Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder that leads to abnormal connections between arteries and veins termed arteriovenous malformations (AVM). ...Mutations in TGFβ pathway members
ALK1
,
ENG
and
SMAD4
lead to HHT. However, a
Smad4
mouse model of HHT does not currently exist. We aimed to create and characterize a
Smad4
endothelial cell (EC)-specific, inducible knockout mouse (
Smad4
f/f
;
Cdh5
-Cre
ERT2
) that could be used to study AVM development in HHT. We found that postnatal ablation of
Smad4
caused various vascular defects, including the formation of distinct AVMs in the neonate retina. Our analyses demonstrated that increased EC proliferation and size, altered mural cell coverage and distorted artery–vein gene expression are associated with
Smad4
deficiency in the vasculature. Furthermore, we show that depletion of
Smad4
leads to decreased
Vegfr2
expression, and concurrent loss of endothelial
Smad4
and
Vegfr2
in vivo leads to AVM enlargement. Our work provides a new model in which to study HHT-associated phenotypes and links the TGFβ and VEGF signaling pathways in AVM pathogenesis.
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.
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