Sphingosine-1-phosphate receptor 3 regulates the transendothelial transport of high-density lipoproteins and low-density lipoproteins in opposite ways.

AIMS: The entry of lipoproteins from blood into the arterial wall is a rate-limiting step in atherosclerosis. It is controversial whether this happens by filtration or regulated transendothelial transport.Because sphingosine-1-phosphate (S1P) preserves the endothelial barrier, we investigated in vivo and in vitro, whether S1P and its cognate S1P-receptor 3 (S1P3) regulate the transendothelial transport of lipoproteins. METHODS AND RESULTS: Compared to apoE-haploinsufficient mice (CTRL), apoE-haploinsufficient mice with additional endothelium-specific knock-in of S1P3 (S1P3-iECKI) showed decreased transport of LDL and Evan's Blue but increased transport of HDL from blood into the peritoneal cave. After 30 weeks of high-fat diet feeding, S1P3-iECKI mice had lower levels of non-HDL-cholesterol and less atherosclerosis than CTRL mice. In vitro stimulation with an S1P3 agonist increased the transport of 125I-HDL but decreased the transport of 125I-LDL through human aortic endothelial cells (HAECs). Conversely, inhibition or knock-down of S1P3 decreased the transport of 125I-HDL but increased the transport of 125I-LDL. Silencing of SCARB1 encoding scavenger receptor B1 (SR-BI) abrogated the stimulation of 125I-HDL transport by the S1P3 agonist. The transendothelial transport of 125I-LDL was decreased by silencing of SCARB1 or ACVLR1 encoding activin-like kinase 1 but not by interference with LDLR. None of the three knock-downs prevented the stimulatory effect of S1P3 inhibition on transendothelial 125I-LDL transport. CONCLUSION: S1P3 regulates the transendothelial transport of HDL and LDL oppositely by SR-BI-dependent and SR-BI-independent mechanisms, respectively. This divergence supports a contention that lipoproteins pass the endothelial barrier by specifically regulated mechanisms rather than passive filtration.

Elevating Endogenous Sphingosine-1-Phosphate (S1P) Levels Improves Endothelial Function and Ameliorates Atherosclerosis in Low Density Lipoprotein Receptor-Deficient (LDL-R-/-) Mice.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid and a constituent of high-density lipoprotein (HDL) exerting several atheroprotective effects in vitro. However, the few studies addressing anti-atherogenic effects of S1P in vivo have led to disparate results. We here examined atherosclerosis development in low-density lipoprotein receptor (LDL-R)-deficient (LDL-R-/-) mice with elevated endogenous S1P levels. METHODS AND RESULTS: Sub-lethally irradiated LDL-R-/- mice were transplanted with bone marrow deficient in sphingosine kinase 2 (SphK2), which led to the elevation of S1P concentrations in erythrocytes, plasma and HDL by approximately 1.5- to 2.0-fold in SphK2-/-/LDL-R-/- mice. Afterwards, mice were fed a Western diet for 14 weeks. Elevation of endogenous S1P significantly reduced atherosclerotic lesion formation by approximately half without affecting the plasma lipid profile. Furthermore, the macrophage content of atherosclerotic lesions and lipopolysaccharide-induced monocyte recruitment to the peritoneal cavity were reduced in SphK2-/-/LDL-R-/- mice. Studies using intra-vital microscopy revealed that endogenous S1P lowered leukocyte adhesion to capillary wall and decreased endothelial permeability to fluorescently labelled LDL. Moreover, SphK2-/-/LDL-R-/- mice displayed decreased levels of vascular cell adhesion molecule 1 in atherosclerotic lesions and in plasma. Studies in vitro demonstrated reduced monocyte adhesion and transport across an endothelial layer exposed to increasing S1P concentrations, murine plasma enriched in S1P or plasma obtained from SphK2-deficient animals. In addition, decreased permeability to fluorescence-labelled dextran beads or LDL was observed in S1P-treated endothelial cells. CONCLUSION: We conclude that raising endogenous S1P levels exerts anti-atherogenic effects in LDL-R-/- mice that are mediated by favourable modulation of endothelial function.

