Sphingolipid Sphingosine 1-phosphate Research Articles

Sphingosine 1-phosphate signaling axis mediates neuropeptide S-induced invasive phenotype of endometriotic cells.

Endometriosis is a chronic gynecological syndrome characterized by endometrial cell invasion of the extra-uterine milieu, pelvic pain and infertility. Treatment relies on either symptomatic drugs or hormonal therapies, even though the mechanism involved in the onset of endometriosis is yet to be elucidated. The signaling of sphingolipid sphingosine 1-phosphate (S1P) is profoundly dysregulated in endometriosis. Indeed, sphingosine kinase (SK)1, one of the two isoenzymes responsible for S1P biosynthesis, and S1P1 , S1P3 and S1P5 , three of its five specific receptors, are more highly expressed in endometriotic lesions compared to healthy endometrium. Recently, missense coding variants of the gene encoding the receptor 1 for neuropeptide S (NPS) have been robustly associated with endometriosis in humans. This study aimed to characterize the biological effect of NPS in endometriotic epithelial cells and the possible involvement of the S1P signaling axis in its action. NPS was found to potently induce cell invasion and actin cytoskeletal remodeling. Of note, the NPS-induced invasive phenotype was dependent on SK1 and SK2 as well as on S1P1 and S1P3 , given that the biological action of the neuropeptide was fully prevented when one of the two biosynthetic enzymes or one of the two selective receptors was inhibited or silenced. Furthermore, the RhoA/Rho kinase pathway, downstream to S1P receptor signaling, was found to be critically implicated in invasion and cytoskeletal remodeling elicited by NPS. These findings provide new information to the understanding of the molecular mechanisms implicated in endometriosis pathogenesis, establishing the rationale for non-hormonal therapeutic targets for its treatment.

Sphingosine-1-phosphate receptor 3 is a non-hormonal target to counteract endometriosis-associated fibrosis

ObjectiveTo study the molecular mechanisms responsible for fibrosis in endometriosis by investigating whether the protein expression levels of sphingosine-1-phosphate receptor 3 (S1PR3), one of the five specific receptors of the bioactive sphingolipid sphingosine 1-phosphate (S1P), correlated with fibrosis extent in endometriotic lesions and which are the cellular mechanisms involved in this process. DesignCase-control laboratory study and cultured endometriotic cells. SubjectsUniversity research institute and university hospital. Patient(s)A total of 33 women, with and without endometriosis, were included in the study. Interventions(s)Endometriotic lesions were obtained from women with endometriosis (endometrioma [OMA], n=8; deep infiltrating endometriosis [DIE], n=15, (urological n=5, gastrointestinal n=6, posterior n=4)) and control endometrium from healthy women, n=10, by means of laparoscopic/hysteroscopic surgery. The expression of S1PR3 was evaluated by immunohistochemistry and the extent of fibrosis by Masson’s trichrome staining. Human cultured epithelial endometriotic 12Z cells were used to evaluate the mechanisms involved in the profibrotic effect of S1PR3 activation. Main Outcome Measure(s)The expression of S1PR3 in endometriotic lesions positively correlated to endometriosis-associated fibrosis. In addition, S1P induced epithelial-mesenchymal transition (EMT)/fibrosis in epithelial endometriotic cells. By RNA interference and pharmacological approaches, the profibrotic effect of S1P was shown to rely on S1PR3, thus unveiling the molecular mechanism implicated in the profibrotic action of the bioactive sphingolipid. Result(s)The protein expression levels of S1PR3 resulted significantly augmented in the glandular sections of OMA and DIE of different localizations in respect to control endometrium and positively correlated with the extent of fibrosis. S1P was shown to have a crucial role in the onset of fibrosis in epithelial endometriotic cells, stimulating the expression of EMT and fibrotic markers. Genetic approaches highlighted that S1PR3 mediates the fibrotic effect of S1P. Downstream of S1PR3, ezrin and ERK1/2 signaling were found critically implicated in the EMT/fibrosis elicited by S1P. Conclusion(s)The S1PR3 receptor may represent a possible innovative pharmacological target for endometriosis.

Sphingosine kinase 1/S1P receptor signaling axis is essential for cellular uptake of Neisseria meningitidis in brain endothelial cells.

