Sphingosine-1-phosphate Receptor 1 Signaling Research Articles

Mitigation of myocardial ischemia/reperfusion–induced chronic heart failure via Shexiang Baoxin Pill–mediated regulation of the S1PR1 signaling pathway

BackgroundWell-defined and effective pharmacological interventions for clinical management of myocardial ischemia/reperfusion (MI/R) injury are currently unavailable. Shexiang Baoxin Pill (SBP), a traditional Chinese medicine Previous research on SBP has been confined to single-target treatments for MI/R injury, lacking a comprehensive examination of various aspects of MI/R injury and a thorough exploration of its underlying mechanisms. PurposeThis study aimed to investigate the therapeutic potential of SBP for MI/R injury and its preventive effects on consequent chronic heart failure (CHF). Furthermore, we elucidated the specific mechanisms involved, contributing valuable insights into the potential pharmacological interventions for the clinical treatment of MI/R injury. MethodsWe conducted a comprehensive identification of SBP components using high-performance liquid chromatography. Subsequently, we performed a network pharmacology analysis based on the identification results, elucidating the key genes influenced by SBP. Thereafter, through bioinformatics analysis of the key genes and validation through mRNA and protein assays, we ultimately determined the centralized upstream targets. Lastly, we conducted in vitro experiments using myocardial and endothelial cells to elucidate and validate potential underlying mechanisms. ResultsSBP can effectively mitigate cell apoptosis, oxidative stress, and inflammation, as well as promote vascular regeneration following MI/R, resulting in improved cardiac function and reduced CHF risk. Mechanistically, SBP treatment upregulates sphingosine-1-phosphate receptor 1 (S1PR1) expression and activates the S1PR1 signaling pathway, thereby regulating the expression of key molecules, including phosphorylated Protein Kinase B (AKT), phosphorylated signal transducer and activator of transcription 3, epidermal growth factor receptor, vascular endothelial growth factor A, tumor necrosis factor-α, and p53. ConclusionThis study elucidated the protective role of SBP in MI/R injury and its potential to reduce the risk of CHF. Furthermore, by integrating downstream effector proteins affected by SBP, this research identified the upstream effector protein S1PR1, enhancing our understanding of the pharmacological characteristics and mechanisms of action of SBP. The significance of this study lies in providing compelling evidence for the use of SBP as a traditional Chinese medicine for MI/R injury and consequent CHF prevention.

Role of the S1P Signaling Pathway in the Pathogenesis of Angioimmunoblastic T-Cell Lymphoma

Peripheral T cell lymphomas (PTCLs) are a group of aggressive lymphoid malignancies originating from mature T cells. Angioimmunoblastic T-cell lymphomas (AITL) represent 20% to 30% of all PTCL diagnoses and are associated with autoimmune features, poor response to chemotherapy, and dismal prognosis. Molecular profiling of AITL has identified T-follicular helper (TFH) cells as the cell of origin of AITL, while mutational analysis has identified frequent alterations in epigenetic regulators ( TET2, DNMT3A, and IDH2) and elements of the TCR pathway as drivers of AITL transformation. Our group identified the highly recurrent RHOA G17V mutation as a defining hallmark of AITL and other T-cell lymphomas of TFH-cell origin (Palomero et al., 2014). Using a novel conditional knockin mouse model, we demonstrated that expression of Rhoa G17V in CD4+ T-cells induces TFH cell specification and promotes AITL lymphomagenesis in the context of loss of Tet2 (Cortes et al., 2018). Recently, we have identified that Rhoa G17V regulates the expression of the sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) one of the five members of the receptor family of S1P, which plays an essential role in immune response and lymphocyte trafficking. During thymic T-cell development, expression of Rhoa G17V in CD4+ cells inhibited S1PR1 downregulation after the double positive stage leading to altered positive selection, reduced CD4+ single positive cell numbers and premature thymic egress of autorreactive T-cells which results in the development of systemic inflammation and autoimmunity. In our Tet2 −/− RHOA G17V AITL tumor model, constitutive activation of S1P receptors supported activation of the STAT3 and NFkB pathway and enhanced migration of malignant cells to peripheral organs. Mechanistically, expression of Rhoa G17V in mature CD4+ T-cells impaired re-phosphorylation of the Ezrin-Radoxin-Moesin (ERM) complex independently of ROCK signaling and led to the activation and increased migration of CD4+ T-cells. Importantly, blockade of S1PR1 signaling by fingolimod induced anti-tumor activity in Tet2 −/− RHOA G17V lymphoma-bearing mice in vivo with significant reductions in tumor burden associated with increased apoptosis and decreased STAT3 phosphorylation. Furthermore, analysis of a panel of primary tumors from AITL patients using a multiplexed staining panel and PhenoImager platform revealed significantly higher S1PR1 expression in AITL tumor cells than normal TFH cells from benign reactive lymph nodes. Our findings highlight the role of S1P signaling pathway in the pathogenesis of RHOA G17V-driven AITLs and the potential therapeutic benefit of targeting this pathway.

