Regulation of angiogenic balance by thrombospondin-1.

血小板反应蛋白-1 调节血管生成平衡。

• 批准号: 7216270
• 负责人: OLGA Valery VOLPERT
• 金额: $ 36.66万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7216270
• 关键词: A, Calcineurin; Affect; Angiogenesis Inducing Agents; Angiogenesis Inhibition; Angiogenesis Inhibitors; Angiogenic Factor; Antibodies; Apoptosis; Apoptosis Inhibitor; Binding; Biochemical; Biological; Biological Assay; CASP8 and FADD-like apoptosis regulating protein; Calcineurin; Cell Death; Cell Survival; Cessation of life; Chromatin; Cyclin A; Data; E2F1 gene; EMSA; Endothelial Cells; Endothelium; Epithelial; Equilibrium; Event; Fibroblast Growth Factor; Fibroblast Growth Factor 2; Genes; Immunoprecipitation; In Vitro; Induction of Apoptosis; Interleukin-2; Knockout Mice; MAPK11 gene; MAPK14 gene; MYB gene; Mediator of activation protein; Molecular; Molecular Target; NF-AT; Northern Blotting; Pathologic; Pathway interactions; Pigments; Proto-Oncogene Proteins c-myb; Regulation; Reporter; Role; Signal Transduction; Small Interfering RNA; Stimulus; TNFRSF6 gene; Testing; Therapeutic Intervention; Thromboplastin; Thrombospondin 1; Tissue Expansion; Tumor Necrosis Factor Ligand Superfamily Member 6; Vascular Endothelial Growth Factors; Western Blotting; angiogenesis; antiangiogenesis therapy; cancer therapy; chromatin immunoprecipitation; cyclooxygenase 2; extracellular; immunocytochemistry; in vivo; inhibitor/antagonist; kinase inhibitor; neutralizing antibody; nuclear factors of activated T-cells; promoter; receptor; research study; response; stress-activated protein kinase 1; transcription factor; tumor

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.

描述(申请人提供)：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。抑制靶分子在内皮细胞重塑的诱导物生成途径中的作用。在最初的提案中，我们证明了抑制血栓反应蛋白-1(TSP1)和色素上皮衍生因子(PEDF)增加了死亡介质EC CD95配体(CD95L)。诱导剂上调死亡受体CD95的表达，CD95结合CD95L并触发细胞凋亡，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，活化T细胞的核因子(NFAT)。NFAT也是平衡血管生成、激活和抑制的分子枢纽。启动子阵列分析证实了TSP1和PEDF引起的几种转录因子的活性变化，包括NFicB、cMyb和Egr-1。这三个都与NFAT形成了共同的网络。我们建议阐明信号和转录事件涉及的NFAT交叉调节的亲血管生成因子和抗血管生成因子。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(VEGF和bFGF)。我们将确定：TSP1和PEDF失活NFAT的上游介质。我们将通过功能检测、免疫沉淀和免疫印迹来评估JNKkinases、p38和GSK在体外的作用。我们将分析抑制剂对NFAT近端激活物、钙调神经磷酸酶A(CNA)及其调节剂、钙动员和DSCR-1水平和活性的影响。NFATc2和CNA基因缺失的小鼠将被用于确认它们的功能作用。TSP1和PEDF对NFAT靶点的调节我们将通过Western和Northern blotting筛选已知的NFAT靶标Bcl2、Cyclins A和Cyclins 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抗体进行评估，并在使用si-RNA或中和抗体的体外血管生成试验中进行评估。NFicB在TSP1和PEDF信号中的作用。免疫细胞化学和EMSA将证实NFicB的激活。将使用生化抑制剂和/或结构性活性IKB(kB*)来证明其功能重要性。在FasL调控中的作用将通过使用抑制剂和芯片来检测。基因敲除的小鼠将被用来验证体内NFicB的贡献。E2F1、Egr-1和c-Myb参与NFAT的作用和调节。EMSA将证实由TSP1或PEDF引起的Egr-1和c-Myb活性的变化。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，激活的内皮细胞有可能被抑制剂诱导细胞凋亡。因此，我们将描述抑制物和刺激物的共同分子靶点，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的治疗干预提供新的靶点。