RESUMO
The early blood vessels of the embryo and yolk sac in mammals develop by aggregation of de novo-forming angioblasts into a primitive vascular plexus, which then undergoes a complex remodeling process. Angiogenesis is also important for disease progression in the adult. However, the precise molecular mechanism of vascular development remains unclear. It is therefore of great interest to determine which genes are specifically expressed in developing endothelial cells (ECs). Here, we used Flk1-deficient mouse embryos, which lack ECs, to perform a genome-wide survey for genes related to vascular development. We identified 184 genes that are highly enriched in developing ECs. The human orthologs of most of these genes were also expressed in HUVECs, and small interfering RNA knockdown experiments on 22 human orthologs showed that 6 of these genes play a role in tube formation by HUVECs. In addition, we created Arhgef15 knockout and RhoJ knockout mice by a gene-targeting method and found that Arhgef15 and RhoJ were important for neonatal retinal vascularization. Thus, the genes identified in our survey show high expression in ECs; further analysis of these genes should facilitate our understanding of the molecular mechanisms of vascular development in the mouse.
Assuntos
Biomarcadores/metabolismo , Embrião de Mamíferos/metabolismo , Endotélio Vascular/metabolismo , Perfilação da Expressão Gênica , Genoma , Neovascularização Fisiológica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Células Cultivadas , Embrião de Mamíferos/citologia , Endotélio Vascular/citologia , Feminino , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/fisiologia , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Técnicas Imunoenzimáticas , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Retina/citologia , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rho de Ligação ao GTPRESUMO
Blood vessel development and network patterning are controlled by several signaling molecules, including VEGF, FGF, TGF-ß, and Ang-1,2. Among these, the role of VEGF-A signaling in vessel morphogenesis is best understood. The biological activity of VEGF-A depends on its reaction with specific receptors Flt1 and Flk1. Roles of VEGF-A signaling in endothelial cell proliferation, migration, survival, vascular permeability, and induction of tip cell filopodia have been reported. In this study, we have generated Flt1-tdsRed BAC transgenic (Tg) mice to monitor Flt1 gene expression during vascular development. We show that tdsRed fluorescence is observed within blood vessels of adult mice and embryos, indicative of retinal angiogenesis and tumor angiogenesis. Flt1 expression recapitulated by Flt1-tdsRed BAC Tg mice overlapped well with Flk1, while Flt1 was expressed more abundantly in endothelial cells of large blood vessels such as dorsal aorta and presumptive stalk cells in retina, providing a unique model to study blood vessel development.