Superoxide potently induces ceramide formation in glomerular endothelial cells

Recent evidence suggests that the sphingolipid-derived second messenger ceramide and oxidative stress are intimately involved in apoptosis induction. Here we report that exposure of microcapillary glomerular endothelial cells to superoxide-generating substances, including hypoxanthine/xanthine oxidase and the redox cyclers DMNQ and menadione results in a dose-dependent and delayed increase in the lipid signaling molecule ceramide. Long-term incubation of endothelial cells for 2-30 h with either DMNQ or hypoxanthine/xanthine oxidase leads to a continuous increase in ceramide levels. In contrast, short-term stimulation for 1 min up to 1 h had no effect on ceramide formation. The DMNQ-induced delayed ceramide formation is dose-dependently inhibited by reduced glutathione, whereas oxidized glutathione was without effect. Furthermore, N-acetylcysteine completely blocks DMNQ-induced ceramide formation. All superoxide-generating substances were found to dose-dependently trigger endothelial cell apoptosis. In addition, glutathione and N-acetylcysteine also prevented superoxide-induced apoptosis and implied that ceramide represents an important mediator of superoxide-triggered cell responses like apoptosis.     

Superoxide dismutase protects against apoptosis and alveolar enlargement induced by ceramide

The molecular events leading to emphysema development include generation of oxidative stress and alveolar cell apoptosis. Oxidative stress upregulates ceramides, proapoptotic signaling sphingolipids that trigger further oxidative stress and alveolar space enlargement, as shown in an experimental model of emphysema due to VEGF blockade. As alveolar cell apoptosis and oxidative stress mutually interact to mediate alveolar destruction, we hypothesized that the oxidative stress generated by ceramide is required for its pathogenic effect on lung alveoli. To model the direct lung effects of ceramide, mice received ceramide intratracheally (Cer(12:0) or Cer(8:0); 1 mg/kg) or vehicle. Apoptosis was inhibited with a general caspase inhibitor. Ceramide augmentation shown to mimic levels found in human emphysema lungs increased oxidative stress, and decreased, independently of caspase activation, the lung superoxide dismutase activity at 48 h. In contrast to their wild-type littermates, transgenic mice overexpressing human Cu/Zn SOD were significantly protected from ceramide-induced superoxide production, apoptosis, and air space enlargement. Activation of lung acid sphingomyelinase in response to ceramide treatment was abolished in the Cu/Zn SOD transgenic mice. Since cigarette smoke-induced emphysema in mice is similarly ameliorated by the Cu/Zn SOD overexpression, we hypothesized that cigarette smoke may induce ceramides in the mouse lung. Utilizing tandem mass spectrometry, we documented increased lung ceramides in adult mice exposed to cigarette smoke for 4 wk. In conclusion, ceramide-induced superoxide accumulation in the lung may be a critical step in ceramide's proapoptotic effect in the lung. This work implicates excessive lung ceramides as amplifiers of lung injury through redox-dependent mechanisms.     

Role of plasma membrane coenzyme Q on the regulation of apoptosis.

Serum withdrawal is a model to study the mechanisms involved in the induction of apoptosis caused by mild oxidative stress. Apoptosis induced by growth factors removal was prevented by the external addition of antioxidants such as ascorbate, alpha-tocopherol and coenzyme Q (CoQ). CoQ is a lipophilic antioxidant which prevents oxidative stress and participates in the regeneration of alpha-tocopherol and ascorbate in the plasma membrane. We have found an inverse relationship between CoQ content in plasma membrane and lipid peroxidation rates in leukaemic cells. CoQ10 addition to serum-free culture media prevented both lipid peroxidation and cell death. Also, CoQ10 addition decreased ceramide release after serum withdrawal by inhibition of magnesium-dependent plasma membrane neutral-sphingomyelinase. Moreover, CoQ10 addition partially blocked activation of CPP32/caspase-3. These results suggest CoQ of the plasma membrane as a regulator of initiation phase of oxidative stress-mediated serum withdrawal-induced apoptosis.     

