Role of Hmbox1 in endothelial differentiation of bone-marrow stromal cells by a small molecule.

Bone marrow stromal cells (BMSCs) play critical roles in repairing endothelium damage. However, the mechanisms underlying BMSC differentiation into vascular endothelial cells (VECs) is not well understood. We aimed to find new factors involved in this process by exploiting a novel chemical inducer in a gene microarray assay. We first identified a novel benzoxazine derivative (6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine; ABO) that can induce BMSC differentiation to VECs in a capillary-like tube formation assay, promote analysis of endothelial cell-specific marker expression, and facilitate uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated low-density lipoprotein (Dil-Ac-LDL). Microarray analysis of BMSCs treated with ABO for 4 h revealed changes in only a handful of genes. The only one upregulated was homeobox-containing 1 (Hmbox1) gene, whereas six genes, including IP-10 and others, were downregulated. The upregulation of Hmbox1 and downregulation of IP-10 were confirmed by RT-PCR, quantitative PCR (qPCR), and Western blot analysis. It is reported that IP-10 could suppresse EC differentiation into capillary structures. In this study ABO could not induce BMSC differentiation to VECs in the presence of IP-10. Small interfering RNA knockdown of Hmbox1 blocked ABO-induced BMSC differentiation and increased the level of IP-10 but decreased Ets-1. Thus, ABO is a novel inducer for BMSC differentiation to VECs, and Hmbox1 is a key factor in the differentiation. IP-10 and Ets-1 might be relevant targets of Hmbox1 in BMSC differentiation to VECs. These findings provide information on a novel target and a new platform for further investigating the gene control of BMSC differentiation to VECs.