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 7

S1PR2 mediates migration inhibition in macrophages.

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S1PR2 mediates migration inhibition in macrophages. (A and B) Effects of...
(A and B) Effects of S1P and MCP-1 on transwell migration in serum-starved peritoneal macrophages isolated from S1pr2+/+Apoe–/– and S1pr2–/–Apoe–/– mice. Macrophages were placed in the upper chamber. Various concentrations of S1P were added to either the lower chamber (A) or the upper chamber (B) in the presence or absence of MCP-1 (100 ng/ml) in the lower chamber. *P < 0.05 compared with the value in the absence of S1P (n = 4 each). (C) Effects of S1P and MCP-1 on Rac activity. Peritoneal macrophages were not treated or treated with either S1P (0.1 μM) alone for 10 minutes, MCP-1 alone (100 ng/ml) for 3 minutes, or the combination of S1P and MCP-1. In the combination treatment, S1P was added 7 minutes before the addition of MCP-1. Amounts of GTP-bound Rac were determined by the pulldown assay (n = 3 each). *P < 0.05, compared with nontreated macrophages; #P < 0.05, compared with MCP-1–treated macrophages. (D) Western blot analysis of VLA-4 (integrin α4β1), LFA-1 (integrin αLβ2), and CCR2 expression on peritoneal macrophages, with α-tubulin as internal control. (E) Macrophage homing assay. Peritoneal macrophages isolated from S1pr2+/+GFP-Tg and S1pr2–/–GFP-Tg mice were injected via the tail vein into S1pr2+/+Apoe–/– mice fed HCD (n = 3 each). Sections of the aortic sinus were made and GFP-expressing cells infiltrating into the intima were observed under a fluorescent microscope (asterisks show the lumen), and GFP-positive cells were counted. Sections were counterstained with DAPI. Representative images (left) and quantified data are shown (right). **P < 0.01. Scale bars: 50 μm. Data are expressed as mean ± SEM.