Effective Prevention of Arterial Thrombosis with Albumin-Thrombin Inhibitor Conjugates.

Thromboprophylaxis is indicated in patients at an elevated risk of developing thrombotic disorders, typically using direct oral anticoagulants or low-molecular-weight heparins. We postulated that transient thromboprophylaxis (days-weeks) could be provided by a single dose of an anticoagulant engineered for prolonged pharmacokinetics. In the present work, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (PPACK) was used as a model anticoagulant to test the hypothesis that conjugation of thrombin inhibitors to the surface of albumin would provide durable protection against thrombotic insults. Covalent conjugates were formed between albumin and PPACK using click chemistry, and they were tested in vitro using a thrombin activity assay and a clot formation assay. Thromboprophylactic efficacy was tested in mouse models of arterial thrombosis, both chemically induced (FeCl3) and following ischemia-reperfusion (transient middle cerebral artery occlusion; tMCAO). Albumin-PPACK conjugates were shown to have nanomolar potency in both in vitro assays, and following intravenous injection had prolonged circulation. Conjugates did not impact hemostasis (tail clipping) or systemic coagulation parameters in normal mice. Intravenous injection of conjugates prior to FeCl3-induced thrombosis provided significant protection against occlusion of the middle cerebral and common carotid arteries, and injection immediately following ischemia-reperfusion reduced stroke volume measured 3 days after injury by ∼40% in the tMCAO model. The data presented here provide support for the use of albumin-linked anticoagulants as an injectable, long-circulating, safe thromboprophylactic agent. In particular, albumin-PPACK provides significant protection against thrombosis induced by multiple mechanisms, without adversely affecting hemostasis.

Gasdermin E-derived caspase-3 inhibitors effectively protect mice from acute hepatic failure.

Programmed cell death (PCD), including apoptosis, apoptotic necrosis, and pyroptosis, is involved in various organ dysfunction syndromes. Recent studies have revealed that a substrate of caspase-3, gasdermin E (GSDME), functions as an effector for pyroptosis; however, few inhibitors have been reported to prevent pyroptosis mediated by GSDME. Here, we developed a class of GSDME-derived inhibitors containing the core structure of DMPD or DMLD. Ac-DMPD-CMK and Ac-DMLD-CMK could directly bind to the catalytic domains of caspase-3 and specifically inhibit caspase-3 activity, exhibiting a lower IC50 than that of Z-DEVD-FMK. Functionally, Ac-DMPD/DMLD-CMK substantially inhibited both GSDME and PARP cleavage by caspase-3, preventing apoptotic and pyroptotic events in hepatocytes and macrophages. Furthermore, in a mouse model of bile duct ligation that mimics intrahepatic cholestasis-related acute hepatic failure, Ac-DMPD/DMLD-CMK significantly alleviated liver injury. Together, this study not only identified two specific inhibitors of caspase-3 for investigating PCD but also, more importantly, shed light on novel lead compounds for treating liver failure and organ dysfunctions caused by PCD.

The Effectiveness of Anti-Apoptotic Agents to Preserve Primordial Follicles and Prevent Tissue Damage during Ovarian Tissue Cryopreservation and Xenotransplantation.

The purpose of this study is to investigate the effectiveness of sphingosine-1-phosphate (S1P) and Z-VAD-FMK (Z-VAD) as anti-apoptotic agents to preserve ovarian function and prevent tissue damage during ovarian tissue cryopreservation and transplantation. This study consisted of two steps, in vitro and in vivo. In the first step, human ovarian tissues were cryopreserved using slow-freezing media alone, S1P, or Z-VAD (control, S1P, Z-VAD group); based on the outcomes in these groups, Z-VAD was selected for subsequent xenotransplantation. In the second step, human frozen/thawed ovarian tissues were grafted into fifty mice divided into three groups: slow-freezing/thawing and transplantation without an anti-apoptotic agent (Trans-control) and xenotransplantation with or without Z-VAD injection (Trans-Z-VAD-positive and Trams-Z-VAD-negative groups, respectively). In the first step, the Z-VAD group had a significantly higher primordial follicular count than the S1P (p = 0.005) and control groups (p = 0.04). Transplanted ovarian tissues were obtained 4 weeks after transplantation (second step). Angiogenesis was significantly increased in the Z-VAD-negative (p = 0.03) and -positive (p = 0.04) groups compared to the control group. This study demonstrated that slow-freezing and transplantation with Z-VAD is an effective method for preserving primordial follicle counts, decreasing double-strand DNA breaks, and increasing angiogenesis in a mouse model. Further molecular and clinical studies are needed to confirm these results.

