Protection against TNF-induced liver parenchymal cell apoptosis during endotoxemia by a novel caspase inhibitor in mice

Excessive apoptotic cell death is implicated in a growing number of acute and chronic disease states. Caspases are critical for the intracellular signaling pathway leading to apoptosis. The aim of this investigation was to evaluate the efficacy and the mechanism of action of the novel caspase inhibitor CV1013 in a well-characterized model of TNF-induced apoptosis. Administration of 700 mg/kg galactosamine/100 microg/kg endotoxin (Gal/ET) induced hepatocellular apoptosis in C3Heb/FeJ mice as indicated by increased caspase-3 activity (706% above controls) and enhanced DNA fragmentation (3400% above controls) at 6 h. In addition, apoptosis was aggravated by the neutrophil-induced injury at 7 h (ALT activities: 4220 +/- 960 U/L and 48 +/- 4% necrosis). All animals died 8-12 h after Gal/ET treatment from shock and liver failure. A dose of 10 or 1 mg/kg of CV1013 administered three times (3, 4.5, and 5.5 h after Gal/ET) effectively prevented caspase-3 activation and parenchymal cell apoptosis at 6 h as well as the subsequent neutrophil-induced aggravation of the injury at 7 h after Gal/ET treatment. Animals treated with 10 mg/kg CV1013 survived for 24 h without liver injury. CV1013 reduced the processing of caspase-3 and caspase-8. This suggests that CV1013 may have inhibited the small amount of active caspase-8 generated at the receptor level. Because of the multiple amplification loops used to activate the entire caspase cascade, blocking the initial intracellular signal by CV1013 was highly effective in preventing apoptotic cell death. CV1013 has therapeutic potential for disease states with excessive apoptosis.     

A novel and selective Na+/Ca2+ exchange inhibitor, SEA0400, improves ischemia/reperfusion-induced renal injury

We evaluated the effects of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline), a novel and selective Na+/Ca2+ exchange inhibitor, on ischemic acute renal failure. Ischemic acute renal failure in rats was induced by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after the contralateral nephrectomy. SEA0400 administration (0.3, 1 and 3 mg/kg, i.v.) before ischemia dose-dependently attenuated the ischemia/reperfusion-induced renal dysfunction and histological damage such as tubular necrosis. SEA0400 pretreatment at the higher dose suppressed the increment of renal endothelin-1 content after reperfusion. The ischemia/reperfusion-induced renal dysfunction was also overcome by post-ischemia treatment with SEA0400 at 3 mg/kg, i.v. In in vitro study, SEA0400 (0.2 and 1 microM) protected cultured porcine tubular cells (LLC-PK1) from hypoxia/reoxygenation-induced cell injury. These findings support the view that Ca2+ overload via the reverse mode of Na+/Ca2+ exchange, followed by endothelin-1 overproduction, plays an important role in the pathogenesis of ischemia/reperfusion-induced renal injury. The possibility exists that a selective Na+/Ca2+ exchange inhibitor such as SEA0400 is useful as effective therapeutic agent against ischemic acute renal failure in humans.     

Attenuation of renal ischemia-reperfusion injury by trimetazidine: evidence of an in vivo antioxidant effect

Renal ischemia-reperfusion injury constitutes the most common pathogenic factor for acute renal failure and is the main contributor to renal dysfunction in allograft recipients and revascularization surgeries. Many studies have demonstrated that reactive oxygen species play an important role in ischemic acute renal failure. The aim of the present study was to investigate the effects of the synthetic antiischemic agent trimetazidine in a rat model of renal ischemia-reperfusion injury. Renal ischemia-reperfusion was induced by clamping the unilateral renal artery for 45 min followed by 24 h of reperfusion. Trimetazidine (2.5 mg/kg i.p.) was administered 24 and 12 h prior to renal artery occlusion and the same dose was given intravenously 1 h before inducing ischemia. Tissue lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS) in kidney homogenates. Renal function was assessed by estimating serum creatinine, blood urea nitrogen (BUN), creatinine and urea clearance. Renal morphological alterations were assessed by histopathological examination of hematoxylin-eosin stained sections of the kidneys. Ischemia-reperfusion produced elevated levels of TBARS and deteriorated the renal function as assessed by increased serum creatinine, BUN and decreased creatinine and urea clearance compared with sham operated rats. The ischemic kidneys of rats showed severe hyaline casts, epithelial swelling, proteinaceous debris, tubular necrosis, medullary congestion and hemorrhage. Trimetazidine markedly reduced elevated levels of TBARS and significantly attenuated renal dysfunction and morphological changes in rats subjected to renal ischemia-reperfusion. These results clearly demonstrate the in vivo antioxidant effect and the therapeutic potential of trimetazidine, an anti-ischemic agent, in attenuating renal ischemia-reperfusion injury.     

