Sphingosine-1-phosphate Lyase Inhibitor Research Articles

Sphingosine-1-phosphate promotes osteogenesis by stimulating osteoblast growth and neovascularization in a vascular endothelial growth factor-dependent manner.

Sphingosine-1-phosphate (S1P) plays multiple roles in bone metabolism and regeneration. Here, we have identified a novel S1P-regulated osteoanabolic mechanism functionally connecting osteoblasts (OBs) to the highly specialized bone vasculature. We demonstrate that S1P/S1PR3 signaling in OBs stimulates vascular endothelial growth factor a (VEGFa) expression and secretion to promote bone growth in an autocrine and boost osteogenic H-type differentiation of bone marrow endothelial cells in a paracrine manner. VEGFa-neutralizing antibodies and VEGF receptor inhibition by axitinib abrogated OB growth in vitro and bone formation in male C57BL/6J in vivo following S1P stimulation and S1P lyase inhibition, respectively. Pharmacological S1PR3 inhibition and genetic S1PR3 deficiency suppressed VEGFa production, OB growth in vitro, and inhibited H-type angiogenesis and bone growth in male mice in vivo. Together with previous work on the osteoanabolic functions of S1PR2 and S1PR3, our data suggest that S1P-dependent bone regeneration employs several nonredundant positive feedback loops between OBs and the bone vasculature. The identification of this yet unappreciated aspect of osteoanabolic S1P signaling may have implications for regular bone homeostasis as well as diseases where the bone microvasculature is affected such as age-related osteopenia and posttraumatic bone regeneration.

Pharmacological elevation of sphingosine-1-phosphate by S1P lyase inhibition accelerates bone regeneration after post-traumatic osteomyelitis.

Posttraumatic osteomyelitis and the ensuing bone defects are a debilitating complication after open fractures with little therapeutic options. We have recently identified potent osteoanabolic effects of sphingosine-1-phosphate (S1P) signalling and have now tested whether it may beneficially affect bone regeneration after infection. We employed pharmacological S1P lyase inhibition by 4-deoxypyrodoxin (DOP) to raise S1P levels in vivo in an unicortical long bone defect model of posttraumatic osteomyelitis in mice. In a translational approach, human bone specimens of clinical osteomyelitis patients were treated in organ culture in vitro with DOP. Bone regeneration was assessed by μCT, histomorphometry, immunohistology and gene expression analysis. The role of S1P receptors was addressed using S1PR3 deficient mice. Here, we present data that DOP treatment markedly enhanced osteogenesis in posttraumatic osteomyelitis. This was accompanied by greatly improved osteoblastogenesis and enhanced angiogenesis in the callus accompanied by osteoclast-mediated bone remodelling. We also identified the target of increased S1P to be the S1PR3 as S1PR3-/- mice showed no improvement of bone regeneration by DOP. In the human bone explants, bone mass significantly increased along with enhanced osteoblastogenesis and angiogenesis. Our data suggest that enhancement of S1P/S1PR3 signalling may be a promising therapeutic target for bone regeneration in posttraumatic osteomyelitis.

Sphingosine-1-Phosphate as Lung and Cardiac Vasculature Protecting Agent in SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may cause severe respiratory illness with high mortality. SARS-CoV-2 infection results in a massive inflammatory cell infiltration into the infected lungs accompanied by excessive pro-inflammatory cytokine production. The lung histology of dead patients shows that some areas are severely emphysematous, with enormously dilated blood vessels and micro-thromboses. The inappropriate inflammatory response damaging the pulmonary interstitial arteriolar walls suggests that the respiratory distress may come in a large part from lung vasculature injuries. It has been recently observed that low plasmatic sphingosine-1-phosphate (S1P) is a marker of a worse prognosis of clinical outcome in severe coronavirus disease (COVID) patients. S1P is an angiogenic molecule displaying anti-inflammatory and anti-apoptotic properties, that promote intercellular interactions between endothelial cells and pericytes resulting in the stabilization of arteries and capillaries. In this context, it can be hypothesized that the benefit of a normal S1P level is due to its protective effect on lung vasculature functionality. This paper provides evidence supporting this concept, opening the way for the design of a pharmacological approach involving the use of an S1P lyase inhibitor to increase the S1P level that in turn will rescue the lung vasculature functionality.

