benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Ischemia

Mesenchymal stem/stromal cells (MSCs) offer great promise in the treatment of ischemic injuries, including stroke, heart infarction, and limb ischemia. However, poor cell survival after transplantation remains a major obstacle to achieve effective MSC therapies. To improve cell survival and retention, we transplanted human bone marrow MSCs with or without a specific prosurvival factor (PSF) cocktail consisting of IGF1, Bcl-X

Topics: Amino Acid Chloromethyl Ketones; Animals; bcl-X Protein; Bone Marrow Cells; Bone Marrow Transplantation; Cell Line; Collagen; Cyclosporine; Drug Combinations; Enzyme Inhibitors; Extremities; Humans; Insulin-Like Growth Factor I; Ischemia; Laminin; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred NOD; Mice, SCID; Neovascularization, Physiologic; Pinacidil; Proteoglycans; Regenerative Medicine

Acute kidney injury (AKI) induced by renal ischemia is a common clinical problem associated with a high morbidity and mortality. The present study investigated whether necroptosis was present in an in vitro renal ischemia model and whether the addition of necrostatin-1 (Nec-1) has a protective effect. In addition, whether autophagy was inhibited following the use of Nec-1 was also examined. When apoptosis was inhibited by z-VAD‑fmk and energy was depleted with antimycin A for 1 h, the morphological abnormalities of human proximal tubular epithelial (HK-2) cells were markedly attenuated, and the cell viability was significantly improved following incubation with Nec-1. LC3-II/I ratios and LC3-II/GAPDH ratios demonstrated a statistically significant decrease in the Nec-1 + tumor necrosis factor (TNF)-α + z-VAD-fmk + antimycin A (1 h) group compared with the control group. In conclusion, the present study suggested that necroptosis was present in HK-2 cells subjected to TNF-α stimulation and energy depletion. Nec-1 inhibits a caspase‑independent necroptotic pathway involving autophagy and may have therapeutic potential to prevent and treat renal ischemic injury.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Autophagy; Cell Line; Cell Survival; Disease Models, Animal; Epithelial Cells; Humans; Imidazoles; Indoles; Ischemia; Microtubule-Associated Proteins; Necrosis; Protective Agents; Tumor Necrosis Factor-alpha

Renal tubular cell death in ischemia-reperfusion does not follow the classical apoptosis or necrosis phenotype. We characterized the morphological and biochemical features of injured tubular epithelial cells in ischemic acute kidney injury (AKI).. Ischemic AKI was induced in rats by 60 min of ischemia followed by 24 h of reperfusion. Light and electron microscopic TUNEL (LM-TUNEL and EM-TUNEL), gel electrophoresis of extracted DNA, and caspase-3 involvement were examined during the development of death.. Damaged tubular epithelial cells with condensed and LM-TUNEL-positive (+) nuclei were prominent at 12 and 18 h after reperfusion with DNA 'ladder' pattern on gel electrophoresis. EM-TUNEL+ cells were characterized by nuclei with condensed and clumping chromatin, whereas the cytoplasm showed irreversible necrosis. The protein levels and activity of caspase-3 did not increase in kidneys after reperfusion. In addition, caspase inhibitor (ZVAD-fmk) failed to inhibit DNA fragmentation and prevent tubular epithelial cell death in ischemic AKI.. Caspase-3-independent internucleosomal DNA fragmentation occurs in injured tubular epithelial cells undergoing irreversible necrosis in ischemic AKI. The manner of this cell death may be identical to the cell death termed apoptotic necrosis, aponecrosis, or necrapoptosis. Ischemia-reperfusion injury activates caspase-3-independent endonuclease, which in turn induces irreversible damage of tubular epithelial cells, and may contribute to the initiation and development of AKI.

Topics: Acute Kidney Injury; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Western; Caspase 3; DNA Fragmentation; Electrophoresis, Agar Gel; Epithelial Cells; In Situ Nick-End Labeling; Ischemia; Kidney; Kidney Tubules; Male; Microscopy, Electron; Necrosis; Nucleosomes; Rats; Rats, Wistar; Reperfusion Injury

