cytochrome-c-t and Ischemia

Cardiolipin is a unique phospholipid which is almost exclusively located at the level of the inner mitochondrial membrane where it is biosynthesized. This phospholipid is known to be intimately involved in several mitochondrial bioenergetic processes. In addition, cardiolipin also has active roles in several of the mitochondrial-dependent steps of apoptosis and in mitochondrial membrane dynamics. Alterations in cardiolipin structure, content and acyl chains composition have been associated with mitochondrial dysfunction in multiple tissues in several physiopathological conditions, including ischemia/reperfusion, different thyroid states, diabetes, aging and heart failure. Cardiolipin is particularly susceptible to ROS attack due to its high content of unsaturated fatty acids. Oxidative damage to cardiolipin would negatively impact the biochemical function of the mitochondrial membranes altering membrane fluidity, ion permeability, structure and function of components of the mitochondrial electron transport chain, resulting in reduced mitochondrial oxidative phosphorylation efficiency and apoptosis. Diseases in which mitochondrial dysfunction has been linked to cardiolipin peroxidation are described. Ca(2+), particularly at high concentrations, appears to have several negative effects on mitochondrial function, some of these effects being linked to CL peroxidation. Cardiolipin peroxidation has been shown to participate, together with Ca(2+), in mitochondrial permeability transition. In this review, we provide an overview of the role of CL peroxidation and Ca(2+) in mitochondrial dysfunction and disease.

Topics: Animals; Calcium; Cardiolipins; Cytochromes c; Ischemia; Lipid Peroxidation; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Reactive Oxygen Species

Positive changes were more pronounced in patients with chronic ischemia of the lower limbs treated with energostim alone and in combination with trental in comparison with patients receiving trental monotherapy. The best effect was attained in patients treated with energostim in combination with trental.

Topics: Adult; Aged; Aged, 80 and over; Arteriosclerosis; Coronary Disease; Cytochromes c; Drug Combinations; Echocardiography; Female; Humans; Inosine; Ischemia; Laser-Doppler Flowmetry; Leg; Male; Microcirculation; Middle Aged; NAD; Pentoxifylline

Selenomethionine (SeMet) is known to alleviate ischemia-reperfusion (I/R) injury. However, its details of action have not been thoroughly elucidated in mice with intestinal I/R injury. In this study, intestinal I/R injury mice models were established, and ELISAs were performed to determine the levels of redox factors, including glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA), in mice intestinal tissues. Furthermore, several apoptosis-related markers, such as cytochrome c (Cyt-c), Bcl-2, and Bax, were detected using qPCR and Western blotting, while caspase-3 was detected using Western blotting alone. The results showed that SeMet alleviated I/R damage by increasing GSH-Px, CAT, and SOD levels and reducing MDA levels. Our data demonstrated that SeMet reduced I/R injury and inhibited the expression of Cyt-c, Bax, and caspase-3. SeMet also increased the expression of Bcl-2 in the intestinal tissues of mice. In addition, the TUNEL assay results showed that SeMet mitigated apoptosis in the villi cells of the intestinal mucosa. The findings also revealed that I/R could lead to increased apoptosis levels and that SeMet alleviated I/R-induced apoptosis by mediating the Bax/cytochrome C/caspase-3 apoptotic signaling pathways in the intestinal I/R injury mice models. Thus, SeMet inhibited apoptosis and resulted in an increase of Bcl-2 levels; downregulated the expression of Bax, Cyt-c, and caspase-3; and alleviated the intestinal ischemia injury in mice. The I/R injury increased the cytosolic Bax, Cyt-c, and caspase-3 levels and significantly decreased Bcl-2 expression levels in the I/R group, compared to the Sham group. However, the levels of all markers were reversed post-SeMet pre-treatment.

Topics: Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; Cytochromes c; Glutathione Peroxidase; Ischemia; Mice; Proto-Oncogene Proteins c-bcl-2; Reperfusion Injury; Selenomethionine; Superoxide Dismutase

The adaptation of mitochondrial homeostasis to ischemic injury is not fully understood. Here, we studied the role of dynamin-related protein 1 (Drp1) in this process. We found that mitochondrial morphology was altered in the early stage of ischemic injury while mitochondrial dysfunction occurred in the late stage of ischemia. Drp1 appeared to inhibit mitophagy by upregulating mito-Clec16a, which suppressed mito-Parkin recruitment and subsequently impaired the formation of autophagosomes in vascular tissues after ischemic injury. Moreover, ischemia-induced Drp1 activation enhanced apoptosis through inducing mitochondrial translocation of BAX and thereby increasing release of Cytochrome C to activate caspase-3/-9 signalling. Furthermore, Drp1 mediated metabolic disorders and inhibited the levels of mitochondrial glutathione to impair free radical scavenging, leading to further increases in ROS and the exacerbation of mitochondrial dysfunction after ischemic injury. Together, our data suggest a critical role for Drp1 in ischemic injury.

Topics: Animals; Apoptosis; Autophagosomes; Cell Line, Tumor; Cytochromes c; Dynamins; Glutathione; Ischemia; Mitochondria; Mitochondrial Dynamics; Mitophagy; Rats, Sprague-Dawley; Reperfusion Injury

