To address whether adult microvessel AV identity is defined by the originating vessel identity or if adult vessels hold the potential to change identity, we used an implant system comprised of defined types of isolated microvessel segments to evaluate the ability of microvessels of a single identity to form a normal microcirculation following neovascu- larization. We have previously shown that the use of all three general types of microvessels (arterioles, capillaries, and venules) in this implant system results in the formation of a complete, functional microvascu- lature, derived from the isolate, after implantation. This microvascular bed is formed through a regimented process where microvessel fragments undergo angiogenesis, post-angiogenesis neovascular re- modeling and network maturation [16,17]. We hypothesized that if new vessel identity is pre-determined by the parent microvessel identity, then the formation of a proper microvascular network should be compromised in the implants if solely one microvessel type (e.g. arterioles) was used. Conversely, the development of a typical, hierarchical vascular tree from this single specified microvessel source would indicate that pre-determination either does not occur or is over- ridden during the post-angiogenesis adaptation. Results Isolation of arteriole segments with magnetic beads yields a functional, highly enriched arterial fraction To enrich isolates for segments of arterial identity, the rodent brain vasculature was perfused with magnetic beads (average Arterial-Venous Plasticity in Adult 11 mm OD) prior to microvessel segment isolation. Due to their size and the delivery method (via the left common carotid artery), the beads lodge in the arterial side of the vasculature (Fig. 1A) allowing for arterioles to be separated from venules and capillaries using a magnet following normal vessel isolation (Fig. 1B). Ar- terial purity was determined by performing the isolation from mice containing a Histone H2B-GFP transgene under control of the indigenous Efnb2 promoter [18]. This allowed us to clearly distinguish vessels of arterial identity (GFP-positive) from other vessels (GFP-negative). With this modified approach, the isolation was significantly enriched for artery-side microvessels (97.161% arterioles versus 66.363% in the total isolate containing all micro- vessel types) (Fig. 1C). Magnetic beads remain in the isolated arterioles; however, they do not impair the angiogenic potential of the isolated microvessels (Fig. 1D, E). Segments of arterial identity give rise to a competent, hierarchical microcirculation To assess vessel identity specification in the adult, we cha- racterized the vessel types formed in an implanted microvascular construct (MVC) in which greater than 97% of the microvessels are arterioles (Fig. 2). As a comparison to these arteriole-only microvascular constructs (aMVC), we prepared MVCs derived from a total isolate (tMVCs) in which all microvessel types (roughly 2/3 arterioles and 1/3 capillaries/venules) were present. After 6 weeks of implantation, both aMVCs and tMVCs contained newly Figure 1. Isolation of arteriole segments with magnetic beads yields a functional, highly enriched arterial fraction. A and B, Isolated brain microvessel segments preloaded with magnetic beads prior to (A) and following (B) extraction with a magnet. Scale bar = 200 mm. C, Repeated use of a magnet to pull out bead-loaded microvessels highly enriches the isolate for segments expressing the Efnb2-GFP reporter. (n = 3), *p,0.05 by Student’s t-test. Phase (D) and fluorescence (E) images of cultured, bead-loaded microvessel segments with sprouting neovessels (arrows). Scale bar = 100 mm. doi:10.1371/journal.pone.0027332.g001 PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27332