Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM + HDL contained S1P, whereas ApoM − HDL did not. Moreover, HDL in Apom −/− mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P-human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM + HDL induced S1P 1 receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM − HDL did not. Importantly, lack of S1P in the HDL fraction of Apom −/− mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1P 1 receptor on endothelial cells, is a vasculoprotective constituent of HDL.endothelial function | crystal structure | sphingolipids | vascular permeability | atherosclerosis S phingosine-1-phosphate (S1P), the phosphorylated metabolite of D-sphingosine, binds to five G protein-coupled receptors (S1P 1 -S1P 5 ) and regulates a plethora of biological actions (1-6). In particular, the prototypical S1P 1 receptor is essential for vascular maturation during development and promotes endothelial cell migration, angiogenesis, and barrier functions (7-9). Thus, S1P is required for maintenance of the barrier property of the lung endothelium (10). Plasma S1P, which is derived from several cellular sources (11,12), is associated with HDL (∼65%) and albumin (∼35%) (3, 5). HDLinduced vasorelaxation as well as barrier-promoting and prosurvival actions on the endothelium have been attributed to S1P signaling (2, 4, 13). Hence, much of the endothelium-protective actions of HDL may result from the actions of S1P on the endothelial S1P receptors. However, the molecular nature of the S1P binding to HDL and interaction with S1P receptors has not been characterized.Apolipoprotein M (apoM) is a lipocalin that resides mainly in the plasma HDL fraction (14). The retained hydrophobic NH 2 -terminal signal peptide anchors apoM in the phospholipid layer of the lipoprotein and prevents filtration of the ∼22-kDa protein in the kidney (15). The biological functions of apoM are understood only partly. Studies in apoM gene-modified mice suggest that apoM has antiatherogenic effects, possibly related in part to apoM's ability to increase cholesterol efflux from macrophage foam cells, to increased preβ-HDL formation, and to antioxidative effects (16)(17)(18). The recent elucidation of the crystal structure of human recombinant apoM (r-apoM) demonstrated a typical lipocalin fold characterized by an eightstranded antiparallel β-barrel enclosing an internal binding pocket that probably facilitates binding of small lipophilic ligands (19).Indeed, r-apoM expressed in Escherichia coli was found to co...

SUMMARY During angiogenesis, nascent vascular sprouts fuse to form vascular networks enabling efficient circulation. Mechanisms that stabilize the vascular plexus are not well understood. Sphingosine 1-phosphate (S1P) is a blood-borne lipid mediator implicated in the regulation of vascular and immune systems. Here we describe a mechanism by which the G protein-coupled S1P receptor-1 (S1P1) stabilizes the primary vascular network. A gradient of S1P1 expression from the mature regions of the vascular network to the growing vascular front was observed. In the absence of endothelial S1P1, adherens junctions are destabilized, barrier function is breached, and flow is perturbed resulting in abnormal vascular hypersprouting. Interestingly, S1P1 responds to S1P as well as laminar shear stress to transduce flow-mediated signaling in endothelial cells both in vitro and in vivo. These data demonstrate that blood flow and circulating S1P activate endothelial S1P1 to stabilize blood vessels in development and homeostasis.

The sphingosine 1-phosphate receptor 1 (S1P1) is abundant in endothelial cells, where it regulates vascular development and microvascular barrier function. In investigating the role of endothelial cell S1P1 in adult mice, we found that the endothelial S1P1 signal was enhanced in regions of the arterial vasculature experiencing inflammation. The abundance of proinflammatory adhesion proteins, such as ICAM-1, was enhanced in mice with endothelial cell–specific deletion of S1pr1 and suppressed in mice with endothelial cell–specific overexpression of S1pr1, suggesting a protective function of S1P1 in vascular disease. The chaperones ApoM+HDL (HDL) or albumin bind to sphingosine 1-phosphate (S1P) in the circulation; therefore, we tested the effects of S1P bound to each chaperone on S1P1 signaling in cultured human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to ApoM+HDL-S1P, but not to albumin-S1P, promoted the formation of a cell surface S1P1–β-arrestin 2 complex and attenuated the ability of the proinflammatory cytokine TNFα to activate NF-κB and increase ICAM-1 abundance. Although S1P bound to either chaperone induced MAPK activation, albumin-S1P triggered greater Gi activation and receptor endocytosis. Endothelial cell–specific deletion of S1pr1 in the hypercholesterolemic Apoe−/− mouse model of atherosclerosis enhanced atherosclerotic lesion formation in the descending aorta. We propose that the ability of ApoM+HDL to act as a biased agonist on S1P1 inhibits vascular inflammation, which may partially explain the cardiovascular protective functions of HDL.