Inhibition of Synovial Macrophage Pyroptosis Alleviates Synovitis and Fibrosis in Knee Osteoarthritis.

Increasing evidence has shown that macrophage pyroptosis in different tissues participates in chronic aseptic inflammation and is related to tissue fibrosis. Our last studies also revealed the vital role of synovial fibroblast pyroptosis in the onset and development of knee osteoarthritis (KOA). In this study, we aimed to investigate whether synovial macrophage pyroptosis did occur and whether this form of cell death should be related to synovitis and fibrosis of KOA. In the synovial tissue of KOA model rats, we observed a decrease of caspase1, NLRP3, ASC, and GSDMD caused by macrophage depletion in both the mRNA and protein expressions. Besides, rats treated with the specific caspase1 inhibitor Ac-YVAD-CMK showed less inflammatory reaction and fibrosis, not only in the expression of proinflammatory factors IL-1ß, IL-18, and HMGB1 and fibrosis markers TGF-ß, PLOD2, COL1A1, and TIMP1 but also in the observation of HE staining, Sirius Red staining, and the transverse diameters of the right knees. Subsequently, we established an LPS+ATP-induced model in macrophages mimicking the inflammatory environment of KOA and inducing macrophage pyroptosis. Macrophages transfected with caspase1 siRNA showed reduced cell death; meanwhile, the relative expression of pyroptosis-related proteins were also downregulated. In addition, the level of fibrotic markers in synovial fibroblasts were significantly decreased after coculture with siRNA GSDMD-transfected macrophages. To conclude, synovial macrophage pyroptosis may occur in the pathological processes of KOA and inhibition of synovial macrophage pyroptosis alleviates synovitis and fibrosis in KOA model rats.

Critical role of NLRP3-caspase-1 pathway in age-dependent isoflurane-induced microglial inflammatory response and cognitive impairment.

BACKGROUND: Elderly patients are more likely to suffer from postoperative cognitive dysfunction (POCD) after surgery and anesthesia. Except for declined organ function, the particular pathogenesis of POCD in elderly patients remains unknown. This study is carried out to determine the critical role of the NOD-like receptor protein 3 (NLRP3)-caspase-1 pathway in isoflurane-induced cognitive impairment. METHODS: Young (6-8 months old) and aged (14 months old) healthy male C57BL/6 mice were exposed to 1.5% isoflurane for 2 h. Some mice received intraperitoneal injection of Ac-YVAD-cmk (8 mg/kg), a specific inhibitor of caspase-1, 30 min before the isoflurane exposure. Morris water maze test was carried out 1 week after the isoflurane anesthesia. Brain tissues were harvested 24 h after the isoflurane anesthesia. Western blotting was carried out to detect the expression of NLRP3, interleukin (IL)-1ß, and IL-18 in the hippocampus. Mouse microglial cell line BV-2 and primary microglial cultures were primed by lipopolysaccharide for 30 min before being exposed to isoflurane. NLRP3 was downregulated by RNA interference. RESULTS: Compared to young mice, aged mice had an increased expression of NLRP3 in the hippocampus. Isoflurane induced cognitive impairment and hippocampal inflammation in aged mice but not in young mice. These effects were attenuated by Ac-YVAD-cmk pretreatment (P < 0.05). Isoflurane activated NLRP3-caspase-1 pathway and increased the secretion of IL-18 and IL-1ß in cells pretreated with lipopolysaccharide but not in cells without pretreatment. Downregulation of NLRP3 attenuated the activation of NLRP3 inflammasome by isoflurane. CONCLUSIONS: NLRP3 priming status in aged mouse brain may be involved in isoflurane-induced hippocampal inflammation and cognitive impairment.