Autophagy is a dynamic recycling process responsible for the breakdown of misfolded proteins and damaged organelles, providing nutrients and energy for cellular renovation and homeostasis. Loss of autophagy is associated with cardiovascular diseases. Caveolin-3 (Cav-3), a muscle-specific isoform, is a structural protein within caveolae and is critical to stress adaptation in the heart. Whether Cav-3 plays a role in regulating autophagy to modulate cardiac stress responses remains unknown. In the present study, we used HL-1 cells, a cardiac muscle cell line, with stable Cav-3 knockdown (Cav-3 KD) and Cav-3 overexpression (Cav-3 OE) to study the impact of Cav-3 in regulation of autophagy. We show that traditional stimulators of autophagy (i.e., rapamycin and starvation) result in upregulation of the process in Cav-3 OE cells while Cav-3 KD cells have a blunted response. Cav-3 coimmunoprecipitated with beclin-1 and Atg12, showing an interaction of caveolin with autophagy-related proteins. In the heart, autophagy may be a major regulator of protection from ischemic stress. We found that Cav-3 KD cells have a decreased expression of autophagy markers [beclin-1, light chain (LC3-II)] after simulated ischemia and ischemia-reperfusion (I/R) compared with WT, whereas OE cells showed increased expression. Moreover, Cav-3 KD cells showed increased cell death and higher level of apoptotic proteins (cleaved caspase-3 and cytochrome c) with suppressed mitochondrial function in response to simulated ischemia and I/R, whereas Cav-3 OE cells were protected and had preserved mitochondrial function. Taken together, these results indicate that autophagy regulates adaptation to cardiac stress in a Cav-3-dependent manner.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Caspase 3; Caveolae; Caveolin 3; Cytochromes c; Heart; Ischemia; Mice; Myocardial Reperfusion Injury; Myocytes, Cardiac; Reperfusion

Demyelination is one of the most important pathological factors of spinal cord injury. Oligodendrocyte apoptosis is involved in triggering demyelination. However, fewer reports on pathological changes and mechanism of demyelination have been presented from compressed spinal cord injury (CSCI). The relative effect of oligodendrocyte apoptosis on CSCI-induced demyelination and the mechanism of apoptosis remain unclear.. In this study, a custom-designed model of CSCI was used to determine whether or not demyelination and oligodendrocyte apoptosis occur after CSCI. The pathological changes in axonal myelinated fibers were investigated by osmic acid staining and transmission electron microscopy. Myelin basic protein (MBP), which is used in myelin formation in the central nervous system, was detected by immunofluorescence and Western blot assays. Oligodendrocyte apoptosis was revealed by in situ terminal-deoxytransferase-mediated dUTP nick-end labeling. To analyze the mechanism of oligodendrocyte apoptosis, we detected caspase-12 [a representative of endoplasmic reticulum (ER) stress], cytochrome c (an apoptotic factor and hallmark of mitochondria), and inhibitor of DNA binding 2 (Id2, an oligodendrocyte lineage gene) by immunofluorescence and Western blot assays.. The custom-designed model of CSCI was successfully established. The rats were spastic, paralyzed, and incontinent. The Basso, Beattie, and Bresnahan (BBB) locomotor rating scale scores were decreased as time passed. The compressed spinal cord slices were ischemic. Myelin sheaths became swollen and degenerative; these sheaths were broken down as time passed after CSCI. MBP expression was downregulated after CSCI and consistent with the degree of demyelination. Oligodendrocyte apoptosis occurred at 1 day after CSCI and increased as caspase-12 expression was enhanced and cytochrome c was released. Id2 was distributed widely in the white matter. Id2 expression increased with time after CSCI.. Demyelination occurred after CSCI and might be partly caused by oligodendrocyte apoptosis, which was positively correlated with ER-mitochondria interactions and enhanced Id2 expression after CSCI in rats.

Topics: Animals; Apoptosis; Axons; Blotting, Western; Caspase 12; Cytochromes c; Demyelinating Diseases; Endoplasmic Reticulum; Fluorescent Antibody Technique; In Situ Nick-End Labeling; Inhibitor of Differentiation Protein 2; Ischemia; Lumbar Vertebrae; Microscopy, Electron, Transmission; Mitochondria; Myelin Basic Protein; Oligodendroglia; Organelles; Rats; Rats, Sprague-Dawley; Spinal Cord Compression

Uterine artery occlusion by laparoscopy (UAOL) has been used for the treatment of uterine fibroids and beneficial effects to patients have been shown in clinical studies since 2000. Fibroid cells are more susceptible to apoptosis than myometrial cells under hypoxic conditions, but the molecular mechanisms underlying this effect remain unclear. The aim of this study was to investigate the role of intracellular calcium (Ca(2+)) release mediated by Ca(2+) channel inositol 1,4,5 trisphosphate receptor1 (IP3R1)/ryanodine receptor1 (RYR1) in the apoptosis of uterine fibroid cells under hypoxia.. We compared the expressions of IP3R1 and RYR1 in fibroid and surrounding myometrial tissue from 20 patients before UAOL. After 6h treatment under hypoxia (1% O2) with or without Ca(2+) channel blockers (heparin or/and ruthenium red), the intracellular Ca(2+) concentration, cytochrome c (Cytc) protein and cell apoptosis were determined.. IP3R1 and RYR1 mRNA and protein levels were significantly higher in fibroid than in myometrial tissues. Under hypoxic conditions, Ca(2+) concentration in fibroid cells was significantly higher than in myometrial cells (Ca(2+): 82.69±16.92nmol/L vs 46.14±9.11nmol/L, P<0.05), and Cytc increased similarly in fibroid cells. These increases in Ca(2+) concentration, Cytc and cell apoptosis were significantly reversed by calcium blocker in fibroid cells.. This study demonstrated that intracellular calcium release mediated by IP3R1/RYR1 could induce apoptosis in uterine fibroid cells under hypoxic conditions, and was responsible for the susceptibility to apoptosis of fibroid cells under UAOL.

Topics: Adult; Apoptosis; Calcium; Calcium Channel Blockers; Cell Hypoxia; Cells, Cultured; Cytochromes c; Female; Gene Expression; Humans; Inositol 1,4,5-Trisphosphate Receptors; Ischemia; Laparoscopy; Leiomyoma; Middle Aged; Myometrium; RNA, Messenger; Ryanodine Receptor Calcium Release Channel; Uterine Artery; Uterine Neoplasms; Uterus

Global increases in small ubiquitin-like modifier (SUMO)-2/3 conjugation are a neuroprotective response to severe stress but the mechanisms and specific target proteins that determine cell survival have not been identified. Here, we demonstrate that the SUMO-2/3-specific protease SENP3 is degraded during oxygen/glucose deprivation (OGD), an in vitro model of ischaemia, via a pathway involving the unfolded protein response (UPR) kinase PERK and the lysosomal enzyme cathepsin B. A key target for SENP3-mediated deSUMOylation is the GTPase Drp1, which plays a major role in regulating mitochondrial fission. We show that depletion of SENP3 prolongs Drp1 SUMOylation, which suppresses Drp1-mediated cytochrome c release and caspase-mediated cell death. SENP3 levels recover following reoxygenation after OGD allowing deSUMOylation of Drp1, which facilitates Drp1 localization at mitochondria and promotes fragmentation and cytochrome c release. RNAi knockdown of SENP3 protects cells from reoxygenation-induced cell death via a mechanism that requires Drp1 SUMOylation. Thus, we identify a novel adaptive pathway to extreme cell stress in which dynamic changes in SENP3 stability and regulation of Drp1 SUMOylation are crucial determinants of cell fate.

