Leukotrienes (LT), products of the 5-lipoxygenase (5-LO) pathway, and their four receptors (LTRs) participate in inflammatory tissue reactions, immune responses and atherosclerosis. Here we present evidence that LTs also contribute to angiogenesis processes in Embryoid Bodies (EBs) derived from the mouse embryonic stem cell line CCE.

Our results demonstrated that 5-LO+ cells appeared first at day 6 of EB differentiation. 5-LO+/CD68+ macrophages were prominent at day 10. Also 5-LO+/CD45+ Leukocytes were detected after day 10 of culture. Real-time PCR and Western Blot analysis revealed that all constituents of the 5-LO pathway including 5-LO, 5-LO activating protein (FLAP), LTA₄-hydrolase, LTC₄-synthase and the four LT receptors (LTRs) were markedly induced during the same time window. HPLC analyses indicated a synthesis of LTB₄ and LTD₄ in conformity with the induction of the 5-LO pathway. To obtain evidence for a functional impact of the 5-LO/LTR system in the EB, we used shRNA-technique to create a FLAP knockdown cell line. Analysis of these cells and EBs showed a significant downregulation of PECAM-1 expression in a time window between day 8 and day 12 of EB differentiation. These results were consistent to the observed effect of the FLAP-inhibitor MK591. Therefore we suggest that the 5-Lipoxygenase pathway participate in angiogenesis during the differentiation of murine EBs.