Ceramide selectively inhibits apoptosis-associated events in NGF-deprived sympathetic neurons

Ceramide manifests both neurotoxic and neuroprotective properties depending on the experimental system. Ito and Horigome previously reported that ceramide delays apoptosis in a classic model of developmental programmed cell death, i.e. sympathetic neurons undergoing NGF deprivation.1 Here, we investigated the actions of ceramide upon the biochemical and genetic changes that occur in NGF deprived neurons. We correlate ceramide's neuroprotective actions with the ability of ceramide to antagonize NGF deprivation-induced oxidative stress and c-jun induction, both of which contribute to apoptosis in this model. However, ceramide did not block NGF deprivation-induced declines in RNA and protein synthesis, suggesting that ceramide does not slow all apoptosis-related events. Overall, these results are significant in that they show that ceramide acts early in the death cascade to antagonize two events necessary for NGF-deprivation induced neuronal apoptosis. Moreover, these results dissociate declines in neuronal function, i.e. macromolecular synthesis, from the neuronal death cascade.     

Long-chain ceramide is a potent inhibitor of the mitochondrial permeability transition pore

The sphingolipid ceramide has been implicated in mediating cell death that is accompanied by mitochondrial functional alterations. Moreover, ceramide has been shown to accumulate in mitochondria upon induction of apoptotic processes. In this study, we sought to evaluate the effects of natural, highly hydrophobic long-chain ceramides on mitochondrial function in vitro. Ceramide in a dodecane/ethanol delivery system inhibited the opening of the mitochondrial permeability transition pore (PTP) induced by either oxidative stress, SH group cross-linking, or high Ca(2+) load, suggesting that the inhibitory point is at a level at which major PTP regulatory pathways converge. Moreover, ceramide had no effect on well known mitochondrial components that modulate PTP activity, such as cyclophilin D, voltage-dependent anion channel, adenine nucleotide transporter, and ATP synthase. The inhibitory effect of ceramide on PTP was not stereospecific, nor was there a preference for ceramide over dihydroceramide. However, the effect of ceramide on PTP was significantly influenced by the fatty acid moiety chain length. These studies are the first to show that long-chain ceramide can influence PTP at physiologically relevant concentrations, suggesting that it is the only known potent natural inhibitor of PTP. These results suggest a novel mechanism of ceramide regulation of mitochondrial function.     

Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells

Nitric oxide and reactive oxygen species play a critical role in photoreceptor apoptosis. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. Here, we demonstrate that the sphingolipid ceramide is the key mediator of oxidative stress-induced apoptosis in 661W retinal photoreceptor cells. Treatment of 661W cells with the nitric oxide donor, sodium nitroprusside, activates acid sphingomyelinase. As a result, sphingomyelin is hydrolysed, which leads to an increase in the concentration of ceramide. We also show that ceramide is responsible for the activation of the mitochondrial apoptotic pathway in 661W photoreceptor cells and subsequent activation of the caspase cascade. Furthermore, we show for the first time that ceramide is responsible for the increased Ca2+ levels in the mitochondria and cytosol that precedes activation of the calpain-mediated apoptotic pathway. Additionally, we provide evidence that ceramide also activates the endolysosomal protease cathepsin D pathway. In summary, our findings show that ceramide controls the cell death decisions in photoreceptor cells and highlight the relevance of acid sphingomyelinase as a potential therapeutic target for the treatment of retinal pathologies.     

