Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this critical process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development using both in vitro imaging and biochemistry as well as in vivo models. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 results in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrate that Rab10 is Notch responsive, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune basement membrane secretion during blood vessel development.

Despite the centrality of Delta/Notch signaling to activate lateral inhibition and define tip/stalk cell specification during angiogenesis, many mechanisms are still incompletely understood. Here, we report that the proteins Epsin homology domain protein 2 (EHD2) and EHD2 binding partner 1 (EHBP1) are required for proper angiogenic signaling in endothelial cells (ECs). EHBP1 and EHD2 localize to Delta-like ligand 4 (Dll4) and actin at adherens junctions. Importantly, we demonstrate that Dll4 is internalized via caveolin-dependent endocytosis, which is reliant on both EHBP1 and EHD2. In the absence of EHBP1 and EHD2, Dll4 endocytosis is significantly impaired, leading to blood vessel defects both in vitro and in vivo. We propose a model wherein EHBP1 and EHD2 regulate Dll4 endocytosis by anchoring its caveolar endocytic pit to the actin cytoskeleton. Overall, this provides mechanistic detail of how Dll4 is endocytosed during Notch activation.

Objective: Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear about how this system is regulated. Our objective was to determine the involvement of Epsin 15 Homology Domain Containing 2 (EHD2) in delta-like ligand 4 (Dll4) endocytosis during Notch activation. Approach and Results: Using both in vivo and in vitro models, we demonstrate that EHD2 is a novel modulator of Notch activation in endothelial cells through controlling endocytosis of Dll4. In vitro, EHD2 localized to plasma membrane-bound Dll4 and caveolae. Chemical disruption of caveolae complexes resulted in EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization. Reduced Dll4 internalization blunted Notch activation in endothelial cells. In vivo, EHD2 is primarily expressed in the vasculature, colocalizing with junctional marker VE-cadherin and Dll4. Knockout of EHD2 in zebrafish produced a significant increase in dysmorphic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. Conclusions: Overall, we demonstrate that EHD2 is necessary for Dll4 transcytosis and downstream Notch activation.

SUMMARYDuring angiogenesis, endothelial cells secrete proteins that make up a planar protein network surrounding blood vessels termed basement membrane (BM). Collagen type IV (Col IV) is a BM protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. To date, little is known about how endothelial cells mediate intracellular transport and selective secretion of Col IV. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 resulted in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrated that Rab10 activation is downstream of Notch signaling, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Overall, our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune BM secretion during blood vessel development.

A correction to this paper has been published: https://doi.org/10.1007/s10456-021-09801-w