Pyrrolidine dithiocarbamate reduces renal dysfunction and injury caused by ischemia/reperfusion of the rat kidney

Dithiocarbamates can modulate the expression of genes associated with inflammation or development of ischemia/reperfusion injury. Here, we investigate the effects of pyrrolidine dithiocarbamate, an inhibitor of nuclear factor (NF)-kappaB activation, on the renal dysfunction and injury caused by ischemia/reperfusion of the rat kidney. Bilateral clamping of renal pedicles (45 min) followed by reperfusion (6 h) caused significant renal dysfunction and marked renal injury. Pyrrolidine dithiocarbamate (100 mg/kg, administered i.v.) significantly reduced biochemical and histological evidence of renal dysfunction and injury caused by ischemia/reperfusion of the rat kidney. Furthermore, pyrrolidine dithiocarbamate markedly reduced the expression of inducible nitric oxide synthase (iNOS) protein and significantly reduced serum levels of nitric oxide. Finally, pyrrolidine dithiocarbamate inhibited the activation of NF-kappaB by preventing its translocation from the cytoplasm into the nuclei of renal cells. These results demonstrate that pyrrolidine dithiocarbamate reduces renal ischemia/reperfusion injury and that dithiocarbamates may provide beneficial actions against ischemic acute renal failure.     

Inhibitors of calpain activation (PD150606 and E-64) and renal ischemia-reperfusion injury

Calpain activation has been implicated in the development of ischemia-reperfusion (I-R) injury. Here we investigate the effects of two inhibitors of calpain activity, PD150606 and E-64, on the renal dysfunction and injury caused by I-R of rat kidneys in vivo. Male Wistar rats were administered PD150606 or E-64 (3mg/kg i.p.) or vehicle (10%, v/v, DMSO) 30min prior to I-R. Rats were subjected to bilateral renal ischemia (45min) followed by reperfusion (6h). Serum and urinary biochemical indicators of renal dysfunction and injury were measured; serum creatinine (for glomerular dysfunction), fractional excretion of Na(+) (FE(Na), for tubular dysfunction) and urinary N-acetyl-beta-d-glucosaminidase (NAG, for tubular injury). Additionally, kidney tissues were used for histological analysis of renal injury, immunohistochemical analysis of intercellular adhesion molecule-1 (ICAM-1) expression and nitrotyrosine formation. Renal myeloperoxidase (MPO) activity (for polymorphonuclear leukocyte infiltration) and malondialdehyde (MDA) levels (for tissue lipid peroxidation) were determined. Both PD150606 and E-64 significantly reduced the increases in serum creatinine, FE(Na) and NAG caused by renal I-R, indicating attenuation of renal dysfunction and injury and reduced histological evidence of renal damage caused by I-R. Both PD150606 and E-64 markedly reduced the evidence of oxidative stress (ICAM-1 expression, MPO activity, MDA levels) and nitrosative stress (nitrotyrosine formation) in rat kidneys subjected to I-R. These findings provide the first evidence that calpain inhibitors can reduce the renal dysfunction and injury caused by I-R of the kidney and may be useful in enhancing the tolerance of the kidney against renal injury associated with aortovascular surgery or renal transplantation.     

Protective effect of nitric oxide on ischemia/reperfusion-induced renal injury and endothelin-1 overproduction

To elucidate the role of nitric oxide (NO) in the pathogenesis of ischemic acute renal failure, we examined the effects of (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409) and N(G)-nitro-L-arginine methyl ester (L-NAME) as a NO donor and a non-selective NO synthase inhibitor on ischemia/reperfusion-induced renal injury and renal endothelin-1 content. Ischemic acute renal failure was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. At 24 h after reperfusion, renal function in untreated acute renal failure rats markedly decreased and histological examination revealed severe renal damage. In addition, increases in renal endothelin-1 contents were evident in the acute renal failure rats at 2, 6, and 24 h after reperfusion, respectively. Pretreatment with FK409 (1 or 3 mg/kg, i.v.) attenuated ischemia/reperfusion-induced renal dysfunction, histological damage, and endothelin-1 overproduction after reperfusion. In contrast, pretreatment with L-NAME (1 or 10 mg/kg, i.v.) aggravated renal injuries of acute renal failure rats at 24 h after reperfusion, and the effect is accompanied by further increases in the renal endothelin-1 content at 2 and 6 h, but not at 24 h, after reperfusion. These results suggest that suppressive effects of NO on the renal endothelin-1 overproduction induced by ischemia/reperfusion in an early phase are probably responsible for the protective effect of NO against ischemic acute renal failure.     