A role for manganese superoxide dismutase in apoptosis after photosensitization

The oxidative stress triggered by photodynamic therapy (PDT) involves generation of cytotoxic reactive oxygen species, including superoxide radical, accumulation of de novo-generated ceramide, and induction of apoptosis. Since PDT with the photosensitizer phthalocyanine Pc 4 induces mitochondrial damage and the superoxide scavenger manganese superoxide dismutase (MnSOD) is localized to mitochondria, here we tested genetically the role of MnSOD in apoptosis and ceramide accumulation after photosensitization with Pc 4. Jurkat cells overexpressing wild-type MnSOD were protected from Pc 4-PDT-initiated apoptosis, but not from increased ceramide response to Pc 4-PDT. In Jurkat cells overexpressing mutant MnSOD, however, DEVDase activation and ceramide formation were promoted post-Pc 4-PDT. Similarly, in MnSOD-null cells, Pc 4-PDT-induced apoptosis, as well as ceramide accumulation, were enhanced compared to their normal counterparts. The data show that MnSOD affects sensitivity of cells to Pc 4-PDT-initiated apoptosis, and partly ceramide accumulation, suggesting that the processes are superoxide-mediated.     

Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction

One of the earliest changes observed in retinal microvessels in diabetic retinopathy is the selective loss of intramural pericytes. We tested the hypothesis that AGE might be involved in the disappearance of retinal pericytes by apoptosis and further investigated the signaling pathway leading to cell death. Chronic exposure of pericytes to methylglyoxal-modified bovine serum albumin (AGE-BSA) (3 microM) leads to a 3-fold increase of apoptosis (8.9 +/- 1.1%), associated with an increase in cellular ceramide (185 +/- 12%) and diacylglycerol (194 +/- 9%) levels. Ceramide formation was almost inhibited (95%) by an acidic sphingomyelinase inhibitor, desipramine (0.3 microM). Dual inhibition of ceramide (95%) and diacylglycerol (80%) production was observed with a phosphatidylcholine-phospholipase C inhibitor, D609 (9.4 microM). Taken together, these results suggest activation of phosphatidylcholine-phospholipase C coupled to acidic sphingomyelinase. However, both inhibitors only partially protected pericytes against apoptosis, suggesting another apoptotic pathway independent of diacylglycerol/ceramide production. Treatments with various antioxidants completely inhibited pericyte apoptosis, suggesting oxidative stress induction during this apoptotic process. Inhibition of diacylglycerol/ceramide production by N-acetyl-L-cysteine suggests that oxidative stress acts upstream of the two metabolic pathways. AGE treated with metal chelators were also able to induce pericyte apoptosis, suggesting a specific effect of AGE on intracellular oxidative stress independent of redox-active metal ions bound to AGE. In conclusion, these results identify new biochemical targets involved in pericyte loss, which can provide new therapeutic perspectives in diabetic retinopathy.     

Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice

Alveolar cell apoptosis is involved in the pathogenesis of emphysema, a prevalent disease primarily caused by cigarette smoking. We report that ceramide, a second messenger lipid, is a crucial mediator of alveolar destruction in emphysema. Inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis, oxidative stress and emphysema caused by blockade of the vascular endothelial growth factor (VEGF) receptors in both rats and mice. Emphysema was reproduced with intratracheal instillation of ceramide in naive mice. Excessive ceramide triggers a feed-forward mechanism mediated by activation of secretory acid sphingomyelinase, as suggested by experiments with neutralizing ceramide antibody in mice and with acid sphingomyelinase-deficient fibroblasts. Concomitant augmentation of signaling initiated by a prosurvival metabolite, sphingosine-1-phosphate, prevented lung apoptosis, implying that a balance between ceramide and sphingosine-1-phosphate is required for maintenance of alveolar septal integrity. Finally, increased lung ceramides in individuals with smoking-induced emphysema suggests that ceramide upregulation may be a crucial pathogenic element and a promising target in this disease that currently lacks effective therapies.     

