Accelerated Reendothelialization, Increased Neovascularization and Erythrocyte Extravasation after Arterial Injury in BAMBI 2/2 Mice Nicolas Guillot 1 , Dmitrij Kollins 1 , Juan J. Badimon 2 , Detlef Schlondorff 1 * . , Randolph Hutter 2. 1 Department of Medicine, Mount Sinai School of Medicine, New York, New York, United States of America, 2 Cardiovascular Institute, Mount Sinai School of Medicine, New York, New York, United States of America Abstract Background: Intimal injury rapidly activates TGFb and enhances vascular repair by the growth of endothelial (EC) and vascular smooth muscle cells (VSMC). The response to the TGFb family of growth factors can be modified by BAMBI (BMP, Activin, Membrane Bound Inhibitor) acting as a non-signaling, competitive antagonist of TGFb type I receptors such as ALK 1 and 5. In vivo the effect of BAMBI will depend on its cell-specific expression and of that of the ALK type receptors. We recently reported EC restricted BAMBI expression and genetic elimination of BAMBI resulting in an in vitro and in vivo phenotype characterized by endothelial activation and proliferation involving alternative pathway activation by TGFb through ALK 1. Methodology/Principal Findings: To test the hypothesis that BAMBI modulates arterial response to injury via its effects on endothelial repair and arterial wall neovascularization we used a model of femoral arterial denudation injury in wild type (WT) and BAMBI 2/2 mice. Arterial response was evaluated at 2 and 4 weeks after luminal endothelial denudation of femoral arteries. The BAMBI 2/2 genotype mice showed accelerated luminal endothelial repair at 2 weeks and a highly unusual increase in arterial wall neovascularization compared to WT mice. The exuberant intimal and medial neovessel formation with BAMBI 2/2 genotype was also associated with significant red blood cell extravasation. The bleeding into the neointima at 2 weeks transiently increased it’s area in the BAMBI 2/2 genotype despite the faster luminal endothelial repair in this group. Vascular smooth muscle cells were decreased at 2 weeks in BAMBI 2/2 mice, but comparable to wild type at 4 weeks. Conclusions/Significance: The absence of BAMBI results in a highly unusual surge in arterial wall neovascularization that surprisingly mimiks features of intra-plaque hemorrhage of advanced atheroma in a mechanical injury model. This suggests important effects of BAMBI on arterial EC homeostasis that need to be further studied in a model of inflammatory atherosclerosis. Citation: Guillot N, Kollins D, Badimon JJ, Schlondorff D, Hutter R (2013) Accelerated Reendothelialization, Increased Neovascularization and Erythrocyte Extravasation after Arterial Injury in BAMBI 2/2 Mice. PLoS ONE 8(3): e58550. doi:10.1371/journal.pone.0058550 Editor: Ryuichi Morishita, Osaka University Graduate School of Medicine, Japan Received July 25, 2012; Accepted February 6, 2013; Published March 1, 2013 Copyright: ß 2013 Guillot 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 the National Institute of Diabetes and Digestive and Kidney Diseases R01-DK081420-01 and RO1-DK081420-02S1 to DS. 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: detlef.schlondorff@mssm.edu . These authors contributed equally to this work. Introduction Arterial injury induces a rapid activation of Transforming Growth Factor b (TGFb) signaling in the arterial wall. TGFb signaling regulates vascular repair via the modulation of endothe- lial cell (EC) and vascular smooth muscle cell (VSMC) migration and proliferation [1]. TGFb overexpression has been shown to enhance neointimal hyperplasia (IH), whereas inhibiting TGFb can attenuate neointimal growth [2,3]. In EC TGFb modulation takes place predominantly through ALK-1 and non-canonical pathways to promote proliferation [4], while in VSMC, TGFb acts likely via ALK-5 signaling [4–6]. The novel molecule BAMBI (BMP, Activin, Membrane Bound Inhibitor) in turn modifies the cellular response to the TGFb family of growth factors by acting as a non-signaling, competitive antagonist of TGFb type I receptors such as ALK 1 and 5 [7,8]. The specific effect of BAMBI on TGFb signaling is dependent on the combination of TGFb receptor and BAMBI expression on cells. EC express specifically ALK 1 and we recently reported EC restricted BAMBI expression [9]. Consistent with the coexpression of ALK 1 and BAMBI in endothelial cells, genetic elimination of BAMBI resulted in a phenotype characterized by in vitro and in vivo endothelial activa- tion and proliferation [9]. We also showed that in EC this phenotype could be mechanistically attributed to enhanced alternative TGFb signaling through ALK 1 and ERK2 and SMAD 1/5 activation after BAMBI elimination [9]. To explore the effect of BAMBI on arterial response to injury we performed femoral arterial (FA) endothelial denudating injury [10,11] in wild- type (WT) and BAMBI 2/2 mice. The effects of BAMBI on re- endothelialization, arterial wall neovascularization and neointima PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e58550