Sphingosine-1-phosphate Signaling Research Articles

New mechanism for mesenchymal stem cell microvesicle to restore lung permeability: intracellular S1P signaling pathway independent of S1P receptor-1

BackgroundMicrovesicles (MVs) derived from human bone marrow mesenchymal stem cell (MSC) were demonstrated to restore lung protein permeability and attenuate acute lung injury. In our previous study, we found that MSC MV increased sphingosine-1-phosphate (S1P) kinase1 mRNA levels in injured human lung microvascular endothelial cells (HLMVEC) significantly. However, the role of S1P signaling in MSC MV to restore lung protein permeability is unknown.MethodsIn this study, we hypothesized that MSC MV might restore lung permeability in part through increasing intracellular S1P signaling pathway in injured HLMVEC independent of S1P receptors. We used the transwell co-culture system to study the effect of MSC MV on protein permeability of Lipopolysaccharide (LPS) damaged HLMVEC.ResultsOur results showed that LPS significantly increased the permeability of HLMVEC to FITC-dextran (70 kDa) within 24 h. MSC MV restores this permeability and, to a large extent, prevents the cytoskeleton protein F-actin from recombining into “actin stress fibers,” and restores the positions of tight junctions and adhesion junctions in the damaged HLMVEC. This therapeutic effect of MSC MV was related to the increase in the S1P level in injured HLMVEC and was not eliminated when adding the antagonist of S1P receptor, suggesting that MSC MV to restore lung permeability was independent of S1P receptors on HLMVEC. Laser confocal further observed that Ca2+ mobilization and Rac1 activation in LPS injured HLMVEC were increased in parallel with the increase in intracellular S1P level after MSC MV treatment.ConclusionsIn short, MSC MV partially restored protein permeability across HLMVEC through the intracellular S1P signaling pathway independent of S1P receptor-1.

S1P/S1PR1 signaling differentially regulates the allogeneic response of CD4 and CD8 T cells by modulating mitochondrial fission.

Graft-versus-host disease (GVHD) significantly contributes to patient morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HSCT). Sphingosine-1-phosphate (S1P) signaling is involved in the biogenetic processes of different immune cells. In the current study, we demonstrated that recipient sphingosine kinase 1 (Sphk1), but not Sphk2, was required for optimal S1PR1-dependent donor T-cell allogeneic responses by secreting S1P. Using genetic and pharmacologic approaches, we demonstrated that inhibition of Sphk1 or S1PR1 substantially attenuated acute GVHD (aGVHD) while retaining the graft-versus-leukemia (GVL) effect. At the cellular level, the Sphk1/S1P/S1PR1 pathway differentially modulated the alloreactivity of CD4+ and CD8+ T cells; it facilitated T-cell differentiation into Th1/Th17 cells but not Tregs and promoted CD4+ T-cell infiltration into GVHD target organs but was dispensable for the CTL activity of allogeneic CD8+ T cells. At the molecular level, the Sphk1/S1P/S1PR1 pathway augmented mitochondrial fission and increased mitochondrial mass in allogeneic CD4+ but not CD8+ T cells by activating the AMPK/AKT/mTOR/Drp1 pathway, providing a mechanistic basis for GVL maintenance when S1P signaling was inhibited. For translational purposes, we detected the regulatory efficacy of pharmacologic inhibitors of Sphk1 and S1PR1 in GVHD induced by human T cells in a xenograft model. Our study provides novel mechanistic insight into how the Sphk1/S1P/S1PR1 pathway modulates T-cell alloreactivity and validates Sphk1 or S1PR1 as a therapeutic target for the prevention of GVHD and leukemia relapse. This novel strategy may be readily translated into the clinic to benefit patients with hematologic malignancies and disorders.

Proton-driven alternating access in a spinster lipid transporter

Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. Here, we employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60TM2:Arg289TM7) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange.

Sphingosine-1-Phosphate (S1P) Lyase Inhibition Aggravates Atherosclerosis and Induces Plaque Rupture in ApoE-/-Mice.

