Oxidative Stress-induced Death Research Articles

Pituitary adenylate cyclase-activating polypeptide protects astroglial cells against oxidative stress-induced apoptosis

Oxidative stress, associated with a variety of disorders including neurodegenerative diseases, results from accumulation of reactive oxygen species (ROS). Oxidative stress is not only responsible for neuron apoptosis, but can also provoke astroglial cell death. Numerous studies indicate that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuron survival, but nothing is known regarding the action of PACAP on astroglial cell survival. Thus, the purpose of the present study was to investigate the potential glioprotective effect of PACAP on H(2)O(2)-induced astrocyte death. Pre-treatment of cultured rat astrocytes with nanomolar concentrations of PACAP prevented cell death provoked by H(2)O(2) (300 μM), whereas vasoactive intestinal polypeptide was devoid of protective activity. The effect of PACAP on astroglial cell survival was abolished by the type 1 PACAP receptor antagonist, PACAP6-38. The protective action of PACAP was blocked by the protein kinase A inhibitor H89, the protein kinase C inhibitor chelerythrine and the mitogen-activated protein (MAP)-kinase kinase (MEK) inhibitor U0126. PACAP stimulated glutathione formation, and blocked H(2)O(2)-evoked ROS accumulation and glutathione content reduction. In addition, PACAP prevented the decrease of mitochondrial activity and caspase 3 activation induced by H(2)O(2). Taken together, these data indicate for the first time that PACAP, acting through type 1 PACAP receptor, exerts a potent protective effect against oxidative stress-induced astrocyte death. The anti-apoptotic activity of PACAP on astrocytes is mediated through the protein kinase A, protein kinase C and MAPK transduction pathways, and can be accounted for by inhibition of ROS-induced mitochondrial dysfunctions and caspase 3 activation.

2-Ethoxy-4,5-diphenyl-1,3-oxazine-6-one activates the Nrf2/HO-1 axis and protects against oxidative stress-induced neuronal death

Apoptosis or programmed cell death has been suggested as an important mode of neurodegeneration in Alzheimer's disease pathogenesis. The present study explored the neuroprotective effect of 2-ethoxy-4,5-diphenyl-1,3-oxazine-6-one (EDPOO) against H 2O 2-induced cell death in rat pheochromocytoma (PC12) cells. We found that H 2O 2 triggered a range of cellular cascades which leads to cell death, whereas pretreatment of the cells with this oxazine derivative attenuated the extent of apoptosis, as assessed by MTT assay, acridine orange/ethidium bromide staining and caspase-3 expression assay. We further showed that EDPOO exerts its neuroprotective effect by enhancing Hsp-70 level, stabilizing Nrf2 and upregulation of HO-1 and γ-GCS. Moreover, this oxazine derivative regulated cellular redox status via antioxidant enzyme upregulation. The neuroprotective effect of this compound may provide a new potential application for the treatment of neurodegenerative diseases.

Big Mitogen-Activated Protein Kinase 1 Protects Cultured Rat Aortic Smooth Muscle Cells From Oxidative Damage

Oxidative stress is considered a major mediator of arteriosclerosis. In vascular smooth muscle cells, oxidative stress-induced cell death (including apoptosis) is probably related to arterial calcification in arteriosclerosis. Big mitogen-activated protein kinase-1 / extracellular signal-regulated kinase 5 (BMK1/ERK5) is a newly identified member of the mitogen-activated protein kinases family. Like Src tyrosine kinase, BMK1/ERK5 is known to be sensitive to oxidative stress; however, its pathophysiological significance is poorly understood. In this study, we investigated the involvement of BMK1 and Src in H(2)O(2)-induced cell death using cultured rat aortic smooth muscle cells (RASMCs). Cell apoptosis was evaluated by using the TdT-mediated dUTP nick end labeling (TUNEL) method, and BMK1 and Src activities were determined by Western blotting. The main results are as follows: 1) BMK1 and Src were activated by H(2)O(2) in a time- and concentration-dependent manner in RASMCs; 2) BMK1 activation by H(2)O(2) was attenuated both in Src-knockdown RASMCs and in RASMCs pretreated with 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a Src family kinases inhibitor; and 3) H(2)O(2)-induced cell death was increased in BMK1- and Src-knockdown RASMCs as well as in PP2-treated RASMCs. These findings suggested that Src and BMK1 may play defensive and resistive roles against oxidative stress-induced death in RASMCs.

