W146

Cell experiment:

Endothelial progenitor cells (EPCs) proliferation is examined by the colorimetric MTS assay. EPCs are seeded in 96-well plates in quintuplicate at a density of 1×104/well and cultured in synchronized DMEM/H (supplemented with 0.5% bovine serum albumin (BSA), and lacking fetal bovine serum (FBS) and endothelial cell growth supplement (ECGS)). After culturing for 12 h, EPCs are incubated in 20% FBS-supplemented DMEM/H with S1P (0, 200, 800, and 1,400 nM). MTS (20 mL/well) is added into the culture medium to incubate for another 8 hrs at 37°C. The optical density (OD) at 490 nm is recorded using a 96-well plate reader. The potent S1PR1 antagonists like W146 (10 μM), and S1PR3 antagonists are added into culture medium for 30 min before the addition of S1P. Simultaneously, cells are counted by detecting the effect of S1P treatment on EPC proliferation[2].

Animal experiment:

Mice[3]Mice (4-6-week-old) are used. All mice go through 2-week adaptation period and are used for experiments at 6-8 weeks of age. Mice are IP injected with W146, and measured for Kit+/Sca-1+/Lin- hematopoietic stem progenitor cells (KSL-HSPC) mobilization by CFU-G/M analysis. Flow cytometry analysis of blood samples from mice pretreated with 5 mg/kg of body weight W146, W140, JTE013, or Cay10444, followed by AMD3100 (ADM) administration. Mice injected with W140 (an inactive enantiomer of W146), JTE013 (S1P2 selective antagonist), or Cay10444 (S1P3 selective antagonist) are used as controls[3].

References:

[1]. M Germana Sanna, et al. Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo. Nat Chem Biol. 2006 Aug;2(8):434-41. Epub 2006 Jul 9.
[2]. Hang Wang, et al. Sphingosine-1-phosphate promotes the proliferation and attenuates apoptosis of Endothelial progenitor cells via S1PR1/S1PR3/PI3K/Akt pathway. Cell Biol Int. 2018 May 23.
[3]. Jingjing Liu, et al. 3-amino-4-(3-hexylphenylamino)-4-oxobutyl phosphonic acid (W146), a Selective Antagonist of Sphingosine-1-phospahte Receptor Subtype 1, Enhances AMD3100-stimulated Mobilization of Hematopoietic Stem Progenitor Cells in Animals. J Biochem Pharmacol Res. 2013 Dec; 1(4): 197–203.