Sphingosine-1-phosphate Levels Research Articles

Abstract 3072: BXQ-350: A novel biologic that allosterically activates glucosylcerebrosidase and demonstrates signs of activity in cancer patients

Abstract Background: Ceramides, glucosylceramides and sphingosine-1-phosphate (S1P) are key bioactive signaling molecules. Ceramides are proapoptotic and mitigate chemoresistance while glucosylceramides and S1P promote cancer cell proliferation, activate multiple oncogenic pathways, promote chemoresistance and stimulate a pro-tumoral microenvironment. Many studies have shown a better prognosis and longer survival for patients with high ceramides levels, while high S1P and glucosylceramides levels are associated with shorter survival and worst prognosis. Levels of ceramides, glucosylceramides and S1P are controlled by enzymes involved in sphingolipid metabolism, including glucosylcerebrosidase (Gcase) and sphingosine kinase (Sphk). Methods: BXQ-350 is a nanovesicle of Saposin C, an allosteric activator of enzymes involved in sphingolipid metabolism; preclinically, BXQ-350 was investigated in different in vitro and in vivo models. Clinically, BXQ-350 was investigated in a first-in-human Phase 1 safety dose and escalation study in cancer patients with recurrent advanced solid malignancies (NCT02859857). Results: Preclinical results demonstrated that BXQ-350 allosterically activates Gcase in vitro, in cells and in vivo in a significant and dose-dependent manner; as expected, in vivo, Gcase activation results in lower levels of glucosylceramides, as much as 2/3 lower for picomolar concentrations of BXQ-350. An increase in ceramides and a decrease in S1P are also observed. By impacting glucosylceramides, ceramides and S1P, BXQ-350 rebalances the S1P/ceramide rheostat in favor of an antitumoral state and homeostasis. By lowering S1P and glucosylceramides, BXQ-350 modulates the innate and adaptive immune response and repolarizes macrophages towards the antitumoral M1 phenotype, inhibits differentiation and activity of immunosuppressor of MDSCs, and promotes the proliferation of CD4+ and CD8+ Tcells as well as their activity. Clinical results showed BXQ-350 was safe and well-tolerated (no DLT, no MTD). Also, 13 patients (~17.8% of evaluable patients) had a clinical benefit up to cycle 6 and beyond (PR, SD). Among patients with PFS > 6 months, there were 4 recurrent CRC patients: 1 patient had a PFS of ~12 months, 2 of ~18 months, and 1 is still on study after 6 years. Furthermore, there were signs that BXQ-350 may alleviate symptoms of CIPN as 4 out of 10 patients with chronic CIPN at time of enrollment experienced an improvement of their symptoms. Conclusions: Preclinical and clinical results demonstrated that BXQ-350 activates Gcase and modulates sphingolipid metabolism leading to a novel anticancer MOA that had not been clinically tested. Additional studies are ongoing to further elucidate BXQ-350’s MOA. Citation Format: Gilles H. Tapolsky, Charlie Cruze, Robin Furnish, Michael Gazda, Timothy Stephens, Lexy Stamper, Nikhil Wilkins, Ray Takigiku. BXQ-350: A novel biologic that allosterically activates glucosylcerebrosidase and demonstrates signs of activity in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3072.

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.

Alteration in the number, morphology, function, and metabolism of erythrocytes in high-altitude polycythemia.

