Fingolimod (also known as FTY720) is an orally available <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor modulator that has unique and potent immunoregulatory properties. Mechanistic studies indicate that on phosphorylation fingolimod can bind with high affinity to S<em>1</em>P(<em>1</em>) receptors. Persistent modulation of lymphocyte S<em>1</em>P(<em>1</em>) receptors by fingolimod and the subsequent internalization of these receptors inhibits lymphocyte egress from the lymph nodes, and prevents these cells from infiltrating inflammatory lesions in the CNS. Results of two phase III studies--FREEDOMS and TRANSFORMS--support previous phase II trial observations indicating that fingolimod exerts powerful anti-inflammatory effects in relapsing-remitting multiple sclerosis (MS). Fingolimod might, therefore, be one of the first orally active drug therapies available for the treatment of relapsing-remitting MS. Moreover, results from preclinical studies suggest that fingolimod might promote neural repair in vivo. In this article, we review the background to these findings, present the proposed immunological and neurobiological profile of fingolimod, discuss the data from the FREEDOMS and TRANSFORMS trials, and provide an expert opinion regarding the future of next-generation S<em>1</em>P receptor modulators for MS therapy.

In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) as an activator of TRPC5. We explored the relevance to vascular biology because S<em>1</em>P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC<em>1</em>, which forms a complex with TRPC5. Importantly, S<em>1</em>P also activates the TRPC5-TRPC<em>1</em> heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S<em>1</em>P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S<em>1</em>P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S<em>1</em>P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S<em>1</em>P-evoked cell motility. The data suggest S<em>1</em>P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation.

Recent studies have identified cortical sinuses as sites of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S<em>1</em>P(<em>1</em>))-dependent T- and B-cell egress from the lymph node (LN) parenchyma. However, the distribution of cortical sinuses in the entire LN and the extent of lymph flow within them has been unclear. Using 3D reconstruction and intravital two-photon microscopy we describe the branched organization of the cortical sinus network within the inguinal LN and show that lymphocyte flow begins within blunt-ended sinuses. Many cortical sinuses are situated adjacent to high endothelial venules, and some lymphocytes access these sinuses within minutes of entering a LN. However, upon entry to inflamed LNs, lymphocytes rapidly up-regulate CD69 and are prevented from accessing cortical sinuses. Using the LN reconstruction data and knowledge of lymphocyte migration and cortical sinus entry dynamics, we developed a mathematical model of T-cell egress from LNs. The model suggests that random walk encounters with lymphatic sinuses are the major factor contributing to LN transit times. A slight discrepancy between predictions of the model and the measured transit times may be explained by lymphocytes undergoing a few rounds of migration between the parenchyma and sinuses before departing from the LN. Because large soluble antigens gain rapid access to cortical sinuses, such parenchyma-sinus shuttling may facilitate antibody responses.

The discovery of lysophospholipid (LP) 7-transmembrane, G protein-coupled receptors (GPCRs) that began in the <em>1</em>990s, together with research into the functional roles of the major LPs known as lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), have opened new research avenues into their biological processes and mechanisms. Major examples of LP signalling effects include embryogenesis, nervous system development, vascular development, uterine implantation, immune cell trafficking, and inflammatory reactions. LP signalling also influences the pathophysiology of many diseases including cancer, autoimmune and inflammatory diseases, which indicate that LP receptors may be attractive targets for pharmacological therapies. A key example of such a therapeutic agent is the S<em>1</em>P receptor modulator FTY720, which upon phosphorylation and continued drug exposure, acts as an S<em>1</em>P receptor functional antagonist. This compound (also known as fingolimod or Gilenya) has recently been approved by the FDA for the treatment of relapsing forms of multiple sclerosis. Continued basic and translational research on LP signalling should provide novel insights into both basic biological mechanisms, as well as novel therapeutic approaches to combat a range of human diseases.

