Plasma Sphingosine-1-phosphate Research Articles

Enhanced platelet sensitization is accompanied by increased expression of the transporter MRP4 and elevated plasma S1P levels in mild COVID-19 convalescents

Viral infections can lead to platelet activation and hemostatic complications. However, the extent to which platelet reactivity remains altered after convalescence, contributing to long-term health impairments as observed after COVID-19 is not yet fully understood. Therefore, we conducted a cohort study (DRKS00025217) to determine platelet function in individuals convalesced from mild COVID-19. Assays were performed ex vivo with blood from convalescents at 2–15 weeks and 6–10 months after convalescence, focusing on platelet aggregation, activation markers, and thrombin formation. In addition, two other potentially relevant factors for platelet function were examined: the immunomodulatory mediator sphingosine-1-phosphate (S1P) and the platelet expression of the transporter MRP4 (ABCC4). Our findings indicate that robust platelet functions, including platelet aggregation determined by light transmission aggregometry, and thrombin formation, were not altered in convalescents compared to matched control individuals. However, an elevation in subtle platelet activation markers, such as P-selectin surface expression and activation of glycoprotein IIb/IIIa, was observed 2–15 weeks after convalescence. This was accompanied by an increased expression of MRP4 in platelets and significantly elevated levels of S1P in platelet-poor plasma. Our findings suggest increased platelet sensitization and a pro-inflammatory state even after convalescence from mild COVID-19, pointing toward MRP4 and S1P as associated factors.

Sphingosine-1-phosphate reduces arterial thrombosis via enhanced endothelial thrombomodulin expression by S1PR1 signaling

Abstract Background Sphingosine 1-phosphate (S1P) is an important lipid mediator in the cardiovascular system. Numerous links between S1P and blood coagulation have been described. However, results regarding the role of S1P in coagulation are inconclusive. A physiological antithrombotic regulator expressed by endothelial cells (EC) is thrombomodulin (TM). To date, the impact of S1P on TM-expression is unknown. Methods We evaluated S1P effects on TM-expression and subsequent platelet adhesion and thrombus formation. For this, we conducted cell culture with human umbilical cord venous endothelial cells (HUVECs), flow cytometry and flow chamber experiments. Additionally, in vivo arterial thrombus formation in mice was analyzed. Results Sphingosine kinase-1 deficient mice (SphK1-/-) have about 70% less circulating plasma S1P and show a significant reduction in TM expression on the aorta as compared to their littermates (WT: 207.2 ± 45.8 pg/g Aorta vs. SphK1-/- 124.5 ± 40.2 pg/g Aorta, p<0.0001). In contrast, S1P incubation of 24 hours significantly increased TM-expression on HUVECs by about 30 percent. This effect was abolished by S1PR1-inhibition with W146. Treatment with S1P reduced platelet adhesion from whole blood to endothelial cells in the flow chamber experiments (Con: 100.00 ± 28.09% vs. S1P: 80.91 ± 22.85%, p<0.0001). This effect could be reversed both by S1PR1 inhibition and with a TM-neutralizing antibody. P-selectin-expression on platelets was decreased after incubation with TM suggesting (Ctrl: 62.79 ± 13.30% vs. TM: 43.47 ± 11.36%, p=0.0222). Finally, to assess the in vivo effects of our findings, arterial thrombus formation in mice was performed. TM inhibited in vivo thrombus formation. Occlusion of the carotid artery could not be detected during the 30 minutes recording. In contrast, SphK1-/- mice with low S1P levels showed decreased times to first occlusion. Treatment with TM in these animals was able to reverse this effect. Conclusion We can conclude that the main findings of our study were (i) S1P increased endothelial TM-expression, (ii) which subsequently leads to reduced platelet adhesion and (iii) inhibition of arterial thrombus formation in vivo.