Sphingosine 1 phosphate lyase inhibition rescues cognition in diabetic mice by promoting anti-inflammatory microglia

Sphingosine-1-phosphate (S1P) is emerging as a crucial sphingolipid modulating neuroinflammation and cognition. S1P levels in the brain have been found to be decreased in cognitive impairment. S1P lyase (S1PL) is the key enzyme in metabolizing S1P and has been implicated in neuroinflammation. This study evaluated the effect of S1PL inhibition on cognition in type 2 diabetic mice. Fingolimod (0.5 mg/kg and 1 mg/kg) rescued cognition in high-fat diet and streptozotocin-induced diabetic mice, as evident in the Y maze and passive avoidance test. We further evaluated the effect of fingolimod on the activation of microglia in the pre-frontal cortex (PFC) and hippocampus of diabetic mice. Our study revealed that fingolimod inhibited S1PL and promoted anti-inflammatory microglia in both PFC and hippocampus of diabetic mice as it increased Ym-1 and arginase-1. The levels of p53 and apoptotic proteins (Bax and caspase-3) were elevated in the PFC and hippocampus of type 2 diabetic mice which fingolimod reversed. The underlying mechanism promoting anti-inflammatory microglial phenotype was also explored in this study. TIGAR, TP53-associated glycolysis and apoptosis regulator, is known to foster anti-inflammatory microglia and was found to be downregulated in the brain of type 2 diabetic mice. S1PL inhibition decreased the levels of p53 and promoted TIGAR, thereby increasing anti-inflammatory microglial phenotype and inhibiting apoptosis in the brain of diabetic mice. Our study reveals that S1PL inhibition could be beneficial in mitigating cognitive deficits in diabetic mice.

S1P lyase inhibition prevents lung injury following high pressure-controlled mechanical ventilation in aging mice

Ventilator-induced Lung Injury (VILI) is characterized by hypoxia, inflammatory cytokine influx, loss of alveolar barrier integrity, and decreased lung compliance. Aging influences lung structure and function and is a predictive factor in the severity of VILI; however, the mechanisms of aging that influence the progression or increased susceptibility remain unknown. Aging impacts immune system function and may increase inflammation in healthy individuals. Recent studies suggest that the bioactive sphingolipid mediator sphingosine-1-phosphate (S1P) and the enzyme that degrades it S1P lyase (SPL) may be involved in lung pathologies including acute lung injury. It is unknown whether aging influences S1P and SPL expression that have been implicated in lung inflammation, injury, and cell apoptosis. We hypothesized that aging and injurious mechanical ventilation synergistically impair S1P levels and enhance S1P lyase (SPL) expression that amplifies alveolar barrier damage and diminishes pulmonary function. Young (2–3 mo) and old (20–25 mo) C57BL/6 mice were mechanically ventilated for 2 h using pressure-controlled mechanical ventilation (PCMV) at 45 cmH2O and 35 cmH2O, respectively. We assessed the impact of aging and PCMV on several indications of acute lung injury, immune cell recruitment, S1P levels and SPL activity. Furthermore, we evaluated the protective effects of inhibiting SPL by tetrahydroxybutylimidazol (THI) administration on the negative outcomes associated with aging and mechanical injury. PCMV exacerbated lung injury in old mice and increased neutrophil influx that was further exacerbated due to aging. SPL expression increased in the young and old ventilated mice and the old nonventilated group. THI treatment reduced several of the indicators of lung injury and resulted in elevated S1P levels in lung tissue and plasma from mice that were injured from mechanical ventilation. CD80 and CD206 activation markers of alveolar and interstitial macrophages were also influenced by THI. SPL inhibition may be a viable therapeutic approach for patients requiring mechanical ventilation by preventing or regulating the exaggerated inflammatory response and reducing lung injury.

Is Sphingosine-1-Phosphate a Regulator of Tumor Vascular Functionality?

