Impact of the arachidonic acid/leukotriene signaling pathway for vasculogenesis and leukocyte differentiation of mouse embryonic stem cells

Abstract

Embryonic stem (ES) cells are able to differentiate into several kinds of cells, such as endothelial and haematopoietic cells. Leukotrienes (LTs) are important mediators of inflammation and are derived from cell membrane released- arachidonic acid (AA) after the cells are stimulated by inflammatory stress or other factors. Previous studies have already shown that LTs involve in the inflammatory response that contributes significantly to local vascular formation. Moreover, the inflammatory response is also accompanied by massive leukocyte accumulation. However, it remains elusive whether LTs have any effect on vasculogenesis or leukocyte differentiation of ES cells in response to inflammation. Therefore, our project focused the mechanisms of how LTs participate in vasculogenesis and leukocyte differentiation. To detected vasculogenesis of embryonic bodies (EBs), endothelial markers CD31, VE-cadherin and fetal liver kinase 1 (Flk-1) were measured by western blot. To further confirm the new vascular structure formation, positive CD31 were measured by immunohistochemistry (IHC). For leukopoiesis detection, leukocyte markers CD18, CD45 and CD68 were measured by IHC. Our results showed that AA dose-dependently stimulated vasculogenesis of ES cell by upregulating endothelial markers. Furthermore a stimulation of leukopoiesis was observed by increasing leukocyte markers. Inhibition of 5-lipoxygenase activating protein (FLAP) which is a key enzyme in LT synthesis with either AM643 or REV5901 significantly reduced vascular structure formation. Furthermore FLAP inhibition abolished leukopoiesis achieved upon treatment with AA. Exogenous addition of LTs partially restored vasculogenesis inhibited by the FLAP inhibitors. Downstream of LT signaling pathway, the leukotrienes B4 (LTB4) receptor 1 antagonist U75302 and the LTB4 receptor 2 antagonist LY255283 as well as the CYsLT blocker BAY-u9773 inhibited vasculogenesis of ES cells. AA treatment of differentiating ES cells increased intracellular reactive oxygen species (ROS) generation. Inhibition of ROS generation by either the free radical scavengers or the NADPH oxidase inhibitor VAS2870 downregulated vasculogenesis of ES cells and inhibited the pro-vasculogenic effect of AA. In conclusion, our study uncovers mechanisms underlying the effects of AA-derived LT synthesis on vasculogenesis and leukocyte differentiation of ES cells as well as AA-increased ROS production on vasculogenesis.