Topics: Animals; Apoptosis; Cell Death; Cell Line; Cysteine Endopeptidases; Cytochromes c; Cytosol; Dynamins; eIF-2 Kinase; Embryo, Mammalian; Gene Expression Regulation; Glucose; GTP Phosphohydrolases; Humans; Ischemia; Mice; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Models, Biological; Mutation; Neurons; Oxygen; Rats; Small Ubiquitin-Related Modifier Proteins; Sumoylation

Cannabinoid type 2 (CB2) receptor agonists can protect myocardium against ischemia/reperfusion (I/R) injury although the underlying mechanism remains unclear. Here we report the antiapoptotic effect of CB2 receptor agonist, JWH133, during myocardial ischemia/reperfusion injury and potential underlying mechanisms.. Ischemia was performed by blocking left coronary artery of rat for 30 min. After ischemia for 30 min, the rat heart was reperfused for 120 min by loosing the ligation of blocking left coronary artery. JWH133 (20 mg/kg), a CB2 receptor selective agonist, or vehicles were injected intravenously 5 minutes before ischemia. Infarct size of myocardium was assessed by histological stain, myocardial apoptosis index (AI) was determined by TUNEL, and mitochondrial membrane potential (∆Ψm) was measured by flow cytometry. Western blots were performed to measure the cytochrome c release, cleaved caspase 3, cleaved caspase 9 and PI3K/Akt kinase phosphorylation.. JWH133 significantly reduced the infarct size and AI of myocardium suffering I/R compared to vehicle-treated group. Further mechanistic study revealed that activation of CB2 receptor by JWH133 inhibited the loss of ΔΨm, reduction of the cleaved caspases-3 and -9, release of mitochondrial cytochrome c to the cytosol, and increase of phosphorylated Akt. These JWH133-mediated effects could be totally abrogated by PI3K inhibitor wortmanin or CB2 receptor antagonist AM630.. Our results demonstrate that activation of CB2 receptor by JWH133 prevent apoptosis during ischemia/reperfusion through inhibition of the intrinsic mitochondria-mediated apoptotic pathway and involvement of the PI3K/Akt signal pathway.

Topics: Androstadienes; Animals; Apoptosis; Cannabinoids; Caspase 3; Caspase 9; Cytochromes c; Indoles; Ischemia; Male; Membrane Potential, Mitochondrial; Mitochondria; Myocardial Reperfusion Injury; Myocardium; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Signal Transduction; Wortmannin

The aim of this study was to investigate the effect of 17β-estradiol (E2) on hepatocyte apoptosis after reduced-size hepatic ischemia/reperfusion (I/R) injury and its mechanism. A rat model of reduced-size hepatic I/R injury was established. Sprague-Dawley rats were randomly allocated into sham, I/R, and E2 + I/R group. 17β-Estradiol (4 mg/kg) or the vehicle was administered i.p. 1 h before ischemia and immediately after operation. For each group, 10 rats were used to investigate the survival during a week after reperfusion. Blood samples and liver tissues were obtained in the remaining animals after 3, 6, 12, and 24 h of reperfusion to assess serum aspartate aminotransferase and alanine aminotransferase levels, liver tissue malondialdehyde concentration, superoxide dismutase activity, and histopathologic changes. Apoptosis ratio; expression of cytochrome c, Bcl-2, and Bax proteins; and enzymatic activities of caspase 9 and caspase 3 were performed in the samples at 12 h after reperfusion. The serum aspartate aminotransferase and alanine aminotransferase levels and tissue malondialdehyde concentration were increased in the I/R group, whereas the increase was significantly reduced by E2. The superoxide dismutase activity, depressed by I/R injury, was elevated back to normal levels by treatment with E2. Severe hepatic damage was observed by light microscopy in the I/R group, whereas administration of E2 resulted in tissue and cellular preservation. Furthermore, E2 inhibited hepatocellular apoptosis by upregulating the ratio of Bcl-2 and Bax expression, reduced cytosolic cytochrome c level, and decreased caspase 9 and caspase 3 activities. The 7-day survival rate was significantly higher in the E2 + I/R group than in the I/R group. These results indicated that E2 protects liver tissues from reduced-size hepatic I/R injury by suppressing mitochondrial apoptotic pathways.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cytochromes c; Cytosol; Estradiol; Ischemia; Liver; Male; Malondialdehyde; Mitochondria; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Transaminases

Brain ischemia has major consequences leading to the apoptosis of astrocytes and neurons. Glucose-regulated protein 78 (GRP78) known for its role in endoplasmic reticulum stress alleviation was discovered on several cell surfaces acting as a receptor for signaling pathways. We have previously described peptides that bind cell surface GRP78 on endothelial cells to induce angiogenesis. We have also reported that ADoPep1 binds cardiomyocytes to prevent apoptosis of ischemic heart cells. In this study we describe the effect of hypoxia on astrocytes and neurons cell surface GRP78. Under hypoxic conditions, there was an increase of more than fivefold in GRP78 on cell surface of neurons while astrocytes were not affected. The addition of the GRP78 binding peptide, ADoPep1, to neurons decreased the percentage of GRP78 positive cells and did not change the percent of astrocytes. However, a significant increase in early and late apoptosis of both astrocytes and neurons under hypoxia was attenuated in the presence of ADoPep1. Intravitreal administration of ADoPep1 to mice in a model of optic nerve crush significantly reduced retinal cell loss after 21 days compared to the crush-damaged eyes without treatment or by control saline vehicle injection. Histological staining demonstrated reduced GRP78 after ADoPep1 treatment. The mechanism of peptide neuroprotection was demonstrated by the inhibition of hypoxia induced caspase 3/7 activity, cytochrome c release and p38 phosphorylation. This study is the first report on hypoxic neuronal and astrocyte cell surface GRP78 and suggests a potential therapeutic target for neuroprotection.

