OBJECTIVE

FTY720 is a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor modulator that showed efficacy in phase II and III clinical trials in patients with multiple sclerosis (MS). FTY720 inhibits lymphocyte egress from secondary lymphoid organs into the peripheral circulation, thereby reducing the number of circulating naïve and central memory T cells, but not effector memory T cells in blood. Little is known to which of these memory T-cell subsets interleukin <em>1</em>7 (IL-<em>1</em>7)-producing T cells (Th<em>1</em>7 cells) belong, which are considered to be key mediators of inflammation in MS, and how they are affected by treatment with FTY720. In this study, we determined the phenotype and frequency of Th<em>1</em>7 cells in blood of untreated, FTY720-treated, and interferon-beta (IFNbeta)-treated patients with MS and healthy donors.

METHODS

In a prospective observational study, circulating T cells were phenotypically characterized and Th<em>1</em>7 cells enumerated in T-cell subsets ex vivo. Production of IL-<em>1</em>7 upon activation and expression of the Th<em>1</em>7-specific transcription factor RORC2 was assessed in vitro.

RESULTS

Th<em>1</em>7 cells were found primarily within central memory T cells in all study populations. FTY720 treatment reduced blood central memory T cells, including RORC2+ and IL-<em>1</em>7-producing T cells, by >90%. FTY720 did not per se affect IL-<em>1</em>7 production when added to activated T cells in vitro.

CONCLUSIONS

Phenotypic Th<em>1</em>7 cells are defined by a central memory T-cell phenotype. FTY720 reduces these Th<em>1</em>7 cells in blood. This is presumably because central memory T cells are retained by FTY720 in secondary lymphoid organs.

The available data concerning the ability of ceramide and other simple sphingolipids to segregate laterally into rigid, gel-like domains in a fluid bilayer has been reviewed. Ceramides give rise to rigid ceramide-enriched domains when their N-acyl chain is longer than C<em>1</em>2. The high melting temperature of hydrated ceramides, revealing a tight intermolecular interaction, is probably responsible for their lateral segregation. Ceramides compete with cholesterol for the formation of domains with lipids such as sphingomyelin or saturated phosphatidylcholines; under these conditions displacement of cholesterol by ceramide involves a transition from a liquid-ordered to a gel-like phase in the domains involved. When ceramide is generated in situ by a sphingomyelinase, instead of being premixed with the other lipids, gel-like domain formation occurs as well, although the topology of the domains may not be the same, the enzyme causing clustering of domains that is not detected with premixed ceramide. Ceramide-<em>1</em>-<em>phosphate</em> is not likely to form domains in fluid bilayers, and the same is true of <em>sphingosine</em> and of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. However, <em>sphingosine</em> does rigidify pre-existing gel domains in mixed bilayers.

Endothelial dysfunction and increased arterial stiffness contribute to multiple vascular diseases and are hallmarks of cardiovascular aging. To investigate the effects of aging on shear stress-induced endothelial nitric oxide (NO) signaling and aortic stiffness, we studied young (3-4 mo) and old (22-24 mo) rats in vivo and in vitro. Old rat aorta demonstrated impaired vasorelaxation to acetylcholine and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, while responses to sodium nitroprusside were similar to those in young aorta. In a customized flow chamber, aortic sections preincubated with the NO-sensitive dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, were subjected to steady-state flow with shear stress increase from 0.4 to 6.4 dyn/cm(2). In young aorta, this shear step amplified 4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence rate by 70.6 +/- <em>1</em>3.9%, while the old aorta response was significantly attenuated (23.6 +/- <em>1</em><em>1</em>.3%, P < 0.05). Endothelial NO synthase (eNOS) inhibition, by N(G)-monomethyl-l-arginine, abolished any fluorescence rate increase. Furthermore, impaired NO production was associated with a significant reduction of the phosphorylated-Akt-to-total-Akt ratio in aged aorta (P < 0.05). Correspondingly, the phosphorylated-to-total-eNOS ratio in aged aortic endothelium was markedly lower than in young endothelium (P < 0.00<em>1</em>). Lastly, pulse wave velocity, an in vivo measure of vascular stiffness, in old rats (5.99 +/- 0.<em>1</em>9<em>1</em> m/s) and in N(omega)-nitro-l-arginine methyl ester-treated rats (4.96 +/- 0.<em>1</em><em>1</em>8 m/s) was significantly greater than that in young rats (3.64 +/- 0.068 m/s, P < 0.00<em>1</em>). Similarly, eNOS-knockout mice demonstrated higher pulse wave velocity than wild-type mice (P < 0.00<em>1</em>). Thus impaired Akt-dependent NO synthase activation is a potential mechanism for decreased NO bioavailability and endothelial dysfunction, which likely contributes to age-associated vascular stiffness.

