Fig 1: Sortilin evokes endothelial dysfunction through the S1P-signaling pathway.(A) NOX activity (n = 4) and (B) ASMase activity (n = 8) in HUVECs treated with vehicle (Ctrl) or sortilin. RFU, relative fluorescence units. (C) Acetylcholine-evoked vasorelaxation in WT mesenteric arteries exposed to vehicle or pretransfected with siRNA against ASMase (Si-ASMase) before sortilin; scrambled siRNA (Si-SCR) was used as control (n = 6). ASMase protein levels after siRNA silencing. (D) Effect of si-ASMase on NOX activity in WT mesenteric arteries exposed to sortilin (n = 4). (E) Acetylcholine-evoked vasorelaxation in WT mesenteric arteries exposed to vehicle or sortilin or pretreated with either K145 or SK1-I before sortilin (n = 5). (F) Effects of K145 and SK1-I on NOX activity in sortilin-stimulated HUVECs (n = 3). (G) LC-MS/MS quantification of extracellular S1P levels in vehicle- or sortilin-treated HUVECs (n = 7). (H) Representative immunoblots and densitometric analyses of 3 independent experiments evaluating S1P1 and S1P3 expression in WT mesenteric arteries exposed to vehicle or sortilin. (I) Acetylcholine-evoked vasorelaxation in WT mesenteric arteries exposed to vehicle or sortilin or pretreated with W146 or TY52156 before sortilin (n = 6). (J and K) NOX activity in (J) HUVECs treated with vehicle or sortilin or pretreated with W146 or TY52156 and in (K) S1P3–/– mesenteric arteries exposed to vehicle or sortilin (n = 4–5 replicates from 3 independent experiments). (L) Acetylcholine-evoked vasorelaxation in mesenteric arteries from WT and S1P3–/– mice exposed to vehicle or sortilin (n = 5). Data are represented as mean ± SD. Unpaired Student’s t test (A, B, G, and H); 1-way (D, F, J, and K) or 2-way (C, E, I, and L) ANOVA followed by Bonferroni’s post hoc test. *P < 0.0001 versus vehicle or SK1-I plus sortilin at the same acetylcholine concentration (as indicated by color code) (E); *P < 0.0001 versus vehicle or TY52156 plus sortilin at the same acetylcholine concentration (as indicated by color code) (I).