Angiogenesis and Tumor Growth

我们关注了3个相关领域。1)我们继续探索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在发育中的内皮细胞中选择性表达，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。我们生成过表达Dll4蛋白的原代内皮细胞，发现Dll4在VEGF-A作用下降低内皮细胞的增殖和迁移反应。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生物反应降低的原因。与Dll4通过Notch信号传导一致，我们发现在过表达Dll4的内皮细胞中显著诱导转录因子HEY2的表达，并且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淋巴源性肿瘤的生长。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。我们已经观察到，许多实验性癌症和其他类型的肿瘤对肿瘤血管中Dll4/Notch信号的抑制作用无反应。目前的研究主要集中在肿瘤相关的Dll4和其他Notch配体的作用，以及肿瘤相关的Notch受体的作用。在这些实验中，我们分析了肿瘤细胞表达Notch配体对肿瘤血管新生发芽的影响。此外，我们正在探索Notch在环境表达的Notch配体诱导的肿瘤细胞中激活的促血管生成作用。相反，我们正在分析Notch信号在肿瘤源性Notch配体诱导的肿瘤浸润性髓细胞中的促血管生成作用。我们的目标是确定基于肿瘤血管中Dll4/Notch信号的抗肿瘤治疗方法的反应性和耐药性机制。2)我们已经探索了neuropilin-1 （Npn1）作为血管内皮生长因子（VEGF）和3类信号素（分别调节内皮和神经元功能的蛋白家族）的肝素结合形式共享的受体的作用。先前的研究表明，与Npn1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Npn1与VEGF或Sema3A结合的机制，以及Sema3A与Npn1的结合如何影响内皮细胞功能。我们已经鉴定出Sema3A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npn1结合形式的VEGF阻断了Sema3A的所有这些活性。VEGF-A可与Sema3A竞争内皮细胞结合，并可促进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，Npn1内化需要Npn1与VEGF受体的配体桥接，Sema3A可以促进Npn1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Npn1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，我们已经探索了靶向Npn1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、寡核苷酸和其他杂交分子，它们可以诱导Npn1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”。这种内化诱导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，寡鸟苷核苷酸，在体外和体内视网膜新生血管模型中都显示出明确的功效。3)我们继续研究ephrinB配体及其EphB受体如何在新形成的血管中协调内皮/内皮/周细胞组装。EphrinB配体是表面结合的；因此b型Eph/Ephrin相互作用中的受体-配体相互作用涉及邻近细胞。B ephrin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，B ephrin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信号在内皮细胞存活调节中的潜在作用。我们发现，沉默EphrinB的表达或酪氨酸磷酸化缺陷突变体EphrinB的表达（包含所有酪氨酸残基的替代，阻止尾部磷酸化，并作为内源性WT ephrin的显性阴性抑制剂）会导致内皮细胞死亡。这种结果不能通过添加外源性VEGFA或FGF2来阻止。生化和遗传学实验表明，JNK3/MAPK10介导了这种死亡，EphrinB2酪氨酸磷酸化依赖的信号通路是MAPK10/JNK3表达的调节剂。因此，JNK3的沉默可以防止EphrinB信号缺陷的内皮细胞的细胞死亡。与这些结果一致，EphrinB2基因缺陷小鼠的视网膜血管系统经历与JNK3激活相关的细胞死亡。这些结果为支持EphrinB作为抑制血管生成的治疗靶点的作用提供了额外的证据。4)我们对信号蛋白6a （Sema6A）在血管内皮中的潜在活性进行了早期观察。我们现在发现跨膜Sema6A在内皮细胞中表达，并响应外源性和内源性VEGF，通过调节VEGFR2信号调节内皮细胞的生存和生长，并通过自分泌VEGFR信号维持内皮细胞的活力。Sema6A在原代内皮细胞中的沉默会促进细胞死亡，而外源性VEGF- a或FGF2无法挽救细胞死亡，这是由于内源性VEGF促生存信号的丧失。小鼠组织分析表明，Sema6A在血管生成和重塑血管中表达。具有Sema6A零突变的小鼠在与内皮细胞死亡增加相关的玻璃体血管复杂性和视网膜血管发育异常减少方面表现出显著缺陷。与对照组相比，sema6a缺失的成年小鼠表现出肿瘤、基质细胞和脉络膜血管生成的减少。在这些研究之前，Sema6A已知在神经系统发育中起重要作用。我们现在已经发现它也调节血管发育和成人血管生成。

We have focused on 3 related areas. 1) We have continued to explore the role of Delta4 (Dll4), an endothelial-specific membrane-bound ligand for Notch1 and Notch4, as a regulator of endothelial cell function. Dll4 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 reduces endothelial cell proliferative and migratory responses in response to VEGF-A. We identified reduced VEGF receptor 2 and Npn-1 expression in Dll4-overexpressing endothelial cells as responsible for reduced biological responses to VEGF-A. Consistent with Dll4 signaling through Notch, we found that 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 that Dll4 can markedly reduce tumor angiogenesis and the growth of 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. We have observed that a number of experimental carcinomas and other tumor types are unresponsive to the inhibitor effects of Dll4/Notch signaling in the tumor vasculature. Current studies are focused on characterizing the role of tumor-associated Dll4, and other Notch ligands, as well as the roles of tumor-associated Notch receptors. In these experiments, we are analyzing the effects of Notch ligand expression by tumor cells on angiogenic sprouting of tumor vessels. In addition, we are exploring the pro-angiogenic effects of Notch activation in the tumor cells induced by environmentally-expressed Notch ligands. Conversely, we are analyzing the pro-angiogenic effects of Notch signaling in tumor-infiltrating myeloid cells induced by tumor-derived Notch ligands. Our goal is to define the mechanisms of responsiveness and resistance to anti-tumor therapeutic approaches based on Dll4/Notch signaling in the tumor vasculature. 