Complement 1q-binding protein inhibits the mitochondrial permeability transition pore and protects against oxidative stress-induced death

Opening of the MPT (mitochondrial permeability transition) pore is a critical event in mitochondrial-mediated cell death. However, with the exception of CypD (cyclophilin D), the exact molecular composition of the MPT pore remains uncertain. C1qbp (complement 1q-binding protein) has recently been hypothesized to be an essential component of the MPT pore complex. To investigate whether C1qbp indeed plays a critical role in MPT and cell death, we conducted both gain-of-function and loss-of-function experiments in MEFs (mouse embryonic fibroblasts). We first confirmed that C1qbp is a soluble protein that localizes to the mitochondrial matrix in mouse cells and tissues. Similarly, overexpression of C1qbp in MEFs using an adenovirus resulted in its exclusive localization to mitochondria. To our surprise, increased C1qbp protein levels actually suppressed H2O2-induced MPT and cell death. Antithetically, knockdown of endogenous C1qbp with siRNA (small interfering RNA) sensitized the MEFs to H2O2-induced MPT and cell death. Moreover, we found that C1qbp could directly bind to CypD. Therefore C1qbp appears to act as an endogenous inhibitor of the MPT pore, most likely through binding to CypD, and thus protects cells against oxidative stress.

Oxidized NADH Oxidase Inhibits Activity of an ATP/NAD Kinase from a Thermophilic Archaeon

NADH oxidases (NOXs) are important enzymes in detoxifying oxidative stress and regenerating oxidized pyridine nucleotides. In the present study, a NOX from Thermococcus kodakarensis KOD1 (NOXtk) was recombinantly expressed in Escherichia coli and purified to homogeneity. NOXtk displayed NADH oxidase activity that was inhibited by oxidization. Under physiological conditions, unoxidized and oxidized NOXtk formed dimers and hexamers, respectively. Mutating the single cysteine residue Cys45 to alanine (NOXtkC45A) decreased NADH oxidase activity without affecting dimerization or hexamerization, suggesting that oligomerization does not occur through disulfide bond formation. Pull-down assay results indicated that an ATP/NAD kinase from T. kodakarensis KOD1 (ANKtk) binds to NOXtk. Use of several assays revealed that ANKtk can only bind to oxidized hexameric NOXtk, through which it inhibits ANKtk activity. Because ANKtk converts NADH to NADPH (an important factor in oxidative stress protection), a model based on in vitro result was proposed in which NOXtk hexamerization under oxic conditions inhibits both NOXtk and ANKtk activities, thereby sensitizing cells to oxidative stress-induced death.

Isoflurane Preconditioning Elicits Competent Endogenous Mechanisms of Protection from Oxidative Stress in Cardiomyocytes Derived from Human Embryonic Stem Cells

Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients that is relatively inaccessible for research purposes. We tested whether anesthetic-induced preconditioning (APC) with isoflurane elicits competent protective mechanisms in hESC-derived cardiomyocytes against oxidative stress to be used as a model of human cardiomyocytes for studying preconditioning. H1 hESC cell line was differentiated into cardiomyocytes using growth factors activin A and bone morphogenetic protein-4. Living ventricular hESC-derived cardiomyocytes were identified using a lentiviral vector expressing a reporter gene (enhanced green fluorescent protein) driven by a cardiac-specific human myosin light chain-2v promoter. Mitochondrial membrane potential, reactive oxygen species production, opening of mitochondrial permeability transition pore, and survival of hESC-derived cardiomyocytes were assessed using confocal microscopy. Oxygen consumption was measured in contracting cell clusters. Differentiation yielded a high percentage (∼85%) of cardiomyocytes in beating clusters that were positive for cardiac-specific markers and exhibited action potentials resembling those of mature cardiomyocytes. Isoflurane depolarized mitochondria, attenuated oxygen consumption, and stimulated generation of reactive oxygen species. APC protected these cells from oxidative stress-induced death and delayed mitochondrial permeability transition pore opening. APC elicits competent protective mechanisms against oxidative stress in hESC-derived cardiomyocytes, suggesting the feasibility to use these cells as a model of human cardiomyocytes for studying APC and potentially other treatments/diseases. Our differentiation protocol is very efficient and yields a high percentage of cardiomyocytes. These results also suggest a promising ability of APC to protect and improve engraftment of hESC-derived cardiomyocytes into the ischemic heart.

Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death

Oxidative stress can induce cytotoxicity in neurons, which plays an important role in the etiology of neuronal damage and degeneration. This study sought to determine the cellular and biochemical mechanisms underlying resveratrol's protective effect against oxidative neuronal death. Cultured HT22 cells, an immortalized mouse hippocampal neuronal cell line, were used as an in vitro model, and oxidative stress and neurotoxicity were induced in these neuronal cells by exposure to high concentrations of glutamate. Resveratrol strongly protected HT22 cells from glutamate-induced oxidative cell death. Resveratrol's neuroprotective effect was independent of its direct radical scavenging property, but instead was dependent on its ability to selectively induce the expression of mitochondrial superoxide dismutase (SOD2) and, subsequently, reduce mitochondrial oxidative stress and damage. The induction of mitochondrial SOD2 by resveratrol was mediated through the activation of the PI3K/Akt and GSK-3β/β-catenin signaling pathways. Taken together, the results of this study show that up-regulation of mitochondrial SOD2 by resveratrol represents an important mechanism for its protection of neuronal cells against oxidative cytotoxicity resulting from mitochondrial oxidative stress.

The purinergic P2Y 13 receptor activates the Nrf2/HO-1 axis and protects against oxidative stress-induced neuronal death

Although extracellular purines may have both trophic and apoptotic functions in the brain depending on the targeted purine receptor and cell type, little is known about the role of specific purine receptors on neurons. In this study, we demonstrate that both ADP and its stable analogue 2-methyl-thio-ADP (2MeSADP) induce up-regulation of the cytoprotective protein heme oxygenase-1 (HO-1). Selective inhibition of 2MeSADP-responsive receptors P2Y 1 and P2Y 13 with their respective antagonists MRS2179 and MRS2211 and the use of pertussis toxin demonstrated a role of the purinergic P2Y 13 receptor in this response. Moreover, luciferase assays demonstrated that ectopic expression of the P2Y 13 receptor in neuroblastoma N2A cells resulted in 2MeSADP-dependent induction of antioxidant response elements from the HO-1 promoter. The transcription factor Nrf2 was critical for HO-1 activation and translocated from the cytosol to the nucleus in response to 2MeSADP. In cerebellar granule neurons (CGNs) derived from Nrf2-knockout mice this purine did not activate the Nrf2/HO-1 axis and did not protect against H 2O 2-induced cell death. The relevance of HO-1 in 2MeSADP-induced neuroprotection was further demonstrated by the evidence that HO-1 inhibition with tin protoporphyrin (SnPP) prevented protection against H 2O 2-induced oxidative stress and cell death. These observations reveal a previously unrecognized role in protection against oxidative stress by extracellular purines acting on the metabotropic P2Y 13 receptor and provide new perspectives for neuroprotective therapies.

