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Blood, Vol. 112, Issue 1, 73-81, July 1, 2008
Role of ephrinB2 expression in endothelial cells during arteriogenesis: impact on smooth muscle cell migration and monocyte recruitment
Blood Korff et al.
112: 73
Supplemental materials for: Korff et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 989 KB)
- Figure S1. Expression of ephrinB2 in stretch-stimulated endothelial cells (JPG, 83.6 KB)
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Different types of endothelial cells were exposed to cyclic stretch (15%, 0.5 Hz, 3 hours). (A) EphrinB2 protein abundance (top) as well as mRNA expression (bottom) analyses revealed that cyclic stretch up-regulates ephrinB2 in arterial (human aortic endothelial cells HAoEC) and venous (human saphenous vein endothelial cells HSaVEC) endothelial cells. β-Actin as well as the house keeping gene RPL32 were used as internal standards, respectively. (B) Similar results were observed upon prolonged exposure of HAoEC and HSaVEC to cyclic stretch (15%, 0.5 Hz, 18 hours; CD31 was used as an internal standard).
- Figure S2. The left figure shows a Western blot analysis of protein lysates collected from PAEC used in this study (JPG, 70 KB)
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Mock and EphB4-transtected PAEC do not express ephrinB2. EphrinB2 or ΔephrinB2-transfected cells over-express a full size and a truncated variant of ephrinB2, respectively. The latter lacks the intracellular domain and thus is not capable of transducing a reverse signal. EphB4 is only detected in the EphB4-transfected PAEC (right panel). The specific binding capacity of these cells was analyzed by adding dimeric soluble ephrinB2/Fc and EphB4/Fc (right figure). These proteins were then detected via their Fc-Tag using specific fluorescence-labeled antibodies. All ephrinB2 over-expressing but not mock-transfected PAEC bind the soluble receptor EphB4 (red staining). EphB4-transfected PAEC correspondingly bind the soluble ligand ephrinB2. For further details, see references 4 and 5.
- Figure S3. Expression of EphB receptors in monocytes (JPG, 108 KB)
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THP-1 cells and human monocytes express several types of EphB receptors which were not affected by MCP-1 stimulation (6 hours, 100 ng/ml). In contrast, ephrinB2 is expressed only in THP-1 cells and strongly increased upon MCP-1 stimulation.
- Figure S4. Immunfluorescence analysis of ephrinB2 in human placenta arteries and porcine aortic endothelial cell (PAEC) spheroids (JPG, 62.6 KB)
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EphrinB2 staining (red Cy3 fluorescence) can be detected at the luminal surface in endothelial cells (A and B, arrowheads; image B represents a detail enlargement of A; scale bars equal 20 µm). Confocal imaging of a longitudinally sectioned human placental artery indicated an apical and lateral localization of ephrinB2 (C-F; scale bars equal 20 µm). Due to the orientation of the blood vessel, the confocal plain focuses from left to right on the basal and then on the apical (i.e., luminal) compartment of the endothelial cell monolayer (illustrated in C). In the basal endothelial cell compartment only CD31 staining can be detected (D and F, left part of the artery) whereas ephrinB2 staining is localized in the apical compartment (E and F, right part of the artery). Cross sections of mock-transfected (G) or ephrinB2-transfected (H) PAEC spheroids were stained for ephrinB2. Strong apical ephrinB2 staining was detected in ephrinB2-PAEC spheroids (H, arrow; scale bar equals 50 µm) whereas only a weak signal was monitored in mock-PAEC spheroids (G).
REFERENCES
1. Füller T., Korff T., Kilian A., Dandekar G., Augustin H.G. Forward EphB4 signaling in endothelial cells controls cellular repulsion and segregation from ephrinB2 positive cells. J. Cell Sci. 2003;116:2461-2470.
2. Korff T., Dandekar G., Pfaff D., Füller T., Goettsch W., Morawietz H., Schaffner F., Augustin H.G. Endothelial ephrinB2 is controlled by microenvironmental determinants and associates context-dependently with CD31. Arterioscler Thromb Vasc Biol. 2006;26:468-474.
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