Effects of SV40 T antigen transduction on human corneal endothelial cell wound healing in vitro
Ching-Hsi Hsiao1, Woo-Jin Sah, Koichi Soya
1Department of Ophthalmology, Ocular Cell Transplantation Laboratory, University of California San Francisco, San Francisco, CA 94143-0730, USA.
Objective:
The aim of this study was to investigate the endothelial wound healing effects of SV40 large T and small t antigen transduction on cultured human corneal endothelial cells (HCEC).
Methods:
Human corneal endothelial cells were infected with either mock solution, Ad green fluorescent protein (GFP), or Ad SV40 T/t antigen/GFP, then mechanically wounded 48 h later. The endothelial wound healing rate was quantified by an analysis of the photographs taken every 12 h after wounding. The characteristics of Ad SV40 T/t Ag/GFP-infected human corneal endothelial cells were evaluated with cell morphology, cell density, contact inhibition, and cytoskeletal features using rhodamine phalloidin to stain F-actin. DNA synthesis was assessed using 5-Bromo-2'-deoxy-uridine (BrdU) labeling.
Results:
Wound healing rates in the first 12 and 24 h after wounding were significantly faster in the Ad SV40 T/t antigen/GFP-infected group than the other two groups. In all three groups, the morphology, cell density, and cytoskeletal features of cells at confluency was similar and contact inhibition retained. There were no differences in the pattern of F-actin and endothelial cell density 4 d after wound closure. However, during the process of wound healing, prominent stress fibers in migrating cells near the wound edge were noted in normal cells at 36 h after wounding, whereas the Ad SV40 T/t Ag/GFP-infected cells showed similar changes as early as 12 h after wounding. BrdU staining results revealed that the Ad SV40 T/t antigen/GFP-infected group had labeled cells showing DNA synthesis in the wound area at 12 h after wounding, while no labeled cells were found in the other two groups.
Conclusions:
In an in vitro model, transduction of human corneal endothelial cells using a recombinant adenoviral vector expressing SV40 T/t antigen enhanced both the wound healing rate and proliferative capacity, especially in the first 12 h after wounding, and the characteristic morphologic features of the infected cells were maintained.