Abnormal Lymphatic Sphingosine-1-Phosphate Signaling Aggravates Lymphatic Dysfunction and Tissue Inflammation.

Lymphedema is a global health problem with no effective drug treatment. Enhanced T-cell immunity and abnormal lymphatic endothelial cell (LEC) signaling are promising therapeutic targets for this condition. Sphingosine-1-phosphate (S1P) mediates a key signaling pathway required for normal LEC function, and altered S1P signaling in LECs could lead to lymphatic disease and pathogenic T-cell activation. Characterizing this biology is relevant for developing much needed therapies. Human and mouse lymphedema was studied. Lymphedema was induced in mice by surgically ligating the tail lymphatics. Lymphedematous dermal tissue was assessed for S1P signaling. To verify the role of altered S1P signaling effects in lymphatic cells, LEC-specific S1pr1-deficient (S1pr1LECKO) mice were generated. Disease progression was quantified by tail-volumetric and -histopathologic measurements over time. LECs from mice and humans, with S1P signaling inhibition, were then cocultured with CD4 T cells, followed by an analysis of CD4 T-cell activation and pathway signaling. Last, animals were treated with a monoclonal antibody specific to P-selectin to assess its efficacy in reducing lymphedema and T-cell activation. Human and experimental lymphedema tissues exhibited decreased LEC S1P signaling through S1P receptor 1 (S1PR1). LEC S1pr1 loss-of-function exacerbated lymphatic vascular insufficiency, tail swelling, and increased CD4 T-cell infiltration in mouse lymphedema. LECs, isolated from S1pr1LECKO mice and cocultured with CD4 T cells, resulted in augmented lymphocyte differentiation. Inhibiting S1PR1 signaling in human dermal LECs promoted T-helper type 1 and 2 (Th1 and Th2) cell differentiation through direct cell contact with lymphocytes. Human dermal LECs with dampened S1P signaling exhibited enhanced P-selectin, an important cell adhesion molecule expressed on activated vascular cells. In vitro, P-selectin blockade reduced the activation and differentiation of Th cells cocultured with shS1PR1-treated human dermal LECs. P-selectin-directed antibody treatment improved tail swelling and reduced Th1/Th2 immune responses in mouse lymphedema. This study suggests that reduction of the LEC S1P signaling aggravates lymphedema by enhancing LEC adhesion and amplifying pathogenic CD4 T-cell responses. P-selectin inhibitors are suggested as a possible treatment for this pervasive condition.

Dialogue between VE-Cadherin and Sphingosine 1 Phosphate Receptor1 (S1PR1) for Protecting Endothelial Functions.