Topics: Animals; Apoptosis; Astrocytes; Cell Hypoxia; Cells, Cultured; Cytochromes c; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Ischemia; Mice; Mice, Inbred C57BL; Neurons; Oligopeptides; Optic Nerve; Phosphorylation; Signal Transduction

We tested the hypothesis that obesity reduced circulating number of endothelial progenitor cells (EPCs), angiogenic ability, and blood flow in ischemic tissue that could be reversed after obesity control.. 8-week-old C57BL/6J mice (n=27) were equally divided into group 1 (fed with 22-week control diet), group 2 (22-week high fat diet), and group 3 (14-week high fat diet, followed by 8-week control diet). Critical limb ischemia (CLI) was induced at week 20 in groups 2 and 3. The animals were sacrificed at the end of 22 weeks.. Heart weight, body weight, abdominal fat weight, serum total cholesterol level, and fasting blood sugar were highest in group 2 (all p<0.001). The numbers of circulating EPCs (C-kit/CD31+, Sca-1/KDR + and CXCR4/CD34+) were lower in groups 1 and 2 than in group 3 at 18 h after CLI induction (p<0.03). The numbers of differentiated EPCs (C-kit/CD31+, CXCR4/CD34+ and CD133+) from adipose tissue after 14-day cultivation were also lowest in group 2 (p<0.001). Protein expressions of VCAM-1, oxidative index, Smad3, and TGF-β were higher, whereas the Smad1/5 and BMP-2, mitochondrial cytochrome-C SDF-1α and CXCR4 were lower in group 2 than in groups 1 and 3 (all p<0.02). Immunofluorescent staining of CD31+ and vWF + cells, the number of small vessel (<15 μm), and blood flow through Laser Doppler scanning of ischemic area were lower in group 2 compared to groups 1 and 3 on day 14 after CLI induction (all p<0.001).. Obesity suppressed abilities of angiogenesis and recovery from CLI that were reversed by obesity control.

Topics: Adipose Tissue; Animals; Biomarkers; Cell Movement; Cytochromes c; Cytosol; Endothelial Cells; Fibrosis; Fluorescent Antibody Technique; Hindlimb; Inflammation; Ischemia; Laser-Doppler Flowmetry; Male; Mice; Mitochondria; Neovascularization, Physiologic; Obesity; Oxidative Stress; Regional Blood Flow; Stem Cells

Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia-ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.

Topics: Animals; Animals, Newborn; Apoptosis; Binding Sites; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochromes c; Disease Models, Animal; Hypoxia-Ischemia, Brain; Ischemia; Mice; Neuroprotective Agents; Oligopeptides; Quinolines; Rats

Recently, nitrite has been rediscovered as a physiologically relevant storage reservoir of nitric oxide in blood and it can readily be converted to nitric oxide under hypoxic and acidic conditions. In this study, the authors evaluated the therapeutic efficacy of nitrite on reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle.. The authors used a vascular pedicle isolated rat cremaster model that underwent 4 hours of warm ischemia followed by 2 hours or 17 hours of reperfusion. At 5 minutes before reperfusion, normal saline, sodium nitrite (0.20 μM/minute/kg), or nitrite mixed with 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (potassium salt) (0.2 mg/minute/kg) was infused into the microcirculation of ischemic cremaster by means of intraarterial infusion. Ischemia-reperfusion-induced microcirculatory alterations were measured after 2 hours of reperfusion. Microvasculature of the cremaster muscle including the vascular pedicle was harvested to determine the mitochondrial dysfunction. The blood concentration of methemoglobin was also measured to determine the toxicity of nitrite.. The authors found that nitrite significantly attenuated ischemia-reperfusion-induced vasoconstriction, arteriole stagnation, and capillary no-reflow in the early phase of reperfusion and the depolarization of mitochondrial membrane potential and cytochrome c release in the late phase of reperfusion. Nitrite-induced protection was significantly blocked by a nitric oxide scavenger (potassium salt). The methemoglobin results showed that the doses of nitrite we used in the present study were safe.. The supplementation of a low dose of nitrite, directly into the microcirculation of ischemic muscle through local intraarterial infusion, significantly attenuated ischemia-reperfusion-induced microcirculatory alterations in vivo and mitochondrial dysfunction in vitro in the microvasculature of skeletal muscle.

Topics: Analysis of Variance; Animals; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Ischemia; Male; Membrane Potentials; Methemoglobin; Microcirculation; Mitochondria, Muscle; Muscle, Skeletal; Nitrites; Random Allocation; Rats; Rats, Sprague-Dawley; Reference Values; Reperfusion Injury

Bromodeoxyuridine incorporates into DNA during mitosis. A long-term stability of the incorporated BrdU is important for the recovery of BrdU-labeled cells. For testing the stability of BrdU incorporation into DNA we pulse-labeled mesenchymal stem cells with BrdU and observed these cells in vitro over 4 weeks. During this time the BrdU-signal was permanently decreasing. Starting with cells containing evenly stained BrdU-nuclei, so-called filled cells, already 3 days after BrdU removal we detected cells containing so-called segmented and punctated BrdU-signals. The number of those labeled cells continuously increased over time. Interestingly, the loss of BrdU in the nucleus was accompanied by an increasing labeling of the cytosol. Further, we injected BrdU intraperitoneally into rats after ischemia and detected BrdU-positive cells in the hippocampus 3 and 23 days after the last BrdU injection. While after 3 days most of the BrdU-positive cells in the hippocampus displayed a filled BrdU-signal, 23 days after BrdU removal an increased number of segmented and punctated BrdU-positive nuclei was detected. The gradual degradation of the BrdU-signal was not caused by cell death. The consequence of this BrdU degradation would be an underestimation of cell proliferation and an overestimation of cell death of newly generated cells.

