Protective actions of a new thromboxane synthetase inhibitor in arachidonate induced sudden death

A new thromboxane synthetase inhibitor, OKY-046, at doses of 0.5 and 1.0 mg/kg prevented mortality induced by sodium arachidonate in 100% of the rabbits studied. Sodium arachidonate at a dose of 1.25 mg/kg uniformly decreased mean arterial blood pressure to values approximately 0 mm Hg, stopped respiration and produced sudden death within 3-5 minutes in all rabbits studied. OKY-046 prevented all these sequelae of the sodium arachidonate. Untreated rabbits challenged with sodium arachidonate develop large increases in circulating thromboxane B2 (TxB2) and 6-keto PGF1 alpha of about 12- to 18-fold. In contrast, OKY-046 prevented the increase in TxB2 concentrations and the pulmonary thrombosis, but did not block the rise in 6-keto PGF1 alpha following arachidonate injection. These results suggest that the protective mechanism of OKY-046 in arachidonate induced sudden death is via selective inhibition of thromboxane synthesis.     

Hispidulin protection against hepatotoxicity induced by bromobenzene in mice

The effects of the natural flavonoid hispidulin (6-methoxy-5, 7, 4′-trihydroxyflavone) on bromobenzene-induced hepatotoxicity in mice were investigated. We found a correlation between liver injury and hepatic lipid peroxidation besides a strong liver glutathione depletion due to the toxicant. Hispidulin at doses between 50 and 150 mg/kg i.p. compared favourably with the reference compound N-acetyl-L-cysteine for inhibition of liver injury and lipid peroxidation. The flavonoid at the highest dose tested was also able to counteract reduced glutathione depletion induced by bromobenzene in starved mice. These hepatoprotective effects can be related to the antioxidant properties of hispidulin.     

Pre-conditioning induced by carbon monoxide provides neuronal protection against apoptosis

Carbon monoxide (CO) is an endogenous product of mammalian cells generated by heme-oxygenase, presenting anti-apoptotic properties in several tissues. The present work demonstrates the ability of small amounts of exogenous CO to prevent neuronal apoptosis induced by excitotoxicity and oxidative stress in mice primary culture of cerebellar granule cells. Additionally, our data show that endogenous CO is a heme-oxygenase product critical for its anti-apoptotic activity. Despite being neuroprotective, CO also induces reactive oxygen species generation in neurons. These two phenomena suggest that CO induces pre-conditioning (PC) to prevent cell death. The role of several PC mediators, namely soluble guanylyl cyclase, nitric oxide (NO) synthase, and ATP-dependent mitochondrial K channel (mitoK(ATP)) was addressed. Inhibition of soluble guanylyl cyclase or NO synthase activity, or closing of mitoK(ATP) abolishes the protective effect conferred by CO. In addition, CO treatment triggers cGMP and NO production in neurons. Opening of mitoK(ATP), which appears to be critical for CO prevention of apoptosis, might be a later event. We also demonstrated that reactive oxygen species generation and de novo protein synthesis are necessary for CO PC effect and neuroprotection. In conclusion, CO induces PC and prevents neuronal apoptosis, therefore constituting a novel and promising candidate for neuroprotective therapies.     

Neuroprotection against neuroblastoma cell death induced by depletion of mitochondrial glutathione

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.     

Pyruvate protection against endothelial cytotoxicity induced by blockade of glucose uptake

We have previously demonstrated that the redox reactant pyruvate prevents apoptosis in the oxidant model of bovine pulmonary artery endothelial cells (BPAEC), and that the anti-apoptotic mechanism of pyruvate is mediated in part via the mitochondrial matrix compartment. However, cytosolic mechanisms for the cytoprotective feature of pyruvate remain to be elucidated. This study investigated the pyruvate protection against endothelial cytotoxicity when the glycolysis inhibitor 2-deoxy-D-glucose (2DG) was applied to BPAEC. Millimolar 2DG blocked the cellular glucose uptake in a concentration- and time-dependent manner with >85% inhibition at > or =5 mM within 24 h. The addition of 2DG evoked BPAEC cytotoxicity with a substantial increase in lipid peroxidation and a marked decrease in intracellular total glutathione. Exogenous pyruvate partially prevented the 2DG-induced cell damage with increasing viability of BPAEC by 25-30%, and the total glutathione was also modestly increased. In contrast, 10 mM L-lactate, as a cytosolic reductant, had no effect on the cytotoxicity and lipid peroxidation that are evoked by 2DG. These results suggest that 2DG toxicity may be a consequence of the diminished potential of glutathione antioxidant, which was partially restored by exogenous pyruvate but not L-lactate. Therefore, pyruvate qualifies as a cytoprotective agent for strategies that attenuate the metabolic dysfunction of the endothelium, and cellular glucose oxidation is required for the functioning of the cytosolic glutathione/NADPH redox system.     

