Synthesis, radiosynthesis and biochemical evaluation of fluorinated analogues of sphingosine-1-phosphate receptor 3 specific antagonists using PET.

Sphingosine-1-phosphate and its receptors (S1PRs) are involved in several diseases such as auto immunity, inflammation and cardiovascular disorders. The S1P analogue fingolimod (Gilenya®) is currently in use for the treatment of relapsing multiple sclerosis. S1PRs are also promising targets for clinical molecular imaging in vivo. The organ distribution of individual S1PRs can be potentially achieved by using S1PR subtype-specific (radiolabeled) chemical probes. Here, we report our efforts on synthesis and in vivo potency determination of new ligands for the S1P receptor 3 (S1P3) based on the S1P3 antagonist TY-52156 and in validation of a potential imaging tracer in vivo using Positron Emission Tomography (PET) after 18F-labelling. A p-fluorophenyl derivative exhibited excellent S1P3 antagonist activity in vitro, good serum stability, and medium lipophilicity. In vivo biodistribution experiments using 18F-PET exhibited significant uptake in the myocardium suggesting potential applications in cardiac imaging.

Sphingosine-1-Phosphate (S1P) Lyase Inhibition Aggravates Atherosclerosis and Induces Plaque Rupture in ApoE-/-Mice.

Altered plasma sphingosine-1-phosphate (S1P) concentrations are associated with clinical manifestations of atherosclerosis. However, whether long-term elevation of endogenous S1P is pro- or anti-atherogenic remains unclear. Here, we addressed the impact of permanently high S1P levels on atherosclerosis in cholesterol-fed apolipoprotein E-deficient (ApoE-/-) mice over 12 weeks. This was achieved by pharmacological inhibition of the S1P-degrading enzyme S1P lyase with 4-deoxypyridoxine (DOP). DOP treatment dramatically accelerated atherosclerosis development, propagated predominantly unstable plaque phenotypes, and resulted in frequent plaque rupture with atherothrombosis. Macrophages from S1P lyase-inhibited or genetically deficient mice had a defect in cholesterol efflux to apolipoprotein A-I that was accompanied by profoundly downregulated cholesterol transporters ATP-binding cassette transporters ABCA1 and ABCG1. This was dependent on S1P signaling through S1PR3 and resulted in dramatically enhanced atherosclerosis in ApoE-/-/S1PR3-/- mice, where DOP treatment had no additional effect. Thus, high endogenous S1P levels promote atherosclerosis, compromise cholesterol efflux, and cause genuine plaque rupture.

Dapagliflozin reduces thrombin generation and platelet activation: implications for cardiovascular risk reduction in type 2 diabetes mellitus.

