Arterial-Venous Plasticity in Adult Figure 3. Mature microvessels derived from arterial segments express arterial and venous markers. A–C, Representative confocal images (Z-projections) of vessel networks in MVCs derived from arterial-isolates obtained from Efnb2-reporter mice implanted for 6 weeks expressing the arterial reporter (Efnb2 in green) and filled with a blood tracer (perfusion in red). Scale bar = 100 mm. D, The % of perfused microvessels (red) positive for the Efnb2-reporter (green). * p,0.05 by Student’s t-test. E and F, Histology sections of 6 week-aMVC implants derived from an Ephb4- reporter transgenic mouse. Explants were sectioned and incubated with the b-galactosidase substrate X-gal to produce a blue precipitate as a marker of Ephb4 expression (arrows in E, scale bar = 50 mm.) or no precipitate indicating an Ephb4-reporter negative vessel (arrows in F, scale bar = 25 mm). doi:10.1371/journal.pone.0027332.g003 Indeed, microvessels in 6 week implanted aMVCs assembled using microvessels isolated from the Ephb4-reporter mouse confirmed the presence of venous-identity vessels (Fig. 3E, F). Because the perfused vessels originated from donor vessels (and consistent with our previous findings [16]), we can rule out the possibility that the EphrinB2- negative, EphB4-positive vessels present in the MVCs originated from the host vasculature. Additionally, while it’s pos- sible that the non-arteriole contaminants in the aMVCs gave rise to the non-arteriole vessels in the resulting mature microvascular tree, the very small fraction of these contaminants (,3%) makes this seem unlikely. Therefore, these findings indicate that parent microvessels of a single identity were capable of deriving a complete microvasculature comprised of all microvessel types. From this, we conclude that vessel identity in new microvascular networks generated during adult neovascularization is not pre- determined. Changes in vessel identity coincides with sprouting angiogenesis The development of venules (and capillaries) from arterial mi- crovessels indicates that a change in vessel identity occurred during the neovascularization process. Because sprouting neovessels in the implants lose the morphological features of their respective parent microvessel fragments (i.e. display uniform, narrow diameters and lack perivascular cell coverage [16]), we hypothesized that identity changes were initiated during sprouting angiogenesis. Therefore, we assessed vessel identity in tMVCs in the sprouting angiogenic phase through the use of reporter mice-isolated vessels. An advantage of using tMVC is that it allows us to see changes in vessel identity in all vessels types, not only in arterioles. Indeed, nearly 100% of the angiogenic neovessels in implanted tMVCs were positive for Efnb2- or Ephb4-reporter expression (Fig. 4A–C) using both brain- (not shown) and adipose-derived isolates sug- gesting that regardless of vessel type, vessel sprouts undergo vessel identity changes during sprouting angiogenesis. Co-expression of the reporters in single, angiogenic neovessels was corroborated by specific immunostaining of Efnb2-reporter neovessels for EphB4 (Fig. 4D). This is consistent with a previous study in which EphrinB2 and EphB4 were reported to be co-expressed in retinal endothelial sprouts [19] and indicates that neovessels generated during sprouting angiogenesis do not retain the identity distinction of the parent microvessels and may even display a mixed AV identity. While the reporter activity in the transgenic mice identifies active Efnb2 and Ephb4 promoters, it does not reflect actual levels of these proteins in vascular cells. Given that the angiogenic neovessels in the MVC have few morphological characteristics of either an arteriole or venule [16] yet have active vessel-type gene promoter activity (i.e. the reporters), we questioned whether or not endogenous identity marker proteins are actually present in the vascular cells. Western blots of lysates prepared from fresh isolates of mature, specified microvessels and angiogenic neovessels from tMVCs showed that while the reporter experiments indicate that nearly all of the neovessels of the implanted MVC have an active Efnb2 promoter (reporter expression and Fig. 4E), total EphrinB2 protein expression was significantly reduced (Fig. 5A). Further- more, the phosphorylation status of EphrinB2, thought important in activation and the positive regulation of endothelial cell and perivascular cell interactions [20], was significantly lower in an- giogenic neovessels than in mature, specified vessels (Fig. 5B). A similar downward trend was also observed with EphB4 transcript, COUP-TFII (another venous marker [21]) protein and transcript (Figs. 5 and 4E), and Jag1 transcript (Fig. 4E). Therefore, while, the continued activity of both arterial and venous marker gene promoters in the sprouting neovessels suggests that the change in PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27332