector showed significant disruption of the actin cytoskeleton within 4 hours after exposure, which persisted at 24 hours. In MOCAS, the AdV vector alone had no effect on outflow facility, but enhanced the response to C3rec at 4 hours.

Conclusions: Coadministration of viral vectors enhances the ability of C3 transferase to disrupt actin stress fiber formation in HTM cells and increase outflow facility in MOCAS. Viral vectors potentially could be used to increase the bioavailability of proteins for cells that are difficult to transfect.

Figure 1

Actin organization in confluent monolayers of differentiated primary HTM cells 4 hours after transfection with C3cs. Cells were treated for 4 hours with vehicle (A), or with 2 (B), 5 (C) or 10 (D) μg/mL C3cs. In the presence of the vehicle alone or 2 μg/mL C3cs, well-formed actin stress fibers (arrowheads) were observed throughout the culture. As the concentration of C3cs was increased, fewer actin stress fibers were observed in the cytoplasm (arrows). At the highest concentration (10 μg/mL), very few actin stress fibers were observed and mainly cortical actin filaments appeared intact, suggesting that the cells were less well spread. Cells were double-labeled with alexa-488 phalloidin and Hoescht 33342. Magnification: ×40.

Figure 2

Actin organization in confluent monolayers of differentiated primary HTM cells receiving C3rec with or without viral vectors. (A) At 4 hours after transfection, no change in actin organization was seen in cells receiving media, virus, or C3rec alone, but cells receiving C3rec and virus simultaneously showed significant actin reorganization and cell rounding. (B) At 24 hours after transfection, cells receiving media or virus alone still showed no change in actin organization, but cells receiving C3rec now showed significant actin reorganization and cell rounding. Cells receiving C3rec and virus simultaneously showed the most widespread actin reorganization and cell rounding. The percentage of cells in each condition exhibiting actin disruption is shown in Table 1.

Figure 3

Graphical representation of the outflow facility results from Tables 1 and 2 for paired anterior segments. Data are the ratio ± SEM of treated to control after correction for baseline (i.e., [C3rec + AdV/BL]/[C3rec/BL] or [AdV/BL]/[untreated/BL] for each of the various time points). *Significantly different from 1.0 by the 2-tailed paired t-test.

Figure 4

H&E staining showing gross morphology of segments at 24 hours after the various treatment regimens. (A, B) Untreated control (Cont) and AdV treated segments from MOCAS ocm-12-08. There were no apparent qualitative morphological differences between paired control and AdV-treated segments in terms of cellularity of the juxtacanalicular regions and along the collagen beams, the integrity of Schlemm's canal (SC), and the organization of the collagen beams. (C, D) The C3rec- and C3rec + AdV–treated segments from MOCAS ocm-12-32. There was reduced cellularity, disorganized beams, and discontinuity of the inner wall of SC. These alterations appeared to be more prominent in the AdV + C3rec segment than in the C3rec segment. TM, trabecular meshwork.