Regulation of angiogenic balance by thrombospondin-1.

描述（申请人提供）：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。抑制剂的目标分子在诱导产生的途径重塑EC。在最初的研究中，我们发现血栓反应蛋白-1 （TSP1）和色素上皮衍生因子（PEDF）抑制剂会增加EC CD95配体（CD95L），这是一种死亡介质。诱导剂上调死亡受体CD95，与CD95L结合，引发细胞凋亡，从而阻断血管生成。这就是EC如何通过血管生成抑制剂/刺激来平衡细胞凋亡和生存。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还作为平衡血管生成激活和抑制的分子枢纽。启动子阵列分析发现，由于TSP1和PEDF， NFicB、cMyb和Egr-1等转录因子的活性发生了变化。这三者与NFAT形成共同的网络。我们建议阐明促血管生成因子和抗血管生成因子在NFAT交叉调控中的信号和转录事件。我们将使用两种不相关的抑制剂，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（VEGF和bFGF）。我们将确定：TSP1和PEDF使NFAT失活的上游介质。我们将通过激酶抑制剂、免疫沉淀和western blotting的功能测定来评估JNKkinases、p38和GSK在体外的作用。我们将分析抑制剂对NFAT近端激活剂、钙调磷酸酶A （calcalineurin A, CnA）及其调节剂、Ca++动员和dsr -1水平和活性的影响。将使用NFATc2和CnA缺失的小鼠来确认它们的功能作用。TSP1和PEDF对NFAT靶点的调控。我们将通过Western和Northern blotting筛选已知的NFAT靶点Bcl-2、细胞周期蛋白A和E、c-FLIP、环氧化酶-2 （Cox-2）、白细胞介素和组织因子。确定的靶点将通过EMSA和ChIP（染色质免疫沉淀）与NFATc2抗体进行评估，并在体外血管生成实验中使用si-RNA或中和抗体进行评估。NFicB在TSP1和PEDF信号中的作用。NFicB激活将通过免疫细胞化学和EMSA证实。将使用生化抑制剂和/或组成的活性IkB （kB*）来证明功能重要性。使用抑制剂和ChIP检测FasL调控的作用。敲除小鼠将用于验证体内NFicB的贡献。E2F1， Egr-1和c-Myb参与NFAT的作用和调节。TSP1或PEDF引起的Egr-1和c-Myb活性的变化将通过EMSA确认。Egr和c-Myb基因缺失的小鼠将被用来评估它们在TSP1或PEDF抗血管生成中的生物学作用。将使用EMSA、IP和ChlP研究与NFAT的相互作用。越来越多的证据表明，激活的内皮细胞可以通过抑制剂诱导细胞凋亡。因此，我们将描述抑制剂和刺激物共同的分子靶标，并确定参与它们相互作用的主要转录因子。这些研究将为使用天然抑制剂进行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.