MOLECULAR APPROACH TO PREMATURE EJACULATION: A PILOT STUDY ON S1P SERUM LEVELS AND S1PR1, S1PR2, S1PR3 POLYMORPHISMS

Objective: Sphingosine-1-phosphate (S1P) and its receptors are involved in various sexual functions, particularly in smooth muscle regulation and vascular responses. However, the role of S1P and its receptors in premature ejaculation (PE) remains unclear. This study investigates the relationship between single nucleotide polymorphisms (SNPs) in the S1PR1, S1PR2, and S1PR3 genes and plasma S1P levels in individuals with PE. Materials and Methods: The study included 100 individuals with PE and 100 healthy controls recruited from urology and psychiatry clinics. DNA was isolated from blood samples, and PCR was used to identify SNPs in the S1PR1 (rs2038366), S1PR2 (rs56357614), and S1PR3 (rs7022797) genes. Plasma S1P levels were measured using ELISA. Results: A significant association was observed between the heterozygous GT genotype of the S1PR1 gene and an increased risk of PE (OR 2.25, 95% CI 1.215–4.168, p = 0.0099). No significant associations were found between S1PR2 or S1PR3 polymorphisms and PE. Plasma S1P levels were significantly lower in the PE group (median 253.25 ng/L) compared to the control group (median 430.82 ng/L) (p < 0.001). Conclusion: S1PR1 gene polymorphism and reduced plasma S1P levels may be linked to the pathophysiology of PE. In contrast, S1PR2 and S1PR3 do not appear to be associated. Further research with larger samples is needed to confirm these findings.

Effect of phospholipid transfer protein on plasma sphingosine-1-phosphate

Plasma phospholipid transfer protein (PLTP) is a risk factor for human coronary artery disease (CAD). Sphingosine-1-phosphate (S1P), carried by high-density lipoprotein (HDL), is a potent lipid mediator and is also associated with CAD. Previous studies indicate that Pltp knockout (KO) (germline) mice have decreased circulating S1P without influencing apoM, a major S1P carrier on HDL. We then hypothesized that, like apoM, PLTP is another S1P carrier. We established inducible Pltp-KO, germline Apom-KO, and Pltp/Apom double KO mice and measured plasma lipoprotein and S1P levels under chow and a Western diet. We found that PLTP deficiency and the double deficiency have a similar effect on HDL reduction, while apoM deficiency has no such effect. Importantly, we found that all mice have about 50% reduction in plasma S1P levels, compared to wild type mice, and PLTP deficiency significantly reduces apoM levels (about 40%), while apoM deficiency has no effect on PLTP activity, indicating that PLTP depletion reduces S1P through HDL reduction and there is no additive effect with the double deficiency. To further evaluate this HDL-reduction-mediated effect, we overexpressed PLTP which also caused a reduction of HDL. We found that the overexpression significantly reduces S1P and apoM as well as apoA-I, a major apolipoprotein on HDL. Furthermore, we found that albumin (another reported S1P carrier) deficiency in mice has no effect on plasma S1P. We also found that the influence of PLTP on HDL may not require its direct binding to the particle. In conclusion, PLTP is not a direct S1P carrier. PLTP depletion or overexpression in adulthood dramatically reduces plasma S1P through HDL reduction. ApoM, but not albumin, deficiency reduces plasma S1P levels.

Zonation, ligand and dose dependence of S1PR1 signalling in blood and lymphatic vasculature.

Circulating levels of sphingosine 1-phosphate (S1P), an HDL-associated ligand for endothelial cell (EC) protective S1P receptor-1 (S1PR1), are reduced in disease states associated with endothelial dysfunction. Yet as S1PR1 has high affinity for S1P and can be activated by ligand-independent mechanisms and EC-autonomous S1P production, it is unclear if relative reductions in circulating S1P impact endothelial function. It is also unclear how EC S1PR1 insufficiency, whether induced by ligand deficiency or by S1PR1-directed immunosuppressive therapy, affects different vascular subsets. We here fine-map the zonation of S1PR1 signalling in the murine blood and lymphatic vasculature, superimpose cell type-specific and relative deficiencies in S1P production to define ligand source- and dose-dependence, and correlate receptor engagement to essential functions. In naïve blood vessels, despite broad expression, EC S1PR1 engagement was restricted to resistance-size arteries, lung capillaries and high-endothelial venules (HEV). Similar zonation was observed for albumin extravasation in EC S1PR1 deficient mice, and brain extravasation was reproduced with arterial EC-selective S1pr1 deletion. In lymphatic EC, S1PR1 engagement was high in collecting vessels and lymph nodes and low in terminal capillaries that drain tissue fluids. While EC S1P production sustained S1PR1 signaling in lymphatics and HEV, hematopoietic cells provided ∼90% of plasma S1P and sustained signaling in resistance arteries and lung capillaries. S1PR1 signaling and endothelial function were both surprisingly sensitive to reductions in plasma S1P with apparent saturation around 50% of normal levels. S1PR1 engagement did not depend on sex or age, but modestly increased in arteries in hypertension and diabetes. Sphingosine kinase (Sphk)-2 deficiency also increased S1PR1 engagement selectively in arteries, which could be attributed to Sphk1-dependent S1P release from perivascular macrophages. This study highlights vessel subtype-specific S1PR1 functions and mechanisms of engagement and supports the relevance of S1P as circulating biomarker for endothelial function.

