In response to a graded decrease in O2, HIF-1 stabilisation was recognized in ECFCs at the lower O2 levels of 3 and 1% with no HIF stabilisation detectable at the higher oxygen concentrations of 5 and 10% (Fig.?1). DMOG-treated cells inhibited ECFC tube formation. This effect was reversed by the addition of PlGF neutralising antibody to the conditioned medium, confirming the direct part of PlGF with this effect. Conclusions This study deepens our understanding of the response of ECFCs to hypoxia and also identifies a novel and important part for PlGF in regulating the vasculogenic potential of ECFCs. Electronic supplementary material The online version of this article (doi:10.1186/s13287-016-0430-0) contains supplementary material, which is available to authorized users. =? =? / test or ANOVA and were plotted as mean??standard deviation (SD) unless otherwise indicated. Results ECFCs were isolated from umbilical wire blood of normal term babies as explained AZD8835 previously [24]. The endothelial character of these cells was confirmed by the manifestation of PECAM (CD31) and melanoma metastasis-associated surface molecule (MUC18, CD146) (Additional file 1: Number S1). In addition, isolated ECFCs did not communicate the leukocyte markers CD45 and the myeloid marker CD14, which confirmed that these INHBB cells were not hematopoietic in nature (Additional file 1: Number S1). To examine the effects of low oxygen on these cells, ECFCs were grown in a range of oxygen tensions from 21% (atmospheric oxygen, 760?mmHg) to 10, 5, 3 and 1% (36?mmHg, Fig.?1a). HIF-1 immunoreactivity was only recognized at 3% O2, with the highest levels of HIF-1 recognized at 1% (Fig.?1a). The inhibition of prolyl-hydroxylases and build up of cytoplasmic HIF-1 at 1% O2 was extremely quick, and was recognized within 5?min of hypoxia exposure (Fig.?1b). A time-dependent increase in HIF-1 build up occurred in ECFCs, with the strongest build up present after 4?h (240?min) of hypoxia exposure. Based on these data, all subsequent hypoxia exposure experiments were carried out at 1% O2 for a minimum of 4C5?h. Open in a separate windows Fig. 1 HIF-1 stabilisation in ECFCs happens rapidly at 1% O2. ECFCs were cultivated in 12% FCS-supplemented EBM-2 medium. a ECFCs were cultured in the AZD8835 indicated oxygen pressure for 5?h. Protein lysates were probed using HIF-1 (hypoxia inducible element 1 alpha Build up of both HIF-1 and HIF-2 isoforms were observed in ECFCs produced in 1% O2 for 8, 24 and 48?h (Fig.?2a). In comparison with ECFCs produced in 21% O2 (160?mmHg), cells grown in 1% O2 demonstrated a marked increase in HIF-1 immunoreactivity in the cytosol, with punctate staining in the nuclei evident in the 5?h time point (Fig.?2b). This suggested the HIF-1 subunit was translocating to the nucleus as part of AZD8835 the standard transcriptional response to hypoxia AZD8835 [25]. This was further corroborated from the improved manifestation of HIF-1 regulated transcripts ANGPTL4 and SLC2A1 (also known as GLUT1) in ECFCs produced in 1% O2 (Fig.?2c). Therefore, exposure to low oxygen concentrations induces a typical hypoxia response in terms of HIF-1 transcription element build up and gene transcription in ECFCs. Open in a separate windows Fig. 2 ECFC reactions to 1% O2. ECFCs were cultivated in 12% FCS-supplemented EBM-2 medium and managed in either 21 or 1% O2 for the indicated occasions. a Protein lysates isolated from time-matched ECFCs produced under normal conditions (21% O2) or hypoxic conditions (1% O2) were probed with HIF-1 (hypoxia inducible element 1.