Introduction: High-altitude polycythemia (HAPC) is a common chronic high-altitude disease characterized by significantly increased erythrocyte, hemoglobin (Hb), and hematocrit values and decreased arterial oxygen saturation. The mechanisms underlying HAPC development are unclear; we aimed to investigate this in an HAPC rat model. Methods: Twelve Sprague-Dawley rats were divided into control and HAPC groups. The HAPC group was exposed to hypobaric hypoxia. This HAPC model was assessed using routine blood tests and blood gas analyses. Bone marrow, peripheral blood reticulocytes (RETs), and peripheral blood erythrocyte apoptosis were measured using flow cytometry. Erythrocyte osmotic fragility (EOF) tests were conducted. Abnormal erythrocytes were counted using electron microscopy. Plasma-free hemoglobin, 5'-nucleotidase (CD73), adenosine, erythrocyte cytosolic adenosine, sphingosine-1-phosphate (S1P), and 2,3-bisphosphoglycerate (BPG) levels were measured using enzyme-linked immunosorbent assays. Erythrocyte metabolic pathway-related protein [adenosine A2B receptor (ADORA2B), erythrocyte equilibrative nucleoside transporter 1 (eENT1), sphingosine kinase 1 (SPHK1), phospho-SPHK1, bisphosphoglycerate mutase (BPGM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] levels were assessed by Western blotting. Results: The HAPC rat model was successfully established (Hb > 210g/L). Indices of bone marrow and peripheral blood RET proportions were significantly higher in the HAPC than the control group (p = 0.04 and p < 0.001, respectively). The proportion of peripheral blood erythrocytes in early apoptosis was significantly lower in the HAPC than the control group (p < 0.001). Vesicular erythrocyte and acanthocyte proportions were significantly higher in the HAPC than the control group (p < 0.001 and p = 0.019, respectively). The EOF tests revealed that 50% erythrocyte hemolysis occurred at 4.0-4.5 and 4.5-5.0g/L NaCl in the control and HAPC groups, respectively. Plasma-free hemoglobin, CD73, adenosine, erythrocyte cytosolic adenosine, S1P, and 2,3-BPG levels and ADORA2B, eENT1, phospho-SPHK1, S1P, BPGM, and GAPDH erythrocyte expression levels (all p ≤ 0.02) were significantly higher in the HAPC than the control group. Conclusion: In model rats, an HAPC-related erythrocyte increase was associated with enhanced bone marrow hematopoietic function and reduced erythrocyte apoptosis, whereas numerous abnormal erythrocytes, increased EOF, and reduced hemolysis resistance were associated with erythrocyte metabolism. CD73/adenosine/S1P/2,3-BPG and eENT1/adenosine/BPGM/2,3-BPG metabolic pathways in erythrocytes were activated in HAPC rats, facilitating oxygen release. These findings further reveal the intrinsic HAPC mechanism and forms a basis for future development of preventive and therapeutic strategies for HAPC.

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-β signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-β signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1β as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

SPHK1/S1P/S1PR pathway promotes the progression of peritoneal fibrosis by mesothelial-mesenchymal transition.

Long-term exposure to non-physiologically compatible dialysate inevitably leads to peritoneal fibrosis (PF) in patients undergoing peritoneal dialysis (PD), and there is no effective prevention or treatment for PF. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid produced after catalysis by sphingosine kinase (SPHK) 1/2 and activates signals through the S1P receptor (S1PR) via autocrine or paracrine. However, the role of SPHK1/S1P/S1PR signaling has never been elucidated in PF. In our research, we investigated S1P levels in peritoneal effluents and demonstrated the role of SPHK1/S1P/S1PR pathway in peritoneal fibrosis. It was found that S1P levels in peritoneal effluents were positively correlated with D/P Cr (r = 0.724, p < .001) and negatively correlated with 4 h ultrafiltration volume (r = -0.457, p < .001). S1PR1 and S1PR3 on peritoneal cells were increased after high glucose exposure invivo and invitro. Fingolimod was applied to suppress S1P/S1PR pathway. Fingolimod restored mouse peritoneal function by reducing interstitial hyperplasia, maintaining ultrafiltration volume, reducing peritoneal transport solute rate, and mitigating the protein expression changes of fibronectin, vimentin, α-SMA, and E-cadherin induced by PD and S1P. Fingolimod preserved the morphology of the human peritoneal mesothelial cells, MeT-5A, and moderated the mesothelial-mesenchymal transition (MMT) process. We further delineated that SPHK1 was elevated in peritoneal cells after high glucose exposure and suppression of SPHK1 in MeT-5A cells reduced S1P release. Overexpression of SPHK1 in MeT-5A cells increased S1P levels in the supernatant and fostered the MMT process. PF-543 treatment, targeting SPHK1, alleviated deterioration of mouse peritoneal function. In conclusion, S1P levels in peritoneal effluent were correlated with the deterioration of peritoneal function. SPHK1/S1P/S1PR pathway played an important role in PF.

Syndecan-1 as a predictor of vulnerable atherosclerotic plaques.

