HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3

Abstract

HDL is a major atheroprotective factor, but the mechanisms underlying this effect are still obscure. HDL binding to scavenger receptor-BI has been shown to activate eNOS, although the responsible HDL entities and signaling pathways have remained enigmatic. Here we show that HDL stimulates NO release in human endothelial cells and induces vasodilation in isolated aortae via intracellular Ca2+ mobilization and Akt-mediated eNOS phosphorylation. The vasoactive effects of HDL could be mimicked by three lysophospholipids present in HDL: sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and lysosulfatide (LSF). All three elevated intracellular Ca2+ concentration and activated Akt and eNOS, which resulted in NO release and vasodilation. Deficiency of the lysophospholipid receptor S1P3 (also known as LPB3 and EDG3) abolished the vasodilatory effects of SPC, S1P, and LSF and reduced the effect of HDL by approximately 60%. In endothelial cells from S1P3-deficient mice, Akt phosphorylation and Ca2+ increase in response to HDL and lysophospholipids were severely reduced. In vivo, intra-arterial administration of HDL or lysophospholipids lowered mean arterial blood pressure in rats. In conclusion, we identify HDL as a carrier of bioactive lysophospholipids that regulate vascular tone via S1P3-mediated NO release. This mechanism may contribute to the vasoactive effect of HDL and represent a novel aspect of its antiatherogenic function.

Figure 1

HDL induces vasodilation in aortae from rats and mice in an eNOS-dependent manner. (a) Thoracic aortic rings from WKY rats were precontracted with PE (1 × 10⁶ mol/l, arrows), and direct relaxation responses to HDL (0.5 mg/ml) or HDL and L-NAME (50 μmol/l) were evaluated. Shown are representative tracings from one experiment of 16. (b) Cumulative findings (mean ± SEM) for maximal relaxation in response to 0.5 mg/ml HDL in the presence of L-NAME (50 μmol/l), SKF-525A ((SKF, 50 μmol/l), or indomethacin (Indo, 10 μmol/l) (n = 6 each). *P < 0.01 vs. HDL. (c) HUVECs loaded with DAF-2DA were stimulated with 0.5 mg/ml HDL in the absence or presence of L-NAME. Cells were fixed and fluorescence was evaluated under a fluorescence microscope. Shown are representative results (n = 5). (d) Dose response of the vasodilatory effect of HDL (n = 3). (e) Thoracic aortic rings from WT 129/C57BL/6 mice and eNOS–/– mice were precontracted with PE, and direct relaxation responses to HDL (0.5 mg/ml) were measured. Shown are representative tracings from one experiment of six.

Figure 2

HDL induces NO release and vasodilation via Akt-mediated eNOS phosphorylation in endothelial cells and in aortic segments. (a) Left panel: [³²P]orthophosphate-labeled HUVECs were stimulated with HDL (0.5 mg/ml) in the presence or absence of LY294002 (10 μmol/l). Immunoprecipitated eNOS (ip) was analyzed by autoradiography (n = 3), and amounts of immunoprecipitated protein were detected by Western blotting. Phosphorylation of Akt at Ser⁴⁷³ was determined in cell lysates with a phosphospecific antibody (n = 5). Right panel: Time-dependence of HDL-induced eNOS and Akt phosphorylation at Ser¹¹⁷⁷ and Ser⁴⁷³, respectively, as analyzed by densitometry (n = 3). (b) Following precontraction of thoracic aortic rings from WKY rats with PE (1 × 10^–6 mol/l, arrows), direct relaxation responses to HDL (0.5 mg/ml) in the absence or presence of LY294002 (10 μmol/l) were evaluated. Shown are original tracings from one experiment of eight. (c) Aortic segments perfused with 0.5 mg/ml HDL were fixed and immunostained for phospho-Ser¹¹⁷⁷-eNOS. Arrows indicate phospho-eNOS staining in the endothelial lining (original magnification, ×200). (d) Fura2-AM–loaded HUVECs were stimulated with 1 mg/ml HDL in the presence or absence of BAPTA-2AM (20 μmol/l) or Ni²⁺ (5 mM). [Ca²⁺]i was measured by fluorescence spectroscopy. Original tracings from representative experiments were superimposed for comparison. (e) HUVECs loaded with DAF-2DA and preincubated with BAPTA-2AM (20 μmol/l) or Ni²⁺ (5 mmol/l) were stimulated with HDL (1 mg/ml) and observed under a fluorescence microscope. Shown are representative results for one experiment of three.

