N-Myc Downstream-Regulated Gene 2 Induces Angiogenesis

Abstract

Angiogenesis is the process of generating new capillary blood vessels from pre-existing vessels and a fundamental process in the normal development, reproduction, and wound healing. Since neovascularization in physiological processes is tightly regulated by a balance of angiogenic and anti-angiogenic factors, disrupted balance effect plays a leading role in the progress of diseases such as tumor growth, rheumatoid arthritis, and various blood vesselrelated pathology. Tumor growth relies on angiogenesis to receive an adequate supply of nutrients and oxygen. In addition, the newly formed blood vessels provide a way for tumor cells to enter the circulation and to metastasize to distant organs. Therefore, treatment of cancer with antiangiogenic agents will control cancer cell growth and metastasis. Among a variety of angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have a prominent activity in tumor metastasis and mortality. Specially, VEGF expression is induced in various cancers by several stimuli and its transcriptional activation is mediated by hypoxia-inducible factor-1 (HIF1). Since angiogenesis requires a multi-step mechanism, the process is mediated by a wide range of angiogenic regulators. In an effort to search for endogenous angiogenic factors, we found that N-myc downstream-regulated gene 2 (NDRG2) is involved in angiogenesis. Human NDRG2 belongs to a family of NDRG. The human NDRG family includes four identified members, NDRG1, NDRG2, NDRG3, and NDRG4, which are highly homologous except for in the Cand N-terminal regions. NDRG2 initially identified as a candidate tumor suppressor gene since its expression is reduced in several cancer cells. It has also been suggested that NDRG2 may have roles in cell differentiation, Alzheimer’s disease, and neurite overgrowth. However, NDRG2 activity related to angiogenesis was not yet reported. Since endothelial cell migration through extracellular matrix is one of essential steps for neovascularization, we tested the effect of conditioned medium derived from NDRG2-transfected cells (NDRG2-CM) on endothelial cell migration. To establish cell line expressing NDRG2, human embryonic kidney (HEK) 293 cells were transfected with NDRG2 expression plasmid and transfected cells stably expressing NDRG2 were selected as described in experimental section. After expression of NDRG2 was confirmed by Western blotting analysis, conditioned medium (CM) from the selected cells was obtained. In chemotaxis chamber, human umbilical vein endothelial cells (HUVECs) were treated with CM from control transfected cells and stably transfected cells for 2 h and HUVECs that had been migrated through membrane pores were counted under microscope. NDRG2-CM showed significantly induced migratory effect, compared to control-CM (Fig. 1A and B). Therefore, these data indicate that NDRG2-CM has the biological activity that can induce endothelial cell migration. We next examined the ability of NDRG2-CM to promote the mesh-like structures of HUVECs on matrigel. Matrigel is generally used for studying HUVEC attachment, migration and differentiation. The extent of mesh-like structure was more enhanced in HUVECs influenced in NDRG2-CM than that of control CM (Fig. 2A and B). This result indicates that NDRG2-CM has the ability of inducing tube formation of endothelial cells. We then measured the protein level of VEGF in NDRG2-