Nuclear morphology and c‐Jun N‐terminal kinase 1 expression differentiate serum‐starved oxidative stress signalling from hydrogen peroxide‐induced apoptosis in retinal neuronal cell line

Oxidative stress induced by serum starvation and H₂O₂ exposure, both triggers apoptosis in retinal neuronal cell line RGC‐5 (retinal ganglion cell‐5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC‐5 under these conditions. Sub‐confluent cells were treated either with H₂O₂ or maintained in SFM (serum‐free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC‐5 by 72 h serum starvation and 500 M H₂O₂ exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H₂O₂ treatment. Serum starvation did not alter JNK1 (c‐Jun N‐terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho‐JNK) was evident. Conversely, H₂O₂ exposure blocked the expression and activation of JNK1 to phospho‐JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC‐5 cells may follow different signalling pathways when challenged with serum starvation and H₂O₂.     

Sitagliptin‐mediated preservation of endothelial progenitor cell function via augmenting autophagy enhances ischaemic angiogenesis in diabetes

Recently, the dipeptidyl peptidase‐4 (DPP‐4) inhibitor sitagliptin, a major anti‐hyperglycaemic agent, has received substantial attention as a therapeutic target for cardiovascular diseases via enhancing the number of circulating endothelial progenitor cells (EPCs). However, the direct effects of sitagliptin on EPC function remain elusive. In this study, we evaluated the proangiogenic effects of sitagliptin on a diabetic hind limb ischaemia (HLI) model in vivo and on EPC culture in vitro. Treatment of db/db mice with sitagliptin (Januvia) after HLI surgery efficiently enhanced ischaemic angiogenesis and blood perfusion, which was accompanied by significant increases in circulating EPC numbers. EPCs derived from the bone marrow of normal mice were treated with high glucose to mimic diabetic hyperglycaemia. We found that high glucose treatment induced EPC apoptosis and tube formation impairment, which were significantly prevented by sitagliptin pretreatment. A mechanistic study found that high glucose treatment of EPCs induced dramatic increases in oxidative stress and apoptosis; pretreatment of EPCs with sitagliptin significantly attenuated high glucose‐induced apoptosis, tube formation impairment and oxidative stress. Furthermore, we found that sitagliptin restored the basal autophagy of EPCs that was impaired by high glucose via activating the AMP‐activated protein kinase/unc‐51‐like autophagy activating kinase 1 signalling pathway, although an autophagy inhibitor abolished the protective effects of sitagliptin on EPCs. Altogether, the results indicate that sitagliptin‐induced preservation of EPC angiogenic function results in an improvement of diabetic ischaemia angiogenesis and blood perfusion, which are most likely mediated by sitagliptin‐induced prevention of EPC apoptosis via augmenting autophagy.     

Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases,p38 and JNK

Poor survival of mesenchymal stem cells (MSCs) compromised the efficacy of stem cell therapy for myocardial infarction. The increase of exogenous reactive oxygen species (ROS) in infracted heart is one of the important factors that challenged the survival of donor MSCs. In the study we aimed to evaluate the effect of oxidative stress on the cell death of MSCs and investigate its mechanisms in order to help with the identification of new biological compounds to reduce donor cells damage. Apoptosis of MSCs were evaluated with Hoechst 33342 staining and flow cytometry analysis. The mitochondrial membrane potential of MSCs was analyzed with JC‐1 staining. Signaling pathways involved in H₂O₂ induced apoptosis were analyzed with Western blot. H₂O₂ induced apoptosis of MSCs in a dose‐ and time‐dependent manner. H₂O₂ induced apoptosis of MSCs via both endoplasmic reticulum (ER) and mitochondrial pathways rather than extrinsic apoptosis pathway. H₂O₂ caused transient rather than sustained activation of p38 and JNK with no effect on ERK1/2 pathway. P38 was involved in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis. P38 directed both ER stress and mitochondria death pathway in the early apoptosis. In conclusion, exogenous ROS was a major factor to induce apoptosis of MSCs. Both ER stress and mitochondria death pathway were involved in the apoptosis of MSCs. H₂O₂ activated p38 that directed the above two pathways in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis of MSCs. J. Cell. Biochem. 111: 967–978, 2010. © 2010 Wiley‐Liss, Inc.     

