Enhanced FTY720-mediated lymphocyte homing requires G alpha i signaling and depends on beta 2 and beta 7 integrin

The immunomodulatory drug FTY720 interferes with sphingosine-1-phosphate (S1P) receptor signaling leading to lymphocyte retention in secondary lymphoid organs and consequently to profound lymphopenia in the peripheral blood. The molecular mechanisms transduced by S1P receptors upon being triggered by its native ligand, S1P, or by FTY720, are largely unknown. In this study we analyze the role of beta2 and beta7 integrin and their ligands ICAM-1, VCAM-1, and MadCAM-1 on lymphocyte homing in the presence of FTY720. We demonstrate that this drug facilitates homing of lymphocytes single-deficient of either beta2 or beta7 integrin but not of beta2-deficient lymphocytes, which in addition were blocked by anti-beta7 integrin Abs. Enhanced lymphocyte homing is preceded by increased adherence of integrin-deficient as well as wild-type lymphocytes to high endothelial venules (HEV) in FTY720-treated animals. Elevated adherence to HEV requires intact lymphocyte Galphai signaling that cannot be stably imprinted on lymphocytes even after prolonged exposure to FTY720. Thus, FTY720 influences lymphocyte homeostasis not only by suppressing lymphocyte egress from lymph nodes but also by facilitating lymphocyte homing across HEV in an integrin-dependent fashion.     

Production and release of sphingosine 1-phosphate and the phosphorylated form of the immunomodulator FTY720

The bioactive lipid molecule sphingosine 1-phosphate (S1P) binds to specific cell surface receptors and regulates several cellular processes. S1P is abundant in plasma, and physiologically its most important target cells are lymphocytes and vascular endothelial cells. S1P plays a pivotal role in the immune system by regulating lymphocyte egress from the thymus and secondary lymphoid organs. The immunomodulator FTY720 impairs this egress, causing lymphopenia. Platelets had long been considered to be the major source of plasma S1P, however recent studies revealed the importance of erythrocytes as a major supply. The sphingosine analog FTY720 is a prodrug, and FTY720 phosphate (FTY720-P) its functional form. Although both erythrocytes and platelets can produce S1P, only platelets synthesize and release FTY720-P. This review will focus on the recent advances in our understanding of the metabolism and release of S1P and FTY720-P, especially in platelets and erythrocytes.     

FTY720 blocks egress of T cells in part by abrogation of their adhesion on the lymph node sinus

Egress of lymphocytes from lymphoid tissues is a complex process in which Gαi-mediated signals play a decisive role. We show here that although FTY720, an agonist of the sphingosine 1-phosphate (S1P)(1) receptor, induces S1P(1) receptor internalization sufficiently in the presence or absence of Gαi2 or Gαi3, the drug blocks egress of wild-type (WT) and Gαi3-deficent T cells, but not Gαi2-deficient T cells, in both WT and Gαi2-deficient hosts. Intravital imaging of lymph nodes revealed that all three groups of T cells approached and engaged cortical sinusoids similarly in the presence or absence of FTY720. The cells also entered and departed the sinus at an almost identical frequency in the absence of the drug. However, after engagement of the sinus, most WT and Gαi3-deficient T cells retracted and migrated back into the parenchyma in FTY720-treated animals, due to a failure of the cells to establish adhesion on the sinus, whereas Gαi2-deficient T cells adhered firmly on the sinus, which prevented their retraction, facilitating their transmigration of the lymphatic endothelial barrier. These data confirm egress of Gαi2(-/-) T cells independent of S1P-mediated chemotaxis and failure of FTY720 to close lymphatic stromal channels and argue for the first time, to our knowledge, that FTY720 induces lymphopenia in part by impairing T cell adhesion to the sinus in a manner dependent on Gαi2.     

