The 5-lipoxygenase pathway regulates vasculogenesis in differentiating mouse embryonic stem cells

It is well established that leukotrienes (LTs), products of the 5-lipoxygenase (5-LO) pathway, participate in inflammatory tissue reactions and immune responses. In the present study, the impact of the 5-LO pathway on vasculogenesis of mouse embryonic stem (ES) cells was investigated.

Immunohistochemistry studies demonstrated that 5-LO⁺ cells first appeared at day 6 of embryoid body (EB) formation from ES cells. 5-LO⁺/CD68⁺ as well as 5-LO⁺/CD45⁺ cells were prominent at day 10 of EB differentiation. Real-time PCR and western blot analysis revealed all constituents of the 5-LO pathway. High performance liquid chromatography analyses indicated the synthesis of LTB₄ and LTD₄ in conformity with induction of the 5-LO pathway. Furthermore, Flk-1⁺/CD105⁺ cells displayed calcium- (Ca²⁺) transients in response to LTB₄, whereas CD11b⁺ cells responded to LTD₄. Treatment of EBs with LTB₄ and LTD₄ resulted in phosphorylation of the mitogen-activated protein kinase ERK1/2. Pharmacological inhibition of the 5-LO pathway and stable shRNA targeting of 5-LO-activating protein decreased capillary cell areas positive for PECAM-1.

Our data demonstrate that the 5-LO pathway emerges early during ES cell differentiation into cells of the myeloid lineage and that LTs play an until now unrecognized role in vascular development of ES cells.