The endothelial cells (EC) of established blood vessels in adults remain extraordinarily quiescent in the sense that they are not actively proliferating, but they fulfill the necessary role to control the permeability of their monolayer that lines the interior of blood vessels. The cell-cell junctions between ECs in the endothelium comprise tight junctions and adherens homotypic junctions, which are ubiquitous along the vascular tree. Adherens junctions are adhesive intercellular contacts that are crucial for the organization of the EC monolayer and its maintenance and regulation of normal microvascular function. The molecular components and underlying signaling pathways that control the association of adherens junctions have been described in the last few years. In contrast, the role that dysfunction of these adherens junctions has in contributing to human vascular disease remains an important open issue. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid mediator found at high concentrations in blood which has important roles in the control of the vascular permeability, cell recruitment, and clotting that follow inflammatory processes. This role of S1P is achieved through a signaling pathway mediated through a family of G protein-coupled receptors designated as S1PR1. This review highlights novel evidence for a direct linkage between S1PR1 signaling and the mediation of EC cohesive properties that are controlled by VE-cadherin.

Pharmacological sphingosine-1 phosphate receptor 1 targeting in cigarette smoke-induced emphysema in mice.

Primarily caused by chronic cigarette smoking (CS), emphysema is characterized by loss of alveolar cells comprising lung units involved in gas exchange and inflammation that culminate in airspace enlargement. Dysregulation of sphingolipid metabolism with increases of ceramide relative to sphingosine-1 phosphate (S1P) signaling has been shown to cause lung cell apoptosis and is emerging as a potential therapeutic target in emphysema. We sought to determine the impact of augmenting S1P signaling via S1P receptor 1 (S1P1) in a mouse model of CS-induced emphysema. DBA2 mice were exposed to CS for 4 or 6 mo and treated with pharmacological agonists of S1P1: phosphonated FTY720 (FTY720-1S and 2S analogs; 0.01-1.0 mg/kg) or GSK183303A (10 mg/kg). Pharmacological S1P1 agonists ameliorated CS-induced lung parenchymal apoptosis and airspace enlargement as well as loss of body weight. S1P1 agonists had modest inhibitory effects on CS-induced airspace inflammation and lung functional changes measured by Flexivent, improving lung tissue resistance. S1P1 abundance was reduced in chronic CS-conditions and remained decreased after CS-cessation or treatment with FTY720-1S. These results support an important role for S1P-S1P1 axis in maintaining the structural integrity of alveoli during chronic CS exposure and suggest that increasing both S1P1 signaling and abundance may be beneficial to counteract the effects of chronic CS exposure.

S1PR1 signaling in cancer: A current perspective.

Sphingosine-1-phosphate receptor 1 (S1PR1) is a G-protein coupled receptor for the bioactive lysosphingolipid sphingosine 1-phosphate (S1P). S1PR1 belongs to the sphingosine-1-phosphate receptor subfamily comprising five members (S1PR1-5). It has prominent roles in regulating endothelial cell cytoskeletal structure, cell migration, immunomodulation, vasculogenesis during embryogenesis, T cell egress and Multiple sclerosis. This review is addressing the role of S1PR1 in tumorigenesis and therapeutic opportunities to target S1PR1 in cancer.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

Sphingosine 1-phosphate-regulated transcriptomes in heterogenous arterial and lymphatic endothelium of the aorta.

Despite the medical importance of G protein-coupled receptors (GPCRs), in vivo cellular heterogeneity of GPCR signaling and downstream transcriptional responses are not understood. We report the comprehensive characterization of transcriptomes (bulk and single-cell) and chromatin domains regulated by sphingosine 1-phosphate receptor-1 (S1PR1) in adult mouse aortic endothelial cells. First, S1PR1 regulates NFκB and nuclear glucocorticoid receptor pathways to suppress inflammation-related mRNAs. Second, S1PR1 signaling in the heterogenous endothelial cell (EC) subtypes occurs at spatially-distinct areas of the aorta. For example, a transcriptomically distinct arterial EC population at vascular branch points (aEC1) exhibits ligand-independent S1PR1/ß-arrestin coupling. In contrast, circulatory S1P-dependent S1PR1/ß-arrestin coupling was observed in non-branch point aEC2 cells that exhibit an inflammatory gene expression signature. Moreover, S1P/S1PR1 signaling regulates the expression of lymphangiogenic and inflammation-related transcripts in an adventitial lymphatic EC (LEC) population in a ligand-dependent manner. These insights add resolution to existing concepts of endothelial heterogeneity, GPCR signaling and S1P biology.

