Inhibition of Caspase Activity in Retinal Cell Apoptosis Induced by Various Stimuli In Vitro

Abstract

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.

Apoptotic cell death represents ordered biochemical events, which are initiated in response to disparate stimuli and proceed differently in diverse cell types. Apoptosis has been implicated in the death of retinal cells during both retinal differentiation and degeneration. ^{1 2} In several diseases such as glaucoma, age-related macular degeneration, and diabetic retinopathy, which comprise the leading causes of blindness worldwide, ^{3 4 5 6 7} retinal cell death is thought to be preceded by an apoptotic mechanism. In addition, photoreceptor degeneration in animal models of retinitis pigmentosa occurs through apoptosis. ^{8 9 10} 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 the caspase activation that mediates apoptotic retinal cell death. For example, upregulation of a CPP32-like protease was observed in the retina after optic nerve transsection, and this was shown to be prevented by caspase inhibitor treatment ¹¹ ; however, the relevance of an axotomy model to clinical disease is limited. 

We therefore sought to study caspase activation in retinal cells in response to various stimuli that are thought to be relevant to numerous neuroretinal disorders. In glaucomatous optic neuropathy, for example, retinal ganglion cell apoptosis has been identified in experimental glaucoma models ^{12 13} and postmortem studies of human eyes with glaucoma. ^{14 15} Multiple causative factors thought to be associated with glaucomatous optic neuropathy, which might initiate apoptotic cell death, include chronic retinal ischemia ¹⁶ and excitotoxicity. ^{17 18} There also is considerable evidence which suggests that an aberrant autoimmunity may contribute to optic neuropathy in glaucoma, ^{19 20} and some patients with glaucoma have elevated titers of serum antibodies to heat shock proteins. ²¹ Furthermore, antibodies against small heat shock proteins, such as heat shock protein 27 (hsp27), can trigger cell death in retinal cells, in vitro and ex vivo, through an apoptotic mechanism. ²²  

We studied the apoptotic signaling cascade in cultured retinal cells exposed to several apoptotic stimuli, including simulated ischemia, excitotoxicity, and hsp27 antibody. The function of these stimuli in the apoptotic process was examined by studying the activation of caspases and alterations in the bcl-2 family of proteins in cultured retinal cells. Here, we present in vitro evidence that the apoptotic process in retinal cells induced by different stimuli share a common mechanism involving caspase-3 and poly-(ADP ribose) polymerase (PARP) cleavage, but differ in their ability to activate caspase-8, which only occurred during apoptosis induced by hsp27 antibody. Furthermore, treatment with caspase inhibitors effectively blocks the apoptotic process in retinal cells exposed to different apoptotic stimuli. 

Materials and Methods

Cell Culture

We used an immortalized rat retinal cell line (E1A.NR3) that contains cells expressing antigens specific for photoreceptors, bipolar cells, ganglion cells, and retinal glial cells, ²³ which has been used successfully in several previous studies. ^{22 24} The retinal cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% each of nonessential amino acids, l-glutamine, vitamins, and antibiotics. ²³ All tissue culture reagents were purchased from Gibco (Grand Island, NY). 

Experimental Design

The retinal cells were plated either on 75-cm² cell culture flasks (Costar, Cambridge, MA) at a density of 20 × 10⁴ cells/flask at a final volume of 12 ml or on 6-well plates (Costar) at a density of 3 × 10⁴ cells/well at a final volume of 2 ml/well. Cells grown to approximate confluence then were incubated in different conditions for 24 hours. For simulated ischemia, cells were exposed to reduced oxygen tensions in medium lacking glucose. Hypoxia was maintained by placing the cultures in an airtight perfusion chamber with a controlled flow of 95% N₂/5% CO₂. To examine the effects of excitotoxins, cells were incubated in the presence of glutamate receptor agonists, including N-methyl-d-aspartate (NMDA, 100 μM) and a non-NMDA subtype,α -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA, 100μ M) (Sigma, St. Louis, MO). To examine the effects of antibody to hsp27, cells were similarly incubated in the presence of monoclonal antibody against hsp27 (100 μg/ml; Stress Gen, Victoria, BC, Canada). These are optimum conditions to induce apoptosis in E1A.NR3 cell line, based on previous concentration-response experiments. ²²  