Invasion of brain endothelial cells (BECs) is central to the pathogenicity of Neisseria meningitidis infection. Here, we established a key role for the bioactive sphingolipid sphingosine-1-phosphate (S1P) and S1P receptor (S1PR) 2 in the uptake process. Quantitative sphingolipidome analyses of BECs infected with N. meningitidis revealed elevated S1P levels, which could be attributed to enhanced expression of the enzyme sphingosine kinase 1 and its activity. Increased activity was dependent on the interaction of meningococcal type IV pilus with the endothelial receptor CD147. Concurrently, infection led to increased expression of the S1PR2. Blocking S1PR2 signaling impaired epidermal growth factor receptor (EGFR) phosphorylation, which has been shown to be involved in cytoskeletal remodeling and bacterial endocytosis. Strikingly, targeting S1PR1 or S1PR3 also interfered with bacterial uptake. Collectively, our data support a critical role of the SphK/S1P/S1PR axis in the invasion of N. meningitidis into BECs, defining a potential target for adjuvant therapy.

The Role of Sphingosine-1-Phosphate Receptor 2 in Mouse Retina Light Responses.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) acts as a ligand for a family of G protein-coupled S1P receptors (S1PR1-5) to participate in a variety of signaling pathways. However, their specific roles in the neural retina remain unclear. We previously showed that S1P receptor subtype 2 (S1PR2) is expressed in murine retinas, primarily in photoreceptors and bipolar cells, and its expression is altered by retinal stress. This study aims to elucidate the role of S1PR2 in the mouse retina. We examined light responses by electroretinography (ERG), structural differences by optical coherence tomography (OCT), and protein levels by immunohistochemistry (IHC) in wild-type (WT) and S1PR2 knockout (KO) mice at various ages between 3 and 6 months. We found that a- and b-wave responses significantly increased at flash intensities between 400~2000 and 4~2000 cd.s/m2, respectively, in S1PR2 KO mice relative to those of WT controls at baseline. S1PR2 KO mice also exhibited significantly increased retinal nerve fiber layer (RNFL) and outer plexiform layer (OPL) thickness by OCT relative to the WT. Finally, in S1PR2 KO mice, we observed differential labeling of synaptic markers by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). These results suggest a specific involvement of S1PR2 in the structure and synaptic organization of the retina and a potential role in light-mediated functioning of the retina.

Sphingosine 1-phosphate elicits a ROS-mediated proinflammatory response in human endometrial stromal cells via ERK5 activation.

Endometriosis is a chronic gynecological disease affecting ~10% women in the reproductive age characterized by the growth of endometrial glands and stroma outside the uterine cavity. The inflammatory process has a key role in the initiation and progression of the disorder. Currently, there are no available early diagnostic tests and therapy relies exclusively on symptomatic drugs, so that elucidation of the complex molecular mechanisms involved in the pathogenesis of endometriosis is an unmet need. The signaling of the bioactive sphingolipid sphingosine 1-phosphate (S1P) is deeply dysregulated in endometriosis. S1P modulates a variety of fundamental cellular processes, including inflammation, neo-angiogenesis, and immune responses acting mainly as ligand of a family of G-protein-coupled receptors named S1P receptors (S1PR), S1P1-5 . Here, we demonstrated that the mitogen-activated protein kinase ERK5, that is expressed in endometriotic lesions as determined by quantitative PCR, is activated by S1P in human endometrial stromal cells. S1P-induced ERK5 activation was shown to be triggered by S1P1/3 receptors via a SFK/MEK5-dependent axis. S1P-induced ERK5 activation was, in turn, responsible for the increase of reactive oxygen species and proinflammatory cytokine expression in human endometrial stromal cells. The present findings indicate that the S1P signaling, via ERK5 activation, supports a proinflammatory response in the endometrium and establish the rationale for the exploitation of innovative therapeutic targets for endometriosis.