Simple SummaryDespite substantial theoretical and experimental support for using vascular normalization as cancer therapy, effectively achieving this strategy in the clinic remains complex. In the present paper, we propose a novel potential approach for the induction of tumor vascular normalization, reduction of hypoxia, and improvement of conventional treatment in cancer patients. This approach consists of the pharmacological modulation of a patient’s plasma S1P levels which through a PDGF signaling can enhance tumor vasculature functionality and reduce hypoxia. This approach is proposed following a clinical observation in pancreatic adenocarcinoma patients and pre-clinical data in different in vivo tumor models, and is supported by a review of the literature describing the biological role of S1P in vascular functionality regulation.Increasing evidence indicates that tumor vasculature normalization could be an appropriate strategy to increase therapies’ efficacy in solid tumors by decreasing hypoxia and improving drug delivery. We searched for a novel approach that reduces hypoxia and enhances chemotherapy efficacy in pancreatic adenocarcinoma which is characterized by disrupted blood vasculature associated with poor patient survival. Clinical significance of plasma levels of the angiogenic lipid sphingosine-1-phosphate (S1P) was assessed at baseline in 175 patients. High plasma S1P concentration was found to be a favorable prognostic/predictive marker in advanced/metastatic pancreatic adenocarcinoma patients treated by gemcitabine alone but not in patients receiving a combination gemcitabine and PDGFR-inhibitor. In pancreatic adenocarcinoma PDX models, oral administration of an S1P lyase inhibitor (LX2931) significantly increased plasma S1P levels, decreased tumor expression of the hypoxia marker (CA IX), and enhanced chemotherapy efficacy when combined with gemcitabine treatment. The direct effect of S1P on tumor oxygenation was assessed by administration of S1P onto tumor-grafted CAM model and measuring intra-tumoral pO2 using a tissue oxygen monitor. S1P increased pO2 in a tumor-CAM model. Thus, increasing plasma S1P is a promising strategy to decrease tumor hypoxia and enhance therapy efficacy in solid tumors. S1P may act as a tumor vasculature normalizer.

Nuclear Sphingosine-1-phosphate Lyase Generated ∆2-hexadecenal is A Regulator of HDAC Activity and Chromatin Remodeling in Lung Epithelial Cells.

Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, is generated from sphingosine by sphingosine kinases (SPHKs) 1 and 2 and is metabolized to ∆2-hexadecenal (∆2-HDE) and ethanolamine phosphate by S1P lyase (S1PL) in mammalian cells. We have recently demonstrated the activation of nuclear SPHK2 and the generation of S1P in the nucleus of lung epithelial cells exposed to Pseudomonas aeruginosa. Here, we have investigated the nuclear localization of S1PL and the role of ∆2-HDE generated from S1P in the nucleus as a modulator of histone deacetylase (HDAC) activity and histone acetylation. Electron micrographs of the nuclear fractions isolated from MLE-12 cells showed nuclei free of ER contamination, and S1PL activity was detected in nuclear fractions isolated from primary lung bronchial epithelial cells and alveolar epithelial MLE-12 cells. Pseudomonas aeruginosa-mediated nuclear ∆2-HDE generation, and H3/H4 histone acetylation was attenuated by S1PL inhibitors in MLE-12 cells and human bronchial epithelial cells. In vitro, the addition of exogenous ∆2-HDE (100-10,000 nM) to lung epithelial cell nuclear preparations inhibited HDAC1/2 activity, and increased acetylation of Histone H3 and H4, whereas similar concentrations of S1P did not show a significant change. In addition, incubation of ∆2-HDE with rHDAC1 generated five different amino acid adducts as detected by LC-MS/MS; the predominant adduct being ∆2-HDE with lysine residues of HDAC1. Together, these data show an important role for the nuclear S1PL-derived ∆2-HDE in the modification of HDAC activity, histone acetylation, and chromatin remodeling in lung epithelial cells.

A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity.

Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a Km value of 2.68 μM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.

S1P lyase inhibition protects against sepsis by promoting disease tolerance via the S1P/S1PR3 axis