To examine the caspase cascade, retinal cells also were incubated under several stress conditions in the presence or absence of different concentrations of caspase inhibitors. One of the inhibitors we used, boc-aspartyl(Ome)-fluoromethylketone (BAF, B-D-FMK; Enzyme System Products, Livermore, CA) is a cell-permeable, nonselective inhibitor of caspases. It has been shown to inhibit caspase-3 and PARP cleavage as well as Fas-mediated death in a dose-dependent manner and thus prevents neuronal death in experimental systems. ^{25 26 27 28 29 30} In addition, we used the caspase inhibitor CBZ-Ile-Glu(Ome)-Thr-Asp-(Ome)-fluoromethylketone (Z-IETD-FMK; Enzyme System Products), which is a cell-permeable, selective inhibitor of caspase-8. ³¹ Control cells were maintained in a medium in the absence of drugs or antibody to hsp27 and incubated in a tissue culture incubator with humidified atmosphere of 5% CO₂ and 95% air at 37°C. 

After incubation, the cells were washed with phosphate-buffered saline solution and labeled using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique ³² for morphologic analysis of apoptosis and quantification of apoptotic cells. In addition, the cells were used in western blot analysis and a protease activity assay. The experiments described below were repeated three times for each condition. 

Morphologic Analysis of Apoptosis

An in situ cell death detection kit (Boehringer Mannheim, Mannheim, Germany) was used to identify the apoptotic cells by TUNEL technique according to the instructions of the manufacturer. Briefly, after fixation, permeabilization, and blocking steps, air-dried cells were incubated with a mixture of fluorescein-labeled nucleotides and terminal deoxynucleotidyl transferase for 1 hour. Terminal deoxynucleotidyl transferase catalyzes the polymerization of labeled nucleotides to free 3′-OH terminals of DNA fragments. Cells incubated with fluorescein-labeled nucleotide mixture without the presence of terminal deoxynucleotidyl transferase served as a negative control. Cells previously treated with DNase I (1 mg/ml) to induce breaks in the DNA strands served as a positive control. 

For quantitative analysis of apoptosis, TUNEL-positive cells were counted in triplicate wells under fluorescence microscope (Olympus, Tokyo, Japan). The percentage of apoptosis was calculated using the total number of cells in these wells determined using a Coulter counter after trypsinization of the cells. 

Western Blot Analysis

After washing the cells with phosphate-buffered saline, they were lysed in sample buffer (1% SDS, 100 mM dithiothreitol (DTT), 60 mM Tris, pH 6.8, 0.001% bromophenol blue). Protein concentrations were determined using the BCA method (Sigma). The samples were boiled for 5 minutes before subjecting them to electrophoresis. 

Samples (50 μg of total protein) were separated by electrophoresis in 10% to 15% sodium dodecyl sulfate polyacrylamide gels at 160 V for 1 hour and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, Marlboro, MA) using a semi-dry transfer system (BioRad, Hercules, CA). After transfer, membranes were blocked in a buffer (50 mM Tris-HCl, 154 mM NaCl, 0.1% Tween-20, pH 7.5) containing 5% nonfat dry milk for 1 hour and then overnight in the same buffer containing a dilution of primary antibody and sodium azide. Primary antibodies were monoclonal antibodies to caspase-8 or PARP or polyclonal antibodies to caspase-3, bcl-2, or bax (Pharmingen, San Diego, CA) and were used at a dilution of 1:1000. After several washes and the second blocking for 20 minutes, the membranes were incubated with a dilution of secondary antibodies conjugated with horseradish peroxidase (Fisher Scientific, Pittsburgh, PA) at 1:2000 for 1 hour. Immunoreactive bands were visualized by enhanced chemiluminescence using commercial reagents (Amersham Life Science, Arlington Heights, IL). 

