FTY720/Fingolimod mitigates paclitaxel-induced Sparcl1-driven neuropathic pain and breast cancer progression.

Paclitaxel is among the most active chemotherapy drugs for the aggressive triple negative breast cancer (TNBC). Unfortunately, it often induces painful peripheral neuropathy (CIPN), a major debilitating side effect. Here we demonstrate that in naive and breast tumor-bearing immunocompetent mice, a clinically relevant dose of FTY720/Fingolimod that targets sphingosine-1-phosphate receptor 1 (S1PR1), alleviated paclitaxel-induced neuropathic pain. FTY720 also significantly attenuated paclitaxel-stimulated glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and expression of the astrocyte-secreted synaptogenic protein Sparcl1/Hevin, a key regulator of synapse formation. Notably, the formation of excitatory synapses containing VGluT2 in the spinal cord dorsal horn induced by paclitaxel was also inhibited by FTY720 treatment, supporting the involvement of astrocytes and Sparcl1 in CIPN. Furthermore, in this TNBC mouse model that mimics human breast cancer, FTY720 administration also enhanced the anti-tumor effects of paclitaxel, leading to reduced tumor progression and lung metastasis. Taken together, our findings suggest that targeting the S1P/S1PR1 axis with FTY720 is a multipronged approach that holds promise as a therapeutic strategy for alleviating both CIPN and enhancing the efficacy of chemotherapy in TNBC treatment.

Efficacy and Safety of Sphingosine 1-Phosphate Receptor Modulators for Ulcerative Colitis: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

BACKGROUND AND AIMS: Sphingosine 1-phosphate receptor modulators (S1PRMs) are an effective treatment for ulcerative colitis (UC). This review summarizes all available randomized trial data on the efficacy and safety of S1PRM therapy. METHODS: Multiple publication databases were systematically searched for randomized control trials (RCTs) of adults with moderate to severe UC treated with S1PRMs. Random effects meta-analysis was performed. The risk of bias was assessed using the Cochrane Risk-of-Bias 2 tool, and the overall quality of evidence was rated using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) approach. RESULTS: We identified 7 RCTs (1737 patients) involving the use of S1PRMs for moderate to severe UC. During induction, S1PRM therapy was efficacious when compared with placebo for clinical remission [RR: 2.65 (95% CI: 2.00, 3.53)], clinical response [RR: 1.68 (95% CI: 1.48, 1.91)], endoscopic improvement [RR: 2.17 (95% CI: 1.76, 2.68)], endoscopic normalization [RR: 2.56 (95% CI: 1.58, 3.83)], mucosal healing [RR: 2.88 (95% CI: 1.94, 4.26)], and histologic remission [RR: 2.42 (95% CI: 1.60, 3.66)]. Similar results were seen throughout the maintenance peroid, although fewer data were available to pool; notably, both sustained [RR: 3.57 (95% CI: 1.23, 10.35)] and steroid-free [RR: 2.92 (95% CI: 1.35, 6.33)] remission were significantly increased by S1PRM. There were no significant differences in adverse events [RR: 1.02 (95% CI: 0.90, 1.15)] and infections [RR: 1.15 (95% CI: 0.82, 1.60)] between S1PRM and placebo. CONCLUSION: Pooling of RCT data confirms that S1PRM therapy is both effective and safe for patients with moderate to severe UC.

Taurine-conjugated bile acids and their link to hepatic S1PR2 play a significant role in hepatitis C-related liver disease.

BACKGROUND: Bile acids mediate gut-liver cross-talk through bile acid receptors. Serum, hepatic, and microbial bile acid metabolism was evaluated in HCV-compensated chronic liver disease. METHODS: Patients underwent liver biopsy; portal and peripheral blood were obtained before (HCVi), and 6 months after sustained virologic response (SVR), splenic blood was obtained only after SVR. The fecal microbiome and liver transcriptome were evaluated using RNA-Seq. Twenty-four bile acids were measured in serum, summed as free, taurine-conjugated bile acids (Tau-BAs), and glycine-conjugated bile acids. RESULTS: Compared to SVR, HCVi showed elevated conjugated bile acids, predominantly Tau-BA, compounded in HCVi cirrhosis. In the liver, transcription of bile acids uptake, synthesis, and conjugation was decreased with increased hepatic spillover into systemic circulation in HCVi. There was no difference in the transcription of microbial bile acid metabolizing genes in HCVi. Despite an overall decrease, Tau-BA remained elevated in SVR cirrhosis, mainly in splenic circulation. Only conjugated bile acids, predominantly Tau-BA, correlated with serum proinflammatory markers and hepatic proinflammatory pathways, including NLRP3 and NFKB. Among hepatic bile acid receptors, disease-associated conjugated bile acids showed the strongest association with hepatic spingosine-1-phosphate receptor 2 (S1PR2). CONCLUSIONS: Enhanced expression of hepatic S1PR2 in HCVi and HCVi-cirrhosis and strong associations of S1PR2 with Tau-BAs suggest pathological relevance of Tau-BA-hepatic S1PR2 signaling in chronic liver disease. These findings have therapeutic implications in chronic liver diseases.