Topics: Animals; Apoptosis; Bone Marrow Cells; Bromodeoxyuridine; Caspase 3; Cell Count; Cell Proliferation; Cells, Cultured; Cytochromes c; Doublecortin Domain Proteins; Enzyme Inhibitors; Hippocampus; Ischemia; Mesenchymal Stem Cells; Microtubule-Associated Proteins; Neuropeptides; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Statistics, Nonparametric; Staurosporine; Time Factors

Although there is growing awareness of the beneficial potential of onion intake to lower the risk of cardiovascular disease, there is little information about the effect of onion on ischemic heart injury, one of the most common cardiovascular diseases.. This study investigates the effect of the methanol-soluble extract of onion on ischemic injury in heart-derived H9c2 cells in vitro and in rat hearts in vivo. The underlying mechanism is also investigated.. To evaluate the effect of onion on ischemia-induced cell death, LDH release and TUNEL-positivity were assessed in H9c2 cells, and the infarct size was measured in a myocardial infarct model. To investigate the mechanism of the cardioprotection by onion, the reactive oxygen species (ROS) level and the mitochondrial membrane potential (DeltaPsi(m)) were measured using an imaging technique; the caspase-3 activity was assayed, and Western blotting was performed to examine cytochrome c release in H9c2 cells.. The methanolic extract of onion had a preventive effect on ischemia/hypoxia-induced apoptotic death in H9c2 cells in vitro and in rat heart in vivo. The onion extract (0.05 g/ml) inhibited the elevation of the ROS, mitochondrial membrane depolarization, cytochrome c release and caspase-3 activation during hypoxia in H9c2 cells. In the in vivo rat myocardial infarction model, onion extract (10 g/kg) significantly reduced the infarct size, the apoptotic cell death of the heart and the plasma MDA level.. In conclusion, the results of this study suggest that the methanolic extract of onion attenuates ischemia/hypoxia-induced apoptosis in heart-derived H9c2 cells in vitro and in rat hearts in vivo, through, at least in part, an antioxidant effect.

Topics: Animals; Antioxidants; Apoptosis; Blotting, Western; Caspase 3; Cell Hypoxia; Coronary Vessels; Cytochromes c; Humans; In Situ Nick-End Labeling; Ischemia; Malondialdehyde; Membrane Potential, Mitochondrial; Myocytes, Cardiac; Onions; Plant Extracts; Rats; Reactive Oxygen Species

To evaluate development of brain injury after hind limbs ischemia/reperfusion (LI/R) in rats, and the effect of MK801 on the brain injury following LI/R.. The limbs ischemia/reperfusion model was established in rats. The MDA contents were evaluated in each group, apoptotic cells were detected with TUNEL, the expression of apoptosis-associated protein, such as bcl-2, cytoC and caspase-3 were determined with immunohistochemistry and Western-blot.. The contents of MDA in brain tissue increased significantly following LI/R. The expression of bcl-2, cytoC, Caspase-3 was increased than those in the control group (P < 0.01) following LI/R significantly. The expression of Caspase-3 was increased 24 h after the onset of reperfusion. The expression of Caspase-3, bcl-2 gene was quite obvious in the midbrain red nucleus region. MK801 inhibited the expression of bcl-2, cytoC, Caspase-3 obviously.. The excessive apoptosis and apoptosis-associated factors could play an important role in the brain injury following LI/R in rat, MK801 might decrease the production of free radical and the excite toxicity of glutamate, inhibit the expression of apoptosis associated protein and reduce the occurrence of apoptosis.

Topics: Animals; Apoptosis; Brain Injuries; Caspase 3; Cytochromes c; Dizocilpine Maleate; Extremities; Ischemia; Male; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reperfusion Injury

In the last decade, various groups have found evidence of nitric oxide production by mitochondrial nitric oxide synthase (mNOS) in a range of experimental models. However, little is known about the role of mNOS in renal ischemia-reperfusion (I/R) injury and its possible involvement in the apoptotic pathway. We analyzed the role of mNOS in apoptosis promotion in rat kidney I/R and its direct implication through experiments in which isolated kidney mitochondria were subjected to hypoxia/reoxygenation. Results showed that neuronal NOS located in the inner mitochondrial membrane is upregulated during renal I/R and that this upregulation, together with the increase in nitric oxide production, is involved in the generation of intramitochondrial peroxynitrite, which in turn leads to cytochrome c release and apoptosis induction in renal I/R.

Topics: Animals; Apoptosis; Biomarkers; Blotting, Western; Cytochromes c; Densitometry; Enzyme Inhibitors; Ischemia; Kidney; Male; Mitochondria; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type I; Nitrites; Peroxynitrous Acid; Rats; Rats, Wistar; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Up-Regulation

In ischemia-reperfusion injuries, elevated calcium and reactive oxygen species (ROS) induce mitochondrial permeability transition (mPT), which plays a pivotal role in mediating damages and cell death. Inhibition of mPT decreases necrotic cell death; however, during reperfusion, the continuous production of ROS may contribute to the temporary opening of the pore and thus the onset of the delayed apoptotic cell death. Based on amiodarone structure, we developed the first SOD-mimetic mPT inhibitor (HO-3538) that can eliminate ROS in the microenvironment of the permeability pore. In isolated mitochondria, HO-3538 inhibited mPT and the release of proapoptotic mitochondrial proteins. It had a ROS scavenging effect and antiapoptotic effect in a cardiomyocyte line and it diminished release of mitochondrial proapoptotic proteins. Furthermore, HO-3538 significantly enhanced the recovery of mitochondrial energy metabolism and functional cardiac parameters; decreased infarct size, lipid peroxidation, and protein oxidation; and suppressed necrotic as well as apoptotic cell death pathways in Langendorff-perfused hearts. In these respects it was somewhat superior to its two constituents, amiodarone and a pyrrol-derivative free radical scavenger. These data suggest that the SOD-mimetic mPT inhibitors are ideal candidates for drug development for the alleviation of postinfarct myocardial injuries.

