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.     

Mitogen-activated protein kinases modulate H(2)O(2)-induced apoptosis in primary rat alveolar epithelial cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant-mediated ATII cell apoptosis using in vitro models of H(2)O(2)-stress. H(2)O(2), delivered either as a bolus or as a flux, lead to time- and concentration-dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H(2)O(2) concentrations and production rates in the physiological range (1 microM) and peaked at 100 microM H(2)O(2). Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H(2)O(2) for maximal responses. H(2)O(2)-induced apoptosis was preceded by rapid activation of all three classes of mitogen-activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro-apoptotic role for JNK pathway and an anti-apoptotic role for ERK- and p38-initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant-related lung injury.     

Leptin protects H9c2 rat cardiomyocytes from H2O2-induced apoptosis

Obesity is a known risk factor for induction of myocardial infarction, but, paradoxically, may also confer a protective effect against subsequent remodeling leading to heart failure. In this study, we investigated the effect of leptin, the product of the obese (ob) gene, on cardiomyocyte apoptosis, a well-characterized component of cardiac remodeling after myocardial infarction. Exposing H9c2 cells to H(2)O(2) decreased cell viability, and this was attenuated by pretreating cells with leptin for 1 h, but not 24 h. Leptin also attenuated the ability of H(2)O(2) to increase phosphatidylserine exposure and annexin V binding. Further investigation of underlying mechanisms of leptin's protective effect demonstrated that the H(2)O(2)-induced decrease in mitochondrial membrane potential (Psi) leading to cytochrome c release was attenuated by leptin pretreatment, and this was associated with reduced translocation of the pro-apoptotic Bax protein to the mitochondrial membrane. Finally, leptin prevented H(2)O(2)-induced increases in caspase-3 cleavage and activity, although again 24 h leptin pretreatment did not confer significant protection. In summary, we have demonstrated that acute leptin pretreatment mediates anti-apoptotic effects in H9c2 rat cardiomyocytes, which may be of significance in clarifying the direct impact of leptin on the heart.     

Protective effect of salidroside against H2O2-induced cell apoptosis in primary culture of rat hippocampal neurons

Salidroside, a phenylpropanoid glycoside separated from a medicinal plant Rhodiola rosea, has been documented to have protective effects on neuronal cells in vitro. This study investigated whether salidroside was able to extend its unique neuroprotection to primary cultured rat hippocampal neurons against hydrogen peroxide (H₂O₂)-induced cell damage. Cell viability tests and cell apoptosis assays confirmed that salidroside pretreatment attenuated H₂O₂-stimulated apoptotic cell death in primary culture of hippocampal neurons in a concentration-dependent manner. The measurements of caspase-3 activity, nitric oxide (NO) production, and NO synthase (NOS) activity suggest that the protection of salidroside, shown in this study, might be mediated by inhibiting caspase-3 activity, and antagonizing NO production and NOS activity during H₂O₂ stimulation. Perhaps, this study might contribute to the development of salidroside as a broad-spectrum agent for preventing and/or treating neuronal damage in neurodegenerative disorders.     

Early determinants of H2O2-induced endothelial dysfunction

Reactive oxygen species (ROS) can stimulate nitric oxide (NO(*)) production from the endothelium by transient activation of endothelial nitric oxide synthase (eNOS). With continued or repeated exposure, NO(*) production is reduced, however. We investigated the early determinants of this decrease in NO(*) production. Following an initial H(2)O(2) exposure, endothelial cells responded by increasing NO(*) production measured electrochemically. NO(*) concentrations peaked by 10 min with a slow reduction over 30 min. The decrease in NO(*) at 30 min was associated with a 2.7-fold increase in O(2)(*-) production (p < 0.05) and a 14-fold reduction of the eNOS cofactor, tetrahydrobiopterin (BH(4), p < 0.05). Used as a probe for endothelial dysfunction, the integrated NO(*) production over 30 min upon repeated H(2)O(2) exposure was attenuated by 2.1-fold (p = 0.03). Endothelial dysfunction could be prevented by BH(4) cofactor supplementation, by scavenging O(2)(*-) or peroxynitrite (ONOO(-)), or by inhibiting the NADPH oxidase. Hydroxyl radical (()OH) scavenging did not have an effect. In summary, early H(2)O(2)-induced endothelial dysfunction was associated with a decreased BH(4) level and increased O(2)(*-) production. Dysfunction required O(2)(*-), ONOO(-), or a functional NADPH oxidase. Repeated activation of the NADPH oxidase by ROS may act as a feed forward system to promote endothelial dysfunction.     