Cell autonomous apoptosis defects in acid sphingomyelinase knockout fibroblasts

A body of evidence suggests that stress-induced sphingomyelin hydrolysis to the second messenger ceramide initiates apoptosis in some cells. Although studies using lymphoblasts from Niemann-Pick disease patients or acid sphingomyelinase (ASMase)-deficient mice have provided genetic support for this hypothesis, these models have not been universally accepted as definitive. Here, we show that mouse embryonic fibroblasts (MEFs) prepared from asmase mice manifest cell autonomous defects in apoptosis in response to several stresses. In particular, asmase(-/-) MEFs failed to generate ceramide and were totally resistant to radiation-induced apoptosis but remained sensitive to staurosporine, which did not induce ceramide. asmase(-/-) MEFs were also partially resistant to tumor necrosis factor alpha/ actinomycin D and serum withdrawal. Thus, resistance to apoptosis in asmase(-/-) MEFs was not global but rather stress type specific. Most importantly, the sensitivity to stress could be restored in the asmase(-/-) MEFs by administration of natural ceramide. Overcoming apoptosis resistance by natural ceramide is evidence that it is the lack of ceramide, not ASMase, that determines apoptosis sensitivity. The ability to rescue the apoptotic phenotype without reversing the genotype by the product of the enzymatic deficiency provides proof that ceramide is obligate for apoptosis induction in response to some stresses.     

Niemann-Pick human lymphoblasts are resistant to phthalocyanine 4-photodynamic therapy-induced apoptosis.

Stress-induced activation of sphingomyelinase (SMase) leading to generation of ceramide, a lipid mediator, has been associated with apoptosis in several malignant and nonmalignant cell lines. Photodynamic therapy (PDT), with the phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N(CH3)2], is an oxidative stress associated with increased ceramide generation and subsequent induction of apoptosis in various cell types. We assessed the role of SMase in photocytotoxicity. Normal human lymphoblasts accumulated ceramide and underwent apoptosis after Pc 4-PDT. In contrast, Niemann-Pick disease (NPD) lymphoblasts, which are deficient in acid sphingomyelinase (ASMase) activity, failed to respond to Pc 4-PDT with ceramide accumulation and apoptosis, suggesting that ASMase may be a Pc 4-PDT target. NPD lymphoblasts were exposed to exogenous bacterial sphingomyelinase (bSMase) to test whether these defects are reversible. Treatment of NPD cells with bSMase itself led to elevated ceramide formation, which did not translate into induction of apoptosis. However, a combination of Pc 4-PDT + bSMase induced a significant apoptotic response. Thus, the combined treatment of Pc 4-PDT + bSMase, rather than bSMase alone, was required to restore apoptosis in NPD cells. These data support the hypothesis that SMase is a proapoptotic factor determining responsiveness of cells to Pc 4-PDT.     

Chemistry and biochemistry of lipid peroxidation products

Abstract

Oxidative stress and resulting lipid peroxidation is involved in various and numerous pathological states including inflammation, atherosclerosis, neurodegenerative diseases and cancer. This review is focused on recent advances concerning the formation, metabolism and reactivity towards macromolecules of lipid peroxidation breakdown products, some of which being considered as ‘second messengers’ of oxidative stress. This review relates also new advances regarding apoptosis induction, survival/proliferation processes and autophagy regulated by 4-hydroxynonenal, a major product of omega-6 fatty acid peroxidation, in relationship with detoxication mechanisms. The use of these lipid peroxidation products as oxidative stress/lipid peroxidation biomarkers is also addressed.     

UVA radiation stimulates ceramide production: relationship to oxidative stress and potential role in ERK, JNK, and p38 activation

Exposure of human keratinocytes to UVA radiation induced an increase in ceramide (CER) intracellular content, with a dose-dependent effect within the range of 4-9 J/cm(2). The production of CER reached a maximum 2 h after UVA irradiation. The increase of CER was proportional to the intracellular content of reactive oxygen species, was prevented by the antioxidant vitamin E, and enhanced by the prooxidant buthionine-sulfoximine, suggesting the involvement of an oxidative stress. UVA decreased both neutral and acid sphingomyelinase activities measured in vitro. A direct cleavage of sphingomyelin to CER by UVA, recently described, was not observed under our experimental conditions. We also show that, downstream of CER, UVA activated the Ser/Thr kinases ERK, JNK, and p38. Since ceramide has been shown to play a role in stress kinase activation, our results provide a possible mechanism for UVA-induced activation of stress kinases via ceramide formation. However, the actual mechanisms whereby CER is produced in cultured cells under UVA exposure remain to be specified.     