Topics: Amiodarone; Animals; Apoptosis; Cytochromes c; Humans; Ischemia; Jurkat Cells; Magnetic Resonance Spectroscopy; Mice; Mitochondria; Myocardial Infarction; Necrosis; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase

Cardiolipin peroxidation plays a critical role in mitochondrial cytochrome c release and subsequent apoptotic process. Mitochondrial pore transition (MPT) is considered as an important step in this process. In this work, the effect of peroxidized cardiolipin on MPT induction and cytochrome c release in rat heart mitochondria was investigated. Treatment of mitochondria with micromolar concentrations of cardiolipin hydroperoxide (CLOOH) resulted in a dose-dependent matrix swelling, DeltaPsi collapse, release of preaccumulated Ca2+ and release of cytochrome c. All these events were inhibited by cyclosporin A and bongkrekic acid, indicating that peroxidized cardiolipin behaves as an inducer of MPT. Ca2+ accumulation by mitochondria was required for this effect. ANT (ADP/ATP translocator) appears to be involved in the CLOOH-dependent MPT induction, as suggested by the modulation by ligands and inhibitors of adenine nucleotide translocator (ANT). Together, these results indicate that peroxidized cardiolipin lowers the threshold of Ca2+ for MPT induction and cytochrome c release. This synergistic effect of Ca2+ and peroxidized cardiolipin on MPT induction and cytochrome c release in mitochondria, might be important in regulating the initial phase of apoptosis and also may have important implications in those physiopathological situations, characterized by both Ca2+ and peroxidized cardiolipin accumulation in mitochondria, such as aging, ischemia/reperfusion and other degenerative diseases.

Topics: Aging; Animals; Apoptosis; Calcium; Cardiolipins; Cell Membrane Permeability; Cytochromes c; Dose-Response Relationship, Drug; Ischemia; Lipid Peroxidation; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Rats

We previously showed that "ischemia" (abrupt cessation of flow) leads to rapid membrane depolarization and increased generation of reactive oxygen species (ROS) in lung microvascular endothelial cells. This response is not associated with anoxia but, rather, reflects loss of normal shear stress. This study evaluated whether a similar response occurs in aortic endothelium. Plasma membrane potential and production of ROS were determined by fluorescence microscopy and cytochrome c reduction in flow-adapted rat or mouse aorta or monolayer cultures of rat aortic endothelial cells. Within 30 s after flow cessation, endothelial cells that had been flow adapted showed plasma membrane depolarization that was inhibited by pretreatment with cromakalim, an ATP-sensitive K(+) (K(ATP)) channel agonist. Flow cessation also led to ROS generation, which was inhibited by cromakalim and the flavoprotein inhibitor diphenyleneiodonium. Aortic endothelium from mice with "knockout" of the K(ATP) channel (K(IR)6.2) showed a markedly attenuated change in membrane potential and ROS generation with flow cessation. In aortic endothelium from mice with knockout of NADPH oxidase (gp91(phox)), membrane depolarization was similar to that in wild-type mice but ROS generation was absent. Thus rat and mouse aortic endothelial cells respond to abrupt flow cessation by K(ATP) channel-mediated membrane depolarization followed by NADPH oxidase-mediated ROS generation, possibly representing a cell-signaling response to altered mechanotransduction.

Topics: Animals; Aorta; Cell Membrane; Cells, Cultured; Cytochromes c; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Ischemia; Male; Mechanotransduction, Cellular; Membrane Potentials; Mice; Mice, Knockout; NADPH Oxidases; Potassium Channels, Inwardly Rectifying; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Stress, Mechanical

Bcl-2-associated death protein (Bad), a member of the Bcl family, directs astrocytes in primary cultures to enter or resist apoptosis during ischemia in vitro. Under ischemia, Bad was the only Bcl family member whose expression was upregulated significantly during the early stages of an ischemic insult. Increased endogenous Bad was translocated from the cytoplasm to mitochondria to induce apoptosis in astrocytes. Concurrently, ischemia also induced Bad phosphorylation specifically on Ser112 to promote survival. This site-specific phosphorylation of Bad was mediated by an early activation of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) intracellular signaling pathway. This study demonstrates that ischemia-induced Bad plays a dual role in determining whether astrocytes enter or resist apoptosis after an ischemic insult.

Topics: Animals; Animals, Newborn; Apoptosis; Astrocytes; bcl-Associated Death Protein; Blotting, Western; Carrier Proteins; Cells, Cultured; Cerebral Cortex; Cytochromes c; Enzyme Induction; Fluorescent Dyes; Gene Expression Regulation; Green Fluorescent Proteins; Immunohistochemistry; Immunoprecipitation; In Situ Nick-End Labeling; Ischemia; Mice; Mice, Inbred ICR; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Mutagenesis, Site-Directed; Neurons; Phosphatidylinositol 3-Kinases; Phosphorylation; Time Factors; Transfection

Ischemic injuries are associated with several pathological conditions, including stroke and myocardial infarction. Several studies have indicated extensive apoptotic cell death in the infarcted area as well as in the penumbra region of the infarcted tissue. Studies with transgenic animals suggest that the mitochondrion-mediated apoptosis pathway is involved in ischemia-related cell death. This pathway is triggered by activation of pro-apoptotic Bcl-2 family members such as Bax. Here, we have identified and synthesized two low molecular weight compounds that block Bax channel activity. The Bax channel inhibitors prevented cytochrome c release from mitochondria, inhibited the decrease in the mitochondrial membrane potential, and protected cells against apoptosis. The Bax channel inhibitors did not affect the conformational activation of Bax or its translocation and insertion into the mitochondrial membrane in cells undergoing apoptosis. Furthermore, the compounds protected neurons in an animal model of global brain ischemia. The protective effect in the animal model correlated with decreased cytochrome c release in the infarcted area. This is the first demonstration that Bax channel activity is required in apoptosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain; Cell Death; Cell Line; Cell Separation; Cytochromes c; Dose-Response Relationship, Drug; Electrophysiology; Flow Cytometry; Gerbillinae; HeLa Cells; Hippocampus; Humans; Ischemia; Lipids; Liposomes; Mice; Mitochondria; Models, Chemical; Neurons; Protein Conformation; Protein Structure, Quaternary; Recombinant Proteins; Reperfusion; Time Factors