In Vitro Caspase-3 Assay

Caspase-3–like (YAMA/CPP32) protease activity was measured in a fluorometric assay by measuring the extent of cleavage of the fluorometric peptide substrate as previously described. ^{30 33} Briefly, the cells were washed with phosphate-buffered saline and lysed in buffer A (10 mM Hepes, pH 7.4, 42 mM KCl, 5 mM MgCl₂, 1 mM DTT, 0.5% CHAPS, 1 mM PMSF, and 1 μg/ml leupeptin). Lysate was then combined in a 96-well plate with buffer B (25 mM Hepes, pH 7.5, 1 mM EDTA, 3 mM DTT, 0.1% CHAPS, and 10% sucrose) containing Ac-Asp-Glu-Val-Asp-7-amino-4-trifluoro-methyl coumarin (Ac-DEVD-AMC) fluorometric substrate (50 μM). Positive controls included purified recombinant caspase-3 (0.1 μg; Upstate Biotechnology, Lake Placid, NY). Fluorescence was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm in a fluorescent plate reader at different time points up to 180 minutes. The protease activity was expressed as picomoles of substrate per milligram of protein per minute, as calculated using the linear range of the assay. 

Results

Induction and Inhibition of Retinal Cell Apoptosis

Apoptosis was induced in cultured retinal cells after incubation in the presence of simulated ischemia, excitotoxins, or hsp27 antibody for 24 hours. Although control cells incubated under normal condition retained normal morphology, retinal cells incubated under different stress conditions exhibited specific morphologic changes of apoptotic cell death, including cell body shrinkage and compaction of the nucleus. In addition, apoptotic cells exhibited bright labeling of fragmented nuclear DNA by the TUNEL technique (Fig. 1) . Quantitative analysis of retinal cells revealed that 15% to 30% of the cells were TUNEL-positive in cultures incubated in the presence of different stimuli. After incubation under identical stress conditions in the presence of the caspase inhibitor, B-D-FMK (50 μM), the apoptosis rate decreased to 4% to 5% of the cell population, which was similar to the apoptosis rate in control cells (3% to 4%) (Fig. 2) . 

Involvement of Caspases in Retinal Cell Apoptosis and Inhibition of Caspase Activity by Caspase Inhibitors

To examine the role of the caspase cascade in retinal apoptosis induced by different stimuli, lysates of retinal cells incubated in the presence of simulated ischemia, excitotoxins, or hsp27 antibody were used in western blot analysis. Western blot analysis demonstrated cleavage of caspase-3 and PARP in retinal cells incubated under all the stress conditions studied. The presence of caspase-3 activation was assessed by the observation of the 17-kDa subunit that was derived from the cleavage of 32-kDa pro-enzyme caspase-3. PARP was cleaved from its 116-kDa form to an 85-kDa residual fragment, characteristic of cells in the process of apoptosis. However, among different stimuli studied, only incubation in the presence of antibody against hsp27 caused cleavage and hence activation of caspase-8. Western blot analysis revealed a 55-kDa immunoreactive band corresponding to caspase-8 and approximately 30- and 20-kDa cleaved products using the lysates of retinal cells incubated with hsp27 antibody. No cleavage of caspase-8, caspase-3, or PARP was detected using the extracts of the control retinal cells maintained in a medium in the absence of drugs or antibody to hsp27 and incubated in a tissue culture incubator with humidified atmosphere of 5% CO₂ and 95% air at 37°C (Fig. 3) . 

In addition to caspases, we also examined the bcl-2 family of proteins, which modulate caspase activation. Western blot analysis revealed the contribution of the members of bcl-2 family to retinal cell apoptosis induced by different stimuli, including simulated ischemia, excitotoxins, and hsp27 antibody. In retinal cells going to apoptosis regardless of the stimuli used to induce apoptotic cell death, bcl-2 expression was lower and bax expression was higher in comparison to control retinal cells (Fig. 4) . 

To examine the effect of caspase inhibitors on caspase cleavage, retinal cells were incubated under stress conditions in the presence of caspase inhibitors. Western blot analysis revealed that treatment with caspase inhibitors prevented the cleavage of caspases and PARP in retinal cells incubated under different stress conditions, including simulated ischemia, excitotoxins, and hsp27 antibody. The nonselective caspase inhibitor BAF (B-D-FMK) (50 μM) inhibited caspase-3 and PARP cleavage but not prominent caspase-8 cleavage. Z-IETD-FMK (20 μM), a selective caspase-8 inhibitor, inhibited the cleavage of caspase-8 and only partially inhibited the cleavage of caspase-3 and PARP. This observation suggests the presence of an additional pathway during the apoptosis induced by hsp27 antibody, which includes caspase-8 activation (Fig. 5) . 

