Plasma Sphingosine-1-phosphate Research Articles

Plasma Sphingolipids in Acute Pancreatitis.

Acute pancreatitis (AP) is a prevalent gastrointestinal disorder associated with systemic inflammatory response syndrome and, in the case of severe AP, a mortality rate ranging from 36% to 50%. Standard clinical treatment of AP includes intensive hydration, analgesia, and management of complications. Unfortunately, the direct treatment of AP at the level of its molecular pathomechanism has not yet been established. Recent studies indicate that the sphingolipid signaling pathway may be one of the important factors contributing to the development of inflammation in pancreatic diseases. In the current study, we sought to investigate this promising route. We examined the plasma sphingolipid profile of 44 patients with acute pancreatitis, dividing them into three groups: mild, moderate and severe AP. Samples were collected from these groups at days 1, 3 and 7 following their hospital admission. We demonstrated significant changes in blood plasma sphingolipids in relation to the time course of AP. We also found an inhibition of de novo ceramide synthesis in mild and moderate AP. However, the most important and novel finding was a significant elevation in sphingosine-1-phosphate (S1P) (a downstream metabolite of ceramide) in mild AP, as well as a dramatic reduction in the lipid molecule content in the early stage (days 1 and 3) of severe AP. This strongly indicates that plasma S1P could serve as a prognostic marker of AP severity.

Unexpected profile of sphingolipid contents in blood and bone marrow plasma collected from patients diagnosed with acute myeloid leukemia

BackgroundImpaired apoptotic pathways in leukemic cells enable them to grow in an uncontrolled way. Moreover, aberrations in the apoptotic pathways are the main factor of leukemic cells drug resistance.MethodsTo assess the presence of potential abnormalities that might promote dysfunction of leukemic cells growth, HPLC system was used to determine sphingosine (SFO), sphinganine (SFA), sphingosine-1-phosphate (S1P) and ceramide (CER) concentration in the blood collected from patients diagnose with acute myeloblastic leukemia (AML; n = 49) and compare to values of control (healthily) group (n = 51). Additionally, in AML group concentration of SFO, SFA, S1P and CER was determined in bone marrow plasma and compared to respective values in blood plasma. The concentration of S1P and CER binding protein – plasma gelsolin (GSN) was also assessed in collected samples using immunoblotting assay.ResultsWe observed that in AML patients the average SFO, SFA and CER concentration in blood plasma was significantly higher (p < 0.001) compare to control group, when blood plasma S1P concentration was significantly lower (p < 0.001). At the same time the CER/S1P ratio in AML patient (44.5 ± 19.4) was about 54% higher compare to control group (20.9 ± 13.1). Interestingly the average concentration of S1P in blood plasma (196 ± 13 pmol/ml) was higher compare to its concentration in plasma collected from bone marrow (154 ± 21 pmol/ml).ConclusionsWe hypothesize that changes in profile of sphingolipids concentration and some of their binding protein partners such as GSN in extracellular environment of blood and bone marrow cells in leukemic patients can be targeted to develop new AML treatment method(s).

Apolipoprotein M mediates sphingosine-1-phosphate efflux from erythrocytes

Sphingosine-1-phosphate (S1P) is a bioactive lipid implicated in e.g. angiogenesis, lymphocyte trafficking, and endothelial barrier function. Erythrocytes are a main source of plasma S1P together with platelets and endothelial cells. Apolipoprotein M (apoM) in HDL carries 70% of plasma S1P, whereas 30% is carried by albumin. The current aim was to investigate the role of apoM in export of S1P from human erythrocytes. Erythrocytes exported S1P more efficiently to HDL than to albumin, particularly when apoM was present in HDL. In contrast, export of sphingosine to HDL was unaffected by the presence of apoM. The specific ability of apoM to promote export of S1P was independent of apoM being bound in HDL particles. Treatment with MK-571, an inhibitor of the ABCC1 transporter, effectively reduced export of S1P from human erythrocytes to apoM, whereas the export was unaffected by inhibitors of ABCB1 or ATPase. Thus, ABCC1 could be involved in export of S1P from erythrocytes to apoM.

Mfsd2b is essential for the sphingosine-1-phosphate export in erythrocytes and platelets.

