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Impressum
In vitro results about the use of CD95 ligand as an apoptotic inductor in glaucomatous filtering procedures
1Hueber A., 1Welsandt G., 1Mietz H., 1Jordan J. F., 2Weller M., 1Esser P. J.
1Zentrum für Augenheilkunde der Universität zu Köln, Joseph-Stelzmann-Str. 9, 50931 Köln, Deutschland; 2Neurologische Klinik, Eberhard-Karls-Universität Tübingen, Hoppe-Seyler-Str. 3, 72076 Tübingen
Objective: Apoptosis allows for the safe disposal of cells without
damaging the surrounding tissue. It is a morphologically defined type
of cell death that is commonly observed during development, e.g., in the
immune and central nervous system and is often referred to as programmed
or physiological cell deaths. Unspecific toxic side effects by the use
of antimetabolites (e.g. 5-fluorouracil or mitomycin-C) in glaucomatous
filtering procedures may be eliminated by induction of apoptosis by CD95
ligand.
Methods: For cell culture experiments we used human tenon fibroblasts,
which are thought to contribute significantly to failures in glaucoma
surgery, treated with different concentrations of CD95 ligand. Cytotoxity
was measured with the cristal violet assay. Apoptosis was investigated
using in situ DNA end labelling (TUNEL). Caspase activity was measured
using fluogenic caspase-3 (Ac-DEVD-AMC) or caspase-1 (Ac-YVAD-AMC) substrate
and western blot for the detection of active fragments.
Results: After exposure of human tenon's fibroblasts to CD95 ligand
for 17h we found a dose dependent killing (LD 50 = 160U). At this concentration
we also detected apoptotic cells using in situ DNA end labelling (TUNEL).
We found a strong dose dependent induction of caspase-3, but not caspase-1
activity, after 10h treatment with CD95 ligand (10U-120U). The active
fragment of caspase-3, but not of caspase-1, could be detected by western
blot.
Conclusions: We demonstrate, that the CD95 ligand induced cell
death in cultured human tenon fibroblasts is apoptotic and mediated by
the caspase-3 pathway. These results suggest that it may be possible to
use CD95 ligand in glaucomatous filtering procedures.
Unterstützung durch die DFG (Mi 347/5-1, Es 82/5-3, He 840/6-3),
Köln Fortune Programm und Retinovit Stiftung