Hepatic vascular control is used by many surgeons to prevent massive hemorrhage during hepatectomy. However, this may carry a risk of ischemic damage to the hepatocytes. Another major drawback of intraoperative occlusion of the hepatoduodenal ligament is portal stasis with resultant intestinal congestion which may cause adverse effects on the intestinal functions. CD44 is a transmembrane glycoprotein present in many types of epithelial cells. By mediating the attachment of dividing crypt cells to the basal lamina via hyaluronan, CD44 is considered to play a role in maintaining the intestinal villus integrity. Apoptosis is a pathway of cell death orchestrated by a family of proteases called caspases. ZVAD-fmk is a cell-permeable irreversible inhibitor of caspase and might block the processing of many caspases. This study is designed with the purpose to evaluate the impact of intraoperative occlusion of the hepatoduodenal ligament on hepatocyte and intestine functions and also to evaluate the potential influence of ZVAD-fmk on the hepatocyte and intestine functions.. Male Sprague-Dawley rats were randomized to 5 groups. Group 1(C) underwent sham operation. Group 2 (HDL30) underwent occluding the hepatoduodenal ligament by for 30 minutes. Group 3 (HDL 15) underwent occluding the hepatoduodenal ligament by for 15 minutes, releasing for 5 minutes, underwent occlusion for another 15 minutes. Group 4 (ZHDL30) first received ZVAD-fmk, then underwent occluding the hepatoduodenal ligament by for 30 minutes. Group 5 (ZHDL15) first received ZVAD-fmk, then underwent occluding the hepatoduodenal ligament for 15 minutes, releasing for 5 minutes, underwent occlusion for another 15 minutes. After removing the temporary occlusion, liver tissue and proximal jejunum were harvested. Hepatocyte and intestine apoptosis were quantitated using the TUNEL method. CD 44 status of jejunum were determined by immunohistochemical staining.. Hepatocyte apoptosis was significantly increased in group (HDL30) and group (HDL15) when compared with group (C). ZVAD-fmk effectively attenuated this phenomenon in both groups. There was no significant difference between group (HDL30) and group (HDL15). Jejunal apoptosis was significantly increased in group (HDL30) and group (HDL15) when compared with group (C). ZVAD-fmk effectively attenuated this phenomenon in both groups. There was no significant difference between group (HDL30) and group (HDL15). CD44 expression on jejunum was significantly increased in group (HDL30) and group (HDL15) when compared with group (C). ZVAD-fmk failed to effectively diminish this phenomenon.. Occlusion of the hepatoduodenal ligament significantly increased both hepatocyte and jejunal apoptosis and pretreatment with ZVAD-fmk could effectively diminish such phenomenon. CD44 expression on jejunum was also significantly increased by intraoperative occlusion of the hepatoduodenal ligament, yet pretreatment with ZVAD-fmk failed to show significant effect on such phenomenon.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Hepatectomy; Hepatocytes; Hyaluronan Receptors; Immunoenzyme Techniques; In Situ Nick-End Labeling; Intestinal Mucosa; Intestines; Ischemia; Jejunum; Ligaments; Liver; Male; Portal System; Rats; Rats, Sprague-Dawley

Acute brain ischaemia (stroke) causes a central area of coagulation necrosis. Peripheral to it and after a few hours, apoptosis causes neurons throughout the entire area to die progressively. However, this sequence of events is related to the reperfusion of regenerated capillaries or collateral circulation, and is considered to be potentially salvageable. Similar findings have been reported in the retina after ischaemia-reperfusion injury in rats. In the present study, we intended to investigate whether delayed cell death is involved in neuronal injuries to the inner retina during chronic retinal ischaemia.. Experimental branch retinal vein occlusion (BRVO) was induced in miniature pigs using indirect argon laser. The eyes were prelevated at 4, 24 and 48 hours and at 1 and 3 weeks following BRVO. The caspase inhibitor Z-VAD was injected intravitreally 24 hours after BRVO. Affected retinas were examined 24 hours later for any protective effect from apoptotic cell death. Histological examination with cresyl violet staining and TUNEL (TdT-mediated dUTP-biotin nick-end labelling) was performed on the samples.. A progressive oedema of the nerve fibre, ganglion cell and inner plexiform layers, related to a widely diffused cell necrosis, was observed in the affected territory within 4-24 hours after BRVO. This was followed by a wave of apoptosis localized at the periphery of the affected territory, which peaked approximately 48 hours after BRVO and was associated with a diffuse oedema of the inner nuclear layer. A progressive atrophy of the inner retina was observed 1-3 weeks after BRVO. Injection of the caspase inhibitor Z-VAD (24 hours after BRVO) decreased the amount of apoptotic cell bodies 48 hours after BRVO.. This study shows that although necrosis is the predominant form of neuronal death in the early phase, massive delayed neuronal cell death caused by apoptosis occurs on a widespread basis as a result of chronic ischaemia after BRVO in the retina. Further studies are needed to evaluate the possibility of rescuing retinal neurons from death by neuroprotective treatments.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Atrophy; Caspase Inhibitors; Caspases; Chronic Disease; Cysteine Proteinase Inhibitors; Edema; In Situ Nick-End Labeling; Injections; Ischemia; Microcirculation; Necrosis; Neuroprotective Agents; Retina; Retinal Diseases; Retinal Vein Occlusion; Retinal Vessels; Swine; Swine, Miniature; Time Factors; Vitreous Body