Organic solvent-induced proximal tubular cell toxicity via caspase-3 activation

BACKGROUND: Occupational exposure to solvents may be associated with development and progression of tubulo-interstitial fibrosis and chronic renal failure. However, the cellular mechanisms by which this occurs remain elusive. MATERIALS AND METHODS: The cytotoxicity of proximal tubular cells (LLC-PK1) exposed to 106 mg/mL of p-xylene or 92 mg/mL of toluene was compared with untreated controls using cell viability (MTS assay) and caspase-3 activity, with or without caspase-3 selective inhibitor. RESULTS: Both compounds reduced cell viability and increased caspase-3 activation (P < 0.005). Inhibition of caspase-3 by the selective inhibitor DEVD-CHO prevented injury (P < 0.001) and inhibited solvent-induced caspase-3 activation (P < 0.005). CONCLUSION: Inhibition of caspase-3, the critical caspase in the apoptosis process, prevents cell injury in LLC-PK1. This suggests that caspase-3 may play a pivotal role in solvent-induced proximal tubular cell injury.     

Single-dose intravenous simvastatin treatment attenuates renal injury in an experimental model of ischemia-reperfusion in the rat

The effect of acute pretreatment with a single dose of simvastatin (1 mg/kg, i.v.; 30 min before ischemia) on renal dysfunction caused by ischemia-reperfusion (I/R) injury in the rat was investigated. I/R injury was induced by clamping both renal vascular pedicles for 45 min, followed by 4 h of reperfusion with saline (2 ml/kg per hour). Simvastatin significantly improved both parameters of glomerular and tubular dysfunction (e.g., creatinine levels and fractional excretion of Na(+), respectively) and especially improved the histological score, compared to control I/R-injured rats treated with saline or 10% DMSO only.     

Inhibition of protein tyrosin phosphatase improves vascular endothelial dysfunction

The study has been designed to investigate the effect of Bis-(maltolato) oxovanadium (BMOV), an inhibitor of protein tyrosin phosphatase (PTPase), in diabetes mellitus and hyperhomocysteinemia induced vascular endothelial dysfunction. Streptozotocin (55 mg kg(-1), i.v.) and methionine (1.7% w/w, p.o., 4 weeks) were administered to rats to produce diabetes mellitus (serum glucose >140 mg dl(-1)) and hyperhomocysteinemia (serum homocysteine>10 microM), respectively. Vascular endothelial dysfunction was assessed using isolated aortic ring preparation, electron microscopy of thoracic aorta and serum concentration of nitrite/nitrate. Serum thiobarbituric acid reactive substances (TBARS) were estimated to assess oxidative stress. Atorvastatin has been employed in the present study as standard drug to improve vascular endothelial dysfunction. BMOV (0.2 mg/ml in drinking water) or atorvastatin (30 mg kg(-1), p.o.) in diabetic and hyperhomocysteinemic rats significantly reduced serum glucose and homocysteine concentration. BMOV or atorvastatin markedly improved acetylcholine induced endothelium dependent relaxation, vascular endothelial lining, serum nitrite/nitrate concentration and serum TBARS in diabetic and hyperhomocysteinemic rats. However, this ameliorative effect of BMOV has been prevented by l-NAME (25 mg kg(-1), i.p.), an inhibitor of NOS or by glibenclamide (5 mg kg(-1), i.p.), a blocker of ATP sensitive K(+) channels. Therefore, it may be concluded that BMOV induced inhibition of PTPase may activate eNOS due to opening of ATP sensitive K(+) channels and consequently reduce oxidative stress to improve vascular endothelial dysfunction.     