Concomitant overexpression of triple antioxidant enzymes selectively increases circulating endothelial progenitor cells in mice with limb ischaemia

Endothelial progenitor cells (EPCs) are a group of heterogeneous cells in bone marrow (BM) and blood. Ischaemia increases reactive oxygen species (ROS) production that regulates EPC number and function. The present study was conducted to determine if ischaemia‐induced ROS differentially regulated individual EPC subpopulations using a mouse model concomitantly overexpressing superoxide dismutase (SOD)1, SOD3 and glutathione peroxidase. Limb ischaemia was induced by femoral artery ligation in male transgenic mice with their wild‐type littermate as control. BM and blood cells were collected for EPCs analysis and mononuclear cell intracellular ROS production, apoptosis and proliferation at baseline, day 3 and day 21 after ischaemia. Cells positive for c‐Kit⁺/CD31⁺ or Sca‐1⁺/Flk‐1⁺ or CD34⁺/CD133⁺ or CD34⁺/Flk‐1⁺ were identified as EPCs. ischaemia significantly increased ROS production and cell apoptosis and decreased proliferation of circulating and BM mononuclear cells and increased BM and circulating EPCs levels. Overexpression of triple antioxidant enzymes effectively prevented ischaemia‐induced ROS production with significantly decreased cell apoptosis and preserved proliferation and significantly increased circulating EPCs level without significant changes in BM EPC populations, associated with enhanced recovery of blood flow and function of the ischemic limb. These data suggested that ischaemia‐induced ROS was differentially involved in the regulation of circulating EPC population.     

Saikosaponin-D Reduces H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced PC12 Cell Apoptosis by Removing ROS and Blocking MAPK-Dependent Oxidative Damage

Neuronal oxidative stress (OS) injury has been proven to be associated with many neurodegenerative diseases, and thus, antioxidation treatment is an effective method for treating these diseases. Saikosaponin-D (SSD) is a sapogenin extracted from Bupleurum falcatum and has been shown to have many pharmacological activities. The main purpose of this study was to investigate whether and how SSD protects PC12 cells from H₂O₂-induced apoptosis. The non-toxic level of SSD significantly mitigated the H₂O₂-induced decrease in cell viability, reduced the apoptosis rate, improved the nuclear morphology, and reduced caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage. Additionally, exogenous H₂O₂-induced apoptosis by damaging the intracellular antioxidation system. SSD significantly slowed the H₂O₂-induced release of malonic dialdehyde (MDA) and lactate dehydrogenase and increased the activity of superoxide dismutase (SOD) and the total antioxidant capacity, thereby reducing apoptosis. More importantly, SSD effectively blocked H₂O₂-induced phosphorylation of extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK), and specific inhibitors of ERK, JNK, and p38-reduced OS injury and apoptosis, suggesting that SSD reduces OS injury and apoptosis via MAPK signalling pathways. Finally, we confirmed that SSD significantly reduced H₂O₂-induced reactive oxygen species (ROS) accumulation, and the ROS inhibitor blocked the apoptosis caused by MAPK activation and cellular oxidative damage. In short, our study confirmed that SSD reduces H₂O₂-induced PC12 cell apoptosis by removing ROS and blocking MAPK-dependent oxidative damage.     

Hydrogen peroxide-induced neuronal apoptosis is associated with inhibition of protein phosphatase 2A and 5, leading to activation of MAPK pathway

Oxidative stress-induced neuronal apoptosis is a prominent feature found in neurodegenerative disorders. However, how oxidative stress induces neuronal apoptosis is not well understood. To address this question, undifferentiated and differentiated neuronal cell lines (PC12 and SH-SY5Y) were exposed to hydrogen peroxide (H₂O₂), a major oxidant generated when oxidative stress occurs. We observed that H₂O₂ induced generation of reactive oxygen species (ROS), leading to apoptosis of the cells in a concentration- and time-dependent manner. H₂O₂ rapidly activated the mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38. Inhibition of Erk1/2, JNK or p38 with kinase inhibitors (U0126, SP600125 or PD169316, respectively), downregulation of Erk1/2 or p38 using RNA interference, or expression of dominant negative c-Jun partially prevented H₂O₂-induced apoptosis. Pretreatment with N-acetyl-l-cysteine (NAC) scavenged H₂O₂-induced ROS, blocking activation of MAPKs and cell death. Furthermore, we found that H₂O₂-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented H₂O₂-activation of Erk/12, JNK and p38, as well as cell death. Similar results were observed in primary murine neurons as well. The results suggest that H₂O₂-induction of ROS inhibit PP2A and PP5, leading to activation of Erk1/2, JNK and p38 pathways thereby resulting in neuronal apoptosis. Our findings suggest that inhibitors of MAPKs (JNK, Erk1/2 and p38), activators of phosphatases (PP2A and PP5) or antioxidants may have potentials to prevent and treat oxidative stress-induced neurodegenerative diseases.     

