Flt-1 (VEGFR-1) Regulation of Endothelial Cell Sprouting and Vessel Morphogenesis

描述(申请人提供)：血管网络通过几个过程发展，包括血管生成，原始血管的新生形成，以及血管生成，从先前存在的血管萌发出新的血管。血管内皮生长因子-A(VEGF)及其受体Flt-1和Flk-1在控制这些过程中起着关键作用。血管内皮生长因子通过Flk-1的信号转导作用已经被很好地描述，但Flt-1在调节血管形成中的作用尚不清楚。Flt-1主要通过产生一种可溶性的Flt-1亚型来调节血管分支，该亚型在很大程度上扮演着配体接收器的角色。此外，最近的证据表明，Notch信号与血管内皮生长因子途径相互作用，指导血管发育，尽管这种相互作用的确切性质尚不清楚。因此，将被检验的假设是，Flt-1通过提供空间线索来调节血管形态发生，以引导内皮细胞(EC)丝状足和芽的形成，而Notch信号调节Flt-1下游的活性，以建立适当的芽延伸模式，进而血管分支。为了实现这些目标，将利用分化的胚胎干细胞培养和小鼠视网膜发育中的血管形成模型。在这两种模型中，通过对Flt-1表达的基因修饰，将扰乱VEGF信号转导。在确定了Flt-1在这些模型中表达的空间分布后，将在EC丝状足突延伸、尖端细胞形成、血管萌发引导以及整个血管分支和形态方面对Flt-1突变血管和野生型(WT)血管进行比较。为了确定Notch信号如何调节Flt-1在引导血管萌发中的作用，将在WT和Flt-1突变的ES细胞和视网膜血管模型中建立Notch信号相对于EC丝状伪足和血管萌发的位置。Notch信号将通过使用抑制剂或中和抗体从基因上受到干扰。开发中的船只将如上所述地进行分析。从这些研究中，将获得关于Flt-1如何调节血管内皮生长因子信号从而发生适当的EC丝状足突延伸、芽形成和血管分支的知识，以及Notch信号如何协调Flt-1的活性以产生适当的分支血管网络。这项研究的见解将对制定治疗策略，在治疗某些病理条件下，如肿瘤生长和糖尿病视网膜病变时干扰血管生长至关重要。此外，这项研究将在设计临床相关的刺激血管形成的治疗方法方面提供参考，这些治疗方法适用于患有血管闭塞疾病的患者，如冠心病和外周血管疾病。

DESCRIPTION (provided by applicant): Vascular networks develop through several process including vasculogenesis, the de novo formation of primitive vessels, and angiogenesis, the sprouting of new vessels from pre-existing ones. Vascular endothelial growth factor-A (VEGF) and its receptors, Flt-1 and Flk-1, play critical roles in controlling these processes. VEGF signaling effects via Flk-1 have been well characterized, but the role of Flt-1 in regulating vessel formation remains unclear. Flt-1 regulates vessel branching, primarily by producing a soluble Flt-1 isoform that acts largely as a ligand sink. Furthermore, recent evidence suggests that Notch signaling interacts with VEGF pathways to guide developing vessels, although the exact nature of this interaction is not well understood. Thus, the hypotheses that will be tested are that Flt-1 regulates vessel morphogenesis by providing spatial cues to guide endothelial cell (EC) filopodia and sprout formation, and that Notch signaling modulates downstream Flt-1 activity to establish the proper pattern of sprout extension and in turn vessel branching. To achieve these goals, models of vessel formation in differentiated embryonic stem (ES) cell cultures and in mouse retina development will be utilized. In both models, VEGF signaling will be disrupted through genetic modification of Flt-1 expression. After characterizing the spatial distribution of Flt- 1 expression in these models, Flt-1 mutant and wild-type (WT) vessels will be compared with regard to EC filopodia extension, tip cell formation, vessel sprout guidance, and overall vessel branching and morphology. To characterize how Notch signaling regulates Flt-1 in guiding vessel sprouts, the location of Notch signaling relative to EC filopodia and vessel sprouts will be established in both the WT and Flt-1 mutant ES cell and retina vessel models. Notch signaling will be disrupted genetically and through the use of inhibitors or neutralizing antibodies. Developing vessels will be analyzed as described above. From these studies, knowledge will be gained regarding how Flt-1 regulates VEGF signaling such that proper EC filopodia extension, sprout formation and vessel branching occur, and how Notch signaling coordinates Flt-1 activity to yield properly branched vascular networks. Insights from this study will be essential in the development of therapeutic strategies to disrupt blood vessel growth in treating certain pathological conditions such as tumor growth and diabetic retinopathy. Furthermore, this study will be informative in the design of clinically relevant treatments for stimulating blood vessel formation in patients suffering from vascular occlusive diseases such as coronary heart disease and peripheral vascular disease.