Analysis of Lipid GPCR Molecular Interactions by Proximity Labeling.

G-protein-coupled receptors (GPCRs) are hepta-helical transmembrane proteins that mediate various intracellular signaling events in response to their specific ligands including many lipid mediators. Although analyses of GPCR molecular interactions are pivotal to understanding diverse intracellular signaling events, affinity purification of interacting proteins by a conventional co-immunoprecipitation method is challenging due to the hydrophobic nature of GPCRs and their dynamic molecular interactions. Proximity labeling catalyzed by a TurboID system is a powerful technique for defining the molecular interactions of target proteins in living cells. TurboID and miniTurbo (a modified version of TurboID) are engineered biotin ligases that biotinylate neighboring proteins in a promiscuous manner. When fused with a target protein and expressed in living cells, TurboID or miniTurbo mediates the biotin labeling of the proteins with close proximity to the target protein, allowing efficient purification of the biotinylated proteins followed by a shot-gun proteomic analysis. In this chapter, we describe a step-by-step protocol for the labeling of GPCR neighboring proteins by TurboID or miniTurbo, purification of the biotin-labeled proteins, and subsequent sample preparation for proteomic analysis. We utilized S1PR1 as a model GPCR, a receptor for a bioactive lipid molecule sphingosine 1-phosphate (S1P) that plays various roles in physiological and pathological conditions. This analysis pipeline enables the mapping of interacting proteins of lipid GPCRs in living cells.

Icanbelimod (CBP-307), a next-generation Sphingosine-1-phosphate receptor modulator, in healthy men: pharmacokinetics, pharmacodynamics, safety, and tolerability in a randomized trial in Australia.

Background: Icanbelimod (formerly CBP-307) is a next-generation S1PR modulator, targeting S1PR1. In this first-in-human study, icanbelimod was investigated in healthy men in Australia. Methods: Participants were randomized 3:1, double-blind, to icanbelimod or placebo in four single-dose cohorts (0.1 mg, 0.25 mg, 0.5 mg [n=8 per cohort], 2.5 mg [n=4]) or for 28-days once-daily treatment in two cohorts (0.15 mg, 0.25 mg [n=8 per cohort]). Participants in the 0.25-mg cohort received 0.1 mg on Day 1. Treatments were administered orally after fasting; following one-week washout, icanbelimod was administered after breakfast in the 0.5-mg cohort. Results: Icanbelimod exposure increased rapidly and dose-dependently with single and multiple dosing (Tmax 4-7 hours). Lymphocyte counts decreased rapidly after single (-11%, 0.1 mg; -40%, 0.25 mg; -71%, 0.5 mg; -77%, 2.5 mg) and multiple doses (-49%, 0.15 mg; -75%, 0.25 mg), and recovered quickly, 7 days after dosing. After single-dose 0.5 mg, although a high-fat breakfast versus fasting did not affect maximal decrease, lymphocyte counts tended to be lower after breakfast across most timepoints up to 72 hours. Twenty-eight participants (63.6%) experienced mainly mild treatment-emergent adverse events (TEAEs). After single-dose icanbelimod, the most common TEAEs were headache (28.6%, n=6) and dizziness (19.0%, n=4). Three participants experienced transient bradycardia, with one serious, following single-dose 2.5 mg icanbelimod. After multiple-dose icanbelimod, the most common TEAEs were headache (50.0%, n=6) and lymphopenia (41.7%, n=5), and two participants withdrew due to non-serious TEAEs. Up-titration attenuated heart rate reductions. Conclusion: Icanbelimod was well-tolerated up to 0.5 mg and effectively reduced lymphocyte counts. Clinical trial registration: ClinicalTrials.gov, identifier NCT02280434.b.