Association of plasma sphingosine-1-phosphate levels with disease severity and prognosis after intracerebral hemorrhage.

Sphingosine-1-phosphate (S1P) is a signaling lipid involved in many biological processes, including inflammatory and immune regulatory responses. The study aimed to determine whether admission S1P levels are associated with disease severity and prognosis after spontaneous intracerebral hemorrhage (ICH). Data of 134 patients with spontaneous ICH and 120 healthy controls were obtained from Biological Resource Sample Database of Intracerebral Hemorrhage at the First Affiliated Hospital of Zhengzhou University. Plasma S1P levels were measured. Regression analyses were used to analyze the association between S1P levels and admission and 90-day modified Rankin scale (mRS) scores. Receiver operating characteristic (ROC) curves assessed the predictive value of S1P levels for ICH severity and prognosis. Patients with ICH exhibited elevated plasma S1P levels compared to the control group (median 286.95 vs. 239.80 ng/mL, p < 0.001). When divided patients into mild-to-moderate and severe groups according to their mRS scores both at admission and discharge, S1P levels were significantly elevated in the severe group compared to the mild-to-moderate group (admission 259.30 vs. 300.54, p < 0.001; 90-day 275.24 vs. 303.25, p < 0.001). The patients were divided into three groups with different concentration gradients, which showed significant statistical differences in admission mRS scores (3 vs. 4 vs. 5, p < 0.001), 90-day mRS scores (2.5 vs. 3 vs. 4, p < 0.001), consciousness disorders (45.5% vs. 68.2% vs. 69.6%, p = 0.033), ICU admission (29.5% vs. 59.1% vs. 89.1%, p < 0.001), surgery (15.9% vs. 47.7% vs. 82.6%, p < 0.001), intraventricular hemorrhages (27.3% vs. 61.4% vs. 65.2%, p < 0.001) and pulmonary infection (25% vs. 47.7% vs. 84.8%, p < 0.001). Multivariate analysis displayed that S1P level was an independent risk factor for disease severity (OR = 1.037, 95% CI = 1.020-1.054, p < 0.001) and prognosis (OR = 1.018, 95% CI = 1.006-1.030, p = 0.003). ROC curves revealed a predictive value of S1P levels with an area under the curve of 0.7952 (95% CI = 0.7144-0.8759, p < 0.001) for disease severity and 0.7105 (95% CI = 0.6227-0.7983, p < 0.001) for prognosis. Higher admission S1P is associated with worse initial disease severity and 90-day functional outcomes in intracerebral hemorrhage.

Preservation of recipient plasma sphingosine-1-phosphate levels reduces transfusion-related acute lung injury.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Kidney derived apolipoprotein M and its role in acute kidney injury.

Aim: Apolipoprotein M (apoM) is mainly expressed in liver and in proximal tubular epithelial cells in the kidney. In plasma, apoM associates with HDL particles via a retained signal peptide and carries sphingosine-1-phosphate (S1P), a small bioactive lipid. ApoM is undetectable in urine from healthy individuals but lack of megalin receptors in proximal tubuli cells induces loss of apoM into the urine. Besides this, very little is known about kidney-derived apoM. The aim of this study was to address the role of apoM in kidney biology and in acute kidney injury. Methods: A novel kidney-specific human apoM transgenic mouse model (RPTEC-hapoMTG) was generated and subjected to either cisplatin or ischemia/reperfusion injury. Further, a stable transfection of HK-2 cells overexpressing human apoM (HK-2-hapoMTG) was developed to study the pattern of apoM secretion in proximal tubuli cells. Results: Human apoM was present in plasma from RPTEC-hapoMTG mice (mean 0.18μM), with a significant increase in plasma S1P levels. In vitro apoM was secreted to both the apical (urine) and basolateral (blood) compartment from proximal tubular epithelial cells. However, no differences in kidney injury score was seen between RPTEC-hapoMTG and wild type (WT) mice upon kidney injury. Further, gene expression of inflammatory markers (i.e., IL6, MCP-1) was similar upon ischemia/reperfusion injury. Conclusion: Our study suggests that kidney-derived apoM is secreted to plasma, supporting a role for apoM in sequestering molecules from excretion in urine. However, overexpression of human apoM in the kidney did not protect against acute kidney injury.