Macrophage polarization contributes to a number of human pathologies. This is exemplified for tumor-associated macrophages (TAMs), which display a polarized M2 phenotype, closely associated with promotion of angiogenesis and suppression of innate immune responses. We present evidence that induction of apoptosis in tumor cells and subsequent recognition of apoptotic debris by macrophages participates in the macrophage phenotype shift. During coculture of human primary macrophages with human breast cancer carcinoma cells (MCF-7) the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-alpha and interleukin (IL) 12-p70 production, but increased formation of IL-8 and -10. Alternative macrophage activation required tumor cell death because a coculture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. Interestingly, phenotype alterations were achieved with conditioned media from apoptotic tumor cells, arguing for a soluble factor. Knockdown of sphingosine kinase (Sphk) 2, but not Sphk1, to attenuate S1P formation in MCF-7 cells, restored classical macrophage responses during coculture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P suppressed nuclear factor (NF)-kappaB signaling. These findings suggest that tumor cell apoptosis-derived S1P contributes to macrophage polarization.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) protects neonatal rat cardiac myocytes from hypoxic damage through unknown signaling pathways. We tested the hypothesis that S<em>1</em>P-induced cardioprotection requires activation by the epsilon-isoform of protein kinase C (PKC epsilon) by subjecting hearts isolated from PKC epsilon knockout mice and wild-type mice to 20 min of global ischemia and 30 min of reperfusion. Pretreatment with a 2-min infusion of <em>1</em>0 nM S<em>1</em>P improved recovery of left ventricular developed pressure (LVDP) in both wild-type and PKC epsilon knockout hearts and reduced the rise in LV end-diastolic pressure (LVEDP) and creatine kinase (CK) release. Pretreatment for 2 min with <em>1</em>0 nM of the ganglioside GM-<em>1</em> also improved recovery of LVDP and suppressed CK release in wild-type hearts but not in PKC epsilon knockout hearts. Importantly, GM-<em>1</em> but not S<em>1</em>P, increased the proportion of PKC epsilon localized to particulate fractions. Our results suggest that GM-<em>1</em>, which enhances endogenous S<em>1</em>P production, reduces cardiac injury through PKC epsilon-dependent intracellular pathways. In contrast, extracellular S<em>1</em>P induces equivalent cardioprotection through PKC epsilon-independent signaling pathways.

Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum--<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF)--in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S<em>1</em>P and PDGF in a serum-free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri-potentiality.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lipid mediator produced from sphingomyelin by the sequential enzymatic actions of sphingomyelinase, ceramidase, and <em>sphingosine</em> kinase. Five subtypes of cell surface G-protein-coupled receptors, S<em>1</em>P(<em>1</em>-5), mediate the actions of S<em>1</em>P in various organs systems, most notably cardiovascular, immune, and central nervous systems. S<em>1</em>P is enriched in blood and lymph but is present at much lower concentrations in interstitial fluids of tissues. This vascular S<em>1</em>P gradient is important for the regulation of trafficking of various immune cells. FTY720, which was recently approved for the treatment of relapsing-remitting multiple sclerosis, potently sequesters lymphocytes into lymph nodes by functionally antagonizing the activity of the S<em>1</em>P(<em>1</em>) receptor. S<em>1</em>P also plays critical roles in the vascular barrier integrity, thereby regulating inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Recent studies have also revealed the involvement of S<em>1</em>P signaling in coagulation and in tumor necrosis factor α-mediated signaling. This review highlights the importance of S<em>1</em>P signaling in these inflammatory processes as well as the contribution of each receptor subtype, which exhibits both cooperative and redundant functions.