One of the earliest effects of hypoxia on neuronal function is to produce a run-down of synaptic transmission, and more prolonged hypoxia results in neuronal death. An increase in the permeability of the outer mitochondrial membrane, controlled by BCL-2 family proteins, occurs in response to stimuli that trigger cell death. By patch clamping mitochondrial membranes inside the presynaptic terminal of a squid giant synapse, we have now found that several minutes of hypoxia trigger the opening of large multiconductance channels. The channel activity is induced concurrently with the attenuation of synaptic responses that occurs under hypoxic conditions. Hypoxia-induced channels are inhibited by NADH, an agent that inhibits large conductance channels produced by a pro-apoptotic fragment of BCL-xL in these synaptic mitochondria. The appearance of hypoxia-induced channels was also prevented by the caspase/cysteine protease inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-fmk), which inhibits proteolysis of BCL-xL during hypoxia. Both NADH and Z-VAD-fmk reduced significantly the rate of decline of synaptic responses during hypoxia. Our results indicate that an increase in outer mitochondrial channel activity is a very early event in the response of neurons to hypoxia and suggest that this increase in activity may contribute to the decline in synaptic function during hypoxia.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-X Protein; Cell Proliferation; Enzyme Inhibitors; Hypoxia; Intracellular Membranes; Ischemia; Membrane Potentials; Mitochondria; Mollusca; NAD; Neurons; Patch-Clamp Techniques; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Synapses; Time Factors

Progressive liver dysfunction contributes significantly to the development of multiple organ failure after trauma/hemorrhage. This study tested the relative impact of necrotic and apoptotic cell death in a graded model of hemorrhagic shock (mean arterial blood pressure=35+/-5 mmHg for 1, 2, or 3 h, followed by 2 h, 1 h, or no resuscitation, respectively) in rats. Prolonged periods of hemorrhagic hypotension (3 h) were paralleled by a profound decrease of hepatic ATP levels and occurrence of pericentral necrosis. Resuscitation after shorter periods of hemorrhagic hypotension resulted in restoration of tissue ATP whereas hepatocellular function as assessed by indocyanine green clearance remained depressed (49.9+/-1.6 mL/(min x kg) at baseline, 28.8+/-1.2 mL/(min x kg) after 2 h of resuscitation; P<0.05). Under these conditions, induction of caspase activity and DNA fragmentation were observed in pericentral hepatocytes that could be prevented by the radical scavenger tempol. Pretreatment with z-Val-Ala-Asp(O-methyl)-flouromethylketone prevented de novo expression of caspase-generated cytokeratin 18, DNA fragmentation, and depression of hepatocellular indocyanine green clearance. These data suggest that prolonged low flow/hypoxia induces ATP depletion and pericentral necrosis and restoration of oxygen supply and ATP levels after shorter periods of low flow ischemia propagate programmed cell death or "pericentral apoptosis."

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Apoptosis; Caspase Inhibitors; Cyclic N-Oxides; Cysteine Proteinase Inhibitors; Hepatocytes; Hypotension; Indocyanine Green; Ischemia; Liver; Male; Necrosis; Rats; Rats, Sprague-Dawley; Resuscitation; Shock, Hemorrhagic; Spin Labels

Understanding the mechanisms of injury associated with cardiac arrest is essential for defining strategies aimed at improving preservation and function of kidneys harvested in non-heart-beating (NHB) donors.. We standardized a model of NHB donors in rats and studied the kinetics and types (apoptosis vs. necrosis) of renal cell death developing during cold storage. Using quantitative polymerase chain reaction, immunoblotting, and caspase inhibition, we also studied the molecular pathways regulating renal cell death in this model.. The kinetics and extent of cell death developing in cortical tubules during cold storage were found to be increased in non-heart-beating (NHB) kidneys. Apoptosis of cortical tubules predominated in NHB kidneys exposed to 10 hr of cold storage, whereas necrosis increased after longer periods of cold ischemia. Shortly after cardiac arrest, a rapid up-regulation of Bax and Hsp 70 was found at the protein level in NHB kidneys. After 24 hr of cold storage, induction of Bax was maintained, whereas protein levels of Hsp70 returned to levels comparable to heart-beating (HB) controls. Also, mRNA levels of Bax were found to increase during cold storage in NHB kidneys. Cortical cell death was found to be largely caspase-independent but responsive to hydroxyl-radical scavenging with dimethyl sulfoxide (DMSO).. Cardiac arrest promotes activation of death-inducing molecules such as Bax and is associated with increased development of caspase-independent renal cell death during cold storage. Developing strategies, such as free radical scavenging, aimed at inhibiting cell death during cold storage, could prove useful for improving preservation of NHB kidneys.

Topics: Amino Acid Chloromethyl Ketones; Animals; bcl-2-Associated X Protein; bcl-X Protein; Caspase Inhibitors; Caspases; Cell Death; Cold Temperature; Cysteine Proteinase Inhibitors; Heart Arrest; HSP70 Heat-Shock Proteins; Ischemia; Kidney Cortex; Kidney Transplantation; Male; Necrosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Inbred F344; Tumor Suppressor Protein p53

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blood Urea Nitrogen; Caspases; Chemotaxis, Leukocyte; Cysteine Proteinase Inhibitors; Cytokines; Depression, Chemical; Drug Administration Schedule; Epidermal Growth Factor; Humans; In Situ Nick-End Labeling; Insulin-Like Growth Factor I; Ischemia; Kidney; Male; Mice; Neoplasm Proteins; Nephritis; Peroxidase; Protein Processing, Post-Translational; Recombinant Proteins; Reperfusion Injury; RNA-Binding Proteins