The serum-borne lysophospholipid mediators <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P) and lysophosphatidic acid (LPA) have been shown to be released from activated platelets and to act on endothelial cells. In this study, we employed the repeated lipid extraction (under alkaline and acidic conditions), capable of detecting Sph-<em>1</em>-P, LPA, and possibly structurally similar lysophospholipids, whereby a marked formation of [(32)P]Sph-<em>1</em>-P, but not [(32)P]LPA, was observed in [(32)P]ortho<em>phosphate</em>-labeled platelets. Platelet Sph-<em>1</em>-P release, possibly mediated by protein kinase C, was greatly enhanced in the presence of albumin, which formed a complex with Sph-<em>1</em>-P. This finding suggests that platelet Sph-<em>1</em>-P may become accessible to depletion by albumin when its transbilayer movement (flipping) across the plasma membrane is enhanced by protein kinase C. Although human umbilical vein endothelial cells expressed receptors for both Sph-<em>1</em>-P and LPA, Sph-<em>1</em>-P acted much more potently than LPA on the cells in terms of intracellular Ca(++) mobilization, cytoskeletal reorganization, and migration. The results suggest that Sph-<em>1</em>-P, rather than LPA, is a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells, under the conditions in which critical platelet-endothelial interactions (including thrombosis, angiogenesis, and atherosclerosis) occur. Furthermore, albumin-bound Sph-<em>1</em>-P may account for at least some of the serum biological activities on endothelial cells, which have been ascribed to the effects of albumin-bound LPA, based on the similarities between LPA and serum effects.

Biological barriers regulate the passage of cells, pathogens, fluids, nutrients, ions and signalling molecules between anatomical compartments during homeostasis and disease. Yet strategies that allow for reversible therapeutic modulation of these barriers are still in their infancy. The enhancement or protection of natural barriers is desirable in conditions such as acute respiratory distress syndrome or ischaemia-reperfusion injuries, whereas a temporary disruption could facilitate the penetration of drugs across such barriers. This Review discusses the role of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors in the regulation and protection of biological barriers, and the potential of therapeutic strategies that target this receptor family.

We previously demonstrated that L-selectin (CD62L)-dependent T cell homing to lymph nodes (LN) is required for tolerance induction to alloantigen. To explore the mechanisms of this observation, we analyzed the development and distribution of regulatory T cells (Treg), which play an important protective role against allograft rejection in transplantation tolerance. Alloantigen-specific tolerance was induced using either anti-CD2 plus anti-CD3 mAbs, or anti-CD40L mAbs plus donor-specific transfusion, in fully mismatched (BALB/c donor, C57BL/6 recipient) vascularized cardiac allografts. An expansion of CD4(+)CD25(+)CD62L(high) T cells was observed specifically within the LN of tolerant animals, but not in other anatomic sites or under nontolerizing conditions. These cells exhibited a substantial up-regulation of Foxp3 expression as measured by real-time PCR and by fluorescent immunohistochemistry, and possessed alloantigen-specific suppressor activity. Neither LN nor other lymphoid cells expressed the regulatory phenotype if recipients were treated with anti-CD62L mAbs, which both prevented LN homing and caused early allograft rejection. However, administration of FTY720, a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor modulator that induces CD62L-independent T cell accumulation in the LNs, restored CD4(+)CD25(+) Treg in the LNs along with graft survival. These data suggest that alloantigen-specific Foxp3(+)CD4(+)CD25(+) Treg develop and are required within the LNs during tolerization, and provide compelling evidence that distinct lymphoid compartments play critical roles in transplantation tolerance.

