The endosomal\xa0RIN2/Rab5C machinery\xa0prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis

Lanette Kempers,Yuki Wakayama,Antonius L Van Boxtel,Dirk De Korte,Charita Furumaya,Anne-Marieke D Van Stalborch,Jaap D Van Buul,Iris M De Cuyper,Wiebke Herzog,Dirk Geerts,Aldo Jongejan,Marije Kat,Coert Margadant,Ivo Van Der Bijl,Marvin Hubert

doi:10.1007/s10456-021-09788-4

Abstract

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.

Highlights

Sprouting angiogenesis is crucial for a range of pathophysiological processes including embryonic development, wound healing, tissue remodeling, cancer, and cardiovascular disease [1]
VEGF receptor 2 (VEGFR2) degradation, human umbilical vein endothelial cells (HUVECs) were deprived of growth factors overnight and stimulated with vascular endothelial growth factor (VEGF), which resulted in a rapid decrease of up to 40% of VEGFR2 levels in 30 min (Fig. 1f), consistent with previous reports [16, 22]
Because VEGF signaling and the expression of certain VEGF targets is important for the induction and maintenance of tip cells during sprouting angiogenesis, we addressed whether the reduced sprouting observed in the absence of Rab5C is due to differences in VEGF-induced tip cell specification

Summary

Introduction

Sprouting angiogenesis is crucial for a range of pathophysiological processes including embryonic development, wound healing, tissue remodeling, cancer, and cardiovascular disease [1]. A key pro-angiogenic event is the interaction of vascular endothelial growth factor A (VEGF-A, further referred to as VEGF) with VEGF receptor 2 (VEGFR2), which promotes endothelial proliferation, survival, and migration through activation of the extracellular-signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) pathways [2]. VEGF/VEGFR2 signaling induces a pro-angiogenic gene expression program and stimulates the selection of specialized ‘tip cells’, which have a distinct morphology and transcriptional signature, and promote the guidance of nascent sprouts [3,4,5,6,7]. VEGF-dependent specification of tip and stalk cells is fine-tuned by activation of Notch signaling, which downregulates VEGFR2 expression to prevent uncontrolled sprouting [7,8,9,10], as well as by the decoy receptor VEGFR1, which sequesters VEGF away from VEGFR2 [11,12,13]. VEGFR2 is recycled back to the cell-surface in recycling compartments, containing either Rab or Rab11 [14, 16, 17]

Methods

Results

Conclusion