Nitric oxide attenuates H(2)O(2)-induced endothelial barrier dysfunction: mechanisms of protection

Nitric oxide (.NO) attenuates hydrogen peroxide (H(2)O(2))-mediated injury in porcine pulmonary artery endothelial cells (PAECs) and modulates intracellular levels of cGMP and cAMP. We hypothesized that.NO attenuates H(2)O(2)-induced PAEC monolayer barrier dysfunction through cyclic nucleotide-dependent signaling mechanisms. To examine this hypothesis, cultured PAEC monolayers were treated with H(2)O(2), and barrier function was measured as transmonolayer albumin clearance. H(2)O(2) caused significant PAEC barrier dysfunction that was attenuated by intracellular as well as extracellular.NO generation.NO increased PAEC cGMP and cAMP levels, but treatment with inhibitors of soluble guanylate cyclase or protein kinase G did not abrogate.NO-mediated barrier protection. In contrast, H(2)O(2) decreased protein kinase A activity, and inhibiting protein kinase A abrogated the protective effect of.NO. H(2)O(2)-induced barrier dysfunction was not associated with decreased levels of cGMP or cAMP. 3-Isobutyl-1-methylxanthine and the cGMP analog 8-bromo-cGMP had little effect on H(2)O(2)-mediated endothelial barrier dysfunction, whereas 8-bromo-cAMP plus 3-isobutyl-1-methylxanthine was protective. These results indicate that.NO modulates vascular endothelial barrier function through cAMP-dependent signaling mechanisms.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death

PAR [poly(ADP-ribose)] is a structural and regulatory component of multiprotein complexes in eukaryotic cells. PAR catabolism is accelerated under genotoxic stress conditions and this is largely attributable to the activity of a PARG (PAR glycohydrolase). To overcome the early embryonic lethality of parg-knockout mice and gain more insights into the biological functions of PARG, we used an RNA interference approach. We found that as little as 10% of PARG protein is sufficient to ensure basic cellular functions: PARG-silenced murine and human cells proliferated normally through several subculturing rounds and they were able to repair DNA damage induced by sublethal doses of H2O2. However, cell survival following treatment with higher concentrations of H2O2 (0.05-1 mM) was increased. In fact, PARG-silenced cells were more resistant than their wild-type counterparts to oxidant-induced apoptosis while exhibiting delayed PAR degradation and transient accumulation of ADP-ribose polymers longer than 15-mers at early stages of drug treatment. No difference was observed in response to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, suggesting a specific involvement of PARG in the cellular response to oxidative DNA damage.     

Cyclosporin A inhibits H2O2-induced apoptosis of human fibroblasts

Several clinical studies have shown that cyclosporin A (CsA) is effective for treating a variety of chronic inflammatory and autoimmune diseases. Because reactive oxygen species are believed to play a key role in the development of these diseases, causing cell apoptosis, we investigated whether CsA inhibits H2O2-induced apoptosis. Preincubation of human fibroblasts with CsA dose-dependently decreased H2O2-induced apoptosis. Apoptosis suppression by CsA was correlated with the prevention of mitochondrial dysfunction and caspase activation. Thus, our results suggest that the inhibition of apoptosis by CsA may at least partly contribute to the anti-inflammatory effect of CsA.     

Evidence for the association of ultraviolet-C and H(2)O(2)-induced apoptosis with acid sphingomyelinase activation

We measured the temperature dependence of oxygen evolution in thylakoids from tobacco using mass spectrometry and high resolution polarography. We determined the initial S-state distribution and the efficiency of the transition between these states including the probability of the O(2) yield through a fast mode. We observed discontinuous changes of the parameters at the temperatures 11 degrees C, 15 degrees C and 21 degrees C. Due to the mass spectroscopy data we think that the irregularity observed at 11 degrees C is due to conformational changes within the water catalytic site. We show that the different contributions of the slow and fast modes of oxygen evolution and of the water molecule exchange are correlated and that their behavior can be explained in terms of the H(2)O accessibility to the water splitting enzyme.     