Transplantation of miR-193b-3p-Transfected BMSCs Improves Neurological Impairment after Traumatic Brain Injury through S1PR3-Mediated Regulation of the PI3K/AKT/mTOR Signaling Pathway.

The aim of the present study was to explore the molecular mechanisms by which miR-193b-3p-trans-fected bone marrow mesenchymal stem cells (BMSCs) transplantation improves neurological impairment after traumatic brain injury (TBI) through sphingosine-1-phosphate receptor 3 (S1PR3)-mediated regulation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway at the cellular and animal levels. BMSCs were transfected with miR-193b-3p. A TBI cell model was established by oxygen-glucose deprivation (OGD)-induced HT22 cells, and a TBI animal model was established by controlled cortical impact (CCI). Cell apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL), and cell activity was detected by a cell counting kit 8 (CCK-8) assay. Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of related proteins and genes. In this study, transfection of miR-193b-3p into BMSCs significantly enhanced BMSCs proliferation and differentiation. Transfection of miR-193b-3p reduced the levels of the interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-alpha (TNF-α) inflammatory factors in cells and mouse models, and it inhibited neuronal apoptosis, which alleviated OGD-induced HT22 cell damage and neural function damage in TBI mice. Downstream experiments showed that miR-193b-3p targeting negatively regulated the expression of S1PR3, promoted the activation of the PI3K/AKT/mTOR signaling pathway, and inhibited the levels of apoptosis and inflammatory factors, which subsequently improved OGD-induced neuronal cell damage and nerve function damage in TBI mice. However, S1PR3 overexpression or inhibition of the PI3K/AKT/mTOR signaling pathway using the IN-2 inhibitor weakened the protective effect of miR-193b-3p-transfected BMSCs on HT22 cells. Transplantation of miR-193b-3p-transfected BMSCs inhibits neurological injury and improves the progression of TBI in mice through S1PR3-mediated regulation of the PI3K/AKT/mTOR pathway.

Exploring the interactions of JAK inhibitor and S1P receptor modulator drugs with the human gut microbiome: Implications for colonic drug delivery and inflammatory bowel disease.

The gut microbiota is a complex ecosystem, home to hundreds of bacterial species and a vast repository of enzymes capable of metabolising a wide range of pharmaceuticals. Several drugs have been shown to affect negatively the composition and function of the gut microbial ecosystem. Janus Kinase (JAK) inhibitors and Sphingosine-1-phosphate (S1P) receptor modulators are drugs recently approved for inflammatory bowel disease through an immediate release formulation and would potentially benefit from colonic targeted delivery to enhance the local drug concentration at the diseased site. However, their impact on the human gut microbiota and susceptibility to bacterial metabolism remain unexplored. With the use of calorimetric, optical density measurements, and metagenomics next-generation sequencing, we show that JAK inhibitors (tofacitinib citrate, baricitinib, filgotinib) have a minor impact on the composition of the human gut microbiota, while ozanimod exerts a significant antimicrobial effect, leading to a prevalence of the Enterococcus genus and a markedly different metabolic landscape when compared to the untreated microbiota. Moreover, ozanimod, unlike the JAK inhibitors, is the only drug subject to enzymatic degradation by the human gut microbiota sourced from six healthy donors. Overall, given the crucial role of the gut microbiome in health, screening assays to investigate the interaction of drugs with the microbiota should be encouraged for the pharmaceutical industry as a standard in the drug discovery and development process.

Pseudoirreversible inhibition elicits persistent efficacy of a sphingosine 1-phosphate receptor 1 antagonist.

Sphingosine 1-phosphate receptor 1 (S1PR1), a G protein-coupled receptor, is required for lymphocyte trafficking, and is a promising therapeutic target in inflammatory diseases. Here, we synthesize a competitive S1PR1 antagonist, KSI-6666, that effectively suppresses pathogenic inflammation. Metadynamics simulations suggest that the interaction of KSI-6666 with a methionine residue Met124 in the ligand-binding pocket of S1PR1 may inhibit the dissociation of KSI-6666 from S1PR1. Consistently, in vitro functional and mutational analyses reveal that KSI-6666 causes pseudoirreversible inhibition of S1PR1, dependent on the Met124 of the protein and substituents on the distal benzene ring of KSI-6666. Moreover, in vivo study suggests that this pseudoirreversible inhibition is responsible for the persistent activity of KSI-6666.