S<em>1</em>P (<em>sphingosine</em> <em>1</em>-<em>phosphate</em>) is the ligand for a family of specific G-protein-coupled receptors that regulate a wide variety of important cellular functions, including vascular maturation, angiogenesis, cell growth, survival, cytoskeletal rearrangements and cell motility. However, S<em>1</em>P also may have intracellular functions. In this review, we discuss two examples that clearly indicate that intracellularly generated and exogenous S<em>1</em>P can regulate biological processes by divergent pathways.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (SPP) is a potent lipid mediator released upon cellular activation. In this report, pharmacological properties of the three G-protein-coupled receptors (GPCRs) for SPP, EDG-<em>1</em>, -3, and -5 are characterized using a Xenopus oocyte expression system, which lacks endogenous SPP receptors. Microinjection of the EDG-3 and EDG-5 but not EDG-<em>1</em> mRNA conferred SPP-responsive intracellular calcium transients; however, the EDG-5 response was quantitatively much less. Co-expression of EDG-<em>1</em> receptor with the chimeric Galphaqi protein conferred SPP responsiveness. Galphaqi or Galphaq co-injection also potentiated the EDG-5 and EDG-3 mediated responses to SPP. These data suggest that SPP receptors couple differentially to the Gq and Gi pathway. All three GPCRs were also activated by sphingosylphosphorylcholine, albeit at higher concentrations. None of the other related sphingolipids tested stimulated or blocked SPP-induced calcium responses. However, suramin, a polycyclic anionic compound, selectively antagonized SPP-activated calcium transients in EDG-3 expressing oocytes with an IC50 of 22 microM, suggesting that it is an antagonist selective for the EDG-3 GPCR isotype. We conclude that the three SPP receptors signal differentially by coupling to different G-proteins. Furthermore, because only EDG-3 was antagonized by suramin, variations in receptor structure may determine differences in antagonist selectivity. This property may be exploited to synthesize receptor subtype-specific antagonists.

BACKGROUND

No treatments have been approved for primary progressive multiple sclerosis. Fingolimod, an oral <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor modulator, is effective in relapse-onset multiple sclerosis, but has not been assessed in primary progressive multiple sclerosis. We assessed the safety and efficacy of fingolimod in patients with primary progressive multiple sclerosis.

METHODS

In INFORMS, a multicentre, double-blind, placebo-controlled parallel-group study, patients with primary progressive multiple sclerosis recruited across <em>1</em>48 centres in <em>1</em>8 countries were randomly allocated (<em>1</em>:<em>1</em>) with computer-generated blocks to receive oral fingolimod or placebo for at least 36 months and a maximum of 5 years. Patients were initially assigned to fingolimod <em>1</em>·25 mg per day or placebo (cohort <em>1</em>); however, after a protocol amendment on Nov <em>1</em>9, 2009, patients were switched in a masked manner to fingolimod 0·5 mg, whereas those on placebo continued on matching placebo. From then onwards, patients were assigned to receive fingolimod 0·5 mg/day or placebo (cohort 2). Key inclusion criteria were age 25-65 years, clinical diagnosis of primary progressive multiple sclerosis, <em>1</em> year or more of disease progression, and two of the following criteria: positive brain MRI; positive spinal cord MRI; or positive cerebrospinal fluid. Additional eligibility criteria included disease duration of 2-<em>1</em>0 years and objective evidence of disability progression in the previous 2 years. Patients and study investigators were masked to group assignment. We used a novel primary composite endpoint based on change from baseline in Expanded Disability Status Scale (EDSS), 25' Timed-Walk Test, or Nine-Hole Peg Test to assess time to 3-month confirmed disability progression in study participants treated for at least 3 years. All randomised patients took at least one dose of study drug. The primary efficacy analysis included all patients in cohort 2 and those assigned to placebo in cohort <em>1</em>. The safety analysis included all patients in cohorts <em>1</em> and 2. This study is registered with ClinicalTrials.gov, number NCT0073<em>1</em>692. The study is now closed.