Abstract 4384: Differential expression and function of peroxiredoxin genes in normal vs. cancerous breast epithelial cell lines

Abstract Peroxiredoxins are thiol-specific antioxidants that reduce a wide variety of cellular peroxides and protect cells from oxidative stress-induced damage and death. These proteins have been implicated in numerous disease processes associated with increased oxidative stress, including cancer. We sought to compare peroxiredoxin expression between the normal MCF10A and cancerous MCF7 breast epithelial cell lines and examine the effect of Prdx1 and Prdx6 suppression in each line. Prdx1 and Prdx6 suppression was achieved through transient transfection of siRNA, and the relative expression of all six mammalian peroxiredoxin genes was determined by real time PCR and western blotting. We also examined the effect on peroxide-induced reactive oxygen species (ROS) accumulation and cytotoxicity. We demonstrated that MCF7 cells express significantly higher levels of Prdxs 1-5, while Prdx6 levels are comparable in the two lines. We achieved approximately 90% mRNA suppression of Prdx1 and Prdx6 in both lines using siRNA. Interestingly, Prdx1 suppression in both lines led to a marked reduction in Prdx2 expression and significant induction of Prdx4 expression. In addition, several Prdx genes were upregulated in MCF10 cells when either Prdx1 or Prdx6 were suppressed. We further demonstrated that Prdx1 and Prdx6 suppression caused a differential increase in peroxide-induced ROS levels and cytotoxicity in the two lines, and that simultaneous knock-down of both genes in MCF10 cells leads to a much more pronounced effect than in MCF7 cells. Together, these data suggest coordinated and complex regulatory interactions between the peroxiredoxin genes, and implicate a potentially different function for these proteins in transformed vs. untransformed cells. The elevated expression of peroxiredoxins in MCF7 cells may also represent a cellular adaptation to the cancerous state. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4384.

The compound isolated from the leaves of Phyllostachys nigra protects oxidative stress-induced retinal ganglion cells death

This study was performed to determine whether the compound isolated from Phyllostachys nigra could attenuate oxidative stress in transformed retinal ganglion cells (RGC-5 cells) death.RGC-5 cells in culture were given two different insults such as l-buthionine-(S,R)-sulfoximine (BSO) plus glutamate for 24h or hydrogen peroxide for 24h, after which cell survival were measured. Among the four systematic fractions tested, ethyl acetate fraction showed a significantly higher inhibition which was in a concentration dependent manner. Eight compounds were isolated from ethyl acetate fraction of P. nigra using preparative RP-HPLC and Sephadex LH-20 column chromatography. Their chemical structures were elucidated by chemical and spectral analysis as isoorientin (1), orientin (2), vitexin (3), cis-coumaric acid (4), p-coumaric acid (5), luteolin 6-C-(6′′-O-trans-caffeoylglucoside) (6), vittariflavone (7) and tricin (8).The luteolin 6-C-(6′′-O-trans-caffeoylglucoside) (compound 6) significantly attenuated the negative effects of BSO plus glutamate or hydrogen peroxide to RGC-5 cells. Treatment of the RGC-5 cells with compound 6 reduced the reactive oxygen species (ROS) as quantified using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). This compound also replenished the reduced glutathione level.In conclusion, these results implicate that compound 6 isolated from P. nigra could be used as a leading compounds for retinal disease via anti-oxidative effects.

DJ-1 protects the nigrostriatal axis from the neurotoxin MPTP by modulation of the AKT pathway

Loss-of-function DJ-1 (PARK7) mutations have been linked with a familial form of early onset Parkinson disease. Numerous studies have supported the role of DJ-1 in neuronal survival and function. Our initial studies using DJ-1-deficient neurons indicated that DJ-1 specifically protects the neurons against the damage induced by oxidative injury in multiple neuronal types and degenerative experimental paradigms, both in vitro and in vivo. However, the manner by which oxidative stress-induced death is ameliorated by DJ-1 is not completely clear. We now present data that show the involvement of DJ-1 in modulation of AKT, a major neuronal prosurvival pathway induced upon oxidative stress. We provide evidence that DJ-1 promotes AKT phosphorylation in response to oxidative stress induced by H(2)O(2) in vitro and in vivo following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Moreover, we show that DJ-1 is necessary for normal AKT-mediated protective effects, which can be bypassed by expression of a constitutively active form of AKT. Taken together, these data suggest that DJ-1 is crucial for full activation of AKT upon oxidative injury, which serves as one explanation for the protective effects of DJ-1.

