Regulation of angiogenic balance by thrombospondin-1.

血管生成的诱导剂促进内皮细胞（EC）存活，而抑制剂引起细胞凋亡。抑制剂 靶分子在诱导剂产生的途径重塑EC。在最初的提案中， 抑制剂血小板反应蛋白-1（TSP 1）和色素上皮衍生因子（PEDF）增加EC CD 95 配体（CD 95 L），死亡介质。诱导剂上调死亡受体CD 95，其结合CD 95 L， 引发细胞凋亡从而阻断血管生成这就是EC如何通过血管生成平衡凋亡和存活 抑制剂/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， 活化T细胞因子（NFAT）。NFAT还充当平衡血管生成激活和 抑制作用启动子阵列分析确定了由于TSP 1和TSP 2引起的几种转录因子的活性变化。 PEDF包括NFicB、cMyb和Egr-1。所有这三个组成共同的网络与NFAT。我们建议澄清 参与促血管生成和抗血管生成的NFAT交叉调节的信号传导和转录事件 因素我们将使用两种非相关的抑制剂，TSP 1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（VEGF和bFGF）。我们将确定： TSP 1和PEDF是NFAT失活的上游介质。我们将评估 JNK激酶、p38和GSK在体外通过激酶抑制剂、免疫沉淀和 蛋白质印迹法。我们将分析抑制剂对NFAT近端激活剂的水平和活性的影响， 钙调磷酸酶A（CnA）及其调节剂，Ca++动员和DSCR-1。NFATc 2和CnA无效的小鼠将 用于确认其功能作用。 TSP 1和PEDF对NFAT靶点的调节。我们将筛选已知的NFAT靶点Bcl-2、细胞周期蛋白A和 Western和北方印迹法检测E、c-FLIP、环氧合酶-2（考克斯-2）、白细胞介素和组织因子。 将通过EMSA和ChIP（染色质免疫沉淀）与NFATc 2评价确认的靶标 抗体，并用si-RNA或中和抗体在体外血管生成测定中评估。 NFicB在TSP 1和PEDF信号传导中的作用。NFicB激活将通过免疫细胞化学确认， EMSA功能重要性将使用生化抑制剂和/或组成型活性物质来证明。 IkB（kB*）。将使用抑制剂和ChIP检查FasL调节中的作用。敲除小鼠将 用于验证体内NFicB贡献。 E2 F1、Egr-1和c-Myb参与NFAT的作用和调节。Egr-1和c-Myb的变化 由TSP 1或PEDF引起的活性将由EMSA确认。Egr和c-Myb无效的小鼠将用于 通过TSP 1或PEDF评价其在抗血管生成中的生物学作用。与NFAT的互动将是 使用EMSA、IP和ChIP进行了研究。越来越多的证据表明， 准备好被抑制剂诱导凋亡。 因此，我们将描绘共同的抑制剂和刺激分子的目标，并确定主要的转录 影响其相互作用的因素。这些研究将产生新的治疗干预目标， 天然抑制剂

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.