Cardiovascular disease remains a major global health concern, with atherosclerosis (AS) being a significant contributor. Vulnerable plaques play a critical role in acute cardiovascular events. Syndecan-1 (SDC-1), a vital membrane proteoglycan in the vascular endothelial glycocalyx, is believed to be associated with plaque progression. However, its precise relationship with severity and vulnerability of atherosclerotic plaque remains unclear. This study aimed to investigate SDC-1 expression and its potential correlation with plaque vulnerability in ApoE-/- atherosclerosis mouse model. Eight-week-old mice were induced into the AS model using a high-fat diet (HFD) and/or partial ligation of the left common carotid artery (PLCA), with a chow diet (CD) control group. After 16weeks, plaques in the aortic root showed the following order: HFD + PLCA group > HFD group > CD + PLCA group > CD group. Immunohistochemistry revealed heightened accumulation of lipid/foam cells and CD68-labeled macrophages in the plaques, elevated vascular endothelial growth factor (VEGF), and matrix Metalloproteinase-9 (MMP-9) in the HFD + PLCA group's plaques, along with reduced collagen and α-SMA-labeled smooth muscle cells, resulting in the highest vulnerability index value. Immunohistofluorescence analysis of frozen plaque sections showed significantly higher SDC-1 expression in the AS mice group compared to the CD group, both positively correlated with plaque vulnerability. Serum analysis demonstrated elevated levels of SDC1, sphingosine 1-phosphate (S1P), and VEGF-A in the AS mice, all positively correlated with plaque vulnerability. Multivariate analysis identified SDC1 as an independent predictor of plaque vulnerability. This study enhances our understanding of plaque vulnerability mechanisms and presents SDC1 as a potential biomarker for atherosclerosis. These findings underscore the importance of addressing modifiable risk factors, such as diet and hemodynamics and suggest the utility of serum SDC1 as a valuable clinical marker. Ultimately, these insights may lead to more effective strategies in combating cardiovascular diseases and improving patient outcomes.

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.

Overexpression of ORMDL3 confers sexual dimorphism in diet-induced non-alcoholic steatohepatitis

ObjectiveThe bioactive sphingolipid metabolites ceramide and sphingosine-1-phosphate (S1P) accumulate with overnutrition and have been implicated in non-alcoholic steatohepatitis (NASH) development. ORMDL3, a negative regulator of the rate-limiting step in ceramide biosynthesis, has been identified as an obesity-related gene. Therefore, we assessed the role of ORMDL3 in diet-induced obesity and development of NASH. MethodsGlobally overexpressing Ormdl3-Flag transgenic mice (ORMDL3TG) were fed a western high-fat, carbohydrate and cholesterol enriched diet, with high fructose-glucose drinking water. Physiological, biochemical and sphingolipidomic analyses were employed to measure the effect of ORMDL3 overexpression on NASH development. ResultsORMDL3TG male but not female mice fed a western high-fat diet and sugar water had exacerbated adipocyte hypertrophy together with increased severity of white adipose inflammation and fibrosis. Hepatic steatosis, dyslipidemia, impaired glucose homeostasis, hyperinsulinemia, and insulin resistance were significantly more severe only in obese ORMDL3TG male mice that accompanied dramatic liver fibrosis, inflammation, and formation of hepatic crown-like structures, which are unique features of human and murine NASH. Obesogenic diet induces ORMDL expression in male mice but reduces it in females. Mechanistically, overexpression of Ormdl3 lowered the levels of S1P and ceramides only in obese female mice and antithetically increased them in tissues of obese males. ORMDL3TG male mice exhibited a much greater induction of the UPR, propagating ER stress that contributed to their early development of NASH. ConclusionsThis study uncovered a previously unrecognized role for ORMDL3 in sexual dimorphism important for the development and progression of NASH.

The role of vitamin D through SphK1/S1P in the regulation of MS progression

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-β, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-β, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

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.

Pro-inflammatory sensitization of platelets after a mild course of COVID-19 infection