Linoleic Acid Derivative DCP-LA Protects Neurons from Oxidative Stress-Induced Apoptosis by Inhibiting Caspase-3/-9 Activation

The present study aimed at understanding the effect of the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) on oxidative stress-induced neuronal death. Sodium nitroprusside (SNP; 1 mM) reduced viability of cultured rat cerebral cortical neurons to 50% of basal levels, but DCP-LA significantly prevented the SNP effect in a concentration (1-100 nM)-dependent manner. In addition, DCP-LA (100 nM) rescued neurons from SNP-induced degradation. SNP (1 mM) activated caspase-3 and -9 in cultured rat cerebral cortical neurons, but DCP-LA (100 nM) abolished the caspase activation. For a mouse model of middle cerebral artery occlusion, oral administration with DCP-LA (1 mg/kg) significantly diminished degraded area due to cerebral infarction. The results of the present study, thus, demonstrate that DCP-LA protects neurons at least in part from oxidative stress-induced apoptosis by inhibiting activation of caspase-3/-9.

Sulforaphane protects immature hippocampal neurons against death caused by exposure to hemin or to oxygen and glucose deprivation

Oxidative stress is a mediator of cell death following cerebral ischemia/reperfusion and heme toxicity, which can be an important pathogenic factor in acute brain injury. Induced expression of phase II detoxification enzymes through activation of the antioxidant response element (ARE)/Nrf2 pathway has emerged as a promising approach for neuroprotection. Little is known, however, about the neuroprotective potential of this strategy against injury in immature brain cells. In this study, we tested the hypothesis that sulforaphane (SFP), a naturally occurring isothiocyanate that is also a known activator of the ARE/Nrf2 antioxidant pathway, can protect immature neurons from oxidative stress-induced death. The hypothesis was tested with primary mouse hippocampal neurons exposed to either O(2) and glucose deprivation (OGD) or hemin. Treatment of immature neurons with SFP immediately after the OGD during reoxygenation was effective in protecting immature neurons from delayed cell death. Exposure of immature hippocampal neurons to hemin induced significant cell death, and both pre- and cotreatment with SFP were remarkably effective in blocking cytotoxicity. RT-PCR analysis indicated that several Nrf2-dependent cytoprotective genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutamate-cysteine ligase modifier subunit (GCLM), which is involved in glutathione biosynthesis, were up-regulated following SFP treatment both in control neurons and following exposure to OGD and hemin. These results indicate that SFP activates the ARE/Nrf2 pathway of antioxidant defense and protects immature neurons from death caused by stress paradigms relevant to those associated with ischemic and traumatic injury to the immature brain.

Neuroprotective Effect of Oligodendrocyte Precursor Cell Transplantation in a Long-Term Model of Periventricular Leukomalacia

Perinatal white matter injury, or periventricular leukomalacia (PVL), is the most common cause of brain injury in premature infants and is the leading cause of cerebral palsy. Despite increasing numbers of surviving extreme premature infants and associated long-term neurological morbidity, our understanding and treatment of PVL remains incomplete. Inflammation- or ischemia/hypoxia-based rodent models, although immensely valuable, are largely restricted to reproducing short-term features of up to 3 weeks after injury. Given the long-term sequelae of PVL, there is a need for subchronic models that will enable testing of putative neuroprotective therapies. Here, we report long term characterization of a neonatal inflammation-induced rat model of PVL. We show bilateral ventriculomegaly, inflammation, reactive astrogliosis, injury to pre-oligodendrocytes, and neuronal loss 8 weeks after injury. We demonstrate neuroprotective effects of oligodendrocyte precursor cell transplantation. Our findings present a subchronic model of PVL and highlight the tissue protective effects of oligodendrocyte precursor cell transplants that demonstrate the potential of cell-based therapy for PVL.