Design, synthesis, and biological evaluation of multiple F-18 S1PR1 radiotracers in rodent and nonhuman primate.

Here we report our design and synthesis of 28 new fluorine-containing compounds as potential F-18 radiotracers for CNS imaging of sphingosine-1-phosphate receptor 1 (S1PR1), and determination of their in vitro binding potency and selectivity toward S1PR1 over other S1PR subtypes. Nine potent and selective compounds, 7c&d, 9a&c, 12b, 15b, and 18a-c with IC50 values ranging from 0.6-12.3 nM for S1PR1 and weak binding toward S1PR2, 3, 4, and 5, were further 18F-radiolabeled to produce [18F]7c&d, [18F]9a&c, [18F]12b, [18F]15b, and [18F]18a-c. Multi-step F-18 radiochemistry procedures were investigated for radiosynthesis of [18F]7c&d and [18F]9a&c, and the presumed intermediates were synthesized and authenticated by analytic HPLC. We then performed nonhuman primate (NHP) PET brain imaging studies for eight radiotracers: [18F]7c&d, [18F]9a, [18F]12b, [18F]15b, and [18F]18a-c. Three radiotracers, [18F]7c, [18F]7d, and [18F]15b, had high NHP brain uptake with standardized uptake values (SUVs) at 2 h post-injection of 2.42, 2.84, and 2.00, respectively, and good brain retention. Our ex vivo biodistribution study in rats confirmed [18F]7d had a high brain uptake with no in vivo defluorination. Radiometabolic analysis of [18F]7c and [18F]7d in rat plasma and brain samples found that [18F]7c has a more favorable metabolic profile than [18F]7d. However, the trend of increased brain uptake precludes [18F]7c as a suitable PET radiotracer for imaging S1PR1 in the brain. Further structural optmization is warranted to identify a highly S1PR1-specific radiotracer with rapid brain uptake kinetics.

S1PR3 inhibition protects against LPS-induced ARDS by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation.

BACKGROUND: Inflammation and endothelial barrier dysfunction are the major pathophysiological changes in acute respiratory distress syndrome (ARDS). Sphingosine-1-phosphate receptor 3 (S1PR3), a G protein-coupled receptor, has been found to mediate inflammation and endothelial cell (EC) integrity. However, the function of S1PR3 in ARDS has not been fully elucidated. METHODS: We used a murine lipopolysaccharide (LPS)-induced ARDS model and an LPS- stimulated ECs model to investigate the role of S1PR3 in anti-inflammatory effects and endothelial barrier protection during ARDS. RESULTS: We found that S1PR3 expression was increased in the lung tissues of mice with LPS-induced ARDS. TY-52156, a selective S1PR3 inhibitor, effectively attenuated LPS-induced inflammation by suppressing the expression of proinflammatory cytokines and restored the endothelial barrier by repairing adherens junctions and reducing vascular leakage. S1PR3 inhibition was achieved by an adeno-associated virus in vivo and a small interfering RNA in vitro. Both the in vivo and in vitro studies demonstrated that pharmacological or genetic inhibition of S1PR3 protected against ARDS by inhibiting the NF-κB pathway and improving mitochondrial oxidative phosphorylation. CONCLUSIONS: S1PR3 inhibition protects against LPS-induced ARDS via suppression of pulmonary inflammation and promotion of the endothelial barrier by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation, indicating that S1PR3 is a potential therapeutic target for ARDS.

Endothelial cell sphingosine 1-phosphate receptor 1 restrains VE-cadherin cleavage and attenuates experimental inflammatory arthritis.

