Vessel Arterial-Venous Plasticity in Adult Neovascularization Sara S. Nunes*, Harish Rekapally, Carlos C. Chang, James B. Hoying Cardiovascular Innovation Institute, University of Louisville and Jewish Hospital/St. Mary’s Health Care, Louisville, Kentucky, United States of America Abstract Objective: Proper arterial and venous specification is a hallmark of functional vascular networks. While arterial-venous identity is genetically pre-determined during embryo development, it is unknown whether an analogous pre-specification occurs in adult neovascularization. Our goal is to determine whether vessel arterial-venous specification in adult neovascularization is pre-determined by the identity of the originating vessels. Methods and Results: We assessed identity specification during neovascularization by implanting isolated microvessels of arterial identity from both mice and rats and assessing the identity outcomes of the resulting, newly formed vasculature. These microvessels of arterial identity spontaneously formed a stereotypical, perfused microcirculation comprised of the full complement of microvessel types intrinsic to a mature microvasculature. Changes in microvessel identity occurred during sprouting angiogenesis, with neovessels displaying an ambiguous arterial-venous phenotype associated with reduced EphrinB2 phosphorylation. Conclusions: Our findings indicate that microvessel arterial-venous identity in adult neovascularization is not necessarily pre-determined and that adult microvessels display a considerable level of phenotypic plasticity during neovascularization. In addition, we show that vessels of arterial identity also hold the potential to undergo sprouting angiogenesis. Citation: Nunes SS, Rekapally H, Chang CC, Hoying JB (2011) Vessel Arterial-Venous Plasticity in Adult Neovascularization. PLoS ONE 6(11): e27332. doi:10.1371/ journal.pone.0027332 Editor: Aernout Luttun, Katholieke Universiteit Leuven, Belgium Received August 8, 2011; Accepted October 14, 2011; Published November 14, 2011 Copyright: 2011 Nunes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health [http://www.nih.gov grant number EB007556] to JBH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sara.vasconcelos@utoronto.ca Introduction The formation of a new, functional post-natal vascular bed by neovascularization depends on complex vascular adaptation pro- cesses. Typically, immature neovessels formed during sprouting angiogenesis must undergo vascular wall maturation and assemble into an effective perfusion circuit comprised of in-flow, exchange, and out-flow vascular pathways. Important in this process is the structural adaptation of the angiogenesis-derived neovessels, often devoid of perivascular coverage, into vessels with sufficient st- ructural integrity and functional activity to accommodate and regulate blood flows and pressures [1,2]. Just as important as and, perhaps, integral to this structural adaptation is the specification of arterial and venous identity [3]. In the absence of proper arterial- venous (AV) specification, vascular networks are improperly formed and dysfunctional [4]. It is thought that in the embryo AV differentiation is genetically pre-determined based on studies demonstrating that AV-specific proteins such as EphrinB2 and its receptor EphB4 are expressed in arterial and venous cells, respectively, in angioblasts prior to the onset of blood flow and heart beat [5,6,7]. In the embryo, in- duction of AV specific markers expression occurs when VEGF signals via VEGF receptor-2/Neuropilin-1 complex in arterial- fated angioblasts leading to the downstream activation of Notch and ERK signaling pathways and the subsequent expression of the arterial marker EphrinB2. Expression of Notch signaling pathway molecules in veins is suppressed by the transcription factor COUP- TFII, preventing Notch signaling and inducing the expression of the venous marker EphB4 [3,8,9,10,11]. While embryonic AV specification is, in part, pre-determined, environmental cues are also important. Studies in avian embryos have shown that in early stages of development, endothelial cells of either arterial or venous origin could colonize and acquire characteristics of both arteries and veins [12]. Moreover, the expression of arterial- and venous-specific genes changed to reflect the specific type of vessel used as the engraftment site. Interestingly, this plasticity (i.e. ability to change AV identity) was progressively lost at later stages of development [12], suggesting that the more committed/differentiated the cells are the less likely they are to change phenotype. Compartmentalized expression of the AV markers EphrinB2 and EphB4 in mature vessels continues into adulthood [13,14], raising the possibility that the parent vessel from which neovessels are originated may influence the identity of the newly forming microvessels. Whether vessel identity during neovascularization in the adult is pre-determined, analogous to that in the embryo, is not known. This is relevant to neo- vascularization therapies in which a complete vascular bed containing arterioles, capillaries and venules must be generated to form a competent vasculature and is particularly important in regenerative therapies where microvessel fragments of different AV identities have been successfully used to treat myocardial infarction [15]. PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27332