Motivation Many patients with chronic open wounds need skin tissue transplantation. Transplantation with donor skin often results in rejection of the donor skin and nowadays transplantation with a tissue engineered scaffold is performed. Successful tissue transplantation and engineering requires adequate vascularization. Even though it is generally accepted that short term hypoxia stimulates angiogenesis, in chronically ischemic/hypoxic tissues and tissue-engineered scaffolds, endothelial cells have reduced angiogenic capacities. Despite the progress in knowledge on inhibiting angiogenesis, little is known about the stimulation. Exposure of endothelial cells to high concentrations of angiogenic growth factors induces chaotic and unstable vascular structures that are not adequately perfused. Therefore, it is important to understand how the induction of angiogenesis during hypoxia can be controlled. Many of the processes induced by hypoxia are mediated by the Hypoxia Inducible Factors, a family of transcription factors that directly induce the expression of many genes. Mainly HIF1α and HIF2α are involved in the regulation of genes under hypoxic conditions. Therefore, this study aims to investigate the putative role of HIF1α and HIF2α in chronic hypoxia-enforced inhibition of vascularization in vitro. Methods Human microvascular endothelial cells (hMVECs) were cultured at 20% oxygen or 1% oxygen for 14 days, without reoxygenation, and transfected with short interfering RNA (si-control, si-HIF1α or si-HIF-2α). The hMVECs were seeded on top of 3D fibrin matrices, and stimulated with the combination of VEGF and TNFα for 6 days to induce tube formation. Results hMVECs cultured at 20% oxygen formed tube-like structures in vitro, whereas those cells that were preconditioned at low (1%) oxygen did not form these structures. Surprisingly, cells transfected with si-HIF2α were able to form tube-like structures at 1% oxygen while the cells transfected with si-HIF1α or si-control did not (n=3). Conclusion Clearly, the formation of tube-like structures was inhibited under chronic hypoxia. However, transfection of chronic hypoxic cells with si-HIF2α restored the sprouting of endothelial cells. This indicates that counter intuitively HIF2α can impair tube formation in vitro. It is, therefore, important to further unravel its actions by using genome-wide RNA-sequence analysis and determine which transcription factor(s) and pathways downstream of HIF2α are involved.