High density lipoprotein (HDL)-associated sphingosine 1-phosphate (S1P) inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression.

BACKGROUND AND AIMS: Macrophage apoptosis is critically involved in atherosclerosis. We here examined the effect of anti-atherogenic high density lipoprotein (HDL) and its component sphingosine-1-phosphate (S1P) on apoptosis in RAW264.7 murine macrophages. METHODS: Mitochondrial or endoplasmic reticulum-dependent apoptosis was induced by exposure of macrophages to etoposide or thapsigargin/fukoidan, respectively. RESULTS: Cell death induced by these compounds was inhibited by S1P as inferred from reduced annexin V binding, TUNEL staining, and caspase 3, 9 and 12 activities. S1P induced expression of the inhibitor of apoptosis protein (IAP) family proteins cIAP1, cIAP2 and survivin, but only the inhibitor of survivin expression YM155 and not the cIAP1/2 blocker GDC0152 reversed the inhibitory effect of S1P on apoptosis. Moreover, S1P activated signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2) and the stimulatory effect of S1P on survivin expression and inhibitory effects on apoptosis were attenuated by STAT3 or JAK2 inhibitors, S3I-201 or AG490, respectively. The effects of S1P on STAT3 activation, survivin expression and macrophage apoptosis were emulated by HDL, HDL lipids, and apolipoprotein (apo) M-containing HDL, but not by apoA-I or HDL deprived of S1P or apoM. In addition, JTE013 and CAY10444, S1P receptor 2 and 3 antagonists, respectively, compromised the S1P and HDL capacities to stimulate STAT3 activation and survivin expression, and to inhibit apoptosis. CONCLUSIONS: HDL-associated S1P inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression. The suppression of macrophage apoptosis may represent a novel mechanism utilized by HDL to exert its anti-atherogenic effects.

Apolipoprotein E interrupts interleukin-1beta signaling in vascular smooth muscle cells.

OBJECTIVES: Apolipoprotein E (apoE) exerts antiatherogenic effects but precise mechanisms remain unclear. We here investigated the effect of apoE on intracellular signaling by interleukin-1beta (IL-1beta), a proinflammatory cytokine present in atherosclerotic lesions. METHODS AND RESULTS: IL-1beta-induced expression and activation of inducible nitric oxide synthase and cyclooxygenase-2 were inhibited by apoE in vascular smooth muscle cells (VSMCs). These inhibitory effects were linked to the suppression of both NF-kappaB and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1beta signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1beta signaling intermediates revealed that NF-kappaB transactivation was inhibited by apoE in MyD88- and IRAK1- but not in TRAF6-transfected cells. Furthermore, apoE prevented IRAK1 phosphorylation and IRAK1-TRAF6 but not MyD88-IRAK1 complex formation. Inhibitory effects of apoE on IL-1beta signaling were abolished after silencing LDL receptor-related protein-1 (LRP1) expression with siRNA. In addition, inhibitors of adenylyl cyclase and protein kinase A (PKA) restored IL-1beta signaling in apoE-treated VSMCs, whereas apoE stimulated PKA activity. ApoE inhibited VSMC activation in response to IL-18 but not to tumor necrosis factor-alpha or polyinosinic:polycytidylic acid. CONCLUSION: ApoE targets IRAK-1 activation and thereby interrupts IL-1beta and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.

Apolipoprotein A-I activates Cdc42 signaling through the ABCA1 transporter.

