Echinomycin also known as NSC has

Echinomycin (also known as NSC-13502) has recently been used as a strong HIF-1α inhibitor that acts by blocking HIF-1 DNA binding and transcriptional activity. Also, echinomycin inhibits the binding of HIF-1 to the endogenous HRE of the VEGF (Low et al., 1986; May et al., 2004; Kong et al., 2005; Vlaminck et al., 2007).
Results
Discussion Oxygen sensing is vital for proper cardiovascular formation, and oxygen tension is likely an important determinant of hematopoietic and endothelial cell production (Cameron et al., 2008). Several groups have already reported that hypoxia, defined as low oxygen tension (1–5% O2), affects mesodermal and hemangioblast lineage specification during early embryogenesis (Ramirez-Bergeron et al., 2004; Adelman et al., 1999; Fehling et al., 2003). In our previous study, the PECAM-positive endothelial cell population was mainly restricted to the center region of attached hEBs and the efficiency of differentiation of these apexbio dilution into endothelial cells was about 6 times higher than in cells from the outgrowth region of attached hEBs (Kim et al., 2007). We hypothesized that hypoxia, which existed in the center region of hEBs, could be a key inducer of differentiation, and consequently, we investigated here the effect of hypoxia on the differentiation of hESCs into vascular lineage cells. Furthermore, this study provides insight into the release of several angiogenic factors, which could improve the vascular lineage differentiation of hypoxic hEBs. To be functionally active, HIF-1α must translocate to the nucleus, dimerize with ARNT and transactivate hypoxia-responsive genes by binding to hypoxia response elements (HREs) located in the promoter or enhancer regions of hypoxia-inducible genes (Ramirez-Bergeron et al., 2004). Here, via immunostaining, we verified the translocation of HIF-1α into the nucleus by hypoxia. Also, we found that the expression of HIF-1α and pro-angiogenic factors was increased by hypoxia. Therefore, we conclude that representative pro-angiogenic factors, such as VEGF and bFGF, are downstream of HIF-1α signaling. Also, up-regulated VEGF expression could act as a survival factor for hypoxic hEBs because VEGF165 is a known survival factor for ES cells under hypoxic conditions and can prevent apoptosis in response to both short- (<24h) and long-term (>24h) hypoxia (Levenberg et al., 2007). This fact suggests that the lack of massive apoptosis in hypoxic hEBs may be due to up-regulated VEGF expression. To further investigate the effect of hypoxia on the differentiation of hESCs, we treated cells with echinomycin, a representative HIF-1α inhibitor. Before testing the effect of echinomycin, we optimized the concentration of echinomycin through several pilot studies; we determined that 20nM for 12h was the optimal dose to avoid cytotoxicity and cell cycle arrest. We found that echinomycin blocked the expression of HIF-1α and downstream pro-angiogenic factor and down-regulated vascular lineage differentiation. This result suggests that hypoxia enhances vascular lineage differentiation by up-regulating HIF-1α and angiogenic factor expression. In all experiments in this study, we measured four angiogenic factors (VEGF, bFGF, Ang-1 and PDGF-BB) as well as HIF-1α expression; of these factors, VEGF and bFGF were the most up-regulated by hypoxia. VEGF is a multifunctional cytokine that also plays an important role in endothelial and hematopoietic differentiation of ESCs as well as angiogenesis and vasculogenesis (Pearson et al., 2008). bFGF has been supplemented in culture medium to sustain the undifferentiated status of hESCs and maintain their self-renewal capacity (Dell\'Era et al., 2003). bFGF has also been shown to play a role in the development of endothelial cells and cardiomyocytes and to preserve the long-term repopulating ability of hematopoietic stem cells in vitro (de Haan et al., 2003). HIF-1α target genes include those encoding VEGF and bFGF. Consequently, we asked whether up-regulated VEGF and bFGF expression was important in stimulating vascular lineage differentiation. We treated normoxic hEBs with commercially available VEGF or bFGF for 10days to induce differentiation. Treating hEBs with VEGF or bFGF increased differentiation into vascular lineage cells to the same extent as hypoxia. These results clearly demonstrate that addition of VEGF or bFGF during hESC differentiation promotes vascular lineage cell development, even in normoxic conditions. The roles of VEGF and bFGF during hESC differentiation are already well known; however, we have not only confirmed the direct effect of VEGF and bFGF treatment on the differentiation of hESCs but also confirmed that up-regulation of VEGF and bFGF by hypoxia permitted vascular lineage differentiation.