Lysophosphatidylcholine reduces the organ injury and dysfunction in rodent models of gram-negative and gram-positive shock

1. Lysophosphatidylcholine (LPC) modulates the inflammatory response and reduces mortality in animal models of sepsis. Here, we investigate the effects of LPC from synthetic (sLPC) and natural, soy bean derived LPC, (nLPC) sources on the organ injury/dysfunction caused by systemic administration of lipopolysaccharide (LPS) or peptidoglycan (PepG) and lipoteichoic acid (LTA). 2. Rats were subjected to (i) endotoxaemia (LPS 6 mg kg(-1) i.v.) and treated with sLPC (1-100 mg kg(-1)), (ii) endotoxaemia and treated with nLPC (10 mg kg(-1)) or (iii) gram-positive shock (PepG 10 mg kg(-1) and LTA 3 mg kg(-1) i.v.) and treated with sLPC (10 mg kg(-1)). 3. Endotoxaemia or gram-positive shock for 6 h resulted in increases in serum makers of renal dysfunction and liver, pancreatic and neuromuscular injury. 4. Administration of sLPC, at 1 or 2 h after LPS, dose dependently (1-10 mg kg(-1)) reduced the organ injury/dysfunction. High doses of sLPC (30 and 100 mg kg(-1)) were shown to be detrimental in endotoxaemia. sLPC also afforded protection against the organ injury/dysfunction caused by gram-positive shock. nLPC was found to be protective in endotoxaemic animals. 5. The beneficial effects of sLPC were associated with an attenuation in circulating levels of interleukin-1beta (IL-1beta). 6. In conclusion, LPC dose and time dependently reduces the organ injury and circulating IL-1beta levels caused by gram-negative or gram-positive shock in the rat. Thus, we speculate that appropriate doses of LPC may be useful in reducing the degree of organ injury and dysfunction associated with shock of various aetiologies.     

Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury

Ischaemia-reperfusion injury causes cell death by both necrosis and apoptosis. Caspase activation is a major event in apoptosis. We therefore examined the effect of caspase inhibitors during reperfusion upon myocardial infarction. Rat isolated hearts were subjected to 35 min coronary occlusion and 120 min reperfusion. Treatment groups were perfused with caspase inhibitors during early reperfusion. We assessed a non-selective caspase inhibitor (Z-VAD. fmk, 0.1 microM), a caspase-8 inhibitor (Z-IETD.fmk, 0.07 microM), a caspase-9 inhibitor (Z-LEHD.fmk, 0.07 microM) and a caspase-3 inhibitor (Ac-DEVD.cmk, 0.07 microM). All caspase inhibitors limited infarct size (infarct-risk ratio per cent: control 38.5+/-2.6; Z-VAD. fmk 24.6+/-3.4; Z-LEHD.fmk 19.3+/-2.4; Z-IETD.fmk 23.0+/-5.4; Ac-DEVD.cmk 27.8+/-3.3; P<0.05 when compared with control value, 1-way ANOVA). We conclude that caspase inhibition during early reperfusion protects myocardium against lethal reperfusion injury.     

Inhibition of HtrA2/Omi ameliorates heart dysfunction following ischemia/reperfusion injury in rat heart in vivo

High temperature requirement A2 (HtrA2)/Omi is a mitochondrial serine protease that is released into the cytosol from mitochondria and in turn promotes caspase activation by proteolyzing inhibitor of apoptosis proteins. Here we asked whether treatment with an HtrA2/Omi inhibitor, 5-[5-(2-nitrophenyl)furfuryliodine]-1,3-diphenyl-2-thiobarbituric acid (UCF-101), restores heart dysfunction following ischemia/reperfusion injury in vivo. Rats underwent a 30-min ischemia by occluding the left anterior descending artery, followed by 24 h reperfusion. UCF-101 (0.75 or 1.5 micromol/kg, i.p.) was administered 10 min before reperfusion. UCF-101 treatment significantly recovered the mean arterial blood pressure and ameliorated contractile dysfunction of the left ventricle 72 h after reperfusion with concomitant reduction of infarct size. Cardio-protection mediated by UCF-101 was correlated with reduced X-linked inhibitor of apoptosis protein (XIAP) degradation and inhibition of Caspase-9, Caspase-3, and Caspase-7 processing. Furthermore, UCF-101 prevented loss of membrane integrity by inhibiting fodrin breakdown in cardiomyocytes. UCF-101-induced cytoprotection was also correlated with reduced Fas ligand expression and inhibition of FLIP degradation following ischemia/reperfusion. These results suggest that UCF-101 rescues cardiomyocytes from ischemia/reperfusion injury by inhibiting XIAP degradation and Fas/Fas-ligand-induced apoptosis, thereby ameliorating ischemia/reperfusion-induced myocardial dysfunction.     