AIMS/HYPOTHESIS: People with diabetes have an increased cardiovascular risk with an accelerated development of atherosclerosis and an elevated mortality rate after myocardial infarction. Therefore, cardioprotective effects of glucose-lowering therapies are of major importance for the pharmacotherapy of individuals with type 2 diabetes. For sodium-glucose cotransporter 2 inhibitors (SGLT2is), in addition to a reduction in blood glucose, beneficial effects on atherosclerosis, obesity, renal function and blood pressure have been observed. Recent results showed a reduced risk of worsening heart failure and cardiovascular deaths under dapagliflozin treatment irrespective of the diabetic state. However, the underlying mechanisms are yet unknown. Platelets are known drivers of atherosclerosis and atherothrombosis and disturbed platelet activation has also been suggested to occur in type 2 diabetes. Therefore, the present study investigates the impact of the SGLT2i dapagliflozin on the interplay between platelets and inflammation in atherogenesis. METHODS: Male, 8-week-old LDL-receptor-deficient (Ldlr-/-) mice received a high-fat, high-sucrose diabetogenic diet supplemented without (control) or with dapagliflozin (5 mg/kg body weight per day) for two time periods: 8 and 25 weeks. In a first translational approach, eight healthy volunteers received 10 mg dapagliflozin/day for 4 weeks. RESULTS: Dapagliflozin treatment ameliorated atherosclerotic lesion development, reduced circulating platelet-leucocyte aggregates (glycoprotein [GP]Ib+CD45+: 29.40 ± 5.94 vs 17.00 ± 5.69 cells, p < 0.01; GPIb+lymphocyte antigen 6 complex, locus G+ (Ly6G): 8.00 ± 2.45 vs 4.33 ± 1.75 cells, p < 0.05) and decreased aortic macrophage infiltration (1.31 ± 0.62 vs 0.70 ± 0.58 ×103 cells/aorta, p < 0.01). Deeper analysis revealed that dapagliflozin decreased activated CD62P-positive platelets in Ldlr-/- mice fed a diabetogenic diet (3.78 ± 1.20% vs 2.83 ± 1.06%, p < 0.01) without affecting bleeding time (85.29 ± 37.27 vs 89.25 ± 16.26 s, p = 0.78). While blood glucose was only moderately affected, dapagliflozin further reduced endogenous thrombin generation (581.4 ± 194.6 nmol/l × min) × 10-9 thrombin vs 254.1 ± 106.4 (nmol/l × min) × 10-9 thrombin), thereby decreasing one of the most important platelet activators. We observed a direct inhibitory effect of dapagliflozin on isolated platelets. In addition, dapagliflozin increased HDL-cholesterol levels. Importantly, higher HDL-cholesterol levels (1.70 ± 0.58 vs 3.15 ± 1.67 mmol/l, p < 0.01) likely contribute to dapagliflozin-mediated inhibition of platelet activation and thrombin generation. Accordingly, in line with the results in mice, treatment with dapagliflozin lowered CD62P-positive platelet counts in humans after stimulation by collagen-related peptide (CRP; 88.13 ± 5.37% of platelets vs 77.59 ± 10.70%, p < 0.05) or thrombin receptor activator peptide-6 (TRAP-6; 44.23 ± 15.54% vs 28.96 ± 11.41%, p < 0.01) without affecting haemostasis. CONCLUSIONS/INTERPRETATION: We demonstrate that dapagliflozin-mediated atheroprotection in mice is driven by elevated HDL-cholesterol and ameliorated thrombin-platelet-mediated inflammation without interfering with haemostasis. This glucose-independent mechanism likely contributes to dapagliflozin's beneficial cardiovascular risk profile.

Potent anti-inflammatory properties of HDL in vascular smooth muscle cells mediated by HDL-S1P and their impairment in coronary artery disease due to lower HDL-S1P: a new aspect of HDL dysfunction and its therapy.

Dysfunctional HDL is associated with coronary artery disease (CAD), but its effect on inflammation in vascular smooth muscle cells (VSMCs) in atherosclerosis is unknown. We investigated the effect of healthy human HDL and CAD-HDL on TNF-α-driven inflammation in VSMCs and examined whether HDL-associated sphingosine-1-phosphate (HDL-S1P) could modulate inflammation with the aim of designing novel HDL-based anti-inflammatory strategies. Healthy human HDL, human CAD-HDL, and mouse HDL were isolated by ultracentrifugation, S1P was measured by liquid chromatography-tandem mass spectrometry, and TNF-α-induced inflammation was characterized by gene expression and analysis of NF-κB-dependent signaling. Mechanisms of S1P interference with TNF-α were assessed by S1P receptor antagonists, mouse knockouts, and short interfering RNA. We observed that healthy HDL potently inhibited the induction of TNF-α-stimulated inflammatory genes, such as iNOS (inducible NO synthase) and MMP9 (matrix metalloproteinase 9), a process that was entirely dependent on HDL-S1P, as evidenced by loss-of-function using S1P-less HDL and mimicked by genuine S1P. Inhibition was based on suppression of TNF-α-activated Akt signaling resulting in reduced IkBαSer32 and p65Ser534 NF-κB phosphorylation based on a persistent phosphatase and tensin homolog activation by S1P through the S1P receptor 2. Intriguingly, S1P suppressed inflammation even hours after initial TNF-α stimulation. The anti-inflammatory effect of healthy HDL correlated with HDL-S1P content and was superior to that of CAD-HDL featuring lower HDL-S1P. Nevertheless, therapeutic loading of HDL with S1P completely restored the anti-inflammatory capacity of CAD-HDL and greatly boosted that of both healthy and CAD-HDL. Suppression of inflammation by HDL-S1P defines a novel pathophysiologic characteristic that distinguishes functional from dysfunctional HDL. The anti-inflammatory HDL function can be boosted by S1P-loading and exploited by S1P receptor-targeting to prevent and even turn off ongoing inflammation.-Keul, P., Polzin, A., Kaiser, K., Gräler, M., Dannenberg, L., Daum, G., Heusch, G., Levkau, B. Potent anti-inflammatory properties of HDL in vascular smooth muscle cells mediated by HDL-S1P and their impairment in coronary artery disease due to lower HDL-S1P: a new aspect of HDL dysfunction and its therapy.