In rheumatoid arthritis, inflammatory mediators extravasate from blood into joints via gaps between endothelial cells (ECs), but the contribution of ECs is not known. Sphingosine 1-phosphate receptor 1 (S1PR1), widely expressed on ECs, maintains the vascular barrier. Here, we assessed the contribution of vascular integrity and EC S1PR1 signaling to joint damage in mice exposed to serum-induced arthritis (SIA). EC-specific deletion of S1PR1 or pharmacological blockade of S1PR1 promoted vascular leak and amplified SIA, whereas overexpression of EC S1PR1 or treatment with an S1PR1 agonist delayed SIA. Blockade of EC S1PR1 induced membrane metalloproteinase-dependent cleavage of vascular endothelial cadherin (VE-cadherin), a principal adhesion molecule that maintains EC junctional integrity. We identified a disintegrin and a metalloproteinase domain 10 (ADAM10) as the principal VE-cadherin "sheddase." Mice expressing a stabilized VE-cadherin construct had decreased extravascular VE-cadherin and vascular leakage in response to S1PR1 blockade, and they were protected from SIA. Importantly, patients with active rheumatoid arthritis had decreased circulating S1P and microvascular expression of S1PR1, suggesting a dysregulated S1P/S1PR1 axis favoring vascular permeability and vulnerability. We present a model in which EC S1PR1 signaling maintains homeostatic vascular barrier function by limiting VE-cadherin shedding mediated by ADAM10 and suggest this signaling axis as a therapeutic target in inflammatory arthritis.

Bile acid-sensitive human norovirus strains are susceptible to sphingosine-1-phosphate receptor 2 inhibition.

Human noroviruses (HuNoVs) are a diverse group of RNA viruses that cause endemic and pandemic acute viral gastroenteritis. Previously, we reported that many HuNoV strains require bile or bile acid (BA) to infect human jejunal intestinal enteroid cultures. BA was not essential for the replication of a pandemic-causing GII.4 HuNoV strain. We found the hydrophobic BA glycochenodeoxycholic acid (GCDCA) promotes the replication of the BA-dependent strain GII.3 in jejunal enteroids. Furthermore, we found that inhibition of the G-protein-coupled BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), by JTE-013, reduced GII.3 infection dose-dependently and inhibited GII.3 cellular uptake in enteroids. Herein, we sought to determine whether S1PR2 is required for other BA-dependent HuNoV strains, the BA-independent GII.4, and whether S1PR2 is required for BA-dependent HuNoV infection in HIEs from other small intestinal segments. We found a second S1PR2 inhibitor, GLPG2938, reduces GII.3 infection dose-dependently, and an S1PR2 agonist (CYM-5520) enhances GII.3 replication in the absence of GCDCA. GII.3 replication also is abrogated in the presence of JTE-013 and CYM-5520. JTE-013 inhibition of S1PR2 in jejunal HIEs reduces GI.1, GII.3, and GII.17 (BA-dependent) but not GII.4 Sydney (BA-independent) infection, providing additional evidence of strain-specific differences in HuNoV infection. Finally, GII.3 infection of duodenal, jejunal, and ileal lines derived from the same individual is reduced with S1PR2 inhibition, indicating a common mechanism of BA-dependent infection among multiple segments of the small intestine. Our results support a model where BA-dependent HuNoVs exploit BA effects on S1PR2 to infect the entire small intestine.IMPORTANCEHuman noroviruses (HuNoVs) are important viral human pathogens that cause both outbreaks and sporadic gastroenteritis. These viruses are diverse, and many strains are capable of infecting humans. Our previous studies have identified strain-specific requirements for hydrophobic bile acids (BAs) to infect intestinal epithelial cells. Moreover, we identified a BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), required for infection by a BA-dependent strain. To better understand how various HuNoV strains enter and infect the small intestine and the role of S1PR2 in HuNoV infection, we evaluated infection by additional HuNoV strains using an expanded repertoire of intestinal enteroid cell lines. We found that multiple BA-dependent strains, but not a BA-independent strain, all require S1PR2 for infection. In addition, BA-dependent infection requires S1PR2 in multiple segments of the small intestine. Together, these results indicate that S1PR2 has value as a potential therapeutic target for BA-dependent HuNoV infection.

Microglia-mediated pericytes migration and fibroblast transition via S1P/S1P3/YAP signaling pathway after spinal cord injury.

