The down-regulation of EphrinB2 in angiogenic neovessels observed in the MVCs is in contrast with other reports describing an increase in EphrinB2 mRNA in vascular cells and neovessels associated with angiogenesis [14,31]. This difference likely reflects the degree of pro-angiogenic activity in the different experimental models. In the microvascular construct, angiogenic sprouting and neovascularization occurs spontaneously in a fairly rarefied en- vironment (collagen I gels and no added factors), as opposed to cultured endothelial cells in MatrigelTM (a complex, pro- angiogenic matrix) treated with angiogenic factors[31] or ischemic tissues in which cytokines and other growth factors are significantly elevated as in these other studies [14,31]. Regardless, we show that angiogenic neovessels, either in culture or implanted, do express EphrinB2 and EphB4, albeit at reduced levels. If the presence of EphrinB2 and EphB4 (as well as other identity markers) reflects some level of specification, then it may be that the vascular system in the implanted microvascular constructs is more plastic than vascular systems in native tissues and that the tissue microenvi- ronment contributes significantly to developing microvascular network character. This is relevant for neovascularization-based regenerative therapies, such as with the MVCs [15], where adap- tation to the new tissue environment is important for the gener- ation of a functional vascular bed. VEGF-induced cell signaling has been implicated in AV spe- cification during embryo development [32], suggesting that VEGF could play a role in AV specification in adult neovascularization. However, we have previously shown that VEGF is not responsible for angiogenic sprouting in the MVCs [16,33], suggesting a dif- ferent mechanism for the regulation of EphrinB2 and EphB4 expression and AV identity specification in our implant system. In the MVCs, the re-appearance of distinct vessel types (defined by morphologies and marker expression) coincides with the appear- ance of blood flow and perivascular cell recruitment[16]. Elevated shear stress induces arterial differentiation and mediates micro- vascular remodeling [34,35,36] suggesting that it may be a key stimulus. However, pulsatility and not shear stress amplitude may be more important in initiating arterial-venous specification in remodeling microvasculatures [37]. Certainly the pivotal role of perivascular cells and Ephrin-mediated mural cell activity in vessel maturation may also determine specific vessel phenotype [20,24]. Perivascular cells are essential for the formation of functional blood vessels [38] by contributing to vessel stabilization and maturation [39,40,41]. Indeed, the association of perivascular cells to the neovessel may mark the end of vessel immaturity and initiate structural adaptation[42]. Direct endothelial-perivascular cell-cell interactions are regulated by Ephrin/Eph signaling [30] and conditional deletion of the EphrinB2 gene in perivascular cells results in fragile and dysfunctional microvessels [43]. Finally, Eph- rinB2 expression in adult perivascular cells is restricted to the arterial compartment of the vasculature [13,14] suggesting a possi- ble role for perivascular cells in establishing an arterial phenotype. Therefore, AV specification in adult neovascularization may be driven by hemodynamic forces and perivascular cell activity, associated with the specific needs of the tissue, rather than a pre- determined program. In our experiments, sprouting angiogenesis occurred from ar- terioles. It is generally considered that angiogenesis arises pri- marily from capillaries and post-capillary venules in vivo, par- ticularly in tumors [44]. Whether or not angiogenesis occurs from arterioles in native tissues, as suggested by our findings, is not known since no systematic study has been performed. It is possible that the isolation procedure whereby individual microvessels are fragmented exposing cells of the microvessel wall to the matrix environment stimulates angiogenesis and thus may be unique to Arterial-Venous Plasticity in Adult our system. However, similar such severing or breaking of micro- vessels, of all types, may occur in situations of wounding and injury. Regardless, our findings indicate that, as with capillaries and venules, arterioles are capable of undergoing sprouting angiogenesis. Methods All animal experiments were performed in compliance with institutional guidelines, conform with US National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by University of Louisville Institutional Animal Care and Use Committee procedures and policies (IACUC #10105 and #10106 protocols). Transgenic mouse and rat strains Efnb2-reporter mouse. B6;129S4-Efnb2tm2Sor/J mice contain a histone H2B-GFP transgene inserted within the arterial identity [14] Efnb2 gene [18]. Ephb4-reporter mouse. B6;129S7-Ephb4tm1And/J mice contain the Tau-LacZ transgene within the Ephb4 gene and under control of the Ephb4 promoter[6] and used to locate microvessels of venous identity[7]. In both cases, homozygous mice die before birth. However, heterozygous animals develop normally and were used as reporter for the specific gene activity. Offspring from het x het breedings were genotyped using standard PCR techniques according to Jackson Laboratories instructions. EphrinB2-reporter primers: 59 - 39 AAG TTC ATC TGG ACC ACC G and TCC TTG AAG AAG ATG GTG CG. EphB4- reporter primers: 59 - 39 ATC GTT GAG AGG CCC TCG AC (common), GTG CTA TTG GTC CGA AGT GTT (wild type) and CTG AGC ATG ATC TTC CAT CAC (mutant)2. GFP transgenic rat. SD-Tg(GFP)2BalRrrc rats contain an EGFP transgene driven by the Ubc promoter integrated into a single locus on chromosome 14 [45]. Except where indicated, these rats served as the source of isolated microvessel segments. Isolation of brain microvessel fragments of arterial identity (aMF) To isolate aMF, magnetic beads (11.3 mm mean diameter, ranging from 10 to 13.9 mm; SPHEROTM Carboxyl Magnetic particles, CM-100-10, Spherotech, Lake Forest, IL) were delivered to the brain vasculature via a PE 50 catheter introduced into the left common carotid. Deep anesthesia was achieved by subcuta- neous injection of a mixture of Ketamine (40 mg/Kg), Xylasine (10 mg/Kg) and Acepromazine (2 mg/Kg). Intraoperative mon- itoring of adequate anesthesia is done by toe pinchs. The animal (mouse or rat) was placed supine and the fur over the lower jaw down to the clavicle was removed with a depilator. Following a midline incision, the left common carotid was exposed from the internal/external branch point down to the proximal-most po- sition in the neck. The proximal end was ligated, the distal end was temporarily ligated, and the catheter introduced towards the brain through a small incision in the common carotid wall. Bead suspensions (75 mg/4 ml and 19 mg/1 ml in heparinized saline for rat and mouse, respectively) were manually pushed through the catheter using a syringe. Prior to use, beads were incubated overnight with rocking in 1% BSA/PBS (Sigma). In the rat, the vasculature was flushed with 2 ml of heparinized saline before delivery of the beads. No such flush was done with the mouse vasculature. Following the delivery of beads, the brain cortex was harvested, minced, digested with collagenase (2 mg/ml, Worthing- ton) and pelleted as previously described6. The digestate pellet was suspended in 15% Dextran 100,000 to 200,000 MW (Sigma, St. Louis, MO, USA) and centrifuged at 4000 xg at 4uC for 20 min. PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27332