2) We have explored the role of neuropilin-1 (Npn1) 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 Npn1 dictates the choice of signal transduction; plexins tranduce semaphorin signaling and VEGF receptors transduce VEGF signaling. We have now examined the mechanisms underlying Npn1 binding to VEGF or Sema3A, and how the engagement of Npn1 by Sema3A affects endothelial cell function. We have identified Sema3A as an inhibitor of endothelial cell adhesion, survival and proliferation and formation of vascular-like structures. Furthermore, we have found that Npn1-binding forms of VEGF block all these activities of Sema3A. 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 Npn1 internalization requires ligand bridging of Npn1 and VEGF receptors, and that Sema3A can promote Npn1 internalization, but requires a significantly higher concentration than VEGF-A. Thus, our results unveil an essential role for Npn1 as a sensor and priority setter for endothelial cell responses to conflicting signals. In additional studies, we have explored the possibility of targeting Npn1 for internalization as a tool to regulate endothelial cell responses to VEGF. In so doing, we have identified a group of polysaccharides, oligonucleotides, and other hybrid molecules that can induce Npn1 internalization and can thus serve as inhibitors of angiogenesis. We have named these compounds as "internalization inducers". Such internalization-inducing compounds could be useful as therapeutics to reduce angiogenesis. One such synthetic compound,an oligoguanosine nucleotide, has shown clear efficacy both in vitro and in an in vivo model of retinal neovascularization. 3) We have continued investigations on how ephrinB ligands and their EphB receptors orchestrate endothelial/endothelial/pericyte assembly in newly-formed vessels. EphrinB ligands are surface-bound; thus receptor-ligand interactions in the B-type Eph/Ephrin interactions involve adjacent cells. In addition to activating their cognate EphB receptors, B Ephrins 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 cells survival. We have found that silencing EphrinB expression or expression of a tyrosine-phosphorylation-deficient mutant EphrinB (contains substitutions of all tyrosine residues that prevent tail phosphorylation and acts as a dominant-negative inhibitor of endogenous WT ephrin) causes endothelial cell death. Such outcome cannot e prevented by the addition of exogenous VEGFA or FGF2. Biochemical and genetic experiments have revealed that such death is mediated by JNK3/MAPK10, and that EphrinB2 tyrosine phosphorylation-dependent signaling serves as a modulator of MAPK10/JNK3 expression. Thus, the silencing of JNK3 prevents cell death in endothelial cells, which are EphrinB signaling-deficient. Consistent with these results, the retinal vasculature in mice genetically-deficient of EphrinB2 undergoes cell death in association with JNK3 activation. These results provide additional evidence supporting a role for EphrinB as a therapeutic target for inhibition of angiogenesis. 4)We have pursued earlier observations on the potential activities of semaphorin6A (Sema6A) in the vascular endothelium. We now found that transmembrane Sema6A is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating VEGFR2 signaling in response to exogenous and endogenous VEGF, which contributes to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6A in primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of pro-survival signaling from endogenous VEGF. Analyses of mouse tissues demonstrate that Sema6A is expressed in angiogenic and remodeling vessels. Mice with null mutations of Sema6A exhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, Matrigel and choroidal angiogenesis compared to controls. Prior to these studies, Sema6A was known to play important roles in development of the nervous system. We have now discovered that it also regulates vascular development and adult angiogenesis.