Regulation of ABCA1-mediated cholesterol efflux by sphingosine-1-phosphate signaling in macrophages.

Sphingolipid and cholesterol metabolism are closely associated at the structural, biochemical, and functional levels. Although HDL-associated sphingosine-1-phosphate (S1P) contributes to several HDL functions, and S1P signaling regulates glucose and lipid metabolism, no study has addressed the involvement of S1P in cholesterol efflux. Here, we show that sphingosine kinase (Sphk) activity was induced by the LXR agonist 22(R)-hydroxycholesterol and required for the stimulation of ABCA1-mediated cholesterol efflux to apolipoprotein A-I. In support, pharmacological Sphk inhibition and Sphk2 but not Sphk1 deficiency abrogated efflux. The involved mechanism included stimulation of both transcriptional and functional ABCA1 regulatory pathways and depended for the latter on the S1P receptor 3 (S1P3). Accordingly, S1P3-deficient macrophages were resistant to 22(R)-hydroxycholesterol-stimulated cholesterol efflux. The inability of excess exogenous S1P to further increase efflux was consistent with tonic S1P3 signaling by a pool of constitutively generated Sphk-derived S1P dynamically regulating cholesterol efflux. In summary, we have established S1P as a previously unrecognized intermediate in LXR-stimulated ABCA1-mediated cholesterol efflux and identified S1P/S1P3 signaling as a positive-feedback regulator of cholesterol efflux. This constitutes a novel regulatory mechanism of cholesterol efflux by sphingolipids.

Hepatocyte nuclear factor 1A deficiency causes hemolytic anemia in mice by altering erythrocyte sphingolipid homeostasis.

The hepatocyte nuclear factor (HNF) family regulates complex networks of metabolism and organ development. Human mutations in its prototypical member HNF1A cause maturity-onset diabetes of the young (MODY) type 3. In this study, we identified an important role for HNF1A in the preservation of erythrocyte membrane integrity, calcium homeostasis, and osmotic resistance through an as-yet unrecognized link of HNF1A to sphingolipid homeostasis. HNF1A-/- mice displayed microcytic hypochromic anemia with reticulocytosis that was partially compensated by avid extramedullary erythropoiesis at all erythroid stages in the spleen thereby excluding erythroid differentiation defects. Morphologically, HNF1A-/- erythrocytes resembled acanthocytes and displayed increased phosphatidylserine exposure, high intracellular calcium, and elevated osmotic fragility. Sphingolipidome analysis by mass spectrometry revealed substantial and tissue-specific sphingolipid disturbances in several tissues including erythrocytes with the accumulation of sphingosine as the most prominent common feature. All HNF1A-/- erythrocyte defects could be simulated by exposure of wild-type (WT) erythrocytes to sphingosine in vitro and attributed in part to sphingosine-induced suppression of the plasma-membrane Ca2+-ATPase activity. Bone marrow transplantation rescued the anemia phenotype in vivo, whereas incubation with HNF1A-/- plasma increased the osmotic fragility of WT erythrocytes in vitro. Our data suggest a non-cell-autonomous erythrocyte defect secondary to the sphingolipid changes caused by HNF1A deficiency. Transcriptional analysis revealed 4 important genes involved in sphingolipid metabolism to be deregulated in HNF1A deficiency: Ormdl1, sphingosine kinase-2, neutral ceramidase, and ceramide synthase-5. The considerable erythrocyte defects in murine HNF1A deficiency encourage clinical studies to explore the hematological consequences of HNF1A deficiency in human MODY3 patients.

Hepatocyte Nuclear Factor 1A Is a Cell-Intrinsic Transcription Factor Required for B Cell Differentiation and Development in Mice.

