Angiogenesis and Tumor Growth

我们关注了3个相关领域。1)我们探索了Notch1和Notch4的内皮特异性膜结合配体Delta4 （Dll4）作为内皮细胞功能调节剂的作用。Dll4是Notch的一种细胞表面配体，在发育中的内皮中选择性表达，是正常血管发育所必需的。出生后，Dll4在血管生成内皮中表达，特别是在肿瘤血管中。我们制备了过表达Dll4蛋白的原代内皮细胞，发现Dll4过表达选择性地降低内皮细胞对VEGF-A的增殖和迁移反应。我们发现，在过表达dll4的内皮细胞中，VEGF受体2和Npn-1的表达减少是导致VEGF- a反应缺陷的原因。与Dll4通过Notch信号传导一致，转录因子HEY2在过表达Dll4的内皮细胞中被显著诱导表达，一种γ分泌酶抑制剂显著重建了被Dll4抑制的内皮细胞增殖。因此，这些研究已经确定Notch配体Dll4是VEGF-A生物活性的选择性抑制剂，可下调VEGF-A主要信号受体VEGFR-2和共受体Npn-1。在利用临床前癌症模型的其他实验中，我们探索了利用Dll4作为内皮细胞中Notch信号的激活剂来抑制血管生成和肿瘤生长的可能性。在建立的小鼠异种和同基因肿瘤模型中，我们已经证明Dll4可以显著减少肿瘤血管生成和肿瘤生长，特别是在淋巴源性肿瘤中。对Dll4抗肿瘤作用机制的研究表明，这些作用至少部分归因于肿瘤微环境和肿瘤血管中的Notch激活，导致VEGFR2表达减少和肿瘤血液灌注减少。目前的研究主要集中在进一步确定Dll4在减少肿瘤新生血管中的作用，特别关注Dll4对细胞外基质沉积和与周细胞相互作用的影响。2)我们已经探索了神经匹林-1 （Npn-1）作为血管内皮生长因子（VEGF）的肝素结合形式和3类信号素（分别调节内皮和神经元功能的蛋白家族）共享的受体的作用。先前的研究表明，与Npn-1结合的配体决定了信号转导的选择；丛蛋白传递信号蛋白信号，VEGF受体传递VEGF信号。我们现在研究了Npn-1与VEGF或Sema3A结合的机制，以及Sema3A与Npn-1的结合如何影响内皮细胞功能。我们已经鉴定出Sema 3A是内皮细胞粘附、存活和增殖以及血管样结构形成的抑制剂。此外，我们发现npn -1结合形式的VEGF阻断了Sema3A的所有这些活性。我们发现VEGF-A可以与Sema3A竞争内皮细胞结合，并且可以促进Npn-1从细胞表面内化。VEGF- a与内皮细胞结合的生化分析表明，Npn-1的内化需要Npn-1与VEGF受体的配体桥接。我们还发现Sema3A可以促进Npn-1的内化，但需要的浓度明显高于VEGF-A。因此，我们的研究结果揭示了Npn-1作为内皮细胞对冲突信号反应的传感器和优先级设置者的重要作用。在其他研究中，我们探索了靶向Npn-1内化作为调节内皮细胞对VEGF反应的工具的可能性。在这样做的过程中，我们已经确定了一组多糖和其他杂交分子，可以诱导Npn-1内化，从而可以作为血管生成的抑制剂。我们将这些化合物命名为“内化诱导剂”。我们已经探索了多种内化诱导化合物，这些化合物可以作为减少血管生成的治疗药物。一种这样的合成化合物在视网膜新生血管的体内模型中显示出明确的功效。3)我们研究了ephrinB配体及其EphB受体如何在新形成的血管中协调内皮细胞/周细胞的组装。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，它们还可以作为信号分子发挥作用。Eph受体是酪氨酸激酶与其膜锚定的ephrin配体相互作用。在我们之前的研究中，我们已经证明内皮细胞中Eph B受体的信号传导对血管结构的组装至关重要。我们现在已经研究了Eph/ephrin信号在内皮细胞/周细胞组装调节中的潜在作用。血管生成的关键步骤包括周细胞向外血管壁的募集，这是一个稳定和强化血管结构的过程。间充质干细胞（MSC）与内皮细胞相互作用后可分化为周细胞。利用骨髓来源的间充质干细胞，我们已经确定了间充质干细胞在体外细胞外基质依赖的管形成和体内基质血管生成实验中与内皮细胞相互作用，从而使间充质干细胞以时间依赖和空间受限的方式与内皮细胞建立接触。P-ephrinB在内皮细胞形态发生过程中被激活，导致体内毛细血管网络稳定和新血管形成，包括生理性视网膜血管形成、病理性视网膜新生血管形成、伤口愈合和肿瘤发生。特异性EphB肽抑制剂、可溶性重组蛋白EphrinB2 - fc、沉默EphrinB2表达和抑制Jak2活性可抑制这种激活。EphrinB2细胞内信号的重要性已经从诱变研究中出现，在诱变研究中，所有在EphrinB2细胞内区域的假定磷酸化位点都发生了突变，而细胞外区域是完整的，能够通过EphB受体进行信号传导。在体内实验中，在细胞外基质的背景下，将MSC- gfp皮下注射到免疫受损小鼠中，结果表明，在内皮细胞/MSC物理相互作用的部位存在P-ephrinB反向信号。这些实验确定了ephrinB反向信号在内皮细胞/周细胞相互作用中的作用，并表明ephrinB信号是调节生理性和病理性血管生成的潜在治疗靶点。综上所述，Dll4及其靶分子neuropilin1和ephrinB是血管生成（包括肿瘤血管生成）的关键调节因子。我们探索了它们在血管生成过程中的活性和相关作用，确定了它们作为调节肿瘤血管生成的潜在靶点，并开发了可能用作血管生成抑制剂的化合物。

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.