Figure 3

HDL-associated lysophospholipids induce vasorelaxation in isolated arteries and NO production and eNOS phosphorylation in endothelial cells. (a) Following precontraction of thoracic aortic rings from WKY rats with PE (1 0 10^–6 mol/l), measurements were taken of direct relaxation responses to HDL (0.5 mg/ml), the HDL-lipid fraction (Lipid, equivalent to 0.5 mg/ml HDL), the HDL protein fraction (Protein, equivalent to 0.5 mg/ml HDL), apoAI (0.1 mg/ml), cholesterol (Chol, 10 μmol/l), phosphatidylcholine (PC, 10 μmol/l), and sphingomyelin (Sm, 10 μmol/l). Cumulative findings (mean ± SEM) for maximal relaxation in eight experiments are shown (*P < 0.01 vs. HDL). (b) HPLC profile of S1P and dihydro-S1P separated on a reverse-phase C18 column after a two-step lipid extraction and derivatization with o-phthaldialdehyde (upper left panel) is shown beside a representative HPLC chromatogram of HDL after addition of dihdro-S1P (50 pmol) before extraction procedures (upper right panel). HPLC chromatogram of o-phthaldialdehyde derivatives of SPC: 0.5 μmol SPC standard (lower left panel) and HDL (lower right panel). arb U, arbitrary units. (c) Following precontraction of thoracic aortic rings from WT 129/C57BL/6 mice (WT) or eNOS–/– mice with PE (1 × 10^–6 mol/l), direct relaxation responses to HDL (0.5 mg/ml), SPC (10 μmol/l), LSF (10 μmol/l), and S1P (10 μmol/l) were tested. Cumulative findings (mean ± SEM) for maximal relaxation in eight experiments are shown (*P < 0.01 vs. WT). (d) Following precontraction of thoracic aortic rings from WKY rats with PE, direct relaxation responses to different doses of SPC, S1P, and LSF were measured. Cumulative findings (mean ± SEM) for maximal relaxation in eight experiments are shown.

Figure 4

Vasodilatory effects of HDL, SPC, S1P, and LSF in vivo. Original tracing of MAP in a rat with endothelin-induced elevation of MAP (500 ng bolus + 1,000 ng/h intravenously) after intra-aortic injection (arrows) of HDL (1 mg), S1P (200 nmol), SPC (200 nmol), LSF (200 nmol), or acetylcholine (ACH, 200 nmol).

Figure 5

Lysophospholipid signaling mediates HDL-, SPC-, S1P-, and LSF-induced Akt and eNOS phosphorylation as well as [Ca²⁺]i increase. (a) HUVECs were stimulated with HDL (0.5 mg/ml) or with 10 μmol/l each (left) or 0.5–5 μmol/l each (right) SPC, S1P, and LSF, with or without preincubation with 100 ng/ml PTX for 16 hours. “PTX control” indicates PTX treatment alone. Cell lysates were analyzed for phospho–Ser⁴⁷³-Akt (p-Ser⁴⁷³-Akt) and phospho–Ser¹¹⁷⁷-eNOS by Western blotting. Loading controls for total eNOS and total Akt content are shown. All results are representative of one experiment of three. (b) HUVECs loaded with DAF-2DA were stimulated with SPC, S1P, and LSF (10 μmol/l each) and observed under a fluorescence microscope. Shown are representative results for one experiment of three. (c) Fura2-AM–loaded HUVECs were stimulated with SPC, S1P, and LSF (10 μmol/l each), and [Ca²⁺]i was measured by fluorescence spectroscopy. Original tracings from representative experiments were superimposed for comparison. (d) Concentration dependence of [Ca²⁺]i increase in HUVECs stimulated with SPC, S1P, and LSF measured as described in c.

Figure 6

Role of S1P₃ in the vasorelaxation induced by HDL and HDL-associated lysophospholipids. (a) Following precontraction of thoracic aortic rings from WT and S1P₃–/– mice with PE (1 × 10^–6 mol/l), direct relaxation responses to HDL (0.5 mg/ml), SPC (10 μmol/l), LSF (10 μmol/l), and S1P (10 μmol/l) were tested. Cumulative findings (mean ± SEM) for maximal relaxation in eight studies are shown (*P < 0.01 vs. same treatment in WT animals). (b) Vasorelaxation in response to different acetylcholine (Ach) concentrations in aortic rings from WT mice compared with S1P₃–/– mice. (c) Fura2-AM–loaded mouse cardiac endothelial cells from WT and S1P₃–/– mice were stimulated with HDL (1 mg/ml) or SPC, LSF, and S1P (10 μM each), respectively. [Ca²⁺]i was measured by fluorescence spectroscopy. Bar graph shows increases in [Ca²⁺]i calculated from three separate determinations (n = 3). (d) Mouse cardiac endothelial cells from WT and S1P₃–/– mice were stimulated with HDL (1 mg/ml), SPC (10 μM), or S1P (10 μM), and Akt phosphorylation after 15 and 30 minutes was measured using phosphospecific antibodies. In S1P₃–/– endothelial cells, Akt phosphorylation was also measured after stimulation with serum for 30 minutes as a control to exclude an overall inability of the cells to activate Akt.

Figure 7

Male and female HDLs are equally potent at inducing vasodilation, and their effect is similarly reduced in S1P₃–/– mice. Gender-specific S1P content in HDL. (a and b) Original tracings from (a) WT mice and (b) S1P₃–/– mice of relaxation responses to male and female HDL (0.1 mg/ml), respectively, following precontraction of thoracic aortic rings with PE (1 × 10^–6 mol/l, arrows). (c) Cumulative findings (mean ± SEM) for maximal relaxation of male (n = 11) and female (n = 8) HDL in WT and S1P₃–/– mice (*P < 0.05 vs. WT). There was no significant difference between male and female HDL by Mann-Whitney U test. (d) Quantification of S1P in male (n = 11) and female HDL (n = 7).

Figure 8

Model of HDL-induced eNOS activation and vasodilation by the lysophospholipid receptor S1P₃. PI-PLC, phosphatidylinositol-specific phospholipase C. A-I, apoAI; N and C, amino- and caboxy-terminus of SR-BI.