Selective activation of G alpha i mediated signalling of S1P3 by FTY720-phosphate

The immune modulator FTY720 is phosphorylated in vivo to FTY720 phosphate (FTY-P), which activates four sphingosine 1-phosphate (S1P) receptors including S1P₃. Upon activation with S1P, S1P₃ couples to G_i- and G_q-protein-dependent signalling pathways. Here we show that FTY-P selectively activates the S1P₃-mediated and G_i-coupled inhibition of adenylyl cyclase. Contemporaneously, it antagonizes the S1P-induced activation of G_q via S1P₃ in intracellular calcium flux measurements, GTP-binding experiments, and flow cytometric analyses of activation-induced receptor down-regulation. In contrast to S1P, pre-treatment with FTY-P did not desensitize S1P-induced calcium flux or chemotaxis via S1P₃. The lack of receptor desensitization prevented S1P₃-mediated migration to FTY-P. Human umbilical vein endothelial cells express S1P₁ and S1P₃, and respond to S1P and FTY-P by ERK1/2 phosphorylation and by intracellular calcium release in a pertussis toxin-sensitive manner. But whereas a mixture of S1P and FTY-P was not affecting ERK1/2 phosphorylation, the intracellular calcium flux was hampered with increasing amounts of FTY-P, which points to a cross-talk between S1P₁ and S1P₃. FTY-P is therefore one of the rare ligands which bind to a receptor that couples multiple G-proteins but selectively activates only one signalling pathway.     

AKP-11 - A Novel S1P1 Agonist with Favorable Safety Profile Attenuates Experimental Autoimmune Encephalomyelitis in Rat Model of Multiple Sclerosis

Sphingosine-1-phosphate receptor 1 (S1P1) mediated regulation of lymphocyte egress from lymphoid organs is recognized as the mechanism of FTY720 (Fingolimod, Gilenya) efficacy in relapsing-remitting forms of multiple sclerosis (RRMS). In this study we describe a novel S1P1 agonist AKP-11, next generation of S1P1 agonist, with immunomodulatory activities in cell culture model and for therapeutic efficacy against an animal model of MS, i.e. experimental autoimmune encephalomyelitis (EAE) but without the adverse effects observed with FTY720. Like FTY720, AKP-11 bound to S1P1 is internalized and activates intracellular AKT and ERKs cellular signaling pathways. In contrast to FTY720, AKP-11 mediated S1P1 downregulation is independent of sphingosine kinase activity indicating it to be a direct agonist of S1P1. The S1P1 loss and inhibition of lymphocyte egress by FTY720 leads to lymphopenia. In comparison with FTY720, oral administration of AKP-11 caused milder and reversible lymphopenia while providing a similar degree of therapeutic efficacy in the EAE animal model. Consistent with the observed reversible lymphopenia with AKP-11, the S1P1 recycled back to cell membrane in AKP-11 treated cells following its withdrawal, but not with withdrawal of FTY720. Accordingly, a smaller degree of ubiquitination and proteolysis of S1P1 was observed in AKP-11 treated cells as compared to FTY720. Consistent with previous observations, FTY720 treatment is associated with adverse effects of bradycardia and lung vascular leaks in rodents, whereas AKP-11 treatment had undetectable effects on bradycardia and reduced lung vascular leaks as compared to FTY720. Taken together, the data documents that AKP-11 treatment cause milder and reversible lymphopenia with milder adverse effects while maintaining therapeutic efficacy similar to that observed with FTY720, thus indicating therapeutic potential of AKP-11 for treatment of MS and related autoimmune disorders.     

Down-regulation of S1P1 Receptor Surface Expression by Protein Kinase C Inhibition

The sphingosine 1-phosphate receptor type 1 (S1P₁) is important for the maintenance of lymphocyte circulation. S1P₁ receptor surface expression on lymphocytes is critical for their egress from thymus and lymph nodes. Premature activation-induced internalization of the S1P₁ receptor in lymphoid organs, mediated either by pharmacological agonists or by inhibition of the S1P degrading enzyme S1P-lyase, blocks lymphocyte egress and induces lymphopenia in blood and lymph. Regulation of S1P₁ receptor surface expression is therefore a promising way to control adaptive immunity. Hence, we analyzed potential cellular targets for their ability to alter S1P₁ receptor surface expression without stimulation. The initial observation that preincubation of mouse splenocytes with its natural analog sphingosine was sufficient to block Transwell^TM chemotaxis to S1P directed subsequent investigations to the underlying mechanism. Sphingosine is known to inhibit protein kinase C (PKC), and PKC inhibition with nanomolar concentrations of staurosporine, calphostin C, and GF109203X down-regulated surface expression of S1P₁ but not S1P₄ in transfected rat hepatoma HTC₄ cells. The PKC activator phorbol 12-myristate 13-acetate partially rescued FTY720-induced down-regulation of the S1P₁ receptor, linking PKC activation with S1P₁ receptor surface expression. FTY720, but not FTY720 phosphate, efficiently inhibited PKC. Cell-based efficacy was obvious with 10 nm FTY720, and in vivo treatment of mice with 0.3–3 mg/kg/day FTY720 showed increasing concentration-dependent effectiveness. PKC inhibition therefore may contribute to lymphopenia by down-regulating S1P₁ receptor cell surface expression independently from its activation.     

Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720

Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.     

Moesin Controls Clathrin-Mediated S1PR1 Internalization in T Cells

The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation.     

Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 agonists induce ubiquitinylation and proteasomal degradation of the receptor

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.     

High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B,Type I,PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists

HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P) and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI''s C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1) antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.     

The Sphingosine 1-Phosphate Transporter,SPNS2, Functions as a Transporter of the Phosphorylated Form of the Immunomodulating Agent FTY720

FTY720 is a novel immunomodulating drug that can be phosphorylated inside cells; its phosphorylated form, FTY720-P, binds to a sphingosine 1-phosphate (S1P) receptor, S1P₁, and inhibits lymphocyte egress into the circulating blood. Although the importance of its pharmacological action has been well recognized, little is known about how FTY720-P is released from cells after its phosphorylation inside cells. Previously, we showed that zebrafish Spns2 can act as an S1P exporter from cells and is essential for zebrafish heart formation. Here, we demonstrate that human SPNS2 can transport several S1P analogues, including FTY720-P. Moreover, FTY720-P is transported by SPNS2 through the same pathway as S1P. This is the first identification of an FTY720-P transporter in cells; this finding is important for understanding FTY720 metabolism.     

Sphingosine-1-phosphate receptor agonists suppress concanavalin A-induced hepatic injury in mice

T cell-mediated immune responses play a critical role in a variety of liver injuries including autoimmune hepatitis. Injection of concanavalin A (Con A) into mice mimics the histological and pathological phenotype of T cell-mediated hepatitis. Recent advances in host immune control of organ transplantation include the development of sphingosine-1-phosphate (S1P) receptor agonists such as FTY720, which alter lymphocyte homing but do not suppress host general immunity. Herein we examined the effect of the new S1P receptor agonist KRP-203 on the Con A-induced liver damage model. In normal liver lymphocytes of BALB/c mice, both FTY720 and KRP203 promoted lymphocyte sequestering from the liver to secondary lymph nodes and significantly reduced the number of liver lymphocytes (p<0.05). Based on this observation, KRP203 was employed in the Con A-induced hepatitis model. KRP203 markedly reduced the number of CD4(+) lymphocytes that infiltrate Con A-treated liver (p<0.05) and successfully reduced serum transaminase elevation (p=0.017), therefore protecting mice from Con A-induced liver injury. Interestingly this homing modulation less occurs in natural hepatic T cell homing through the chemokine receptor, CXCR4. Therefore, S1P receptor agonists preferentially target CXCR4(+)CD4(+) peripheral blood T lymphocytes and suppress the occurrence of Con A-induced hepatitis, suggesting their therapeutic usefulness against T cell-mediated hepatic injury.     

The immunosuppressant FTY720 inhibits tumor angiogenesis via the sphingosine 1-phosphate receptor 1

FTY720, a sphingosine 1-phosphate (S1P) analog, acts as an immunosuppressant through trapping of T cells in secondary lymphoid tissues. FTY720 was also shown to prevent tumor growth and to inhibit vascular permeability. The MTT proliferation assay illustrated that endothelial cells are more susceptible to the anti-proliferative effect of FTY720 than Lewis lung carcinoma (LLC1) cells. In a spheroid angiogenesis model, FTY720 potently inhibited the sprouting activity of VEGF-A-stimulated endothelial cells even at concentrations that apparently had no anti-proliferative effect. Mechanistically, the anti-angiogenic effect of the general S1P receptor agonist FTY720 was mimicked by the specific S1P1 receptor agonist SEW2871. Moreover, the anti-angiogenic effect of FTY720 was abrogated in the presence of CXCR4-neutralizing antibodies. This indicates that the effect was at least in part mediated by the S1P1 receptor and involved transactivation of the CXCR4 chemokine receptor. Additionally, we could illustrate in a coculture spheroid model, employing endothelial and smooth muscle cells (SMCs), that the latter confer a strong protective effect regarding the action of FTY720 upon the endothelial cells. In a subcutaneous LLC1 tumor model, the anti-angiogenic capacity translated into a reduced tumor size in syngeneic C57BL/6 mice. Consistently, in the Matrigel plug in vivo assay, 10 mg/kg/d FTY720 resulted in a strong inhibition of angiogenesis as demonstrated by a reduced capillary density. Thus, in organ transplant patients, FTY720 may prove efficacious in preventing graft rejection as well as tumor development.     

