Arterial-Venous Plasticity in Adult Figure 5. Angiogenic neovessels exhibit reduced arterial and venous marker protein expression. A, Protein expression of EphrinB2 and COUP-TFII in mature microvessels and angiogenic neovessels in tMVCs as described for Fig. 3E. B, EphrinB2 phosphorylation from similar preparations as in A. * p,0.05, n = 3 by Student’s t-test. doi:10.1371/journal.pone.0027332.g005 Discussion We show here that vessel segments of arterial identity are capable of giving rise to the full spectrum of vessel types intrinsic to a functional microcirculation in neovascularization. The findings are consistent with the concept that microvessel AV identity in adult neovascularization is not necessarily pre-determined and that adult microvessels display a considerable level of phenotypic ada- ptation and plasticity. Whether capillaries or venules have similar plastic capabilities is not clear. However, given that sprouting occurs from capillaries and venules as well as arterioles in tMVCs (unpublished observation), it’s likely that non-arteriole types share this same phenotypic plasticity. The absence of pre-determination in adult neovascularization is in contrast to the embryo in which vessel identity, at least during the early phases of vascular for- mation, is pre-determined by a complex molecular program [26]. This may reflect the intrinsic de novo assembly of the embryonic vasculature, which is not prominent in adult neovascularization. A common clinical solution to peripheral revascularization is to use veins as artery grafts [27]. Interestingly, while these vein grafts adapt to the new vascular position (e.g. wall thickening [27]), they do not fully convert to an artery phenotype. For example, saph- enous vein grafts used in patients lost the EphB4 expression (i.e. lost their venous identity) but did not express EphrinB2 (i.e. exhibit an arterial molecular phenotype) [28], suggesting that mature adult vessels have limited plasticity. This in contrast to the findings of our present study in which there was complete identity conversion. This difference in plasticity may reflect an intrinsic plasticity character between large vessels, such as used in vein grafting, and microvessels (the focus of our study). However, given that the microvessels in the implanted construct underwent profound changes to the vessel wall structure intrinsic to angiogenesis and neovascularization [16] while the wall structure is essentially preserved in vein grafts, perhaps complete vessel plasticity in the adult is contingent upon extensive vessel wall restructuring and may only occur during neovascularization. The co-expression of both AV markers in neovessels in MVCs is consistent with a report that shows co-expression of EphrinB2 and EphB4 in tip and stalk cells in retinal sprouts [19] and suggests that changes in identity appear to result in a mixed AV identity. However, the reduced EphrinB2 expression and activation and the absence of morphologies characteristic of specific microvessel identities [16] in the implant system raises the possibility that a true ‘‘mixing’’ of arteriole and venule-specific characters is not really occurring. Instead, the angiogenic neovessel phenotype may reflect a more generic, ambiguous vessel phenotype, which gives way to specific microvessel types as neovascularization progresses. Moreover, growing evidence that vessel marker proteins have roles other than establishing vessel identity in vessel sprouting during vessel remodeling and angiogenesis, such as cell signaling modu- lation and directing cell tip filopodial extension [22,29,30], rein- forces this interpretation. Indeed, EphrinB2 can act as a pro- angiogenic factor in postnatal neovascularization, since it is capable of inducing neovascularization when delivered in a corneal micro- pocket assay [31]. Regardless, our findings indicate that without an additional assessment of vessel maturation, the use of EphrinB2 and EphB4 as vessel identity markers during active neovascular- ization may be problematic. PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27332