Platelet-derived growth factor receptor ß positive (PDGFRß+) pericytes detach from the microvascular wall and migrate into the injury center following spinal cord injury (SCI), which has been widely regarded as the main source of fibrotic scar, but the mechanism of migration and fibroblast transition remains elusive. Here we show the associated spatiotemporal distribution between microglia and pericytes at three and seven days post-injury (dpi). The increased expression of Sphingosine kinase-1 (SPHK1) in microglia significantly raised the concentration of Sphingosine-1-phosphate (S1P) in the spinal cord, which promotes migration and fibroblast transition of pericyte. In vitro experiments, we found the elevated Sphingosine 1-phosphate receptor 3 (S1P3), the S1P/S1PR3 axis inhibited the phosphorylation of YAP and promoted its nuclear translocation, which contributed to the formation of alpha-smooth muscle actin (α-SMA) and collagen type I (COL1) protein, This process can be blocked by an S1P3 specific inhibitor TY52156 in vitro. The S1P/S1P3/YAP pathway might be a potential target for treatment in SCI.

Kaempferol mitigates sepsis-induced acute lung injury by modulating the SphK1/S1P/S1PR1/MLC2 signaling pathway to restore the integrity of the pulmonary endothelial cell barrier.

Sepsis-induced acute lung injury (SALI) is the common complication of sepsis, resulting in high incidence and mortality rates. The primary pathogenesis of SALI is the interplay between acute inflammation and endothelial barrier damage. Studies have shown that kaempferol (KPF) has anti-sepsis properties. Sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) signaling pathway's significance in acute lung damage and S1P receptor 1 (S1PR1) agonists potential in myosin light chain 2 (MLC2) phosphorylation are documented. Whether KPF can regulate the SphK1/S1P/S1PR1/MLC2 signaling pathway to protect the lung endothelial barrier remains unclear. This study investigates the KPF's therapeutic effects and molecular mechanisms in repairing endothelial cell barrier damage in both LPS-induced sepsis mice and human umbilical vein endothelial cells (HUVECs). KPF significantly reduced lung tissue damage and showed anti-inflammatory effects by decreasing IL-6 and TNF-α synthesis in the sepsis mice model. Further, KPF administration can reduce the high permeability of the LPS-induced endothelial cell barrier and alleviate lung endothelial cell barrier injury. Mechanistic studies showed that KPF pretreatment can suppress MLC2 hyperphosphorylation and decrease SphK1, S1P, and S1PR1 levels. The SphK1/S1P/S1PR1/MLC2 signaling pathway controls the downstream proteins linked to endothelial barrier damage, and the Western blot (WB) showed that KPF raised the protein levels. These proteins include zonula occludens (ZO)-1, vascular endothelial (VE)-cadherin and Occludin. The present work revealed that in mice exhibiting sepsis triggered by LPS, KPF strengthened the endothelial barrier and reduced the inflammatory response. The SphK1/S1P/S1PR1/MLC2 pathway's modulation is the mechanism underlying this impact.

SphK1 deficiency ameliorates the development of atherosclerosis by inhibiting the S1P/S1PR3/Rhoa/ROCK pathway.

BACKGROUND AND AIMS: S1P is an important factor regulating the function of the vascular endothelial barrier. SphK1 is an important limiting enzyme for the synthesis of S1P. However, the role of the SphK1/S1P-mediated vascular endothelial barrier function in atherosclerosis has not been fully revealed. This study explored the roles and mechanisms of SphK1 on atherosclerosis in vivo and in vitro. METHODS: In vivo, ApoE-/- and SphK1-/-ApoE-/- mice were fed a high-fat diet to induce atherosclerosis. In vitro, ox-LDL induced HUVECs to establish a cell model. Aortic histological changes were measured by H&E staining, Oil Red O staining, EVG staining, Sirius scarlet staining, immunofluorescence, and Evans Blue Assay. Western blotting was performed to explore the specific mechanism. RESULTS: We validated that deficiency of SphK1 resulted in a marked amelioration of atherosclerosis, as indicated by the decreased lipid accumulation, inflammatory factors, oxidative stress, aortic plaque area, inflammatory factor infiltration, VCAM-1 expression, and vascular endothelial permeability. Moreover, deficiency of SphK1 downregulated the expression of aortic S1PR3, Rhoa, ROCK, and F-actin. The results of administration with the SphK1 inhibitor PF-543 and the S1PR3 inhibitor VPC23019 in vitro further confirmed the conclusion that deficiency of SphK1 reduced S1P level and S1PR3 protein expression, inhibited Rhoa/ROCK signaling pathway, regulated protein expression of F-actin, improved vascular endothelial dysfunction and permeability, and exerted anti-atherosclerotic effects. CONCLUSIONS: This study revealed that deficiency of SphK1 relieved vascular endothelial barrier function in atherosclerosis mice via SphK1/S1P/S1PR signaling pathway.