Synthesis, stability, and implications of phosphothioate agonists of sphingosine-1-phosphate receptors

Phosphothioates may provide metabolic stability when compared to their phosphate counterparts, while retaining the potency and efficacy as agonists at sphingosine-1-phosphate (S1P) G-protein coupled receptors. Unlike their phosphate precursors, phosphothioate compounds with S1P-receptor profiles similar to that of FTY720, an emerging immunomodulator, were shown to evoke prolonged lymphopenia in vivo. Analysis of mouse plasma concentrations for a series of related alcohol/phosphate/phosphothioate compounds showed the conversion of the phosphate to alcohol. These preliminary data highlight the importance of metabolic regulation of S1P receptor ligands.     

A splicing isoform of LPP1, LPP1a, exhibits high phosphatase activity toward FTY720 phosphate

The sphingolipid metabolite sphingosine 1-phosphate (S1P) plays an essential function in the egress of T cells from the thymus and secondary lymphoid organs. The novel immunomodulating agent FTY720 is phosphorylated in vivo to the functional form FTY720 phosphate (FTY720-P), which is structurally similar to S1P. FTY720-P inhibits the S1P-mediated T cell egress as an agonist of S1P receptors. FTY720-P is not stable in plasma and is dephosphorylated to FTY720. In the present study, we investigated activities toward FTY720-P of LPP family members (LPP1, LPP1a, LPP2, and LPP3), which exhibit broad substrate specificity. Of the four, LPP1a, the splicing isoform of LPP1, had the highest activity toward FTY720-P, and the highest affinity. Among blood-facing cells tested, only endothelial cells displayed high phosphatase activity for FTY720-P. Significant levels of LPP1a expression were found in endothelial cells, suggesting that LPP1a is important for the dephosphorylation of FTY720-P in plasma.     

Roles for lysophospholipid S1P receptors in multiple sclerosis

Sphingosine 1-phosphate (S1P) signaling in the treatment of multiple sclerosis (MS) has been highlighted by the efficacy of FTY720 (fingolimod), which upon phosphorylation can modulate S1P receptor activities. FTY720 has become the first oral treatment for relapsing MS that was approved by the FDA in September 2010. Phosphorylated FTY720 modulates four of the five known S1P receptors (S1P(1), S1P(3), S1P(4), and S1P(5)) at high affinity. Studies in human MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have revealed that FTY720 exposure alters lymphocyte trafficking via sequestration of auto-aggressive lymphocytes within lymphoid organs, representing the current understanding of its mechanism of action. These effects primarily involve S1P(1), which is thought to attenuate inflammatory insults in the central nervous system (CNS). In addition, FTY720's actions may involve direct effects on S1P receptor-mediated signaling in CNS cells, based upon the known expression of S1P receptors in CNS cell types relevant to MS, access to the CNS through the blood-brain barrier (BBB), and in vitro studies. These data implicate lysophospholipid signaling--via S1P(1) and perhaps other lysophospholipid receptors--in therapeutic approaches to MS and potentially other diseases with immunological and/or neurological components.     

Functional characterization of sphingosine 1-phosphate receptor agonist in human endothelial cells

Sphingosine 1-phosphate (S1P) is a pleiotropic lysophospholipid mediator involved in many cellular responses, including transient calcium mobilization, activation of MAP kinase signaling, inhibition of adenylyl cyclase and increased cell migration. S1P has been shown to be an effective activator of vascular endothelial cells via the interaction with cell surface G protein-coupled receptors (GPCRs), namely S1P-R (formerly EDG-R). The potent immunomodulator, FTY720, is phosphorylated by sphingosine kinase (SK) to FTY720-P. Recently it was shown that FTY720-P, not FTY720, can bind to four out of five of the S1P-R. In the present study, we evaluated the effects of FTY720, FTY720-P, and analogues of FTY720-P: an active (R)-enantiomer [AFD(R)] and an inactive (S)-enantiomer [AFD(S)], on endothelial cell functions. Treatment of HUVEC with FTY720-P, but not FTY720, lead to a robust transient increase in calcium mobilization, detected using the fluorometric imaging plate reader (FLIPR) assay. Additionally, only the phosphorylated derivative (FTY720-P) stimulated MAPK activation. We also observed complementary activities of S1P and FTY720-P in an established in vitro endothelial morphogenesis (Matrigel tube formation) assay and an in vitro endothelial cell migration assay. Using a potent inhibitor of sphingosine kinase, N,N-dimethylsphingosine (DMS), FTY720's effects were inhibited in the migration assay, suggesting that FTY720-P is the active mediator. The effects of FTY720-P in these assays were inhibited by pre-treatment with PTx (pertussis toxin), indicating the requirement of a Gi-coupled S1P receptor. These findings suggest that agonist of S1P-R are able to regulate important endothelial cell properties, which may lead to a greater insight into vascular functions.     