It has been suggested that the signal transduction initiated by apolipoprotein A-I (apoA-I) activates key proteins involved in cholesterol efflux. ABCA1 serves as a binding partner for apoA-I, but its participation in apoA-I-induced signaling remains uncertain. We show that the exposure of human fibroblasts to ABCA1 ligands (apolipoproteins and amphipathic helical peptides) results in the generation of intracellular signals, including activation of the small G-protein Cdc42, protein kinases (PAK-1 and p54JNK), and actin polymerization. ApoA-I-induced signaling was abrogated by glyburide, an inhibitor of the ABC transporter family, and in fibroblasts from patients with Tangier disease, which do not express ABCA1. Conversely, induction of ABCA1 expression with the liver X receptor agonist, T0901317, and the retinoid X receptor agonist, R0264456, potentiated apoA-I-induced signaling. Similar effects were observed in HEK293 cells overexpressing ABCA1-green fluorescent protein (GFP) fusion protein, but not ABCA1-GFP (K939M), which fails to hydrolyze ATP, or a nonfunctional ABCA1-GFP with a truncated C terminus. We further found that Cdc42 coimmunoprecipitates with ABCA1 in ABCA1-GFP-expressing HEK293 cells exposed to apoA-I but not in cells expressing ABCA1 mutants. We conclude that ABCA1 transduces signals from apoA-I by complexing and activating Cdc42 and downstream kinases and, therefore, acts as a full apoA-I receptor.

Low density lipoproteins inhibit the Na+/H+ antiport in human platelets via activation of p38MAP kinase.

Low density lipoproteins (LDL) inhibit the Na+/H+ antiport and thereby sensitize platelet towards agonist. However, mechanisms underlying the suppressing effect of LDL on Na+/H+ exchange are unclear. We here show that the lowering of intracellular pH and the suppression of the sodium propionate-induced Na+/H+ exchange in the presence of LDL are abolished by SKF86002, a selective inhibitor of p38MAP kinase (p38MAPK). The inhibitory effect of LDL on Na+/H+ exchange was mimicked by H2O2, which directly activates p38MAPK. Exposure of platelets to LDL or H2O2 led to phosphorylation of p38MAPK, its upstream regulator MAP kinase kinase 3/6 (MKK 3/6), and its downstream target heat shock protein 27 (HSP27), and this effect was abrogated in SKF86002-pretreated platelets. In addition, both LDL and H2O2 produced the SKF86002-sensitive phosphorylation of an oligopeptide encompassing p38MAPK phosphorylation sequence derived from NHE-1, a major Na+/H+ exchanger in platelets. We further show that the sensitizing effects of LDL on the thrombin-induced platelet activation, as reflected by aggregation and granule secretion, are abolished in cells pretreated with SKF86002. We conclude that activation of p38MAPK is required for the inhibitory effect of LDL on Na+/H+ antiport and thereby for LDL-dependent sensitization in human platelets.

Involvement of Cdc42 signaling in apoA-I-induced cholesterol efflux.

Cholesterol efflux, an important mechanism by which high density lipoproteins (HDL) protect against atherosclerosis, is initiated by docking of apolipoprotein A-I (apoA-I), a major HDL protein, to specific binding sites followed by activation of ATP-binding cassette transporter A1 (ABCA1) and translocation of cholesterol from intracellular compartments to the exofacial monolayer of the plasma membrane where it is accessible to HDL. In this report, we investigated potential signal transduction pathways that may link apoA-I binding to cholesterol translocation to the plasma membrane and cholesterol efflux. By using pull-down assays we found that apoA-I substantially increased the amount of activated Cdc42, Rac1, and Rho in human fibroblasts. Moreover, apoA-I induced actin polymerization, which is known to be controlled by Rho family G proteins. Inhibition of Cdc42 and Rac1 with Clostridium difficile toxin B inhibited apoA-I-induced cholesterol efflux, whereas inhibition of Rho with Clostridium botulinum C3-exoenzyme exerted opposite effects. Adenoviral expression of a Cdc42(T17N) dominant negative mutant substantially reduced apoA-I-induced cholesterol efflux, whereas dominant negative Rac1(T17N) had no effect. We further found that two downstream effectors of Cdc42/Rac1 signaling, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), are activated by apoA-I. Pharmacological inhibition of JNK but not p38 MAPK decreased apoA-I-induced cholesterol efflux, whereas anisomycin and hydrogen peroxide, two direct JNK activators, could partially substitute for apoA-I in its ability to induce cholesterol efflux. These results for the first time demonstrate activation of Rho family G proteins and stress kinases by apoA-I and implicate the involvement of Cdc42 and JNK in the apoA-I-induced cholesterol efflux.