The hepatocyte NF (HNF) family of transcription factors regulates the complex gene networks involved in lipid, carbohydrate, and protein metabolism. In humans, HNF1A mutations cause maturity onset of diabetes in the young type 3, whereas murine HNF6 participates in fetal liver B lymphopoiesis. In this study, we have identified a crucial role for the prototypical member of the family HNF1A in adult bone marrow B lymphopoiesis. HNF1A(-/-) mice exhibited a clear reduction in total blood and splenic B cells and a further pronounced one in transitional B cells. In HNF1A(-/-) bone marrow, all B cell progenitors-from pre-pro-/early pro-B cells to immature B cells-were dramatically reduced and their proliferation rate suppressed. IL-7 administration in vivo failed to boost B cell development in HNF1A(-/-) mice, whereas IL-7 stimulation of HNF1A(-/-) B cell progenitors in vitro revealed a marked impairment in STAT5 phosphorylation. The B cell differentiation potential of HNF1A(-/-) common lymphoid progenitors was severely impaired in vitro, and the expression of the B lymphopoiesis-promoting transcription factors E2A, EBF1, Pax5, and Bach2 was reduced in B cell progenitors in vivo. HNF1A(-/-) bone marrow chimera featured a dramatic defect in B lymphopoiesis recapitulating that of global HNF1A deficiency. The HNF1A(-/-) lymphopoiesis defect was confined to B cells as T lymphopoiesis was unaffected, and bone marrow common lymphoid progenitors and hematopoietic stem cells were even increased. Our data demonstrate that HNF1A is an important cell-intrinsic transcription factor in adult B lymphopoiesis and suggest the IL-7R/STAT5 module to be causally involved in mediating its function.

Plasma sphingosine-1-phosphate concentrations are associated with systolic heart failure in patients with ischemic heart disease.

OBJECTIVE: Sphingosine-1-Phosphate (S1P) is a bioactive sphingolipid with important functions in immunity, inflammation and cardiovascular biology. S1P is associated with prevalence and severity of coronary artery disease and myocardial infarction. However, its relevance in ischemic cardiomyopathy is unknown. We aimed to investigate associations of plasma S1P and other sphingolipids with the extent of heart failure in patients with ischemic heart disease. METHODS AND RESULTS: 74 patients with ischemic heart disease were investigated in this observational study. Plasma concentrations of S1P, C16 ceramide and sphingomyelin (SM) were measured using liquid chromatography/tandem mass-spectrometry and associated with objective (echocardiography) and subjective (dyspnea) signs of heart failure. Plasma S1P and SM but not C16 ceramide concentrations were negatively associated with left ventricular ejection fraction (LVEF) and dyspnea (ranked by New York Heart Association; LVEF: S1P standardized coefficient beta: -0.25; 95%CI: -273 to -13nM, p=0.03; SM beta: -0.24; 95%CI: -16,310 to -413nM, p=0.04; NYHA: S1P beta: -0.3; 95%CI: -174 to -26nM, p=0.009; SM beta: -0.46; 95%CI: -13,462 to -5013nM, p<0.001). ROC analysis revealed that S1P and SM predicted impaired LVEF with optimal cut-off levels below 843nM and 77µM, respectively. CONCLUSION: S1P is associated with the impairment of LVEF and dyspnea. Considering the major effects of S1P on cardiac and vascular functions in experimental models, we put forward the hypothesis that S1P is causally involved in the pathophysiology of heart failure. Interfering pharmacologically with S1P receptors may have an impact on ischemic cardiomyopathy.

New fluorinated agonists for targeting the sphingosin-1-phosphate receptor 1 (S1P(1)).

The sphingosine-1-phosphate receptor type 1 (S1P1) is involved in fundamental biological processes such as regulation of immune cell trafficking, vascular barrier function and angiogenesis. This Letter presents multistep syntheses of various fluorine substituted 12-aryl analogues of the drug fingolimod (FTY720) and a seven-steps route to 2-amino-17,17-difluoro-2-(hydroxymethyl)heptadecan-1-ol. In vitro and in vivo tests proved all these compounds as potent S1P1 receptor agonists.

Synthesis of new ligands for targeting the S1P1 receptor.

Sphingosine-1-phosphate (S1P) influences various fundamental biological processes by interacting with a family of five G protein-coupled receptors (S1P1-5). FTY720, a sphingosine analogue, which was approved for treatment of relapsing forms of multiple sclerosis, is phosphorylated in vivo and acts as an agonist of four of the five S1P receptor subtypes. Starting from these lead structures we developed new agonists for the S1P1 receptor. The biological activity was tested in vivo and promising ligands were fluorinated at different positions to identify candidates for positron emission tomography (PET) imaging after [(18)F]-labelling. The radioligands shall enable the imaging of S1P1 receptor expression in vivo and thus may serve as novel imaging markers of S1P-related diseases.

Synthesis of fluorinated analogues of sphingosine-1-phosphate antagonists as potential radiotracers for molecular imaging using positron emission tomography.

