Vascular endothelium as a contributor of plasma sphingosine 1-phosphate

Abstract

Sphingosine 1-phosphate (S1P), an abundant lipid mediator in plasma, regulates vascular and immune cells by activating S1P receptors. In this report, we investigated the mechanisms by which high plasma S1P levels are maintained in mice. We found that plasma S1P turns over rapidly with a half-life of approximately 15 minutes, suggesting the existence of a high-capacity biosynthetic source(s). Transplantation of bone marrow from wild-type to Sphk1(-/-)Sphk2(+/-) mice restored plasma S1P levels, suggesting that hematopoietic cells are capable of secreting S1P into plasma. However, plasma S1P levels were not appreciably altered in mice that were thrombocytopenic, anemic, or leukopenic. Surprisingly, reconstitution of Sphk1(-/-)Sphk2(+/-) bone marrow cells into wild-type hosts failed to reduce plasma S1P, suggesting the existence of an additional, nonhematopoietic source for plasma S1P. Adenoviral expression of Sphk1 in the liver of Sphk1(-/-) mice restored plasma S1P levels. In vitro, vascular endothelial cells, but not hepatocytes, secreted S1P in a constitutive manner. Interestingly, laminar shear stress downregulated the expression of S1P lyase (Sgpl) and S1P phosphatase-1 (Sgpp1) while concomitantly stimulating S1P release from endothelial cells in vitro. Modulation of expression of endothelial S1P lyase with small interfering RNA and adenoviral expression altered S1P secretion, suggesting an important role played by this enzyme. These data suggest that the vascular endothelium, in addition to the hematopoietic system, is a major contributor of plasma S1P.

Figure 1

High turnover of S1P in plasma. A and B, Functional similarity between C17- and C18-S1P. A, MEECs were stimulated at indicated concentrations of C17- and C18-S1P for 5 minutes, and phosphorylation of MAPK was analyzed by immunoblot analysis. B, Serum-starved HEK293 cells expressing S1P1R-GFP were treated with FBS or 100 nmol/L C17- or C18-S1P for 30 minutes, fixed, and imaged by a confocal microscope. C, Rapid disappearance of S1P in plasma. C17-S1P (1.5 nmol) complexed with 4% mouse serum albumin was injected intravenously, C17-S1P in plasma was quantified, and the half-life of C17-S1P was calculated (n=3). *Differs from 5 minutes (P<0.01).

Figure 2

Hematopoietic and nonhematopoietic sources of plasma S1P. A, Plasma S1P levels in wild-type and Sphk1^−/− Sphk2^+/− Sphk(3N) mice. B, Reciprocal bone marrow transplants in wild-type and Sphk(3N) mice. Wild-type mice receiving Sphk(3N) bone marrow maintained normal plasma S1P levels (left), whereas wild-type marrow restored plasma S1P levels when transplanted into Sphk(3N) mice.

Figure 3

Thrombocytopenia and anemia do not affect plasma S1P. A and B, Lack of effect of platelet depletion on plasma S1P levels. A, Injection of anti-GPIba antibody (2 μg/g) greatly reduced platelet number, as determined by fluorescence-activated cell-sorting analysis (inset), when compared with control (IgG 2 μg/g) (n=5 per group; **P<0.01). B, Plasma S1P levels in control and anti-GPIba antibody–injected mice. C and D, Lack of effect of PHZ-induced anemia on plasma S1P levels. C, Hematocrit values 48 hours after PHZ treatment (n=4 per group; **P<0.01). D, Plasma S1P levels in control and PHZ-treated mice.

Figure 4

Total bone marrow suppression does not reduce plasma S1P. Mice were subjected to total body irradiation and platelets (A), red blood cells (B), and white blood cells (C) were quantified. D, Plasma S1P levels were quantified by HPLC as described (n=3; *P<0.05, **P<0.01 vs day 0).

Figure 5

Expression of Sphk1 in liver restores plasma S1P in Sphk1^−/− mice. A, Plasma S1P levels in wild-type and Sphk1^−/− mice. B, Adenoviral transduction of Sphk1 into Sphk1^−/− mice restored plasma S1P levels. **P<0.01. C through F, Immunohistochemistry of liver sections stained with an Sphk1 antibody (brown) and counterstained with hematoxylin: wild-type (C), Sphk1^−/− (D), Sphk1^−/−transduced with Sphk1 adenovirus (E), and Sphk1^−/− transduced with GFP adenovirus (F). Note the strong expression of Sphk1 in sinusoidal endothelial cells of liver (E arrows).

Figure 6

S1P release by endothelial cells in vitro. A, S1P formation in intracellular (black) and extracellular compartments (gray). Note that endothelial cells avidly secrete S1P. B, Formation of endogenous S1P in the intracellular compartment (circles) and time-dependent accumulation of S1P in the extracellular compartment (squares) of HUVECs on medium change.

Figure 7

Shear stress induces S1P synthesis and release from MEECs. A, Formation of S1P in the intracellular and extracellular compartments under static (black) and laminar shear-treated (gray) conditions. B, Time-dependent accumulation of S1P in the media of MEECs subjected to laminar shear stress.

Figure 8

Laminar shear stress downregulates S1P lyase expression. A, Quantitative RT-PCR analysis of S1P lyase (Sgpl) transcript in static and shear stress–treated MEECs. Sgpl transcript was normalized to Gapdh mRNA. B, Immunoblot analysis of S1P lyase. MEECs were subjected to laminar shear stress as described in Materials and Methods. Immunoblot analysis on total homogenate was performed with affinity-purified anti-S1P lyase antibody. GAPDH was used as a loading control. Densitometric analysis of immunoblot is shown. C, Downregulation of S1P lyase by double-stranded siRNA in MEECs stimulated intracellular synthesis of [³H]-S1P and its release to extracellular medium (black: scrambled siRNA; gray: S1P lyase si). Down-regulation of S1P lyase by siRNA and corresponding control (scrambled siRNA) is also shown by immunoblot analysis (n=3). D, Transduction of human adeno-Sgpl in HUVECs decreased the formation of [³H]-S1P in intracellular and extracellular compartments (black: AdGFP; gray: AdSgpl). Stimulated expression of S1P lyase is shown by immunoblot analysis (n=3).