Angiogenesis and Tumor Growth

血管生成和肿瘤生长

• 批准号: 7969829
• 负责人: Giovanna Tosato
• 金额: $ 59.23万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7969829
• 关键词: Affect; Angiogenesis Inhibitors; Angiogenic Factor; Area; Binding; Biochemical; Biological Assay; Blood Vessels; Blood capillaries; Bone Marrow; Cancer Model; Cell Adhesion; Cell Proliferation; Cell physiology; Cell surface; Clinic; Conflict (Psychology); Deposition; Development; Drug Delivery Systems; Endothelial Cells; Endothelium; Eph Family Receptors; Ephrin B Receptor; Extracellular Domain; Extracellular Matrix; Goals; Heparin Binding; Hybrids; In Vitro; Ligand Binding; Ligands; Malignant Neoplasms; Membrane; Mesenchymal Stem Cells; Modeling; Mus; Mutagenesis; Mutate; Names; Neurons; Neuropilin-1; Pathologic Neovascularization; Peptides; Pericytes; Pharmaceutical Preparations; Phosphorylation Site; Physiological; Polysaccharides; Process; Protein Family; Receptor Signaling; Recombinant Proteins; Regulation; Relative (related person); Resistance; Retinal Neovascularization; Role; Semaphorin-3; Semaphorin-3A; Signal Transduction; Signaling Molecule; Site; Structure; Surface; Testing; Therapeutic; Time; Tube; Tumor Angiogenesis; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Wound Healing; angiogenesis; blood perfusion; cancer therapy; capillary; delta protein; gamma secretase; in vivo; in vivo Model; inhibitor/antagonist; lymphoid neoplasm; matrigel; neovascularization; notch protein; overexpression; pre-clinical; receptor; reconstitution; research study; response; retina blood vessel structure; selective expression; sensor; therapeutic target; tool; transcription factor; tumor; tumor growth; tumorigenesis

We have focused on 3 related areas. 1) We have explored the role for Delta4 (Dll4), an endothelial specific membrane-bound ligand for Notch1 and Notch4, as a regulator of endothelial cell function. Dll4 is a cell-surface ligand of Notch that is selectively expressed in the developing endothelium and is required for normal vascular development. Post-natally, Dll4 is expressed in the angiogenic endothelium, particularly in the tumor vasculature. We generated primary endothelial cells overexpressing Dll4 protein, and found that Dll4 overexpression reduces endothelial cell proliferative and migratory responses selectively in response to VEGF-A. We identified reduced VEGF receptor 2 and Npn-1 expression in Dll4-overexpressing endothelial cells as responsible for defective responses to VEGF-A. Consistent with Dll4 signaling through Notch, expression of the transcription factor HEY2 was significantly induced in Dll4-overexpressing endothelial cells, and a gamma secretase inhibitor significantly reconstituted endothelial cell proliferation inhibited by Dll4. Thus, these studies have identified the Notch ligand Dll4 as a selective inhibitor of VEGF-A biologic activities down-regulating the principal VEGF-A signaling receptor, VEGFR-2 and co-receptor Npn-1. In additional experiments utilizing pre-clinical cancer models, we have explored the possibility of utilizing Dll4 as an activator of Notch signaling in endothelial cells to inhibit angiogenesis and tumor growth. In xenogeneic and syngeneic tumor models established in mice, we have documented tha Dll4 can markedly reduce tumor angiogenesis and tumor growth, particularly in tumors of lymphoid origin. Studies of the mechanisms for the anti-tumor effects of Dll4 have shown that these are attributable at least in part, to Notch activation in the tumor microenvironment and in the tumor vasculature resulting in reduced VEGFR2 expression and reduced tumor blood perfusion. Current studies are focused in further defining the role of Dll4 in reduced tumor neovascularization, with a particular focus on the effects of Dll4 on extracellular matrix deposition and interactions with pericytes. 