Aortic Valve Stenosis Causes Accumulation of Extracellular Hemoglobin and Systemic Endothelial Dysfunction.

BACKGROUND: Whether aortic valve stenosis (AS) can adversely affect systemic endothelial function independently of standard modifiable cardiovascular risk factors is unknown. METHODS: We therefore investigated endothelial and cardiac function in an experimental model of AS mice devoid of standard modifiable cardiovascular risk factors and human cohorts with AS scheduled for transcatheter aortic valve replacement. Endothelial function was determined by flow-mediated dilation using ultrasound. Extracellular hemoglobin (eHb) concentrations and NO consumption were determined in blood plasma of mice and humans by ELISA and chemiluminescence. This was complemented by measurements of aortic blood flow using 4-dimensional flow acquisition by magnetic resonance imaging and computational fluid dynamics simulations. The effects of plasma and red blood cell (RBC) suspensions on vascular function were determined in transfer experiments in a murine vasorelaxation bioassay system. RESULTS: In mice, the induction of AS caused systemic endothelial dysfunction. In the presence of normal systolic left ventricular function and mild hypertrophy, the increase in the transvalvular gradient was associated with elevated eryptosis, increased eHb and plasma NO consumption; eHb sequestration by haptoglobin restored endothelial function. Because the aortic valve orifice area in patients with AS decreased, postvalvular mechanical stress in the central ascending aorta increased. This was associated with elevated eHb, circulating RBC-derived microvesicles, eryptotic cells, lower haptoglobin levels without clinically relevant anemia, and consecutive endothelial dysfunction. Transfer experiments demonstrated that reduction of eHb by treatment with haptoglobin or elimination of fluid dynamic stress by transcatheter aortic valve replacement restored endothelial function. In patients with AS and subclinical RBC fragmentation, the remaining circulating RBCs before and after transcatheter aortic valve replacement exhibited intact membrane function, deformability, and resistance to osmotic and hypoxic stress. CONCLUSIONS: AS increases postvalvular swirling blood flow in the central ascending aorta, triggering RBC fragmentation with the accumulation of hemoglobin in the plasma. This increases NO consumption in blood, thereby limiting vascular NO bioavailability. Thus, AS itself promotes systemic endothelial dysfunction independent of other established risk factors. Transcatheter aortic valve replacement is capable of limiting NO scavenging and rescuing endothelial function by realigning postvalvular blood flow to near physiological patterns. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT05603520. URL: https://www.clinicaltrials.gov; Unique identifier: NCT01805739.

Animal Welfare Aspects in Planning and Conducting Experiments on Rodent Models of Subarachnoid Hemorrhage.

Subarachnoid hemorrhage is an acute life-threatening cerebrovascular disease with high socio-economic impact. The most frequent cause, the rupture of an intracerebral aneurysm, is accompanied by abrupt changes in intracerebral pressure, cerebral perfusion pressure and, consequently, cerebral blood flow. As aneurysms rupture spontaneously, monitoring of these parameters in patients is only possible with a time delay, upon hospitalization. To study alterations in cerebral perfusion immediately upon ictus, animal models are mandatory. This article addresses the points necessarily to be included in an animal project proposal according to EU directive 2010/63/EU for the protection of animals used for scientific purposes and herewith offers an insight into animal welfare aspects of using rodent models for the investigation of cerebral perfusion after subarachnoid hemorrhage. It compares surgeries, model characteristics, advantages, and drawbacks of the most-frequently used rodent models-the endovascular perforation model and the prechiasmatic and single or double cisterna magna injection model. The topics of discussing anesthesia, advice on peri- and postanesthetic handling of animals, assessing the severity of suffering the animals undergo during the procedure according to EU directive 2010/63/EU and weighing the use of these in vivo models for experimental research ethically are also presented. In conclusion, rodent models of subarachnoid hemorrhage display pathophysiological characteristics, including changes of cerebral perfusion similar to the clinical situation, rendering the models suited to study the sequelae of the bleeding. A current problem is low standardization of the models, wherefore reporting according to the ARRIVE guidelines is highly recommended. Animal welfare aspects of rodent models of subarachnoid hemorrhage. Rodent models for investigation of cerebral perfusion after subarachnoid hemorrhage are compared regarding surgeries and model characteristics, and 3R measures are suggested. Anesthesia is discussed, and advice given on peri- and postanesthetic handling. Severity of suffering according to 2010/63/EU is assessed and use of these in vivo models weighed ethically.