We then performed fluorometric analysis using retinal cells incubated under different stress conditions to measure the cleavage of Ac-DEVD-AMC, which reflects caspase-3–like activity. In accordance with the results of western blot analysis, the amount of DEVD-AMC cleaving activity was increased in retinal cells incubated under different apoptotic stimuli (range, 11.0–24.8 pmol/mg protein/min) compared to control cells (range, 3–4.6 pmol/mg protein/min). Approximately 2.1 to 3.4 times increase in the DEVD-AMC cleaving activity in retinal cells incubated in the presence of different stimuli is shown in Figure 6 . Treatment of retinal cells with caspase inhibitors inhibited caspase-3–like activity in a concentration-dependent manner (Fig. 7) . The caspase-3–like activity in retinal cells was reduced by ∼ 70% with 50 μM of B-D-FMK (range, 4.3–7.2 pmol/mg protein/min). However, treatment of retinal cells with 20 μM of the selective inhibitor of caspase-8, Z-IETD-FMK, resulted in decreased caspase-3–like activity only in retinal cells incubated with hsp27 antibody (∼40%). This observation suggests that hsp27 antibody-mediated retinal cell apoptosis involves caspase-8 activation (Figs. 6 7) . 

Discussion

A variety of death commitment signals in mammalian cells converge to activate the executioner proteolysis cascade that play a critical role in initiating and sustaining the biochemical events that result in apoptotic cell death. The proteolysis is mediated by a conserved group of caspases that are related to mammalian interleukin 1β–converting enzyme and to nematode CED-3. ^{34 35} Caspases exist as proenzymes that are proteolytically processed to their active forms in response to an apoptosis-inducing stimulus. Activated caspases cleave each other’s precursors into mature, active enzymes in a proteolytic cascade similar to complement activation or blood clotting. For example, after cell-surface death receptors such as tumor necrosis factor-α-1 or the CD95/Fas are activated, their cytoplasmic tails bind to downstream adapter proteins, such as FADD (Fas-associated death domain protein). The Fas–FADD complex then binds a receptor-interacting protein (FLICE) that contains an interleukin 1β–converting enzyme–related protease domain (caspase-8) and can initiate the caspase cascade directly. ^{36 37 38} Although this pathway of caspase cascade eventually induces caspase-3 activation, caspase-3 also can be activated by other upstream mediators, which are unrelated to caspase-8. As a consequence, PARP, the major substrate of caspase-3, is cleaved, which catalyzes the poly(ADP-ribosyl)ation of various nuclear proteins with NAD as substrate and contributes to cell death by depleting the cell of NAD and ATP. ^{39 40} In addition, caspases contribute to apoptosis through direct disassembly of cell structures, including nuclear lamina and cytoskeleton. ⁴¹  

Our in vitro observations revealed that the apoptotic machinery in retinal cells is regulated, in part by a proteolytic cascade. Although all the stimuli studied, including simulated ischemia, excitotoxins, and hsp27 antibody, eventually caused the cleavage of caspase-3 and PARP, caspase-8 was cleaved only during apoptosis induced by hsp27 antibody. This observation suggests a different upstream signaling pathway for hsp27 antibody–mediated apoptosis, despite similar downstream effector events to those induced by other stimuli. 

We also observed that treatment of retinal cells with compounds capable of inhibiting multiple proteins in the caspase family can block the downstream apoptosis. One of the inhibitors we used, B-D-FMK, is a reactive derivative of aspartic acid, the amino acid locus that caspases cleave distally. Because of its membrane permeability, it is effective in living cells, and it has been shown to inhibit caspase-3 and PARP cleavage as well as Fas-mediated death in a dose-dependent manner and thereby to prevent neuronal death. ^{25 26 27 28 29 30} In accordance with these findings, our studies revealed that B-D-FMK and in part, Z-IETD-FMK, a specific inhibitor of caspase-8, ³¹ can prevent the execution of apoptosis in retinal cells exposed to different apoptotic stimuli. 