Altered plasma sphingosine-1-phosphate (S1P) concentrations are associated with clinical manifestations of atherosclerosis. However, whether long-term elevation of endogenous S1P is pro- or anti-atherogenic remains unclear. Here, we addressed the impact of permanently high S1P levels on atherosclerosis in cholesterol-fed apolipoprotein E-deficient (ApoE−/−) mice over 12 weeks. This was achieved by pharmacological inhibition of the S1P-degrading enzyme S1P lyase with 4-deoxypyridoxine (DOP). DOP treatment dramatically accelerated atherosclerosis development, propagated predominantly unstable plaque phenotypes, and resulted in frequent plaque rupture with atherothrombosis. Macrophages from S1P lyase-inhibited or genetically deficient mice had a defect in cholesterol efflux to apolipoprotein A-I that was accompanied by profoundly downregulated cholesterol transporters ATP-binding cassette transporters ABCA1 and ABCG1. This was dependent on S1P signaling through S1PR3 and resulted in dramatically enhanced atherosclerosis in ApoE−/−/S1PR3−/− mice, where DOP treatment had no additional effect. Thus, high endogenous S1P levels promote atherosclerosis, compromise cholesterol efflux, and cause genuine plaque rupture.

Sphingosine 1-phosphate signaling in perivascular cells enhances inflammation and fibrosis in the kidney.

Chronic kidney disease (CKD), characterized by sustained inflammation and progressive fibrosis, is highly prevalent and can eventually progress to end-stage kidney disease. However, current treatments to slow CKD progression are limited. Sphingosine 1-phosphate (S1P), a product of sphingolipid catabolism, is a pleiotropic mediator involved in many cellular functions, and drugs targeting S1P signaling have previously been studied particularly for autoimmune diseases. The primary mechanism of most of these drugs is functional antagonism of S1P receptor-1 (S1P1) expressed on lymphocytes and the resultant immunosuppressive effect. Here, we documented the role of local S1P signaling in perivascular cells in the progression of kidney fibrosis using primary kidney perivascular cells and several conditional mouse models. S1P was predominantly produced by sphingosine kinase 2 in kidney perivascular cells and exported via spinster homolog 2 (Spns2). It bound to S1P1 expressed in perivascular cells to enhance production of proinflammatory cytokines/chemokines upon injury, leading to immune cell infiltration and subsequent fibrosis. A small-molecule Spns2 inhibitor blocked S1P transport, resulting in suppression of inflammatory signaling in human and mouse kidney perivascular cells in vitro and amelioration of kidney fibrosis in mice. Our study provides insight into the regulation of inflammation and fibrosis by S1P and demonstrates the potential of Spns2 inhibition as a treatment for CKD and potentially other inflammatory and fibrotic diseases that avoids the adverse events associated with systemic modulation of S1P receptors.

A new chapter in lipid signaling and kidney fibrosis.

The perivascular sphingosine 1-phosphate signaling axis may be an emerging therapeutic target for treating chronic kidney disease (Tanaka et al.).

Targeting the Sphingolipid Rheostat in Gliomas.

Gliomas are highly aggressive cancer types that are in urgent need of novel drugs and targeted therapies. Treatment protocols have not improved in over a decade, and glioma patient survival remains among the worst of all cancer types. As a result, cancer metabolism research has served as an innovative approach to identifying novel glioma targets and improving our understanding of brain tumors. Recent research has uncovered a unique metabolic vulnerability in the sphingolipid pathways of gliomas that possess the IDH1 mutation. Sphingolipids are a family of lipid signaling molecules that play a variety of second messenger functions in cellular regulation. The two primary metabolites, sphingosine-1-phosphate (S1P) and ceramide, maintain a rheostat balance and play opposing roles in cell survival and proliferation. Altering the rheostat such that the pro-apoptotic signaling of the ceramides outweighs the pro-survival S1P signaling in glioma cells diminishes the hallmarks of cancer and enhances tumor cell death. Throughout this review, we discuss the sphingolipid pathway and identify the enzymes that can be most effectively targeted to alter the sphingolipid rheostat and enhance apoptosis in gliomas. We discuss each pathway’s steps based on their site of occurrence in the organelles and postulate novel targets that can effectively exploit this vulnerability.

Sphingosine-1-phosphate receptor modulators in stroke treatment.