Identification of Glomerular and Plasma Apolipoprotein M as Novel Biomarkers in Glomerular Disease

Dysregulation of sphingolipid and cholesterol metabolism contributes to the pathogenesis of glomerular diseases (GDs). Apolipoprotein M (ApoM) promotes cholesterol efflux and modulates the bioactive sphingolipid sphingosine-1-phosphate (S1P). Glomerular ApoM expression is decreased in patients with focal segmental glomerulosclerosis (FSGS). We hypothesized that glomerular ApoM deficiency occurs in GD and that ApoM expression and plasma ApoM correlate with outcomes. Patients with GD from the Nephrotic Syndrome Study Network (NEPTUNE) were studied. We compared glomerular mRNA expression of ApoM (gApoM), sphingosine kinase 1 (SPHK1), and S1P receptors 1 to 5 (S1PR1-5) in patients (n= 84) and controls (n= 6). We used correlation analyses to determine associations between gApoM, baseline plasma ApoM (pApoM), and urine ApoM (uApoM/Cr). We used linear regression to determine whether gApoM, pApoM, and uApoM/Cr were associated with baseline estimated glomerular filtration rate (eGFR) and proteinuria. Using Cox models, we determined whether gApoM, pApoM, and uApoM/Cr were associated with complete remission (CR) and the composite of end-stage kidney disease (ESKD) or≥40% eGFR decline. gApoM was reduced (P< 0.01) and SPHK1 and S1PR1 to 5 expression was increased (P< 0.05) in patients versus controls, consistent with ApoM/S1P pathway modulation. gApoM positively correlated with pApoM in the overall cohort (r= 0.34, P< 0.01) and in the FSGS (r= 0.48, P< 0.05) and minimal change disease (MCD) (r= 0.75, P< 0.05) subgroups. Every unit decrease in gApoM and pApoM (log2) was associated with a 9.77 ml/min per 1.73 m2 (95% confidence interval [CI]: 3.96-15.57) and 13.26 ml/min per 1.73 m2 (95% CI: 3.57-22.96) lower baseline eGFR, respectively (P< 0.01). From Cox models adjusted for age, sex, or race, pApoM was a significant predictor of CR (hazard ratio [HR]: 1.85; 95% CI: 1.06-3.23). pApoM is a potential noninvasive biomarker of gApoM deficiency and strongly associates with clinical outcomes in GD.

Sphingosine 1-Phosphate as Essential Signaling Molecule in Inflammatory Skin Diseases.

Sphingolipids are crucial molecules of the mammalian epidermis. The formation of skin-specific ceramides contributes to the formation of lipid lamellae, which are important for the protection of the epidermis from excessive water loss and protect the skin from the invasion of pathogens and the penetration of xenobiotics. In addition to being structural constituents of the epidermal layer, sphingolipids are also key signaling molecules that participate in the regulation of epidermal cells and the immune cells of the skin. While the importance of ceramides with regard to the proliferation and differentiation of skin cells has been known for a long time, it has emerged in recent years that the sphingolipid sphingosine 1-phosphate (S1P) is also involved in processes such as the proliferation and differentiation of keratinocytes. In addition, the immunomodulatory role of this sphingolipid species is becoming increasingly apparent. This is significant as S1P mediates a variety of its actions via G-protein coupled receptors. It is, therefore, not surprising that dysregulation in the signaling pathways of S1P is involved in the pathophysiological conditions of skin diseases. In the present review, the importance of S1P in skin cells, as well as the immune cells of the skin, is elaborated. In particular, the role of the molecule in inflammatory skin diseases will be discussed. This is important because interfering with S1P signaling pathways may represent an innovative option for the treatment of inflammatory skin diseases.

Image-Based Longitudinal Characterization of Corneal Wound to Understand the Role of Sphingosine-1-Phosphate.

Since its discovery, the bioactive sphingolipid sphingosine 1-phosphate (S1P) has been shown to involve in a myriad of cellular and physiological processes. In the process of tissue healing, S1P plays an important role in both normal and pathological healing, leading to fibrosis in multiple tissues including the cornea. Cornea covers the anterior portion of the eye and is responsible for the refraction of light. Corneal transparency is essential to obtain a clear vision, and a proper wound healing process is necessary for a clear cornea. Even though S1P is indicated to be a critical player in corneal fibrosis, we lack a detailed understanding of the role of S1P signaling in corneal wound healing and fibrosis. Herein, we describe a methodology to characterize the in-vivo wound healing process of the cornea using an easy and affordable imaging-based assay. This gives a consistent and easy way to characterize the wound and also the longitudinal healing process.

Angiogenesis Model of Cornea to Understand the Role of Sphingosine 1-Phosphate.