Sphingosine-1-phosphate (S1P), a potent signalling lipid secreted by red blood cells and platelets, plays numerous biologically significant roles. However, the identity of its long-sought exporter is enigmatic. Here we show that the major facilitator superfamily transporter 2b (Mfsd2b), an orphan transporter, is essential for S1P export from red blood cells and platelets. Comprehensive lipidomic analysis indicates a dramatic and specific accumulation of S1P species in Mfsd2b knockout red blood cells and platelets compared with that of wild-type controls. Consistently, biochemical assays from knockout red blood cells, platelets, and cell lines overexpressing human and mouse Mfsd2b proteins demonstrate that Mfsd2b actively exports S1P. Plasma S1P level in knockout mice is significantly reduced by 42-54% of that of wild-type level, indicating that Mfsd2b pathway contributes approximately half of the plasma S1P pool. The reduction of plasma S1P in knockout mice is insufficient to cause blood vessel leakiness, but it does render the mice more sensitive to anaphylactic shock. Stress-induced erythropoiesis significantly increased plasma S1P levels and knockout mice were sensitive to these treatments. Surprisingly, knockout mice exhibited haemolysis associated with red blood cell stomatocytes, and the haemolytic phenotype was severely increased with signs of membrane fragility under stress erythropoiesis. We show that S1P secretion by Mfsd2b is critical for red blood cell morphology. Our data reveal an unexpected physiological role of red blood cells in sphingolipid metabolism in circulation. These findings open new avenues for investigating the signalling roles of S1P derived from red blood cells and platelets.

Effects of apolipoprotein M in uremic atherosclerosis

Background and aimsChronic kidney disease is characterized by uremia and causes premature death, partly due to accelerated atherosclerosis. Apolipoprotein (apo) M is a plasma carrier protein for the lipid sphingosine-1-phosphate (S1P). The Apom-S1P complex associates with HDL, and may contribute to its anti-atherosclerotic effects. The role of Apom/S1P in atherosclerosis is presently controversial and has not been explored in a uremic setting. We aimed to explore whether plasma concentrations of Apom/S1P are altered by uremia and whether Apom overexpression or deficiency affects classical and uremic atherosclerosis. MethodsMild to moderate uremia was induced by subtotal nephrectomy (NX) in 86–92 Apoe-deficient mice that were either Apom-wild type, Apom-deficient, or overexpressed Apom (∼10 fold). The effects of uremia on plasma Apom/S1P and atherosclerosis were evaluated and compared to non-nephrectomized controls. ResultsUremia increased plasma Apom by ∼25%, but not S1P. Plasma S1P was elevated by ∼300% in mice overexpressing Apom, and decreased by ∼25% in Apom-deficient mice. Apom overexpression augmented aortic root atherosclerosis and plasma cholesterol. In contrast, aortic arch atherosclerosis was unaffected by the Apom genotype. There was no effect of Apom-deficiency or Apom overexpression on uremic atherosclerosis. ConclusionsThis study highlights the complexity of Apom/S1P in atherosclerosis and challenges the notion that the Apom/S1P complex is anti-atherogenic, at least in Apoe-deficient mice.

Determination of Sphingosine-1-Phosphate in Human Plasma Using Liquid Chromatography Coupled with Q-Tof Mass Spectrometry.

Evidence suggests that high-density lipoprotein (HDL) components distinct from cholesterol, such as sphingosine-1-phosphate (S1P), may account for the anti-atherothrombotic effects attributed to this lipoprotein. The current method for the determination of plasma levels of S1P as well as levels associated with HDL particles is still cumbersome an assay method to be worldwide practical. Recently, a simplified protocol based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the sensitive and specific quantification of plasma levels of S1P with good accuracy has been reported. This work utilized a triple quadrupole (QqQ)-based LC-MS/MS system. Here we adapt that method for the determination of plasma levels of S1P using a quadrupole time of flight (Q-Tof) based LC-MS system. Calibration curves were linear in the range of 0.05 to 2 µM. The lower limit of quantification (LOQ) was 0.05 µM. The concentration of S1P in human plasma was determined to be 1 ± 0.09 µM (n = 6). The average accuracy over the stated range of the method was found to be 100 ± 5.9% with precision at the LOQ better than 10% when predicting the calibration standards. The concentration of plasma S1P in the prepared samples was stable for 24 h at room temperature. We have demonstrated the quantification of plasma S1P using Q-Tof based LC-MS with very good sensitivity, accuracy, and precision that can used for future studies in this field.