Endothelial sphingosine 1-phosphate receptors promote vascular normalization and antitumor therapy

Sphingosine 1-phosphate receptor-1 (S1PR1) is essential for embryonic vascular development and maturation. In the adult, it is a key regulator of vascular barrier function and inflammatory processes. Its roles in tumor angiogenesis, tumor growth, and metastasis are not well understood. In this paper, we show that S1PR1 is expressed and active in tumor vessels. Murine tumor vessels that lack S1PR1 in the vascular endothelium (S1pr1 ECKO) show excessive vascular sprouting and branching, decreased barrier function, and poor perfusion accompanied by loose attachment of pericytes. Compound knockout of S1pr1, 2, and 3 genes further exacerbated these phenotypes, suggesting compensatory function of endothelial S1PR2 and 3 in the absence of S1PR1. On the other hand, tumor vessels with high expression of S1PR1 (S1pr1 ECTG) show less branching, tortuosity, and enhanced pericyte coverage. Larger tumors and enhanced lung metastasis were seen in S1pr1 ECKO, whereas S1pr1 ECTG showed smaller tumors and reduced metastasis. Furthermore, antitumor activity of a chemotherapeutic agent (doxorubicin) and immune checkpoint inhibitor blocker (anti-PD-1 antibody) were more effective in S1pr1 ECTG than in the wild-type counterparts. These data suggest that tumor endothelial S1PR1 induces vascular normalization and influences tumor growth and metastasis, thus enhancing antitumor therapies in mouse models. Strategies to enhance S1PR1 signaling in tumor vessels may be an important adjunct to standard cancer therapy of solid tumors.

Myeloid sphingosine-1-phosphate receptor 1 is important for CNS autoimmunity and neuroinflammation

The critical role of sphingosine-1-phosphate (S1P) signaling in lymphocyte trafficking is well recognized, however, the contribution of myeloid cell-S1P signaling in neuroimmunity is less well understood. We previously reported that C57BL/6J mice harboring phosphorylation defective S1P receptor 1 (S1P1) (with mutated serines in the carboxyl terminus, leading to impaired receptor internalization) [S1P1(S5A)] developed severe, TH17-dominant experimental autoimmune encephalomyelitis. In this study, we demonstrate that S1P1-mediated TH17 polarization is not an intrinsic T cell effect, but dependent on sustained S1P1 signaling in myeloid cells. First, utilizing the S1P1(S5A) mice in the EAE model, we observed that S1P1 activated and enhanced antigen presentation function in myeloid cells. Second, sequential phosphorylation of STAT3 occurred in dendritic cells, monocytes, and macrophages/microglia during neuroinflammation. Third, we show that pro-inflammatory (CD45hiCD11b+Ly6Chi) monocytes contribute to TH17 differentiation and neuroinflammation by regulating IL-6 expression. Finally, results from experiments utilizing myeloid cell-specific S1P1 overexpression (S1pr1f/stop/f:LysMCre) mice demonstrate that myeloid cell S1P1 directly contributes to severity of neuroinflammation. These findings reveal the critical contribution of myeloid-S1P1 signaling in CNS autoimmunity.

RETRACTED ARTICLE: Combination of sphingosine-1-phosphate receptor 1 (S1PR1) agonist and antiviral drug: a potential therapy against pathogenic influenza virus

