Regulation of angiogenic balance by thrombospondin-1.

描述（由申请方提供）：血管生成诱导剂促进内皮细胞（EC）存活，抑制剂导致细胞凋亡。抑制剂靶向分子在诱导剂产生的途径重塑EC。在最初的提议中，我们表明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），一种死亡介质。诱导剂上调死亡受体CD 95，其结合CD 95 L并触发细胞凋亡，从而阻断血管生成。这就是EC如何通过血管生成抑制剂/刺激平衡凋亡和存活。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，即活化T细胞核因子（NFAT）。NFAT还充当平衡血管生成激活和抑制的分子枢纽。启动子阵列分析鉴定了由于TSP 1和PEDF引起的几种转录因子的活性变化，包括NFicB、cMyb和Egr-1。所有这三个组成共同的网络与NFAT。我们建议阐明信号和转录事件参与NFAT交叉调节的促和抗血管生成因子。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的NFAT失活的上游介质。我们将评估JNK激酶，p38和GSK在体外的激酶抑制剂，免疫沉淀和蛋白质印迹功能测定的贡献。我们将分析抑制剂对NFAT近端激活剂、钙调磷酸酶A（CnA）及其调节剂、Ca++动员和DSCR-1的水平和活性的影响。将使用NFATc 2和CnA无效的小鼠来确认其功能作用。TSP 1和PEDF对NFAT靶点的调节。我们将通过Western和北方印迹法筛选已知的NFAT靶点Bcl-2、细胞周期蛋白A和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）使用NFATc 2抗体评价确认的靶标，并在体外血管生成试验中使用si-RNA或中和抗体进行评估。NFicB在TSP 1和PEDF信号传导中的作用。将通过免疫细胞化学和EMSA确认NFicB活化。将使用生化抑制剂和/或组成型活性IkB（kB*）证明功能重要性。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。由TSP 1或PEDF引起的Egr-1和c-Myb活性变化将通过EMSA确认。将使用Egr和c-Myb缺失的小鼠来评价它们在TSP 1或PEDF的抗血管生成中的生物学作用。将使用EMSA、IP和ChIP研究与NFAT的相互作用。 越来越多的证据表明，激活的内皮细胞是由抑制剂诱导凋亡。因此，我们将描绘共同的抑制剂和刺激的分子靶点，并确定参与其相互作用的主要转录因子。这些研究将为使用天然抑制剂的治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): 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 upstream mediators of 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, Calcineurin 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 and c-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-angiogenesis 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.