Endothelial cell decisions integrate soluble and matrix-bound VEGF gradients

项目摘要(请参阅说明)： 血管内皮生长因子(VEGF)家族是一类重要的细胞因子，参与控制血管存活和新生血管的生长。最近，血管内皮生长因子抑制剂已被FDA批准用于治疗血管增多的疾病(癌症和黄斑变性)，而增加血管内皮生长因子的表达是治疗周围动脉疾病和冠状动脉疾病等缺血性疾病的主要研究途径，尽管临床试验如下 然而，这并不成功。作为病因或治疗靶点，至少有70种疾病涉及到血管内皮生长因子。因此，对血管内皮生长因子信号的调控对于许多治疗策略至关重要。最近的证据表明，体内更多的血管内皮生长因子结合在细胞外基质上，而不是自由扩散，这些基质结合的血管内皮生长因子亚型控制着体内的分支频率和网络密度。通过基质分子将血管内皮生长因子呈递给受体可能 启动一种不同的信号传导模式，但传统的体外细胞培养模型不能区分可溶性的 血管内皮生长因子信号来源于基质结合的血管内皮生长因子信号。在这个方案中，我们开发了一个组合的实验计算 分离可溶性和基质结合的血管内皮生长因子信号的方法。使用新的或可用的不结合血管内皮生长因子的纤维连接蛋白突变体(目标1)，我们将使用最先进的微模板技术在具有血管内皮生长因子结合能力梯度的底物上电镀内皮细胞(目标2)。体外的增殖、迁移和存活分析将明确表明，在这些关键的内皮细胞决策中，可溶性血管内皮生长因子还是基质结合的血管内皮生长因子是更有效的刺激因子(目标2)。然后，我们建议使用体内转基因技术来增加血管内皮生长因子 梯度和阻断体内基质结合的血管内皮生长因子对受体酪氨酸激酶的呈递(目标3)。这将检验体外培养内皮细胞是否是体内内皮细胞决策的良好模型。

PROJECT SUMMARY (See Instmctions): 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 extracellular 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 experimentalcomputational approach to separate the soluble and matrix-bound VEGF signals. Using novel or available fibronectin mutants that do not bind VEGF (Aim 1), we will use state-of-the-art micropatteming 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.