Inhibition of acid sphingomyelinase by ambroxol prevents SARS-CoV-2 entry into epithelial cells.

The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)-methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [14C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019.

Interferon regulatory factor 8 regulates expression of acid ceramidase and infection susceptibility in cystic fibrosis.

Most patients with cystic fibrosis (CF) suffer from acute and chronic pulmonary infections with bacterial pathogens, which often determine their life quality and expectancy. Previous studies have demonstrated a downregulation of the acid ceramidase in CF epithelial cells resulting in an increase of ceramide and a decrease of sphingosine. Sphingosine kills many bacterial pathogens, and the downregulation of sphingosine seems to determine the infection susceptibility of cystic fibrosis mice and patients. It is presently unknown how deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) connects to a marked downregulation of the acid ceramidase in human and murine CF epithelial cells. Here, we employed quantitative PCR, western blot analysis, and enzyme activity measurements to study the role of IRF8 for acid ceramidase regulation. We report that genetic deficiency or functional inhibition of CFTR/Cftr results in an upregulation of interferon regulatory factor 8 (IRF8) and a concomitant downregulation of acid ceramidase expression with CF and an increase of ceramide and a reduction of sphingosine levels in tracheal and bronchial epithelial cells from both human individuals or mice. CRISPR/Cas9- or siRNA-mediated downregulation of IRF8 prevented changes of acid ceramidase, ceramide, and sphingosine in CF epithelial cells and restored resistance to Pseudomonas aeruginosa infections, which is one of the most important and common pathogens in lung infection of patients with CF. These studies indicate that CFTR deficiency causes a downregulation of acid ceramidase via upregulation of IRF8, which is a central pathway to control infection susceptibility of CF cells.

Multiparametric MRI identifies subtle adaptations for demarcation of disease transition in murine aortic valve stenosis.

Aortic valve stenosis (AS) is the most frequent valve disease with relevant prognostic impact. Experimental model systems for AS are scarce and comprehensive imaging techniques to simultaneously quantify function and morphology in disease progression are lacking. Therefore, we refined an acute murine AS model to closely mimic human disease characteristics and developed a high-resolution magnetic resonance imaging (MRI) approach for simultaneous in-depth analysis of valvular, myocardial as well as aortic morphology/pathophysiology to identify early changes in tissue texture and critical transition points in the adaptive process to AS. AS was induced by wire injury of the aortic valve. Four weeks after surgery, cine loops, velocity, and relaxometry maps were acquired at 9.4 T to monitor structural/functional alterations in valve, aorta, and left ventricle (LV). In vivo MRI data were subsequently validated by histology and compared to echocardiography. AS mice exhibited impaired valve opening accompanied by significant valve thickening due to fibrotic remodelling. While control mice showed bell-shaped flow profiles, AS resulted not only in higher peak flow velocities, but also in fragmented turbulent flow patterns associated with enhanced circumferential strain and an increase in wall thickness of the aortic root. AS mice presented with a mild hypertrophy but unaffected global LV function. Cardiac MR relaxometry revealed reduced values for both T1 and T2 in AS reflecting subtle myocardial tissue remodelling with early alterations in mitochondrial function in response to the enhanced afterload. Concomitantly, incipient impairments of coronary flow reserve and myocardial tissue integrity get apparent accompanied by early troponin release. With this, we identified a premature transition point with still compensated cardiac function but beginning textural changes. This will allow interventional studies to explore early disease pathophysiology and novel therapeutic targets.

Antiviral and Anti-Inflammatory Activities of Fluoxetine in a SARS-CoV-2 Infection Mouse Model.