Preconditioning with hydrogen peroxide (H2O2) or ischemia in H2O2-induced cardiac dysfunction

To assess whether allantoin levels in serum and urine are influenced by exhaustive and moderate exercise and whether allantoin is a useful indicator of exercise-induced oxidative stress in humans, we made subjects perform exhaustive and moderate (100% and 40% VO₂max) cycling exercise and examined the levels of allantoin, thiobarbituric acid reactive substances (TBARS) and urate in serum and urine. Immediately after exercise at 100% VO₂max, the serum allantoin/urate ratio was significantly elevated compared with the resting levels while the serum urate levels was significantly elevated 30 min after exercise. The serum TBARS levels did not increase significantly compared with the resting levels. Urinary allantoin excretion significantly increased during 60 min of recovery after exercise, however, urinary urate excretion decreased significantly during the same period. The urinary allantoin/urate ratio also rapidly increased during 60 min of recovery after exercise. Urinary TBARS excretion decreased during the first 60 min of the recovery period and thereafter significantly increased during the latter half of the recovery period. On the contrary, after 40% VO₂max of exercise, no significant changes in the levels of urate, allantoin and TBARS in serum or urine were observed. These findings suggest that allantoin levels in serum and urine may reflect the extent of oxidative stress in vivo and that the allantoin which appeared following exercise may have originated not from urate formed as a result of exercise but from urate that previously existed in the body. Furthermore, these findings support the view that allantoin in serum and urine is a more sensitive and reliable indicator of in vivo oxidative stress than lipid peroxidation products measured as TBARS.     

H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury

Non-phagocytic NAD(P)H oxidases have been implicated as major sources of reactive oxygen species in blood vessels. These oxidases can be activated by cytokines, thereby generating O(2), which is subsequently converted to H(2)O(2) and other oxidant species. The oxidants, in turn, act as important second messengers in cell signaling cascades. We hypothesized that reactive oxygen species, themselves, can activate the non-phagocytic NAD(P)H oxidases in vascular cells to induce oxidant production and, consequently, cellular injury. The current report demonstrates that exogenous exposure of non-phagocytic cell types of vascular origin (smooth muscle cells and fibroblasts) to H(2)O(2) activates these cell types to produce O(2) via an NAD(P)H oxidase. The ensuing endogenous production of O(2) contributes significantly to vascular cell injury following exposure to H(2)O(2). These results suggest the existence of a feed-forward mechanism, whereby reactive oxygen species such as H(2)O(2) can activate NAD(P)H oxidases in non-phagocytic cells to produce additional oxidant species, thereby amplifying the vascular injury process. Moreover, these findings implicate the non-phagocytic NAD(P)H oxidase as a novel therapeutic target for the amelioration of the biological effects of chronic oxidant stress.     

Adenosine A2B receptors play an important role in bone homeostasis

Bone homeostasis is a finely regulated mechanism involving different molecular pathways including adenosine signaling. The aim of this study is to determine the bone phenotype of adenosine A2B receptor knockout (A2BRKO) mice and to measure their ability to form new bone. Moreover, we analyzed the functionality of osteoclasts and osteoblasts from A2BRKO mice. Microcomputed tomography (μCT) analysis revealed a decrease of bone substance, bone mineral density, and trabecular number in A2BRKO mice compared to the WT mice at the same age. We measured the new bone formation by injecting fluorescent markers: it was reduced in femur and tibia of A2BRKO mice compare to the WT. A2BRKO young mice have fewer osteoblasts and an increase of osteoclasts was measured in the hind limbs of young and adult mice. A2BRKO osteoclasts are also more active in vitro, showing an increase of pit formation in dentin discs. Surprisingly in mature osteoblasts from A2BRKO mice, we measured an increase of calcified matrix production, collagen deposition, and alkaline phosphatase activity. These results demonstrate that A2BR on osteoblasts and osteoclasts regulate bone homeostasis.     