Caspase inhibitor zVAD-fmk protects against acute pancreatitis-associated lung injury via inhibiting inflammation and apoptosis.

BACKGROUND/OBJECTIVES: Pulmonary apoptosis is an important pathogenic mechanism of acute lung injury induced by many factors. This study aims to investigate whether the caspase inhibitor zVAD-fmk has a protective effect against lung injury in the severe acute pancreatitis model (SAP) in rats. METHODS: Seventy-two Sprague-Dawley rats were randomly divided into Sham, SAP, and SAP + zVAD-fmk groups. The SAP model was established by injection of 5% sodium taurocholate into the pancreatic duct. Animals were sacrificed at 3 h, 6 h, 12 h, and 24 h after operation and then HE staining analysis was performed to assess the lung injury. ELISA was used to detect the activity of myeloperoxidase (MPO) and the concentrations of tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). Western blotting was used to detect the expression of cleaved caspase-3 in the lung tissues. RESULTS: Rats in SAP group showed obvious lung injury through pathologic examination. Pretreatment with zVAD-fmk significantly inhibited a post-SAP increase in the activation of MPO, TNF-α, IL-1ß, and caspase-3, and decreased lung injury induced by SAP as determined by the pathologic score. CONCLUSION: Our results suggest that apoptosis plays an important role in acute pancreatitis-associated lung injury (APALI), and inhibition of caspase activity may represent a new therapeutic approach for the treatment of APALI.

Delayed, long-term administration of the caspase inhibitor Q-VD-OPh reduced brain injury induced by neonatal hypoxia-ischemia.

Apoptosis contributes greatly to the morphological and biochemical features of cell death after neonatal cerebral hypoxia-ischemia (HI), making this mode of cell death a promising therapeutic target. We previously showed that 10 mg/kg of the caspase inhibitor Q-VD-OPh at the onset of and immediately after HI on postnatal day 9 reduced brain infarct volume. In this study, delayed administration of Q-VD-OPh, 12 and 36 h after HI, decreased HI-induced caspase-3 activity (DEVD cleavage) by 23% and diminished the levels of the proinflammatory chemokines CCL2 (MCP-1) and CCL3 (MIP-1α) by 29.3 and 29.1%, respectively, but not the levels of the anti-inflammatory cytokines IL-4 and IL-10. Long-term administration of Q-VD-OPh initiated at 12 h after HI, and continued at 24-hour intervals for 2 weeks, reduced total brain tissue loss by 31.3% from 41.5±3.1 mm3 in the vehicle group to 28.5±3.0 mm3 in the Q-VD-OPh group when evaluated 16 weeks after HI (p=0.004). Q-VD-OPh treatment also ameliorated the loss of sensorimotor function, as evaluated by a cylinder rearing test (Q-VD-OPh: 30.8±4.3% vs. vehicle: 59.7±6.3% in nonimpaired forepaw preference) 3 weeks after HI, and reduced HI-induced hyperactivity, as measured in an open field test (Q-VD-OPh: 4,062±198 cm vs. vehicle: 4,792±205 cm in distance moved) 7 weeks after the insult. However, the functional protection was no longer observed when analyzed again at later time points. The mechanisms underlying the discrepancy between sustained morphological protection and transient functional protection remain to be elucidated.

Apoptosis, necrosis, and autophagy in mouse intestinal damage after 15-Gy whole body irradiation.