Mobilization studies in mice deficient in sphingosine kinase 2 support a crucial role of the plasma level of sphingosine-1-phosphate in the egress of hematopoietic stem progenitor cells.

Sphingosine-1-phosphate (S1P) is a bioactive lipid involved in cell signaling and, if released from cells, also plays a crucial role in regulating the trafficking of lympho-hematopoietic cells, including primitive hematopoietic stem/progenitor cells (HSPCs). It has been demonstrated that S1P chemoattracts HSPCs, and its level in peripheral blood creates a gradient directing egress of these cells during mobilization. In this paper we analyzed hematopoiesis in mice deficient in sphingosine kinase 2 (Sphk2-KO mice) and studied the effect of this mutation on plasma S1P levels. We found that Sphk2-KO mice have normal hematopoiesis, and, in contrast to Sphk1-KO mice, the circulating S1P level is highly elevated in these animals and correlates with the fact that HSPCs in Sphk2-KO animals, also in contrast to Sphk1-KO animals, show enhanced mobilization. These results were recapitulated in wild type (WT) animals employing an Sphk2 inhibitor. We also administered an inhibitor of the S1P-degrading enzyme S1P lyase, known as tetrahydroxybutylimidazole (THI), to WT mice and observed that this resulted in an increase in S1P level in PB and enhanced mobilization of HSPCs. In sum, our results support a crucial role for S1P gradients in blood plasma in the mobilization process and indicate that small-molecule inhibitors of Sphk2 and Sgpl1 could be employed as mobilization-facilitating compounds. At the same time, further studies are needed to explain the unexpected effect of Sphk2 inhibition on increasing S1P levels in plasma.

Reduction in serum sphingosine 1-phosphate concentration in malaria.

Sphingosine 1-phosphate (S1P) is a lipid mediator formed by the metabolism of sphingomyelin which is involved in the endothelial permeability and inflammation. Although the plasma S1P concentration is reportedly decreased in patients with cerebral malaria, the role of S1P in malaria is still unclear. The purpose of this study was to examine the impact of malaria on circulating S1P concentration and its relationship with clinical data in malaria patients. Serum S1P levels were measured in 29 patients with P. vivax, 30 patients with uncomplicated P. falciparum, and 13 patients with complicated P. falciparum malaria on admission and on day 7, compared with healthy subjects (n = 18) as control group. The lowest level of serum S1P concentration was found in the complicated P. falciparum malaria group, compared with P. vivax, uncomplicated P. falciparum patients and healthy controls (all p < 0.001). In addition, serum S1P level was positively correlated with platelet count, hemoglobin and hematocrit levels in malaria patients. In conclusions, low levels of S1P are associated with the severity of malaria, and are correlated with thrombocytopenia and anemia. These findings highlight a role of S1P in the severity of malaria and support the use of S1P and its analogue as a novel adjuvant therapy for malaria complications.

Endurance training selectively increases high-density lipoprotein-bound sphingosine-1-phosphate in the plasma.

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid that is found in relatively high concentration in human plasma. Erythrocytes, endothelial cells, and activated platelets are the main sources of circulating S1P. The majority of plasma S1P is transported bound to high-density lipoprotein (HDL) and albumin. In recent years, HDL-bound S1P attracted much attention due to its cardioprotective and anti-atherogenic properties. We have previously found that endurance-trained athletes are characterized by higher plasma S1P concentration compared to untrained individuals. This finding prompted us to examine the effect of endurance training on S1P metabolism in blood. Thirteen healthy, untrained, male subjects completed an 8-week training program on a rowing ergometer. Three days before the first, and 3days after the last training session, blood samples were drawn from an antecubitalvein. We found that total plasma S1P concentration was increased after the training. Further analysis of different plasma fractions showed that the training selectively elevated HDL-bound S1P. This effect was associated with activation of sphingosine kinase in erythrocytes and platelets and enhanced S1P release from red blood cells. We postulate that increase in HDL-bound S1P level is one of the mechanisms underlying beneficial effects of regular physical activity on cardiovascular diseases.

