Tie-2-Expression Controls the Radio-Sensitivity of Hematopoietic Stem/Progenitor Cells.
Authors: Takahashi Kenji , Monzen Satoru , Kashiwakura Ikuo 
Published: October 2019

Abstract
Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (Tie-2) and its ligand, angiopoietin-1 (Ang-1) play an important role in the remodeling and maturation the vessels. However, Tie-2 expressed in hematopoietic stem cells has been reported to interact with Ang-1 on stromal osteoblasts in the bone marrow niche. This interaction leads to tight adhesion of hematopoietic stem cells to stromal cells, resulting in the maintenance of long-term repopulating activity of hematopoietic stem cells. Although the high radio-sensitivity of hematopoietic stem/progenitor cells is a serious cause of radiation damage in accidentally irradiated victims, the relationship of Tie-2/Ang-1-signals relate to radio-sensitivity is unclear. In addition, a diagnosis of specific radio-sensitivity in patients with malignant diseases allows radio- and/or chemo-therapy to be performed more effectively. Therefore, the radio-protection activity and the possible association of to radio-sensitivity in hematopoietic stem/progenitor of Tie-2/Ang-1 signaling was evaluated. This study was approved by the Committee of Medical Ethics of Hirosaki University School of Medicine. CD34+ hematopoietic stem/progenitor cells were isolated using a magnet sorting kit from human placental and umbilical cord blood units (n = 33) at the end of full-term deliveries after obtaining informed consent from the mothers. Each purified CD34+ fraction individually (81.3 ± 13.7%) was analyzed for the expression of Tie-2 using a flow cytometer. The range of Tie-2-expression was 1.6 ∼ 12.4% (mean = 5.13%). Based on the Tie-2-positive rate, all 33 fractions were classified into CD34+/Tie-2low fraction (Tie-2 positive rate; < 5%, n = 17) or CD34+/Tie-2high fraction (Tie-2 positive rate; ≥ 5%, n = 16). The CD34+ cells were exposed to X-rays at 2 Gy by an X-rays generator (150 kVp, 20 mA, 0.8 ∼ 0.95 Gy/min). The radio-sensitivity of the hematopoietic stem/progenitor cells of each specimen was measured using clonogenic assays. Specifically, the BFU-E, CFU-GM and CFU-GEMM of each sample were assessed in triplicate using a 14-day culture assay in a methylcellulose medium with 5 growth factors (EPO; 4 U/ml, SCF and IL-3; 100 ng/ml, G-CSF and GM-CSF; 10 ng/ml). In addition, CFU-Meg was assessed in triplicate using a 14-day plasma clot technique with 2 growth factors (TPO; 50 ng/ml, SCF; 100 ng/ml). Unexpectedly, the total CFC number decreased with the expression of Tie-2 in CD34+ cells and the total CFC number in CD34+/Tie-2high fractions was lower than that of CD34+/Tie-2low fractions. Moreover, the survival of total CFC at 2 Gy and Tie-2-expression were positively correlated in the CD34+ cells and the total CFC number in CD34+/Tie-2high fractions was more resistant to X-rays than that of CD34+/Tie-2low fractions. Furthermore, to clarify the interaction radio-sensitivity and Tie-2-expression, the effect of Ang-1 on the radio-protective activity in X-irradiated CD34+ cells was examined. However, no protective effect was observed. Although previous reports have showed that CD34+/Tie-2+ cells were more primitive than CD34+/Tie-2− cells, these results suggested that Tie-2-expression appeared to enhance the radio-resistant ability of CD34+ cells and that it was a marker for a radio-sensitivity. Since the direct activation by Ang-1 was not associated with the survival of X-irradiated CD34+ cells, another signaling mechanism may thus be involved in the radio-resistance activity of Tie-2.