Endothelial dysfunction is a critical factor in many cardiovascular diseases, including hypertension. Although lipid signaling has been implicated in endothelial dysfunction and cardiovascular disease, specific molecular mechanisms are poorly understood. Here we report that Nogo-B, a membrane protein of the endoplasmic reticulum, regulates endothelial sphingolipid biosynthesis with direct effects on vascular function and blood pressure. Nogo-B inhibits serine palmitoyltransferase, the rate-limiting enzyme of the de novo sphingolipid biosynthetic pathway, thereby controlling production of endothelial sphingosine 1-phosphate and autocrine, G protein–coupled receptor–dependent signaling by this metabolite. Mice lacking Nogo-B either systemically or specifically in endothelial cells are hypotensive, resistant to angiotensin II–induced hypertension and have preserved endothelial function and nitric oxide release. In mice that lack Nogo-B, pharmacological inhibition of serine palmitoyltransferase with myriocin reinstates endothelial dysfunction and angiotensin II–induced hypertension. Our study identifies Nogo-B as a key inhibitor of local sphingolipid synthesis and shows that autocrine sphingolipid signaling within the endothelium is critical for vascular function and blood pressure homeostasis.

Sphingosine 1-phosphate (S1P), a sphingolipid mediator, regulates various cellular functions via high-affinity G protein-coupled receptors, S1P1-5. The S1P-S1P receptor signaling system plays important roles in lymphocyte trafficking and maintenance of vascular integrity, thus contributing to the regulation of complex inflammatory processes. S1P is enriched in blood and lymph while maintained low in intracellular or interstitial fluids, creating a steep S1P gradient that is utilized to facilitate efficient egress of lymphocytes from lymphoid organs. Blockage of the S1P-S1P receptor signaling system results in a marked decrease in circulating lymphocytes because of a failure of lymphocyte egress from lymphoid organs. This provides a basis of immunomodulatory drugs targeting S1P1 receptor such as FTY720, an immunosuppressive drug approved in 2010 as the first oral treatment for relapsing–remitting multiple sclerosis. The S1P-S1P receptor signaling system also plays important roles in maintenance of vascular integrity since it suppresses sprouting angiogenesis and regulates vascular permeability. Dysfunction of the S1P-S1P receptor signaling system results in various vascular defects, such as exaggerated angiogenesis in developing retina and augmented inflammation due to increased permeability. Endothelial-specific deletion of S1P1 receptor in mice fed high-fat diet leads to increased formation of atherosclerotic lesions. This review highlights the importance of the S1P-S1P receptor signaling system in inflammatory processes. We also describe our recent findings regarding a specific S1P chaperone, apolipoprotein M, that anchors to high-density lipoprotein and contributes to shaping the endothelial-protective and anti-inflammatory properties of high-density lipoprotein.

Sphingosine 1-phosphate (S1P) is a lipid mediator produced from sphingomyelin by the sequential enzymatic actions of sphingomyelinase, ceramidase, and sphingosine kinase. Five subtypes of cell surface G-protein-coupled receptors, S1P1–5, mediate the actions of S1P in various organs systems, most notably cardiovascular, immune, and central nervous systems. S1P is enriched in blood and lymph but is present at much lower concentrations in interstitial fluids of tissues. This vascular S1P gradient is important for the regulation of trafficking of various immune cells. FTY720, which was recently approved for the treatment of relapsing-remitting multiple sclerosis, potently sequesters lymphocytes into lymph nodes by functionally antagonizing the activity of the S1P1 receptor. S1P also plays critical roles in the vascular barrier integrity, thereby regulating inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Recent studies have also revealed the involvement of S1P signaling in coagulation and in tumor necrosis factor α-mediated signaling. This review highlights the importance of S1P signaling in these inflammatory processes as well as the contribution of each receptor subtype, which exhibits both cooperative and redundant functions.

G2A is a G protein-coupled receptor that is predominantly expressed in lymphoid tissues and macrophages. G2A can be induced by diverse stimuli to cause cell cycle arrest in the G 2 /M phase in pro-B and T cells. G2A is also expressed in macrophages within atherosclerotic lesions, suggesting G2A involvement in atherosclerosis. Recently, G2A was discovered to possess proton-sensing ability. In this paper, we report another function of G2A, that is, as a receptor for 9-hydroxyoctadecadienoic acid (9-HODE) and other oxidized free fatty acids. G2A, expressed in CHO-K1 or HEK293 cells, showed 9-HODE-induced intracellular calcium mobilization, inositol phosphate accumulation, inhibition of cAMP accumulation, [35 S]guanosine 5-3-O-(thio)triphosphate binding, and MAP kinase activation. Furthermore, G2A was activated by various oxidized derivatives of linoleic and arachidonic acids, but it was weakly activated by cholesteryl-9-HODE. Oxidized phosphatidylcholine (1-palmitoyl-2-linoleoyl) when hydrolyzed with phospholipase A 2 also evoked intracellular calcium mobilization in G2A-expressing cells. These results indicate that G2A is activated by oxidized free fatty acids produced by oxidation and subsequent hydrolysis of phosphatidylcholine or cholesteryl linoleate. Thus, G2A might have a biological role in diverse pathological conditions including atherosclerosis.

Metabolic response by F-FDG was effective in predicting efficacy and survival at 1 month after nivolumab treatment.