The bioactive sphingolipid sphingosine 1-phosphate (S1P) and its five cognate receptors (S1PR1-5) have been implicated to play important role in multiple aspects of human physiology and diseases. The S1P-S1PR1 signaling axis in endothelial cells is crucial for establishing flow competent blood vessels. The role of S1P in neovascular pathology is of great interest and is evolving as a promising target for treatment. Here we describe an easy and affordable in vivo model of corneal neovascularization using an alkali chemical burn to the cornea. This method gives a consistent and easy-to-quantitate procedure for neovascularization and angiogenesis studies.

Sphingosine-1-phosphate improves outcome of no-reflow acute myocardial infarction via sphingosine-1-phosphate receptor 1.

Therapeutic options targeting post-ischaemic cardiac remodelling are sparse. The bioactive sphingolipid sphingosine-1-phosphate (S1P) reduces ischaemia/reperfusion injury. However, its impact on post-ischaemic remodelling independently of its infarct size (IS)-reducing effect is yet unknown and was addressed in this study. Acute myocardial infarction (AMI) in mice was induced by permanent ligation of the left anterior descending artery (LAD). C57Bl6 were treated with the S1P lyase inhibitor 4-deoxypyridoxine (DOP) starting 7days prior to AMI to increase endogenous S1P concentrations. Cardiac function and myocardial healing were assessed by cardiovascular magnetic resonance imaging (cMRI), murine echocardiography, histomorphology, and gene expression analysis. DOP effects were investigated in cardiomyocyte-specific S1P receptor 1 deficient (S1PR1 Cardio Cre+) and Cre- control mice and S1P concentrations measured by LC-MS/MS. IS and cardiac function did not differ between control and DOP-treated groups on day one after LAD-ligation despite fourfold increase in plasma S1P. In contrast, cardiac function was clearly improved and myocardial scar size reduced, respectively, on Day 21 in DOP-treated mice. The latter also exhibited smaller cardiomyocyte size and reduced embryonic gene expression. The benefit of DOP treatment was abolished in S1PR1 Cardio Cre+. S1P improves cardiac function and myocardial healing post AMI independently of initial infarct size and accomplishes this via the cardiomyocyte S1PR1. Hence, in addition to its beneficial effects on I/R injury, S1PR1 may be a promising target in post-infarction myocardial remodelling as adjunctive therapy to revascularization as well as in patients not eligible for standard interventional procedures.

S1P lyase inhibition improves post-ischemic cardiac remodeling independently of infarct size via S1P receptor 1

Abstract Background Revascularization of the infarct vessel is the golden standard in acute myocardial infarction (AMI). Even in Western countries, more than ten percent of AMI patients are so-called “latecomers” and it is controversial if revascularization of the infarct vessel is beneficial in these patients. Current guidelines even discourage revascularization of the infarcted artery if symptom onset was &amp;gt;48 hours and the patient is asymptomatic. These patients are at high risk for cardiovascular events and heart failure with reduced ejection fraction (HFrEF). HFrEF has an enormous socio-economic impact, high morbidity, and mortality. Purpose Therapeutic options targeting post-ischemic cardiac remodeling are sparse. The bioactive sphingolipid sphingosine-1-phosphate (S1P) reduces ischemia/reperfusion injury when administered in advance. However, its impact on post-ischemic remodeling independently of its infarct size (IS)-reducing effect is yet unknown. Methods Acute myocardial infarction (AMI) in mice was induced by permanent ligation of the left anterior descending artery (LAD). C57Bl/6J were treated with the S1P lyase inhibitor 4-deoxypyridoxine (DOP) starting seven days prior to AMI to increase endogenous S1P concentrations. Cardiac function and myocardial healing were assessed by cardiovascular magnetic resonance imaging (cMRI), histomorphology and gene expression analysis. DOP effects were investigated in cardiomyocyte-specific S1P receptor 1 deficient (S1PR1 Cardio Cre+ and Cre− control) mice, and S1P concentrations measured by LC-MS/MS. Results S1P concentrations in plasma before induction of AMI were increased fourfold by DOP (Control 0.97±0.09μM [n=6] vs. DOP 3,80±0,09μM [n=6]). Scar size determined by MRI, as well as ejection fraction (EF), did not differ 24 h post AMI. In contrast, after 21 days, there was a clear difference between the two groups (scar size vehicle: 19.3±6.2% vs. DOP 13.4±5.7%; EF: Vehicle: 26.4±8.7% vs. DOP 38.2±11.8%). In addition, in the remote area 21 days post AMI in the DOP-treated animals, a reduced gene expression of brain natriuretic peptide, atrial natriuretic peptide and collagen 1a2. Finally, cardiomyocyte diameter in the remote myocardium was 21% smaller in DOP-treated (Vehicle: 21.95±1.59μm vs. DOP 17.35±0.77μm). The benefit of DOP-treatment was abolished in cardiomyocyte-specific S1PR1-deficient mice. Conclusion S1P improves cardiac function and myocardial healing post AMI independently of initial infarct size via S1PR1. Hence, in addition to its beneficial effects on I/R injury, S1PR1 may be a promising target in post-infarction myocardial remodeling as adjunctive therapy to revascularization and in patients who are not eligible for standard interventional procedures. Funding Acknowledgement Type of funding sources: None.