S1PR2 participates in intestinal injury in severe acute pancreatitis by regulating macrophage pyroptosis.

Background: Severe acute pancreatitis (SAP) is an inflammatory disorder affecting the gastrointestinal system. Intestinal injury plays an important role in the treatment of severe acute pancreatitis. In this study, we mainly investigated the role of S1PR2 in regulating macrophage pyroptosis in the intestinal injury of severe acute pancreatitis. Methods: The SAP model was constructed using cerulein and lipopolysaccharide, and the expression of S1PR2 was inhibited by JTE-013 to detect the degree of pancreatitis and intestinal tissue damage in mice. Meanwhile, the level of pyroptosis-related protein was detected by western blot, the level of related mRNA was detected by PCR, and the level of serum inflammatory factors was detected by ELISA. In vitro experiments, LPS+ATP was used to construct the pyroptosis model of THP-1. After knockdown and overexpression of S1PR2, the pyroptosis proteins level was detected by western blot, the related mRNA level was detected by PCR, and the level of cell supernatant inflammatory factors were detected by ELISA. A rescue experiment was used to verify the sufficient necessity of the RhoA/ROCK pathway in S1PR2-induced pyroptosis. Meanwhile, THP-1 and FHC were co-cultured to verify that cytokines released by THP-1 after damage could regulate FHC damage. Results: Our results demonstrated that JTE-013 effectively attenuated intestinal injury and inflammation in mice with SAP. Furthermore, we observed a significant reduction in the expression of pyroptosis-related proteins within the intestinal tissue of SAP mice upon treatment with JTE-013. We confirmed the involvement of S1PR2 in THP-1 cell pyroptosis in vitro. Specifically, activation of S1PR2 triggered pyroptosis in THP-1 cells through the RhoA/ROCK signaling pathway. Moreover, it was observed that inflammatory factors released during THP-1 cell pyroptosis exerted an impact on cohesin expression in FHC cells. Conclusion: The involvement of S1PR2 in SAP-induced intestinal mucosal injury may be attributed to its regulation of macrophage pyroptosis.

Re-emergence of T lymphocyte-mediated synaptopathy in progressive multiple sclerosis.

Background: Secondary progressive multiple sclerosis (SPMS) is defined by the irreversible accumulation of disability following a relapsing-remitting MS (RRMS) course. Despite treatments advances, a reliable tool able to capture the transition from RRMS to SPMS is lacking. A T cell chimeric MS model demonstrated that T cells derived from relapsing patients exacerbate excitatory transmission of central neurons, a synaptotoxic event absent during remitting stages. We hypothesized the re-emergence of T cell synaptotoxicity during SPMS and investigated the synaptoprotective effects of siponimod, a sphingosine 1-phosphate receptor (S1PR) modulator, known to reduce grey matter damage in SPMS patients. Methods: Data from healthy controls (HC), SPMS patients, and siponimod-treated SPMS patients were collected. Chimeric experiments were performed incubating human T cells on murine cortico-striatal slices, and recording spontaneous glutamatergic activity from striatal neurons. Homologous chimeric experiments were executed incubating EAE mice T cells with siponimod and specific S1PR agonists or antagonists to identify the receptor involved in siponimod-mediated synaptic recovery. Results: SPMS patient-derived T cells significantly increased the striatal excitatory synaptic transmission (n=40 synapses) compared to HC T cells (n=55 synapses), mimicking the glutamatergic alterations observed in active RRMS-T cells. Siponimod treatment rescued SPMS T cells synaptotoxicity (n=51 synapses). Homologous chimeric experiments highlighted S1P5R involvement in the siponimod's protective effects. Conclusion: Transition from RRMS to SPMS involves the reappearance of T cell-mediated synaptotoxicity. Siponimod counteracts T cell-induced excitotoxicity, emphasizing the significance of inflammatory synaptopathy in progressive MS and its potential as a promising pharmacological target.

The Species Effect: Differential Sphingosine-1-Phosphate Responses in the Bone in Human Versus Mouse.