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that can influence a broad range of biological processes through its binding to five distinct G-protein-coupled receptors. S1P receptor modulators are a new group of immunosuppressive agents currently used in the immunotherapy of multiple sclerosis. Inflammation following stroke can exacerbate neuronal injury. Given that S1P signaling is linked to multiple immune processes, therapies targeting the S1P axis may be suitable for treating stroke. In this review, we outline S1P metabolism and S1P receptors, discuss the mechanisms of action of S1P receptor modulators in lymphocyte migration and their direct action on cells of the central nervous system, and provide a concise summary of the efficacy of S1P receptor modulators in animal studies and clinical trials on treatments for stroke.

Sphingosine 1-phosphate attenuates neuronal dysfunction induced by amyloid-β oligomers through endocytic internalization of NMDA receptors.

Soluble oligomers arising from the aggregation of the amyloid beta peptide (Aβ) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Prefibrillar oligomers of the 42-residue form of Aβ (Aβ42 O) show membrane-binding capacity and trigger the disruption of Ca2+ homeostasis, a causative event in neuron degeneration. Since bioactive lipids have been recently proposed as potent protective agents against Aβ toxicity, we investigated the involvement of sphingosine 1-phosphate (S1P) signalling pathway in Ca2+ homeostasis in living neurons exposed to Aβ42 O. We show that both exogenous and endogenous S1P rescued neuronal Ca2+ dyshomeostasis induced by toxic Aβ42 O in primary rat cortical neurons and human neuroblastoma SH-SY5Y cells. Further analysis revealed a strong neuroprotective effect of S1P1 and S1P4 receptors, and to a lower extent of S1P3 and S1P5 receptors, which activate the Gi -dependent signalling pathways, thus resulting in the endocytic internalization of the extrasynaptic GluN2B-containing N-methyl-D-aspartate receptors (NMDARs). Notably, the S1P beneficial effect can be sustained over time by sphingosine kinase-1 overexpression, thus counteracting the down-regulation of the S1P signalling induced by Aβ42 O. Our findings disclose underlying mechanisms of S1P neuronal protection against harmful Aβ42 O, suggesting that S1P and its signalling axis can be considered promising targets for therapeutic approaches for AD.

Visualizing Sphingosine-1-Phosphate Receptor 1(S1P1) Signaling During Central Nervous System De- and Remyelination.

Multiple sclerosis (MS) is an inflammatory-demyelinating disease of the central nervous system (CNS) mediated by aberrant auto-reactive immune responses. The current immune-modulatory therapies are unable to protect and repair immune-mediated neural tissue damage. One of the therapeutic targets in MS is the sphingosine-1-phosphate (S1P) pathway which signals via sphingosine-1-phosphate receptors 1-5 (S1P1-5). S1P receptors are expressed predominantly on immune and CNS cells. Considering the potential neuroprotective properties of S1P signaling, we utilized S1P1-GFP (Green fluorescent protein) reporter mice in the cuprizone-induced demyelination model to investigate in vivo S1P - S1P1 signaling in the CNS. We observed S1P1 signaling in a subset of neural stem cells in the subventricular zone (SVZ) during demyelination. During remyelination, S1P1 signaling is expressed in oligodendrocyte progenitor cells in the SVZ and mature oligodendrocytes in the medial corpus callosum (MCC). In the cuprizone model, we did not observe S1P1 signaling in neurons and astrocytes. We also observed β-arrestin-dependent S1P1 signaling in lymphocytes during demyelination and CNS inflammation. Our findings reveal β-arrestin-dependent S1P1 signaling in oligodendrocyte lineage cells implying a role of S1P1 signaling in remyelination.

Sphingosine-1-Phosphate (S1P) and S1P Signaling Pathway Modulators, from Current Insights to Future Perspectives.