The coronavirus disease 2019 (COVID-19) pandemic continues to cause significant morbidity and mortality worldwide. Since a large portion of the world's population is currently unvaccinated or incompletely vaccinated and has limited access to approved treatments against COVID-19, there is an urgent need to continue research on treatment options, especially those at low cost and which are immediately available to patients, particularly in low- and middle-income countries. Prior in vitro and observational studies have shown that fluoxetine, possibly through its inhibitory effect on the acid sphingomyelinase/ceramide system, could be a promising antiviral and anti-inflammatory treatment against COVID-19. In this report, we evaluated the potential antiviral and anti-inflammatory activities of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and against variants of concern in vitro, i.e., SARS-CoV-2 ancestral strain, Alpha B.1.1.7, Gamma P1, Delta B1.617 and Omicron BA.5. Fluoxetine, administrated after SARS-CoV-2 infection, significantly reduced lung tissue viral titres and expression of several inflammatory markers (i.e., IL-6, TNFα, CCL2 and CXCL10). It also inhibited the replication of all variants of concern in vitro. A modulation of the ceramide system in the lung tissues, as reflected by the increase in the ratio HexCer 16:0/Cer 16:0 in fluoxetine-treated mice, may contribute to explain these effects. Our findings demonstrate the antiviral and anti-inflammatory properties of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and its in vitro antiviral activity against variants of concern, establishing fluoxetine as a very promising candidate for the prevention and treatment of SARS-CoV-2 infection and disease pathogenesis.

Sphingosine kills bacteria by binding to cardiolipin.

Sphingosine is a long-chain sphingoid base that has been shown to have bactericidal activity against many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli We have previously demonstrated that sphingosine is present in nasal, tracheal, and bronchial epithelial cells and constitutes a central element of the defense of the airways against bacterial pathogens. Here, using assorted lipid-binding and cell biology assays, we demonstrate that exposing P. aeruginosa and S. aureus cells to sphingosine results in a very rapid, i.e. within minutes, permeabilization of the bacterial plasma membrane, resulting in leakiness of the bacterial cells, loss of ATP, and loss of bacterial metabolic activity. These alterations rapidly induced bacterial death. Mechanistically, we demonstrate that the presence of the protonated NH2 group in sphingosine, which is an amino-alcohol, is required for sphingosine's bactericidal activity. We also show that the protonated NH2 group of sphingosine binds to the highly negatively-charged lipid cardiolipin in bacterial plasma membranes. Of note, this binding was required for bacterial killing by sphingosine, as revealed by genetic experiments indicating that E. coli or P. aeruginosa strains that lack cardiolipin synthase are resistant to sphingosine, both in vitro and in vivo We propose that binding of sphingosine to cardiolipin clusters cardiolipin molecules in the plasma membrane of bacteria. This clustering results in the formation of gel-like or even crystal-like structures in the bacterial plasma membrane and thereby promotes rapid permeabilization of the plasma membrane and bacterial cell death.

Sphingosine prevents binding of SARS-CoV-2 spike to its cellular receptor ACE2.

Sphingosine has been shown to prevent and eliminate bacterial infections of the respiratory tract, but it is unknown whether sphingosine can be also employed to prevent viral infections. To test this hypothesis, we analyzed whether sphingosine regulates the infection of cultured and freshly isolated ex vivo human epithelial cells with pseudoviral particles expressing SARS-CoV-2 spike (pp-VSV-SARS-CoV-2 spike) that served as a bona fide system mimicking SARS-CoV-2 infection. We demonstrate that exogenously applied sphingosine suspended in 0.9% NaCl prevents cellular infection with pp-SARS-CoV-2 spike. Pretreatment of cultured Vero epithelial cells or freshly isolated human nasal epithelial cells with low concentrations of sphingosine prevented adhesion of and infection with pp-VSV-SARS-CoV-2 spike. Mechanistically, we demonstrate that sphingosine binds to ACE2, the cellular receptor of SARS-CoV-2, and prevents the interaction of the receptor-binding domain of the viral spike protein with ACE2. These data indicate that sphingosine prevents at least some viral infections by interfering with the interaction of the virus with its receptor. Our data also suggest that further preclinical and finally clinical examination of sphingosine is warranted for potential use as a prophylactic or early treatment for coronavirus disease-19.

Acid Ceramidase Rescues Cystic Fibrosis Mice from Pulmonary Infections.

