Ras/Raf1-dependent signal in sphingosine-1-phosphate-induced tube formation in human coronary artery endothelial cells

doi:10.1016/S0006-291X(03)01065-9

Biochemical and Biophysical Research Communications

Volume 306, Issue 4, 11 July 2003, Pages 924-929

https://doi.org/10.1016/S0006-291X(03)01065-9 Get rights and content

Abstract

Since we recently reported that high density lipoprotein, which contains the bioactive lipid sphingosine-1-phosphate (S1P) [Arterioscler. Thromb. Vasc. Biol. 23 (2003) 802], induced human coronary artery endothelial cell (HCEC) tube formation mediated by a Ras/Raf/ERK (extracellular signal-activated kinase) pathway, we thought that it would be very important to evaluate whether the signal in S1P-induced tube formation is Ras-dependent or -independent. In an in vitro model of HCEC tube formation on a matrix gel, S1P-induced tube formation. ERK1/2 inhibitor (PD98059) and pertussis toxin (PTX) suppressed S1P-induced tube formation. S1P activated phospho(p)-ERK1/2, while dominant-negative RasN17 blocked S1P-induced p-ERK1/2. Moreover, RasN17 inhibited S1P-induced tube formation. S1P activated Ras/Raf1 by Ras pull-down assay and this effect was inhibited by PTX. These results demonstrate that Ras/Raf1-dependent ERK activation mediated by PTX-sensitive G protein-coupled receptors may be a potent signal in S1P-induced HCEC tube formation.

Section snippets

Materials and methods

Materials. The following antibodies and reagents were generously provided as indicated or purchased: a specific inhibitor of MEK, PD98059, 2-(2^′-amino-3^′-methoxyphenyl) oxanaphthalen-4-one (New England BioLabs); sphingosine-1-phosphate (S1P) and pertussis toxin (PTX) (Sigma); and phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴) antibody and ERK1/2 antibody (Cell Signaling Technology).

Cell culture. HCECs were purchased from Clonetics (San Diego, CA). HCECs were cultured in media supplemented with 5% FBS,

S1P induces tube formation and its effect is blocked by pertussis toxin and ERK1/2 inhibitor

We first examined the ability of S1P to stimulate and stabilize tube formation with HCECs cultured on Matrigel. As shown in Fig. 1B, S1P led to the formation of a capillary-like structure on the Matrigel surface; this proangiogenic effect was maximal after 8 h. We optimized the dose for the maximum dose required to induce tube formation in initial experiments, in which the dose–response showed that the maximum effective dose of S1P was 1 μM (not shown).

Activation of the Ras effector ERK1/2

Discussion

The present results demonstrate that S1P can induce Ras-dependent ERK activation through PTX-sensitive GPCRs and that Ras plays a critical role in S1P-induced angiogenesis in HCECs. Our results identified Ras as a key player in the angiogenic action of S1P.

While recent reports have suggested that the inhibition of Ras might be an effective anti-angiogenic therapy [6], [15], it is unclear whether Ras plays a direct role in S1P-induced signal transduction. However, several lines of evidence

Acknowledgements

This work was supported by the Takeda Science Foundation, Fukuoka University School of Medicine, the Eboshi Association, and the Japan Research Foundation for Clinical Pharmacology.

