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Inhibition of caspase activity in retinal cell apoptosis induced by various stimuli in vitro
1Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Purpose:
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.
Insights
Caspase activation drives retinal cell apoptosis from various stimuli. Inhibiting caspases effectively blocks this cell death, offering potential therapeutic strategies for neuroretinal diseases.
Area of Science:
- Neuroscience
- Cell Biology
- Ophthalmology
Background:
- Neuronal apoptosis involves cysteine aspartic acid proteases (caspases).
- Caspase activation in retinal apoptosis is not well understood.
- Retinal cell apoptosis can be triggered by ischemia, excitotoxicity, and anti-hsp27 antibodies.
Purpose of the Study:
- Investigate caspase activation in retinal cell apoptosis induced by diverse stimuli.
- Determine if caspase inhibition can prevent retinal cell apoptosis.
Main Methods:
- Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay for apoptosis.
- Western blot and fluorometric assays to assess caspase activity.
- Western blot analysis of bcl-2 and bax expression.
Main Results:
- A common caspase cascade (caspase-3, PARP cleavage) occurred in apoptosis regardless of stimulus.
- Caspase-8 activation was specific to hsp27 antibody-induced apoptosis.
- Retinal apoptosis involved decreased bcl-2 and increased bax expression.
- Caspase inhibitors (B-D-FMK, Z-IETD-FMK) blocked apoptosis.
Conclusions:
- Retinal cell apoptosis shares a common executioner pathway.
- Caspase activation is a critical step in retinal apoptosis.
- Specific caspase inhibitors effectively block retinal cell death.
- These findings suggest therapeutic potential for neuroretinal diseases.