Evaluation of plasma sphingosine 1-phosphate, hepcidin and cardiovascular damage biomarkers (cardiac troponin I and homocysteine) in rats infected with brucellosis and vaccinated (Rev-1, RB-51).

Brucellosis is known as one of important zoonosis. Studying the histological and biochemical effects of the disease could help to increase our knowledge about it. The aim of the present study was to evaluate changes of plasma parameters after intraperitoneal injection of two species of Brucella (Brucella melitensis and Brucella abortus) and two vaccines (Rev-1, RB-51) in the rat. Forty male rats were divided into five groups (n=8 in each group). Two groups received suspensions of Brucella abortus and Brucella melitensis and two other groups were injected intraperitoneally with two mentioned vaccines and the last group received only distilled water. The results showed a significant increase in sphingosine 1-phosphate, Malondialdehyde, hepcidin, homocysteine, cardiac troponin I and copper levels and a considerable decrease in the levels of iron and zinc (P≤0.01) in infected groups compared to the control animals. In vaccinated groups, hepcidin was increased but other parameters were not changed in comparison to the control group. It can be concluded that increase of homocysteine and cardiac troponin I in brucellosis could be a warning for cardiac adverse effects. Besides, increase of sphingosine 1-phosphate probably indicates its stimulant and modulatory effects in anti- Brucellosis biochemical pathways of the host.

Involvement of Band3 in the efflux of sphingosine 1-phosphate from erythrocytes.

Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator that is thought to be involved in various diseases. Although the main source of S1P in the plasma is erythrocytes, how S1P is exported from erythrocytes has not been elucidated. When we differentiated K562 cells into erythroblast-like cells with sodium butyrate, we observed that the efflux of S1P was increased without increased expression of previously proposed S1P transporters, while the expression levels of Band3 were increased. Therefore, in this study, we investigated the involvement of Band 3, the most characteristic membranous transporter for erythrocytes, in S1P efflux, using 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid, disodium salt (H2DIDS), which is an inhibitor of Band3. First, we treated human washed erythrocytes with H2DIDS and found that H2DIDS decreased the S1P levels in the supernatant, while it increased the cellular S1P contents. Next, when we injected H2DIDS into mice, the plasma S1P level was significantly decreased. Finally, when we overexpressed or suppressed Band3 in K562 cells, S1P efflux was enhanced or decreased, respectively, while the overexpression of Band3 in HEK293 cells did not modulate S1P efflux. These results suggested the possible involvement of Band3 in the transport of S1P, a multi-functional bioactive phospholipid, from erythrocytes.

Low sphingosine-1-phosphate plasma levels are predictive for increased mortality in patients with liver cirrhosis

Background & aimThe association of circulating sphingosine-1-phosphate (S1P), a bioactive lipid involved in various cellular processes, and related metabolites such as sphinganine-1-phosphate (SA1P) and sphingosine (SPH) with mortality in patients with end-stage liver disease is investigated in the presented study. S1P as a bioactive lipid mediator, is involved in several cellular processes, however, in end-stage liver disease its role is not understood.MethodsThe study cohort consisted of 95 patients with end-stage liver disease and available information on one-year outcome. The median MELD (Model for end-stage liver disease) score was 12.41 (Range 6.43–39.63). The quantification of sphingolipids in citrated plasma specimen was performed after methanolic protein precipitation followed by hydrophilic interaction liquid chromatography and tandem mass spectrometric detection.ResultsS1P and SA1P displayed significant correlations with the MELD score. Patients with circulating S1P levels below the lowest tertile (110.68 ng/ml) showed the poorest one-year survival rate of only 57.1%, whereas one-year survival rate in patients with S1P plasma levels above 165.67 ng/ml was 93.8%. In a multivariate cox regression analysis including platelet counts, concentrations of hemoglobin and MELD score, S1P remained a significant predictor for three-month and one-year mortality.ConclusionsLow plasma S1P concentrations are highly significantly associated with prognosis in end-stage liver disease. This association is independent of the stage of liver disease. Further studies should be performed to investigate S1P, its role in the pathophysiology of liver diseases and its potential for therapeutic interventions.

Sphingosine-1-phosphate and its receptors in anti-neutrophil cytoplasmic antibody-associated vasculitis.

