Effects of SV40 T antigen transduction on human corneal endothelial cell wound healing in vitro.

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.

CD40 expression in normal human cornea and regulation of CD40 in cultured human corneal epithelial and stromal cells.

PURPOSE: To determine whether CD40-CD40 ligand (CD40L) interaction plays a role in corneal inflammatory responses, the expression of CD40 and CD40L on normal human cornea was investigated. In addition, using cultured human corneal epithelial (HCE) and human corneal stromal (HCS) cells, the regulation of CD40 expression in human corneal cells investigated, including that induced by proinflammatory cytokines such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. METHODS: Frozen optimal cutting temperature (OCT) compound-embedded sections of corneal tissues obtained from 18 normal human corneas were examined by an immunoperoxidase staining technique with anti-CD40 and anti-CD40L monoclonal antibodies (mAbs). Also, cultured HCE and HCS cells, with IFN-gamma (250-1000 U/mL) or TNF-alpha (500-4000 U/mL) treatment for 1 to 4 days and with no treatment, were stained by the immunofluorescence technique with mAbs and analyzed by flow cytometry. RESULTS. The area of positive staining for CD40 showed a topographical difference. The limbal epithelial cells were predominantly positive for CD40. Positive staining was also found to a lesser extent on the cells in the basal layer of peripheral corneal epithelium. Epithelial cells of the central cornea showed no immunoreactivity for CD40. Corneal stromal cells were negative for CD40 in most of the donor tissues (positive: 5 of the 18 corneas). Endothelial cells were distinctly negative for CD40. Cultured HCE cells were also positive but decreased in positive cell number with lengthening culture period. None or less than 5% of the cultured HCS cells were CD40 positive. IFN-gamma enhanced CD40 expression on both cell types. In contrast, TNF-alpha enhanced CD40 on HCE but not on HCS cells. No component cells of normal human cornea or cultured HCE and HCS cells showed immunoreactivity for CD40L. CONCLUSIONS: In the human cornea, CD40 is expressed predominantly on limbal epithelial cells and also on cultured HCE cells with high proliferative potential. In addition, the expression of CD40 is induced on cultured HCE and HCS cells differentially by proinflammatory cytokines, such as IFN-gamma and TNF-alpha.