H2O2-induced block of glycolysis as an active ADP-ribosylation reaction protecting cells from apoptosis.

H2O2 treatment on U937 cells leads to the block of glycolytic flux and the inactivation of glyceraldehyde-3-phosphate-dehydrogenase by a posttranslational modification (possibly ADP-ribosylation). Glycolysis spontaneously reactivates after 2 h of recovery from oxidative stress; thereafter cells begin to undergo apoptosis. The specific ADP-ribosylation inhibitor 3-aminobenzamide inhibits the stress-induced inactivation of glyceraldehyde-3-phosphate-dehydrogenase and the block of glycolysis; concomitantly, it anticipates and increases apoptosis. Exogenous block of glycolysis (i.e., by culture in glucose-free medium or with glucose analogs or after NAD depletion), turns the transient block into a stable one: this results in protection from apoptosis, even when downstream cell metabolism is kept active by the addition of pyruvate. All this evidence indicates that the stress-induced block of glycolysis is not the result of a passive oxidative damage, but rather an active cell reaction programmed via ADP-ribosylation for cell self-defense.     

H(2)O(2)-induced Ca(2+) overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway

Generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury and Ca(2+) overload has not been fully characterized. The increase in Na(+)/H(+) exchanger (NHE-1) activity observed during I/R injury is an attractive candidate to link increased ROS production with Ca(2+) overload. We have shown that low doses of H(2)O(2) increase NHE-1 activity in an extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined the effect of low doses of H(2)O(2) on intracellular Ca(2+) in fura 2-loaded, spontaneously contracting neonatal rat ventricular myocytes. H(2)O(2) induced a time- and concentration-dependent increase in diastolic intracellular Ca(2+) concentration that was blocked by inhibition of ERK1/2 activation with 5 microM U-0126 (88%) or inhibition of NHE-1 with 5 microM HOE-642 (50%). Increased NHE activity was associated with phosphorylation of the NHE-1 carboxyl tail that was blocked by U-0126. These results suggest that H(2)O(2) induced Ca(2+) overload is partially mediated by NHE-1 activation secondary to phosphorylation of NHE-1 by the ERK1/2 MAP kinase pathway.     

Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).     

Estradiol protects adipose tissue-derived stem cells against H(2) O(2) -induced toxicity

Oxidative stress is associated with various pathophysiological processes, including cell survival, adhesion, apoptosis, and cancer. In the present study, we aimed to evaluate the effects of H₂O₂‐induced toxicity on adipose tissue–derived stem cells (ADSCs) and whether 17β‐estradiol (E₂) has protective effects on these cells. ADSCs derived from adult Sprague–Dawley rats were pretreated with different doses of E₂ for 24 h and then exposed to 200 µM H₂O₂ for 4 h. Incubation of ADSCs with H₂O₂‐decreased cell viability in a concentration‐dependent fashion (p < 0.0001), whereas pretreatment of these cells with E₂ significantly reversed toxicity (p < 0.05), inhibited apoptotic changes, and decreased lipid peroxidation (p < 0.0005). Our findings suggest that E₂ protects ADSCs from oxidative‐induced cell death, and therefore, it may be used to improve the survival rate and regenerative capacity of stem cells. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:301–307, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21421     

Characterization of H2O2-induced acute apoptosis in cultured neural stem/progenitor cells

In the present study, we characterized hydrogen peroxide (H₂O₂)-induced cell apoptosis and related cell signaling pathways in cultured embryonic neural stem/progenitor cells (NS/PCs). Our data indicated that H₂O₂ induced acute cell apoptosis in NS/PC in concentration- and time-dependent manners and selectively, it transiently increased PI3K-Akt and Mek-Erk1/2 in a dose-dependent manner. Inhibition of PI3K-Akt with wortmannin, a PI3-K inhibitor, was found to significantly increase H₂O₂-induced acute apoptosis and dramatically decrease basal pGSK3β levels. The level of pGSK3β remained unchanged with H₂O₂ exposure. We conclude that the transient activation of PI3K-Akt signaling delays the H₂O₂-induced acute apoptosis in cultured NS/PCs in part through maintaining the basal pGSK3β level and activating other downstream effectors.