2) We have explored the role of neuropilin-1 (Npn-1) as a receptor shared by heparin-binding forms of vascular endothelial growth factor (VEGF) and class 3 semaphorins, protein families that regulate endothelial and neuronal function, respectively. Previous studies have shown that ligand binding to Npn-1 dictates the choice of signal transduction; plexins tranduce semaphorin signaling and VEGF receptors transduce VEGF signaling. We have now examined the mechanisms underlying Npn-1 binding to VEGF or Sema3A, and how the engagement of Npn-1 by Sema3A affects endothelial cell function. We have identified Sema 3A as an inhibitor of endothelial cell adhesion, survival and proliferation and formation of vascular-like structures. Furthermore, we have found that Npn-1-binding forms of VEGF block all these activities of Sema3A. We found that VEGF-A can compete with Sema3A for endothelial cell binding, and can promote Npn-1 internalization from the cell surface. Biochemical analysis of VEGF-A binding to endothelial cells revealed that Npn-1 internalization requires ligand bridging of Npn-1 and VEGF receptors. We also found that Sema3A can promote Npn-1 internalization, but requires a significantly higher concentration than VEGF-A. Thus, our results unveil an essential role for Npn-1 as a sensor and priority setter for endothelial cell responses to conflicting signals. In additional studies, we have explored the possibility of targeting Npn-1 for internalization as a tool to regulate endothelial cell responses to VEGF. In so doing, we have identified a group of polysaccharides and other hybrid molecules that can induce Npn-1 internalization and can thus serve as inhibitors of angiogenesis. We have named these compounds as "internalization inducers". We have explored a variety of internalization-inducing compounds that could be useful as therapeutics to reduce angiogenesis. One such synthetic compound has shown clear efficacy in an in vivo model of retinal neovascularization. 3) We have studied how ephrinB ligands and their EphB receptors orchestrate endothelial/pericyte assembly in newly-formed vessels. EphrinB ligands are surface-bound; in addition to activating their cognate EphB receptors, they can function as signaling molecules when engaged by the receptor through "reverse signaling". Eph receptors are tyrosine kinases interacting with their membrane-anchored ephrin ligands. In our previous studies, we have demonstrated that signaling by Eph B receptors in endothelial cells is critical to assembly into vascular structures. We have now investigated the potential role of Eph/ephrin signaling in the regulation of endothelial/pericytes assembly. A critical step in angiogenesis consists of the recruitment of pericytes to the outer vessel wall, a process that stabilizes and fortifies vessels structure. Mesenchymal stem cells (MSC) can differentiate into pericytes upon interaction with endothelial cells. Using bone marrow-derived MSC, we have established that MSC interact with endothelial cells during extracellular matrix-dependent tube formation in vitro and matrigel angiogenesis assay in vivo, such that MSC establish contact with endothelial cells in a time-dependent and spatially-constrained manner. P-ephrinB is activated in the course of endothelial cell morphogenic processes leading to capillary network stabilization and new vessel formation in vivo, including physiological retinal vessel formation, pathological retinal neovascularization, wound healing and tumorigenesis. This activation is inhibited by specific EphB peptide inhibitors, by a soluble recombinant protein ephrinB2-Fc, by silencing expression of EphrinB2 and by inhibiting Jak2 activity. The importance of EphrinB2 intracellular signaling has emerged from mutagenesis studies in which all the putative phosphorylation sites within the EphrinB2 intracellular domain were mutated, whereas the extracellular domain was intact and capable of signaling through the EphB receptors. In vivo experiments, in which MSC-GFP were injected subcutaneously into immuno-compromised mice in the context of extracellular matrix, show that P-ephrinB reverse signaling is present at those sites where endothelial cells/MSC physically interact. These experiments establish a role for ephrinB reverse signaling in endothelial/pericyte interactions, and suggest that ephrinB signaling is a potential therapeutic target for modulation of physiologic and pathologic angiogenesis. In summary, Dll4 and its target molecules neuropilin1 and ephrinB are critical regulators of angiogenesis, including tumor angiogenesis. We have explored their activities and relative roles during the angiogenic process, have identified them as potential targets for modulation of tumor angiogenesis, and developed compounds that could be useful as angiogenesis inhibitors.