HPLC-MS/MS Analysis for Sphingosine 1-Phosphate after the Dephosphorylation by Hydrogen Fluoride

Sphingosine 1-phosphate (S1P) is a signaling lipid molecule involved in various cellular processes. It is important to develop a quantitative method for S1P to determine endogenous levels and to investigate its functions. As S1P is a tiny lipid component of most biological samples, highly sensitive analysis by LC-MS/MS is required. The main challenge in S1P analysis by chromatography is peak-broadening due to the presence of a polar phosphate and the fact that S1P is indeed a zwitterion itself. In this study, we used hydrogen fluoride (HF) to efficiently remove a phosphate and then analyzed the surrogate, sphingosine, as a sharp peak by LC-ESI-MS/MS. We optimized the dephosphorylation reaction in terms of temperature and reaction time. Multiple reaction monitoring (MRM) for a dephosphorylated form of S1P and C17-S1P as an internal standard at m/z transition 300.4 &gt; 282.4 (quantification ion), 300.4 &gt; 262.4 (qualification ion), 286.3 &gt; 268.2 (internal standard) was conducted. This method was validated by essential parameters such as specificity, linearity, range, LOQ, LOD, accuracy, precision, and repeatability. To confirm this new method, we quantified S1P levels in various serum products (100.0~284.4 nM). In the sample pretreatment conditions for extracting S1P, the concern about potential sphingosine contamination in serum was negligible. The dephosphorylation efficiency by this method was about two-fold higher than that of alkaline phosphatase (APase). To apply the method in vivo, we analyzed S1P in plasma and kidney tissues obtained from a chronic kidney disease (CKD) mouse model. S1P levels were increased only in CKD kidney tissue but not in plasma. In conclusion, by applying the dephosphorylation step with HF, we established a new, sensitive LC-MS/MS quantitative method for S1P that can be applied to biological samples.

Construction of Plasma Sphingosine 1-Phosphate Expression Level and Risk Nomogram in Patients with Ischemic Stroke

Construction of Plasma Sphingosine 1-Phosphate Expression Level and Risk Nomogram in Patients with Ischemic Stroke

The effect of normobaric hypoxia on acute exercise-induced changes in blood sphingoid base-1-phosphates metabolism in cyclists.

Extracellular sphingosine-1-phosphate (S1P) emerged as an important regulator of muscle function. We previously found that plasma S1P concentration is elevated in response to acute exercise and training. Interestingly, hypoxia, which is commonly utilized in training programs, induces a similar effect. Therefore, the aim of the current study was to determine the effect of normobaric hypoxia on exercise-induced changes in blood sphingolipid metabolism. Fifteen male competitive cyclists performed a graded cycling exercise until exhaustion (GE) and a simulated 30 km individual time trial (TT) in either normoxic or hypoxic (FiO2 = 16.5%) conditions. Blood samples were taken before the exercise, following its cessation, and after 30 min of recovery. We found that TT increased dihydrosphingosine-1-phosphate (dhS1P) concentration in plasma (both HDL- and albumin-bound) and blood cells, as well as the rate of dhS1P release from erythrocytes, regardless of oxygen availability. Plasma concentration of S1P was, however, reduced during the recovery phase, and this trend was augmented by hypoxia. On the other hand, GE in normoxia induced a selective increase in HDL-bound S1P. This effect disappeared when the exercise was performed in hypoxia, and it was associated with reduced S1P level in platelets and erythrocytes. We conclude that submaximal exercise elevates total plasma dhS1P concentration via increased availability of dihydrosphingosine resulting in enhanced dhS1P synthesis and release by blood cells. Maximal exercise, on the other hand, induces a selective increase in HDL-bound S1P, which is a consequence of mechanisms not related to blood cells. We also conclude that hypoxia reduces post-exercise plasma S1P concentration.

The Fracture Risk Assessment Tool Probability and Trabecular Bone Score Mediate the Relationship between Sphingosine 1-phosphate Levels and Fracture Risk