Female sterility resulting from oocyte destruction is an unfortunate, and in many cases inevitable, consequence of chemotherapy. We show that unfertilized mouse oocytes exposed to therapeutic levels of the antitumor drug, doxorubicin (DXR), undergo apoptosis; however, fertilized oocytes do not initiate apoptosis, but enter cell-cycle arrest, when treated with DXR. Apoptosis induced by DXR in oocytes is blocked by <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, an inhibitor of ceramide-promoted cell death. Oocytes from Bax-deficient, but not p53-null, female mice display complete resistance to DXR-induced apoptosis in vivo and in vitro. Pretreatment of oocytes with a specific peptide inhibitor of caspases also abrogates the apoptotic response to DXR. These findings indicate that oocyte destruction caused by chemotherapy can be prevented by manipulation of apoptosis-associated signaling pathways.

The possibilities to preserve fertility in women exposed to chemotherapy are: in vitro fertilization plus embryo cryopreservation, ovarian cryopreservation, unfertilized ova cryopreservation, and the administration of a gonadotropin-releasing hormone (GnRH) agonist. Because none of these methods is ideal, combination of several methods should be considered. Because the chances of preserving gonadal function following combined-modality treatment are significantly better for girls than for boys, simulation of a prepubertal milieu was applied only to women of reproductive age. The administration of GnRH agonists to women with Hodgkin's disease, breast cancer, and other malignancies, or to patients with lupus nephropathy, in parallel with chemotherapy, by others and by us, has demonstrated a significantly lower rate of premature ovarian failure in survivors than in nonrandomized controls. Several prospective, randomized studies are ongoing. A recent meta-analysis found that the administration of a GnRH agonist, in addition to chemotherapy, to patients with breast cancer was associated with less recurrence and superior survival. Several possibilities to explain the beneficial effect of GnRH agonists to minimize chemotherapy-associated gonadotoxicity are suggested: (a) The hypogonadotropic milieu decreases the number of primordial follicles entering the differentiation stage, which is more vulnerable to chemotherapy; (b) The hypoestrogenic state decreases ovarian perfusion and delivery of chemotherapy to the ovaries; (c) A direct effect of the GnRH agonist on the ovary occurs independently of the gonadotropin level; (d) GnRH agonists may upregulate an intragonadal antiapoptotic molecule such as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>; (e) The GnRH agonist may protect ovarian germline stem cells.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid with important functions in the immune system. S<em>1</em>P levels are regulated by the balance between its synthesis through <em>sphingosine</em> kinases and its degradation by S<em>1</em>P lyase. S<em>1</em>P signals through plasma membrane G-protein-coupled receptors (S<em>1</em>PR<em>1</em>-S<em>1</em>PR5) or acts directly on intracellular targets. Although it has long been known that the S<em>1</em>P-S<em>1</em>PR<em>1</em> axis mediates T cell egress from lymphoid organs, recent studies have revealed intrinsic functions of S<em>1</em>P and its receptors in both innate and adaptive immune systems that are independent of immune cell trafficking. Here I summarize recent advances in understanding of the roles of S<em>1</em>P and S<em>1</em>P receptors in inflammatory and allergic responses and lymphocyte differentiation, which directly contribute to the regulation of inflammatory and autoimmune diseases. I also describe strategies to target S<em>1</em>P and S<em>1</em>P receptors for immune-mediated diseases, particularly the immunosuppressant FTY720 (fingolimod), which has recently become the first oral therapy for relapsing multiple sclerosis.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) ligation of endothelial differentiation gene-<em>1</em> receptor coupled to the heterotrimeric G protein, Gi, promotes endothelial barrier strengthening via Rac-dependent assembly of adherens junctions (AJs). However, the mechanism of Rac activation induced by S<em>1</em>P stimulation remains unclear. In live endothelial cells expressing GFP-Rac, we observed that S<em>1</em>P induced the translocation of Rac to intercellular junctions, resulting in junctional sealing. We investigated the role of intracellular Ca2+ in signaling Rac activation and the enhancement of endothelial barrier function. We observed that S<em>1</em>P activated the release of Ca2+ from endoplasmic reticulum stores, and subsequent Ca2+ entry via lanthanum-sensitive store-operated Ca2+ channels (SOC) after store depletion. Inhibition of Gi, phospholipase C, or inositol tris<em>phosphate</em> receptor prevented the S<em>1</em>P-activated increase in intracellular Ca2+ as well as Rac activation, AJ assembly, and enhancement of endothelial barrier. Chelation of intracellular Ca2+ with BAPTA blocked S<em>1</em>P-induced Rac activation, indicating the requirement for Ca2+ in the response. Inhibition of SOC by lanthanum or transient receptor potential channel <em>1</em> (TRPC<em>1</em>), a SOC constituent, by TRPC<em>1</em> antibody, failed to prevent S<em>1</em>P-induced Rac translocation to junctions and AJ assembly. Thus, our results demonstrate that S<em>1</em>P promotes endothelial junctional integrity by activating the release of endoplasmic reticulum-Ca2+, which induces Rac activation and promotes AJ annealing.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lysosphingophospholipid that has been implicated in the regulation of vital biological processes. Abundant evidence indicates that S<em>1</em>P acts as both an intracellular messenger and an extracellular ligand for a family of five specific G protein-coupled S<em>1</em>P receptors (S<em>1</em>PRs). Cellular levels of S<em>1</em>P are tightly regulated in a spatio-temporal manner through its synthesis catalyzed by <em>sphingosine</em> kinases (SphKs) and degradation by S<em>1</em>P lyase (SPL) and specific S<em>1</em>P phosphohydrolases. Over the past decade, the identification and cloning of genes encoding S<em>1</em>P metabolizing enzymes has increased rapidly. Overexpression and deletion of these enzymes has provided important insights into the intracellular and the "inside-out" functions of S<em>1</em>P. The purpose of this review is to summarize the current knowledge of S<em>1</em>P metabolizing enzymes, their enzymatic properties, and their roles in the control of cellular functions by S<em>1</em>P.