A single exposure to cocaine or immobilization stress provides extremely long-lasting, selective protection against sudden cardiac death from tetracaine

Exposure of rats to one injection of cocaine (35 mg/kg, i.p.) or a single four-hour period of immobilization protected them from the virtually instantaneous death but not from the later, seizure-related death seen in untreated controls following administration of the local anesthetic, tetracaine, 1-4 weeks later. These data suggest that when appropriately timed, strong sympathomimetic stimulation--whether generated by an environmental stressor or a drug--can provide long-lasting protection against the sudden cardiac death potential of local anesthetics. As such, they provide a means for understanding why the incidence of sudden cardiac arrest from one such agent--cocaine itself--is not higher and suggest that an individual's stress history may play a key role in determining vulnerability to the cardiotoxic effect of this compound.     

Sulforaphane protects human chondrocytes against cell death induced by various stimuli

Chondrocyte cell death can contribute to cartilage degeneration in articular diseases, such as osteoarthritis (OA). Sulforaphane (SFN), a natural compound derived from cruciferous aliment, is well known as an anti-carcinogen, but according to recent evidence it also shows cytoprotective effects on a variety of non-tumoral cells. Therefore we have tested the ability of SFN to protect chondrocytes from cell death in vitro. Treatment of growing monolayer cultures of human C-28/I2 chondrocytes with SFN in the low micro-molecular range for a few days, reduced cell growth without affecting cell survival or inducing apoptosis. However it decreased cell death in C-28/I2 chondrocytes exposed to stimuli previously reported to promptly trigger apoptosis, that is, the cytokine tumor necrosis factor-α (TNF) plus cycloheximide (CHX) or the polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM) plus CHX. In particular pre-treatment with SFN reduced effector and initiator caspase activities and the associated activation of JNK kinases. SFN exerted a cytoprotective action even versus H(2)O(2) , which differently from the previous stimuli induced cell death without producing an evident caspase activation. SFN pre-treatment also prevented caspase activation in three-dimensional micromass cultures of OA chondrocytes stimulated with growth-related oncogene α (GROα), a pro-apoptotic chemokine. The suppression of caspase activation in micromasses appeared to be related to the inhibition of p38 MAPK phosphorylation. In conclusion, the present work shows that low micro-molecular SFN concentrations exert pro-survival and anti-apoptotic actions and influence signaling pathways in a variety of experimental conditions employing chondrocyte cell lines and OA chondrocytes treated with a range of death stimuli.     

Antigen-specific protection against graft-vs-host induced immune deficiency

A severe, antigen-nonspecific, and long-lasting immune-deficient state can be induced in healthy, adult immune-competent F1 hybrid mice by a single i.v. injection of parental T lymphocytes. The present report demonstrates that this graft-vs-host-induced immune deficiency (GVHID) can be prevented in an antigen-specific way by immunization of the F1 mice with allogeneic cells before induction of GVHID. Thus, spleen cells from (A X B)F1 mice primed with allogeneic cells from strain C and then injected with parental spleen cells from A did not generate cytotoxic T lymphocyte responses to trinitophenyl-modified self cells or to allogeneic cells from third party strains D or E. However, spleen cells from the same mice generated normal levels of cytotoxic T lymphocyte activity to allogeneic cells from C, the strain used for immunization. Furthermore, mice exposed to murine cytomegalovirus before induction of GVHID were resistant to a subsequent challenge with murine cytomegalovirus, whereas GVHID mice that received only the murine cytomegalovirus challenge all died. These findings are discussed with respect to the possibilities that primed and unprimed T helper cells may be differentially susceptible to the suppressive effects of GVH.     