Sphingomyelin hydrolysis during apoptosis

Sphingolipid breakdown products are now being recognized as important players in apoptosis. Ceramide, which is considered to serve as second messenger, is mainly generated by hydrolysis of the membrane sphingophospholipid sphingomyelin (SM) through the action of a sphingomyelinase (SMase). However, little is known about the localization and regulation of this phenomenon. Here, we summarize the current knowledge on the function of SM hydrolysis in apoptosis signaling. In particular, the present review focuses on the role of neutral sphingomyelinase (N-SMase) in the generation of the proapoptotic ceramide. This enzyme is regulated by several mechanisms, including the tumor necrosis factor (TNF) receptor-associated protein FAN (for factor associated with N-SMase activation) and oxidative stress. These observations place SMase activation and SM hydrolysis as early events in the apoptosis signaling cascade.     

Survival Response to Increased Ceramide Involves Metabolic Adaptation through Novel Regulators of Glycolysis and Lipolysis

The sphingolipid ceramide elicits several stress responses, however, organisms survive despite increased ceramide but how they do so is poorly understood. We demonstrate here that the AKT/FOXO pathway regulates survival in increased ceramide environment by metabolic adaptation involving changes in glycolysis and lipolysis through novel downstream targets. We show that ceramide kinase mutants accumulate ceramide and this leads to reduction in energy levels due to compromised oxidative phosphorylation. Mutants show increased activation of Akt and a consequent decrease in FOXO levels. These changes lead to enhanced glycolysis by upregulating the activity of phosphoglyceromutase, enolase, pyruvate kinase, and lactate dehydrogenase to provide energy. A second major consequence of AKT/FOXO reprogramming in the mutants is the increased mobilization of lipid from the gut through novel lipase targets, CG8093 and CG6277 for energy contribution. Ubiquitous reduction of these targets by knockdown experiments results in semi or total lethality of the mutants, demonstrating the importance of activating them. The efficiency of these adaptive mechanisms decreases with age and leads to reduction in adult life span of the mutants. In particular, mutants develop cardiac dysfunction with age, likely reflecting the high energy requirement of a well-functioning heart. The lipases also regulate physiological triacylglycerol homeostasis and are important for energy metabolism since midgut specific reduction of them in wild type flies results in increased sensitivity to starvation and accumulation of triglycerides leading to cardiac defects. The central findings of increased AKT activation, decreased FOXO level and activation of phosphoglyceromutase and pyruvate kinase are also observed in mice heterozygous for ceramide transfer protein suggesting a conserved role of this pathway in mammals. These data reveal novel glycolytic and non-autonomous lipolytic pathways in response to increased ceramide for sustenance of high energy demanding organ functions like the heart.     

Ceramide response post-photodamage is absent after treatment with HA14-1

The oxidative stress induced by photodynamic therapy using the phthalocyanine Pc 4 (PDT) can lead to apoptosis, and is accompanied by photodamage to Bcl-2 and accumulation of de novo ceramide. Similar to PDT, the oxidative stress inducer and Bcl-2 inhibitor HA14-1 triggers apoptosis. To test the specificity of the ceramide response, Jurkat cells were exposed to an equitoxic dose of HA14-1. Unlike PDT, HA14-1 did not induce accumulation of de novo ceramide, although levels of sphingomyelin, phosphatidylserine and phosphatidylethanolamine were below control values after either treatment. In contrast to PDT, (i) the transient inhibition of serine palmitoyltransferase induced by HA14-1 was associated with the initial decrease in de novo ceramide, and (ii) HA14-1-initiated inhibition of sphingomyelin synthase and glucosylceramide synthase did not result in accumulation of de novo ceramide. These results show that the ceramide response to PDT is not induced by another pro-apoptotic stimulus, and may be unique to PDT as described here.     

