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℃. 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].

W146 is a selective antagonist of sphingosine-1-phosphate receptor 1 (S1PR1) with an EC50 value of 398 nM.

W146 is a S1PR1 antagonist with a Ki of ~70-80 nM[1]. Compared with the S1P treated group, the S1P attenuated TUNEL labeling index of EPCs is significantly increased by pretreatment with W146. Furthermore, pretreatment with W146 significantly increases activated cleaved caspase-3 levels. The reduced EPCs apoptosis which induced by S1P is completely abolished after treatment with W146[2].

Injections of W146, W140, JTE013, or Cay10444 do not alter the basal WBC count in mice. It is found that a significant increase in KSL-HSPC mobilization when mice are pretreated with W146, compared to that in mice pretreated with dextran followed by AMD3100 administration. Moreover, pre-treatment of W146 shows approximately 8-fold increase of KSL-HSPC mobilization, measured by the CFU-G/M colony forming assays, compared to that in mice treated with AMD3100 alone. The W146-mediated augmentation of KSL-HSPC mobilization is specific, because pretreatment of mice with W140 is unable to produce any effect on AMD3100-stimulated KSL-HSPC mobilization[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.