Neurological Consequences of Sphingosine Phosphate Lyase Insufficiency.

In 2017, an inborn error of metabolism caused by recessive mutations in SGPL1 was discovered. The disease features steroid-resistant nephrotic syndrome, adrenal insufficiency, and neurological defects. The latter can include sensorineural hearing loss, cranial nerve defects, peripheral neuropathy, abnormal brain development, seizures and/or neurodegeneration. SGPL1 encodes the pyridoxal-5’-phosphate (PLP) dependent enzyme sphingosine phosphate lyase (SPL), and the condition is now referred to as SPL insufficiency syndrome (SPLIS). SPL catalyzes the final step in the degradative pathway of sphingolipids in which the bioactive sphingolipid sphingosine-1-phosphate (S1P) is irreversibly degraded to a long chain aldehyde and phosphoethanolamine (PE). SPL guards the only exit point for sphingolipid metabolism, and its inactivation leads to accumulation of various types of sphingolipids which have biophysical roles in plasma membrane rafts and myelin, and signaling roles in cell cycle progression, vesicular trafficking, cell migration, and programmed cell death. In addition, the products of the SPL reaction have biological functions including regulation of autophagic flux, which is important in axonal and neuronal integrity. In this review, the neurological manifestations of SPLIS will be described, and insights regarding the neurological consequences of SPL insufficiency from the study of brain-specific SPL knockout mice and Drosophila SPL mutants will be summarized.

Silence of S1PR4 Represses the Activation of Fibroblast-like Synoviocytes by Regulating IL-17/STAT3 Signaling Pathway.

Rheumatoid arthritis (RA) is a chronic inflammatory disease with persistent inflammation and progressive joint damage. However, the underlying pathological mechanisms of RA are still unclear. Fibroblast‑like synoviocytes (FLSs) play an important role in the pathogenesis of RA by the regulation of proliferation and apoptosis, and the release of multiple pro-inflammatory factors. The lipid mediator sphingosine-1-phosphate receptor 4 (S1PR4) is one of the sphingolipid sphingosine-1-phosphate (S1P) receptors, which affects the function of immune cells. However, the role of S1PR4 in the activation of FLSs and the development of RA is unknown. In this study, we found that the expression of S1PR4 was significantly increased in RA-FLSs. The silence of S1PR4 decreases the proliferation, migration, proinflammation, and promotes the apoptosis of RA-FLSs, accompanied with repressing interleukin-17 (IL-17)/signal transducer and activator of transcription 3 (STAT3) signal pathway. However, the regulatory effects of S1PR4 silencing on RA-FLSs were partly abolished by additional recombinant human (rh) IL-17A treatment. Therefore, our study demonstrated that S1PR4 silencing might inhibit proliferation, migration, proinflammation, and promote apoptosis of RA-FLSs partly by repressing IL-17, which suggests that inhibitors for S1PR4 might be a potentially promising strategy for the treatment of RA.

FTY720 decreases ceramides levels in the brain and prevents memory impairments in a mouse model of familial Alzheimer's disease expressing APOE4.