MUC1 oncoprotein promotes autophagy in a survival response to glucose deprivation

Tumor cells survive under conditions of nutrient deprivation by mechanisms that are not fully understood. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and blocks oxidative stress-induced death. The present studies show that MUC1 inhibits the induction of necrosis in response to the deprivation of glucose. MUC1 suppressed glucose deprivation-induced increases in reactive oxygen species (ROS) and thereby depletion of ATP and cell death. Cells respond to oxidative stress and energy depletion with the induction of autophagy. Our results demonstrate that MUC1 blocks depletion of ATP and sustains growth of glucose-deprived cells by a mechanism sensitive to the autophagy inhibitor, 3-methyladenine. Silencing expression of ATG7, a protein essential for the formation of autophagic vacuoles, also attenuated the MUC1-sustained increases in ATP and growth in response to glucose deprivation. Moreover, we found that MUC1 stimulates AMPK activation and thereby promotes lysosomal turnover of LC3-II, a marker of starvation-induced autophagic activity. These results indicate that MUC1 suppresses glucose deprivation-induced increases in ROS and thereby promotes ATP production and survival. The findings also indicate that the overexpression of MUC1 as found in human cancers could provide a survival advantage in microenvironments with low glucose levels.

FOXO3a-dependent regulation of Pink1 (Park6) mediates survival signaling in response to cytokine deprivation

Loss-of-function mutations of phosphatase/tensin homolog deleted on chromosome 10 (PTEN)-induced putative kinase 1 (Pink1) (also known as Park6) identified in familial forms of Parkinson's disease (PD) are associated with compromised mitochondrial function. Emerging data suggest that Pink1 is an essential pro-survival factor that is induced in response to oxidative stress. However, the mechanisms regulating Pink1 expression under stress conditions remain unknown. Forkhead box, subgroup O (FOXO) transcription factors carry out distinct biological functions in response to different extracellular signals. Notably, FOXO factors possess evolutionarily conserved roles in protecting cells from oxidative stress-induced death. Here we report that the FOXO family member FOXO3a controls Pink1 transcription in both mouse and human cells subjected to growth factor deprivation and that this regulation is exerted through evolutionarily conserved FOXO binding elements. Induction of Pink1 by FOXO3a is crucial for survival signals in lymphocytes, as depletion of Pink1 sensitizes these cells to death induced by deprivation of an essential growth factor. Our data reveal that the role of FOXO factors in protecting cells from growth factor deprivation-triggered apoptosis has been underestimated and that FOXOs mediate this protection by transactivating anti-apoptotic effectors like Pink1. Given the essential role of Pink1 in combating cell death, our findings may help to dissect the mechanisms by which FOXO proteins function as anti-oxidative stress factors.

Inhibition of JAK2 protects renal endothelial and epithelial cells from oxidative stress and cyclosporin A toxicity

Cyclosporin A is an immunosuppressant drug widely used in solid organ transplantation, but it has nephrotoxic properties that promote oxidative stress. The JAK2/STAT pathway has been implicated in both cell protection and cell injury; therefore, we determined a role of JAK2 in oxidative stress-mediated renal cell injury using pathophysiologically relevant oxidative challenges. The AG490 JAK2 inhibitor and overexpression of a dominant negative JAK2 protein protected endothelial and renal epithelial cells in culture against peroxide, superoxide anion and cyclosporin A induced cell death while reducing intracellular oxidation in cells challenged with peroxide and cyclosporin A. The decrease in Bcl2 expression and caspase 3 activation, induced by oxidative stress, was prevented by AG490. In mouse models of ischemia/reperfusion and cyclosporin A nephrotoxicity, AG490 decreased peritubular capillary and tubular cell injury. Our study shows that JAK2 inhibition is a promising renoprotective strategy defending endothelial and tubular cells from cyclosporin A- and oxidative stress-induced death.

SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

BackgroundSERPINA3K, an extracellular serine proteinase inhibitor (serpin), has been shown to have decreased levels in the retinas of diabetic rats, which may contribute to diabetic retinopathy. The function of SERPINA3K in the retina has not been investigated.Methodology/Principal FindingsThe present study identified a novel function of SERPINA3K, i.e. it protects retinal cells against oxidative stress-induced cell death including retinal neuronal cells and Müller cells. Flow-cytometry showed that the protective effect of SERPINA3K on Müller cells is via reducing oxidation-induced necrosis. Measurements of intracellular calcium concentration showed that SERPINA3K prevented the intracellular calcium overload induced by H2O2. A similar protective effect was observed using a calcium chelator (BAPTA/AM). Further, SERPINA3K inhibited the phosphorylation of phospholipase C (PLC)-gamma1 induced by H2O2. Likewise, a specific PLC inhibitor showed similar protective effects on Müller cells exposed to H2O2. Furthermore, the protective effect of SERPINA3K was attenuated by a specific PLC activator (m-3M3FBS). Finally, in a binding assay, SERPINA3K displayed saturable and specific binding on Müller cells.Conclusion/SignificanceThese results for the first time demonstrate that SERPINA3K is an endogenous serpin which protects cells from oxidative stress-induced cells death, and its protective effect is via blocking the calcium overload through the PLC pathway. The decreased retinal levels of SERPINA3K may represent a new pathogenic mechanism for the retinal Müller cell dysfunction and neuron loss in diabetes.

Protection of peroxiredoxin II on oxidative stress-induced cardiomyocyte death and apoptosis

Peroxiredoxin II, a cytosolic isoform of the antioxidant enzyme family, has been implicated in cancer-associated cell death and apoptosis, but its functional role in the heart remains to be elucidated. Interestingly, the expression levels of peroxiredoxin II were decreased in mouse hearts upon ischemia-reperfusion, while they were elevated in two genetically modified hyperdynamic hearts with phospholamban ablation or protein phosphatase 1 inhibitor 1 overexpression. To delineate the functional significance of altered peroxiredoxin II expression, adenoviruses encoding sense or antisense peroxiredoxin II were generated; cardiomyocytes were infected, and then subjected to H(2)O(2) treatment to mimic oxidative stress-induced cell death and apoptosis. H(2)O(2) stimulation resulted in a significant decrease of endogenous peroxiredoxin II expression, along with reduced cell viability in control cells. However, overexpression of peroxiredoxin II significantly protected from H(2)O(2)-induced apoptosis and necrosis, while downregulation of this enzyme promoted the detrimental effects of oxidative stress in cardiomyocytes. The beneficial effects of peroxiredoxin II were associated with increased Bcl-2 expression, decreased expression of Bax and attenuated activity of caspases 3, 9 and 12. Furthermore, there were no significant alterations in the expression levels of the other five isoforms of peroxiredoxin, as well as active catalase or glutathione peroxidase-1 after ischemia-reperfusion or H(2)O(2) treatment. These findings suggest that peroxiredoxin II may be a unique antioxidant in the cardiac system and may represent a potential target for cardiac protection from oxidative stress-induced injury.

Candidaalbicans cells lacking CaMCA1-encoded metacaspase show resistance to oxidative stress-induced death and change in energy metabolism

Candidaalbicans, an opportunistic pathogen, can undergo programmed cell death upon various stimuli, including oxidative stress. In this study, we showed that deletion of CaMCA1, a homologue of Saccharomyces cerevisiae metacaspase YCA1, could both attenuated oxidative stress-induced cell death and caspase activation. Compared to wild-type strain, Camca1Δ mutant showed higher accumulation of trehalose and transcription of the genes related to trehalose biosynthesis (TPS2 and TPS3) under the condition of oxidative stress. Furthermore, lower intracellular ATP concentration and mitochondrial membrane potential, less endogenous reactive oxygen species (ROS) generation were observed in Camca1Δ mutant. Our results suggest that CaMCA1 might mediate the sensitiveness to oxidative stress by affecting energy metabolism in C. albicans.