Mechanism of cell death of rat cardiac fibroblasts induced by serum depletion

Serum starvation has recently been shown to cause cell death of cardiac fibroblasts and increased synthesis of extracellular matrix proteins in the surviving cells. In the present study, events occurring in the dying cells were investigated. Cultured adult rat cardiac fibroblasts were exposed to serum-free medium. Cell number was measured using a Coulter Counter Channelyzer. The activity of the extracellular signal-regulated or mitogen-activated protein kinases (ERK1/2, p42/p44^MAPK), the p38 kinase (p38^MAPK), the c-Jun N-terminal kinases (p46/p54^JNK), and Akt kinase was assessed by Western blotting and phospho-specific antibodies. Caspase 7-cleavage was investigated by Western blotting and specific antibodies. Caspase 3 activity was measured by detection of its cleaved substrate. The appearance of necrosis was studied by inclusion of trypan blue. Apoptosis was assessed by DNA ladder formation. The mRNA expression of Bax and Bcl-2 was investigated by quantitative real-time PCR. Serum withdrawal led to the death of 26% of cultured isolated cardiac fibroblasts during the first 5 h. The activity of the p42/p44^MAPK as well as of Akt kinase was partially reduced. For p46/p54^JNK and p38^MAPK, elevated phosphorylation was measured. Inhibition of p46/p54^JNK and p38^MAPK activity by SB202190 did not affect the decrease in cell number. Cleavage of caspase7 was detected after 90 min. However, no activation of caspase 3 was measured. DNA fragmentation was not found after serum depletion. Trypan blue staining, however, was observed in 16% of the cells after 5 h. The mRNA levels of both Bax and Bcl-2 were increased after 30 min. These results indicate the appearance of necrosis during serum starvation in cardiac fibroblasts. However, some processes typical of apoptosis were also detected.     

Rhizobacterially induced protection of watermelon against Didymella bryoniae

Aims: To identify rhizobacteria from the Mekong Delta of Vietnam, which can systemically protect watermelon against Didymella bryoniae and elucidate the mechanisms involved in the protection conferred by isolate Pseudomonas aeruginosa 23_1‐1. Methods and Results: Bacteria were isolated from watermelon roots and their antagonistic ability tested in vitro. Of 190 strains, 68 were able to inhibit D. bryoniae by production of antibiotics. Four strains were able to reduce foliar infection by D. bryoniae when applied to watermelon seeds before sowing. Strain Ps. aeruginosa 23_1‐1 was chosen for investigations of the mechanisms involved in protection and ability to control disease under field conditions. In the field, the bacterium was able to significantly reduce disease in two consecutive seasons and increase yield. Furthermore, it colonized watermelon plants endophytically, with higher numbers in plants infected by D. bryoniae than in noninoculated plants. To elucidate the mechanisms involved in protection, the infection biology of the pathogen was studied in bacterially treated and control plants. Pseudomonas aeruginosa 23_1‐1 treatment inhibited pathogen penetration and this was associated with hydrogen peroxide accumulation, increased peroxidase activity and occurrence of new peroxidase isoforms, thus indicating that resistance was induced. Conclusions: The endophytic bacterium Ps. aeruginosa 23_1‐1 can control D. bryoniae in watermelon by antibiosis and induced resistance under greenhouse and field conditions. Significance and Impact of the Study: These findings suggest that rhizobacteria from native soils in Vietnam can be used to control gummy stem blight of watermelon through various mechanisms including induction of resistance.     

Cellular protection of morin against the oxidative stress induced by hydrogen peroxide

Flavonoids are a class of secondary metabolites abundantly found in fruits and vegetables. In addition, flavonoids have been reported as potent antioxidants with beneficial effects against oxidative stress-related diseases such as cancer, aging, and diabetes. The present study was carried out to investigate the cytoprotective effects of morin (2′,3,4′,5,7-pentahydroxyflavone), a member of the flavonoid group, against hydrogen peroxide (H₂O₂)-induced DNA and lipid damage. Morin was found to prevent the cellular DNA damage induced by H₂O₂ treatment, which is shown by the inhibition of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation (a modified form of DNA base), inhibition of comet tail (a form of DNA strand breakage), and decrease of nuclear phospho histone H2A.X expression (a marker for DNA strand breakage). In addition, morin inhibited membrane lipid peroxidation, which is detected by inhibition of thiobarbituric acid reactive substance (TBARS) formation. Morin was found to scavenge the intracellular reactive oxygen species (ROS) generated by H₂O₂ treatment in cells, which is detected by a spectrofluorometer, flow cytometry, and confocal microscopy after staining of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Morin also induces an increase in the activity of catalase and protein expression. The results of this study suggest that morin protects cells from H₂O₂-induced damage by inhibiting ROS generation and by inducing catalase activation.     

