This result suggests that the VEGF Trap is biologically active in the injured cord and that, surprisingly, endogenous VEGF alters EC plasticity in a pattern that appears to modulate the functional status of neovascular beds in and around the injury site, as luminal IB4 affinity appears to be related to the maturation state of newly formed blood vessels in the injured spinal cord (Benton (Melder em et al. /em , 1996) as well as induce disruption of the endothelial glycocalyx (Fu and Shen, 2003). tissue is regulated by multiple effectors and is not limited by endogenous VEGF activation of affected spinal microvessels. by interruption of malignant neovascularization and is currently in clinical trials for this application (Rudge isolectin B4 (FITC-IB4, L-9381; Sigma, St. Louis, MO) was delivered systemically by intravenous injection the right external jugular vein and allowed to circulate for 15 minutes. Mice were then transcardially perfused with 10 ml of saline followed by 15 ml of 4% paraformaldehyde (PFA). Spinal cords were dissected and longitudinally sectioned at 20 Nm on a cryostat, slide-mounted, and stored at ?80C until use. Immunohistochemical analyses and quantitative assessment of microvascular density-To determine vascular density in injury epicenters, vascular endothelial cells (ECs) were identified using a monoclonal rat anti-PECAM-1 antibody (#550274, 1: 50, BD Pharmingen, San Diego, CA). SCI epicenters were identified by quantification of extravascular laminin deposition using polyclonal rabbit anti-laminin (L9393, 1: 100, Sigma, St. Louis, MO)(Benton = ?5.61, df = 7, P 0.001) and penumbral zones (= ?5.94, df = 7, P 0.001) expressed luminal affinity for IB4 (Fig. 2B). This result suggests that the VEGF Trap is usually biologically active in the injured cord and that, surprisingly, endogenous VEGF alters EC plasticity in a pattern that appears to modulate the functional status of neovascular beds in and around the injury site, as luminal IB4 affinity appears to be related to the maturation state of newly formed blood vessels in the injured spinal cord (Benton (Melder em et al. /em , 1996) as well as induce disruption of the endothelial glycocalyx (Fu and Shen, 2003). Current results would most directly support this latter explanation. Fundamentally, present data spotlight the complexity of vascular regulation after SCI, a precedent for which exists in peripheral tissue pathology. Specifically, comparable micro-vascular resistance to VEGF-R1 and VEGF-R2 antagonism in pathologic angiogenesis is currently of intense investigation, especially in the context of tumor neovascularization (Shojaei and Ferrara, 2008; Bergers and Hanahan, 2008). To date, several explanations for this phenomenon have been proposed, which fall under the conceptual categories of adaptive/evasive resistance and intrinsic non-responsiveness (Bergers and Hanahan, 2008). Several of these possibilities are quite plausible in the context of SCI. For example, several option pro-angiogenic pathways are induced in solid tumors and appear to contribute to the circumvention of an absolute dependence on VEGF for neovascularization. These include fibroblast growth factor (Fgf) (Casanovas em et al. /em , 2005), interleukin 8 (IL8) (Mizukami em et al. /em , 2005), and platelet derived growth factor-alpha (PDGFA) (Fernando em et al. /em , 2008). Importantly, all of these pro-angiogenic cytokines are upregulated in injured/inflamed spinal tissue (Tripathi and McTigue, 2008; Sun em et al. /em , 2008; Ishizu em et al. /em , 2005). It is possible that a comparable redirection of the endogenous angiogenic response CHR-6494 may occur in SCI, with increased activation of these angiogenic pathways CHR-6494 in affected microvascular ECs. Unbiased transcriptional screening of ECs isolated from tumor microvasculature has identified a number of novel regulators of tumor neovascularization (St em et al. /em , 2000). Of those identified, Delta-like ligand 4 (DLL4) was robustly enriched in tumor ECs, suggesting a role for Notch signaling in vascular activation in solid tumors. Indeed, this pathway appears to be quite relevant to tumor neovascularization refractory to VEGF blockade (for review see Thurston and Kitajewski, 2008). Blockade of DLL4 reduces tumor size by disrupting microvascular function and had additive effects when combined with anti-VEGF therapy (Ridgway em et al. /em , 2006; Noguera-Troise em et al. /em , 2006). Interestingly, spinal microvascular ECs express detectable levels of DLL4 acutely following SCI (unpublished observations) suggesting NR4A2 a comparable co-stimulatory role for Notch in angiogenesis in the injured spinal cord. Currently, studies are underway to determine to what extent Notch activation regulates EC survival and/or plasticity following SCI. Finally, in many pathoangiogenic contexts it is likely that lack of complete efficacy of VEGF-R1 and CR2 blockade CHR-6494 may be due to incomplete suppression of VEGF signaling in activated ECs. This is most likely due to expression of neuropilin1 (NRP1) receptor,.