Apoptotic cells (AC) are rapidly engulfed by professional phagocytes such as macrophages to avoid secondary necrosis and thus inflammation. Recognition of AC polarizes macrophages toward an anti-inflammatory phenotype, which shows homology to an alternatively activated M2 macrophage. However, mechanistic details provoking these phenotype alterations are incompletely understood. Here, we demonstrate a biphasic up-regulation of heme oxygenase-<em>1</em> (HO-<em>1</em>), a protein that bears an antiapoptotic as well as an anti-inflammatory potential, in primary human macrophages, which were exposed to the supernatant of AC. Although the first phase of HO-<em>1</em> induction at 6 h was accomplished by AC-derived <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) acting via S<em>1</em>P receptor <em>1</em>, the second wave of HO-<em>1</em> induction at 24 h was attributed to autocrine signaling of vascular endothelial growth factor A (VEGFA), whose expression and release were facilitated by S<em>1</em>P. Whereas VEGFA release from macrophages was signal transducer and activator of transcription (STAT) <em>1</em>-dependent, vascular endothelial growth factor itself triggered STAT<em>1</em>/STAT3 heterodimer formation, which bound to and activated the HO-<em>1</em> promoter. Knockdown of HO-<em>1</em> proved its relevance in facilitating enhanced expression of the antiapoptotic proteins Bcl-2 and Bcl-X(L), as well as the anti-inflammatory adenosine receptor A(2A). These findings suggest that HO-<em>1</em>, which is induced by AC-derived S<em>1</em>P, is critically involved in macrophage polarization toward an M2 phenotype.

OBJECTIVE

The purpose of this study was to define heterogeneity in the molecular profile of lipids, including sphingomyelin and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, among physicochemically-defined HDL subpopulations and potential relevance to antiatherogenic biological activities of dense HDL3.

RESULTS

The molecular profile of lipids (cholesteryl esters, phospholipids, sphingomyelin, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>) in physicochemically-defined normolipidemic HDL subpopulations was determined by high-performance liquid chromatography and gas chromatography. As HDL particle size and molecular weight decreased with increment in density, molar lipid content diminished concomitantly. On a % basis, sphingomyelin abundance diminished in parallel with progressive increase in HDL density from HDL2b (<em>1</em>2.8%) to HDL3c (6.2%; P<0.00<em>1</em>); in contrast, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> was preferentially enriched in small HDL3 (40 to 50 mmol/mol HDL) versus large HDL2 (<em>1</em>5 to 20 mmol/mol HDL; P<0.0<em>1</em>). Small HDL3c was equally enriched in LpA-I particles relative to LpA-I:A-II. The <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>/sphingomyelin ratio correlated positively with the capacities of HDL subspecies to attenuate apoptosis in endothelial cells (r=0.73, P<0.00<em>1</em>) and to retard LDL oxidation (r=0.58, P<0.0<em>1</em>).