The protection mediated by the bioactive sphingolipid sphingosine-1-phosphate (S1P) declines during Alzheimer's disease (AD) progression, especially in patients carrying the apolipoprotein E ε4 (APOE4) isoform. The drug FTY720 mimics S1P bioactivity, but its efficacy in treating AD is unclear. Two doses of FTY720 (0.1mg / kg and 0.5mg / kg daily) were given by oral gavage for 15 weeks to transgenic mouse models of familial AD carrying human apolipoprotein E (APOE) APOE3 (E3FAD) or APOE4 (E4FAD). After 12 weeks of treatment, animals were subjected to behavioral tests for memory, locomotion, and anxiety. Blood was withdrawn at different time points and brains were collected for sphingolipids analysis by mass spectrometry, gene expression by RT-PCR and Aβ quantification by ELISA. We discovered that low levels of S1P in the plasma is associated with a higher probability of failing the memory test and that FTY720 prevents memory impairments in E4FAD. The beneficial effect of FTY720 was induced by a shift of the sphingolipid metabolism in the brain towards a lower production of toxic metabolites, like ceramide d18:1/16:0 and d18:1/22:0, and reduction of amyloid-β burden and inflammation. In conclusion, we provide further evidence of the druggability of the sphingolipid system in AD.

Bedside to benchtop translational development: Targeting the S1P/ceramide axis may alleviate symptoms of chemical induced peripheral neuropathy.

e15045 Background: Chemical Induced Peripheral Neuropathy (CIPN) is a debilitating and serious side-effect associated with many chemotherapeutic treatments. Often, CIPN is a dose-limiting toxicity, and its effects can be long-lasting in some patients. The pathology of CIPN is thought to involve not only cytotoxicity of neuronal cells, but also inflammation and immune responses. Hence, it is not surprising that the sphingolipid sphingosine-1-phosphate (S1P) is thought to be a key contributor to CIPN. Methods: BXQ-350, a nanovesicle comprised of recombinantly expressed Saposin C and dioleoylphosphatidylserine, has broad anticancer activity. It modulates sphingolipid metabolism and signaling, lowers S1P and increases pro-apoptotic ceramides, and induces an anti-tumoral immune response. BXQ-350 was investigated in a Phase 1 dose-escalation safety study of all-comer cancer patients with advanced solid malignancies, including high grade gliomas ( NCT02859857 )*. Multiple secondary parameters were included to characterize BXQ-350’s pharmacokinetics, efficacy profile and to elucidate potential biomarkers. Results: Interestingly, several patients with established CIPN spontaneously reported improvement of their neuropathy-related symptoms following BXQ-350 administration. Plasma samples from those patients revealed changes in circulating levels of sphingolipids and cytokines, including reduction of circulating levels of S1P and of IL-6, IL-8, cytokines, following BXQ-350 dosing. These properties of BXQ-350 were subsequently investigated in a murine oxaliplatin-CIPN preclinical model; results showed a dose-dependent prevention/resolution of CIPN, which also correlated with decreasing systemic S1P levels. Additional plasma based CIPN specific biomarkers were investigated, and preclinical results suggest that BXQ-350 may also favorably inhibit specific immune cells that favor CPIN. Conclusions: These preclinical and clinical observations related to CIPN, coupled with the fact that BXQ-350 acts synergistically with many chemotherapies and radiation, warrant further its investigation into preventing or improving CIPN related symptoms in cancer patients. Clinical trial information: 02859857.

Keep a Little Fire Burning-The Delicate Balance of Targeting Sphingosine-1-Phosphate in Cancer Immunity.

The sphingolipid sphingosine-1-phosphate (S1P) promotes tumor development through a variety of mechanisms including promoting proliferation, survival, and migration of cancer cells. Moreover, S1P emerged as an important regulator of tumor microenvironmental cell function by modulating, among other mechanisms, tumor angiogenesis. Therefore, S1P was proposed as a target for anti-tumor therapy. The clinical success of current cancer immunotherapy suggests that future anti-tumor therapy needs to consider its impact on the tumor-associated immune system. Hereby, S1P may have divergent effects. On the one hand, S1P gradients control leukocyte trafficking throughout the body, which is clinically exploited to suppress auto-immune reactions. On the other hand, S1P promotes pro-tumor activation of a diverse range of immune cells. In this review, we summarize the current literature describing the role of S1P in tumor-associated immunity, and we discuss strategies for how to target S1P for anti-tumor therapy without causing immune paralysis.