Sphingosine-1-phosphate (S1P) and S1P receptors (S1PR) are bioactive lipid molecules that are ubiquitously expressed in the human body and play an important role in the immune system. S1P-S1PR signaling has been well characterized in immune trafficking and activation in both innate and adaptive immune systems. Despite this knowledge, the full scope in the pathogenesis of autoimmune disorders is not well characterized yet. From the discovery of fingolimod, the first S1P modulator, until siponimod, the new molecule recently approved for the treatment of secondary progressive multiple sclerosis (SPMS), there has been a great advance in understanding the S1P functions and their involvement in immune diseases, including multiple sclerosis (MS). Modulation on S1P is an interesting target for the treatment of various autoimmune disorders. Improved understanding of the mechanism of action of fingolimod has allowed the development of the more selective second-generation S1PR modulators. Subtype 1 of the S1PR (S1PR1) is expressed on the cell surface of lymphocytes, which are known to play a major role in MS pathogenesis. The understanding of S1PR1’s role facilitated the development of pharmacological strategies directed to this target, and theoretically reduced the safety concerns derived from the use of fingolimod. A great advance in the MS treatment was achieved in March 2019 when the Food and Drug Association (FDA) approved Siponimod, for both active secondary progressive MS and relapsing–remitting MS. Siponimod became the first oral disease modifying therapy (DMT) specifically approved for active forms of secondary progressive MS. Additionally, for the treatment of relapsing forms of MS, ozanimod was approved by FDA in March 2020. Currently, there are ongoing trials focused on other new-generation S1PR1 modulators. This review approaches the fundamental aspects of the sphingosine phosphate modulators and their main similarities and differences.

Slit-Robo signalling establishes a Sphingosine-1-phosphate gradient to polarise fin mesenchyme.

Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identifies a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the apical ectodermal ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate the coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.

Signal Transduction and Gene Regulation in the Endothelium.

Extracellular signals act on G-protein-coupled receptors (GPCRs) to regulate homeostasis and adapt to stress. This involves rapid intracellular post-translational responses and long-lasting gene-expression changes that ultimately determine cellular phenotype and fate changes. The lipid mediator sphingosine 1-phosphate (S1P) and its receptors (S1PRs) are examples of well-studied GPCR signaling axis essential for vascular development, homeostasis, and diseases. The biochemical cascades involved in rapid S1P signaling are well understood. However, gene-expression regulation by S1PRs are less understood. In this review, we focus our attention to how S1PRs regulate nuclear chromatin changes and gene transcription to modulate vascular and lymphatic endothelial phenotypic changes during embryonic development and adult homeostasis. Because S1PR-targeted drugs approved for use in the treatment of autoimmune diseases cause substantial vascular-related adverse events, these findings are critical not only for general understanding of stimulus-evoked gene regulation in the vascular endothelium, but also for therapeutic development of drugs for autoimmune and perhaps vascular diseases.

Expression of sphingosine-1-phosphate receptor 2 is correlated with migration and invasion of human colon cancer cells: A preliminary clinical study.

Sphingosine-1-phosphate (S1P) is a bioactive phospholipid that serves as a potent mediator of cell proliferation, differentiation and apoptosis by binding to S1P receptors (S1PRs). S1P signalling is involved in the pathogenesis of numerous types of disease, including cancer. To the best of our knowledge, however, little is known about the expression patterns of S1PRs and their role in human colorectal cancer (CRC) cell migration and invasion. The aim of the present study was to investigate the role of S1P signalling in the metastasis of colon cancer cells and the expression of S1PRs in patients with CRC. The protein and mRNA expression levels of S1PRs and sphingosine kinases (SPHKs) in 55 patients with CRC were detected by western blotting (WB), immunohistochemical (IHC) analysis and reverse transcription-quantitative PCR. The levels of S1P in serum from patients and healthy individuals were quantified by ELISA. S1PRs antagonists JTE013, FTY720 and S1PR2-small interfering (si)RNA were used to determine the role of S1PR2 in human CRC LOVO and SW480 cell lines. Migration and invasion assays were performed for functional analysis. The levels of S1P in serum were significantly increased in patients with CRC compared with healthy individuals. The relative mRNA expression levels of S1PR2 were significantly downregulated in tumour compared with normal tissue, whereas S1PR1 and SPHK1 were upregulated. WB showed that 58% (32/55 cases) of patients presented downregulated S1PR2 protein expression. IHC analysis indicated that expression of S1PR2 was lower in tumour than in normal tissue in 65.5% (36/55 cases) of patients. Exogenous addition of S1P promoted migration and invasion in the different cell types. S1P stimulated the migration and invasion of SW480 cells. The inhibition of S1PR2 by JTE013 or S1PR2-siRNA significantly promoted the migration and invasion of SW480 cells, while FTY720 reversed these effects. The present study indicated that expression levels of S1PRs, particularly S1PR2, were associated with migration and invasion of CRC cells. The present findings revealed a novel mechanism by which S1P inhibited tumour cell migration and invasion via a S1PR2-dependent pathway, suggesting that S1PR2 may be a therapeutic target for treatment of colon cancer.