C5a plays a crucial role in anti-neutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. Our previous studies found that the interaction between sphingosine-1-phosphate (S1P) and C5a plays an important role in the ANCA-mediated activation of neutrophils. In the current study, the expression levels of S1P in plasma and its receptors (S1PR1-5) in kidneys were analysed in patients with ANCA-associated vasculitis (AAV). Plasma samples from 32 AAV patients in active stage and 20 AAV patients in remission were collected. The plasma levels of S1P were determined by an enzyme-linked immunosorbent assay (ELISA). The expression of S1PR1-5 in the renal specimens from 24 AAV patients was detected by immunohistochemistry. The associations of the plasma levels of S1P and renal expression of S1PRs with clinical and pathological parameters were analysed. The level of plasma S1P was significantly higher in AAV patients in active stage than it was in both patients in remission and in normal controls. Correlation analysis showed that the plasma levels of S1P correlated with the initial serum creatinine levels (r = 0.502, P = 0.003) and inversely correlated with the estimated glomerular filtration rate (eGFR; r = -0.358, P = 0.044) in AAV patients. Double-labelling immunofluorescence assay suggested that S1PR1-5 were expressed on endothelial cells in the glomeruli and that S1PR1, 4 and 5 were expressed on neutrophils. In AAV patients, the circulating S1P levels were elevated and the renal expression of S1PR2-5 was upregulated. The levels of circulating S1P and the renal expression of S1PR were associated with the renal involvement and disease activity of AAV.

S1P in HDL promotes interaction between SR-BI and S1PR1 and activates S1PR1-mediated biological functions: calcium flux and S1PR1 internalization

HDL normally transports about 50-70% of plasma sphingosine 1-phosphate (S1P), and the S1P in HDL reportedly mediates several HDL-associated biological effects and signaling pathways. The HDL receptor, SR-BI, as well as the cell surface receptors for S1P (S1PRs) may be involved partially and/or completely in these HDL-induced processes. Here we investigate the nature of the HDL-stimulated interaction between the HDL receptor, SR-BI, and S1PR1 using a protein-fragment complementation assay and confocal microscopy. In both primary rat aortic vascular smooth muscle cells and HEK293 cells, the S1P content in HDL particles increased intracellular calcium concentration, which was mediated by S1PR1. Mechanistic studies performed in HEK293 cells showed that incubation of cells with HDL led to an increase in the physical interaction between the SR-BI and S1PR1 receptors that mainly occurred on the plasma membrane. Model recombinant HDL (rHDL) particles formed in vitro with S1P incorporated into the particle initiated the internalization of S1PR1, whereas rHDL without supplemented S1P did not, suggesting that S1P transported in HDL can selectively activate S1PR1. In conclusion, these data suggest that S1P in HDL stimulates the transient interaction between SR-BI and S1PRs that can activate S1PRs and induce an elevation in intracellular calcium concentration.

Involvement of CETP (Cholesteryl Ester Transfer Protein) in the Shift of Sphingosine-1-Phosphate Among Lipoproteins and in the Modulation of its Functions.

Sphingosine-1-phosphate (S1P) is a vasoprotective lipid mediator. About two thirds of plasma S1P rides on high-density lipoprotein (HDL), and several pleiotropic properties of HDL have been ascribed to S1P. In human subjects, CETP (cholesteryl ester transfer protein) greatly influences HDL quantities. In this study, we attempted to elucidate the roles of CETP in the metabolism of S1P. We overexpressed CETP in mice that lacked CETP and found that CETP overexpression decreased the HDL level but failed to modulate the levels of S1P and apolipoprotein M (apoM), a carrier of S1P, in the total plasma. We observed, however, that the distribution of S1P and apoM shifted from HDL to apoB-containing lipoproteins. When we administered C17S1P bound to apoM-containing lipoprotein, C17S1P and apoM were rapidly transferred to apoB-containing lipoproteins in CETP-overexpressing mice. When HDL containing C17S1P was mixed with low-density lipoprotein ex vivo, C17S1P shifted to the low-density lipoprotein fraction independent of the presence of CETP. Concordant with these results, apoM was distributed mainly to the same fraction as apo AI in a CETP-deficient subject, although apoM was also detected in apo AI-poor fractions in a corresponding hypercholesterolemia subject. About the bioactivities of S1P carried on each lipoprotein, S1P riding on apoB-containing lipoproteins induced the phosphorylation of Akt (AKT8 virus oncogene cellular homolog) and eNOS (endothelial nitric oxide synthase) in human umbilical vein endothelial cells, and CETP overexpression increased insulin secretion and sensitivity, which was inhibited by an S1P receptor 1 or 3 antagonist. CETP modulates the distribution of S1P among lipoproteins, which affects the bioactivities of S1P.

