Shear Stress Increases Expression of the Arterial Endothelial Marker EphrinB2 in Murine ES Cells via the VEGF-Notch Signaling Pathways

Tomomi Masumura, Kimiko Yamamoto, Nobutaka Shimizu, Syotaro Obi, Joji Ando

Arterioscler Thromb Vasc Biol, doi: 10.1161/ATVBAHA.109.193185

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. (Arterioscler Thromb Vasc Biol. 2009;29:00-00.)

C-reactive protein exerts angiogenic effects on vascular endothelial cells and modulates associated signalling pathways and gene expression.

Turu MM, Slevin M, Matou S, West D, Rodriguez C, Luque A, Grau-Olivares M, Badimon L, Martinez-Gonzalez J, Krupinski J.

BMC Cell Biol. 2008 Sep 2;9(1):47.

ABSTRACT:
BACKGROUND: Formation of haemorrhagic neovessels in the intima of developing atherosclerotic plaques is thought to significantly contribute to plaque instability resulting in thrombosis. C-reactive protein (CRP) is an acute phase reactant whose expression in the vascular wall, in particular, in reactive plaque regions, and circulating levels increase in patients at high risk of cardiovascular events. Although CRP is known to induce a pro-inflammatory phenotype in endothelial cells (EC) a direct role on modulation of angiogenesis has not been established.
RESULTS: Here, we show that CRP is a powerful inducer of angiogenesis in bovine aortic EC (BAEC) and human coronary artery EC (HCAEC). CRP, at concentrations corresponding to moderate/high risk (1-5 microg/ml), induced a significant increase in proliferation, migration and tube-like structure formation in vitro and stimulated blood vessel formation in the chick chorioallantoic membrane assay (CAM). CRP treated with detoxi-gel columns retained such effects. Western blotting showed that CRP increased activation of early response kinase-1/2 (ERK1/2), a key protein involved in EC mitogenesis. Furthermore, using TaqMan Low-density Arrays we identified key pro-angiogenic genes induced by CRP among them were vascular endothelial cell growth factor receptor-2 (VEGFR2/KDR), platelet-derived growth factor (PDGF, notch family transcription factors (Notch1 and Notch3), cysteine-rich angiogenic inducer 61 (CYR61/CCN1) and inhibitor of DNA binding/differentiation-1 (ID1).
CONCLUSION: This data suggests a role for CRP in direct stimulation of angiogenesis and therefore may be a mediator of neovessel formation in the intima of vulnerable plaques.

TNF primes endothelial cells for angiogenic sprouting by inducing a tip cell phenotype.

Sainson RC, Johnston DA, Chu HC, Holderfield MT, Nakatsu MN, Crampton SP, Davis J, Conn E, Hughes CC.

Blood, 15 May 2008, Vol. 111, No. 10, pp. 4997-5007.

Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2- to 3-day pulse stimulates angiogenesis by inducing an endothelial “tip cell” phenotype. TNF induces the known tip cell genes platelet-derived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFkappaB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) of tip cells sprouting in vitro. Thus, in angiogenesis, the temporal expression of TNF is critical: it delays angiogenesis initially by blocking signaling through VEGFR2, but in addition by inducing a tip cell phenotype through an NFkappaB-dependent pathway, it concomitantly primes endothelial cells (ECs) for sprouting once the initial inflammatory wave has passed.

Haemodynamics determined by a genetic programme govern asymmetric development of the aortic arch

Kenta Yashiro, Hidetaka Shiratori & Hiroshi Hamada

Nature Vol 450 | 8 November 2007 | doi:10.1038/nature06254

Laterality of the internal organs of vertebrates is determined by asymmetric Nodal signalling in the lateral plate mesoderm. A deficiency of such signalling results in heterotaxia syndrome, characterized by anomalous laterality of visceral organs and complex congenital heart conditions1. Pitx2, the transcription factor induced by the Nodal signal, regulates left–right asymmetric morphogenesis. The cellular and molecular bases of asymmetric morphogenesis remain largely unknown, however. Here we show that ablation of unilateral Pitx2 expression in mice impairs asymmetric remodelling of the branchial arch artery (BAA) system, resulting in randomized laterality of the aortic arch. Pitx2-positive cells were found not to contribute to asymmetrically remodelled arteries. Instead, Pitx2 functions in the secondary heart field and induces a dynamic morphological change in the outflow tract of the heart, which results in the provision of an asymmetric blood supply to the sixth BAA. This uneven distribution of blood flow results in differential signalling by both the platelet-derived growth factor receptor and vascular endothelial growth factor receptor 2. The consequent stabilization of the left sixth BAA and regression of its right counterpart underlie left-sided formation of the aortic arch. Our results therefore indicate that haemodynamics, generated by a Pitx2-induced morphological change in the outflow tract, is responsible for the asymmetric remodelling of the great arteries.