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Impressum
Death receptors in human microvascular and choroidal endothelial cells
Esser P., Luther T., Esser J. M., Kociok N., Welsandt G., Schraermeyer U.
University of Cologne, Germany, Josef Stelzmann Str.9 50931 Köln
Objective: Central element of the clinical pathology of diabetic
retinopathy is the dysfunction of the endothelium that leads to the formation
of acellular capillaries and retinal hypoxia. Here we sought to investigate
the presence of newly discovered ligand-receptor-systems from the TNF-receptor
superfamily, i.e. Apo3-L, Apo2-L and its receptors Trail-R1 to R2 in human
microvascular endothelial (HMEC) and human choroidal endothelial cells
(CEC), which are thought to play a major role in the development of neovascular
retinal diseases.
Methods: The preparation of CEC was performed using LEA lectin
coated dynabeads. Total RNA was prepared by guanidium/phenol extraction
from HMEC and CEC. The presence of APO-3L, Apo2-L and TRAIL-receptor 1,2
(DR4, DR5) 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, and 2.5 ng of total RNA. Negative controls were performed
by omitting RT during the first strand synthesis. Amplification products
were separated on a 2% agarose gel and visualized with ethidiumbromide.
Cell culture assays were performed using the cristal-violet assay.
Results: Specific bands for Apo2-L and TRAIL-receptors 1,2 as well
as APO3L could be detected by RT-PCR in both cell lines. The negative
controls did not display a signal. In our semiquantitative assay, the
serial dilutions revealed a strong presence of specific mRNA for all positive
samples. CEC were rather resistant to the action of APO-2L alone, however,
this resistance was overcome by the addition of Prostaglandin F2-alpha.
Conclusion: The presence of several death receptors in HMEC and
CEC has implications for future studies of therapeutic strategies aiming
at inhibition of endothelial cell loss. Similar to CD95L, TRAIL activates
rapid apoptosis in many types of fast proliferating cells while most normal
cells appear to be resistant to TRAIL's cytotoxic action. Further investigations
need to clarify whether the expression of these receptors and ligands
are of biological significance in retinal diseases.