An aqueous extract of Zingiber officinale Roscoe protects mouse primary hepatic cells against hydrogen peroxide-induced oxidative stress

Hepatocytes exposed to an oxidative stressor such as hydrogen peroxide (H₂O₂) are potentially sensitized to cell death; thus, reactive oxygen species (ROS) are considered to be critical mediators of liver damage. Zingiber officinale Roscoe (ZO), also known as ginger, is cultivated commercially in China, India, Korea, and other parts of the world. In addition, it is used as a spice and flavoring agent and is also purported to possess a number of medicinal properties. In the present study, we examined the protective effect of ZO against cell damage caused by H₂O₂-induced oxidative stress. ZO reduced H₂O₂-induced apoptotic signals and the levels of intracellular ROS. ZO pretreatment also increased the phosphorylation of c-Jun, and JNK kinase. The expression of heme oxygenase-1 (HO-1) and heat shock protein 72 (HSP72) were increased by ZO pretreatment more than H₂O₂ treatment. In addition, siRNA-mediated knockdown of HO-1 and HSP72 decreased protective effect of ZO pretreatment. Our data suggest that ZO decreases ROS levels and the expressions of HO-1 and HSP72 are involved in the hepatocyte protective function of ZO against H₂O₂.     

Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210

Abstract

Background. Insulin protects cardiomyocytes from reactive oxygen species (ROS)-induced apoptosis after ischemic/reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from ROS injury. Methods. Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H₂O₂) or insulin at various concentrations for various periods of time, or with insulin and H₂O₂ for various periods of time. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results. The percentage of viable cells was significantly and inversely associated with H₂O₂ concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H₂O₂ or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H₂O₂-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion. Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H₂O₂/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.     

Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in endothelial cells

Reactive oxygen species including H₂O₂ lead vascular endothelial cells (EC) to undergo apoptosis. Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid mediator that elicits various EC responses. We aimed to explore whether and how S1P modulates EC apoptosis induced by H₂O₂. Treatment of cultured bovine aortic EC (BAEC) with H₂O₂ (750 μM for 6 h) led to DNA fragmentation (ELISA), DNA nick formation (TUNEL staining), and cleavage of caspase-3, key features of EC apoptosis. These responses elicited by H₂O₂ were alike markedly attenuated by pretreatment with S1P (1 μM, 30 min). H₂O₂ induced robust phosphorylation of both p38 and JNK MAP kinases. However, pretreatment with S1P decreased phosphorylation of only p38 MAP kinase, but not that of JNK; conversely, an inhibitor of p38 MAP kinase, but not that of JNK, attenuated H₂O₂-induced caspase-3 activation. Thus S1P attenuates H₂O₂-induced apoptosis of cultured BAEC, involving p38 MAP kinase.     

d-β-Hydroxybutyrate inhibited the apoptosis of PC12 cells induced by H2O2 via inhibiting oxidative stress

Oxidative stress has an important role in neurodegenerative diseases and cerebral ischemic injury. It is reported that d-β-hydroxybutyrate (DβHB), the major component of ketone bodies, is neuroprotective in recent studies. Therefore, in the present work the neuroprotective effects of DβHB on H₂O₂-induced apoptosis mediated by oxidative stress was investigated. PC12 cells were exposed to H₂O₂ with different concentrations of H₂O₂ for different times after DβHB pretreatment. MTT assay, apoptotic rates, intracellular reactive oxygen species (ROS) level, GSH content, mitochondrial membrane potential (MMP) and caspase-3 activity were determined. The results showed that DβHB inhibited the decrease of cell viability induced by H₂O₂ in PC12 cells. DβHB decreased the apoptotic rates induced by H₂O₂. The changes of intracellular ROS, GSH, MMP and caspase-3 activity due to H₂O₂ exposure were partially reversed in PC12 cells. So DβHB inhibited the apoptosis of PC12 cells induced by H₂O₂ via inhibiting oxidative stress.     