The pandemic 2009 influenza A H1N1 virus is associated with significant mortality. Targeting S1PR1, which is known to modulate the immune response, provides protection against pathogenic influenza virus. The functional role and molecular mechanism of S1PR1 were analysed by generating inducible endothelial cell-specific S1PR1 knockout mice and assessing the therapeutic efficacy of the selective S1PR1 agonist CYM5442 against acute lung injury (ALI) induced by the 2009 influenza A H1N1 virus. Immune-mediated pulmonary injury is aggravated by the absence of endothelial S1PR1 and alleviated by treatment with CYM-5442, suggesting a protective function of S1PR1 signaling during H1N1 infection. S1PR1 signaling does not affect viral clearance in mice infected with influenza. Mechanistically, the MAPK and NF-kB signaling pathways are involved in the ALI mediated by S1PR1 in infected mice. Combined administration of the S1PR1 agonist CYM-5442 and the antiviral drug oseltamivir provides maximum protection from ALI. Our current study provides insight into the molecular mechanism of S1PR1 mediating the ALI induced by H1N1 infection and indicates that the combination of S1PR1 agonist with antiviral drug could potentially be used as a therapeutic remedy for future H1N1 virus pandemics.

Sphingosine 1-Phosphate Receptor 1 Signaling Maintains Endothelial Cell Barrier Function and Protects Against ImmuneComplex-Induced Vascular Injury.

Immune complex (IC) deposition activates polymorphonuclear neutrophils (PMNs), increases vascular permeability, and leads to organ damage in systemic lupus erythematosus and rheumatoid arthritis. The bioactive lipid sphingosine 1-phosphate (S1P), acting via S1P receptor 1 (S1P1 ), is a key regulator of endothelial cell (EC) barrier function. This study was undertaken to investigate whether augmenting EC integrity via S1P1 signaling attenuates inflammatory injury mediated by ICs. In vitro barrier function was assessed in human umbilical vein endothelial cells (HUVECs) by electrical cell-substrate impedance sensing. Phosphorylation of myosin light chain 2 (p-MLC-2) and VE-cadherin staining in HUVECs were assessed by immunofluorescence. A reverse Arthus reaction (RAR) was induced in the skin and lungs of mice with S1P1 deleted from ECs (S1P1 EC-knockout [ECKO] mice) and mice treated with S1P1 agonists and antagonists. S1P1 agonists prevented loss of barrier function in HUVECs treated with IC-activated PMNs. S1P1 ECKO and wild-type (WT) mice treated with S1P1 antagonists had amplified RAR, whereas specific S1P1 agonists attenuated skin and lung RAR in WT mice. ApoM-Fc, a novel S1P chaperone, mitigated EC cell barrier dysfunction induced by activated PMNs invitro and attenuated lung RAR. Expression levels of p-MLC-2 and disruption of VE-cadherin, each representing manifestations of cell contraction and destabilization of adherens junctions, respectively, that were induced by activated PMNs, were markedly reduced by treatment with S1P1 agonists and ApoM-Fc. Our findings indicate that S1P1 signaling in ECs modulates vascular responses to IC deposition. S1P1 agonists and ApoM-Fc enhance the EC barrier, limit leukocyte escape from capillaries, and provide protection against inflammatory injury. The S1P/S1P1 axis is a newly identified target to attenuate tissue responses to IC deposition and mitigate end-organ damage.

SPHK1-S1PR1-RANKL Axis Regulates the Interactions Between Macrophages and BMSCs in Inflammatory Bone Loss.

Accumulating evidence indicates that the immune and skeletal systems interact with each other through various regulators during the osteoclastogenic process. Among these regulators, the bioactive lipid sphingosine-1-phosphate (S1P), which is synthesized by sphingosine kinase 1/2 (SPHK1/2), has recently been recognized to play a role in immunity and bone remodeling through its receptor sphingosine-1-phosphate receptor 1 (S1PR1). However, little is known regarding the potential role of S1PR1 signaling in inflammatory bone loss. We observed that SPHK1 and S1PR1 were upregulated in human apical periodontitis, accompanied by macrophage infiltration and enhanced expression of receptor activator of NF-κB ligand (RANKL, an indispensable factor in osteoclastogenesis and bone resorption) and increased numbers of S1PR1-RANKL double-positive cells in lesion tissues. Using an in vitro co-culture model of macrophages and bone marrow stromal cells (BMSCs), it was revealed that in the presence of lipopolysaccharide (LPS) stimulation, macrophages could significantly induce SPHK1 activity, which resulted in activated S1PR1 in BMSCs. The activated S1P-S1PR1 signaling was responsible for the increased RANKL production in BMSCs, as S1PR1-blockage abolished this effect. Applying a potent S1P-S1PR1 signaling modulator, Fingolimod (FTY720), in a Wistar rat apical periodontitis model effectively prevented bone lesions in vivo via downregulation of RANKL production, osteoclastogenesis, and bone resorption. Our data unveiled the regulatory role of SPHK1-S1PR1-RANKL axis in inflammatory bone lesions and proposed a potential therapeutic intervention by targeting this cell-signaling pathway to prevent bone loss. © 2018 American Society for Bone and Mineral Research.