Towards the mechanism of protection against indomethacin induced gastro-intestinal ulceration by naloxone

Activities of lysosomal hydrolases have been evaluated in relation to indomethacin and naloxone, using purified lysosomal fractions from rat intestinal mucosa. Indomethacin treatment significantly decreased (p less than 0.001) lysosomal enzyme activities in purified lysosomes, while an increase in the activities was observed in intestinal homogenates. However, indomethacin could not affect lysosomal system in animals pretreated with naloxone, thereby establishing that naloxone neutralises the effect of indomethacin.     

The dawn of a new era in the struggle against sudden cardiac death

The year 2009 is important for Dutch cardiology. It marks not only the 75th anniversary of the Netherlands Society of Cardiology, but also the starting of ICD therapy, 25 years ago. The first ICD implant in the Netherlands was carried out in Utrecht by the cardiac surgeon Dr Penn with the technical assistance of Mr Seah Nisam in April 1984. The patient was referred from Maastricht by Professor Wellens. Referral was needed since Maastricht had no cardiac surgery department at that time.     

Bradykinin protects against brain microvascular endothelial cell death induced by pathophysiological stimuli

The morphological and functional integrity of the microcirculation is compromised in many cardiovascular diseases such as hypertension, diabetes, stroke, and sepsis. Angiotensin converting enzyme inhibitors (ACEi), which are known to favor bradykinin (BK) bioactivity by reducing its metabolism, may have a positive impact on preventing the microvascular structural rarefaction that occurs in these diseases. Our study was designed to test the hypothesis that BK, via B2 receptors (B2R), protects the viability of the microvascular endothelium exposed to the necrotic and apoptotic cell death inducers H₂O₂ and LPS independently of hemodynamics. Expression (RT‐PCR and radioligand binding) and functional (calcium mobilization with fura‐2AM, and p42/p44MAPK and Akt phosphorylation assays) experiments revealed the presence of functional B2R in pig cerebral microvascular endothelial cells (pCMVEC). In vitro results showed that the cytocidal effects of H₂O₂ and LPS on pCMVEC were significantly decreased by a BK pretreatment (MTT and crystal violet tests, annexin‐V staining/FACS analysis), which was countered by the B2R antagonist HOE 140. BK treatment coincided with enhanced expression of the cytoprotective proteins COX‐2, Bcl‐2, and ^Cu/ZnSOD. Ex vivo assays on rat brain explants showed that BK impeded (by ～40%) H₂O₂‐induced microvascular degeneration (lectin‐FITC staining). The present study proposes a novel role for BK in microvascular endothelial protection, which may be pertinent to the complex mechanism of action of ACEi explaining their long‐term beneficial effects in maintaining vascular integrity. J. Cell. Physiol. 222:168–176, 2010. © 2009 Wiley‐Liss, Inc.     

Mechanically induced sudden death in chest wall impact (commotio cordis)

Sudden death due to nonpenetrating chest wall impact in the absence of injury to the ribs, sternum and heart is known as commotio cordis. Although once thought rare, an increasing number of these events have been reported. Indeed, a significant percentage of deaths on the athletic field are due to chest wall impact. Commotio cordis is most frequently observed in young individuals (age 4–18 years), but may also occur in adults. Sudden death is instantaneous or preceded by several seconds of lightheadedness after the chest wall blow. Victims are most often found in ventricular fibrillation, and successful resuscitation is more difficult than expected given the young age, excellent health of the victims, and the absence of structural heart disease. Autopsy examination is notable for the lack of any significant cardiac or thoracic abnormalities.

In an experimental model of commotio cordis utilizing anesthetized juvenile swine, ventricular fibrillation can be produced by a 30 mph baseball strike if the strike occurred during the vulnerable period of repolarization, on the upslope of the T-wave. Energy of the impact object was also found to be a critical variable with 40 mph baseballs more likely to cause ventricular fibrillation than velocities less or greater than 40 mph. In addition, more rigid impact objects and blows directly over the center of the chest were more likely to cause ventricular fibrillation. Peak left ventricular pressure generated by the chest wall blow correlated with the risk of ventricular fibrillation. Activation of the K⁺_ATP channel is a likely cause of the ventricular fibrillation produced by chest wall blows. Successful resuscitation is attainable with early defibrillation.