BackgroundOne-third of all deaths in hospitals are caused by sepsis. Despite its demonstrated prevalence and high case fatality rate, antibiotics remain the only target-oriented treatment option currently available. Starting from results showing that low-dose anthracyclines protect against sepsis in mice, we sought to find new causative treatment options to improve sepsis outcomes.MethodsSepsis was induced in mice, and different treatment options were evaluated regarding cytokine and biomarker expression, lung epithelial cell permeability, autophagy induction, and survival benefit. Results were validated in cell culture experiments and correlated with patient samples.FindingsEffective low-dose epirubicin treatment resulted in substantial downregulation of the sphingosine 1-phosphate (S1P) degrading enzyme S1P lyase (SPL). Consequent accumulation and secretion of S1P in lung parenchyma cells stimulated the S1P-receptor type 3 (S1PR3) and mitogen-activated protein kinases p38 and ERK, reducing tissue damage via increased disease tolerance. The protective effects of SPL inhibition were absent in S1PR3 deficient mice. Sepsis patients showed increased expression of SPL, stable expression of S1PR3, and increased levels of mucin-1 and surfactant protein D as indicators of lung damage.InterpretationOur work highlights a tissue-protective effect of SPL inhibition in sepsis due to activation of the S1P/S1PR3 axis and implies that SPL inhibitors and S1PR3 agonists might be potential therapeutics to protect against sepsis by increasing disease tolerance against infections.FundingThis study was supported by the Center for Sepsis Control and Care (CSCC), the German Research Foundation (DFG), RTG 1715 (to M. H. G. and I. R.) and the National Institutes of Health, Grant R01GM043880 (to S. S.).

Pharmacological inhibition of sphingosine-1-phosphate lyase partially reverses spatial memory impairment in streptozotocin-diabetic rats

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid with strong neuroprotective properties that is important for normal excitability and synaptic transmission in the hippocampal neurons. Considering the above, the aim of the present study was to determine whether increasing brain S1P level is able to reverse spatial memory impairment in streptozotocin-diabetic rats. The experiment was carried out on diabetic (n = 22) and nondiabetic (n = 10) male Wistar rats. Diabetes was induced by a single injection of streptozotocin. Eleven weeks later, 11 diabetic animals received injections of THI (S1P lyase inhibitor) for seven days. During the last five days of the experiment spatial reference memory acquisition and retention were tested in the Morris water maze task. The animals were then anaesthetized and samples of the hippocampus, prefrontal cortex, striatum, and cerebellum were excised. The content of S1P and related sphingolipids was measured using a HPLC method. Diabetes induced a depletion of ceramide in the hippocampus and cerebellum that was associated with impaired spatial memory and learning. Administration of THI to the diabetic animals prevented ceramide depletion in the hippocampus and cerebellum, and induced an increase in S1P content in all examined brain structures. These effects were associated with an improvement in spatial memory. We conclude that pharmacological inhibition of S1P lyase partially reverses the impairment in spatial memory induced by chronic hyperglycemia, and that this effect may be related to the prevention of ceramide depletion in the hippocampus and cerebellum, the increase in brain S1P level, or both.

Inhibition of sphingosine 1-phosphate lyase activates human keratinocyte differentiation and attenuates psoriasis in mice

Sphingosine 1-phosphate (S1P) lyase is an intracellular enzyme that catalyzes the irreversible degradation of S1P and has been suggested as a therapeutic target for the treatment of psoriasis vulgaris. Because S1P induces differentiation of keratinocytes, we examined whether modulation of S1P lyase and altered intracellular S1P levels regulate proliferation and differentiation of human neonatal epidermal keratinocyte (HEKn) cells. To identify the physiological functions of S1P lyase in skin, we inhibited S1P lyase in HEKn cells with an S1P lyase-specific inhibitor (SLI) and with S1P lyase 1 (SGPL1)-specific siRNA (siSGPL1). In HEKn cells, pharmacological treatment with the SLI caused G1 arrest by upregulation of p21 and p27 and induced keratin 1, an early differentiation marker. Similarly, genetic suppression by siSGPL1 arrested the cell cycle at the G1 phase and activated differentiation. In addition, enzyme suppression by siSGPL1 upregulated keratin 1 and differentiation markers including involucrin and loricrin. When hyperproliferation of HEKn cells was induced by interleukin (IL)-17 and IL-22, pharmacologic inhibition of S1P lyase by SLI decreased proliferation and activated differentiation of HEKn cells simultaneously. In addition, SLI administration ameliorated imiquimod-induced psoriatic symptoms including erythema, scaling, and epidermal thickness in vivo. We thus demonstrated that S1P lyase inhibition reduces cell proliferation and induces keratinocyte differentiation, and that inhibition may attenuate psoriasiform changes. Collectively, these findings suggest that S1P lyase is a modulating factor for proliferation and differentiation, and support its potential as a therapeutic target for psoriasis in human keratinocytes.

Agonist-induced activation of the S1P receptor 2 constitutes a novel osteoanabolic therapy for the treatment of osteoporosis in mice.

