Hideru Obinata

Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M

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 S1P1 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 S1P1 receptor on endothelial cells, is a vasculoprotective constituent of HDL.

Flow-Regulated Endothelial S1P Receptor-1 Signaling Sustains Vascular Development

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.

Identification of 9-hydroxyoctadecadienoic acid and other oxidized free fatty acids as ligands of the G protein-coupled receptor G2A.

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 G2/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, [35S]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 A2 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.

Sphingosine 1-phosphate in coagulation and inflammation

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.

HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation

HDL, the good cholesterol, biases the endothelial response to the lipid S1P to protect blood vessels. Maintaining vascular health with HDL Flow through blood vessels subjects endothelial cells to abnormal shear forces at specific locations that trigger inflammation, which contributes to atherosclerotic plaque formation. Galvani et al. found that vascular inflammation and atherosclerosis in mice were suppressed by the endothelial cell receptor S1P1, which is activated by S1P, a lipid mediator that is abundant in blood bound to different chaperone proteins. S1P suppressed inflammation in cultured endothelial cells when bound to the lipoprotein ApoM+HDL. Thus, S1P bound to different chaperones triggered distinct or “biased” signaling pathways, which may also contribute to the protective effect of HDL, commonly called “good cholesterol,” in atherosclerosis. 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.

G2A as a receptor for oxidized free fatty acids.

G2A was identified as a G protein-coupled receptor that can be induced by different classes of DNA-damaging agents and block cell cycle progression in lymphocytes. We recently reported that G2A functions as a receptor for oxidized free fatty acids derived from linoleic and arachidonic acids. When ectopically expressed in CHO cells, G2A mediates intracellular signaling events such as intracellular calcium mobilization and JNK activation in response to oxidized free fatty acids. In human epidermal keratinocytes, G2A mediates the secretion of cytokines including interleukin-6 and -8, and blocks cell cycle progression at the G1 phase in response to ligands. G2A might function as a sensor that monitors the oxidative states and mediates appropriate cellular responses such as secretion of paracrine signals and attenuation of proliferation.

Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1

Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca(2+) for its maximum activity. The K(M) values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 microM, and the V(max) values were 27.3 and 1.62 micromol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.

Nogo-B regulates endothelial sphingolipid homeostasis to control vascular function and blood pressure

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.

Detection of G protein-coupled receptor-mediated cellular response involved in cytoskeletal rearrangement using surface plasmon resonance

G protein-coupled receptors (GPCRs) form a superfamily of cell surface receptors that play fundamental roles in physiology and pathophysiology. Although GPCRs have been the most successful targets for drug discovery, there still remain many orphan GPCRs, which provides opportunities for development of novel drugs. Here, we introduce a new method for evaluation of GPCR activation utilizing a surface plasmon resonance (SPR) sensor. Cells expressing GPCRs were cultured directly on an SPR sensor chip and stimulated with GPCR ligands, resulting in SPR responses that were dependent on the type of G alpha subunits coupling with receptors. Namely G(i)- and/or G(12/13)-coupled receptors evoked SPR responses but G(s)- or G(q)-coupled ones did not. Analyses on the intracellular signal pathways revealed that small G protein Rho/Rac-mediated actin rearrangement plays an important role in the signal transduction pathways leading to the SPR responses. An SPR response was also evoked by insulin-like growth factor-1, which stimulates Rac-dependent stress fiber formation via its receptor-tyrosine kinase. Thus, this method provides a unique opportunity for real-time monitoring of cellular responses involved in cytoskeletal rearrangements, and may be useful in ligand/drug discovery for certain types of receptor, such as G(i)- and G(12/13)-coupled receptors.

Impaired endothelial barrier function in apolipoprotein M–deficient mice is dependent on sphingosine-1-phosphate receptor 1

Apolipoprotein M (ApoM) transports sphingosine‐1‐phosphate (S1P) in plasma, and ApoM‐deficient mice (Apom–/–) have ~50% reduced plasma S1P levels. There are 5 known S1P receptors, and S1P induces adherens junction formation between endothelial cells through the S1P1 receptor, which in turn suppresses vascular leak. Increased vascular permeability is a hallmark of inflammation. The purpose of this study was to explore the relationships between vascular leakage in ApoM deficiency and S1P1 function in normal physiology and in inflammation. Vascular permeability in the lungs was assessed by accumulation of dextran molecules (70 kDa) and was increased ~40% in Apom–/– mice compared to WT (C57Bl6/j) mice. Reconstitution of plasma ApoM/S1P or treatment with an S1P1 receptor agonist (SEW2871) rapidly reversed the vascular leakage to a level similar to that in WT mice, suggesting that it is caused by decreased plasma levels of S1P and reduced S1P1 stimulation. In a carrageenan‐induced model of inflammation, Apom–/– mice had increased vascular leakage compared with that in WT mice. Adenoviral overexpression of ApoM in Apom–/– mice decreased the vascular leakage compared to adenoviral overexpression of green fluorescent protein. The study suggests that vascular leakage of albumin‐sized particles in ApoM deficiency is S1P‐ and S1P1‐dependent and this dependency exacerbates the response to inflammatory stimuli.—Christensen, P. M., Liu, C. H., Swendeman, S. L., Obinata, H., Qvortrup, K., Nielsen, L B., Hla, T., Di Lorenzo, A., Christoffersen, C. Impaired endothelial barrier function in apolipoprotein M‐deficient mice is dependent on sphingosine‐1‐phosphate receptor 1. FASEB J. 30, 2351–2359 (2016). www.fasebj.org