Abstract Background Coronavirus disease-2019 (COVID-19) is a viral respiratory illness caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) which can lead to an increased risk of thromboembolic events but also complications even after recovery from the disease. Multiple factors may enhance the risk of thrombotic events in COVID-19 patients including excessive inflammatory responses and increased platelet activity. To date there are hardly any data on how platelet function is affected after a mild course of infection. Purpose In this study, we investigated whether platelet function is affected after recovery from mild COVID-19 disease. Besides the response of platelets to platelet stimuli, expression of the transporter protein multidrug resistance protein 4 (MRP4) and one of its substrates, the inflammatory lipid mediator sphingosine-1-phosphate (S1P), were measured. Methods Venous blood samples were collected from patients within 2-15 weeks after recovery from a mild SARS-CoV-2 infection (recovered) and matched individuals (controls) after written informed consent. Participants were invited again at 6-9 months after infection to reexamine platelet function. Parameters of platelet aggregation were determined by light transmission aggregometry (LTA) in platelet-rich plasma (PRP). Expression of platelet activation markers were measured by Flow Cytometry (FACS). In addition, concentrations of S1P were determined by LC-MS/MS and MRP4-expression in platelets via Western blotting. Results Within 2-15 weeks after recovery, no alterations in platelet aggregation were observed after stimulation of PRP with various ADP (1, 2, and 10 µM) or collagen (1 and 2.5 µg/mL) concentrations. However, regarding FACS analysis, platelet surface expression of CD62P and PAC1 from convalescents prior activation by ADP (2 µM) and by CRP-XL (0.125 ng/mL) was significantly upregulated in comparison to samples from controls (p&amp;lt;0.05 for both). Intriguingly, S1P levels in platelet-poor plasma (PPP) from COVID-19 convalescents were significantly elevated compared to controls, i.e. 0.75±0.01 vs 0.65±0.02 nmol/mL, respectively (p&amp;lt;0.001). S1P in PRP and serum samples was not altered. Furthermore, platelet MRP4 expression was significantly elevated in COVID-19 convalescents (p&amp;lt;0.05). These differences in S1P and MRP4 levels were only present after 2-15 weeks of recovery, but not 6-9 months after infection. Conclusions While after a mild course of COVID-19 infection robust platelet functions such as aggregation determined by LTA were not affected, subtle differences in platelet activation markers occurred. Enhanced platelet MRP4 expression, which may correlate with the observed elevated S1P in PPP, are in agreement with a pro-inflammatory sensitization of platelets even after a mild COVID-19 infection. The implications of this observation concerning long-term effects of COVID-19 await further studies.

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.

Interpetrosal sphingosine-1-phosphate ratio predicting Cushing's disease tumor laterality and remission after surgery.

Cushing's disease (CD) poses significant challenges in its treatment due to the lack of reliable biomarkers for predicting tumor localization or postoperative clinical outcomes. Sphingosine-1-phosphate (S1P) has been shown to increase cortisol biosynthesis and is regulated by adrenocorticotropic hormone (ACTH). We employed bilateral inferior petrosal sinus sampling (BIPSS), which is considered the gold standard for diagnosing pituitary sources of CD, to obtain blood samples and explore the clinical predictive value of the S1P concentration ratio in determining tumor laterality and postoperative remission. We evaluated 50 samples from 25 patients who underwent BIPSS to measure S1P levels in the inferior petrosal sinuses bilaterally. Serum S1P levels in patients with CD were significantly higher on the adenoma side of the inferior petrosal sinus than on the nonadenoma side (397.7 ± 15.4 vs. 261.9 ± 14.88; P < 0.05). The accuracy of diagnosing tumor laterality with the interpetrosal S1P and ACTH ratios and the combination of the two was 64%, 56% and 73%, respectively. The receiver operating characteristic curve analysis revealed that the combination of interpetrosal S1P and ACTH ratios, as a predictor of tumor laterality, exhibited a sensitivity of 81.82% and a specificity of 75%, with an area under the curve value of 84.09%. Moreover, we observed that a high interpetrosal S1P ratio was associated with nonremission after surgery. Correlation analyses demonstrated that the interpetrosal S1P ratio was associated with preoperative follicle-stimulating hormone (FSH), luteinizing hormone (LH), and postoperative ACTH 8 am levels (P < 0.05). Our study demonstrated a significant association between the interpetrosal S1P ratio and tumor laterality, as well as postoperative remission in CD, suggesting that the interpetrosal S1P ratio could serve as a valuable biomarker in clinical practice.

Targeting the SPHK1/S1P/S1PR2 axis ameliorates GH-secreted pituitary adenoma progression.

Growth hormone-secreted pituitary adenoma (GHPA) is a prominent subtype of pituitary adenoma (PA) associated with progressive somatic disfigurement, various complications, and elevated mortality rates. Existing treatment options have limited efficacy, highlighting the urgent need for novel pharmacological interventions. Previous studies have revealed that sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptors (S1PRs) signalling have critical roles in the tumour microenvironment, but their role in GHPA remains unclear. We performed integrative analyses including bioinformatics analyses, functional studies, and clinical validation to investigate the pathological roles of SPHK1/S1P and evaluated the effectiveness of the S1P receptor 2 (S1PR2) inhibitor JTE-013 in GHPA treatment. SPHK1/S1P signalling is abnormally expressed in patients with GHPA. Knockdown of SPHK1 suppresses S1P-mediated cell proliferation in GH3 Cells. Mechanistically, S1P inhibits apoptosis and autophagy while promoting the secretion of Growth Hormone (GH) by binding to the S1P receptor subtype 2 (S1PR2) in GH3 cells. Moreover, the function of S1PR2 in GH3 cells is mediated by the downstream Akt-Creb pathway. We then identify the S1PR2 as a novel target for therapeutic intervention in GHPA. Systemic administration of the potent and selective S1PR2 antagonist, JTE-013, significantly reduces both tumour size and GH secretion. Importantly, we identify preoperative serum S1P levels as a biomarker predicting poor prognosis in GHPA patients at follow-up. Our study shows that blocking SPHK1/S1P/S1PR2 axis can ameliorate the progression of GHPA, providing evidence of a promising therapeutic target for GHPA.