Sphingolipids in Lung Pathology in the Coronavirus Disease Era: A Review of Sphingolipid Involvement in the Pathogenesis of Lung Damage.

Sphingolipids are bioactive lipids involved in the regulation of cell survival, proliferation, and the inflammatory response. The SphK/S1P/S1PR pathway (S1P pathway) is a driver of many anti-apoptotic and proliferative processes. Pro-survival sphingolipid sphingosine-1-phosphate (S1P) initiates its signaling cascade by interacting with various sphingosine-1-phosphate receptors (S1PR) through which it is able to exert its pro-survival or inflammatory effects. Whereas sphingolipids, including ceramides and sphingosines are pro-apoptotic. The pro-apoptotic lipid, ceramide, can be produced de novo by ceramide synthases and converted to sphingosine by way of ceramidases. The balance of these antagonistic lipids and how this balance manifests is the essence of the sphingolipid rheostat. Recent studies on SARS-CoV-2 have implicated the S1P pathway in the pathogenesis of novel coronavirus disease COVID-19-related lung damage. Accumulating evidence indicates that an aberrant inflammatory process, known as “cytokine storm” causes lung injury in COVID-19, and studies have shown that the S1P pathway is involved in signaling this hyperinflammatory response. Beyond the influence of this pathway on cytokine storm, over the last decade the S1P pathway has been investigated for its role in a wide array of lung pathologies, including pulmonary fibrosis, pulmonary arterial hypertension (PAH), and lung cancer. Various studies have used S1P pathway modulators in models of lung disease; many of these efforts have yielded results that point to the potential efficacy of targeting this pathway for future treatment options. Additionally, they have emphasized S1P pathway’s significant role in inflammation, fibrosis, and a number of other endothelial and epithelial changes that contribute to lung damage. This review summarizes the S1P pathway’s involvement in COVID-19 and chronic lung diseases and discusses the potential for targeting S1P pathway as a therapeutic option for these diseases.

S1P Stimulates Erythropoietin Production in Mouse Renal Interstitial Fibroblasts by S1P1 and S1P3 Receptor Activation and HIF-2α Stabilization.

Erythropoietin (Epo) is the critical hormone for erythropoiesis. In adults, Epo is mainly produced by a subset of interstitial fibroblasts in the kidney, with minor amounts being produced in the liver and the brain. In this study, we used the immortalized renal interstitial fibroblast cell line FAIK F3-5 to investigate the ability of the bioactive sphingolipid sphingosine 1-phosphate (S1P) to stimulate Epo production and to reveal the mechanism involved. Stimulation of cells with exogenous S1P under normoxic conditions (21% O2) led to a dose-dependent increase in Epo mRNA and protein levels and subsequent release of Epo into the medium. S1P also enhanced the stabilization of HIF-2α, a key transcription factor for Epo expression. S1P-stimulated Epo mRNA and protein expression was abolished by HIF-2α mRNA knockdown or by the HIF-2 inhibitor compound 2. Furthermore, the approved S1P receptor modulator FTY720, and its active form FTY720-phosphate, both exerted a similar effect on Epo expression as S1P. The effect of S1P on Epo was antagonized by the selective S1P1 and S1P3 antagonists NIBR-0213 and TY-52156, but not by the S1P2 antagonist JTE-013. Moreover, inhibitors of the classical MAPK/ERK, the p38-MAPK, and inhibitors of protein kinase (PK) C and D all blocked the effect of S1P on Epo expression. Finally, the S1P and FTY720 effects were recapitulated in the Epo-producing human neuroblastoma cell line Kelly, suggesting that S1P receptor-dependent Epo synthesis is of general relevance and not species-specific. In summary, these data suggest that, in renal interstitial fibroblasts, which are the primary source of plasma Epo, S1P1 and 3 receptor activation upregulates Epo under normoxic conditions. This may have a therapeutic impact on disease situations such as chronic kidney disease, where Epo production is impaired, causing anemia, but it may also have therapeutic value as Epo can mediate additional tissue-protective effects in various organs.