Osteoporosis is a worldwide epidemic but pharmacological agents to stimulate new bone formation are scarce. We have shown that increasing tissue levels of sphingosine-1-phosphate (S1P) by blocking its degradation by the S1P lyase has pronounced osteoanabolic effect in mouse osteoporosis models by stimulating osteoblast differentiation through the S1P receptor 2 (S1P2). However, S1P lyase inhibitors have side effects complicating potential clinical use. Here, we tested whether direct S1P2 engagement by the S1P2 agonist CYM5520 exerted osteoanabolic potential in estrogen deficiency-induced osteopenia in mice. We compared its efficacy to LX2931, a novel S1P lyase inhibitor currently tested in rheumatoid arthritis. CYM5520, LX2931 or vehicle were administered to ovariectomized mice for 6 weeks beginning 5 weeks after ovariectomy, Bone mass, cellular composition and mechanical strength were assessed by microCT, histomorphometry and three point bending tests. Plasma markers of bone metabolism were analyzed by ELISA. Therapeutic treatment with CYM5520 and LX2931 clearly increased long bone and vertebral bone mass to impressive 3-5 fold over vehicle in osteopenic ovariectomized mice. As expected, lymphopenia was a side effect of LX2931, whereas none occurred with CYM5520. Consistent with an osteoanabolic effect, CYM5520 increased osteoblast number, osteoid surface and alkaline phosphatase area 2-3 fold over vehicle. Plasma concentrations of the osteoanabolic marker procollagen I C-terminal propeptide were also elevated by CYM5520 and LX2931. LX2931 but not yet CYM5520 increased cortical thickness and mechanical strength without affecting mineral density. Treatment with a pharmacological S1P2 agonist corrected ovariectomy-induced osteopenia in mice by inducing new bone formation thus constituting a novel osteoanabolic approach to osteoporosis.

Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss.

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P2 potently stimulated osteoblastogenesis at the expense of adipogenesis byinversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis byinducing osteoprotegerin through newly discovered p38-GSK3β-β-catenin and WNT5A-LRP5 pathways. Accordingly, S1P2-deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serumS1P levels were positively associated with serumcalcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and thebone formation markerPINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.

Amelioration of colitis through blocking lymphocytes entry to Peyer's patches by sphingosine-1-phosphate lyase inhibitor.

Sphingosine-1-phosphate (S1P) receptor 1, a therapeutic target of the S1P1 agonist FTY720, plays a crucial role in lymphocyte migration and is expressed in several cells including naïve T lymphocytes and endothelial cells. 2-Acetyl-4-tetrahydroxybutyl imidazole (THI), an inhibitor of S1P lyase, exhibits immunomodulatory activity through increasing the S1P concentration in the secondary lymphoid organs, but its effects on colitis remain unclear. This study aimed to clarify how THI affects colitis and migration of naïve T lymphocytes in Peyer's patches (PPs). The effect of THI on gut immunity was investigated by analyzing the dextran sulfate sodium (DSS)-induced murine colitis model, lymphocyte components in thoracic duct lymphocytes (TDLs), and microscopic movement of TDLs in PPs. 2-Acetyl-4-tetrahydroxybutyl imidazole ameliorated DSS-induced colitis histologically by causing a significant decrease in colonic lymphocyte infiltration and expression of mucosal pro-inflammatory cytokines. THI suppressed the inflow of naïve T lymphocytes into the thoracic duct. Microscopic observation of PPs in control animals revealed that many TDLs egressed to the stroma and migrated to lymph capillaries after attaching to the high endothelial venules (HEVs). THI or FTY720 treatment in recipient animals blocked lymphocyte egression from the HEVs to the stroma. This is the first study to clarify the ameliorating effects of THI on DSS-induced colitis. Microscopic observations demonstrated the involvement of HEVs in the egression of S1P-dependent gut-tropic T lymphocytes to lymph capillaries. This S1P lyase inhibitor might become a novel immunosuppressant for inflammatory bowel disease therapy by blocking infiltration of lymphocytes through HEVs into the stroma in PPs.

Sphingolipids in Ventilator Induced Lung Injury: Role of Sphingosine-1-Phosphate Lyase.

