Functional differences between two Tie2 ligands, angiopoietin-1 and-2, in regulation of adult bone marrow hematopoietic stem cells

Objective. Angiopoietin-1 (Ang-1) plays a critical role in the maintenance of hematopoietic stem cells (HSCs) in the bone marrow (BM) through its binding to the Tie2 receptor. Ang-2, another Tie2 ligand, is known to be an antagonist of Tie2/Ang-1 signaling in angiogenesis; however, its function in regulation of HSCs remains unclear. Here, we investigated the functional differences between Ang-1 and Ang-2 in the maintenance of HSCs. Materials and Methods. We treated mouse BM lineage (-)Sea-1(+)c-Kit(+) side population(+) cells with Ang-1 and/or Ang-2, and evaluated angiopoietin function by gene expression analysis, immunocytochemical staining of phosphorylated Akt, a colony-formation assay, and a long-term BM reconstitution assay. Results. Gene expression analysis and BM transplantation assay revealed that Ang-1 upregulated expression of p57, p78, Itgb1, Alcam, Tie2, Hoxb4, and Bmi1 genes in HSCs, while Ang-2 antagonized the effects of Ang-1. Ang-1 enhanced the phosphorylation of Akt, while Ang-2 again reduced the effect of Ang-1. The colony assay demonstrated that neither Ang-1, nor Ang-2 influenced the colony formation of HSCs. BM transplantation assay, following in vitro cultivation of HSCs with angiopoietins, showed that Ang-1 maintained long-term repopulating activity of HSCs, while the addition of Ang-2 interfered drastically with the effects of Ang-1. Conclusion. Gene expression analysis and BM transplantation assay demonstrated that Ang-1 maintained HSC activity in an in vitro culture. In contrast, Ang-2 reversed the effects of Ang-1/Tie2 signaling in the regulation of long-term HSCs. Our data suggest that Ang-1 is a dominant ligand for the Tie2 receptor in long HSCs in BM. (C) 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.