Sphingosine 1-Phosphate Signaling at the Skin Barrier Interface.

Simple SummarySphingosine 1-phosphate is a bioactive phospholipid derived from cell membranes. It is biosynthesized and metabolized within the cell and released extracellularly to trigger various cellular responses via sphingosine 1-phosphate receptors. Keratinocyte and mast cell functions are responsible for skin barrier acquired and innate immunity, and express sphingosine 1-phosphate receptors. These receptors regulate not only cell differentiation and proliferation, but also immune responses such as cell migration and cytokine secretion, through G-protein-mediated signaling pathways. Sphingosine 1-phosphate and its receptor signaling pathways are also involved in several skin diseases along with various receptor agonists and are being investigated as potential therapeutic agents. Here, we focus on the association of sphingosine 1-phosphate and its receptors within the skin barrier interface.Sphingosine 1-phosphate (S1P) is a product of membrane sphingolipid metabolism. S1P is secreted and acts via G-protein-coupled receptors, S1PR1-5, and is involved in diverse cellular functions, including cell proliferation, immune suppression, and cardiovascular functions. Recent studies have shown that the effects of S1P signaling are extended further by coupling the different S1P receptors and their respective downstream signaling pathways. Our group has recently reported that S1P inhibits cell proliferation and induces differentiation in human keratinocytes. There is a growing understanding of the connection between S1P signaling, skin barrier function, and skin diseases. For example, the activation of S1PR1 and S1PR2 during bacterial invasion regulates the synthesis of inflammatory cytokines in human keratinocytes. Moreover, S1P-S1PR2 signaling is involved in the production of inflammatory cytokines and can be triggered by epidermal mechanical stress and bacterial invasion. This review highlights how S1P affects human keratinocyte proliferation, differentiation, immunoreaction, and mast cell immune response, in addition to its effects on the skin barrier interface. Finally, studies targeting S1P-S1PR signaling involved in inflammatory skin diseases are also presented.

Molecular mechanisms underlying the heterogeneous barrier responses of two primary endothelial cell types to sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) signals to enhance or destabilize the vascular endothelial barrier depending on the receptor engaged. Here, we investigated the differential barrier effects of S1P on two influential primary endothelial cell (EC) types, human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs). S1PR1 (barrier protective) and S1PR3 (barrier disruptive) surface and gene expression were quantified by flow cytometry and immunofluorescence, and RT-qPCR, respectively. Functional evaluation of EC monolayer permeability in response to S1P was quantified with transendothelial electrical resistance (TEER) and small molecule permeability. S1P significantly enhanced HUVEC barrier function, while promoting HPMEC barrier breakdown. Immunofluorescence and flow cytometry analysis showed select, S1PR3-high HPMECs, suggesting susceptibility to barrier destabilization following S1P exposure. Reevaluation of HPMEC barrier following S1P exposure under inflamed conditions demonstrated synergistic barrier disruptive effects of pro-inflammatory cytokine and S1P. The role of the Rho-ROCK signaling pathway under these conditions was confirmed through ROCK1/2 inhibition (Y-27632). Thus, the heterogeneous responses of ECs to S1P signaling are mediated through Rho-ROCK signaling, and potentially driven by differences in the surface expression of S1PR3.

Skeletal Muscle and COVID-19: The Potential Involvement of Bioactive Sphingolipids

SARS-CoV-2 virus infection is the cause of the coronavirus disease 2019 (COVID-19), which is still spreading over the world. The manifestation of this disease can range from mild to severe and can be limited in time (weeks) or persist for months in about 30–50% of patients. COVID-19 is considered a multiple organ dysfunction syndrome and the musculoskeletal system manifestations are beginning to be considered of absolute importance in both COVID-19 patients and in patients recovering from the SARS-CoV-2 infection. Musculoskeletal manifestations of COVID-19 and other coronavirus infections include loss of muscle mass, muscle weakness, fatigue or myalgia, and muscle injury. The molecular mechanisms by which SARS-CoV-2 can cause damage to skeletal muscle (SkM) cells are not yet well understood. Sphingolipids (SLs) represent an important class of eukaryotic lipids with structural functions as well as bioactive molecules able to modulate crucial processes, including inflammation and viral infection. In the last two decades, several reports have highlighted the role of SLs in modulating SkM cell differentiation, regeneration, aging, response to insulin, and contraction. This review summarizes the consequences of SARS-CoV-2 infection on SkM and the potential involvement of SLs in the tissue responses to virus infection. In particular, we highlight the role of sphingosine 1-phosphate signaling in order to aid the prediction of novel targets for preventing and/or treating acute and long-term musculoskeletal manifestations of virus infection in COVID-19.