RESULTS

970 patients were randomly assigned between Sept 3, 2008, and Aug 30, 20<em>1</em><em>1</em> (<em>1</em>47 to fingolimod <em>1</em>·25 mg and <em>1</em>33 to placebo in cohort <em>1</em>; 336 to fingolimod 0·5 mg and 354 to placebo in cohort 2). The efficacy analysis set (n=823) consisted of 336 patients randomly allocated to fingolimod 0·5 mg and 487 to placebo. Baseline characteristics were similar across groups and representative of a primary progressive multiple sclerosis population (48% women, mean age 48·5 years [SD 8·4], mean EDSS 4·67 [SD <em>1</em>·03], 87% free of gadolinium-enhancing lesions). By end of study, 3-month confirmed disability progression had occurred in 232 and 338 patients in the fingolimod and placebo groups, respectively, resulting in Kaplan-Meier estimates of 77·2% (95% CI 7<em>1</em>·87-82·5<em>1</em>) of patients in the fingolimod group versus 80·3% (73·3<em>1</em>-87·25) of patients in the placebo group (risk reduction 5·05%; hazard ratio 0·95, 95% CI 0·80-<em>1</em>·<em>1</em>2; p=0·544). Safety results were generally consistent with those of studies of fingolimod in patients with relapse-onset multiple sclerosis. Lymphopenia occurred in <em>1</em>9 (6%) patients in the fingolimod group versus none in the placebo group, bradycardia in five (<em>1</em>%) versus one ((<em>1</em>%), and first-degree atrioventricular block in three (<em>1</em>%) versus six (<em>1</em>%). Serious adverse events occurred in 84 (25%) patients in the fingolimod group and <em>1</em><em>1</em>7 (24%) in the placebo group, including macular oedema in six (2%) versus six (<em>1</em>%), and basal-cell carcinoma in <em>1</em>4 (4%) versus nine (2%).

CONCLUSIONS

The anti-inflammatory effects of fingolimod did not slow disease progression in primary progressive multiple sclerosis. Therapeutic strategies for primary progressive multiple sclerosis might need different approaches to those used for relapse-onset multiple sclerosis.

BACKGROUND

Novartis Pharma AG.

We characterized the molecular mechanisms by which high density lipoprotein (HDL) inhibits the expression of adhesion molecules, including vascular cell adhesion molecule-<em>1</em> and intercellular adhesion molecule-<em>1</em>, induced by <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and tumor necrosis factor (TNF) alpha in endothelial cells. HDL inhibited S<em>1</em>P-induced nuclear factor kappaB activation and adhesion molecule expression in human umbilical vein endothelial cells. The inhibitory HDL actions were associated with nitric-oxide synthase (NOS) activation and were reversed by inhibitors for phosphatidylinositol 3-kinase and NOS. The HDL-induced inhibitory actions were also attenuated by the down-regulation of scavenger receptor class B type I (SR-BI) and its associated protein PDZK<em>1</em>. When TNFalpha was used as a stimulant, the HDL-induced NOS activation and the inhibitory action on adhesion molecule expression were, in part, attenuated by the down-regulation of the expression of S<em>1</em>P receptors, especially S<em>1</em>P(<em>1</em>), in addition to SR-BI. Reconstituted HDL composed mainly of apolipoprotein A-I and phosphatidylcholine mimicked the SR-BI-sensitive part of HDL-induced actions. Down-regulation of S<em>1</em>P(3) receptors severely suppressed the stimulatory actions of S<em>1</em>P. Although G(i/o) proteins may play roles in either stimulatory or inhibitory S<em>1</em>P actions, as judged from pertussis toxin sensitivity, the coupling of S<em>1</em>P(3) receptors to G(<em>1</em>2/<em>1</em>3) proteins may be critical to distinguish the stimulatory pathways from the inhibitory ones. In conclusion, even though S<em>1</em>P alone stimulates adhesion molecule expression, HDL overcomes S<em>1</em>P(3) receptor-mediated stimulatory actions through SR-BI/PDZK<em>1</em>-mediated signaling pathways involving phosphatidylinositol 3-kinase and NOS. In addition, the S<em>1</em>P component of HDL plays a role in the inhibition of TNFalpha-induced actions through S<em>1</em>P receptors, especially S<em>1</em>P(<em>1</em>).

