旁分泌因子Maeg调控血管新生的机制研究

Angiogenesis is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, and migration. Up to date, the functional analysis of Maeg, a member of EGF-like protein family, involved in blood vessel formation in vivo is thus far lacking. Lately, we identified and cloned the zebrafish-secreted factor maeg. Our data shows that maeg was expressed in developing somites during a time window encompassing many key steps in embryonic angiogenesis. Maeg loss of function in zebrafish embryos specifically interrupted sprouting angiogenesis of intersegmental vessels (ISVs). ISVs in maeg mutants grew only halfway through their ventral trajectory and stalled at the boundary between the notochord and neural tube, accompanied with the cell number reduction of ISV. Furthermore, we found that the filopodia number of ISV tip cells was significantly reduced in Egfl6 morphants. Most interestingly, gain of Egfl6 function resulted in hyper-angiogenic phenotype, with ISV appearing H-shaped, Y-shaped, and knot-like structure, which has not been reported in zebrafish before. In addition, our preliminary data indicates that Maeg acts as a ligand of Integrin to regulate the EC behavior. These experimental findings suggest Maeg is a prominent paracrine factor regulating embryonic angiogenesis. In the current project, we propose to investigate the celluar mechanism of maeg regulating vascular development in zebrafish embryo. Moreover we plan to identify the receptor and downstream signaling pathway of Maeg, which is responsible for the vascular phenotype. The outcome of this project will allow us to open a new avenue for target-designing drugs to control the formation of blood vessels in a variety of pathological conditions caused by insufficient or excessive blood supply.

血管新生是一个受到严格调控的复杂而有序的过程。由周围细胞产生的多种分泌因子参与调控血管内皮细胞的分化、增殖和迁移。到目前为止EGF-like家族成员分泌因子Maeg在体内血管新生中的功能研究还未见报道。我们前期工作中克隆了斑马鱼maeg，发现其主要表达在发育中的体节。其表达时间窗对应了胚胎血管形成的多个关键阶段。Maeg基因敲除特异地导致体节间血管芽生的缺陷和内皮端细胞丝状伪足异常。尤其有趣的是，maeg表达上调导致体节间血管过度生长，形成H型、Y型和独特的公路环岛样结构，并伴有血流灌注；且此结构还有继续芽生的趋势。这些前期结果提示maeg是一个重要的调控血管新生的因子。本项目拟进一步分析maeg基因敲除和表达上调后斑马鱼胚胎血管表型产生的细胞生物学机理；结合多种手段鉴定Maeg的受体分子和下游信号通路，阐述其调控血管新生的分子机制。本项目为调控血管新生提供新的理论依据和潜在的药物靶点。

血管新生是一个受到严格调控的复杂而有序的过程。由周围细胞产生的多种分泌因子参与调控血管内皮细胞的分化、增殖和迁移。到目前为止EGF-like家族成员分泌因子Maeg在体内血管新生中的功能研究还未见报道。我们克隆了斑马鱼maeg，通过原位杂交以及原位免疫荧光实验发现其主要表达在发育中的体节。其表达时间窗对应了胚胎血管形成的多个关键阶段。通过基因编辑技术介导的Maeg基因敲除特异地导致体节间血管芽生的缺陷和内皮端细胞丝状伪足显著的减少。此外，maeg表达上调导致体节间血管过度生长，形成H型、Y型和独特的公路环岛样结构，并伴有血流灌注；且这些结构还有继续芽生的趋势。这些结果显示maeg是一个重要的调控血管新生的因子。通过在体实时连续成像分析显示maeg 基因敲除和表达上调后分别导致血管内皮细胞的增殖和迁移的正负方向的调控。此外结合转录组芯片技术、western blot、流式细胞分选等多种手段揭示了Maeg结合其细胞膜定位受体分子itegrin导致下游信号通路PI3K/Akt 和 Mek/ERK的激活，促进血管新生的分子机制。本项目为调控血管新生提供新的理论依据也为肿瘤、糖尿病等疾病的血管新生病变供新潜在的药物靶点。