Caspases as drug targets in ischemic organ injury

Caspases are intracellular cysteine proteases that mediate cell death and inflammation. Caspase-3 is a major mediator of both apoptotic and necrotic cell death. Caspase-1 mediates inflammation though the activation of the cytokines interleukin-1beta (IL-1beta) and interleukin-18 (IL-18). Increases in both caspase-1 and -3 have been described in ischemic injury to various organs including brain, heart and kidney. Both pharmacological inhibitors and genetic approaches have been used to inhibit caspases in vivo. Pancaspase inhibitors protect against ischemic injury in brain, heart and kidney. Pancaspase inhibition also reduces cold preservation injury due to apoptosis in liver endothelial cells and prolongs animal survival after orthotopic liver transplantation. Caspase-1 inhibition or caspase-1 deficiency protects against ischemic injury in brain, heart and kidney models of ischemia. Specifically, impaired IL-18 processing protects caspase-1-deficient mice from ischemic acute renal failure. This review focuses on studies of caspase-1 and pancaspase inhibition in ischemic injury to brain, heart and kidney. In addition, the studies of pancaspase inhibition in cold ischemic injury and organ preservation will be reviewed. The therapeutic potential of caspase inhibition in ischemic injury will be discussed.     

Actinonin, a meprin inhibitor, protects ischemic acute kidney injury in male but not in female rats

We investigated the effects of actinonin, an inhibitor of a matrix-degrading enzyme meprin, on ischemic acute kidney injury in male and female rats, and these were compared with the effects of verapamil, a Ca(2+) channel blocker. Ischemic acute kidney injury was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. At 24 h after reperfusion, renal function and histology of both males and females showed significant deterioration. The degrees of renal dysfunction and histological damage were much more severe in males than in females. Pre-ischemic treatment with actinonin (10 or 30 mg/kg, i.v.) dose-dependently attenuated the ischemia/reperfusion-induced renal injury in male rats, but failed to improve the renal injury in female rats. On the other hand, verapamil (1 mg/kg, i.v.) could efficiently prevent the ischemic acute kidney injury in female rats, as well as male rats. These results indicate that the renoprotective effect of actinonin is male-specific, thereby suggesting that meprin is involved in exacerbation of ischemia/reperfusion-induced renal injury in male rats. The possibility that meprin is a key factor involved in the sex difference in the pathogenesis of ischemic acute kidney injury, warrants further attention.     

Benidipine inhibits apoptosis during ischaemic acute renal failure in rats

We have investigated the effects of benidipine (hydrochloride), a calcium antagonist, against ischaemic acute renal failure in rats. Using histological examination, we studied whether the inhibition of apoptosis was associated with the protective effects of benidipine on the ischaemic renal injury. Acute renal failure was induced by the unilateral clamping of the left renal artery for 60 min, followed by reperfusion and contralateral nephrectomy. Drugs were given intravenously 5 min before the unilateral clamping. Prophylactic administrations of benidipine (10 microg kg(-1), i.v.) significantly ameliorated the development of renal failure as estimated by the measurements of serum creatinine and blood urea nitrogen 24 h after the reperfusion. Amlodipine (besilate, 100 and 300 microg kg(-1), i.v.) tended to attenuate renal dysfunction. Lisinopril (300 and 1000 microg kg(-1), i.v.), an angiotensin converting enzyme inhibitor, was ineffective in this acute renal failure model. Histological examination using the terminal transferase-mediated dUTP-biotin nick end-labelling (TUNEL) method to detect apoptotic cells revealed that the TUNEL-positive tubular epithelium was prominent in the renal cortex 24 h after the reperfusion. The TUNEL-positive cells were significantly reduced by pretreatment with benidipine. The results demonstrate that benidipine can ameliorate the ischaemic acute renal failure in rats and suggest that the renoprotective effect of benidipine was at least partly attributable to the reduction of apoptosis in tubular epithelial cells.     

