Functional Difference of Two Tie2 Ligands, Angiopoietin-1 and -2, in the Regulation of the Adult Bone Marrow Hematopoietic Stem Cells

Majority of the adult HSCs is in quiescent state in cell cycle, and it is likely that maintenance of the HSC quiescence and slow cell-cycle progression are critical for sustaining the self-renewing HSC pool. Indeed, sub-populations of long-term HSC (LT-HSC) are in the quiescent state and have robust reconstitution activity of hematopoiesis. Therefore, it is suggested that quiescence of HSCs is directly linked to the ability for the efficient reconstitution. We previously reported that the side-population (SP) in Lineage⁻ Sca-1⁺Kit⁺ (LSK) cells resided in the BM niche is the quiescent HSC population, and is enriched in the Tie2 receptor-positive fraction in LSK. So far, four types of ligands, angiopoietin (Ang)-1~4, have been identified as the ligands of Tie2. We found that Ang-1/Tie2 signaling induces the quiescence or slow cell cycling of HSCs and contributes to the maintenance of long-term repopulating ability of LT-HSCs (Arai et al. 2004). Ang-2 is dispensable for embryonic angiogenesis but is specifically required for postnatal vascular remodeling and lymphatic patterning. In the hematopoietic system, however, the role of Ang-2 in the regulation of LT-HSCs is still unclear. Here, we investigated the functional difference between Ang-1 and Ang-2 in the maintenance of LT-HSCs. First, we examined the expression of CDK inhibitors, cell adhesion molecules, cytokine receptors, transcription factors, and anti-apoptotic factors induced by the Ang-1 and/or Ang2. LSK-SP cells were cultured with stem cell factor (SCF) in the presence or absence of angiopoietins (Ang-1 and/or Ang-2), and gene expressions were analyzed by high-throughput real-time PCR assays using a novel nanofluidic platform (BioMark^™ Dynamic Array and Digital Array, Fluidigm). We found that Ang-1 upregulated the expression of p57, p18, α4-integrin, β1-integrin, N-cadherin, Tie2, Hoxb4, Bmi-1 and Bax, compared to control. Although Ang-2 also upregulated p57 and Tie2, its effect was smaller than that of Ang-1. In addition, Ang-1-induced upregulations of p57 and p18 were suppressed by the simultaneous addition of Ang-2. Next we examined the effects of Ang-1 and Ang-2 on the long-term engraftment capacity of LSK-SP cells. LSK-SP cells (Ly5.1⁺) were cultured in the serum-free medium containing SCF and thrombopoietin (THPO) with or without angiopoietins (Ang-1 and/or Ang-2). After 7 days of culture, cells were transplanted into the recipient mice (Ly5.2⁺). Percentage of Ly5.1⁺ cells in the mice transplanted with the HSCs cultured with SCF and THPO was 9.8 ± 4.0 % after 3 months of BM transplantation (BMT). Consistent with our previous report, the LSK-SP cells cultured with Ang-1 efficiently maintained long-term repopulation (LTR), and the percentage of Ly5.1⁺ cells in the recipient’s peripheral blood was 40.8 ± 7.7 % after 3 months of BMT. Although Ang-2 also maintained LTR activity of HSCs (14.5 ± 4.7 %), the frequency of donor-derived cells was significantly lower than the case of Ang-1. Furthermore, the effect of Ang-1 was dramatically interfered by the addition of Ang-2 (10.0 ± 4.5 %). These observations suggest that in the BM niche, especially in the endosteal niche, Ang-1 might be the dominant ligand for Tie2 in LT-HSCs and contribute to the maintenance of the stem cell activities. In addition, we isolated osteoblasts (OB) and mesenchymal progenitor cells (MPCs), the major osteoblastic niche components, from endosteum by collagenase treatment of bone fragments and found that osteoblastic niche cells expressed Ang-1 but not Ang-2, which is consistent with the results of qPCR and LTR-assay. We hypothesized that Ang-2 is involved in the release of HSCs from the niche for the mobilization of HSCs. Collectively, Ang-1 plays a role as the key factor for LTR-activity maintenance of LT-HSCs, and Ang-2 has an antagonistic effect against Ang-1/Tie2 signaling.