A recent study of two Drosophila mutant strains that exhibit an age-related retinal degeneration and a human homologue of retinitis pigmentosa has revealed that cells rescued from apoptosis can serve a useful function. ⁴² Preservation of visual function by blockade of apoptosis provides a strong rationale for further exploration of antiapoptotic strategies in the treatment of retinal degenerative diseases. As seen in glaucomatous optic neuropathy retinal apoptosis can be triggered by a broad array of different stimuli; therefore, caspases may be attractive targets to block apoptosis, regardless of the causative event. 

Another intracellular pathway, which modulates cell survival by affecting adapters needed for the activation of caspases, involves bcl-2 family members. ^{43 44} The bcl-2 family of proteins includes both apoptosis-promoting (e.g., bax and bad) and apoptosis-inhibiting (e.g., bcl-2 and bcl-xL) members. ^{45 46 47 48} This is consistent with our observations that during the retinal cell apoptosis induced by all three stimuli, bcl-2 expression decreased and bax expression increased. 

Previous studies of neuronal tissues have shown the contribution of the caspases and/or bcl-2 pathway to the apoptosis induced by different stimuli we examined in retinal cells. For example, the contribution of the caspases in the apoptotic component of ischemia-induced neuronal death has been demonstrated. ^{30 49 50 51 52 53} In addition, glutamate-induced apoptosis of cerebellar granule neurons has been shown to be mediated by a posttranslational activation of caspase-3. ⁵⁴ Moreover, bax promotes apoptotic cell death in neuronal ischemia; bax expression is increased in neurons that die, and the upregulation of bcl-2 expression in surviving neurons protects these neurons from cell death. ^{55 56}  

Cells respond to a variety of stress conditions, including ischemia and excitotoxicity, by adaptive changes that either blunt the death signal or prevent the activation of sensor or effector molecules and thereby limit and/or repair cell damage. One of the components of these cellular response mechanisms is the upregulation of the expression of heat shock proteins. Heat shock proteins, including hsp27, increase cell survival and resistance to apoptosis by affecting both upstream signaling and downstream effector events as shown in neuronal cells as well as in cancer cell lines. ^{57 58 59} Considerable evidence suggests that antibodies against heat shock proteins may confer a loss of protective role of endogenous heat shock proteins and thereby facilitate apoptotic cell death. For example, antibodies that bind heat shock proteins can increase the rate of cell death after certain noxious insults. ⁶⁰ In accordance with these observations, the presence of antibodies to hsp27 recently has been found to correlate with an improved survival in patients with breast cancer. ⁶¹ In addition to the previous observation of the role of antibodies against small heat shock proteins to induce apoptosis in retinal cells, ²² current findings reveal the role of caspase cascade and bcl-2 pathway in the retinal cell apoptosis induced by hsp27 antibody. 

In conclusion, our in vitro observations provide evidence that apoptosis in retinal cells induced by simulated ischemia, excitotoxins, or hsp27 antibody shares a common executioner proteolysis cascade, including caspase-3 and PARP cleavage, but differ in their ability to activate caspase-8, which occurs during apoptosis induced by hsp27 antibody. In addition, during the apoptotic process in retinal cells expression of bcl-2 decreased and expression of bax increased regardless of the cell stressor used to induce apoptosis. An improved understanding the apoptotic process at molecular level can facilitate efforts to rationally manipulate apoptosis for therapeutic gain. Our observations of the protective role of caspase inhibitors during retinal cell apoptosis induced by different stimuli may therefore have some therapeutic implications in future studies. Such a treatment modality may provide a means to rescue and protect retinal neuronal cells from apoptotic cell death induced by multiple causes of injury and thus be useful in the treatment of many neuroretinal diseases, including glaucomatous optic neuropathy. Further evaluation of caspase-independent mechanisms of cell death, as well as the function of cells rescued by caspase inhibitors, will determine the potential of caspase inhibitors for the treatment of retinal cell death. 

 Supported in part by Grant EY12314 (MBW) from National Eye Institute, Bethesda, Maryland, and an unrestricted grant to Washington University School of Medicine, Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness Inc., New York, New York.

 Submitted for publication January 22, 1999; revised June 1, 1999; accepted June 22, 1999.

 Commercial relationships policy: N.