Enterocytes die during high-dose radiation exposure in radiation accidents. The modality of cell death has a profound effect on the therapeutic response. The ilea from mice with 15 Gy total body irradiation (TBI) were drawn, morphological features observed by hematoxylin and eosin staining and transmission electron micrographs. The biochemical features of mouse ileum presented with the structure were cleaved Caspase-3 (apoptosis marker), Light Chain 3 (LC3)-I's conversion to LC3-II (autophagy marker) and high mobility group box chromosomal protein 1's secretion (necrosis marker). Then, the autophagy inhibitor (3-methyladenine), caspase inhibitor (Z-VAD-FMK) or necrosis inhibitor (necrostatin) was used to prevent death. Apoptosis, autophagy and necrosis were all appeared in the ileum, but necrosis had the biggest size; the use of 3-methyladenine and Z-VAD-FMK prolong one day's life of the mice after 15 Gy TBI, necrostatin significantly extended the lifespan of 15 Gy irradiated mice (p < 0.05). The results suggest that the death of enterocytes could not be classified into one type of cell death but rather as 'mixed death.'

Combination of necroptosis and apoptosis inhibition enhances cardioprotection against myocardial ischemia-reperfusion injury.

PURPOSE: Necroptosis has been proposed as a mode of cell death that is a caspase-independent programmed necrosis. We investigated whether necroptosis is involved in myocardial ischemia-reperfusion injury in isolated guinea pig hearts and, if so, whether simultaneous inhibition of necroptosis and apoptosis confers enhanced cardioprotection. METHODS: Isolated perfused guinea pig hearts were subjected to 30 min ischemia and 4 h reperfusion (control = CTL, n = 8). Necrostatin-1 (necroptosis inhibitor, 10 µM), Z-VAD (apoptosis inhibitor, 0.1 µM) and both inhibitors were administered starting 5 min before ischemia and during the initial 30 min of reperfusion (Nec, Z-VAD, Nec + Z-VAD; n = 8 each). Contractile recovery was monitored by left ventricular developed (LVDP) and end-diastolic (LVEDP) pressure. Infarct size was determined by triphenyltetrazolium chloride staining. Tissue samples were obtained after 4 h reperfusion to determine expression of receptor-interacting protein 1 (RIP1) and activated caspase 3 by Western blot analysis. RESULTS: After reperfusion, Nec + Z-VAD had higher LVDP and lower LVEDP compared with CTL. Infarct size was reduced in Nec and Z-VAD compared with CTL. Combination of necroptosis and apoptosis inhibition further reduced infarct size. Expression of activated caspase 3 was not increased in Z-VAD and Nec + Z-VAD compared with Nec and CTL. Expression of RIP1 was preserved in Z-VAD and Nec + Z-VAD compared with CTL, suggesting RIP1-mediated necrosis is involved in myocardial ischemia-reperfusion injury. CONCLUSION: Necroptosis is involved in myocardial ischemia-reperfusion injury, and simultaneous inhibition of necroptosis and apoptosis enhances the cardioprotective effect. These findings may provide a novel, additive strategy for cardioprotection in acute myocardial infarction.

Inhibition of caspase mediated apoptosis restores muscle function after crush injury in rat skeletal muscle.

Although muscle regeneration after injury is accompanied by apoptotic cell death, prolonged apoptosis inhibits muscle restoration. The goal of our study was to provide evidence that inhibition of apoptosis improves muscle function following blunt skeletal muscle injury. Therefore, 24 rats were used for induction of injury to the left soleus muscle using an instrumented clamp. All animals received either 3.3 mg/kg i.p. of the pan-caspase inhibitor Z-valinyl-alanyl-DL: -aspartyl-fluoromethylketone (z-VAD.fmk) (n = 12 animals) or equivalent volumes of the vehicle solution DMSO (n = 12 animals) at 0 and 48 h after trauma. After assessment of the fast twitch and tetanic contraction capacity of the muscle at days 4 and 14 post injury, sampling of muscle tissue served for analysis of cell apoptosis (cleaved caspase 3 immunohistochemistry), cell proliferation (BrdU immunohistochemistry) as well as of muscle tissue area and myofiber diameter (HE planimetric analysis). Muscle strength analysis after 14 days in the z-VAD.fmk treated group revealed a significant increase in relative muscle strength when compared to the DMSO treated group. In contrast to the DMSO treated injured muscle, showing a transient switch towards a fast-twitching muscle phenotype (significant increase of the twitch-to-tetanic force ratio), z-VAD.fmk treated animals showed an enhanced healing process with a faster restoration of the twitch-to-tetanic force ratio towards the physiological slow-twitching muscle phenotype. This enhancement of muscle function was accompanied by a significant decrease of cell apoptosis and cell proliferation at day 4 as well as by a significant increase of muscle tissue area at day 4. At day 14 after injury z-VAD.fmk treated animals presented with a significant increase of myofiber diameter compared to the DMSO treated animals. Thus, z-VAD.fmk could provide a promising option in the anti-apoptotic therapy of muscle injury.