Functional antagonism of sphingosine-1-phosphate receptor 1 prevents cuprizone-induced demyelination.

Recent evidence suggests that the oral drug Fingolimod (FTY720) for relapsing-remitting multiple sclerosis (MS) may act directly on the central nervous system (CNS) and modulate disease pathogenesis and progression in experimental models of MS. However, the specific subtype of sphingosine-1-phosphate (S1P) receptors that mediates the effect of FTY720 on the CNS cells has not been fully elucidated. Here, we report that S1P receptor 1 (S1PR1) is elevated in reactive astrocytes in an autoimmunity independent mouse model of MS and that selective S1PR1 modulation is sufficient to ameliorate the loss of oligodendrocytes and demyelination. The non-selective S1PR modulator, FTY720, or a short-lived S1PR1-specific modulator, CYM5442, was administered daily to mice while on cuprizone diet. Both FTY720- and CYM5422-treated mice displayed a significant reduction in oligodendrocyte apoptosis and astrocyte and microglial activation in comparison to vehicle-treated groups, which was associated with decreased production of proinflammatory mediators and down-regulation of astrocytic S1PR1 protein. Interestingly, S1PR1 modulation during the early phase of cuprizone intoxication was required to suppress oligodendrocyte death and consequent demyelination as drug treatment from 10 days after the initiation of cuprizone feeding was no longer effective. CYM5442 treatment during the brief cuprizone exposure significantly prevented Il-1β, Il-6, Cxcl10, and Cxcl3 induction, resulting in suppression of subsequent reactive gliosis and demyelination. Our study identifies functional antagonism of S1PR1 as a major mechanism for the protective effect of FTY720 in the cuprizone model and suggests pathogenic contributions of astrocyte S1PR1 signaling in primary demyelination and its potential as a therapeutic target for CNS inflammation.

Bioluminescence imaging of G protein-coupled receptor activation in living mice

G protein-coupled receptors (GPCRs), a superfamily of cell-surface receptors involved in virtually all physiological processes, are the major target class for approved drugs. Imaging GPCR activation in real time in living animals would provide a powerful way to study their role in biology and disease. Here, we describe a mouse model that enables the bioluminescent detection of GPCR activation in real time by utilizing the clinically important GPCR, sphingosine-1-phosphate receptor 1 (S1P1). A synthetic S1P1 signaling pathway, designed to report the interaction between S1P1 and β-arrestin2 via the firefly split luciferase fragment complementation system, is genetically encoded in these mice. Upon receptor activation and subsequent β-arrestin2 recruitment, an active luciferase enzyme complex is produced, which can be detected by in vivo bioluminescence imaging. This imaging strategy reveals the dynamics and spatial specificity of S1P1 activation in normal and pathophysiologic contexts in vivo and can be applied to other GPCRs.