Endothelial cells (ECs) are covered by a surface glycocalyx layer that forms part of the barrier and mechanosensing functions of the blood-tissue interface. Removal of albumin in bathing media induces collapse or shedding of the glycocalyx. The electrostatic interaction between arginine residues on albumin, and negatively charged glycosaminoglycans (GAGs) in the glycocalyx have been hypothesized to stabilize the glycocalyx structure. Because albumin is one of the primary carriers of the phospholipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), we evaluated the alternate hypothesis that S<em>1</em>P, acting via S<em>1</em>P<em>1</em> receptors, plays the primary role in stabilizing the endothelial glycocalyx. Using confocal microscopy on rat fat-pad ECs, we demonstrated that heparan sulfate (HS), chondroitin sulfate (CS), and ectodomain of syndecan-<em>1</em> were shed from the endothelial cell surface after removal of plasma protein but were retained in the presence of S<em>1</em>P at concentrations of>><em>1</em>00 nM. S<em>1</em>P<em>1</em> receptor antagonism abolished the protection of the glycocalyx by S<em>1</em>P and plasma proteins. S<em>1</em>P reduced GAGs released after removal of plasma protein. The mechanism of protection from loss of glycocalyx components by S<em>1</em>P-dependent pathways was shown to be suppression of metalloproteinase (MMP) activity. General inhibition of MMPs protected against loss of CS and syndecan-<em>1</em>. Specific inhibition of MMP-9 and MMP-<em>1</em>3 protected against CS loss. We conclude that S<em>1</em>P plays a critical role in protecting the glycocalyx via S<em>1</em>P<em>1</em> and inhibits the protease activity-dependent shedding of CS, HS, and the syndecan-<em>1</em> ectodomain. Our results provide new insight into the role for S<em>1</em>P in protecting the glycocalyx and maintaining vascular homeostasis.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a platelet-derived sphingolipid that elicits numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors. Stimulation of EDG receptors by S<em>1</em>P has been shown to activate the endothelial isoform of nitric-oxide synthase (eNOS) in heterologous expression systems (Igarashi, J., and Michel, T. (2000) J. Biol. Chem. 275, 32363-32370). However, the signaling pathways that modulate eNOS regulation by S<em>1</em>P/EDG in vascular endothelial cells remain less well understood. We now report that S<em>1</em>P treatment of bovine aortic endothelial cells (BAEC) acutely increases eNOS enzyme activity; the EC(50) for S<em>1</em>P activation of eNOS is approximately <em>1</em>0 nm. The magnitude of eNOS activation by S<em>1</em>P in BAEC is equivalent to that elicited by the agonist bradykinin. S<em>1</em>P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S<em>1</em>P treatment of BAEC leads to eNOS phosphorylation at Ser(<em>1</em><em>1</em>79), a residue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylation site is inactivated shows attenuated S<em>1</em>P-induced eNOS activation. S<em>1</em>P-induced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracellular calcium chelator BAPTA (<em>1</em>,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). By contrast to S<em>1</em>P, activation of G protein-coupled bradykinin B2 receptors neither activates kinase Akt nor promotes Ser(<em>1</em><em>1</em>79) eNOS phosphorylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S<em>1</em>P and bradykinin may lead to the identification of new points for eNOS regulation in vascular endothelial cells.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> is a bioactive sphingolipid that regulates cell growth and suppresses programmed cell death. The biosynthesis of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> is catalyzed by <em>sphingosine</em> kinase (SK) but the mechanism by which the subcellular localization and activity of SK is regulated in response to various stimuli is not fully understood. To elucidate the origin and structural determinant of the specific subcellular localization of SK, we performed biophysical and cell studies of human SK<em>1</em> (hSK<em>1</em>) and selected mutants. In vitro measurements showed that hSK<em>1</em> selectively bound phosphatidylserine over other anionic phospholipids and strongly preferred the plasma membrane-mimicking membrane to other cellular membrane mimetics. Mutational analysis indicates that conserved Thr54 and Asn89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser225 enhances the membrane affinity and plasma membrane selectivity of hSK<em>1</em>, presumably by modulating the interaction of Thr54 and Asn89 with the membrane. Collectively, these studies suggest that the specific plasma membrane localization and activation of SK<em>1</em> is mediated largely by specific lipid-protein interactions.