Thrombospondin 1 (TSP-1) is a matricellular protein that inhibits angiogenesis and causes apoptosis in vivo and in vitro in several cancerous cells and tissues. Here we identify TSP-1 as the molecule with the highest induction level at 3 hours of IR injury in rat and mouse kidneys subjected to ischemia/reperfusion (IR) injury using the DNA microarray approach. Northern hybridizations demonstrated that TSP-1 expression was undetectable at baseline, induced at 3 and 12 hours, and returned to baseline levels at 48 hours of reperfusion. Immunocytochemical staining identified the injured proximal tubules as the predominant sites of expression of TSP-1 in IR injury and showed colocalization of TSP-1 with activated caspase-3. Addition of purified TSP-1 to normal kidney proximal tubule cells or cells subjected to ATP depletion in vitro induced injury as demonstrated by cytochrome c immunocytochemical staining and caspase-3 activity. The deleterious role of TSP-1 in ischemic kidney injury was demonstrated directly in TSP-1 null mice, which showed significant protection against IR injury-induced renal failure and tubular damage. We propose that TSP-1 is a novel regulator of ischemic damage in the kidney and may play an important role in the pathophysiology of ischemic kidney failure.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Blotting, Northern; Blotting, Western; Caspase 3; Caspases; CD36 Antigens; Colorimetry; Cytochromes c; DNA, Complementary; Enzyme Activation; Gene Deletion; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Ischemia; Kidney; Kidney Tubules; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Thrombospondin 1; Time Factors

A possible mechanism for D-cis-diltiazem (diltiazem)-mediated improvement of the contractile function of ischemic/reperfused hearts was examined. Thirty-five-min ischemia/60-min reperfusion recovered little the left ventricular developed pressure (LVDP) and decreased myocardial high-energy phosphates (HEPs). Ischemia induced an accumulation of tissue Na+ content, an increase in cytochrome c in the cytosolic fraction, and a decrease in the oxygen consumption rate (OCR) in perfused hearts. Treatment of the heart with 1 microM diltiazem for the last 3-min of pre-ischemia did not affect the decrease in HEPs during ischemia, whereas that with 3 microM partially attenuated the decrease in ATP, suggesting that 3 microM diltiazem exerted energy-sparing effect. Treatment with 1 microM diltiazem enhanced the post-ischemic recovery of LVDP associated with attenuation of the ischemia-induced accumulation of tissue Na+, increase in cytochrome c in the cytosolic fraction, and decrease in myocardial OCR, and restoration of the myocardial HEPs during reperfusion. Combined treatment with diltiazem and a Na+/H+ exchange inhibitor, but not a Na+ channel blocker, facilitated the attenuation of Na+ accumulation in the ischemic heart and the enhancement of the post-ischemic recovery of LVDP. Sodium lactate, a possible metabolite in ischemic hearts, and sodium chloride increased the Na+ concentration in mitochondria, released cytochrome c into incubation medium, and reduced the mitochondrial respiration. Treatment of isolated mitochondria with diltiazem failed to attenuate the sodium lactate- and sodium chloride-induced alterations. These results suggest that the cardioprotection of diltiazem may be exerted via attenuating cytosolic Na+ overload through Na+ channels in the ischemic heart, leading to preservation of mitochondrial functional ability during ischemia, followed by improvement of post-ischemic energy production and contractile recovery.

Topics: Animals; Calcium; Cardiotonic Agents; Cardiovascular Agents; Cytochromes c; Diltiazem; Drug Interactions; Ischemia; Male; Mitochondria; Myocardial Contraction; Myocardial Reperfusion Injury; Myocardium; Oxygen Consumption; Phosphates; Rats; Rats, Wistar; Reperfusion Injury; Respiration; Sodium; Sodium Channel Blockers; Sodium-Hydrogen Exchangers

Apoptosis has been implicated in ischemic renal injury. Thus one strategy of renal protection is to antagonize apoptosis. However, apoptosis inhibitory approaches remain to be fully explored. Zn(2+) has long been implicated in apoptosis inhibition; but systematic analysis of the inhibitory effects of Zn(2+) is lacking. Moreover, whether Zn(2+) blocks renal cell apoptosis following ischemia is unknown. Here, we demonstrate that Zn(2+) is a potent apoptosis inhibitor in an in vitro model of renal cell ischemia. ATP depletion induced apoptosis in cultured renal tubular cells, which was accompanied by caspase activation. Zn(2+) at 10 microM inhibited both apoptosis and caspase activation, whereas Co(2+) was without effect. In ATP-depleted cells, Zn(2+) partially prevented Bax activation and cytochrome c release from mitochondria. In isolated cell cytosol, Zn(2+) blocked cytochrome c-stimulated caspase activation at low-micromolar concentrations. In addition, Zn(2+) could directly antagonize the enzymatic activity of purified recombinant caspases. We conclude that Zn(2+) is a potent inhibitor of apoptosis in renal tubular cells following ATP depletion. Zn(2+) blocks apoptosis at multiple steps including Bax activation, cytochrome c release, apoptosome function, and caspase activation.