Sphingosine-1-phosphate (S1P) receptors play major roles in cardiovascular, immunological and neurological diseases. The recent approval of the sphingolipid drug Fingolimod (Gilenya®), a sphingosine-1-phosphate agonist for relapsing multiple sclerosis, in 2010 exemplifies the potential for targeting sphingolipids for the treatment of human disorders. Moreover, non-invasive in vivo imaging of S1P receptors that are not available till now would contribute to the understanding of their role in specific pathologies and is therefore of preclinical interest. Based on fluorinated analogues of the S1P1 receptor antagonist W146 showing practically equal in vitro potency as the lead structure, the first S1P receptor antagonist [18F]-radiotracer has been synthesized and tested for in vivo imaging of the S1P1 receptor using positron emission tomography (PET). Though the tracer is serum stable, initial in vivo images show fast metabolism and subsequent accumulation of free [18F]fluoride in the bones.

Sphingosine-1-phosphate promotes osteogenesis by stimulating osteoblast growth and neovascularization in a vascular endothelial growth factor-dependent manner.

Sphingosine-1-phosphate (S1P) plays multiple roles in bone metabolism and regeneration. Here, we have identified a novel S1P-regulated osteoanabolic mechanism functionally connecting osteoblasts (OBs) to the highly specialized bone vasculature. We demonstrate that S1P/S1PR3 signaling in OBs stimulates vascular endothelial growth factor a (VEGFa) expression and secretion to promote bone growth in an autocrine and boost osteogenic H-type differentiation of bone marrow endothelial cells in a paracrine manner. VEGFa-neutralizing antibodies and VEGF receptor inhibition by axitinib abrogated OB growth in vitro and bone formation in male C57BL/6J in vivo following S1P stimulation and S1P lyase inhibition, respectively. Pharmacological S1PR3 inhibition and genetic S1PR3 deficiency suppressed VEGFa production, OB growth in vitro, and inhibited H-type angiogenesis and bone growth in male mice in vivo. Together with previous work on the osteoanabolic functions of S1PR2 and S1PR3, our data suggest that S1P-dependent bone regeneration employs several nonredundant positive feedback loops between OBs and the bone vasculature. The identification of this yet unappreciated aspect of osteoanabolic S1P signaling may have implications for regular bone homeostasis as well as diseases where the bone microvasculature is affected such as age-related osteopenia and posttraumatic bone regeneration.

Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates bone growth and regeneration. In the present study, a novel regenerative mechanism was connected to S1P signaling within the bone. Activation of its receptor S1PR3 in bone-forming osteoblasts led to secretion of vascular endothelial growth factor a (VEGFa), the most potent vessel-stimulating factor. This stimulated the development of specialized vessels of the bone marrow, the H-type vessels, that supported overall bone regeneration. These findings foster our understanding of regular bone metabolism and suggest that S1P-based drugs may help treat diseases such as age-related osteopenia and posttraumatic bone regeneration, conditions crucially dependent on functional bone microvasculature.

Sphingosine-1-phosphate receptor 3 promotes recruitment of monocyte/macrophages in inflammation and atherosclerosis.

RATIONALE: The role of sphingosine-1-phosphate (S1P) and its receptors in the pathogenesis of atherosclerosis has not been investigated. OBJECTIVE: We hypothesized that the S1P receptor 3 (S1P(3)) plays a causal role in the pathogenesis of atherosclerosis. METHODS AND RESULTS: We examined atherosclerotic lesion development in mice deficient for S1P(3) and apolipoprotein (Apo)E. Although S1P(3) deficiency did not affect lesion size after 25 or 45 weeks of normal chow diet, it resulted in a dramatic reduction of the monocyte/macrophage content in lesions of S1P(3)(-/-)/ApoE(-/-) double knockout mice. To search for putative defects in monocyte/macrophage recruitment, we examined macrophage-driven inflammation during thioglycollate-induced peritonitis. Elicited peritoneal macrophages were reduced in S1P(3)-deficient mice and expressed lower levels of tumor necrosis factor-α and monocyte chemoattractant protein-1. Bone marrow-derived S1P(3)-deficient macrophages produced less MCP-1 in response to lipopolysaccharide stimulation. In vitro, S1P was chemotactic for wild-type but not S1P(3)-deficient peritoneal macrophages. In vivo, S1P concentration increased rapidly in the peritoneal cavity after initiation of peritonitis. Treatment with the S1P analog FTY720 attenuated macrophage recruitment to the peritoneum. Studies in bone marrow chimeras showed that S1P(3) in both hematopoietic and nonhematopoietic cells contributed to monocyte/macrophage accumulation in atherosclerotic lesions. Finally, S1P(3) deficiency increased the smooth muscle cell content of atherosclerotic lesions and enhanced neointima formation after carotid ligation arguing for an antiproliferative/antimigratory role of S1P(3) in the arterial injury response. CONCLUSIONS: Our data suggest that S1P(3) mediates the chemotactic effect of S1P in macrophages in vitro and in vivo and plays a causal role in atherosclerosis by promoting inflammatory monocyte/macrophage recruitment and altering smooth muscle cell behavior.

