98th Annual Meeting DOG 2000

V 349

The death receptors TRAMP and TRAIL are present in human tenon fibroblasts

J. M. Esser, P. Esser, J. F. Jordan, G. Welsandt, H. Mietz, G. K. Krieglstein,

Purpose: Programmed cell death (Apoptosis) causes cell death without inducing an inflammatory response. In the recent past, several "death-receptors" with the predominant function to induce apoptotic cell death have been identified. Here, we sought to investigate the presence of death-receptors from the TNF-receptor superfamily, i.g, TNF-receptor-related apoptosis-mediating protein (TRAMP), TNF-related apoptosis-inducing ligand (TRAIL, APO-2) receptors-1,2,3,4 and the corresponding ligands in human tenon fibroblasts, which are thought to play a major role in scar formation after glaucoma surgery.

Methods: Total RNA was prepared by guanidium/phenol extraction from cultured human tenon fibroblasts of passage P2. The presence of TRAMP, TRAIL-ligand and TRAIL-receptor 1,2,3,4 mRNA was investigated by RT-PCR using specific PCR primers. The cDNA was diluted with TE buffer corresponding to RT transcripts of 62.5, 12.5, 2.5 and 0.5 ng of total RNA. Negative controls were performed by omitting RT during the first strand synthesis. The amplification products were separated on a 2% agarose gel and visualized with ethidiumbromide.

Results: Specific bands for the chosen primer combinations were detected by RT-PCR in P2 cells for TRAMP and TRAIL-receptors 1,2,3,4. In contrast, no specific signal for TRAIL-ligand was detected. The negative controls did not display a signal. In our semiquantitative assay, all positive samples showed a strong presence of specific mRNA in serial dilutions.

Conclusions: The presence of several death receptors in human tenon fibroblasts has implications for future studies of antiproliferative and proapoptotic therapeutic strategies in the inhibition of scar formation after glaucoma surgery. Similar to CD95L, TRAIL activates rapid apoptosis in many types of fast proliferating cells excluding most normal cells.Therefore, TRAIL may be useful as an antiproliferative agent in the future.

Supported by DFG (Es 82/5-3, Mi 347/5-1, He 840/6-2), Köln Fortune program and Retinovit Foundation
Department of Ophthalmology, University of Cologne, Joseph-Stelzmann Str. 9, D-50931 Köln