27 Effects of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone on frozen - thawed bovine sperm

Semen cryopreservation is critical for appropriate planning of both AI and IVF trials, improving the benefit:cost ratio. However, cryopreservation induces damage in mammalian spermatozoa, resulting in decreased fertility (Medeiros et al. 2002 Theriogenology 57, 327-344). It is known that cryopreservation and thawing induce apoptosis in a variety of cells, including bovine sperm (Anzar et al. 2002 Biol. Reprod. 66, 354-360). Cryotolerance of in vitro-produced bovine embryos was recently improved by inhibiting apoptosis using a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD-FMK), during vitrification and subsequent culture (Pero et al. 2018 Theriogenology 108, 127-135). The aim of this work was to evaluate whether treatment of bovine frozen-thawed sperm with the caspase inhibitor Z-VAD-FMK may prevent aberrant apoptosis and consequently improve sperm viability. Six ejaculates from 12 bulls were used for the trial. Semen was diluted at 37°C with BioXcell extender (BioXcell, West Lebanon, NH) to a final concentration of 30×106 spermatozoa mL−1, and straws were kept at 4°C for 4h and then frozen in an automated system. After thawing, Percoll-separated spermatozoa were incubated at 37°C for 1h with 0, 20, and 100µM ZVAD-FMK. Sperm viability and membrane integrity were assessed by Trypan Blue/Giemsa and hypo-osmotic swelling test, respectively, as previously described (Longobardi et al. 2017 Theriogenology 88, 1-8). Sperm motility was examined by phase contrast microscopy at 40× magnification on a thermoregulated stage at 37°C. Apoptosis was evaluated by TUNEL technique, which assesses DNA fragmentation (Takeda et al. 201561, 185-190). The mitochondrial membrane potential was then assessed by flow cytometric analysis with the mitochondrial probe JC-1 (Garner and Thomas 1999Mol. Reprod. Dev. 53, 222-229). Data were analysed by ANOVA using least significant difference as post-hoc test. The treatment of bovine frozen-thawed sperm with 100µM ZVAD-FMK decreased the percentage of sperm exhibiting DNA fragmentation (17.8±1.1, 13.3±2.8, and 10.5±2.5 with 0, 20, and 100µM ZVAD, respectively; P<0.05). Moreover, both concentrations of ZVAD-FMK increased the percentage of hypo-osmotic swelling test+ sperm, indicating improved membrane integrity compared with the control (60.5±3.5, 70.9±2.1, and 74.3±2.1 with 0, 20, and 100µM ZVAD-FMK, respectively; P<0.01). However, no differences were found in sperm viability (82.3±0.5, 84.6±1.0, and 84.3±2.1 with 0, 20, and 100µM ZVAD-FMK, respectively) and motility (60.0±2.9, 62.5±3.4, and 67.5±2.1 with 0, 20, and 100µM ZVAD-FMK, respectively). Furthermore, no differences were observed among groups in the percentage of sperm exhibiting normal mitochondrial membrane potential (62.4±12.7, 57.9±12.8, and 50.8±8.8 with 0, 20, and 100µM ZVAD, respectively). In conclusion, caspase inhibition with 100µM ZVAD-FMK after thawing was effective in reducing sperm DNA fragmentation and increasing sperm membrane integrity, suggesting a beneficial effect on fertility. However, as the other fertility-related parameters did not improve, further investigations are required to draw definite conclusions.

Caspase Inhibition after Neonatal Ischemia in the Rat Brain

Caspase-3 has been identified as a key protease in the execution of apoptosis and appears to be an important downstream event after hypoxia-ischemia in the immature brain. The efficacy of a pan-caspase inhibitor, boc-aspartyl-(Ome)-fluoromethyl-ketone (BAF), was tested in a model of unilateral focal ischemia with reperfusion in 7-day-old rats. The BAF inhibitor was given intraperitoneally 5 minutes before reperfusion via the carotid artery. This procedure reduced the activity of caspase-3 by 79% but did not induce a significant reduction in infarct volume (23.8 ± 7.5% versus 30.1 ± 6.4%). Animals were distributed in two populations. One population exhibited an infarct, whereas the other appeared to be fully protected. BAF-treated animals exhibiting an infarct mostly displayed necrotic cell death, whereas apoptotic nuclei were observed in untreated or vehicle-treated animals. Repeated dose of BAF (5 minutes before and 9 hours after reperfusion) did not also provide benefit after neonatal ischemia, although a general trend to reduce lesion was observed (20.5 ± 3.7% versus 34.4 ± 5.9%). These findings raise critical questions about the use of peptide ketone apoptotic inhibitors in improving histopathologic outcomes after neonatal stroke.