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

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

Sphingosine-1-phosphate suppresses GLUT activity through PP2A and counteracts hyperglycemia in diabetic red blood cells.

Red blood cells (RBC) are the major carriers of sphingosine-1-phosphate (S1P) in blood. Here we show that variations in RBC S1P content achieved by altering S1P synthesis and transport by genetic and pharmacological means regulate glucose uptake and metabolic flux. This is due to S1P-mediated activation of the catalytic protein phosphatase 2 (PP2A) subunit leading to reduction of cell-surface glucose transporters (GLUTs). The mechanism dynamically responds to metabolic cues from the environment by increasing S1P synthesis, enhancing PP2A activity, reducing GLUT phosphorylation and localization, and diminishing glucose uptake in RBC from diabetic mice and humans. Functionally, it protects RBC against lipid peroxidation in hyperglycemia and diabetes by activating the pentose phosphate pathway. Proof of concept is provided by the resistance of mice lacking the S1P exporter MFSD2B to diabetes-induced HbA1c elevation and thiobarbituric acid reactive substances (TBARS) generation in diabetic RBC. This mechanism responds to pharmacological S1P analogues such as fingolimod and may be functional in other insulin-independent tissues making it a promising therapeutic target.

Revealing concealed cardioprotection by platelet Mfsd2b-released S1P in human and murine myocardial infarction.

Antiplatelet medication is standard of care in acute myocardial infarction (AMI). However, it may have obscured beneficial properties of the activated platelet secretome. We identify platelets as major source of a sphingosine-1-phosphate (S1P) burst during AMI, and find its magnitude to favorably associate with cardiovascular mortality and infarct size in STEMI patients over 12 months. Experimentally, administration of supernatant from activated platelets reduces infarct size in murine AMI, which is blunted in platelets deficient for S1P export (Mfsd2b) or production (Sphk1) and in mice deficient for cardiomyocyte S1P receptor 1 (S1P1). Our study reveals an exploitable therapeutic window in antiplatelet therapy in AMI as the GPIIb/IIIa antagonist tirofiban preserves S1P release and cardioprotection, whereas the P2Y12 antagonist cangrelor does not. Here, we report that platelet-mediated intrinsic cardioprotection is an exciting therapeutic paradigm reaching beyond AMI, the benefits of which may need to be considered in all antiplatelet therapies.

Multi-targeted 1H/19F MRI unmasks specific danger patterns for emerging cardiovascular disorders.

Prediction of the transition from stable to acute coronary syndromes driven by vascular inflammation, thrombosis with subsequent microembolization, and vessel occlusion leading to irreversible myocardial damage is still an unsolved problem. Here, we introduce a multi-targeted and multi-color nanotracer platform technology that simultaneously visualizes evolving danger patterns in the development of progressive coronary inflammation and atherothrombosis prior to spontaneous myocardial infarction in mice. Individual ligand-equipped perfluorocarbon nanoemulsions are used as targeting agents and are differentiated by their specific spectral signatures via implementation of multi chemical shift selective 19F MRI. Thereby, we are able to identify areas at high risk of and predictive for consecutive development of myocardial infarction, at a time when no conventional parameter indicates any imminent danger. The principle of this multi-targeted approach can easily be adapted to monitor also a variety of other disease entities and constitutes a technology with disease-predictive potential.

Sphingosine 1-phosphate levels in plasma and HDL are altered in coronary artery disease.