β1-Blockade Prevents Post-Ischemic Myocardial Decompensation Via β3AR-Dependent Protective Sphingosine-1 Phosphate Signaling

BackgroundAlthough β-blockers increase survival in patients with heart failure (HF), the mechanisms behind this protection are not fully understood, and not all patients with HF respond favorably to them. We recently showed that, in cardiomyocytes, a reciprocal down-regulation occurs between β1-adrenergic receptors (ARs) and the cardioprotective sphingosine-1-phosphate (S1P) receptor-1 (S1PR1). ObjectivesThe authors hypothesized that, in addition to salutary actions due to direct β1AR-blockade, agents such as metoprolol (Meto) may improve post–myocardial infarction (MI) structural and functional outcomes via restored S1PR1 signaling, and sought to determine mechanisms accounting for this effect. MethodsWe tested the in vitro effects of Meto in HEK293 cells and in ventricular cardiomyocytes isolated from neonatal rats. In vivo, we assessed the effects of Meto in MI wild-type and β3AR knockout mice. ResultsHere we report that, in vitro, Meto prevents catecholamine-induced down-regulation of S1PR1, a major cardiac protective signaling pathway. In vivo, we show that Meto arrests post-MI HF progression in mice as much as chronic S1P treatment. Importantly, human HF subjects receiving β1AR-blockers display elevated circulating S1P levels, confirming that Meto promotes S1P secretion/signaling. Mechanistically, we found that Meto-induced S1P secretion is β3AR-dependent because Meto infusion in β3AR knockout mice does not elevate circulating S1P levels, nor does it ameliorate post-MI dysfunction, as in wild-type mice. ConclusionsOur study uncovers a previously unrecognized mechanism by which β1-blockers prevent HF progression in patients with ischemia, suggesting that β3AR dysfunction may account for limited/null efficacy in β1AR-blocker–insensitive HF subjects.

Sphingosine 1-phosphate receptor 1 (S1PR1) agonist CYM5442 inhibits expression of intracellular adhesion molecule 1 (ICAM1) in endothelial cells infected with influenza A viruses.

BackgroundInfluenza A virus infection and its complications effect a large population worldwide. Endothelial cells are an important component in lung inflammation caused by influenza A virus infection. The roles of endothelial sphingosine 1-phophate receptor 1 (S1PR1) in the regulation of molecules involved in leukocyte recruitment during influenza A virus infection still remain unknown. In this report, we tested our hypothesis that S1PR1 agonist CYM5442 inhibits expression of intracellular adhesion molecules 1 (ICAM1) in endothelial cells infected with influenza A virus.MethodsHuman pulmonary microvascular endothelial cells (HPMEC) were infected with influenza A virus H1N1. Expression of cytokines, chemokines, interferons, and cellular adhesion molecules was measured by q-PCR. Expression of ICAM1 was further tested by Western Blotting. A S1PR1 agonist CYM5442 was added to the culture media to assess CYM5442’s inhibitory effects during virus infection.ResultsHPMEC could be infected with H1N1 and responded to produce pro-inflammatory cytokines, chemokines, type I interferons, and cellular adhesion molecules. Addition of CYM5442 in culture media reduced the production of ICAM1 via a dosage- and time-dependent manner. CYM5442 inhibited the activation of nuclear factor (NF)-κB. The regulatory effects of CYM5442 were β-arrestin2-dependent.ConclusionActivated S1PR1 signaling regulates the production of cellular adhesion molecules by inhibiting NF- κB activation via a β-arrestin2-dependent manner.

S1PR1-mediated IFNAR1 degradation modulates plasmacytoid dendritic cell interferon-α autoamplification