A new mouse mutant of the Cdh23 gene with early-onset hearing loss facilitates evaluation of otoprotection drugs.

We report a novel mutation (erlong, erl) of the cadherin 23 (Cdh23) gene in a mouse model for DFNB12 characterized by progressive hearing loss beginning from postnatal day 27 (P27). Genetic and sequencing analysis revealed a 208 T >C transition causing an amino-acid substitution (70S-P). Caspase expression was upregulated in mutant inner ears. Hearing was preserved (up to 35-dB improvement) in pan-caspase inhibitor Z-VAD-FMK-treated mutants compared with untreated mutants (P<0.05). Outer hair cell (OHC) loss in the cochleae of Z-VAD-FMK-treated mutants was significantly reduced compared with those of untreated mice. Thus, the erl mutation can lead to hearing loss through apoptosis. This is the first genetic mouse model of hearing loss shown to respond to otoprotective drug therapy. The short interval from initial hearing loss to deafness (P27-P90) makes this model ideal for screening and validating otoprotective drugs.

Enhanced cell cycle perturbation and apoptosis mediate the synergistic effects of ST1926 and ATRA in neuroblastoma preclinical models.

Retinoic acid therapy is nowadays an important component of treatment for residual disease of stage IV neuroblastoma after multimodal therapy. Nevertheless, arising resistance and treatment toxicity could represent relevant limiting factors. In the present study, we show that retinoic acid enhances the cytostatic and apoptogenic properties of the novel adamantyl retinoid ST1926 in a panel of neuroblastoma cells with different p53 status and caspase 8 expression, resulting in synergistic effects as assessed by Combination Index and Isobologram analysis. Under conditions where the two drugs alone produced no toxic effects, their combination resulted in enhanced G2-M arrest and sub-G1 population as shown by BrdU pulse-chase and labeling experiments. PARP cleavage, caspase 3, 8 and 9 activation and modulation of DR4 and FAS were indicative of enhanced apoptosis triggered by the co-incubation of the two drugs whereas neither ST1926-mediated genotoxic damage nor ATRA-differentiating effects were affected by the combined treatment. Caspase-3 and 8-mediated apoptosis appeared to play an important role in the drugs synergism. In fact, the addition of a pan-caspase inhibitor ZVAD-FMK reverted this effect in SK-N-DZ cells, and synergism was confined to limited drugs doses in HTLA cells not expressing caspase-8. Although not modulated, p53 appeared to enhance cells responsiveness to retinoid/ATRA combination. In vivo studies in the most sensitive neuroblastoma model SK-N-DZ, confirmed enhanced activity of the drugs combination vs single treatments. The study provides important lines of evidence that such a drugs combination could represent a less toxic and more effective approach for maintenance treatment in children with neuroblastoma.

[Inhibition of elicitation of allergic contact dermatitis by topical use of Z-VAD-FMK, a broad caspase inhibitor: experiment in mice].