Previous studies have shown that sphingosine kills a variety of pathogenic bacteria, including Pseudomonas aeruginosa and Staphylococcus aureus Sphingosine concentrations are decreased in airway epithelial cells of cystic fibrosis (CF) mice, and this defect has been linked to the infection susceptibility of these mice. Here, we tested whether the genetic overexpression of acid ceramidase rescues cystic fibrosis mice from pulmonary infections with P. aeruginosa We demonstrate that the transgenic overexpression of acid ceramidase in CF mice corresponds to the overexpression of acid ceramidase in bronchial and tracheal epithelial cells and normalizes ceramide and sphingosine levels in bronchial and tracheal epithelial cells. In addition, the expression of ß1-integrin, which is ectopically expressed on the luminal surface of airway epithelial cells in cystic fibrosis mice, an alteration that is very important for mediating pulmonary P. aeruginosa infections in cystic fibrosis, is normalized in cystic fibrosis airways upon the overexpression of acid ceramidase. Most importantly, the overexpression of acid ceramidase protects cystic fibrosis mice from pulmonary P. aeruginosa infections. Infection of CF mice or CF mice that inhaled sphingosine with P. aeruginosa or a P. aeruginosa mutant that is resistant to sphingosine indicates that sphingosine and not a metabolite kills P. aeruginosa upon pulmonary infection. These studies further support the use of acid ceramidase and its metabolite sphingosine as potential treatments of cystic fibrosis.

Recombinant Acid Ceramidase Reduces Inflammation and Infection in Cystic Fibrosis.

Rationale: In cystic fibrosis the major cause of morbidity and mortality is lung disease characterized by inflammation and infection. The influence of sphingolipid metabolism is poorly understood with a lack of studies using human airway model systems.Objectives: To investigate sphingolipid metabolism in cystic fibrosis and the effects of treatment with recombinant human acid ceramidase on inflammation and infection.Methods: Sphingolipids were measured using mass spectrometry in fully differentiated cultures of primary human airway epithelial cells and cocultures with Pseudomonas aeruginosa. In situ activity assays, Western blotting, and quantitative PCR were used to investigate function and expression of ceramidase and sphingomyelinase. Effects of treatment with recombinant human acid ceramidase on sphingolipid profile and inflammatory mediator production were assessed in cell cultures and murine models.Measurements and Main Results: Ceramide is increased in cystic fibrosis airway epithelium owing to differential function of enzymes regulating sphingolipid metabolism. Sphingosine, a metabolite of ceramide with antimicrobial properties, is not upregulated in response to P. aeruginosa by cystic fibrosis airway epithelia. Tumor necrosis factor receptor 1 is increased in cystic fibrosis epithelia and activates NF-κB signaling, generating inflammation. Treatment with recombinant human acid ceramidase, to decrease ceramide, reduced both inflammatory mediator production and susceptibility to infection.Conclusions: Sphingolipid metabolism is altered in airway epithelial cells cultured from people with cystic fibrosis. Treatment with recombinant acid ceramidase ameliorates the two pivotal features of cystic fibrosis lung disease, inflammation and infection, and thus represents a therapeutic approach worthy of further exploration.

Ceramide and Related Molecules in Viral Infections.

Ceramide is a lipid messenger at the heart of sphingolipid metabolism. In concert with its metabolizing enzymes, particularly sphingomyelinases, it has key roles in regulating the physical properties of biological membranes, including the formation of membrane microdomains. Thus, ceramide and its related molecules have been attributed significant roles in nearly all steps of the viral life cycle: they may serve directly as receptors or co-receptors for viral entry, form microdomains that cluster entry receptors and/or enable them to adopt the required conformation or regulate their cell surface expression. Sphingolipids can regulate all forms of viral uptake, often through sphingomyelinase activation, and mediate endosomal escape and intracellular trafficking. Ceramide can be key for the formation of viral replication sites. Sphingomyelinases often mediate the release of new virions from infected cells. Moreover, sphingolipids can contribute to viral-induced apoptosis and morbidity in viral diseases, as well as virus immune evasion. Alpha-galactosylceramide, in particular, also plays a significant role in immune modulation in response to viral infections. This review will discuss the roles of ceramide and its related molecules in the different steps of the viral life cycle. We will also discuss how novel strategies could exploit these for therapeutic benefit.

Characterization of the small molecule ARC39, a direct and specific inhibitor of acid sphingomyelinase in vitro.

Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM's catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.

Role of 1-Deoxysphingolipids in docetaxel neurotoxicity.