CONCLUSIONS

An elevated <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>/sphingomyelin ratio is an integral feature of small dense HDL3, reflecting enrichment in <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, a key antiapoptotic molecule, and depletion of sphingomyelin, a structural lipid with negative impact on surface fluidity and LCAT activity. These findings further distinguish the structure and antiatherogenic activities of small, dense HDL.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) enhances human pulmonary endothelial monolayer integrity via Rac GTPase-dependent formation of a cortical actin ring (Garcia et al. J Clin Invest <em>1</em>08: 689-70<em>1</em>, 200<em>1</em>). The mechanisms underlying this response are not well understood but may involve rapid redistribution of focal adhesions (FA) as attachment sites for actin filaments. We evaluate the effects of S<em>1</em>P on the redistribution of paxillin, FA kinase (FAK), and the G protein-coupled receptor kinase-interacting proteins (GITs). S<em>1</em>P induced Rac GTPase activation and cortical actin ring formation at physiological concentrations (0.5 microM), whereas 5 microM S<em>1</em>P caused prominent stress fiber formation and activation of Rho and Rac GTPases. S<em>1</em>P (0.5 microM) stimulated the tyrosine phosphorylation of FAK Y(576), and paxillin was linked to FA disruption and redistribution to the cell periphery. Furthermore, S<em>1</em>P induced a transient association of GIT<em>1</em> with paxillin and redistribution of the GIT2-paxillin complex to the cell cortical area without affecting GIT2-paxillin association. These results suggest a role of FA rearrangement in S<em>1</em>P-mediated barrier enhancement via Rac- and GIT-mediated processes.

Lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are potent lipid growth factors with similar abilities to stimulate cytoskeleton-based cellular functions. Their effects are mediated by a subfamily of G protein-coupled receptors (GPCRs) encoded by endothelial differentiation genes (edgs). We hypothesize that large quantities of LPA and S<em>1</em>P generated by activated platelets may influence endothelial cell functions. Using an in vitro wound healing assay, we observed that LPA and S<em>1</em>P stimulated closure of wounded monolayers of human umbilical vein endothelial cells and adult bovine aortic endothelial cells, which express LPA receptor Edg2, and S<em>1</em>P receptors Edg<em>1</em> and Edg3. The two major components of wound healing, cell migration and proliferation, were stimulated individually by both lipids. LPA and S<em>1</em>P also stimulated intracellular Ca(2+) mobilization and mitogen-activated protein kinase (MAPK) phosphorylation. Pertussis toxin partially blocked the effects of both lipids on endothelial cell migration, MAPK phosphorylation, and Ca(2+) mobilization, implicating G(i)/(o)-coupled Edg receptor signaling in endothelial cells. LPA and S<em>1</em>P did not cross-desensitize each other in Ca(2+) responses, suggesting involvement of distinct receptors. Thus LPA and S<em>1</em>P affect endothelial cell functions through signaling pathways activated by distinct GPCRs and may contribute to the healing of wounded vasculatures.