Functional and Molecular Studies of Renal Sphingosine 1 Phosphate Signaling Pathway in IUGR Benjamin Bhunu1 and Suttira Intapad1 Tulane University School of Medicine

Fetal Growth Restriction (FGR) is now regarded as a risk factor for adult renal diseases, however, the precise underlying mechanism that is responsible for this observation remains vague. Sphingosine 1 Phosphate (S1P) is a lysophospholipid with important functions in the regulation of renal physiology and the cardiovascular system. Previously, we demonstrated that in FGR but not control animals, acute activation of Sphingosine 1 Phosphate Receptor 1 (S1PR1) transiently reduces Blood Pressure (BP) in an endothelial nitric oxide synthase dependent manner. We have also shown that both male and female FGR mice have lower nephron number in the kidneys, however, unlike females, male FGR offspring have reduced renal function suggesting sex specific differences in fetal programing of kidney diseases. We have also shown that within 5 minutes of pharmacologically activating S1PR1 with SEW2871, GFR significantly reduces in male control but not IUGR animals. The objective of this study was to test the effect of longer stimulation of S1PR1 agonist on renal function and to determine the distribution of renal S1P signaling molecules. On their 13th day of gestation, pregnant C57BL mice went through a Reduced Uterine Pressure Perfusion (RUPP) or sham surgery to generate FGR and control offspring respectively. At 6 months, measurement of Glomerular Filtration Rate (GFR) and expression of proteins that are involved in S1P signaling was performed using plasma FITC‐Anulin decay, PCR, and immunohistochemistry. For immunohistochemistry, the Integrated Optical Densities (IDO) values from (Diaminobenzidine) DAB‐stained kidney sections were used to statistically quantify protein expression. GFR was lower in male FGR offspring than control (8.85 ±0.55 vs 13.86±1.13 µL/min/kg BW, P˂0.05, FGR vs control males) while in females there was no statistical difference (12.33±0.87 vs 15.92 ±1.94 L/min/kg BW, P˃ 0.05 control females vs FGR). Administration of SEW2871 and measurement of GFR after one hour confirmed that GFR decreases only in control males but not IUGR while in females both FGR and control groups had similar reduction response (6.00±0.46 vs 8.41±0.79 µL/min/kg BW, P˃ 0.05 control males vs FGR) (5. 97±0.48 vs 5.72 ±1.01 µL/min/kg BW, P˃ 0.05 control females vs FGR). Whole kidney transcriptomics showed no statistical differences in the expression profile of S1PR 1‐3 and Sphingosine 1 Phosphate Kinases 1 and 2 (SPHK1‐2) between FGR and control male and female animals. Immunohistochemistry analysis of the kidney medulla and cortex of male animals revealed no difference in the expression profile of S1PR1 in both FGR and control and increased expression of S1PR2 in both the medulla and cortex of FGR animals. S1PR3 was decreased in both the medulla and cortex of FGR mice. SPHK 1 and 2 were not differentially expressed in the medulla of control and FGR animal whereas in the cortex both the two receptors were downregulated in FGR. Our results indicate that acute activation of S1PR1 differentially regulates GFR between male and female FGR. Consequences of the upregulation of S1PR2 and the down regulation of S1PR 3 and SPHK 1‐2 proteins in the kidney of male FGR warrants further investigations.