Reperfusion, not simulated ischemia, initiates intrinsic apoptosis injury in chick cardiomyocytes

Although ischemia-reperfusion (I/R) can initiate apoptosis, the timing and contribution of the mitochondrial/cytochrome c apoptosis death pathway to I/R injury is unclear. We studied the timing of cytochrome c release during I/R and whether subsequent caspase activation contributes to reperfusion injury in confluent chick cardiomyocytes. One-hour simulated ischemia followed by 3-h reperfusion resulted in significant cell death, with most cell death evident during the reperfusion phase and demonstrating mitochondrial cytochrome c release within 5 min after reperfusion. By contrast, cells exposed to prolonged ischemia for 4 h had only marginally increased cell death and no detectable cytochrome c release into the cytosol. Caspase activation could not be detected after ischemia only, but it significantly increased after reperfusion. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, Ac-Asp-Gln-Thr-Asp-H, or benzyloxycarbonyl-Leu-Glu (Ome)-His-Asp-(Ome)-fluoromethyl ketone given only at reperfusion significantly attenuated cell death and resulted in return of contraction. Antixoxidants decreased cytochrome c release, nuclear condensation, and cell death. These results suggest that reperfusion oxidants initiate cytochrome c release within minutes, and apoptosis within hours, significant enough to increase cell death and contractile dysfunction.

Blockade of Caspases Inhibits Amebic Liver Abscess Formation in a Mouse Model of Disease

ABSTRACT We looked at the effect of inhibiting caspases on amebic liver abscess in the mouse model of infection. A dose of the pan-caspase inhibitor benzyloxycarbonyl-V-A-D-O-methyl fluoromethyl ketone (Z-VAD-FMK; R & D Systems) given to SCID mice 2 h prior to direct hepatic inoculation with Entamoeba histolyticatrophozoites, and 12 h after amebic inoculation, reduced the mean liver abscess size by 70% at 24 h compared to a control group. These data indicate that apoptosis plays a significant but not an exclusive role in amebic liver abscess formation in the mouse model.

Caspase-3 Activity and Carbonyl Cyanide m-Chlorophenylhydrazone-induced Apoptosis in HL-60 cells

The role of caspase proteases in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced apoptosis of human promyelocytic HL-60 cells was examined. Treatment of HL-60 cells with micromolar concentrations of CCCP resulted in cell death, with typical apoptotic features such as chromatin condensation, formation of apoptotic bodies, nucleosomal fragmentation of DNA and a distinct increase in caspase-3 activity. The results, however, indicated that full caspase-3 inhibition by the selective inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone (Z-DEVD-FMK) did not prevent cell death, nor did it affect the manifestation of apoptotic hallmarks, including apoptotic bodies formation and nucleosomal DNA fragmentation. The only distinct effect that Z-DEVD-FMK exhibited was to retard the disruption of the plasma membrane. We therefore assume that caspase-3 activity itself is not essential for the manifestation of apoptotic features mentioned above. Similarly, the pan-specific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not prevent cell death. On the contrary, Z-VAD-FMK completely prevented DNA cleavage and apoptotic body formation, but it failed to completely counteract chromatin condensation. Thus, in the presence of Z-VAD-FMK, application of CCCP concentrations that otherwise induced apoptosis, resulted in the appearance of two morphologically different groups of dead cells with intact DNA. The first group included cells with necrotic-like nuclear morphology, and therefore could be taken as being “truly” necrotic in nature, because they had intact DNA. The cells of the second group formed small single-spherical nuclei with condensed chromatin. In spite of having intact DNA, they could not be taken as “truly” necrotic cells. It is evident that in the experimental system, caspase proteases play an essential role in the formation of apoptotic bodies and in the cleavage of nucleosomal DNA, but not in the condensation of chromatin. Therefore, it is likely that the choice between cell death modalities is not solely a matter of the caspase proteases present.

Peroxynitrite-induced apoptosis involves activation of multiple caspases in HL-60 cells

In this study, we show that caspases 2, 3, 6, and 7 were activated during peroxynitrite-induced apoptosis in human leukemia HL-60 cells and that processing of these caspases was accompanied by cleavage of poly(ADP-ribose) polymerase and lamin B. Treatment of cells with DEVD-fluoromethyl ketone (FMK), a selective inhibitor for caspase 3-like proteases, resulted in a marked diminution of apoptotic cells. VAVAD-FMK, an inhibitor of caspase 2, partially inhibited the apoptotic response to peroxynitrite. However, selective inactivation of caspase 6 by VEID-FMK did not affect apoptosis rates. These data suggest that caspase 3-like proteases and caspase 2, but not caspase 6, are required for peroxynitrite-induced apoptosis in this cell type. Moreover, we demonstrate that peroxynitrite treatment stimulated activation of caspases 8 and 9, two initial caspases in the apoptotic signaling pathway, and preincubation of cells with their inhibitor, IETD-FMK, inhibited activation of caspase 3-like proteases and caspase 2 at the concentration that prevents the apoptosis. These observations, together, suggest that caspase 8 and/or caspase 9 mediates activation of caspase 3-like proteases and caspase 2 during the apoptosis induced by peroxynitrite in HL-60 cells.

Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-Permeable Fluorogenic Caspase Substrates

To detect caspase activities in intact apoptotic cells at the single cell level, cell-permeable fluorogenic caspase substrates were synthesized incorporating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitro treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. Dexamethasone induced the following order of appearance of caspase activities as judged by flow cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamined using confocal microscopy. The VEIDase activity appeared before DEVDase in every apoptotic cell treated with dexamethasone. In contrast, anti-Fas stimulation altered this sequence: IETDase was the first measurable caspase activity and DEVDase preceded VEIDase. In an attempt to determine the intracellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl-aspartyl(β-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examined its ability to inhibit previously activated intracellular caspases. However, no significant reductions of these activities were observed. These fluorogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase activation is dependent on the apoptotic stimulus.