Background: The sphingosine 1-phosphate (S1P) concentration is a potential biomarker of osteoporotic fracture and is associated with both the fracture risk assessment tool (FRAX) probability and trabecular bone score (TBS), which are well-known predictors of fracture. We sought to estimate the effect of the S1P concentration on fracture risk using the FRAX probability and TBS as mediators.Methods: Plasma S1P concentrations, FRAX variables, and TBSs were measured in 66 postmenopausal women with fractures and 273 postmenopausal women without fractures. Associations between S1P concentration, FRAX probability, TBS, and fracture risk were analyzed using correlation, logistic regression, and mediation analyses.Results: Subjects in the highest S1P concentration tertile had a higher fracture risk (odds ratio [OR], 5.09; 95% confidence interval [CI], 2.22–11.67) than those in the lowest S1P concentration tertile before adjustment. Subjects in the highest FRAX probability tertile had a higher fracture risk (OR, 14.59; 95% CI, 5.01–42.53) than those in the lowest FRAX probability tertile before adjustment. Subjects in the lowest TBS tertile had a higher fracture risk (OR, 4.76; 95% CI, 2.28–9.93) than those in the highest TBS tertile before adjustment. After adjustment for FRAX probability and TBS, the highest S1P concentration tertile was still associated with a higher fracture risk (OR, 3.13; 95% CI, 1.28–7.66). The FRAX probability and TBS accounted for 32.6% and 21.7%, respectively, of the relationship between the S1P concentration and fracture risk.Conclusions: The relationship between the circulating S1P concentration and fracture risk was partly mediated by the FRAX probability, bone microarchitecture, and other factors.

Lipid-lowering drugs affect lung cancer risk via sphingolipid metabolism: a drug-target Mendelian randomization study.

Background: The causal relationship between lipid-lowering drug (LLD) use and lung cancer risk is controversial, and the role of sphingolipid metabolism in this effect remains unclear. Methods: Genome-wide association study data on low-density lipoprotein (LDL), apolipoprotein B (ApoB), and triglycerides (TG) were used to develop genetic instrumental variables (IVs) for LLDs. Two-step Mendelian randomization analyses were performed to examine the causal relationship between LLDs and lung cancer risk. The effects of ceramide, sphingosine-1-phosphate (S1P), and ceramidases on lung cancer risk were explored, and the proportions of the effects of LLDs on lung cancer risk mediated by sphingolipid metabolism were calculated. Results: APOB inhibition decreased the lung cancer risk in ever-smokers via ApoB (odds ratio [OR] 0.81, 95% confidence interval [CI] 0.70-0.92, p = 0.010), LDL (OR 0.82, 95% CI 0.71-0.96, p = 0.040), and TG (OR 0.63, 95% CI 0.46-0.83, p = 0.015) reduction by 1 standard deviation (SD), decreased small-cell lung cancer (SCLC) risk via LDL reduction by 1 SD (OR 0.71, 95% CI 0.56-0.90, p = 0.016), and decreased the plasma ceramide level and increased the neutral ceramidase level. APOC3 inhibition decreased the lung adenocarcinoma (LUAD) risk (OR 0.60, 95% CI 0.43-0.84, p = 0.039) but increased SCLC risk (OR 2.18, 95% CI 1.17-4.09, p = 0.029) via ApoB reduction by 1 SD. HMGCR inhibition increased SCLC risk via ApoB reduction by 1 SD (OR 3.04, 95% CI 1.38-6.70, p = 0.014). The LPL agonist decreased SCLC risk via ApoB (OR 0.20, 95% CI 0.07-0.58, p = 0.012) and TG reduction (OR 0.58, 95% CI 0.43-0.77, p = 0.003) while increased the plasma S1P level. PCSK9 inhibition decreased the ceramide level. Neutral ceramidase mediated 8.1% and 9.5% of the reduced lung cancer risk in ever-smokers via ApoB and TG reduction by APOB inhibition, respectively, and mediated 8.7% of the reduced LUAD risk via ApoB reduction by APOC3 inhibition. Conclusion: We elucidated the intricate interplay between LLDs, sphingolipid metabolites, and lung cancer risk. Associations of APOB, APOC3, and HMGCR inhibition and LPL agonist with distinct lung cancer risks underscore the multifaceted nature of these relationships. The observed mediation effects highlight the considerable influence of neutral ceramidase on the lung cancer risk reduction achieved by APOB and APOC3 inhibition.

Revealing concealed cardioprotection by platelet Mfsd2b-released S1P in human and murine myocardial infarction

