Inhibition of caspase activity in retinal cell apoptosis induced by various stimuli in vitro.
OBJECTIVE
Although recent work implicates a decisive role for a family of cysteine aspartic acid proteases, termed caspases, as mediators of neuronal apoptosis, little is known about caspase activation that accompanies apoptosis in the retina. The purpose of this study was to investigate caspase activation in retinal cell apoptosis induced by various stimuli, including simulated ischemia, excitotoxicity, and antibody to heat shock protein 27 (hsp27), and to assess whether the inhibition of caspases can block apoptosis in retinal cells induced by different stimuli.


METHODS
Apoptotic cell death induced in cultured retinal cells by simulated ischemia, excitotoxicity, or hsp27 antibody was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique. Changes in the caspase activity were studied using western blot analysis and a fluorometric protease activity assay in the presence or absence of caspase inhibitors. In addition, changes in the expression of bcl-2 and bax were examined by western blot analysis.


RESULTS
The authors' in vitro observations revealed that the apoptotic process in retinal cells induced by different stimuli share a common executioner proteolysis cascade, including caspase-3 and poly(ADP ribose) polymerase cleavage. One exception, however, was that caspase-8 activation was only observed during the apoptosis induced by hsp27 antibody. In retinal cells going to apoptosis regardless of the stimulus, bcl-2 expression was decreased and bax expression was increased. Furthermore, the authors observed that treatment of retinal cells with inhibitors of caspases, including B-D-FMK and Z-IETD-FMK, blocked the apoptotic cell death induced by different stimuli.


CONCLUSIONS
The authors' observations provide a better understanding of the apoptotic process in retinal cells at molecular level and demonstrate an effective blockade of caspase activation with specific inhibitors. These findings may have therapeutic implications in the treatment of neuroretinal diseases, which are characterized by apoptotic cell death.
Tezel
G
G
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Wax
M B
MB
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D015687:Cell Hypoxia; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006360:Heat-Shock Proteins / Q000276:immunology; D020287:In Situ Nick-End Labeling; D016202:N-Methylaspartate / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D012160:Retina / Q000166:cytology / Q000502:physiology*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology; D051028:bcl-2-Associated X Protein
D000911:Antibodies, Monoclonal; C494828:Bax protein, rat; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D006360:Heat-Shock Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D016202:N-Methylaspartate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D011065:Poly(ADP-ribose) Polymerases; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9