Farnesoid X receptor prevents neutrophil extracellular traps via reduced sphingosine-1-phosphate in chronic kidney disease.

Farnesoid X receptor (FXR) activation reduces renal inflammation, but the underlying mechanisms remain elusive. Neutrophil extracellular traps (NETs) are webs of DNA formed when neutrophils undergo specialized programmed cell death (NETosis). The signaling lipid sphingosine-1-phosphate (S1P) stimulates NETosis via its receptor on neutrophils. Here, we identify FXR as a negative regulator of NETosis via repressing S1P signaling. We determined the effects of the FXR agonist obeticholic acid (OCA) in mouse models of adenosine phosphoribosyltransferase (APRT) deficiency and Alport syndrome, both genetic disorders that cause chronic kidney disease. Renal FXR activity is greatly reduced in both models, and FXR agonism reduces disease severity. Renal NETosis and sphingosine kinase 1 (Sphk1) expression are increased in diseased mice, and they are reduced by OCA in both models. Genetic deletion of FXR increases Sphk1 expression, and Sphk1 expression correlates with NETosis. Importantly, kidney S1P levels in Alport mice are two-fold higher than controls, and FXR agonism restores them back to baseline. Short-term inhibition of sphingosine synthesis in Alport mice with severe kidney disease reverses NETosis, establishing a causal relationship between S1P signaling and renal NETosis. Finally, extensive NETosis is present in human Alport kidney biopsies (six male, nine female), and NETosis severity correlates with clinical markers of kidney disease. This suggests the potential clinical relevance of the newly identified FXR-S1P-NETosis pathway. In summary, FXR agonism represses kidney Sphk1 expression. This inhibits renal S1P signaling, thereby reducing neutrophilic inflammation and NETosis.NEW & NOTEWORTHY Many preclinical studies have shown that the farnesoid X receptor (FXR) reduces renal inflammation, but the mechanism is poorly understood. This report identifies FXR as a novel regulator of neutrophilic inflammation and NETosis via the inhibition of sphingosine-1-phosphate signaling. Additionally, NETosis severity in human Alport kidney biopsies correlates with clinical markers of kidney disease. A better understanding of this signaling axis may lead to novel treatments that prevent renal inflammation and chronic kidney disease.

Density of Sphingosine-1-Phosphate Receptors Is Altered in Cortical Nerve-Terminals of Insulin-Resistant Goto-Kakizaki Rats and Diet-Induced Obese Mice

Sphingosine-1-phosphate (S1P) is a phosphosphingolipid with pleiotropic biological functions. S1P acts as an intracellular second messenger, as well as extracellular ligand to five G-protein coupled receptors (S1PR1-5). In the brain, S1P regulates neuronal proliferation, apoptosis, synaptic activity and neuroglia activation. Moreover, S1P metabolism alterations have been reported in neurodegenerative disorders. We have previously reported that S1PRs are present in nerve terminals, exhibiting distinct sub-synaptic localization and neuromodulation actions. Since type 2 diabetes (T2D) causes synaptic dysfunction, we hypothesized that S1P signaling is modified in nerve terminals. In this study, we determined the density of S1PRs in cortical synaptosomes from insulin-resistant Goto-Kakizaki (GK) rats and Wistar controls, and from mice fed a high-fat diet (HFD) and low-fat-fed controls. Relative to their controls, GK rats showed similar cortical S1P concentration despite higher S1P levels in plasma, yet lower density of S1PR1, S1PR2 and S1PR4 in nerve-terminal-enriched membranes. HFD-fed mice exhibited increased plasma and cortical concentrations of S1P, and decreased density of S1PR1 and S1PR4. These findings point towards altered S1P signaling in synapses of insulin resistance and diet-induced obesity models, suggesting a role of S1P signaling in T2D-associated synaptic dysfunction.