OBJECTIVE: To explore the effects of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), a broad caspase inhibitor, on the elicitation of murine allergic contact dermatitis (ACD) and examine the effects on T lymphocytes. METHODS: 1-fluoro-2,4-dinitrobenzene (DNFB) was used to establish the classical murine model of ACD. Different concentrations of Z-VAD-FMK were applied before ear provocation. Several parameters were detected, including ear swelling degree, weight differences and thickness of ear tissue under microscope between 2 ears. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of Th1 cytokines (INF-γ and IL-2) in ear tissues. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect their levels of mRNA and the results were shown as "copies relative to one million housekeeping genes". Local lymph node assay (LLNA) was conducted. Bromodeoxyuridine-flow cytometry was used to detect the proliferation of T lymphocytes in local lymph node and flow cytometry to detect the activation of T lymphocytes. RESULTS: The right ear swelling degree, weight differences and thickness between two ears in the 1.25 mmol/L Z-VAD-FMK group were (12.6 ± 1.2)×10(-2) mm, (3.1 ± 0.2) mg, and (12.1 ± 1.1)×10(-2) mm respectively. And they were all significantly lower than those of the negative control group((17.4 ± 1.6)×10(-2) mm, (4.2 ± 0.3) mg, (16.7 ± 1.5)×10(-2) mm;q = 3.25, 2.98, 3.12, all P < 0.05). The levels of INF-γ and IL-2 in the ear skin lesions of 1.25 mmol/L Z-VAD-FMK group were (856 ± 45) and (167 ± 12) pg/ml respectively and they were both significantly lower than those of the negative control group ((1180 ± 58) and (225 ± 16) pg/ml; q = 3.11, 3.14, both P < 0.05). The mRNA levels of the above two cytokines were 152 ± 12 and 96 ± 8 respectively and they were both significantly lower than those of the negative control group (220 ± 15 and 156 ± 11;q = 3.15, 3.42, both P < 0.05). In LLNA, the mean intensity of BrdU in T lymphocytes of 1.25 mmol/L Z-VAD-FMK-treated group was significantly weaker than that of the negative control group (185 ± 15 vs 298 ± 21, q = 3.02, P < 0.05). The percent of activation markers-positive T lymphocytes of the Z-VAD-FMK group were 7.8% ± 0.7%, 9.8% ± 0.8% and 31.2% ± 2.8% respectively and they were all significantly lower than those of the negative control group (10.5% ± 1.0%, 14.5% ± 1.1%, 46.5% ± 3.2%, q = 3.16, 3.52, 3.11, all P < 0.05). CONCLUSION: Topical use of Z-VAD-FMK prior to ear provocation can suppress the proliferation and activation of T lymphocytes in both skin tissues and local lymph nodes and thus result in the inhibitory effect of allergic contact dermatitis.

Protective effect of caspase inhibition on compression-induced muscle damage.

There are currently no effective therapies for treating pressure-induced deep tissue injury. This study tested the efficacy of pharmacological inhibition of caspase in preventing muscle damage following sustained moderate compression. Adult Sprague-Dawley rats were subjected to prolonged moderate compression. Static pressure of 100 mm Hg compression was applied to an area of 1.5 cm2 in the tibialis region of the right limb of the rats for 6 h each day for two consecutive days. The left uncompressed limb served as intra-animal control. Rats were randomized to receive either vehicle (DMSO) as control treatment (n =8) or 6 mg kg⁻¹ of caspase inhibitor (z-VAD-fmk; n =8) prior to the 6 h compression on the two consecutive days.Muscle tissues directly underneath the compression region of the compressed limb and the same region of control limb were harvested after the compression procedure.Histological examination and biochemical/molecular measurement of apoptosis and autophagy were performed. Caspase inhibition was effective in alleviating the compression-induced pathohistology of muscle. The increases in caspase-3 protease activity, TUNEL index, apoptotic DNA fragmentation and pro-apoptotic factors (Bax, p53 and EndoG) and the decreases in anti-apoptotic factors (XIAP and HSP70) observed in compressed muscle of DMSO-treated animals were not found in animals treated with caspase inhibitor. The mRNA content of autophagic factors (Beclin-1, Atg5 and Atg12) and the protein content of LC3, FoxO3 and phospho-FoxO3 that were down-regulated in compressed muscle of DMSO-treated animals were all maintained at their basal level in the caspase inhibitor treated animals. Our data provide evidence that caspase inhibition attenuates compression-induced muscle apoptosis and maintains the basal autophagy level. These findings demonstrate that pharmacological inhibition of caspase/apoptosis is effective in alleviating muscle damage as induced by prolonged compression.