A major dose-limiting side effect of docetaxel chemotherapy is peripheral neuropathy. Patients' symptoms include pain, numbness, tingling and burning sensations, and motor weakness in the extremities. The molecular mechanism is currently not understood, and there are no treatments available. Previously, we have shown an association between neuropathy symptoms of patients treated with paclitaxel and the plasma levels of neurotoxic sphingolipids, the 1-deoxysphingolipids (1-deoxySL) (Kramer et al, FASEB J, 2015). 1-DeoxySL are produced when the first enzyme of the sphingolipid biosynthetic pathway, serine palmitoyltransferase (SPT), uses L-alanine as a substrate instead of its canonical amino acid substrate, L-serine. In the current investigation, we tested whether 1-deoxySL accumulate in the nervous system following systemic docetaxel treatment in mice. In dorsal root ganglia (DRG), we observed that docetaxel (45 mg/kg cumulative dose) significantly elevated the levels of 1-deoxySL and L-serine-derived ceramides, but not sphingosine-1-phosphate (S1P). S1P is a bioactive sphingolipid and a ligand for specific G-protein-coupled receptors. In the sciatic nerve, docetaxel decreased 1-deoxySL and ceramides. Moreover, we show that in primary DRG cultures, 1-deoxysphingosine produced neurite swellings that could be reversed with S1P. Our results demonstrate that docetaxel chemotherapy up-regulates sphingolipid metabolism in sensory neurons, leading to the accumulation of neurotoxic 1-deoxySL. We suggest that the neurotoxic effects of 1-deoxySL on axons can be reversed with S1P.

Acid sphingomyelinase regulates TH 2 cytokine release and bronchial asthma.

BACKGROUND: Allergic diseases and especially allergic asthma are widespread diseases with high prevalence in childhood, but also in adults. Acid sphingomyelinase (ASM) is a key regulator of the sphingolipid pathway. Previous studies defined the association of ASM with the pathogenesis of TH 1-directed lung diseases like cystic fibrosis and acute lung injury. Here, we define the role of ASM in TH 2-regulated allergic bronchial asthma. METHODS: To determine the role of Asm under baseline conditions, wild-type (WT) and Asm-/- mice were ventilated with a flexiVent setup and bronchial hyperresponsiveness was determined using acetylcholine. Flow cytometry and cytokine measurements in bronchoalveolar lavage fluid and lung tissue were followed by in vitro TH 2 differentiations with cells from WT and Asm-/- mice and blockade of Asm with amitriptyline. As proof of principle, we conducted an ovalbumin-induced model of asthma in WT- and Asm-/-  mice. RESULTS: At baseline, Asm-/- mice showed better lung mechanics, but unaltered bronchial hyperresponsiveness. Higher numbers of Asm-/- T cells in bronchoalveolar lavage fluid released lower levels of IL-4 and IL-5, and these results were paralleled by decreased production of typical TH 2 cytokines in Asm-/- T lymphocytes in vitro. This phenotype could be imitated by incubation of T cells with amitriptyline. In the ovalbumin asthma model, Asm-/- animals were protected from high disease activity and showed better lung functions and lower levels of eosinophils and TH 2 cytokines. CONCLUSION: Asm deficiency could induce higher numbers of TH 2 cells in the lung, but those cells release decreased TH 2 cytokine levels. Hereby, Asm-/- animals are protected from bronchial asthma, which possibly offers novel therapeutic strategies, for example, with ASM blockade.

Signalling Effects Induced by Acid Ceramidase in Human Epithelial Or Leukemic Cell Lines.

BACKGROUND/AIMS: Recent studies indicated that an inhalation treatment of cystic fibrosis mice with acid ceramidase prevents and eliminates infections with Pseudomonas aeruginosa and Stapyhlococcus aureus. Inhalation of acid ceramidase facilitated the elimination of P. aeruginosa in acutely- or chronically-infected mice with cystic fibrosis. Thus, inhalation of acid ceramidase might be a preventive and/or curative treatment for patients with cystic fibrosis suffering from pneumonia. METHODS: We treated cultured epithelial cells or leukemic T-lymphocytes (Jurkat cells) with purified acid ceramidase and determined intracellular signalling events, proliferation and cell survival. Specifically, we measured the activity of AKT, p38-kinase and p70S6-kinase using activation-specific phospho-antibodies in western blot studies. Trypan Blue staining served to analyze proliferation and cell survival. RESULTS: Our studies indicate that treatment of Chang epithelial cells or Jurkat T lymphocytes with purified acid ceramidase results in a dose dependent activation of AKT, p38-kinase and p70S6-kinase, while tyrosine phosphorylation of intracellular proteins remains largely unchanged. Acid ceramidase treatment did not change expression of tight junction proteins such as ZO-1, ZO-2 and occludin. Cellular viability and proliferation were not affected by acid ceramidase treatment. CONCLUSION: Our data suggest that treatment of epithelial cells and lymphocytes with acid ceramidase results in activation of distinct pathways, in particular AKT- and p38K-dependent pathways, while no global activation or cell death was observed.

