Endothelial cell decisions integrate soluble and matrix-bound VEGF gradients

描述（由申请人提供）： 项目摘要：血管内皮生长因子（VEGF）家族由参与控制血管存活和血管生成出芽的关键细胞因子组成。最近，VEGF抑制剂已被FDA批准用于治疗血管过度形成疾病（癌症和黄斑变性），而增加VEGF表达是缺血性疾病如外周动脉疾病和冠状动脉疾病的主要研究途径，尽管临床试验尚未成功。VEGF作为病因或治疗靶点参与至少70种疾病。因此，VEGF信号传导的调节对于许多治疗策略至关重要。最近的证据表明，更多的VEGF结合到细胞外基质在体内比自由扩散，这些基质结合亚型的VEGF控制分支频率和网络密度在体内。通过基质分子将VEGF呈递给受体可以启动不同的信号传导模式，但是传统的体外细胞培养模型不能区分可溶性VEGF信号与基质结合的VEGF信号。在这个建议中，我们开发了一种结合实验计算的方法来分离可溶性和基质结合的VEGF信号。我们将开发一种新的纤连蛋白突变体，不结合VEGF（目标1），我们将使用国家的最先进的微图案化技术，以平板内皮细胞的基板上的梯度VEGF结合能力（目标2）。增殖、迁移和存活的体外测定将明确显示可溶性VEGF或基质结合VEGF是否是这些关键内皮细胞决定的更有效刺激物（目的2）。然后，我们建议使用体内转染技术来增加VEGF梯度，并在体内阻断基质结合的VEGF向受体酪氨酸激酶的呈递（目的3）。这将测试体外内皮细胞培养是否是体内内皮细胞决策的良好模型。 与公共卫生的相关性：血管内皮生长因子（VEGF）的抑制或上调是70多种疾病的治疗靶点，如癌症、冠状动脉疾病和糖尿病视网膜病变。这项研究使用了一种新的计算和实验相结合的方法来引出VEGF如何启动其信号，如果成功的话，将更好地定义如何在治疗上靶向它。

DESCRIPTION (provided by applicant): Project Summary: The vascular endothelial growth factor (VEGF) family consists of critical cytokines that are implicated in controlling vascular survival and angiogenic sprouting. Recently VEGF inhibitors have been approved by the FDA to treat diseases of hypervascularization (cancer and macular degeneration), while increasing VEGF expression is a major research avenue for ischemic diseases such as peripheral artery disease and coronary artery disease, though clinical trials are as yet unsuccessful. VEGF is involved in at least 70 diseases as either cause or therapeutic target. Regulation of VEGF signaling is therefore of critical importance for many therapeutic strategies. Recent evidence suggests that more VEGF is bound to the extra cellular matrix in vivo than freely diffuses, and that these matrix-binding isoforms of VEGF control branching frequency and network density in vivo. The presentation of VEGF to the receptors by a matrix molecule may initiate a distinct signaling mode, but traditional in vitro cell culture models are unable to differentiate the soluble VEGF signal from the matrix-bound VEGF signal. In this proposal, we develop a combined experimental-computational approach to separate the soluble and matrix-bound VEGF signals. We will develop a novel fibronectin mutant that does not bind VEGF (Aim 1) and we will use state-of-the-art micro patterning techniques to plate endothelial cells on a substrate with gradients of VEGF binding ability (Aim 2). In vitro assays of proliferation, migration and survival will definitively show whether soluble VEGF or matrix-bound VEGF is the more potent stimulator of these critical endothelial cell decisions (Aim 2). We then propose to use in vivo transfection techniques to increase VEGF gradients and to block presentation of matrix-bound VEGF to receptor tyrosine kinases in vivo (Aim 3). This will test whether the in vitro endothelial cell culture is a good model of the decision-making of endothelial cells in vivo. Relevance to Public Health: Inhibition or up regulation of vascular endothelial growth factor (VEGF) is a therapeutic target in over 70 diseases as diverse as cancer, coronary artery disease and diabetic retinopathy. The proposed research uses a novel combined computational and experimental method to elicit how VEGF initiates its signals and, if successful, will better define how to target it therapeutically.