T-lymphocytes promote cerebral inflammation, thus aggravating neuronal injury after stroke. Fingolimod, a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor analog, prevents the egress of lymphocytes from primary and secondary lymphoid organs. Based on these findings, we hypothesized fingolimod treatment would reduce the number of T-lymphocytes migrating into the brain, thereby ameliorating cerebral inflammation following experimental intracerebral hemorrhage (ICH). We investigated the effects of fingolimod in two well-established murine models of ICH, implementing intrastriatal infusions of either bacterial collagenase (cICH) or autologous blood (bICH). Furthermore, we tested the long term neurological improvements by Fingolimod in a collagenase-induced rat model of ICH. Fingolimod, in contrast to vehicle administration alone, improved neurological functions and reduced brain edema at 24 and 72 h following experimental ICH in CD-<em>1</em> mice (n=<em>1</em>03; p<0.05). Significantly fewer lymphocytes were found in blood and brain samples of treated animals when compared to the vehicle group (p<0.05). Moreover, fingolimod treatment significantly reduced the expression of intercellular adhesion molecule-<em>1</em> (ICAM-<em>1</em>), interferon-γ (INF-γ), and interleukin-<em>1</em>7 (IL-<em>1</em>7) in the mouse brain at 72 h post-cICH (p<0.05 compared to vehicle). Long-term neurocognitive performance and histopathological analysis were evaluated in Sprague-Dawley rats between 8 and <em>1</em>0 weeks post-cICH (n=28). Treated rats showed reduced spatial and motor learning deficits, along with significantly reduced brain atrophy and neuronal cell loss within the basal ganglia (p<0.05 compared to vehicle). We conclude that fingolimod treatment ameliorated cerebral inflammation, at least to some extent, by reducing the availability and subsequent brain infiltration of T-lymphocytes, which improved the short and long-term sequelae after experimental ICH in rodents.

Various therapeutic advances have led to a paradigm shift in the clinical management of patients with IBD. The introduction of immunosuppressive (such as azathioprine) and biologic agents (such as TNF blockers) has markedly reduced the need to use corticosteroids for therapy. Furthermore, the α4β7 integrin blocker vedolizumab has been introduced for clinical IBD therapy. Moreover, various new inhibitors of cytokines (for example, IL-6-IL-6R and IL-<em>1</em>2-IL-23 blockers or apremilast), modulators of cytokine signalling events (for example, JAK inhibitors or SMAD7 blocker), inhibitors of transcription factors (for example, GATA3 or RORγt) and new anti-adhesion and anti-T-cell-activation and migration strategies (for example, β7 integrin, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors and MAdCAM<em>1</em> inhibitors, regulatory T-cell therapy and stem cells) are currently being evaluated in controlled clinical trials. This Review aims to provide a comprehensive overview about current and future therapeutic approaches for IBD therapy. Furthermore, potential mechanisms of action of these therapeutic approaches and their implications for clinical therapy in IBD are discussed.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a potent lipid mediator, is a ligand for a family of five G protein-coupled receptors (S<em>1</em>P(<em>1</em>-5)) that have been shown to regulate a variety of biological responses important for cancer progression. The cellular level of S<em>1</em>P is low and tightly regulated in a spatio-temporal manner through its synthesis catalyzed by two <em>sphingosine</em> kinases, denoted SphK<em>1</em> and SphK2. Many stimuli activate and translocate SphK<em>1</em> to the plasma membrane by mechanisms that are dependent on its phosphorylation. Much less is known about activation of SphK2. Here we demonstrate that epidermal growth factor (EGF) as well as the protein kinase C activator, phorbol ester, induce rapid phosphorylation of hSphK2 which was markedly reduced by inhibition of MEK<em>1</em>/ERK pathway. Down-regulation of ERK<em>1</em> blocked EGF-induced phosphorylation of SphK2. Recombinant ERK<em>1</em> phosphorylated hSphK2 in vitro and increased its enzymatic activity. ERK<em>1</em> also was found to be in a complex with hSphK2 in vivo. Site-directed mutagenesis indicated that hSphK2 is phosphorylated on Ser-35<em>1</em> and Thr-578 by ERK<em>1</em> and that phosphorylation of these residues is important for EGF-stimulated migration of MDA-MB-453 cells. These studies provide the first clues to the mechanism of agonist-mediated SphK2 activation and enhance understanding of the regulation of SphK2 activity by phosphorylation and its role in movement of human breast cancer cells toward EGF.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase is a widely expressed enzyme that catalyzes the essentially irreversible cleavage of the signaling molecule <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. To investigate whether <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase influences mammalian cell fate decisions, a recombinant human <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase fused to green fluorescent protein was expressed in HEK293 cells. The recombinant enzyme was active, localized to the endoplasmic reticulum, and reduced baseline <em>sphingosine</em> and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> levels. Stable overexpression led to diminished viability under stress, which was attributed to an increase in apoptosis and was reversible in a dose-dependent manner by exogenous <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. In contrast to <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, the products of the lyase reaction had no effect on apoptosis. Lyase enzymatic activity was required to potentiate apoptosis, because cells expressing a catalytically inactive enzyme behaved like controls. Stress increased the amounts of long- and very long-chain ceramides in HEK293 cells, and this was enhanced in cells overexpressing wild type but not catalytically inactive lyase. The ceramide increases appeared to be required for apoptosis, because inhibition of ceramide synthase with fumonisin B<em>1</em> decreased apoptosis in lyase-overexpressing cells. Thus, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase overexpression in HEK293 cells decreases <em>sphingosine</em> and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> amounts but elevates stress-induced ceramide generation and apoptosis. This identifies <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase as a dual modulator of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and ceramide metabolism as well as a regulator of cell fate decisions and, hence, a potential target for diseases with an imbalance in these biomodulators, such as cancer.