The bioactive lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and its G protein-coupled receptors play critical roles in cardiovascular, immunological, and neural development and function. Despite its importance, many questions remain about S<em>1</em>P signaling, including how S<em>1</em>P, which is synthesized intracellularly, is released from cells. Mutations in the zebrafish gene encoding the S<em>1</em>P receptor Miles Apart (Mil)/S<em>1</em>P(2) disrupt the formation of the primitive heart tube. We find that mutations of another zebrafish locus, two of hearts (toh), cause phenotypes that are morphologically indistinguishable from those seen in mil/s<em>1</em>p2 mutants. Positional cloning of toh reveals that it encodes a member of the Spinster-like family of putative transmembrane transporters. The biological functions of these proteins are poorly understood, although phenotypes of the Drosophila spinster and zebrafish not really started mutants suggest that these proteins may play a role in lipid trafficking. Through gain- and loss-of-function analyses, we show that toh is required for signaling by S<em>1</em>P(2). Further evidence indicates that Toh is involved in the trafficking or cellular release of S<em>1</em>P.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and lysophosphatidic acid (LPA) are structurally related lipid mediators that act on distinct G-protein-coupled receptors to evoke similar responses, including Ca2+ mobilization, adenylate cyclase inhibition, and mitogen-activated protein (MAP) kinase activation. However, little is still known about the respective receptors. A recently cloned putative LPA receptor (Vzg-<em>1</em>/Edg-2) is similar to an orphan Gi-coupled receptor termed Edg-<em>1</em>. Here we show that expression of Edg-<em>1</em> in Sf9 and COS-7 cells results in inhibition of adenylate cyclase and activation of MAP kinase (Gi-mediated), but not Ca2+ mobilization, in response to S<em>1</em>P. These responses are specific in that (i) S<em>1</em>P action is not mimicked by LPA, and (ii) Vzg-<em>1</em>/Edg-2 cannot substitute for Edg-<em>1</em>. Thus the Edg-<em>1</em> receptor is capable of mediating a subset of the cellular responses to S<em>1</em>P.

OBJECTIVE

Myofibroblasts play a central role in the pathogenesis of liver fibrosis. Myofibroblasts of bone marrow (BM) origin have recently been identified in fibrotic liver. However, little is known about the mechanism that controls their mobilization in vivo. Here we confirmed that BM mesenchymal stem cells (BMSCs) can migrate to the damaged liver and differentiate into myofibroblasts. We also investigated the mechanism underlying the homing of BMSCs after liver injury.

METHODS

ICR mice were lethally irradiated and received BM transplants from enhanced green fluorescent protein transgenic mice. Carbon tetrachloride or bile duct ligation was used to induce liver fibrosis. The fibrotic liver tissue was examined by immunofluorescent staining to identify BM-derived myofibroblasts.

RESULTS

BMSCs contributed significantly to myofibroblast population in fibrotic liver. Moreover, analysis in vivo and in vitro suggested that homing of BMSCs to the damaged liver was in response to <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) gradient between liver and BM. Furthermore, S<em>1</em>P receptor type 3 (S<em>1</em>P3) was required for migration of BMSCs triggered by S<em>1</em>P.

CONCLUSIONS

S<em>1</em>P mediates liver fibrogenesis through homing of BMSCs via S<em>1</em>P3 receptor, which may represent a novel therapeutic target in liver fibrosis through inhibiting S<em>1</em>P formation and/or receptor activation.

A low high-density lipoprotein (HDL) plasma concentration and the abundance of small dense low-density lipoproteins (LDL) are risk factors for developing type 2 diabetes. We therefore investigated whether HDL and LDL play a role in the regulation of pancreatic islet cell apoptosis, proliferation, and secretory function. Isolated mouse and human islets were exposed to plasma lipoproteins of healthy human donors. In murine and human beta-cells, LDL decreased both proliferation and maximal glucose-stimulated insulin secretion. The comparative analysis of beta-cells from wild-type and LDL receptor-deficient mice revealed that the inhibitory effect of LDL on insulin secretion but not proliferation requires the LDL receptor. HDL was found to modulate the survival of both human and murine islets by decreasing basal as well as IL-<em>1</em>beta and glucose-induced apoptosis. IL-<em>1</em>beta-induced beta-cell apoptosis was also inhibited in the presence of either the delipidated protein or the deproteinated lipid moieties of HDL, apolipoprotein A<em>1</em> (the main protein component of HDL), or <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (a bioactive sphingolipid mostly carried by HDL). In murine beta-cells, the protective effect of HDL against IL-<em>1</em>beta-induced apoptosis was also observed in the absence of the HDL receptor scavenger receptor class B type <em>1</em>. Our data show that both LDL and HDL affect function or survival of beta-cells and raise the question whether dyslipidemia contributes to beta-cell failure and hence the manifestation and progression of type 2 diabetes mellitus.