Abstract Background/Introduction Platelet activation on ruptured or eroded atherosclerotic lesions leads to thrombus formation and ischemic events. Hence, platelet inhibition is the cornerstone of primary and secondary prevention in patients with cardiovascular disease. Recently, non-canonical effects of platelets in multiple disease processes were revealed. However, others have demonstrated the opposite: a protective and regenerative role of platelets during myocardial healing. Sphingosine-1-phosphate (S1P) is an important bioactive lipid released from platelets upon activation and has potent cardioprotective properties in AMI when acutely administered or kept constitutively high due to pharmacological or genetic inhibition of its degradation. In humans, S1P levels in plasma are dynamically regulated during AMI. Purpose We hypothesized that platelets may be an important source of the S1P burst during AMI and that platelet-derived S1P is relevant for the extent of myocardial damage due to AMI. Methods We have addressed these questions in experimental AMI in mice lacking components of the S1P synthesis, release and signaling pathways. In additon, we conducted a hypothesis generating, prospective, monocentric, time-series, translational analysis in 127 patients with ST-elevation myocardial infarction (STEMI) to analyze the association between S1P plasma concentration and infarct size. Results Injection of cell-free supernatant of activated platelets (SNT+) prior AMI in mice led to decreased infarct size after 24 h of reperfusion as compared to supernatant of non-activated platelets (SNT-). This reduction of infarct size by SNT+ was associated with reduced neutrophil accumulation and reduced apoptosis after AMI. S1P-concentrations in SNT+ was 25% higher as compared to SNT-. S1P concentrations in SNT- of mice lacking Sphingosinkinase-1 (SphK1) were 70% lower and did not increase in SNT+. Accordingly, SNT+ of SphK1- deficient platelets failed to reduce infarct size. Deficiency of platelet S1P exporter Mfsd2b shows increased infarct size during in vivo AMI compared to littermate controls. SNT+ of WT mice led to improved outcome after ischemia in a model of Langendorff-perfused hearts. This was blunted by inhibition of S1P receptor 1 and genetic cardiomyocyte deficiency of S1PR1. Platelet S1P release during activation was reduced by P2Y12 inhibition but preserved during GPIIb/IIIa (tirofiban) inhibition. This translated to blunted cardioprotection by SNT+ of cangrelor treated platelets but sustained cardioprotection during tirofiban treatment. Plasma S1P concentrations was associated with cardiovascular death and infarct size in patients with ST-elevation myocardial infarction. Conclusion We have identified the mechanisms underlying platelet-associated cardioprotection and finally, revealed the importance of platelet-released S1P on admission in patients with ST-elevation myocardial infarction (STEMI) regarding to infarct size and long-term prognosis.

Beneficial Role of Erythrocyte Sphingosine 1-Phosphate in Preventing Renal Macrophage Polarization and Fibrosis through Combating HIF-1α-Dependent Creatine Phosphate Shuttle

Introduction: Erythrocytes, the major reservoir and supplier for plasma sphingosine 1 phosphate (S1P), is a potent biolipid signaling via its specific receptors responsible for immune cell trafficking and function. Current evidence has revealed that erythrocyte intracellular S1P has its own function to promote oxygen (O 2) delivery to counteract tissue hypoxia. Aim: To understand whether O 2 delivery mediated by erythrocyte S1P is involved in tissue macrophage polarization and fibrosis. Methods and Results: Genetic ablation of erythrocyte SphK1 (the only enzyme to generate S1P in erythrocyte, eSphK1 -/-) resulted in less O 2 delivery from erythrocytes, thus leading to severe renal hypoxia, damage and fibrosis in angiotensin II induced hypertensive kidney disease mouse model. Surprisingly, although plasma S1P level was reduced to 50% in Ang II-infused eSphK1 -/- mice, these mice displayed preferentially elevated pro-fibrosis M2 macrophages surrounding the hypoxic renal tubules. Surprisingly, there was no significant difference of S1P level in either kidney or renal macrophages between Ang II-infused controls and eSphK1 -/- mice, ruling out the possibility of difference of kidney or macrophage S1P levels responsible for renal M2 macrophage polarization. Metabolically, untargeted high-throughput metabolic profiling in macrophages purified from mouse kidney revealed a substantial reduction of amino acids (AAs) and fatty acids (FAs) but increased creatine. We further validate this in vitro by 13C 6, 15N 4-labeled arginine tracer. Hypoxia (1~2% O 2) promoted increased 13C 2-labeled creatine (arginine downstream metabolite) in murine bone marrow derived macrophages (BMDMs) time dependently. At cellular and molecular level, we further demonstrated that hypoxia signaling via HIF-1a directly enhanced IL-4 induced M2 polarization of BMDMs as well as expression of Ckb (a gene encoding creatine shuttle), which can be reversed by Chrysin (an HIF-1α inhibitor). Preclinical studies revealed that chrysin rescued the severe renal damage in eSphK1 -/-mice manipulated with unilateral ureteral obstruction (UUO) and prevented development of chronic kidney disease (CKD) in the control mice. Translational research validated mouse findings in kidney tissue of CKD patients. Conclusion: Our work has revealed that erythrocyte S1P combats HIF1α-dependent creatine shuttle, renal macrophage polarization and fibrosis and highlighted potential diagnostic and therapeutic possibilities for macrophage polarization and renal fibrosis.