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Later, formation of capillary-like vessels under influence of S1P was reported.49 The S1P-induced tube formation was pertussis toxin–sensitive and depended on activation of either Ras→Raf→ERK1/2 or phosphatidylinositol 3-kinase→Akt→endothelial nitric oxide synthase (eNOS) pathways.50,51 Recently, redistribution of cortactin and formation of cortactin-Arp2/3 complexes were implicated in S1P-induced angiogenesis.52
Numerous epidemiologic and interventional studies have revealed an inverse relationship between plasma concentrations of high-density lipoprotein (HDL) and coronary risk. There are several well-documented HDL functions, which may account for the antiatherogenic effects of this lipoprotein. Recent studies document that HDL serves as a carrier for the bioactive lysosphingolipid sphingosine 1-phosphate (S1P), which determines its functional properties. Generally available databases (eg, PubMed) were used, as well as our own results. An increasing body of evidence indicates that S1P is a mediator of many of the atheroprotective effects of HDL, including the ability to promote vasodilation and angiogenesis and protection against ischemia/reperfusion injury. These latter effects are believed to involve S1P-mediated retardation or suppression of inflammatory processes, such as endothelial expression of adhesion molecules, production of proinflammatory chemokines and cytokines, generation of reactive oxygen species, and cardiomyocyte apoptosis after myocardial infarction. This review article summarizes the evidence that S1P is a component of HDL contributing to the antiatherogenic and cardioprotective potential attributed to this lipoprotein.
Newly developed reconstituted high-density lipoprotein containing sphingosine-1-phosphate induces endothelial tube formation
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The concentration of (POPC/S1P/A–I)rHDL was the same as the maximum effective dose for HCEC proliferation in Fig. 2B. Although 20 μg/ml of (POPC/S1P/A–I)rHDL did not induce tube formation or cell proliferation, this concentration of (POPC/S1P/A–I)rHDL also did not induce apoptosis [% apoptosis without and with (POPC/S1P/A–I)rHDL were 0.9 ± 0.1 and 0.8 ± 0.1%, respectively] and showed G1 arrest [% G1 phase without and with (POPC/S1P/A–I)rHDL were 75 ± 2 and 86 ± 2%, respectively]. Activation of Akt and ERK affects HCEC tube formation in addition to cellular proliferation and cell migration [14]. In addition, NO may be a critical modulator of angiogenesis [25,26].
Reconstituted high-density lipoprotein (rHDL) has been shown to produce a rapid regression of atherosclerosis in animal models and humans. Sphingosine-1-phosphate (S1P), which is a bioactive lipid in HDL, plays a role in mitogenesis, endothelial cell motility, and cell survival, as well as organization and differentiation into a vessel. In this study, we examined the direct role of a newly developed rHDL, [POPC(1-palmitoyl-2-oleoyl phosphatidylcholine)/S1P/apolipoproteinA–I(A–I)]rHDL containing S1P in tube formation in endothelial cells (ECs) as well as cholesterol efflux in macrophage. The effect of (POPC/S1P/A–I)rHDL on cholesterol efflux in macrophage was similar to that of conventional rHDL, (POPC/A–I)rHDL. In addition, (POPC/S1P/A–I)rHDL induced EC proliferation through the activation of phospho-Akt and phospho-extracellular-signal-regulated kinases (p-ERK) 1/2 and EC tube formation, and this effect was blocked by inhibitors of Akt, ERK and endothelial nitric-oxide synthase (eNOS). In addition, (POPC/S1P/A–I)rHDL-induced p-ERK1/2 activation and EC tube formation can be mainly attributed to S1P-stimutated signaling through S1P₂ and S1P₃ as determined by an anti-sense strategy. In conclusion, (POPC/S1P/A–I)rHDL induces cholesterol efflux independently of S1P but has additional S1P-mediated effects on EC tube formation mediated by Akt/ERK/NO through S1P₂ and S1P₃. In the future, these new discs may be useful for the treatment of atherosclerotic and ischemic cardiovascular disease, such as acute coronary syndrome and atherosclerosis obliterans.
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Extracellular sphingosine 1-phosphate (S1P) has been shown to contribute to the action of high density lipoprotein (HDL) on endothelial and smooth muscle cells. We examined the relationship of lipoprotein-associated S1P concentrations with cholesterol (C) and apolipoprotein (apo) contents of lipoprotein and lipoprotein subfractions characterized by capillary isotachophoresis (cITP).
Blood samples were drawn from 16 volunteers. S1P concentrations were quantified by bioassay based on the ability of S1P to stimulate its receptor. cITP was performed using plasma that had been prestained with NBD-ceramide.
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Ras/Raf1-dependent signal in sphingosine-1-phosphate-induced tube formation in human coronary artery endothelial cells

Abstract

Section snippets

Materials and methods

S1P induces tube formation and its effect is blocked by pertussis toxin and ERK1/2 inhibitor

Discussion

Acknowledgements

Biochim. Biophys. Acta

Cell

Mol. Cell.

Methods Enzymol.

J. Biol. Chem.

Eur. J. Cancer

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Endothelial cell apoptosis in angiogenesis and vessel regression

Circ. Res.