Sphingosine-1-phosphate receptor-2 deficiency leads to inhibition of macrophage proinflammatory activities and atherosclerosis in apoE-deficient mice
Fei Wang, … , Makoto Kinoshita, Yoh Takuwa
Fei Wang, … , Makoto Kinoshita, Yoh Takuwa
Published October 18, 2010
Citation Information: J Clin Invest. 2010;120(11):3979-3995. https://doi.org/10.1172/JCI42315.
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Research Article Cardiology

Sphingosine-1-phosphate receptor-2 deficiency leads to inhibition of macrophage proinflammatory activities and atherosclerosis in apoE-deficient mice

Abstract

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that has pleiotropic effects in a variety of cell types including ECs, SMCs, and macrophages, all of which are central to the development of atherosclerosis. It may therefore exert stimulatory and inhibitory effects on atherosclerosis. Here, we investigated the role of the S1P receptor S1PR2 in atherosclerosis by analyzing S1pr2–/– mice with an Apoe–/– background. S1PR2 was expressed in macrophages, ECs, and SMCs in atherosclerotic aortas. In S1pr2–/–Apoe–/– mice fed a high-cholesterol diet for 4 months, the area of the atherosclerotic plaque was markedly decreased, with reduced macrophage density, increased SMC density, increased eNOS phosphorylation, and downregulation of proinflammatory cytokines compared with S1pr2+/+Apoe–/– mice. Bone marrow chimera experiments indicated a major role for macrophage S1PR2 in atherogenesis. S1pr2–/–Apoe–/– macrophages showed diminished Rho/Rho kinase/NF-κB (ROCK/NF-κB) activity. Consequently, they also displayed reduced cytokine expression, reduced oxidized LDL uptake, and stimulated cholesterol efflux associated with decreased scavenger receptor expression and increased cholesterol efflux transporter expression. S1pr2–/–Apoe–/– ECs also showed reduced ROCK and NF-κB activities, with decreased MCP-1 expression and elevated eNOS phosphorylation. Pharmacologic S1PR2 blockade in S1pr2+/+Apoe–/– mice diminished the atherosclerotic plaque area in aortas and modified LDL accumulation in macrophages. We conclude therefore that S1PR2 plays a critical role in atherogenesis and may serve as a novel therapeutic target for atherosclerosis.

Authors

Fei Wang, Yasuo Okamoto, Isao Inoki, Kazuaki Yoshioka, Wa Du, Xun Qi, Noriko Takuwa, Koichi Gonda, Yasuhiko Yamamoto, Ryunosuke Ohkawa, Takumi Nishiuchi, Naotoshi Sugimoto, Yutaka Yatomi, Kunitoshi Mitsumori, Masahide Asano, Makoto Kinoshita, Yoh Takuwa

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Figure 6

The activity of the Rho/ROCK/NF-κB pathway is inhibited in S1pr2-deficient macrophages.

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The activity of the Rho/ROCK/NF-κB pathway is inhibited in S1pr2-deficie...
(A–C) Serum-starved peritoneal macrophages from S1pr2+/+Apoe–/– (white bars) and S1pr2–/–Apoe–/– (black bars) mice were stimulated with S1P (0.1 μM) for 2 minutes (A), 5 minutes (B), or 20 minutes (C) and analyzed for GTP-Rho (A), phosphorylation (Thr850) of MYPT1 (B), and phosphorylation (Ser536) of NF-κB (p65) (C) as described in Methods (n = 3 each). (D) Peritoneal macrophages were stimulated with S1P (0.1 μM) for 6 hours. TNF-α mRNA level was determined by real-time PCR. (E–G) BM-derived macrophages from S1pr2+/+Apoe–/– (white bars) and S1pr2–/–Apoe–/– (black bars) mice were cultured in DMEM containing 10% FBS and treated with either JTE-K1, Y-27632 (Wako), or BAY11-7085 (Merck-Calbiochem) for 6 hours. mRNA expression levels of CD36 (E), ABCA1 (F), and LXRα (G) were determined by real-time PCR (n = 5 each). (H) Foam cell formation assay. Serum-starved BM-derived macrophages from S1pr2+/+Apoe–/– mice were exposed to oxLDL for 6 hours. S1P (0.1 μM), JTE-K1, Y-27632, and/or BAY11-7085 were added 6 hours before adding oxLDL. Quantified data of oil red O–positive cells are shown (n = 3 each). In A–H, the inhibitors (10 μM JTE-K1, 10 μM Y-27632, and 10 μM BAY11-7085) were added 10–30 minutes before S1P addition and present in the medium throughout the subsequent incubation. *P < 0.05; **P < 0.01; ***P < 0.001, as compared with nontreated S1pr2+/+Apoe–/– macrophages. #P < 0.05; ##P < 0.01; ###P < 0.001, as compared with S1P-treated S1pr2+/+Apoe–/– macrophages. Data are expressed as the ratio of the values in treated cells over nontreated cells and are shown as mean ± SEM (n = 5 each).