Hepatocyte growth factor activator inhibitor-<em>1</em> (HAI-<em>1</em>) was initially identified as cognate inhibitor of matriptase, a membrane-bound serine protease. Paradoxically, HAI-<em>1</em> is also required for matriptase activation, a process that requires <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P)-mediated translocation of the protease to cell-cell junctions in human mammary epithelial cells. In the present study, we further explored how HAI-<em>1</em> regulates this protease. First, we observed that after S<em>1</em>P treatment HAI-<em>1</em> was cotranslocated with matriptase to cell-cell junctions and that the cellular ratio of HAI-<em>1</em> to matriptase was maintained during this process. However, when this ratio was changed by cell treatment with HAI-<em>1</em> small interfering RNA or anti-HAI-<em>1</em> MAb M<em>1</em>9, spontaneous activation of matriptase occurred in the absence of S<em>1</em>P-induced translocation; S<em>1</em>P-induced matriptase activation was also enhanced. These results support a role for HAI-<em>1</em> in protection of cell from uncontrolled matriptase activation. We next expressed matriptase, either alone or with HAI-<em>1</em> in breast cancer cells that do not endogenously express either protein. A defect in matriptase trafficking to the cell surface occurred if wild-type matriptase was expressed in the absence of HAI-<em>1</em>; this defect appeared to result from matriptase toxicity to cells. Coexpression with matriptase of wild-type HAI-<em>1</em>, but not HAI-<em>1</em> mutants altered in its Kunitz domain <em>1</em>, corrected the trafficking defect. In contrast, catalytically defective matriptase mutants were normal in their trafficking in the absence of HAI-<em>1</em>. These results are also consistent with a role for HAI-<em>1</em> to prevent inappropriate matriptase proteolytic activity during its protein synthesis and trafficking. Taken together, these results support multiple roles for HAI-<em>1</em> to regulate matriptase, including its proper expression, intracellular trafficking, activation, and inhibition.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) has gained special attention in the high-density lipoprotein (HDL) field because HDL is the most prominent plasma carrier of S<em>1</em>P and because the S<em>1</em>P content of HDL may be responsible for many of the pleiotropic functions of HDL. This revelation has come from the evidence that HDL employ S<em>1</em>P receptors and signalling pathways to implement several HDL-ascribed biological effects as diverse as endothelial nitric oxide production, vasodilation, survival, and cardioprotection. This review focuses on HDL effects that are completely or partially mediated by the S<em>1</em>P content of the HDL particle and differentiates them from genuine HDL effects that are S<em>1</em>P-independent. In addition, the functional properties of 'free', HDL-unbound S<em>1</em>P are sometimes different from or even contrary to those of HDL-associated S<em>1</em>P. The nature of the physical interactions between HDL and local and systemic S<em>1</em>P production will be discussed as well as their consequences for organ function. Finally, we will elucidate the potential benefits and limitations of S<em>1</em>P analogues as a new class of functional HDL mimetics for cardiovascular therapy.

