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


RESULTS
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 LTB(4) and LTD(4) in conformity with induction of the 5-LO pathway. Furthermore, Flk-1(+)/CD105(+) cells displayed calcium- (Ca(2+)) transients in response to LTB(4), whereas CD11b(+) cells responded to LTD(4). Treatment of EBs with LTB(4) and LTD(4) 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.


CONCLUSIONS
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.
Finkensieper
Andreas
A
Cardiology Division, Clinic of Internal Medicine I, Friedrich Schiller University, Erlanger Allee 101, Jena D-07743, Germany. andreas_finkensieper@gmx.net
Kieser
Sophia
S
Bekhite
Mohamed M
MM
Richter
Madeleine
M
Mueller
Joerg P
JP
Graebner
Rolf
R
Figulla
Hans-Reiner
HR
Sauer
Heinrich
H
Wartenberg
Maria
M
D058927:5-Lipoxygenase-Activating Proteins; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D019070:Cell Lineage / Q000502:physiology; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007975:Leukotriene B4 / Q000494:pharmacology; D017998:Leukotriene D4 / Q000494:pharmacology; D015289:Leukotrienes / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology; D008264:Macrophages / Q000166:cytology / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009389:Neovascularization, Pathologic / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D018077:Receptors, Leukotriene / Q000235:genetics / Q000378:metabolism; D018102:Receptors, Leukotriene B4 / Q000235:genetics / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
D058927:5-Lipoxygenase-Activating Proteins; C548593:Alox5ap protein, mouse; D002352:Carrier Proteins; D015289:Leukotrienes; C503704:Ltb4r1 protein, mouse; C412867:Ltb4r2 protein, mouse; D008565:Membrane Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018077:Receptors, Leukotriene; D018102:Receptors, Leukotriene B4; D007975:Leukotriene B4; D017998:Leukotriene D4; C412375:cysteinyl leukotriene receptor 2; D001094:Arachidonate 5-Lipoxygenase; D005982:Glutathione Transferase; D040301:Vascular Endothelial Growth Factor Receptor-2; D048049:Extracellular Signal-Regulated MAP Kinases; D004851:Epoxide Hydrolases; C041116:leukotriene-C4 synthase; C083169:leukotriene D4 receptor; C043134:leukotriene A4 hydrolase