 

Figure 1.

 

Morphologic analysis of apoptotic cell death in retinal cells using TUNEL technique. (A) Phase contrast microscope image of retinal cells incubated under normal condition; (B) phase contrast microscope image of retinal cells incubated under ischemic condition; (C) fluorescence microscope image of control retinal cells stained using TUNEL technique; (D) fluorescence microscope image of retinal cells incubated under ischemic condition and stained using TUNEL technique. Arrows show cells exhibiting positive TUNEL. Original magnification, ×40.

Figure 2.

 

Quantitative analysis of apoptotic cell death in retinal cells using TUNEL technique. The percentage of TUNEL-positive cells incubated under different apoptotic stimuli (white columns) decreased to levels similar to control after incubation in the presence of caspase inhibitor (B-D-FMK) (50 μM) (black columns). Error bars, SD. NMDA, N-methyl-d-aspartate; AMPA,α -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid.

Figure 3.

 

Western blot findings demonstrating caspase and PARP cleavage. Column 1, control retinal cells; column 2, retinal cells incubated under ischemic condition; column 3, retinal cells incubated with NMDA; column 4, retinal cells incubated with AMPA; column 5, retinal cells incubated with antibody against hsp27. Fifty-five–kilodalton immunoreactive band corresponding to caspase-8 and its 30- and 20-kDa cleaved products were observed only using the lysates of retinal cells incubated with hsp27 antibody. However, the cleavage of caspase-3 and PARP was observed using the extracts of retinal cells incubated under all the stress conditions studied, including the presence of ischemia, excitotoxins, and hsp27 antibody. The presence of caspase-3 activation was assessed by the observation of a 17-kDa subunit that was derived from the cleavage of 32-kDa proenzyme caspase-3. PARP was cleaved from its 116-kDa form to an 85-kDa residual fragment characteristic of cells in the process of apoptosis. No cleavage of caspase-8, caspase-3, or PARP was detected using the extracts of the control retinal cells.

Figure 4.

 

Western blot findings demonstrating alterations in the bcl-2 family of proteins. In comparison to the control cells bcl-2 expression was lower and bax expression was higher in retinal cells incubated under different stress conditions that induced apoptotic cell death (column 1, control retinal cells; column 2, retinal cells incubated under ischemic condition; column 3, retinal cells incubated with NMDA; column 4, retinal cells incubated with AMPA; column 5, retinal cells incubated with antibody against hsp27).

Figure 5.

 

Western blot findings using retinal cells treated by caspase inhibitors. Incubation of the retinal cells under different stress conditions in the presence of drugs inhibiting caspases prevented specific caspase cleavage. Column 1, control retinal cells; column 2, retinal cells incubated with antibody against hsp27; column 3, retinal cells incubated with antibody against hsp27 in the presence of caspase inhibitor, B-D-FMK (50 μM); column 4, retinal cells incubated with antibody against hsp27 in the presence of specific caspase-8 inhibitor, Z-IETD-FMK (20 μM). While B-D-FMK inhibited caspase-3 and PARP cleavage but not prominently caspase-8 cleavage, Z-IETD-FMK inhibited the cleavage of caspase-8 and only partially the cleavage of caspase-3 and PARP.

Figure 6.

 

In vitro protease activity assay. Caspase-3–like activity was increased in retinal cells incubated under different stress conditions compared to control cells (white columns) and this increase was blocked by caspase inhibitor, B-D-FMK (50 μM) (black columns). However, treatment with specific caspase-8 inhibitor, Z-IETD-FMK (20 μM) (gray columns) was only effective to decrease caspase-3–like activity in retinal cells incubated with hsp27 antibody. Error bars, SD. NMDA, N-methyl-d-aspartate; AMPA,α -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid.

Figure 7.

 

In vitro protease activity assay. Caspase-3–like activity was blocked in retinal cells incubated with hsp27 antibody by a nonselective caspase inhibitor, B-D-FMK (black circles) and a specific caspase-8 inhibitor, Z-IETD-FMK (white circles) in a concentration-dependent manner. Error bars, SD.

The authors thank Gail M. Seigel for providing E1A.NR3 rat retinal cell line and Eugene M. Johnson, Jr. for his helpful advice.