Hepatopulmonary syndrome is associated with low sphingosine-1-phosphate levels and can be ameliorated by the functional agonist fingolimod

Hepatopulmonary syndrome (HPS) is characterised by a defect in arterial oxygenation induced by pulmonary vascular dilatation in patients with liver disease. Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator, suppresses vasodilation by reducing nitric oxide (NO) production. We investigated the role of S1P in patients with HPS and the role of fingolimod as a therapeutic option in an experimental model of HPS. Patients with cirrhosis with HPS (n= 44) and without HPS (n= 89) and 25 healthy controls were studied. Plasma levels of S1P, NO, and markers of systemic inflammation were studied. In a murine model of common bile duct ligation (CBDL), variations in pulmonary vasculature, arterial oxygenation, liver fibrosis, and inflammation were estimated before and after administration of S1P and fingolimod. Log of plasma S1P levels was significantly lower in patients with HPS than in those without HPS (3.1 ± 1.4 vs. 4.6 ± 0.2; p <0.001) and more so in severe intrapulmonary shunting than in mild and moderate intrapulmonary shunting (p <0.001). Plasma tumour necrosis factor-α (76.5 [30.3-91.6] vs. 52.9 [25.2-82.8]; p= 0.02) and NO (152.9 ± 41.2 vs. 79.2 ± 29.2; p= 0.001) levels were higher in patients with HPS than in those without HPS. An increase in Th17 (p <0.001) and T regulatory cells (p <0.001) was observed; the latter inversely correlated with plasma S1P levels. In the CBDL HPS model, fingolimod restored pulmonary vascular injury by increasing the arterial blood gas exchange and reducing systemic and pulmonary inflammation, resulting in improved survival (p= 0.02). Compared with vehicle treatment, fingolimod reduced portal pressure (p <0.05) and hepatic fibrosis and improved hepatocyte proliferation. It also induced apoptotic death in hepatic stellate cells and reduced collagen formation. Plasma S1P levels are low in patients with HPS and even more so in severe cases. Fingolimod, by improving pulmonary vascular tone and oxygenation, improves survival in a murine CBDL HPS model. A low level of plasma sphingosine-1-phosphate (S1P) is associated with severe pulmonary vascular shunting, and hence, it can serve as a marker of disease severity in patients with hepatopulmonary syndrome (HPS). Fingolimod, a functional agonist of S1P, reduces hepatic inflammation, improves vascular tone, and thus retards the progression of fibrosis in a preclinical animal model of HPS. Fingolimod is being proposed as a potential novel therapy for management of patients with HPS.

Plasma sphingolipid abnormalities in neurodegenerative diseases.

In recent years, there has been increasing evidence that several lipid metabolism abnormalities play an important role in the pathogenesis of neurodegenerative diseases. However, it is still unclear which lipid metabolism abnormalities play the most important role in neurodegenerative diseases. Plasma lipid metabolomics (lipidomics) has been shown to be an unbiased method that can be used to explore lipid metabolism abnormalities in neurodegenerative diseases. Plasma lipidomics in neurodegenerative diseases has been performed only in idiopathic Parkinson's disease (IPD) and Alzheimer's disease (AD), and comprehensive studies are needed to clarify the pathogenesis. In this study, we investigated plasma lipids using lipidomics in individuals with neurodegenerative diseases and healthy controls (CNs). Plasma lipidomics was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in those with IPD, dementia with Lewy bodies (DLB), multiple system atrophy (MSA), AD, and progressive supranuclear palsy (PSP) and CNs. The results showed that (1) plasma sphingosine-1-phosphate (S1P) was significantly lower in all neurodegenerative disease groups (IPD, DLB, MSA, AD, and PSP) than in the CN group. (2) Plasma monohexylceramide (MonCer) and lactosylceramide (LacCer) were significantly higher in all neurodegenerative disease groups (IPD, DLB, MSA, AD, and PSP) than in the CN group. (3) Plasma MonCer levels were significantly positively correlated with plasma LacCer levels in all enrolled groups. S1P, Glucosylceramide (GlcCer), the main component of MonCer, and LacCer are sphingolipids that are biosynthesized from ceramide. Recent studies have suggested that elevated GlcCer and decreased S1P levels in neurons are related to neuronal cell death and that elevated LacCer levels induce neurodegeneration by neuroinflammation. In the present study, we found decreased plasma S1P levels and elevated plasma MonCer and LacCer levels in those with neurodegenerative diseases, which is a new finding indicating the importance of abnormal sphingolipid metabolism in neurodegeneration.