Inhibition of PI-3-K and AKT Amplifies Kv1.3 Inhibitor-Induced Death of Human T Leukemia Cells.

BACKGROUND/AIMS: We have previously shown that inhibition of the mitochondrial Kv1.3 channel results in an initial mitochondrial hyperpolarization and a release of oxygen radicals that mediate mitochondrial depolarization, cytochrome c release and death. Here, we investigated whether inhibition of Kv1.3 channels can also induce cellular resistance mechanisms that counteract the induction of cell death under certain conditions. METHODS: We treated leukemic T cells with the mitochondria-targeted Kv1.3 inhibitor PCARBTP and determined the activity of different kinases associated with cell survival including ZAP70, PI-3-K, AKT, JNK and ERK by measuring the activation-associated phosphorylation of these proteins. Furthermore, we inhibited AKT and JNK and determined the effect of PCARBTP-induced tumor cell death. RESULTS: We demonstrate that treatment of Jurkat T leukemia cells with low doses of the mitochondria-targeted inhibitor of Kv1.3 PCARBTP (0.25 µM or 1 µM) for 10 minutes induced a constitutive phosphorylation/activation of the pro-survival signaling molecules ZAP70, PI-3-K, AKT and JNK, while the phosphorylation/activation of ERK was not affected. Stimulation of Jurkat cells via the TCR/CD3 complex induced an additional activation of a similar pattern of signaling events. Higher doses of the Kv1.3 inhibitor, i.e. 10 µM PCARBTP, reduced the basal phosphorylation/activation of these signaling molecules and also impaired their activation upon stimulation via the TCR/CD3 complex. A low dose of PCARBTP, i.e. 0.25 µM PCARBTP, was almost without any effect on cell death. In contrast, concomitant inhibition of PI-3-K or AKT greatly sensitized Jurkat leukemia cells to the Kv1.3 inhibitor PCARBTP and allowed induction of cell death already at 0.25 µM PCARBTP. CONCLUSION: These studies indicate that Jurkat leukemia cells respond to low doses of the mitochondria-targeted Kv1.3 inhibitor PCARBTP with an activation of survival signals counteracting cell death. Inhibition of these T cell survival signals sensitizes leukemia cells to death induced by mitochondria-targeted Kv1.3 inhibitors. High doses of the Kv1.3 inhibitor inactivate these signals directly permitting death.

Clinical Development of Sphingosine as Anti-Bacterial Drug: Inhalation of Sphingosine in Mini Pigs has no Adverse Side Effects.

BACKGROUND/AIMS: Pulmonary infections with Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus) are of utmost clinical relevance in patients with cystic fibrosis, chronic obstructive pulmonary disease, after trauma and burn, upon ventilation or in immuno-compromised patients. Many P. aeruginosa and S. aureus strains are resistant to many known antibiotics and it is very difficult or often impossible to eradicate the pathogens in patient´s lungs. We have recently shown that the sphingoid base sphingosine very efficiently kills many pathogens, including for instance P. aeruginosa, S. aureus or Acinetobacter baumannii, in vitro. In vivo experiments of our group on cystic fibrosis mice indicated that inhalation of sphingosine prevents or eliminates existing acute or chronic pneumonia with P. aeruginosa or S. aureus in these mice. We also demonstrated that sphingosine is safe to use for inhalation up to high doses, at least in mice. To facilitate development of sphingosine to an anti-bactericidal drug that can be used in humans for inhalation, safety data on non-rodents, larger animals are absolutely required. METHODS: Here, we inhaled mini pigs with increasing doses of sphingosine for 10 days and analyzed the uptake of sphingosine into epithelial cells of bronchi as well as into the trachea and lung and the systemic circulation. Moreover, we measured the generation of ceramide and sphingosine 1-phosphate that potentially mediate inflammation, the influx of leukocytes, epithelial cell death and disruption of the epithelial cell barrier. RESULTS: We demonstrate that inhalation of sphingosine results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea, but not in systemic accumulation. Inhaled sphingosine had no side effects up to very high doses. CONCLUSION: In summary, we demonstrate that inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no systemic or local side effects.

Pharmacological Inhibition of Acid Sphingomyelinase Ameliorates Experimental Autoimmune Encephalomyelitis.

