Sphingosine 1-Phosphate Receptor 2 Negatively Regulates Neointimal Formation in Mouse Arteries

Abstract

Abstract-Neointimal lesion formation was induced in sphingosine 1-phosphate (S1P) receptor 2 (S1P 2 )-null and wild-type mice by ligation of the left carotid artery. After 28 days, large neointimal lesions developed in S1P 2 -null but not in wild-type arteries. This was accompanied with a significant increase in both medial and intimal smooth muscle cell (SMC) replication between days 4 to 28, with only minimal replication in wild-type arteries. S1P 2 -null SMCs showed a significant increase in migration when stimulated with S1P alone and together with platelet-derived growth factor, whereas both wild-type and null SMCs migrated equally well to platelet-derived growth factor. S1P increased Rho activation in wild-type but not in S1P 2 -null SMCs, and inhibition of Rho activity promoted S1P-induced SMC migration. Plasma S1P levels were similar and did not change after surgery. These results suggest that activation of S1P 2 normally acts to suppress SMC growth in arteries and that S1P is a regulator of neointimal development. Key Words: sphingosine 1-phosphate receptors Ⅲ smooth muscle cells Ⅲ neointima S phingosine 1-phosphate (S1P) is a bioactive sphingolipid formed by activation of sphingosine kinases. 1 It exerts pleiotropic effects on many cells by regulating cytoskeletal rearrangement, cell survival, cell migration, cell proliferation, angiogenesis, and vascular development. [2][3][4][5] Recently S1P has received attention as a regulator of the cardiovascular system. In part, this is because there are high levels of S1P in plasma, and a recent report showed that they correlate well with the reoccurrence of vascular events. 6 -10 Further platelets release S1P during their activation, and consequently S1P levels are likely to be high at sites of arterial injury. 11,12 S1P acts through 5 G protein-coupled receptors (S1P 1 to S1P 5 ), although arterial smooth muscle cells (SMCs) express only S1P 1 , S1P 2 , and S1P 3 . 4,13 Initially these receptors were called endothelial cell differentiation gene receptors. 14 Activation of S1P receptors induces coupling to a variety of G proteins, which in turn leads to activation of multiple pathways. In SMCs most work has concentrated on S1P 1 and S1P 2 because they have opposing actions. S1P 1 couples to Gi and leads to activation of extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Rac. 13,15,16 Adult SMCs only weakly express S1P 1 , although it is more highly expressed in pup cells, and this has been linked to their increased ability to migrate and proliferate in response to S1P. 17 S1P 1 is also strongly expressed in SMCs from rat intimal lesions as well as in human atherosclerotic lesions. 17,18 These data have been used to suggest that activation of S1P 1 may induce events leading to restenosis and the formation of arterial lesions. S1P 2 is the main receptor expressed by most adult medial SMCs and couples to Gi, Gq, and G12/13, and its activation by S1P is associated with inhibition of SMC migration. 13,19 This is thought to occur via coupling...

Sphingosine-1-Phosphate Receptor 3 Promotes Recruitment of Monocyte/Macrophages in Inflammation and Atherosclerosis

“…In these, administration of a S1P 1 /S1P 3 antagonist in rats inhibited neointima formation after balloon injury of the carotid 34 and neointima formation after carotid ligation was enhanced in S1P 2 Ϫ/Ϫ mice. 17 The potent inhibition of PDGF-BB-induced migration by S1P and its lack of effect on basal migration that we see in our study confirm previous in vitro reports. 18,28,35 The absent effect of S1P on proliferation has also been described before e. g. for human SMCs, 18 although an enhanced proliferation by S1P has also been described for rat SMCs, where an S1P 1 /S1P 3 inhibitor prevented it but did not alter basal proliferation.…”

“…16 Lesion area and vessel area in the aortic root were calculated from 4 consecutive 5-m sections of 40 m apart over the entire root. The left carotid artery was ligated at the bifurcation as previously described 17 and serially sectioned over its entire length 4 weeks after surgery. Every 10th section starting at the ligation site was stained with H&E and neointimal and medial areas and neointimal cells were quantified by histomorphometry.…”

“…Every 10th section starting at the ligation site was stained with H&E and neointimal and medial areas and neointimal cells were quantified by histomorphometry. Smooth muscle cells (SMCs) were isolated from aortae (6 to 7 aortae per preparation) by enzymatic digestion as described, 17 plated on Vitrogen100, and used for experiments up to the sixth passage. For proliferation studies, SMCs were serum-starved for 24 hours and cultured in 1% FCS in the presence or absence of 1 mol/L S1P and 1 nmol/L platelet-derived growth factor (PDGF), respectively, added every other day for 4 days as described.…”

Sphingosine-1-Phosphate Receptor Subtypes Differentially Regulate Smooth Muscle Cell Phenotype

“…Recent studies by Reidy and colleagues 20,21 provided evidence showing that Figure 5. Inhibition of the S1P1 and S1P3 receptors increases S1P-induced H4Ac and SRF enrichment of the SM␣-actin and SMMHC CArG promoter region.…”

“…19 In contrast, exciting recent studies by Reidy and colleagues revealed that carotid neointimal hyperplasia is increased in response to ligation in mice that are null for the S1P2 receptor and that mouse lines with higher levels of S1P1 mRNA, ie, FVB mice, show increased neointimal hyperplasia compared to C57/Bl6 mice. 20,21 Taken together, the general paradigm that has emerged is that S1P1 regulates SMC proliferation and migration whereas S1P2 has opposing actions. However, given observations by Proia et al 19,22 that S1P receptors play a critical role in vascular development, one must also consider the possibility that enhanced neointimal formation in S1P2 null mice may also be attributable to secondary adaptive changes in gene expression.…”