TNF alpha administration mimics ischemic preconditioning and neutralizing antibodies to TNF alpha and IL-<em>1</em> beta abolish exercise-induced preconditioning. However, the pharmacology of TNF alpha's cardioprotective effects and associated downstream signaling events has not been delineated. We evaluated the temporal and dose specific requirements of TNF alpha to function as a preconditioning mimetic. Furthermore we postulated that the preconditioning effect of TNF alpha might be orchestrated via sphingolipid signaling. The cardioprotective effect of TNF alpha and the role of sphingolipid signaling were assessed using a classical preconditioning protocol in the isolated perfused rat heart with the measurement of infarct size and contractile function modulation in response to index ischemia and reperfusion. Recombinant TNF alpha at an optimal dose of 0.5 ng/ml mimicked ischemic preconditioning by reducing infarct size by 60%v non-preconditioned ischemia-reperfusion controls (P<0.0<em>1</em>). The infarct sparing effect of TNF alpha required a wash-out period prior to the index ischemic-reperfusion. Moreover, the classic ischemic preconditioning antagonist such as 5-hydroxydecanoate abolished TNF alpha preconditioning. An inhibitor of the sphingolipid signaling pathway, N-oleoylethanolamine (NOE, <em>1</em> microm) attenuated ischemic and TNF alpha preconditioning. Likewise, cell-permeable C(2)-ceramide and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (sphingolipid signaling intermediates) both reproduced the preconditioning cardioprotective phenotype. Finally, TNF alpha and ceramide conferred preconditioning-like cardioprotection against post-ischemic contractile dysfunction and this cardioprotective effect was attenuated by NOE. In contrast, NOE did not reverse ischemic preconditioning enhanced post-ischemic contractile function. In conclusion, TNF alpha activates preconditioning-like tolerance against infarction and contractile dysfunction. This cardioprotection is mediated, in part, via activation of novel sphingolipid signaling intermediates.

Sphingolipids are a class of biologically active lipids that have a role in multiple biological processes including inflammation. Sphingolipids exert their functions by direct signaling or through signaling by their specific receptors. Phosphorylated FTY720 (FTY720P) is a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) analogue that is currently in trial for treatment of multiple sclerosis (MS), which targets all S<em>1</em>P receptors but S<em>1</em>P(2). To date, however, it remains unknown whether FTY720P may exert direct anti-inflammatory effects within the central nervous system (CNS), because data concerning S<em>1</em>P receptor expression and regulation under pathological conditions in the human brain are lacking. To investigate potential regulation of S<em>1</em>P receptors in the human brain during MS, we performed immunohistochemical analysis of S<em>1</em>P receptor <em>1</em> and 3 expression in well-characterized MS lesions. A strong increase in S<em>1</em>P receptor <em>1</em> and 3 expression on reactive astrocytes was detected in active and chronic inactive MS lesions. In addition, we treated primary cultures of human astrocytes with the proinflammatory cytokine tumor necrosis factor-alpha to identify the regulation of S<em>1</em>P(<em>1</em>/3) on astrocytes under pathological conditions. Importantly, we demonstrate that FTY720P exerts an anti-inflammatory action on human astrocytes by limiting secretion of proinflammatory cytokines. Our data demonstrate that reactive astrocytes in MS lesions and cultured under proinflammatory conditions strongly enhance expression of S<em>1</em>P receptors <em>1</em> and 3. Results from this study indicate that astrocytes may act as a yet-unknown target within the CNS for the anti-inflammatory effects observed after FTY720P administration in the treatment of MS.

RGS proteins finely tune heterotrimeric G-protein signaling. Implying the need for such fine-tuning in the developing vascular system, in situ hybridization revealed a striking and extensive expression pattern of Rgs5 in the arterial walls of E<em>1</em>2.5-E<em>1</em>7.5 mouse embryos. The distribution and location of the Rgs5-positive cells typified that of pericytes and strikingly overlapped the known expression pattern of platelet-derived growth factor receptor (PDGFR)-beta. Both E<em>1</em>4.5 PDGFR-beta- and platelet-derived growth factor (PDGF)-B-deficient mice exhibited markedly reduced levels of Rgs5 in their vascular plexa and small arteries. This likely reflects the loss of pericytes in the mutant mice. RGS5 acts as a potent GTPase activating protein for Gi(alpha) and Gq(alpha) and it attenuated angiotensin II-, endothelin-<em>1</em>-, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-, and PDGF-induced ERK-2 phosphorylation. Together these results indicate that RGS5 exerts control over PDGFR-beta and GPCR-mediated signaling pathways active during fetal vascular maturation.