Drp1介导线粒体能量代谢拮抗大鼠缺血再灌注肾损伤

目的　探讨线粒体动力相关蛋白1（Drp1）介导线粒体能量代谢参与缺血再灌注肾损伤的分子机制。方法　实验时间2020年10月14日。8～10周龄雄性Wistar大鼠随机分为正常对照组、假手术组、模型组和Drp1抑制剂组,每组5只,每只体质量为0.25～0.30 kg。通过手术建立大鼠肾脏缺血再灌注急性肾损伤模型,Drp1抑制剂组大鼠腹腔注射线粒体分裂抑制剂（mitochondrial division inhibitor 1,Mdivi-1）20 mg/kg体质量,其余各组大鼠注射等体积生理盐水,造模24 h留取血清及肾脏组织。比较组间大鼠血肌酐水平、肾组织病理学改变、肾组织细胞凋亡情况、线粒体超微结构、线粒体ATP酶活力,并建立体外HK-2细胞缺氧复氧模型,经处理后JC-1染色法检测细胞线粒体膜电位变化。采用方差分析。结果　与假手术组比较,模型组大鼠血肌酐明显升高［（41.12±1.895）µmol/L比（48.68±2.065）µmol/L］,肾小管损伤评分升高［（1.29±0.426）分比（6.50±0.577）分］,每高倍镜视野下大鼠肾组织细胞凋亡数量增加［（2.40±0.547）个比（10.20±1.095）个］,电镜下线粒体损伤更明显,线粒体ATP酶活力降低［（6.38±0.321）U/mg prot比（4.18±0.198）U/mg prot］,HK-2细胞缺氧复氧模型中,模型组较假手术组线粒体膜电位降低的细胞比例增多［（9.81±0.251）%比（4.24±0.598）%］,差异均有统计学意义（均P<0.05）。与模型组相比,Drp1抑制剂组大鼠血肌酐下降［（48.68±2.065）µmol/L比（43.28±0.895）µmol/L］,肾小管损伤评分降低［（6.50±0.577）分比（4.50±0.578）分］,每高倍镜视野下大鼠肾组织细胞凋亡数量减少［（10.20±1.095）个比（6.60±1.140）个］,线粒体结构损伤减轻,细胞线粒体ATP酶活力增加［（4.18±0.198）U/mg prot比（5.16±0.628）U/mg prot］,HK-2细胞缺氧复氧模型中,Drp1抑制剂组较模型组线粒体膜电位降低的细胞比例减少［（5.90±0.360）%比（9.81±0.251）%］,差异均有统计学意义（均P<0.05）。结论　选择性抑制Drp1可通过抑制线粒体分裂,有效改善能量代谢障碍,减少细胞凋亡,减轻肾缺血再灌注损伤。     

Chrysotile asbestos causes AEC apoptosis via the caspase activation in vitro and in vivo

To determine whether alveolar epithelial cell (AEC) apoptosis via caspase activation is involved in asbestos-induced lung injury, we examined apoptosis, caspase-3 and-9 activation using chrysotile asbestos exposure models in vitro and in vivo. Apoptotic cells were assessed in A549 cells with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method after 48 hours exposure of chrysotile asbestos (5 to 100 ug/cm2). Asbestos exposure induced a dose-dependent increase of apoptotic cells, and both pretreatment with Z-LEHD-FMK (caspase-3 inhibitor) or Z-DEMK-FMK (caspase-9 inhibitor) significantly suppressed asbestos-induced apoptosis. Expression of cleaved caspase-3 and-9 increased significantly from 18 to at least 48 hours after asbestos exposure. In vivo study, either 1 or 2 mg of chrysotile asbestos were administered into rat lungs intratracheally, and the lungs were obtained 3 days, 1 and 2 weeks, 1, 3 and 6 months after the administration. Asbestos exposure increased the number of apoptotic cells and the activation of cleaved caspase-3 and -9 most at 3 days in a dose dependent manner, and continued to increase them until at least 6 months after asbestos exposure. Apoptotic cells and cleaved caspase-3 and -9 positive cells were mainly observed in AECs. These findings suggest that AEC apoptosis via caspase-3 and -9 activation is involved in asbestos-induced lung injury.     