Ceramide-dependent caspase 3 activation is prevented by coenzyme Q from plasma membrane in serum-deprived cells

Coenzyme Q (CoQ) is the key factor for the activity of the eukaryotic plasma membrane electron transport chain. Consequently, CoQ is essential in the cellular response against redox changes affecting this membrane. Serum withdrawal induces a mild oxidative stress, which produces lipid peroxidation in membranes. In fact, apoptosis induced by serum withdrawal can be prevented by several antioxidants including CoQ. Also, CoQ can maintain cell growth in serum-limiting conditions, whereas plasma membrane redox system (PMRS) inhibitors such as capsaicin, which compete with CoQ, inhibit cell growth and induce apoptosis. To understand how plasma membrane CoQ prevents oxidative stress-induced apoptosis we have studied the induction of apoptosis by serum withdrawal in CEM cells and its modulation by CoQ. Serum-withdrawal activates neutral sphingomyelinase (N-SMase), ceramide release and caspase-3-related proteases. CoQ addition to serum-free cultures inhibited a 60% N-SMase activation, an 80% ceramide release, and a 50% caspase-3 activity induced by serum deprivation. Caspase activation dependent on ceramide release since C 2 -ceramide was only able to mimic this effect in 10% foetal calf serum cultured cells but not in serum-free cultures. Also, in vitro experiments demonstrated that C 2 -ceramide and ceramide-rich lipid extracts directly activated caspase-3. Taken together, our results indicate that CoQ protects plasma membrane components and controls stress-mediated lipid signals by its participation in the PMRS.     

Ceranib‐2‐induced suicidal erythrocyte death

Ceramide is known to trigger apoptosis of nucleated cells and eryptosis of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Besides ceramide, stimulators of eryptosis include increase of cytosolic Ca²⁺‐activity ([Ca²⁺]_i) and oxidative stress. Ceramide is degraded by acid ceramidase and inhibition of the enzyme similarly triggers apoptosis. The present study explored, whether ceramidase inhibitor Ceranib‐2 induces eryptosis. Flow cytometry was employed to quantify phosphatidylserine‐exposure at the cell surface from annexin‐V‐binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3‐fluorescence, reactive oxygen species (ROS) from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. A 48 h exposure of human erythrocytes to Ceranib‐2 significantly increased the percentage of annexin‐V‐binding cells (≥50 μM) and the percentage of hemolytic cells (≥10 μM) without significantly modifying forward scatter. Ceranib‐2 significantly increased Fluo3‐fluorescence, DCF fluorescence and ceramide abundance. The effect of Ceranib‐2 on annexin‐V‐binding was not significantly blunted by removal of extracellular Ca²⁺. Ceranib‐2 triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to increase of ceramide abundance and induction of oxidative stress, but not dependent on Ca²⁺ entry. Copyright © 2016 John Wiley & Sons, Ltd.     

NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis

Endoplasmic reticulum (ER)-induced apoptosis and oxidative stress contribute to several chronic disease processes, yet molecular and cellular mechanisms linking ER stress and oxidative stress in the setting of apoptosis are poorly understood and infrequently explored in vivo. In this paper, we focus on a previously elucidated ER stress-apoptosis pathway whose molecular components have been identified and documented to cause apoptosis in vivo. We now show that nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) and NOX-mediated oxidative stress are induced by this pathway and that apoptosis is blocked by both genetic deletion of the NOX subunit NOX2 and by the antioxidant N-acetylcysteine. Unexpectedly, NOX and oxidative stress further amplify CCAAT/enhancer binding protein homologous protein (CHOP) induction through activation of the double-stranded RNA-dependent protein kinase (PKR). In vivo, NOX2 deficiency protects ER-stressed mice from renal cell CHOP induction and apoptosis and prevents renal dysfunction. These data provide new insight into how ER stress, oxidative stress, and PKR activation can be integrated to induce apoptosis in a pathophysiologically relevant manner.