Fig. 5. HDL-S1P acts as a biased agonist on S1P₁.

(A) HUVECs were transduced with lentivirus encoding GFP-tagged S1P₁ and after serum starvation, were stimulated with TNFα or with human HDL (huHDL), albumin- S1P, or P-FTY720 for the indicated times. Confocal microscopy was performed to assess the localization of GFP-tagged S1P₁. Images are representative of three independent experiments. (B) Serum-starved HUVECs were stimulated with albumin-S1P or HDL-S1P for the indicated times. MAPK phosphorylation was assessed by immunoblot of total lysates. (C) Serum-starved HUVECs were preincubated for 30 min with 3-isobutyl-1-methylxanthine (IBMX), and then stimulated with increasing doses of albumin-S1P, HDL-S1P, or ApoM⁻HDL in the presence of forskolin for 30 min. Results are expressed as picomole of cAMP per microgram of protein. Data represent combined analysis of five separate experiments and are expressed as means ± SD. **P < 0.01 by Friedman test followed by Dunnett’s multiple comparisons.