RESUMEN
Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic 'tip' and 'stalk' cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica , Proteolisis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Animales , Proteínas Portadoras/genética , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Pez Cebra/genética , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Analysis of cardiovascular development in zebrafish embryos has become a major driver of vascular research in recent years. Imaging-based analyses have allowed the discovery or verification of morphologically distinct processes and mechanisms of, e.g., endothelial cell migration, angiogenic sprouting, tip or stalk cell behavior, and vessel anastomosis. In this chapter, we describe the techniques and tools used for confocal imaging of zebrafish endothelial development in combination with general experimental approaches for molecular dissection of involved signaling pathways.
Asunto(s)
Transducción de Señal , Pez Cebra , Animales , Pez Cebra/metabolismo , Morfogénesis , Proteínas de Pez Cebra/metabolismo , Movimiento Celular , Neovascularización FisiológicaRESUMEN
Canonical Wnt signaling is crucial for vascularization of the central nervous system and blood-brain barrier (BBB) formation. BBB formation and modulation are not only important for development, but also relevant for vascular and neurodegenerative diseases. However, there is little understanding of how Wnt signaling contributes to brain angiogenesis and BBB formation. Here we show, using high resolution in vivo imaging and temporal and spatial manipulation of Wnt signaling, different requirements for Wnt signaling during brain angiogenesis and BBB formation. In the absence of Wnt signaling, premature Sphingosine-1-phosphate receptor (S1pr) signaling reduces VE-cadherin and Esama at cell-cell junctions. We suggest that Wnt signaling suppresses S1pr signaling during angiogenesis to enable the dynamic junction formation during anastomosis, whereas later S1pr signaling regulates BBB maturation and VE-cadherin stabilization. Our data provides a link between brain angiogenesis and BBB formation and identifies Wnt signaling as coordinator of the timing and as regulator of anastomosis.