Lanthanum chloride suppresses hydrogen peroxide-enhanced calcification in rat calcifying vascular cells

Lanthanum chloride (LaCl₃) has been shown to retard the progression of established atherosclerotic lesions in animal models, and used as a calcium channel blocker in various cellular experiments. In this study, we assessed the role of lanthanum chloride (LaCl₃) in H₂O₂-enhanced calcification in rat calcifying vascular cells (CVCs) and examined the involvement of MAPK signaling pathways. H₂O₂ induced growth inhibition of CVCs, as well as increases in intracellular levels of calcium and reactive oxygen species, ALP activity, apoptosis and calcium deposition. These effects of H₂O₂ were suppressed by pretreatment of the cells with 1 μM of LaCl₃ for 2 h. In addition, H₂O₂ activated the phosphorylation of ERK1/2, JNK and p38 MAPK, but only the last two were associated with the ALP activity. Our findings demonstrate that H₂O₂-enhanced osteoblastic differentiation and apoptosis are responsible for the increased calcification in rat CVCs, and LaCl₃ can counteract these effects by suppressing the activation of JNK (JNK2, but not JNK1) and p38 MAPK signaling pathway.     

Combination of simvastatin administration and EPC transplantation enhances angiogenesis and protects against apoptosis for hindlimb ischemia

The aim of this present study is to investigate the impacts of combinatorial simvastatin administration and endothelial progenitor cell (EPC) transplantation on therapeutic angiogenesis in an athymic nude mouse model of hind limb ischemia. Athymic nude mice were divided into four groups (n = 10/group): vehicle administration plus PBS injection (control), simvastatin administration plus PBS injection (simvastatin), vehicle administration plus EPC transplantation (EPC), and simvastatin administration plus EPC transplantation (combination). The combination therapy had the greatest laser Doppler blood perfusion imager (LDPI) index and capillary density among the four groups. Importantly, this combination therapy significantly reduced apoptosis of ischemic skeletal muscle cells in part through downregulation of Bax and upregulation of Bcl-2 compared with the other groups. Moreover, the combination therapy exhibited the highest efficacy of increasing the ratio of phospho-Akt to Akt among the four groups. Taken together, the simvastatin and EPC combination therapy promotes powerful angiogenesis in hindlimb ischemia. The combination therapy not only inhibites apoptosis of ischemic skeletal muscle cells partially via downregulation of Bax and upregulation of Bcl-2, but also activates Akt phosphorylation significantly. These efficacies may be mediated by the angiogenic potency of simvastatin, EPCs, and by the beneficial effects of simvastatin on transplanted EPCs as well.     

ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis

Apoptosis signal-regulating kinase (ASK) 1 is activated in response to various cytotoxic stresses including TNF, Fas and reactive oxygen species (ROS) such as H₂O₂, and activates c-Jun NH₂-terminal kinase (JNK) and p38. However, the roles of JNK and p38 signaling pathways during apoptosis have been controversial. Here we show that by deleting ASK1 in mice, TNF- and H₂O₂-induced sustained activations of JNK and p38 are lost in ASK1^–/– embryonic fibroblasts, and that ASK1^–/– cells are resistant to TNF- and H₂O₂-induced apoptosis. TNF- but not Fas-induced apoptosis requires ROS-dependent activation of ASK1–JNK/p38 pathways. Thus, ASK1 is selectively required for TNF- and oxidative stress-induced sustained activations of JNK/p38 and apoptosis.     

Non‐apoptotic function of caspases in a cellular model of hydrogen peroxide‐associated colitis

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)‐associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non‐tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H₂O₂). A population of HCEC survived H₂O₂‐induced oxidative stress via JNK‐dependent cell cycle arrests. Caspases, p21^WAF1 and γ‐H2AX were identified as JNK‐regulated proteins. Up‐regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan‐caspase inhibitor Z‐VAD‐FMK caused up‐regulation of γ‐H2AX, a DNA‐damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G₁‐phase as first response to oxidative stress and increased S‐phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non‐apoptotic function by promoting cells through G₁‐ and S‐phase by overriding the G₁/S‐ and intra‐S checkpoints despite DNA‐damage. This led to the accumulation of cells in the G₂/M‐phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ‐H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase‐dependent proteolytic degradation of the DNA‐damage checkpoint protein ATM that is upstream of γ‐H2AX. As a consequence, undetected DNA‐damage and increased proliferation were found in repeatedly H₂O₂‐exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non‐apoptotic function of caspases. Overexpression of upstream p‐JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.     