Mechanical ventilation (MV) performed in respiratory failure patients to maintain lung function leads to ventilator-induced lung injury (VILI). This study investigates the role of sphingolipids and sphingolipid metabolizing enzymes in VILI using a rodent model of VILI and alveolar epithelial cells subjected to cyclic stretch (CS). MV (0 PEEP (Positive End Expiratory Pressure), 30 mL/kg, 4 h) in mice enhanced sphingosine-1-phosphate lyase (S1PL) expression, and ceramide levels, and decreased S1P levels in lung tissue, thereby leading to lung inflammation, injury and apoptosis. Accumulation of S1P in cells is a balance between its synthesis catalyzed by sphingosine kinase (SphK) 1 and 2 and catabolism mediated by S1P phosphatases and S1PL. Thus, the role of S1PL and SphK1 in VILI was investigated using Sgpl1+/− and Sphk1−/− mice. Partial genetic deletion of Sgpl1 protected mice against VILI, whereas deletion of SphK1 accentuated VILI in mice. Alveolar epithelial MLE-12 cells subjected to pathophysiological 18% cyclic stretch (CS) exhibited increased S1PL protein expression and dysregulation of sphingoid bases levels as compared to physiological 5% CS. Pre-treatment of MLE-12 cells with S1PL inhibitor, 4-deoxypyridoxine, attenuated 18% CS-induced barrier dysfunction, minimized cell apoptosis and cytokine secretion. These results suggest that inhibition of S1PL that increases S1P levels may offer protection against VILI.

2-Acetyl-5-tetrahydroxybutyl imidazole (THI) protects 661W cells against oxidative stress.

Retinal degeneration and in particular retinitis pigmentosa (RP) is associated to ceramide (Cer) accumulation and cell death induction. Cer and sphingosine-1-phosphate (S1P) belong to the sphingolipids class and exert a pro-apoptotic and pro-survival activity, respectively. Our aim is to target sphingolipid metabolism by inhibiting S1P lyase that regulates one of the S1P degradation pathways, to reduce retinal photoreceptor damage. The murine 661W cone-like cell line was pretreated with THI, an inhibitor of S1P lyase and exposed to H2O2-induced oxidative stress. 661W cell viability and apoptosis were evaluated by Trypan Blue and TUNEL assay, respectively. Protein expression of mediators of the survival/death pathway (ERK1/2, Akt, Bcl-2, Bax) was analyzed by Western blotting. RT-PCR was performed to establish HO-1 transcript changes and LC-MS analysis to measure Cer intracellular content. THI rescues inhibitory H2O2-effect on 661W cell viability and impairs H2O2-induced apoptosis by increasing Bcl-2/Bax ratio. THI administration counteracts the oxidative stress effects of H2O2 on 661W cells by activating the Nrf2/HO-1 pathway, regulating ERK and Akt phosphorylation levels, and decreasing Cer intracellular content. We conclude that sphingolipid metabolism manipulation can be considered a therapeutic target to promote photoreceptor survival.

Correction of lung inflammation in a F508del CFTR murine cystic fibrosis model by the sphingosine-1-phosphate lyase inhibitor LX2931.

Progressive lung disease with early onset is the main cause of mortality and morbidity in cystic fibrosis patients. Here we report a reduction of sphingosine-1-phosphate (S1P) in the lung of unchallenged Cftrtm1EUR F508del CFTR mutant mice. This correlates with enhanced infiltration by inducible nitric oxide synthase (iNOS)-expressing granulocytes, B cells, and T cells. Furthermore, the ratio of macrophage-derived dendritic cells (MoDC) to conventional dendritic cells (cDC) is higher in mutant mouse lung, consistent with unprovoked inflammation. Oral application of a S1P lyase inhibitor (LX2931) increases S1P levels in mutant mouse tissues. This normalizes the lung MoDC/cDC ratio and reduces B and T cell counts. Lung granulocytes are enhanced, but iNOS expression is reduced in this population. Although lung LyC6+ monocytes are enhanced by LX2931, they apparently do not differentiate to MoDC and macrophages. After challenge with bacterial toxins (LPS-fMLP) we observe enhanced levels of proinflammatory cytokines TNF-α, KC, IFNγ, and IL-12 and the inducible mucin MUC5AC in mutant mouse lung, evidence of deficient resolution of inflammation. LX2931 does not prevent transient inflammation or goblet cell hyperplasia after challenge, but it reduces MUC5AC and proinflammatory cytokine levels toward normal values. We conclude that lung pathology in homozygous mice expressing murine F508del CFTR, which represents the most frequent mutation in CF patients, is characterized by abnormal behavior of infiltrating myeloid cells and delayed resolution of induced inflammation. This phenotype can be partially corrected by a S1P lyase inhibitor, providing a rationale for therapeutic targeting of the S1P signaling pathway in CF patients.