Erythrocyte Sphingosine 1 Phosphate Is Essential to Combat HIF1α-Dependent Creatine Shuttle, Macrophage Polarization and Fibrosis

Erythrocytes have long been considered as the major reservoir and supplier for plasma sphingosine 1 phosphate (S1P), which is a potent biolipid signaling via its specific receptors responsible for immune cell trafficking and polarization of tissue macrophages. Currently, emerging evidence has revealed that erythrocyte intracellular S1P has its own function to promote oxygen (O2) delivery to counteract tissue hypoxia. However, whether O2 delivery mediated by erythrocyte S1P is involved in tissue macrophage polarization, inflammation and fibrosis remains unrecognized. Here we report that genetic ablation of erythrocyte SphK1 (the only enzyme to generate S1P in erythrocyte eSphK1-/-) resulted in less O2 delivery from erythrocytes, thus leading to severe hypoxic tissue microenvironment, kidney damage and fibrosis in a well-accepted pathological hypoxic model infused with angiotensin II (a potent vasoconstrictor known to be elevated in hypertensive patients). Surprisingly, although plasma S1P levels were reduced to 50% in Ang II-infused eSphK1-/- mice, these mice displayed preferentially elevated pro-fibrosis M2 macrophages surrounding the hypoxic renal tubules (one of the most hypoxic sites with Ang II infusion). Metabolically, untargeted high-throughput metabolic profiling in macrophages purified from mouse kidney revealed a substantial reduction of amino acids (AAs) and fatty acids (FAs) but increased creatine. Further studies demonstrated that no significant differences of S1P level in either kidney or renal macrophages between Ang II-infused controls and eSphK1-/- mice, ruling out the possibility of difference of kidney or macrophage S1P levels responsible for renal M2 macrophage polarization in Ang II-infused eSphK1-/- mice. Instead, we revealed that reduced O2 offload from erythrocytes was previously unrecognized but essential to promote tissue M2 polarization and fibrosis by upregulating creatine shuttle to meet rapid intracellular energy needs when FAs, AAs and O2 were insufficient due to hypoxia in Ang II-infused eSphK1-/- mice. At cellular and molecular level, we demonstrated that hypoxia signaling via HIF-1a directly induced M2 polarization as well as expression of Slc6a8 (a gene encoding creatine transport), M2 specific marker gene and fibrotic marker gene in cultured murine BMDMs. Overall, our work has revealed that erythrocyte S1P combats HIF1α-dependent creatine shuttle, tissue macrophage polarization and fibrosis and highlighted potential diagnostic and therapeutic possibilities for macrophage polarization and tissue fibrosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Plasma S1P and Sphingosine are not Different Prior to Pre-Eclampsia in Women at High Risk of Developing the Disease

Sphingolipids like sphingosine-1-phosphate (S1P) have been implicated in the pathophysiology of pre-eclampsia. We hypothesized that plasma S1P would be increased in women at high risk of developing pre-eclampsia who subsequently develop the disease. Low circulating placental growth factor (PlGF) is known to be associated with development of pre-eclampsia; so further, we hypothesized that increased S1P would be associated with concurrently low PlGF. This was a case-control study using stored maternal blood samples from 14 to 24 weeks of pregnancy, collected from 95 women at increased risk of pre-eclampsia. Pregnancy outcome was classified as uncomplicated, preterm pre-eclampsia (<37 weeks), or term pre-eclampsia. Plasma lipids were extracted and analyzed by ultraperformance liquid chromatography coupled to electrospray ionization MS/MS to determine concentrations of S1P and sphingosine. Median plasma S1P was 0.339 nmol/ml, and median sphingosine was 6.77 nmol/l. There were no differences in the plasma concentrations of S1P or sphingosine in women who subsequently developed pre-eclampsia, no effect of gestational age, fetal sex, ethnicity, or the presence of pre-existing hypertension. There was a correlation between S1P and sphingosine plasma concentration (P < 0.0001). There was no relationship between S1P or sphingosine with PlGF. Previous studies have suggested that plasma S1P may be a biomarker of pre-eclampsia. In our larger study, we failed to demonstrate there are women at high risk of developing the disease. We did not show a relationship with known biomarkers of the disease, suggesting that S1P is unlikely to be a useful predictor of the development of pre-eclampsia later in pregnancy.