Grain and Bean Lysates Improve Function of Endothelial Progenitor Cells from Human Peripheral Blood: Involvement of the Endogenous Antioxidant Defenses

Increased oxidative stress contributes to the functional impairment of endothelial progenitor cells (EPCs), the pivotal players in the servicing of the endothelial cell lining. Several evidences suggest that decreasing oxidative stress by natural compounds with antioxidant properties may improve EPCs bioactivity. Here, we investigated the effects of Lisosan G (LG), a Triticum Sativum grain powder, and Lady Joy (LJ), a bean lysate, on function of EPCs exposed to oxidative stress. Peripheral blood mononuclear cells were isolated and plated on fibronectin-coated culture dishes; adherent cells, identified as early EPCs, were pre-treated with different concentrations of LG and LJ and incubated with hydrogen peroxide (H₂O₂). Viability, senescence, adhesion, ROS production and antioxidant enzymes gene expression were evaluated. Lysate-mediated Nrf-2 (nuclear factor (erythroid-derived 2)-like 2)/ARE (antioxidant response element) activation, a modulator of oxidative stress, was assessed by immunocytochemistry. Lady Joy 0.35–0.7 mg/ml increases EPCs viability; pre-treatment with either LG 0.7 mg/ml and LJ 0.35–0.7 mg/ml protect EPCs viability against H₂O₂-induced injury. LG 0.7 and LJ 0.35–0.7 mg/ml improve EPCs adhesion; pre-treatment with either LG 0.35 and 0.7 mg/ml or LJ 0.35, 0.7 and 1.4 mg/ml preserve adhesiveness of EPCs exposed to H₂O₂. Senescence is attenuated in EPCs incubated with lysates 0.35 mg/ml. After exposure to H₂O₂, LG pre-treated cells show a lower senescence than untreated EPCs. Lysates significantly decrease H₂O₂-induced ROS generation. Both lysates increase glutathione peroxidase-1 and superoxide dismutase-2 (SOD-2) expression; upon H₂O₂ exposure, pre-treatment with LJ allows higher SOD-2 expression. Heme oxigenase-1 increases in EPCs pre-treated with LG even upon H₂O₂ exposure. Finally, incubation with LG 0.7 mg/ml results in Nrf-2 translocation into the nucleus both at baseline and after the oxidative challenge. Our data suggest a protective effect of lysates on EPCs exposed to oxidative stress through the involvement of antioxidant systems. Lisosan G seems to activate the Nrf-2/ARE pathways.     

Activation of mitogen-activated protein kinases is essential for hydrogen peroxide -induced apoptosis in retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells are constantly exposed to oxidative injury while clearing byproducts of photoreceptor turnover, a circumstance thought to be responsible for degenerative retinal diseases. The mechanisms of hydrogen peroxide (H₂O₂)-induced apoptosis in RPE cells are not fully understood. We studied signal transduction mechanisms of H₂O₂-induced apoptosis in the human RPE cell line ARPE-19. Activation of two stress kinases (JNK and p38) occurs during H₂O₂ stimulation, and H₂O₂-mediated cell death was significantly reduced by their specific inhibition. Exposure to a lethal dose of H₂O₂ elicited Bax translocation to the mitochondria and release of apoptosis-inducing factor (AIF) from the mitochondria, both of which were abolished by either JNK- or p38-specific inhibitors. Both H₂O₂-induced cell death and JNK/p38 phosphorylation were partially inhibited by C. difficile toxin B, inhibitor of Rho, Rac, and cdc42. Use of pull-down assays revealed that the small GTPase activated by H₂O₂ is Rac1. This study is the first to demonstrate that H₂O₂ induces a Rac1/JNK1/p38 signaling cascade, and that JNK and p38 activation is important for H₂O₂-induced apoptosis as well as AIF/Bax translocation of RPE cells. Y.-C. Yang and T.-C. Ho contributed equally to the work described herein.