Ang1 Functions as An Autocrine Activating Factor of Tie2 Signaling In Hematopoietic Stem Cells.

Abstract 1548

Hematopoietic stem cells (HSCs) are clonogenic cells that possess the self-renewal capacity to produce more HSCs, as well as the multilineage potential that gives rise to a defined set of mature differentiated progeny for maintenance or repair of the whole blood system. HSCs lie in the hematopoietic niches located along the inner surface of the bone or the sinusoidal endothelium, and are in contact with niche cells. The cell-cell interactions with niche cells are believed to be an important prerequisite to trigger signaling events in HSCs, thereby controlling the balance between HSC self-renewal and differentiation. However, the precise molecular mechanisms regulating niche cell-HSC interactions are not well understood. One of the key molecules for those interactions is Angiopoietin-1 (Ang1). Ang1 is expressed by the niche cells and has been identified as an activating ligand for Tie2 (tyrosine kinase with Ig-like loops and epidermal growth factor homology domains 2). The expression of Tie2 is dominant in HSCs, and Tie2 in HSCs is supposed to be stimulated by Ang1 derived from niche cells. However, Ang1 is also expressed in HSCs. Detailed analysis has shown that Ang1 expression was found to be restricted in long-term HSCs (CD34^-lineage^-Sca-1⁺c-Kit⁺), indicating that Ang1 derived from HSCs plays a role in regulating HSCs. We attempted to elucidate a novel regulating system for HSCs through Ang1-Tie2 signaling by utilizing a hematopoietic cell line in which Tie2 was stably expressed (Ba/F3-Tie2). In Ba/F3-Tie2 cells, Tie2 was found to be phosphorylated on tyrosine residues, even without exogenous addition of Ang1. In the same cells, the expression level of endogenous Ang1 was increased four-fold. When Ang1 expression was down-regulated by transduction with a lentiviral vector expressing short hairpin RNA (shRNA) for Ang1 (shAng1), the phosphorylation of Tie2 was suppressed, suggesting that Tie2 expressed in Ba/F3-Tie2 cells could be stimulated by endogenous Ang1. To mimic the physiological circumstances of the bone marrow, Ba/F3-Tie2 cells were cultured on OP9 stromal cells. Under these culture conditions, the effect of endogenous Ang1 was investigated. Down-regulation of Ang1 by shAng1 demonstrated an approximate 50% reduction in the proliferation of Ba/F3-Tie2 cells on the OP9 cell layer. A HSC-rich population of cells prepared from bone marrow (lineage^-Sca-1⁺c-Kit⁺; LSK) was also analyzed on OP9 cell layers. Similar to the results obtained from the analysis of Ba/F3-Tie2 cells, down-regulation of Ang1 by shAng1 resulted in an approximately 70% decrease in the proliferation of LSK cells cultured on OP9 monolayers. We confirmed that the suppressive effect on HSC proliferation was due to the lack of Ang1 from HSCs by culturing on Ang1-defective OP9 cells. Finally, we performed in vivo analysis to confirm the importance of endogenous Ang1 to HSCs. Ly5.2 LSK cells transduced with the shAng1 expressing vector were transplanted along with Ly5.1xLy5.2 bone marrow cells into lethally irradiated Ly5.1 mice. The Ly5.2 donor-derived cells in the recipient's peripheral blood were monitored every 2 weeks. As expected, shAng1-introduced donor cells were at decreased ratios at week four (mean ratios, 31.5% for control vs. 17.5% for shAng1), and were reduced to an even lower level at week 12 (mean ratios, 27.1% for control vs. 6.79% for shAng1). This phenomenon was also confirmed by histochemical results, where statistically fewer HSCs existed in the bone marrow of recipient mice in which shAng1-introduced HSCs were transplanted, as compared to the control. Altogether, our data suggested that Tie2 in HSCs could be stimulated by the Ang1 produced by the surrounding HSCs, and this possible autocrine regulation might control the functions of HSCs.