Bone marrow endothelial cells secrete thymosin β4 and AcSDKP

Objective: Bone marrow endothelial cells are the essential component of the bone marrow microenvironment. They produce many kinds of cytokines, including stimulators and inhibitors. Many researchers have suggested that in the presence of endothelial cell layer, CD34⁺CD38^- cells are capable of expansion. The ability of the endothelial cell layer to protect hematopoietic stem cells from extensive differentiation may be related to the inhibitors derived from endothelial cells. The aim of the present study was to determine whether the inhibitors thymosin β4 and AcSDKP are elaborated by murine bone marrow endothelial cells. Materials and Methods: Murine bone marrow endothelial cells (mBMECs) were cultured in serum-free conditioned medium. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the differential expression of the thymosin-β gene, and reverse phase high-performance chromatography (HPLC) and mass spectroscopy were used to determine the concentration of thymosin β4 (Tβ4) and AcSDKP in EC lysate and in the medium (mBMEC-CM). Colony-forming unit granulocyte-macrophage (CFU-GM) colony assays were used to examine the effect of components (mw 3-10 kD, <3 kD) of mBMEC-CM, thymosin β4, and AcSDKP on the proliferation of hematopoietic cells. Results: mBMECs expressed Tβ4 mRNA. In EC lysate and mBMEC-CM, Tβ4 and AcSDKP were detected. After adding protease inhibitors, the concentration of Tβ4 in EC lysate increased significantly, while the concentration of AcSDKP decreased. mBMEC-CM (mw 3-10 kD) had no effect on the formation of CFU-GM. However, mBMEC-CM (mw <3 kD) could inhibit the growth of CFU-GM. Tβ4 (10^-11∼10^-7mol/L) and AcSDKP (10^-11∼10^-5mol/L) had dose-dependent inhibitory effects on the growth of CFU-GM. Angiotensin converting enzyme (ACE), the enzyme degrading AcSDKP, could partially eliminate the inhibitory effect of mBMEC-CM (mw <3 kD) on CFU-GM. Conclusion: BMECs express and secrete Tβ4 and AcSDKP. Tβ4 exists in the 3-10 kD component of mBMEC-CM, while AcSDKP exists in the <3 kD component of ECCM. Both components exert inhibitory effects on the proliferation of hematopoietic progenitors.