Immunological response in early brain injury after SAH.

This study summarized the role of inflammation in the early brain injury after subarachnoid hemorrhage. Elevation of cytokines, activation of MMPs and phosphorylation of MAPK contributes to neuronal apoptosis and brain edema. Anti-inflammation may be potential strategy for the prevention and suppression of early brain injury after subarachnoid hemorrhage.

Subarachnoid hemorrhage causes pulmonary endothelial cell apoptosis and neurogenic pulmonary edema in mice.

OBJECTS: Neurogenic pulmonary edema (NPE) is a well-known complication of subarachnoid hemorrhage (SAH), which potentially causes a poor outcome. The aim of this study was to examine if NPE occurs in the endovascular perforation model of SAH in mice and if apoptosis contributes to NPE development after SAH in mice. METHODS: Sham-operated or SAH mice were treated with an intraperitoneal administration of vehicle or an antiapoptotic drug Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) 1 h post-SAH. Pulmonary edema measurements and evaluation of apoptosis occurrence were performed on the lung at 24 h post-SAH. RESULTS: SAH caused NPE, which was associated with apoptosis of pulmonary endothelial cells. Z-VAD-FMK significantly prevented apoptosis and NPE. CONCLUSIONS: Pulmonary endothelial cell apoptosis contributes to the pathophysiology of NPE after SAH in mice.

Induction of programmed cell death by inhibition of AKT with the alkylphosphocholine perifosine in in vitro models of platinum sensitive and resistant ovarian cancers.

PURPOSE: We analyzed the anti-tumor effect and the mechanism of action of perifosine, an orally active alkylphospholipid AKT inhibitor using in vitro models of human ovarian cancer. METHODS: Ovarian cancer cells OAW42, PA-1, SKOV3, and A2780 as well as platinum resistant A2780cis cells were incubated with increasing concentrations of perifosine, with and without multi-caspase inhibitor zVAD-FMK. The effect of a combined treatment with cisplatin and perifosine was investigated in OAW42, SKOV3, A2780 and A2780cis cells. Cytotoxic effects of perifosine were analyzed using crystal violet staining, FACS analysis of DNA content as well as Annexin V/propidium iodide-double staining. The effect of perifosine on AKT phosphorylation was determined by Western blotting. RESULTS: Perifosine displayed anti-tumor activity in all five cell lines, which increased time-dependently. While IC(50) values at 24 h were >40 µM, IC(50) values after 72 h decreased to 10 µM in OAW42 and 25 µM in PA-1 and 30 µm in SKOV3 cells. In platinum resistant A2780cis cells perifosine showed good antiproliferative activity (IC(50) = 3 µm). At adequate doses, perifosine increased cytotoxic effects of cisplatin in OAW42, A2780 and A2780cis cell. Anti-tumor activity of perifosine was not confined to a specific phase of the cell cycle and could not be decreased by the pan-caspase inhibitor zVAD-FMK. AnnexinV/propidium iodide-double staining after treatment with perifosine was not indicative of classical apoptosis. AKT phosphorylation was dose-dependently inhibited by perifosine. CONCLUSIONS: Perifosine showed substantial cytotoxic effects in various in vitro models of ovarian cancer. Since anti-tumor effects were not confined to platinum-sensitive cells perifosine seems to be a good candidate for clinical studies in patients especially with platinum resistant ovarian cancer.

Intracochlear perfusion of leupeptin and z-VAD-FMK: influence of antiapoptotic agents on gunshot-induced hearing loss.

