on October 1, 2009
Published online before print October 1, 2009, doi: 10.1161/ATVBAHA.109.193185
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Submitted on June 15, 2009
Accepted on September 8, 2009
Shear Stress Increases Expression of the Arterial Endothelial Marker EphrinB2 in Murine ES Cells via the VEGF-Notch Signaling Pathways
Tomomi Masumura ;
From the Departments of Biomedical Engineering (T.M., K.Y., N.S., S.O.) and Pediatrics (N.S.), Graduate School of Medicine, University of Tokyo, Japan; PRESTO, Japan Science and Technology Agency (K.Y.), Saitama, Japan; and the Laboratory of Biomedical Engineering (J.A.), School of Medicine, Dokkyo Medical University, Tochigi, Japan.
* To whom correspondence should be addressed. E-mail: jo-ji{at}umin.ac.jp.
Objective—Arterial-venous specification in the embryo has been assumed to depend on the influence of fluid mechanical forces, but its cellular and molecular mechanisms are still poorly understood. Our previous in vitro study revealed that fluid shear stress induces endothelial cell (EC) differentiation by murine embryonic stem (ES) cells. In the present study we investigated whether shear stress regulates the arterial-venous specification of ES-cell-derived ECs.
Methods and Results—When murine ES cell–derived VEGFR2+ ES cells were exposed to shear stress, expression of the arterial EC marker protein ephrinB2 increased dose-dependently. The ephrinB2 mRNA levels also increased in response to shear stress, whereas the mRNA levels of the venous EC marker EphB4 decreased. Notch cleavage and translocation of the Notch intracellular domain (NICD) into the nucleus occurred as early as 30 minutes after the start of shear stress and increased with time. Gamma-Secretase inhibitors (DAPT and L685 458), and the recombinant extracellular domain of the Notch ligand DLL4 abolished the shear stress–induced NICD translocation, and that, in turn, blocked the shear stress–induced upregulation of ephrinB2 expression. In addition, the VEGF receptor kinase inhibitor SU1498 was found to suppress both the shear-stress-induced Notch cleavage and up-regulation of ephrinB2 expression.
Conclusion—Exposure to shear stress induces an increase in expression of ephrinB2 in murine ES cells via VEGF-Notch signaling pathways.
Key words: embryonic stem cells • arterio-venous differentiation • endothelial cells • shear stress • vascular biology