Blunting immunopathology without abolishing host defense is the foundation for safe and effective modulation of infectious and autoimmune diseases. Sphingosine 1-phosphate receptor 1 (S1PR1) agonists are effective in treating infectious and multiple autoimmune pathologies; however, mechanisms underlying their clinical efficacy are yet to be fully elucidated. Here, we uncover an unexpected mechanism of convergence between S1PR1 and interferon alpha receptor 1 (IFNAR1) signaling pathways. Activation of S1PR1 signaling by pharmacological tools or endogenous ligand sphingosine-1 phosphate (S1P) inhibits type 1 IFN responses that exacerbate numerous pathogenic conditions. Mechanistically, S1PR1 selectively suppresses the type I IFN autoamplification loop in plasmacytoid dendritic cells (pDCs), a specialized DC subset, for robust type I IFN release. S1PR1 agonist suppression is pertussis toxin-resistant, but inhibited by an S1PR1 C-terminal-derived transactivating transcriptional activator (Tat)-fusion peptide that blocks receptor internalization. S1PR1 agonist treatment accelerates turnover of IFNAR1, suppresses signal transducer and activator of transcription 1 (STAT1) phosphorylation, and down-modulates total STAT1 levels, thereby inactivating the autoamplification loop. Inhibition of S1P-S1PR1 signaling in vivo using the selective antagonist Ex26 significantly elevates IFN-α production in response to CpG-A. Thus, multiple lines of evidence demonstrate that S1PR1 signaling sets the sensitivity of pDC amplification of IFN responses, thereby blunting pathogenic immune responses. These data illustrate a lipid G-protein coupled receptor (GPCR)-IFNAR1 regulatory loop that balances effective and detrimental immune responses and elevated endogenous S1PR1 signaling. This mechanism will likely be advantageous in individuals subject to a range of inflammatory conditions.

Intravital Two-Photon Imaging of Lymphocytes Crossing High Endothelial Venules and Cortical Lymphatics in the Inguinal Lymph Node.

Lymphocyte recirculation through lymph nodes (LNs) requires their crossing of endothelial barriers present in blood vessels and lymphatics by means of chemoattractant-triggered cell migration. The chemoattractant-chemoattractant receptor axes that predominately govern the trafficking of lymphocytes into, and out of, LNs are CCL19/CCR7 and sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1PR1), respectively. Blood-borne lymphocytes downregulate S1PR1 and use CCR7 signaling to adhere to high endothelial venules (HEVs) for transmigration. During their LN residency, recirculating lymphocytes reacquire S1PR1 and attenuate their sensitivity to chemokines. Eventually lymphocytes exit the LN by entering the cortical or medullary lymphatics, a process that depends upon S1PR1 signaling. Upon entering into the lymph, lymphocytes lose their polarity, downregulate their sensitivity to S1P due to the high concentration of S1P, and upregulate their sensitivity to chemokines. However, many of the details of lymphocyte transmigration across endothelial barriers remain poorly understood. Intravital two-photon imaging with advanced microscope technologies not only allows the real-time observation of immune cells in intact LN of a live mouse, but also provides a means to monitor the interactions between circulating lymphocytes and stromal barriers. Here, we describe procedures to visualize lymphocytes engaging and crossing HEVs, and approaching and crossing the cortical lymphatic endothelium to enter the efferent lymph in live mice.

Gαs Relays Sphingosine-1-Phosphate Receptor 1 Signaling to Stabilize Vascular Endothelial-Cadherin at Endothelial Junctions to Control Mouse Embryonic Vascular Integrity

Sphingosine-1-phosphate receptor 1 (S1PR1), a G protein-coupled receptor (GPCR), controls vascular stability by stabilizing vascular endothelial (VE)-cadherin junctional localization and inhibiting vascular endothelial growth factor receptor 2 (VEGFR2) signaling. However, the molecular mechanisms that link S1PR1 signaling to intracellular effectors remain unknown. In this study, we demonstrate that the heterotrimeric G protein subfamily member Gαs, encoded by GNAS, acts as a relay mediator of S1PR1 signaling to control vascular integrity by stabilizing VE-cadherin at endothelial junctions. The endothelial cell-specific deletion of Gαs in mice causes early embryonic lethality with massive hemorrhage and a disorganized vasculature. The immunostaining results revealed that Gαs deletion remarkably reduces the junctional localization of VE-cadherin, whereas the mural cell coverage of the vessels is not impaired. In addition, we found that Gαs depletion blocks the S1PR1-activation induced VE-cadherin stabilization at junctions, supporting that Gαs acts downstream of S1PR1 signaling. Thus, our results demonstrate that Gαs is an essential mediator to relay S1PR1 signaling and maintain vascular integrity.