Sphingosine-1-phosphate receptor agonism impairs the efficiency of the local immune response by altering trafficking of naive and antigen-activated CD4+ T cells

FTY720 (2-amino-[2-(4-octylphenyl) ethyl]-1,3-propanediol hydrochloride) is an immunosuppressive agent that inhibits allograft rejection. We recently demonstrated that FTY-phosphate, the active metabolite of FTY720, acts as a full agonist for sphingosine-1-phosphate (S1P) receptors. Furthermore, activation of S1P receptors with their natural ligand, S1P, as well as pharmacological ligands leads to lymphopenia, probably due to sequestration of lymphocytes in secondary lymphoid organs. In the present study we used a local Ag-challenged mouse model to examine the effects of FTY720 on T cell activation in the draining lymph node (DLN) and on the release of activated T cells to the peripheral blood compartment. We showed that the number of Ag-activated CD4(+) T cells in the DLN after injection of Ag and CFA into a footpad was dramatically reduced after FTY720 treatment. However, T cell proliferation, both in vitro and in vivo, was not impaired by FTY720. Our results suggest that the reduced efficiency of T cell responses in the DLN in response to a local Ag is probably due to a defective recirculation of naive T cells caused by FTY720 treatment. Furthermore, we found that the numbers of naive and Ag-activated CD4(+) T cells in the peripheral blood of Ag-challenged mice were equally reduced with FTY720 treatment, suggesting that both T cell subsets are sequestered in the DLNs. Thus, FTY720 induces immunosuppression through inhibition of both the recirculation of naive T cells and the release of Ag-activated T cells from the DLN to lymph and to the blood compartment.     

FTY720 Protects Cardiac Microvessels of Diabetes: A Critical Role of S1P1/3 in Diabetic Heart Disease

Background: Diabetes is associated with an increased risk of cardiac microvascular disease. The mechanisms by which this damage occurs are unknown. However, research suggests that signaling through the sphingosine-1-phosphates receptor 1 and 3 (S1P1/3) by FTY720, a sphiongolipid drug that is structually similar to SIP, may play a role in the treatment on cardiac microvascular dysfunction in diabetes. We hypothesized that FTY720 might exert the cardioprotective effects of S1P1 and S1P3 viaprotein kinase C-beta (PKCβ II) signaling pathway.Methodology/Principal Findings: Transthoracic echocardiography was performed to detect the change of cardiac function. Scanning and transmission electron microscope with lanthanum tracer were used to determine microvascular ultrastructure and permeability in vivo. Apoptosis was detected by TUNEL and CD31 dual labeling in paraffin-embedded sections. Laser capture miscrodissection was used to assess cardiac micovascular endothelial cells (CMECs) in vivo. RT-PCR and Western blot analysis were used to determine the mRNA levels and protein expression of S1P1, S1P3, and PKCβ II. In the diabetic rats vs. controls, cardiac capillaries showed significantly higher density; CD31 positive endothelial cells were significantly reduced; the apoptosis index of cardiac endothlial cells was significantly higher. And FTY720 could increase the expressional level of S1P1 and boost S1P3 trasnslocation from membrane to nuclear, then ameliorate cardiac microvascular barrier impairment and pathologic angiogenesis induced by diabetes. In addition, overexpression of PKCβ II significantly decreased the protective effect of FTY720.Conclusions: Our study represents that the deregulation of S1P1 and S1P3 is an important signalresponsible for cardiac microvascular dysfunction in diabetes. FTY720 might be competent to serve as a potential therapeutic approach for diabetic heart disease through ameliorating cardiac microvascular barrier impairment and pathologic angiogenesis, which might be partly dependent on PKCβII-mediated signaling pathway.