Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, Tokyo, Japan.
Exp Hematol. 2010 Feb;38(2):82-9. doi: 10.1016/j.exphem.2009.11.007. Epub 2009 Nov 26.
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.
We treated mouse BM lineage(-)Sca-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.
Gene expression analysis and BM transplantation assay revealed that Ang-1 upregulated expression of p57, p18, 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.
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.
血管生成素-1（Ang-1）通过与其受体 Tie2 结合，在骨髓（BM）中对造血干细胞（HSCs）的维持起着至关重要的作用。另一种 Tie2 配体 Ang-2 被认为是血管生成中 Tie2/Ang-1 信号的拮抗剂；然而，其在调节 HSCs 方面的功能仍不清楚。在这里，我们研究了 Ang-1 和 Ang-2 在维持 HSCs 中的功能差异。
我们用 Ang-1 和/或 Ang-2 处理小鼠 BM 谱系(-)Sca-1(+)c-Kit(+)侧群(+)细胞，并通过基因表达分析、磷酸化 Akt 的免疫细胞化学染色、集落形成测定和长期 BM 重建测定来评估血管生成素的功能。
基因表达分析和 BM 移植实验表明，Ang-1 上调了 HSCs 中 p57、p18、Itgb1、Alcam、Tie2、Hoxb4、Bmi1 基因的表达，而 Ang-2 拮抗了 Ang-1 的作用。Ang-1 增强了 Akt 的磷酸化，而 Ang-2 再次降低了 Ang-1 的作用。集落形成实验表明，Ang-1 和 Ang-2 均不影响 HSCs 的集落形成。在体外培养 HSCs 后进行 BM 移植实验，结果表明 Ang-1 维持 HSCs 的长期重植活性，而添加 Ang-2 则严重干扰了 Ang-1 的作用。
基因表达分析和 BM 移植实验表明，Ang-1 在体外培养中维持 HSC 活性。相反，Ang-2 逆转了 Ang-1/Tie2 信号在调节长期 HSCs 中的作用。我们的数据表明，在 BM 中，Ang-1 是 Tie2 受体在长 HSCs 中的优势配体。