Sphingosine-1-phosphate signalling-a key player in the pathogenesis of Angiotensin II-induced hypertension.

Hypertension is a complex condition involving functional and structural alterations of the microvasculature and an activation of the immune system. T-lymphocytes play a crucial role during the development of hypertension in experimental models, yet the underlying mechanisms remain elusive. Lymphocyte egress from lymph nodes is controlled by sphingosine-1-phosphate (S1P), a natural lipid mediator regulating immune cell and vascular function in health and disease. We therefore investigated the involvement of S1P signalling in the pathogenesis of hypertension. Angiotensin-II (AngII) treatment resulted in high blood pressure (BP) associated to increased plasma S1P and circulating T-cell counts. T-cell egress from lymph nodes was found to be a critical initial step for the onset of hypertension as fingolimod, a S1P-receptor agonist sequestering lymphocytes in the lymph nodes and inducing lymphopenia, blunted BP responses to AngII. Furthermore, activity of S1P-generating enzyme type 2 (SphK2) in haematopoietic cells critically contributed to AngII-induced lymphocyte mobilization from the lymph nodes as SphK2-/- mice and mice where SphK2 was ablated only in the haematopoietic system presented an accumulation of T-cells in mesenteric lymph nodes and a blunted BP response. In addition, deregulation of vascular SphK2 expression associated to a thrombo-inflammatory phenotype of the microvasculature, and to functional alterations of small resistance arteries. The presented results point to a critical involvement of S1P and its signalling axis in the pathogenesis of hypertension. Specifically, SphK2 evolves as key player in immune cell trafficking and vascular dysfunction contributing to the development of overt hypertension.

Paradoxical Association of Postoperative Plasma Sphingosine-1-Phosphate with Breast Cancer Aggressiveness and Chemotherapy.

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that has been shown to serve an important regulatory function in breast cancer progression. This study analyzes plasma S1P levels in breast cancer patients undergoing adjuvant therapy as compared to healthy control volunteers. 452 plasma S1P samples among 158 breast cancer patients, along with 20 healthy control volunteers, were analyzed. Mean S1P levels did not significantly differ between cancer patients and controls. Smoking was associated with higher S1P levels in cancer patients. Baseline S1P levels had weak inverse correlation with levels of the inflammatory mediator interleukin- (IL-) 17 and CCL-2 and positive correlation with tumor necrosis factor alpha (TNF-α). Midpoint S1P levels during adjuvant therapy were lower than baseline, with near return to baseline after completion, indicating a relationship between chemotherapy and circulating S1P. While stage of disease did not correlate with plasma S1P levels, they were lower among patients with Her2-enriched and triple-negative breast cancer as compared to luminal-type breast cancer. Plasma S1P levels are paradoxically suppressed in aggressive breast cancer and during adjuvant chemotherapy, which raises the possibility that postoperative plasma S1P levels do not reflect S1P secretion from resected breast cancer.

Sphingosine-1-Phosphate (S1P) Signaling through S1P Receptor 1 (S1PR1) in Macrophage Contributes to Pain in Sickle Cell Disease