The lysophospholipids <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and lysophosphatidic acid (LPA) stimulate cellular proliferation and affect numerous cellular functions by signaling through G protein-coupled endothelial differentiation gene-encoded (Edg) receptors. S<em>1</em>P and LPA also act as survival factors in many cell types, but have not previously been studied in cardiac myocytes. We incubated neonatal rat cardiac myocytes either in room air/<em>1</em>% CO2 (normoxia) or in an atmosphere of 99% N2/<em>1</em>%CO2 (hypoxia) at 37 degrees C for <em>1</em>8-20 h in the absence of glucose. Cell viability was measured using a calcein ester green fluorescence assay. Under normoxic conditions 88.7+/-<em>1</em>.0% of the cells were viable after <em>1</em>8-20 h. Severe hypoxia reduced viability to 6<em>1</em>.3+/-4.3% (n=6, P<0.05). In myocytes preincubated with either <em>1</em>0 microM S<em>1</em>P or <em>1</em> microM LPA for 2 h, the effects of severe hypoxia on cell viability were prevented resulting in survival equivalent to normoxia. Neither the protein kinase C inhibitor chelethyrine (<em>1</em> microM) nor the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoic acid, (5-HD, <em>1</em>00 microM) had any effect on myocyte survival during severe hypoxia, but both agents completely abolished the ability of S<em>1</em>P to rescue cardiac myocytes from hypoxic cell death. We also tested the effects of dimethyl<em>sphingosine</em> (DMS), which inhibits <em>sphingosine</em> kinase synthesis of S<em>1</em>P. Incubation of neonatal rat cardiac myocytes with <em>1</em>0 microM DMS for 2 h in the presence of serum resulted in 25-30% cell death during <em>1</em>8-20 h of normoxia. DMS-induced cell death was prevented by concurrent preincubation with either S<em>1</em>P or GM-<em>1</em>, a ganglioside that activates <em>sphingosine</em> kinase to increase intracellular levels of S<em>1</em>P. We conclude that both S<em>1</em>P and LPA are cardioprotective for hypoxic neonatal rat ventricular myocytes. S<em>1</em>P acts through cellular membrane receptors by signaling mechanisms involving protein kinase C and mitochondrial K(ATP) channels. Both endogenous and exogenously applied S<em>1</em>P are effective in preventing cell death induced by inhibition of <em>sphingosine</em> kinase.

The novel immunosuppressant FTY720 activates <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors (S<em>1</em>PRs) that affect responsiveness of lymphocytes to chemokines such as stromal cell-derived factor <em>1</em> (SDF-<em>1</em>), resulting in increased lymphocyte homing to secondary lymphoid organs. Since SDF-<em>1</em> and its receptor CXCR4 are also involved in bone marrow (BM) homing of hematopoietic stem and progenitor cells (HPCs), we analyzed expression of S<em>1</em>PRs and the influence of FTY720 on SDF-<em>1</em>/CXCR4-mediated effects in human HPCs. By reverse transcriptase-polymerase chain reaction (RT-PCR), S<em>1</em>PRs were expressed in mobilized CD34+ HPCs, particularly in primitive CD34+/CD38- cells. Incubation of HPCs with FTY720 resulted in prolonged SDF-<em>1</em>-induced calcium mobilization and actin polymerization, and substantially increased SDF-<em>1</em>-dependent in vitro transendothelial migration, without affecting VLA-4, VLA-5, and CXCR4 expression. In nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, the number of CD34+/CD38- cells that homed to the BM after <em>1</em>8 hours was significantly raised by pretreatment of animals and cells with FTY720, tending to result in improved engraftment. In addition, in vitro growth of HPCs (week-5 cobblestone area-forming cells [CAFCs]) was 2.4-fold increased. We conclude that activation of S<em>1</em>PRs by FTY720 increases CXCR4 function in HPCs both in vitro and in vivo, supporting homing and proliferation of HPCs. In the hematopoietic microenvironment, S<em>1</em>PRs are involved in migration and maintenance of HPCs by modulating the effects of SDF-<em>1</em>.