Topics: Adenosine Triphosphate; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; Cells, Cultured; Cytochromes c; Cytosol; Ischemia; Kidney Tubules, Proximal; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Recombinant Proteins; Zinc

Cardiac fibroblasts play an essential role in the physiology of the heart. These produce extracellular matrix proteins and synthesize angiogenic and cardioprotective factors. Although fibroblasts of cardiac origin are known to be resistant to apoptosis and to remain metabolically active in situations compromising cell survival, the underlying mechanisms are unknown. Here, we report that cardiac fibroblasts were more resistant than dermal or pulmonary fibroblasts to mitochondria-dependent cell death. Cytochrome c release was blocked in cardiac fibroblasts but not in dermal fibroblasts treated with staurosporine, etoposide, serum deprivation, or simulated ischemia, precluding caspase-3 activation and DNA fragmentation. Resistance to apoptosis of cardiac fibroblasts correlated with the expression of the anti-apoptotic protein Bcl-2, whereas skin and lung fibroblasts did not express detectable levels of this protein. Bcl-x(L,) Bax, and Bak were expressed at similar levels in cardiac, dermal, and lung fibroblasts. In addition, the death of cardiac fibroblasts during hypoxia was not associated with the cleavage of Bid but rather with Bcl-2 disappearance, suggesting the requirement of the mitochondrial apoptotic machinery to execute death receptor-induced programmed cell death. Knockdown of bcl-2 expression by siRNA in cardiac fibroblasts increased their apoptotic response to staurosporine, serum, and glucose deprivation and to simulated ischemia. Moreover, dermal fibroblasts overexpressing Bcl-2 achieved a similar level of resistance to these stimuli as cardiac fibroblasts. Thus, our data demonstrate that Bcl-2 is an important effector of heart fibroblast resistance to apoptosis and highlight a probable mechanism for promoting survival advantage in fibroblasts of cardiac origin.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspases; Cell Death; Cell Survival; Cytochromes c; Cytosol; DNA Fragmentation; Dose-Response Relationship, Drug; Extracellular Matrix; Fibroblasts; Ischemia; Microscopy, Fluorescence; Myocardium; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Small Interfering; Skin; Staurosporine; Time Factors; Transfection

We describe the isolation and characterization of a new apaf-1-interacting protein (APIP) as a negative regulator of ischemic injury. APIP is highly expressed in skeletal muscle and heart and binds to the CARD of Apaf-1 in competition with caspase-9. Exogenous APIP inhibits cytochrome c-induced activation of caspase-3 and caspase-9, and suppresses cell death triggered by mitochondrial apoptotic stimuli through inhibiting the downstream activity of cytochrome c released from mitochondria. Conversely, reduction of APIP expression potentiates mitochondrial apoptosis. APIP expression is highly induced in mouse muscle affected by ischemia produced by interruption of the artery in the hindlimb and in C2C12 myotubes created by hypoxia in vitro, and the blockade of APIP up-regulation results in TUNEL-positive ischemic damage. Furthermore, forced expression of APIP suppresses ischemia/hypoxia-induced death of skeletal muscle cells. Taken together, these results suggest that APIP functions to inhibit muscle ischemic damage by binding to Apaf-1 in the Apaf-1/caspase-9 apoptosis pathway.

Topics: Amino Acid Sequence; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 9; Caspases; Cytochromes c; HeLa Cells; Humans; Hypoxia; Ischemia; Kidney; Male; Mice; Mice, Inbred C57BL; Mitochondria; Molecular Sequence Data; Muscle, Skeletal; Proteins; RNA, Messenger; Sequence Homology, Amino Acid

Ischemia and reperfusion cause mitochondrial dysfunctions that initiate the mitochondrial apoptosis pathway. They involve the release of cytochrome C and the activation of the caspase cascade but the mechanism(s) leading to cytochrome C release is(are) poorly understood. The aim of this study was to analyse the relation between cytochrome C release and the opening of the permeability transition pore (PTP) during in situ liver ischemia and reperfusion. Liver ischemia was induced for 30, 60 and 120 min and blood re-flow was subsequently restored for 30 and 180 min. Ischemia hugely altered mitochondrial functions, i.e., oxidative phosphorylation and membrane potential, and was accompanied by a time-dependent mitochondrial release of cytochrome C into the cytosol and by activations of caspases-3 and -9. PTP opening was not observed during ischemia, as demonstrated by the absence of effect of an in vivo pre-treatment of rats with cyclosporin A (CsA), a potent PTP inhibitor. Cytochrome C release was due neither to a direct effect of caspases onto mitochondria nor to an interaction of Bax or Bid with the mitochondrial membrane but could be related to a direct effect of oxygen deprivation. In contrast, during reperfusion, CsA pre-treatment inhibits cytochrome C release, PTP opening and caspase activation. At this step, cytochrome C release is likely to occur as a consequence of PTP opening. In conclusion, our study reveals that cytochrome C release, and thus the induction of the mitochondrial cell death pathway, occur successively independently and dependent on PTP opening during liver ischemia and reperfusion, respectively.

Topics: Animals; Caspases; Cyclosporine; Cytochromes c; Drug Interactions; Hypoxia; Ischemia; Liver Diseases; Mitochondria; Mitochondria, Liver; Permeability; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reperfusion

c-Jun N-terminal kinase (JNK) signaling is an important contributor to stress-induced apoptosis, but it is unclear whether JNK and its isoforms (JNK1, JNK2, and JNK3) have distinct roles in cerebral ischemia. Here we show that JNK1 is the major isoform responsible for the high level of basal JNK activity in the brain. In contrast, targeted deletion of Jnk3 not only reduces the stress-induced JNK activity, but also protects mice from brain injury after cerebral ischemia-hypoxia. The downstream mechanism of JNK3-mediated apoptosis may include the induction of Bim and Fas and the mitochondrial release of cytochrome c. These results suggest that JNK3 is a potential target for neuroprotection therapies in stroke.

Topics: Animals; Apoptosis; Brain; Cells, Cultured; Cytochromes c; Enzyme Activation; Glucose; Hippocampus; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; Ischemia; Mice; Mitochondria; Mitogen-Activated Protein Kinase 10; Mitogen-Activated Protein Kinases; Myocardium; Neurons; Oxygen; Protein Isoforms; Protein-Tyrosine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Time Factors; Transcription, Genetic