Sickle cell disease (SCD) is the most common autosomal recessive hemolytic disorder affecting more than 100,000 African-Americans and millions around the world. The severe health complication of patients with SCD is chronic pain, which is the leading cause of SCD patient morbidity and hospitalization. Our proposed research builds on the results from our nonbiased high-throughput metabolomics screen, which revealed erythrocyte sphingosine-1-phosphate (S1P), a bioactive lipid, is significantly elevated in the circulation of humans and mice with SCD. We identified that erythrocyte-sphingosine kinase 1 (Sphk1), the enzyme that generates S1P, promote erythrocyte sickling and the genetic deletion of Sphk1 significantly reduce pain symptoms in SCD mice. S1P is a pleiotropic molecule that signals via five g-protein coupled receptors (GPCRs) known as S1P receptors (S1PR1-5). S1PR1 is widely expressed in many cell types, including neurons and immune cells. We treated SCD mice with FDA approved drug, FTY720, to specifically target S1PR1. We demonstrated that mice had improved mechanical and thermal pain sensitivity. Notably, using flow cytometry, we found that the percentage of macrophages infiltrated to skin and percentage of S1PR1 expression in macrophages in the skin were significantly induced in SCD mice compared to the controls. This implies that elevated S1PR1 in macrophage contributes to chronic pain in SCD mice. To test this possibility, we treated SCD mice with clodronate liposomes to deplete tissue macrophages. We identified that mice had reduced sensitivity to mechanical and thermal stimuli assessments. Altogether, we provide both genetic and pharmacological evidence that elevated plasma S1P signaling via S1PR1 likely on macrophages contributes to chronic pain in SCD. Additionally, S1PR1 is known to mediate pro-inflammatory cytokine secretion in immune cells. We identified that SCD mice have elevated levels of interleukin-6 (IL-6), a pro-inflammatory cytokine, and is likely involved in SCD pain. To study the role of IL-6 in SCD pain, we generated SCD-IL-6-/- mice and observed improved mechanical and thermal pain sensitivity in these mice. From these evidences, we hypothesize that Sphk1 mediate S1P-S1PR1 activation in macrophages and IL-6 induction to promote SCD pain. Our work addresses a knowledge gap regarding an inflammatory mediated mechanism involved in SCD pain and novel therapies to counteract pain in SCD. DisclosuresNo relevant conflicts of interest to declare.

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes

Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be useful for the high-throughput screening of S1P transporter inhibitors using conventional fluorometers.

Abstract 064: Role of Sphingosine-1-phosphate 2 Receptor in Renal Microvascular Function of Renal Ischemia-reperfusion Rats

Renal ischemia-reperfusion (IR) induced acute kidney injury (IR-AKI) is accompanied by increased renal vascular resistance and a dramatic decline in glomerular filtration rate. Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in IR-AKI. Our recent studies established that exogenous S1P is a potent vasoconstrictor in rat afferent arterioles. We postulated that renal IR enhanced sensitivity of afferent arterioles to S1P-induced vasoconstriction via S1P2 receptor activation. The afferent arteriole response to S1P was assessed using the in vitro blood-perfused juxtamedullary nephron preparation. IR rats were induced by 60 min bilateral renal artery occlusion followed by 24 hours reperfusion. Baseline arteriolar diameter decreased significantly in IR (11.9±0.7 vs. 14.7±0.5 μm in sham, n=7, P&lt;0.05). Exogenous S1P evoked concentration-dependent vasoconstriction in sham rats. Increasing S1P (10 -10 -10 -5 M) decreased diameter to 99±1, 95±2, 89±2, 80±3, 58±4, and 34±2% of baseline (n=7), respectively. Renal IR shifted the S1P vasoconstrictor profile to the left. Arteriolar diameter declined to 93±1, 87±1, 76±1, 64±5, 46±5 and 35±4% of baseline (n=7, P&lt;0.05 vs sham at 10 -10 -10 -7 M S1P). S1P2 receptor blockade with JTE-013 markedly increased baseline diameter by 51±13% in IR (P&lt;0.05, vs. 11±3% in shams, n=3-4), suggesting that endogenous S1P exerts a greater influence on arteriolar tonic in IR than in sham. JTE-013 also markedly attenuated the enhanced vasoconstriction to S1P in IR rats. Arteriolar diameter averaged 112±2, 110±1, 114±3, 102±6, 81±6 and 61±10% of control in response to increasing S1P (P&lt;0.05 vs. JTE-013 untreated-IR) similar to JTE-013 treated sham. In contrast, arteriolar responses to Ang II, endothelin-1 or the S1P precursor, sphingosine were unaltered in IR rats. S1P content increased from 1.3±0.4 to 1.9±0.4 pmol/mg protein (~45%) in the cortical tissue of IR rats and 2.2±0.4 to 3.4±0.2 pmol/mg protein (~55%) in the medullary tissue but reduced the plasma S1P concentration from 2.5±0.3 to 1.3±0.1 μM (~50%, P&lt;0.05, n=3). These data establish that renal IR increases sphingolipid metabolites in rat kidneys and enhances renal microvascular S1P signaling, probably via S1P2 receptor activation.