Mouse strain-dependent caspase activation during acetaminophen hepatotoxicity does not result in apoptosis or modulation of inflammation

The mechanisms of acetaminophen (APAP)-mediated hepatic oncotic necrosis have been extensively characterized. However, it was recently demonstrated that fed CD-1 mice have a transient caspase activation which initiates apoptosis. To evaluate these findings in more detail, outbred (Swiss Webster, SW) and inbred (C57BL/6) mice were treated with APAP with or without pan-caspase inhibitor and compared to the apoptosis model of galactosamine (GalN)/endotoxin (ET). Fasted or fed APAP-treated C57BL/6 mice showed no evidence of caspase-3 processing or activity. Interestingly, a minor, temporary increase in caspase-3 processing and activity (150% above baseline) was observed after APAP treatment only in fed SW mice. The degree of caspase-3 activation in SW mice after APAP was minor compared to that observed in GalN/ET-treated mice (1600% above baseline). The pancaspase inhibitor attenuated caspase activation and resulted in increased APAP-induced injury (plasma ALT, necrosis scoring). The caspase inhibitor did not affect apoptosis because regardless of treatment only < 0.5% of hepatocytes showed consistent apoptotic morphology after APAP. In contrast, > 20% apoptotic cells were observed in GalN/ET-treated mice. Presence of the caspase inhibitor altered hepatic glutathione levels in SW mice, which could explain the exacerbation of injury. Additionally, the infiltration of hepatic neutrophils was not altered by the fed state of either mouse strain. Conclusion: Minor caspase-3 activation without apoptotic cell death can be observed only in fed mice of some outbred strains. These findings suggest that although the severity of APAP-induced liver injury varies between fed and fasted animals, the mechanism of cell death does not fundamentally change.     

Differential protection with inhibitors of caspase-8 and caspase-3 in murine models of tumor necrosis factor and Fas receptor-mediated hepatocellular apoptosis

Excessive apoptosis has been implicated in a number of acute and chronic human diseases. The activation of caspases has been shown to be critical for the apoptotic process. The objective of this investigation was to evaluate the beneficial effects and mechanism of action of the caspase-8 inhibitor IETD-CHO and the caspase-3 inhibitor DEVD-CHO against tumor necrosis factor (TNF)-induced hepatocellular apoptosis in vivo and compare these results to effects of the same inhibitors against Fas-induced apoptosis. Treatment of C3Heb/FeJ mice with 700 mg/kg galactosamine/100 microg/kg endotoxin induced parenchymal apoptosis (indicated by caspase-3 activation and morphology) and severe liver injury (indicated by the increase in plasma alanine aminotransferase activities and histology) at 7 h. Treatment with IETD-CHO or DEVD-CHO (10 mg/kg at 3, 4.5, and 5.5 h) significantly attenuated caspase-3 activation and liver injury. Western analysis showed that DEVD-CHO had no effect while IETD-CHO substantially reduced procaspase-3 and procaspase-9 processing. On the other hand, caspase-3 activation and liver injury by the anti-Fas antibody Jo-2 was completely prevented by a single dose of DEVD-CHO and, as previously shown, by IETD-CHO at 90 min. Both inhibitors prevented procaspase-3 and procaspase-9 processing. Thus, there are fundamental differences in the efficacy of caspase inhibitors in these two models. We conclude that Fas may rely exclusively on caspase-8 activation and mitochondria to activate caspase-3, which can process more procaspase-8 and thus propagate the amplification of the apoptotic signal. TNF can activate a similar signaling pathway. However, alternative signaling mechanisms seem to exist, which can compensate if the main pathway is blocked.     

Protective effects of SEA0400, a novel and selective inhibitor of the Na+/Ca2+ exchanger,on myocardial ischemia-reperfusion injuries

The Na(+)/Ca(2+) exchanger (NCX) is involved in myocardial ischemia-reperfusion injuries. We examined the effects of 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a potent and selective inhibitor of NCX, on myocardial ischemia-reperfusion injury models. In canine cardiac sarcolemmal vesicles and rat cardiomyocytes, SEA0400 potently inhibited the Na(+)-dependent 45Ca(2+) uptake with an IC(50) value of 90 and 92 nM, compared with 2-[2-[4-(4-nitrobenzyloxy)phenyl]isothiourea (KB-R7943, 7.0 and 9.5 microM), respectively. In rat cardiomyocytes, SEA0400 (1 and 3 microM) attenuated the Ca(2+) paradox-induced cell death. In isolated rat Langendorff hearts, SEA0400 (0.3 and 1 microM) improved the cardiac dysfunction induced by low-pressure perfusion followed by normal perfusion. In anesthetized rats, SEA0400 (0.3 and 1 mg/kg, i.v.) reduced the incidence of ventricular fibrillation and mortality induced by occlusion of the left anterior descending coronary artery followed by reperfusion. These results suggest that SEA0400 is a most potent NCX inhibitor in the heart and that it has protective effects against myocardial ischemia-reperfusion injuries.