High-density lipoproteins (HDL) are the major plasma carriers for sphingosine 1-phosphate (S1P) in healthy individuals, but their S1P content is unknown for patients with coronary artery disease (CAD). The aim of the study was to determine whether the S1P levels in plasma and HDL are altered in coronary artery disease. S1P was determined in plasma and HDL isolated by ultracentrifugation from patients with myocardial infarction (MI, n = 83), stable CAD (sCAD, n = 95), and controls (n = 85). In our study, total plasma S1P levels were lower in sCAD than in controls (305 vs. 350 pmol/mL). However, normalization to HDL-cholesterol (a known determinant of plasma S1P) revealed higher normalized plasma S1P levels in sCAD than in controls (725 vs. 542 pmol/mg) and even higher ones in MI (902 pmol/mg). The S1P amount contained in isolated HDL from these individuals was lower in sCAD than in controls (S1P per protein in HDL: 132 vs. 153 pmol/mg). The amount of total plasma S1P bound to HDL was lower in sCAD and MI than in controls (sCAD: 204, MI: 222, controls: 335 pmol/mL), while the non-HDL-bound S1P was, accordingly, higher (sCAD: 84, MI: 81, controls: 10 pmol/mL). HDL-bound plasma S1P was dependent on the plasma HDL-C in all groups, but normalization to HDL-C still yielded lower HDL-bound plasma S1P in patients with sCAD than in controls (465 vs. 523 pmol/mg). The ratio of non-HDL-bound plasma S1P to HDL-C-normalized HDL-bound S1P was also higher in both sCAD (0.18 mg/mL) and MI (0.15 mg/mL) than in controls (0.02 mg/mL). Remarkably, levels of non-HDL-bound plasma S1P correlated with the severity of CAD symptoms as graded by Canadian Cardiovascular Score, and discriminated patients with MI and sCAD from controls. Furthermore, a negative association was present between non-HDL-bound plasma S1P and the S1P content of isolated HDL in controls, but was absent in sCAD and MI. Finally, MI patients with symptom duration of less than 12 h had the highest levels of total and normalized plasma S1P, as well as the highest levels of S1P in isolated HDL. The HDL-C-normalized plasma level of S1P is increased in sCAD and even further in MI. This may be caused by an uptake defect of HDL for plasma S1P in CAD, and may represent a novel marker of HDL dysfunction.

Survivin determines cardiac function by controlling total cardiomyocyte number.

BACKGROUND: Survivin inhibits apoptosis and regulates cell division in many organs, but its function in the heart is unknown. METHODS AND RESULTS: We show that cardiac-specific deletion of survivin resulted in premature cardiac death. The underlying cause was a dramatic reduction in total cardiomyocyte numbers as determined by a stereological method for quantification of cells per organ. The resulting increased hemodynamic load per cell led to progressive heart failure as assessed by echocardiography, magnetic resonance imaging, positron emission tomography, and invasive catheterization. The reduction in total cardiomyocyte number in alpha-myosin heavy chain (MHC)-survivin(-/-) mice was due to an approximately 50% lower mitotic rate without increased apoptosis. This occurred at the expense of DNA accumulation because survivin-deficient cardiomyocytes displayed marked DNA polyploidy indicative of consecutive rounds of DNA replication without cell division. Survivin small interfering RNA knockdown in neonatal rat cardiomyocytes also led to polyploidization and cell cycle arrest without apoptosis. Adenoviral overexpression of survivin in cardiomyocytes inhibited doxorubicin-induced apoptosis, induced DNA synthesis, and promoted cell cycle progression. The phenotype of the alphaMHC-survivin(-/-) mice also allowed us to determine the minimum cardiomyocyte number sufficient for normal cardiac function. In human cardiomyopathy, survivin was potently induced in the failing heart and downregulated again after hemodynamic support by a left ventricular assist device. Its expression positively correlated with the mean cardiomyocyte DNA content. CONCLUSIONS: We suggest that the ontogenetically determined cardiomyocyte number may be an independent factor in the susceptibility to cardiac diseases. Through its profound impact on both cardiomyocyte replication and apoptosis, survivin may emerge as a promising new target for myocardial regeneration.

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 S1P(3). Upon activation with S1P, S1P(3) couples to G(i)- and G(q)-protein-dependent signalling pathways. Here we show that FTY-P selectively activates the S1P(3)-mediated and G(i)-coupled inhibition of adenylyl cyclase. Contemporaneously, it antagonizes the S1P-induced activation of G(q) via S1P(3) 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 S1P(3). The lack of receptor desensitization prevented S1P(3)-mediated migration to FTY-P. Human umbilical vein endothelial cells express S1P(1) and S1P(3), 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 S1P(1) and S1P(3). 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.