BACKGROUND/AIMS: Multiple sclerosis (MS) is one of the most common autoimmune disorders of the central nervous system (CNS) and the leading cause of neurological disability among young adults in the Western world. We have previously shown that the acid sphingomyelinase plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. METHODS: We induced adoptively transferred EAE in wildtype and acid sphingomyelinase-deficient mice. In addition, we immunized mice with MOGaa35-55 to induce active EAE and treated the mice with amitriptyline, a functional inhibitor of the acid sphingomyelinase. We investigated symptoms of EAE, blood-brain barrier integrity and neuroinflammation. RESULTS: In the model of adoptively transferred EAE we demonstrate that expression of acid sphingomyelinase in the recipients rather than on transferred encephalitogenic T cells contributes to the clinical development of EAE symptoms. To test if pharmacological targeting of acid sphingomyelinase can be explored for the development of novel therapies for MS, we inhibited acid sphingomyelinase with amitriptyline in mice in which EAE was induced by active immunization. We demonstrate that pharmacological inhibition of acid sphingomyelinase using amitriptyline protects against the development of EAE and markedly attenuates the characteristic detrimental neuroinflammatory response. CONCLUSION: The studies identify the acid sphingomyelinase as a novel therapeutic target for treating MS patients.

Antidepressants act by inducing autophagy controlled by sphingomyelin-ceramide.

Major depressive disorder (MDD) is a common and severe disease characterized by mood changes, somatic alterations, and often suicide. MDD is treated with antidepressants, but the molecular mechanism of their action is unknown. We found that widely used antidepressants such as amitriptyline and fluoxetine induce autophagy in hippocampal neurons via the slow accumulation of sphingomyelin in lysosomes and Golgi membranes and of ceramide in the endoplasmic reticulum (ER). ER ceramide stimulates phosphatase 2A and thereby the autophagy proteins Ulk, Beclin, Vps34/Phosphatidylinositol 3-kinase, p62, and Lc3B. Although treatment with amitriptyline or fluoxetine requires at least 12 days to achieve sphingomyelin accumulation and the subsequent biochemical and cellular changes, direct inhibition of sphingomyelin synthases with tricyclodecan-9-yl-xanthogenate (D609) results in rapid (within 3 days) accumulation of ceramide in the ER, activation of autophagy, and reversal of biochemical and behavioral signs of stress-induced MDD. Inhibition of Beclin blocks the antidepressive effects of amitriptyline and D609 and induces cellular and behavioral changes typical of MDD. These findings identify sphingolipid-controlled autophagy as an important target for antidepressive treatment methods and provide a rationale for the development of novel antidepressants that act within a few days.

Acid Sphingomyelinase Deficiency Ameliorates Farber Disease.

Farber disease is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments for Farber disease are clinically available, and affected patients have a severely shortened lifespan. We have recently reported a novel acid ceramidase deficiency model that mirrors the human disease closely. Acid sphingomyelinase is the enzyme that generates ceramide upstream of acid ceramidase in the lysosomes. Using our acid ceramidase deficiency model, we tested if acid sphingomyelinase could be a potential novel therapeutic target for the treatment of Farber disease. A number of functional acid sphingomyelinase inhibitors are clinically available and have been used for decades to treat major depression. Using these as a therapeutic for Farber disease, thus, has the potential to improve central nervous symptoms of the disease as well, something all other treatment options for Farber disease can't achieve so far. As a proof-of-concept study, we first cross-bred acid ceramidase deficient mice with acid sphingomyelinase deficient mice in order to prevent ceramide accumulation. Double-deficient mice had reduced ceramide accumulation, fewer disease manifestations, and prolonged survival. We next targeted acid sphingomyelinase pharmacologically, to test if these findings would translate to a setting with clinical applicability. Surprisingly, the treatment of acid ceramidase deficient mice with the acid sphingomyelinase inhibitor amitriptyline was toxic to acid ceramidase deficient mice and killed them within a few days of treatment. In conclusion, our study provides the first proof-of-concept that acid sphingomyelinase could be a potential new therapeutic target for Farber disease to reduce disease manifestations and prolong survival. However, we also identified previously unknown toxicity of the functional acid sphingomyelinase inhibitor amitriptyline in the context of Farber disease, strongly cautioning against the use of this substance class for Farber disease patients.