Sphingosine-1-phosphate receptor 3 promotes neointimal hyperplasia in mouse iliac-femoral arteries.

OBJECTIVE: The objective of this study was to define a role for sphingosine-1-phosphate receptor 3 (S1PR3) in intimal hyperplasia. METHODS AND RESULTS: A denudation model of the iliac-femoral artery in wild-type and S1PR3-null mice was used to define a role for S1PR3 in the arterial injury response because we found in humans and mice that expression of S1PR3 was higher in these arteries compared with carotid arteries. At 28 days after surgery, wild-type arteries formed significantly larger lesions than S1PR3-null arteries. Bromodeoxyuridine labeling experiments demonstrated that on injury, wild-type arteries exhibited higher medial as well as intimal proliferation than S1PR3-null arteries. Because S1PR3 expression in vitro was low, we expressed S1PR3 in S1PR3-null smooth muscle cells (SMCs) using retroviral-mediated gene transfer to study the effects of S1PR3 on cell functions and signaling. SMCs expressing S1PR3, but not vector-transfected controls, responded to sphingosine-1-phosphate stimulation with activation of Rac, Erk, and Akt. SMCs expressing S1PR3 also migrated more. CONCLUSIONS: In humans and mice, S1PR3 expression was higher in iliac-femoral arteries compared with carotid arteries. S1PR3 promoted neointimal hyperplasia on denudation of iliac-femoral arteries in mice, likely by stimulating cell migration and proliferation through activation of signaling pathways involving Erk, Akt, and Rac.

Sphingosine 1-phosphate receptor 2 negatively regulates neointimal formation in mouse arteries.

Neointimal lesion formation was induced in sphingosine 1-phosphate (S1P) receptor 2 (S1P2)-null and wild-type mice by ligation of the left carotid artery. After 28 days, large neointimal lesions developed in S1P2-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. S1P2-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 S1P2-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 S1P2 normally acts to suppress SMC growth in arteries and that S1P is a regulator of neointimal development.

Matrix metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury.

Matrix metalloproteinases (MMPs) and, in particular, MMP-9 are important for smooth muscle cell (SMC) migration into the intima. In this study, we sought to determine whether MMP-9 is critical for SMC migration and for the formation of a neointima by using mice in which the gene was deleted (MMP-9(-/-) mice). A denuding injury to the arteries of wild-type mice promoted the migration of medial SMCs into the neointima at 6 days, and a large neointimal lesion was observed after 28 days. In wild-type arteries, medial SMC replication was approximately 8% at day 4, 6% at day 6, and 4% at day 8 and had further decreased to 1% at day 14. Intimal cell replication was 65% at 8 days and had decreased to approximately 10% at 14 days after injury. In MMP-9(-/-) arteries, SMC replication was significantly lower at day 8. In addition, SMC migration and arterial lesion growth were significantly impaired in MMP-9(-/-) arteries. SMCs, isolated from MMP-9(-/-) mouse arteries, showed an impairment of migration and replication in vitro. Thus, our present data indicate that MMP-9 is critical for the development of arterial lesions by regulating both SMC migration and proliferation.

Chlorogenic acid exhibits anti-obesity property and improves lipid metabolism in high-fat diet-induced-obese mice.

This study investigated the efficacy of chlorogenic acid on altering body fat in high-fat diet (37% calories from fat) induced-obese mice compared to caffeic acid. Caffeic acid or chlorogenic acid was supplemented with high-fat diet at 0.02% (wt/wt) dose. Both caffeic acid and chlorogenic acid significantly lowered body weight, visceral fat mass and plasma leptin and insulin levels compared to the high-fat control group. They also lowered triglyceride (in plasma, liver and heart) and cholesterol (in plasma, adipose tissue and heart) concentrations. Triglyceride content in adipose tissue was significantly lowered, whereas the plasma adiponectin level was elevated by chlorogenic acid supplementation compared to the high-fat control group. Body weight was significantly correlated with plasma leptin (r=0.894, p<0.01) and insulin (r=0.496, p<0.01) levels, respectively. Caffeic acid and chlorogenic acid significantly inhibited fatty acid synthase, 3-hydroxy-3-methylglutaryl CoA reductase and acyl-CoA:cholesterol acyltransferase activities, while they increased fatty acid beta-oxidation activity and peroxisome proliferator-activated receptors alpha expression in the liver compared to the high-fat group. These results suggest that caffeic acid and chlorogenic acid improve body weight, lipid metabolism and obesity-related hormones levels in high-fat fed mice. Chlorogenic acid seemed to be more potent for body weight reduction and regulation of lipid metabolism than caffeic acid.

Regulation of arterial lesions in mice depends on differential smooth muscle cell migration: a role for sphingosine-1-phosphate receptors.

The response of mice arteries to injury varies significantly between strains. FVB mice develop large neointimas after injury, whereas very small lesions form in C57BL/6 mice. After injury, platelet interaction with the denuded artery and early smooth muscle (SMC) replication are identical in both strains; however, the migration of SMCs differs significantly. FVB cells readily move into the developing neointima, whereas only the occasional C57BL/6 cells migrate. Injured arteries showed no difference in matrix metalloproteinases (MMP-2 and MMP-9) and plasminogen activator activities. In vitro, sphingosine-1-phosphate (S1P) in combination with platelet-derived growth factor (PDGF) stimulates migration of FVB cells but inhibits migration of C57BL/6 SMCs. Both SMCs migrate equally well to PDGF alone. One explanation is that the SMCs express different S1P receptors. Real-time polymerase chain reaction shows that FVB cells express higher levels of S1P receptor-1 (S1P(1)) compared with C57BL/6 cells, which express higher levels of S1P receptor-2 (S1P(2)). In addition, the migration of C57BL/6 cells can be increased by inhibiting S1P(2), whereas inhibiting S1P(1) expression slows the migration of FVB cells. Taken together these studies suggest that expression of S1P receptors vary within inbred mouse strains and that S1P is critical for SMC migration and lesion formation after injury.