Sphingolipids Imbalanced in Brain Microvessel of Predisposed Reduced Uterine Perfusion Mice

Preeclampsia (PE), a hypertension disorder, is one of the most serious complications that can occur during pregnancy. It affects at least 2‐8% of pregnancies, causing up to 76,000 maternal deaths and 500,000 infant fatalities. PE negatively impacts the vasculature of multiple body systems and has significant neurovascular implications including reversible cerebral vasoconstriction syndrome and ischemic stroke. Although PE resolves after delivery, long term cerebrovascular effects remain. Women who have experienced PE are estimated to have an 80% increased risk of future stroke. Although the consequences of PE are extensive and prevalent, its full etiology is not yet known. It is suspected that cerebrovascular dysfunction plays a significant role in these critical ramifications of PE. Interestingly, the ceramide and sphingosine‐1‐phosphate (S1P) pathway is known to regulate many of the systems that are altered in PE. Specifically, ceramide and S1P are involved in regulation of blood pressure, vascular tone, and vessel permeability in addition to other cell and tissue processes. Because of the significant involvement of ceramide, S1P, and their signaling constituents in endothelial cell and vascular integrity, variations within this pathway may explain the link between PE and cerebrovascular disease. We hypothesize that the ceramide and S1P signaling pathway is linked to dysfunctional brain vasculature in mice that had a PE‐induced pregnancy. This study aims to prove the ceramide and S1P signaling pathway is altered in the cerebral microvasculature of PE murine models. C57BL female mice underwent reduced uterine perfusion pressure (RUPP) surgery at 10 weeks old to simulate PE or sham surgery. Brain microvessels (BMVs) were isolated from these mice dams nine months following delivery. Two step reverse transcription qPCR was performed to assess the mRNA expression of the various ceramide and S1P signaling components. It was found that RUPP mice BMVs express significantly less sphingosine kinase 1 (SphK1) in comparison to sham BMVs. RUPP mice BMVs also demonstrated significantly increased sphingosine‐1‐phosphate lyase 1 (Sgpl1) and phospholipid phosphatase 1 (Plpp1). RUPP mice BMVs exhibited decreased expression of ceramide kinase and sphingosine kinase 2 (Sphk2) as well. SphK1 and Sphk2 are key enzymes in producing S1P, an active signaling lipid responsible for maintaining the blood brain barrier, cerebrovascular integrity, and angiogenesis. Sgpl1 is responsible for the degradation of S1P into its inactive form, therefore contributing to endothelial apoptosis and diminished vessel integrity. Plpp1 catalyzes the production of pro‐apoptotic ceramide and was discovered to promote the metabolism of S1P. Ceramide kinase is a crucial enzyme in producing anti‐apoptotic ceramide‐1‐phosphate. These discrepancies in ceramide and S1P signaling pathway expression may contribute to impaired cerebrovascular health, providing insight into the increased incidence of stroke with PE.

Intracellular Sphingosine‐1‐Phosphate is Involved in Lung Cancer Cells Proliferation

BackgroundSphingosine‐1‐phosphate (S1P) is a membrane‐derived bioactive phospholipid exerting a multitude of effects on respiratory cell physiology and pathology through five S1P receptors (S1PR1‐5). Higher levels of S1P have been registered in a broad range of respiratory diseases, including inflammatory disorders and cancer, although its exact role is still elusive.ObjectivesThe aim of our study was to understand the role of S1P in healthy versus lung adenocarcinoma cells after Toll‐Like Receptors (TLRs) activation, well‐known modulators of sphingolipid metabolism.MethodsWe stimulated healthy fibroblast and lung adenocarcinoma cells with TLRs ligands and S1P, to compare physiological vs pathological S1P role. Pro‐inflammatory cytokines release, proliferation rate and the expression/activity of the mediators involved in the S1P signaling were evaluated.ResultsWe found that the activation of TLR9, but not of TLR4, was involved in the generation of S1P by means of ceramidase and sphingosine kinase I/II (SPHK I/II) activation in tumor cells but not in non‐cancerous cells. Through an inside‐out signaling, newly formed S1P was extruded from the cells through its transporter SPNS2, supporting TLR9/NF‐kB‐mediated TNF‐α release mainly via membrane S1PR3. These results were not observed in healthy fibroblasts, implying that this pathway was mainly involved in pathological conditions. Moreover, we found that S1PR3 and SPHK II were overexpressed in the nucleus of lung adenocarcinoma cells and that were associated to S1P‐mediated proliferation of tumor cells.ConclusionThese results prove that the endogenous TLR9‐induced S1P can on one side favor pro‐inflammatory mechanisms in the tumor microenvironment via the activation of cell surface receptors, but on the other tumor progression via the nuclear S1PR3/SPHK II axis, highlighting a novel molecular mechanism that identifies S1P as one of the crucial mediators for lung carcinogenesis‐associated inflammatory processes.