Scintillation Proximity Assay to Detect the Changes in Cellular Dihydrosphingosine 1-Phosphate Levels.

Compounds that modulate the activity of sphingosine 1-phosphate (S1P)-metabolizing enzymes are expected to be potential therapeutic agents for various diseases. Investigation of their potencies requires not only cell-free but also cell-based assays in which intracellular accumulation/depletion of S1P could be monitored. However, conventional methods have limitations to their simplicity, mainly due to the necessity of a separation process that separates S1P from its related substances. Here, we describe a method utilizing a scintillation proximity assay (SPA) for semi-quantifying intracellular [(3)H]-labeled dihydroS1P ([(3)H]dhS1P), which is also a substrate for S1P-metabolizing enzymes. We found that uncoated yttrium silicate SPA beads could selectively bind to and detect [(3)H]dhS1P rather than [(3)H]dihydrosphingosine (the non-phosphorylated form of [(3)H]dhS1P). Based on this, we developed a novel cell-based assay system which does not require any organic solvent extraction or chromatographic separation, and confirmed its practicality by using siRNA targeting S1P lyase (S1PL) and known S1PL inhibitors as models. Our results demonstrated that this assay is useful for rapid and easy evaluation of S1PL inhibitors, and could be potentially applicable for all compounds that modulate the activity of S1P-metabolizing enzymes.

Sphingosine-1-Phosphate (S1P) Lyase Inhibition Causes Increased Cardiac S1P Levels and Bradycardia in Rats.

Inhibition of the sphingosine-1-phosphate (S1P)-catabolizing enzyme S1P lyase (S1PL) elevates the native ligand of S1P receptors and provides an alternative mechanism for immune suppression to synthetic S1P receptor agonists. S1PL inhibition is reported to preferentially elevate S1P in lymphoid organs. Tissue selectivity could potentially differentiate S1PL inhibitors from S1P receptor agonists, the use of which also results in bradycardia, atrioventricular block, and hypertension. But it is unknown if S1PL inhibition would also modulate cardiac S1P levels or cardiovascular function. The S1PL inhibitor 6-[(2R)-4-(4-benzyl-7-chlorophthalazin-1-yl)-2-methylpiperazin-1-yl]pyridine-3-carbonitrile was used to determine the relationship in rats between drug concentration, S1P levels in select tissues, and circulating lymphocytes. Repeated oral doses of the S1PL inhibitor fully depleted circulating lymphocytes after 3 to 4 days of treatment in rats. Full lymphopenia corresponded to increased levels of S1P of 100- to 1000-fold in lymph nodes, 3-fold in blood (but with no change in plasma), and 9-fold in cardiac tissue. Repeated oral dosing of the S1PL inhibitor in telemeterized, conscious rats resulted in significant bradycardia within 48 hours of drug treatment, comparable in magnitude to the bradycardia induced by 3 mg/kg fingolimod. These results suggest that S1PL inhibition modulates cardiac function and does not provide immune suppression with an improved cardiovascular safety profile over fingolimod in rats.

Bacterial versus human sphingosine-1-phosphate lyase (S1PL) in the design of potential S1PL inhibitors

A series of potential active-site sphingosine-1-phosphate lyase (S1PL) inhibitors have been designed from scaffolds 1 and 2, arising from virtual screening using the X-ray structures of the bacterial (StS1PL) and the human (hS1PL) enzymes. Both enzymes are very similar at the active site, as confirmed by the similar experimental kinetic constants shown by the fluorogenic substrate RBM13 in both cases. However, the docking scoring functions used probably overestimated the weight of electrostatic interactions between the ligands and key active-site residues in the protein environment, which may account for the modest activity found for the designed inhibitors. In addition, the possibility that the inhibitors do not reach the enzyme active site should not be overlooked. Finally, since both enzymes show remarkable structural differences at the access channel and in the proximity to the active site cavity, caution should be taken when designing inhibitors acting around that area, as evidenced by the much lower activity found in StS1PL for the potent hS1PL inhibitor D.