BACKGROUND

Sphingolipids are emerging as important signaling molecules that may be produced by cardiac tissue during ischemic stress or as a consequence of inflammation. Because both inflammation and myocardial ischemia are associated with coronary artery disease (CAD), a study was designed to test the ability of serum sphingolipids to predict obstructive CAD.

METHODS

The study consisted of 308 consecutive patients undergoing coronary angiography for all indications. The primary data points were the assessment of coronary artery stenosis with angiography and the measurements of serum sphingolipids.

RESULTS

In this diverse population, serum <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) was a significant predictor of CAD (P <.00<em>1</em>). Multivariate analysis with logistic regression demonstrated that serum S<em>1</em>P was more predictive of obstructive CAD (odds ratio = 7.6<em>1</em>) than the traditional risk factors (age, sex, family history of CAD, diabetes mellitus, lipid profile, hypertension, etc.). A 3-variable S<em>1</em>PC composite score was derived by combining the power of the S<em>1</em>P marker with the 2 most important risk factors, age and sex. The relationship between the S<em>1</em>PC and CAD scores was continuous and progressive, such that patients with elevated S<em>1</em>PC scores had higher occurrences of obstructive CAD. S<em>1</em>PC was also predictive of disease severity; 53.2% of patients in the fourth S<em>1</em>PC quartile had 2 to 3 vessel CAD, whereas only 5.2% of patients in the first S<em>1</em>PC quartile had 2 to 3 vessel disease (RR = <em>1</em>0.2 for severity).

CONCLUSIONS

Serum S<em>1</em>P is a remarkably strong and robust predictor of both the occurrence and severity of coronary stenosis. An S<em>1</em>P-based composite score may be useful as a novel, non-invasive indicator of obstructive CAD.

OBJECTIVE

Lymphocytes are important players in the pathophysiology of acute ischemic stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thrombo-inflammation, fosters microvascular dysfunction and secondary infarct growth. FTY720, a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor modulator, blocks the egress of lymphocytes from lymphoid organs and has been shown to reduce ischemic neurodegeneration; however, the underlying mechanisms are unclear. We investigated the mode of FTY720 action in models of cerebral ischemia.

METHODS

Transient middle cerebral artery occlusion (tMCAO) was induced in wild-type and lymphocyte-deficient Rag<em>1</em>(-/-) mice treated with FTY720 (<em>1</em> mg/kg) or vehicle immediately before reperfusion. Stroke outcome was assessed 24 hours later. Immune cells in the blood and brain were counted by flow cytometry. The integrity of the blood-brain barrier was analyzed using Evans Blue dye. Thrombus formation was determined by immunohistochemistry and Western blot, and was correlated with cerebral perfusion.

RESULTS

FTY720 significantly reduced stroke size and improved functional outcome in wild-type mice on day <em>1</em> and day 3 after transient middle cerebral artery occlusion. This protective effect was lost in lymphocyte-deficient Rag<em>1</em>(-/-) mice and in cultured neurons subjected to hypoxia. Less lymphocytes were present in the cerebral vasculature of FTY720-treated wild-type mice, which in turn reduced thrombosis and increased cerebral perfusion. In contrast, FTY720 was unable to prevent blood-brain barrier breakdown and transendothelial immune cell trafficking after transient middle cerebral artery occlusion.

CONCLUSIONS

Induction of lymphocytopenia and concomitant reduction of microvascular thrombosis are key modes of FTY720 action in stroke. In contrast, our findings in Rag<em>1</em>(-/-) mice and cultured neurons argue against direct neuroprotective effects of FTY720.