The deterioration of osteoblast-led bone formation and the upregulation of osteoclast-regulated bone resorption are the primary causes of bone diseases, including osteoporosis. Numerous circulating factors play a role in bone homeostasis by regulating osteoblast and osteoclast activity, including the sphingolipid-sphingosine-1-phosphate (S1P). However, to date no comprehensive studies have investigated the impact of S1P activity on human and murine osteoblasts and osteoclasts. We observed species-specific responses to S1P in both osteoblasts and osteoclasts, where S1P stimulated human osteoblast mineralisation and reduced human pre-osteoclast differentiation and mineral resorption, thereby favouring bone formation. The opposite was true for murine osteoblasts and osteoclasts, resulting in more mineral resorption and less mineral deposition. Species-specific differences in osteoblast responses to S1P were potentially explained by differential expression of S1P receptor 1. By contrast, human and murine osteoclasts expressed comparable levels of S1P receptors but showed differential expression patterns of the two sphingosine kinase enzymes responsible for S1P production. Ultimately, we reveal that murine models may not accurately represent how human bone cells will respond to S1P, and thus are not a suitable model for exploring S1P physiology or potential therapeutic agents.

TCF12 Transcriptionally Activates SPHK1 to Induce Osteosarcoma Angiogenesis by Promoting the S1P/S1PR4/STAT3 Axis.

Transcription factor 12 (TCF12) is a known oncogene in many cancers. However, whether TCF12 can regulate malignant phenotypes and angiogenesis in osteosarcoma is not elucidated. In this study, we demonstrated increased expression of TCF12 in osteosarcoma tissues and cell lines. High TCF12 expression was associated with metastasis and poor survival rate of osteosarcoma patients. Knockdown of TCF12 reduced the proliferation, migration, and invasion of osteosarcoma cells. TCF12 was found to bind to the promoter region of sphingosine kinase 1 (SPHK1) to induce transcriptional activation of SPHK1 expression and enhance the secretion of sphingosine-1-phosphate (S1P), which eventually resulted in the malignant phenotypes of osteosarcoma cells. In addition, S1P secreted by osteosarcoma cells promoted the angiogenesis of HUVECs by targeting S1PR4 on the cell membrane to activate the STAT3 signaling pathway. These findings suggest that TCF12 may induce transcriptional activation of SPHK1 to promote the synthesis and secretion of S1P. This process likely enhances the malignant phenotypes of osteosarcoma cells and induces angiogenesis via the S1PR4/STAT3 signaling pathway.

Low shear stress-induced blockage of autophagic flux impairs endothelial barrier and facilitates atherosclerosis in mice.

Atherosclerosis preferentially occurs in areas with low shear stress (LSS) and oscillatory flow. LSS has been demonstrated to correlate with the development of atherosclerosis. The sphingosine 1-phosphate receptor 1 (S1PR1), involving intravascular blood flow sensing, regulates vascular development and vascular barrier function. However, whether LSS affects atherosclerosis via regulating S1PR1 remains incompletely clear. In this study, immunostaining results of F-actin, ß-catenin, and VE-cadherin indicated that LSS impaired endothelial barrier function in human umbilical vein endothelial cells (HUVECs). Western blot analysis showed that LSS resulted in blockage of autophagic flux in HUVECs. In addition, autophagy agonist Rapamycin (Rapa) antagonized LSS-induced endothelial barrier dysfunction, whereas autophagic flux inhibitor Bafilomycin A1 (BafA1) exacerbated it, indicating that LSS promoted endothelial barrier dysfunction by triggering autophagic flux blockage. Notably, gene expression analysis revealed that LSS downregulated S1PR1 expression, which was antagonized by Rapa. Selective S1PR1 antagonist W146 impaired endothelial barrier function of HUVECs under high shear stress (HSS) conditions. Moreover, our data showed that expression of GAPARAPL2, a member of autophagy-related gene 8 (Atg8) proteins, was decreased in HUVECs under LSS conditions. Autophagic flux blockage induced by GAPARAPL2 knockdown inhibited S1PR1, aggravated endothelial barrier dysfunction of HUVECs in vitro, and promoted aortic atherosclerosis in ApoE-/- mice in vivo. Our study demonstrates that autophagic flux blockage induced by LSS downregulates S1PR1 expression and impairs endothelial barrier function. GABARAPL2 inhibition is involved in LSS-induced autophagic flux blockage, which impairs endothelial barrier function via downregulation of S1PR1.