RESUMO
BACKGROUND: Apoptosis very probably plays a key part in endothelial cell loss during corneal storage in organ culture as well as hypothermic storage. However, the mechanisms underlying endothelial apoptosis are poorly understood. The response of a human corneal endothelial cell (HCEC) line to staurosporine, a known inducer of apoptosis, was investigated to gain insights into the intracellular modulators that participate in endothelial cell death. METHODS: Immortalised HCECs were studied after 3, 6, 12, and 24 hours of incubation with 0.2 micro M staurosporine. Cell shedding was monitored. Hoechst 33342 fluorescent DNA staining combined with propidium iodide was used for apoptosis/necrosis quantification and morphological examination. The caspase-3 active form was assessed using western blot, proteolytic activity detection, and immunocytochemistry. The cleaved form of poly(ADP-ribose) polymerase (PARP) was assessed using immunocytochemistry and western blot. The ultrastructural features of cells were screened after 12 hours with staurosporine or vehicle. RESULTS: The specific apoptotic nature of staurosporine induced HCEC death was confirmed. The ultrastructural features of staurosporine treated cells were typical of apoptosis. HCEC shedding and DNA condensation increased with time. Caspase-3 activity was detected as early as 3 hours after exposure with staurosporine, peaking at 12 hours of incubation. The presence of cleaved PARP after 3 hours confirmed caspase-3 activation. CONCLUSIONS: These data suggest strongly that HCEC cell death induced by staurosporine is apoptosis. The main consequence of HCEC apoptosis is shedding. Staurosporine induced apoptosis of endothelial cells involves activation of caspase-3, and could be a useful model to study strategies of cell death inhibition.
