Arterial-Venous Plasticity in Adult Figure 4. Angiogenic neovessels express arterial and venous genetic markers. A–C, Confocal images (Z-projections) of angiogenic neovessels in tMVC implants during the sprouting angiogenic phase (week 1 post-implantation) obtained from (A) Efnb2- or (B) Ephb4-reporter mice. Microvessels were identified by labeling endothelial cells with TRITC-conjugated GS-I lectin (red). Ephb4-driven LacZ expression shown in panel B was detected with an anti-b-galactosidase-FITC antibody. C, Determination of the % fraction of angiogenic neovessels positive for either the Efnb2- or Ephb4-reporter in tMVCs prepared as in A and B. D, Co-expression of the Efnb2-reporter gene and EphB4 in individual angiogenic neovessels in 1 week tMVC implants prepared from Efnb2-reporter mice (green) and immunostained with an anti-EphB4 antibody (red). Scale bar in all panels = 100 mm. E, Real-time PCR measurement of mRNA expression of EphrinB2, EphB4, COUP-TFII, Delta-like4 (Dll4), Jag1 and Notch4 genes in freshly isolated, specified microvessels (mature) and angiogenic neovessels in tMVCs (angiogenic). * p,0.05, n = 3 by Student’s t-test. doi:10.1371/journal.pone.0027332.g004 identity occurring during sprouting angiogenesis appears to result in a mixed AV identity, the apparent reduction in these proteins suggests that the neovessels are losing specific vessel-type character during angiogenesis and acquiring a more generic, ambiguous vascular phenotype. Of course it is possible that, since these proteins serve different functions during vessel sprouting [22] other than establishing vessel identity, these molecules may not serve as effective identity markers in this setting. Interestingly, transcript levels for Dll4 and Notch4, molecules downstream of the VE- GFR2/NRP-1 signaling complex in the arterial specification pathway and therefore also possible markers of arterial identity [3,11,23], were not different in the angiogenic sprouts (Fig. 4E). However, these proteins are also important in establishing tip and stalk cell identity in the angiogenic sprout [24] and mediate sprout branching [25], roles that are not necessarily specific to marking AV identity. PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27332