我们专注于3个相关领域。 1）我们探索了Delta4（DLL4）的作用，Delta4（DLL4）是Notch1和Notch4的内皮特异性膜结合的配体，是内皮细胞功能的调节剂。 DLL4是一种缺口的细胞表面配体，在发育中的内皮中有选择地表达，是正常血管发育所必需的。 在产后，DLL4在血管生成内皮中表达，尤其是在肿瘤脉管系统中。我们产生了过表达DLL4蛋白的原代内皮细胞，发现DLL4过表达可响应VEGF-A选择性地降低内皮细胞增殖和迁移反应。我们确定在过表达DLL4的内皮细胞中降低的VEGF受体2和NPN-1表达是负责对VEGF-A的有缺陷反应。 与DLL4信号通过Notch一致，在过表达DLL4的内皮细胞中，转录因子HEY2的表达显着诱导，并且DLL4抑制了γ泌尿酶抑制剂显着重新组建的内皮细胞增殖。因此，这些研究已将Notch配体DLL4确定为VEGF-A生物学活性的选择性抑制剂，从而下调了主VEGF-A信号受体，VEGFR-2和共受体NPN-1。在利用临床前癌症模型的其他实验中，我们探索了将DLL4用作内皮细胞中Notch信号传导激活因子抑制血管生成和肿瘤生长的可能性。在小鼠中建立的异构和合成性肿瘤模型中，我们记录了THA DLL4可以显着减少肿瘤血管生成和肿瘤生长，尤其是在淋巴样肿瘤中。 DLL4抗肿瘤作用机制的研究表明，这些机制至少部分归因于肿瘤微环境的缺口激活以及肿瘤脉管系统的凹口激活，从而导致VEGFR2表达降低和肿瘤血液灌注降低。目前的研究集中在进一步定义DLL4在减少肿瘤新血管形成中的作用，特别关注DLL4对细胞外基质沉积的影响以及与周细胞的相互作用。 2）我们已经探索了神经蛋白-1（NPN-1）作为血管内皮生长因子（VEGF）（VEGF）和3类闪光蛋白，分别调节内皮和神经元功能的蛋白质家族的肝素结合形式的受体的作用。先前的研究表明，配体与NPN-1结合决定了信号转导的选择。 plexins tranduce semaphorin信号传导和VEGF受体传递VEGF信号传导。现在，我们已经检查了NPN-1与VEGF或SEMA3A结合的机制，以及SEMA3A NPN-1的参与如何影响内皮细胞功能。我们已经将SEMA 3A确定为内皮细胞粘附，生存和增殖以及血管状结构的形成的抑制剂。此外，我们发现NPN-1结合形式的VEGF阻断了SEMA3A的所有这些活动。我们发现VEGF-A可以与SEMA3A竞争内皮细胞结合，并可以促进细胞表面的NPN-1内在化。 VEGF-A与内皮细胞结合的生化分析表明，NPN-1内部化需要NPN-1和VEGF受体的配体桥接。我们还发现SEMA3A可以促进NPN-1内部化，但需要比VEGF-A更高的浓度。因此，我们的结果揭示了NPN-1作为传感器和优先设置器的重要作用，用于内皮细胞对冲突信号的响应。在其他研究中，我们探讨了将NPN-1靶向内在化作为调节内皮细胞反应的工具的可能性。这样，我们已经确定了一组可以诱导NPN-1内在化的多糖和其他杂化分子，因此可以用作血管生成的抑制剂。我们将这些化合物称为“内部化诱导剂”。我们已经探索了各种内在化诱导化合物，这些化合物可以用作减少血管生成的治疗剂。一种这样的合成化合物在视网膜新血管形成体内模型中表现出明显的功效。 3）我们研究了ephrinb配体及其EPHB受体如何在新形成的血管中编排内皮/周细胞组件。 ephrinb配体是表面结合的。除了激活其同源EPHB受体外，当受体通过“反向信号传导”参与时，它们还可以充当信号分子。 EPH受体是酪氨酸激酶与膜锚定的埃弗林配体相互作用。在我们以前的研究中，我们已经证明，EPH B受体在内皮细胞中的信号传导对于组装成血管结构至关重要。现在，我们已经研究了EPH/Ephrin信号传导在调节内皮/周细胞组件中的潜在作用。血管生成的关键步骤是将周细胞募集到外容器壁上，这一过程稳定并构成了血管结构。与内皮细胞相互作用后，间充质干细胞（MSC）可以分化为周细胞。使用骨髓衍生的MSC，我们确定MSC在体内体外和基质基质依赖性管形成期间与内皮细胞相互作用，并在体内与基质凝胶血管生成测定法相互作用，以便MSC以时间依赖性和空间约束的方式与内皮细胞建立与内皮细胞的接触。在内皮细胞形态发生过程的过程中激活了p-磷，从而导致毛细血管网络稳定和体内新血管形成，包括生理视网膜血管形成，病理视网膜新生血管形成，伤口愈合和肿瘤衰变。这种激活被特定的EPHB肽抑制剂抑制，可溶性重组蛋白Ephrinb2-Fc，通过沉默的Ephrinb2表达并抑制JAK2活性。以弗林B2的细胞内信号传导的重要性来自诱变研究，其中ephrinb2内的所有假定的磷酸化位点突变是突变的，而细胞外结构域是完整的，并且能够通过EPHB受体信号传导。在细胞外基质中，将MSC-GFP皮下注射到免疫受损的小鼠中的体内实验表明，p-磷逆信号存在于内皮细胞/MSC物理相互作用的那些位点。这些实验在内皮/周细胞相互作用中为ephrinb反向信号传导确定了作用，并表明ephrinb信号传导是调节生理和病理血管生成的潜在治疗靶标。总之，DLL4及其靶标分子Neuropilin1和Ephrinb是血管生成的关键调节剂，包括肿瘤血管生成。我们已经探索了它们在血管生成过程中的活性和相对作用，并将它们确定为调节肿瘤血管生成的潜在靶标，并开发出可以用作血管生成抑制剂的化合物。