Regulation of angiogenic balance by thrombospondin-1.

血管生成的诱导剂促进内皮细胞(EC)的存活，而抑制剂则导致细胞凋亡。抑制剂 内皮细胞重塑中诱导物生成途径中的靶分子。在最初的提案中，我们展示了 凝血酶敏感蛋白-1(TSP1)和色素上皮衍生因子(PEDF)抑制剂增加EC CD95 配体(CD95L)，死亡调解人。诱导剂上调死亡受体CD95，CD95结合CD95L和 触发细胞凋亡，从而阻止血管生成。这就是EC如何通过血管生成来平衡细胞凋亡和生存 抑制物/刺激物。我们确定了促血管生成因子和抗血管生成因子的另一个共同靶点--核 活化T细胞因子(NFAT)。NFAT还作为分子支点平衡血管生成激活和 抑制力。启动子阵列分析确定了TSP1和TSP1引起的几种转录因子的活性变化 PEDF包括NFicB、cMyb和Egr-1。这三个都与NFAT形成了共同的网络。我们建议澄清 促血管生成和抗血管生成的NFAT交叉调节中的信号和转录事件 各种因素。我们将使用两种无关的抑制物，TSP1和PEDF，以及两种刺激物，血管内皮生长 因子和碱性成纤维细胞生长因子(血管内皮生长因子和碱性成纤维细胞生长因子)。我们将确定： TSP1和PEDF对NFAT失活的上游调节作用。我们将评估以下方面的贡献 JNKkinases，p38和GSK的体外功能测定 Western blotting。我们将分析抑制剂对NFAT近端激活物水平和活性的影响， 钙调神经磷酸酶A(CNA)及其调节剂、钙离子动员和DSCR-1。NFATc2和CNA为空的小鼠将 用来确认它们的职能作用。 TSP1和PEDF对NFAT靶点的调节我们将筛选已知的NFAT靶点Bcl2、Cyclins A和 Western和Northern印迹法检测E、c-flip、环氧合酶-2(COX-2)、白介素类和组织因子。 确认的靶点将通过EMSA和CHIP(染色质免疫沉淀)与NFATc2进行评估 用siRNA或中和抗体在体外血管生成试验中进行评估。 NFicB在TSP1和PEDF信号中的作用。NFicB的激活将通过免疫细胞化学和 紧急救援局。将使用生化抑制剂和/或结构性活性来证明功能重要性 Ikb(kB*)。在FasL调控中的作用将通过使用抑制剂和芯片来检测。基因敲除的老鼠将会是 用于在体内验证NFicB的贡献。 E2F1、Egr-1和c-Myb参与NFAT的作用和调节。Egr-1和c-Myb基因的变化 由TSP1或PEDF引起的活动将由EMSA确认。Egr和c-Myb为空的小鼠将用于 评价它们在TSP1或PEDF抗血管生成中的生物学作用。与NFAT的互动将是 使用EMSA、IP和ChLP进行了研究。越来越多的证据表明，激活的内皮细胞是 准备好接受抑制物诱导的细胞凋亡。 因此，我们将描绘抑制物和刺激物的共同分子靶点，并确定主要转录 它们相互作用所涉及的因素。这些研究将为治疗干预提供新的靶点。 天然的抑制剂。

Inducers of angiogenesis promote endothelial cell (EC) survival, and inhibitors cause apoptosis. Inhibitors target molecules in the inducer-generated pathways in remodeling EC. In the original proposal we showed that inhibitors Thrombospondin-1 (TSP1) and pigment epithelial-derived factor (PEDF) increase EC CD95 ligand (CD95L), a death mediator. Inducers upregulate death receptor, CD95, which binds CD95L and triggers apoptosis, thus blocking angiogenesis. This is how EC balance apoptosis and survival by angiogenic inhibitors/stimuli. We identified another common target of the pro- and anti-angiogenic factors, the nuclear factor of activated T-cells (NFAT). NFAT also acts as molecular pivot balancing angiogenesis activation and inhibition. Promoter array analysis identified activity changes of several transcription factors due to TSP1 and PEDF including NFicB, cMyb and Egr-1. All three form common network with NFAT. We propose to elucidate signaling and transcriptional events involved in the NFAT cross-regulation by the pro- and anti-angiogenic factors. We will use two non-related inhibitors, TSP1 and PEDF, and two stimuli, vascular endothelial growth factor and basic fibroblast growth factor (VEGF and bFGF). We will determine: The upstreammediatorsof NFAT deactivation by TSP1 and PEDF. We will evaluate the contribution of JNKkinases, p38 and GSK in vitro by functional assays with kinase inhibitors, immunoprecipitation and western blotting. We will analyze the effect of inhibitors on the levels and activity of NFAT proximal activator, Cacineurin A (CnA) and its modulators, Ca++ mobilization and DSCR-1. Mice null for NFATc2 and CnA will be used to confirm their functional role. The regulation of NFAT targets by TSP1 and PEDF. We will screen known NFAT targets Bcl-2, cyclins A and E, c-FLIP, cyclooxygenase-2 (Cox-2), interleukins and tissue factor by Western and Northern blotting. Confirmed targets will be evaluated by EMSA and ChIP (chromatin immunoprecipitation) with NFATc2 antibodies and assessed in the in vitro angiogenesis assays with si-RNA or neutralizing antibodies. NFicB role in the TSP1 and PEDF signaling. NFicB activation will be confirmed by immunocytochemistry and EMSA. Functional importance will be demonstrated using biochemical inhibitor and/or constitutive^ active IkB (kB*). Role in the FasL regulation will be examined using inhibitor and by ChIP. Knock-out mice will be used to verify in vivo NFicB contribution. The involvement of E2F1,Egr-1 and c-Myb in NFAT action and regulation. Changes in Egr-1 andc-Myb activity due to TSP1 or PEDF will be confirmed by EMSA. Mice null for Egr and c-Myb will be used to evaluate their biological role in the anti-angiogeniesis by TSP1 or PEDF. The interaction with NFAT will be studied using EMSA, IP, and ChlP.A growing body of evidence suggests that activated endothelium is poised for apoptosis induction by inhibitors. We will thus delineate molecular targets common for the inhibitors and stimuli and identify major transcription factors involved in their interaction. These studies will yield new targets for therapeutic intervention using natural inhibitors.