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

描述（由申请人提供）：血管网络的发展经过几个过程，包括血管发生，原始血管的新生形成和血管生成，从已有的血管中萌发新血管。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关键作用。通过Flk-1介导的VEGF信号传导作用已被很好地表征，但Flt-1在调节血管形成中的作用尚不清楚。Flt-1主要通过产生可溶性Flt-1异构体来调节血管分支，该异构体主要作为配体汇。此外，最近的证据表明Notch信号与VEGF通路相互作用，引导血管发育，尽管这种相互作用的确切性质尚不清楚。因此，将被验证的假设是Flt-1通过提供空间线索来引导内皮细胞（EC）丝状伪足和芽的形成来调节血管形态发生，而Notch信号调节下游Flt-1的活性以建立芽延伸和血管分支的适当模式。为了实现这些目标，分化胚胎干（ES）细胞培养和小鼠视网膜发育中的血管形成模型将被利用。在这两种模型中，通过对Flt-1表达的基因修饰，VEGF信号将被破坏。在对这些模型中Flt-1表达的空间分布进行表征后，将比较Flt-1突变型和野生型（WT）血管在EC丝状足延伸、尖端细胞形成、血管芽引导以及整体血管分支和形态方面的差异。为了描述Notch信号如何调控Flt-1在引导血管芽中的作用，我们将在WT和Flt-1突变ES细胞和视网膜血管模型中建立Notch信号相对于EC丝状足和血管芽的位置。通过使用抑制剂或中和抗体，Notch信号将被遗传破坏。如上所述，将对正在发育的血管进行分析。通过这些研究，我们将了解Flt-1如何调节VEGF信号，从而使EC丝状足延伸、芽形成和血管分支发生，以及Notch信号如何协调Flt-1活性以产生适当的分支血管网络。从这项研究中获得的见解将对治疗策略的发展至关重要，以破坏血管生长，治疗某些病理条件，如肿瘤生长和糖尿病视网膜病变。此外，该研究将为冠心病和外周血管疾病等血管闭塞性疾病患者刺激血管形成的临床相关治疗设计提供信息。

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.