Investigation of Sphingosine‐1‐Phosphate‐induced Vasodilation in the Human Microvasculature

The sphingolipid sphingosine-1-phosphate (S1P) has emerged as a regulator of microvascular tone in animals, however its role in the human microcirculation remains unknown. Pre-clinical studies suggest that S1P-induced activation of two endothelial-specific receptors, S1PR1 and S1PR3,elicits nitric-oxide (NO)-mediated vasodilation. As opposed to S1PR1, the S1PR3 pathway also activates NADPH oxidase (NOX) to produce reactive oxygen species (ROS). We therefore hypothesized that S1P induces vasodilation in the human microvasculature through activation of S1PR1 and S1PR3 in both a ROS-independent and ROS-dependent manner, respectively. Human microvessels (100-200µm in diameter) were prepared for videomicroscopy. Following equilibration, arterioles were pre-constricted with endothelin-1 to 30-70% of their passive diameters. Luminal diameter was measured and recorded in regular intervals (1 min) in response to increasing concentrations of S1P (10-12 to 10-6 M) in the presence or absence of the S1PR1 receptor antagonist W146 (10-5M), S1PR3 antagonist CAY10444 (10-5 M), nitric oxide synthase inhibitor Nω-nitro-ʟ-arginine (L-NAME, 10-4 M), NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO, 10-4 M), polyethylene glycol-catalase (peg-Cat, 500U/ml), NADPH oxidase inhibitor apocynin (3x10-4 M), NOX-2 inhibitor GSK2795039 (10-6 M) or the NOX-4 inhibitor GKT137831 (10−6 M). S1P induced vasodilation in a dose-responsive manner with a maximum dilation of 56.5%±4.9, n=12 (mean±SEM). Dilation was abolished during inhibition of S1PR1 (1.5%±5.2, n=4) and was reduced during inhibition of S1PR3 (19.7%±8.7, n=6). Both L-NAME and c-PTIO inhibited S1P-induced dilation (13.0%±7.5, n=4 and 11.2%±3.3, n=4, respectively). Interestingly, dilation was nearly completely inhibited by peg-Cat (11%±6.6, n=4), apocynin (12.5%±4.4, n=3) and the NOX-4 inhibitor (7.2%±3.1, n=4). Dilation was also partly reduced during inhibition of NOX-2 (25.6%±7.8, n=4). These data suggest that S1P-induced dilation occurs through activation of S1PR1 and S1PR3 through formation of NO and NOX-4-generated H2O2. These translational studies highlight the inter-species variation observed in vascular signaling and provide insight into the mechanism by which S1P regulates microvascular resistance in humans.

Sphingosine 1-phosphate receptors are dysregulated in endometriosis: possible implication in transforming growth factor β–induced fibrosis

To study the molecular mechanisms involved in the appearance of the fibrotic trait in endometriosis by investigating whether the signaling pathway of the bioactive sphingolipid sphingosine 1-phosphate (S1P) was altered in endometriotic lesions. Case-control laboratory study. University research institute and university hospital. A total of 75 women, with and without endometriosis, were included in the study. Endometrial samples were obtained from women affected (n = 15 endometrioma [OMA]; n = 30 deep infiltrating endometriosis [DIE]) and not (n = 30) by endometriosis by means of laparoscopic surgery, followed by clinical and imaging investigation and checking for the expression of fibrosis markers and genes implicated in S1P metabolism and signaling by means of real-time polymerase chain reaction. The role of the S1P signaling axis in endometriosis-associated fibrosis was studied invitro, where RNA interference approaches were used to investigate if S1P synthesis by sphingosine kinases (SKs) and specific S1P receptors (S1PRs) are implicated in the profibrotic effect of the cytokine transforming growth factor (TGF) β1. mRNA expression analysis of S1PR demonstrated a deep dysregulation of S1P signaling in endometriosis, characterized by increased expression of fibrosis markers: S1P1 was transcriptionally more expressed in OMA, and S1P3 and S1P5 mRNA levels were significantly augmented in both OMA and DIE. SK1 and its activating protein calcium- and integrin-binding protein 1 (CIB1) were significantly up-regulated in OMA and DIE. A crucial role for the SK/S1PR axis in the profibrotic effect elicited by TGFβ1 was highlighted invitro. The S1P signaling axis may represent a useful biomarker or innovative pharmacologic target for endometriosis.