The therapeutic efficiency of cochlear infusion of two anti-apoptotic substances: a potent calpain inhibitor, leupeptin and a caspase inhibitor, z-VAD-FMK was evaluated in guinea pigs after a gunshot noise-induced trauma (170 dB SPL). A preliminary study showed that hair cell apoptosis appeared within 7 days of the noise trauma. For each animal, one of the cochleae was perfused directly starting 1 h after the trauma with leupeptin or z-VAD-FMK for 7 days via a mini-osmotic pump whereas the other cochlea was untreated. ABR threshold shifts were measured over a 14-day recovery period. The functional hearing study was supplemented by histological analysis. Two days after the trauma significant differences were observed between threshold shifts in the z-VAD-FMK-treated and the non-treated ears. Cochleograms showed that hair cell losses were significantly lower in z-VAD-FMK-treated ears. Regarding the leupeptin treatment, no significant difference between treated and non-treated ears was observed. This work indicates that early direct infusion of z-VAD-FMK into the cochlea accelerates hearing recovery and reduces hair cell loss after gunshot noise-induced trauma. These results suggest that the gunshot noise-induced trauma may involve the caspase pathway rather than the calpain pathway in the apoptotic process.

Caspase-1 inhibition ameliorates murine acute graft versus host disease by modulating the Th1/Th17/Treg balance.

Our previous studies have implicated Caspase-1 signaling in driving the proinflammatory state of acute graft versus host disease (aGVHD). Therefore, we aimed to elucidate the mechanism of Caspase-1 in in murine models of aGVHD through specific inhibition of its activity with the decoy peptide Ac-YVAD-CMK. We transplanted bone marrow from donor C57BL/6 (H-2b) mice into recipient BALB/c (H-2Kd) mice and randomized the recipients into the following treatment cohorts: (1) allogeneic hematopoietic stem cell transplantation and splenic cell infusion control (PBS group); (2) low dose Ac-YVAD-CMK (AC low group); (3) and high dose Ac-YVAD-CMK (AC high group). Indeed, we observed that Caspase-1 inhibition by Ac-YVAD-CMK ameliorated pathological damage and inflammation in the liver, lungs, and colon elicited by aGVHD. This was associated with reduced mortality secondary to aGVHD. Mechanistically, we found that Caspase-1 inhibition modulated donor T cell expansion, restored the balance of Th1/Th17/Treg subsets, and markedly decreased serum levels and aGVHD target organ mRNA expression of IL-1ß, IL-18, and HMGB1. Thus, we demonstrate that inhibition of Caspase-1 by Ac-YVAD-CMK mitigates murine aGVHD by regulating Th1/Th17/Treg balance and attenuating its characteristic proinflammatory state.

Cysteine protease inhibitors cure an experimental Trypanosoma cruzi infection.

Trypanosoma cruzi is the causative agent of Chagas' disease. The major protease, cruzain, is a target for the development of new chemotherapy. We report the first successful treatment of an animal model of Chagas' disease with inhibitors designed to inactivate cruzain. Treatment with fluoromethyl ketone-derivatized pseudopeptides rescued mice from lethal infection. The optimal pseudopeptide scaffold was phenylalanine-homophenylalanine. To achieve cure of infection, this pseudopeptide scaffold was incorporated in a less toxic vinyl sulfone derivative. N-methyl piperazine-Phe-homoPhe-vinyl sulfone phenyl also rescued mice from a lethal infection. Six of the treated mice survived over nine months, three without further treatment. Three mice that had entered the chronic stage of infection were retreated with a 20-d regimen. At the conclusion of the experiments, five of the six mice had repeated negative hemacultures, indicative of parasitological cure. Studies of the effect of inhibitors on the intracellular amastigote form suggest that the life cycle is interrupted because of inhibitor arrest of normal autoproteolytic cruzain processing at the level of the Golgi complex. Parasites recovered from the hearts of treated mice showed the same abnormalities as those treated in vitro. No abnormalities were noted in the Golgi complex of host cells. This study provides proof of concept that cysteine protease inhibitors can be given at therapeutic doses to animals to selectively arrest a parasitic infection.