Rationale:: Endothelial Notch signaling is critical for early vascular development and survival. Yet, previously described mice lacking endothelial a disintegrin and metalloproteinase 10 (ADAM10), a key regulator of Notch signaling, survived into adulthood with organ-specific vascular defects. These findings raised questions about whether these vascular defects were related to Notch signaling or other functions of ADAM10. Objective:: The aims of the study are to determine whether compensatory or redundant functions of ADAM17 in Notch signaling can explain the survival of Adam10 ΔEC mice, explore the contribution of different Tie2-Cre transgenes to the differences in survival, and establish whether the Adam10 ΔEC vascular phenotypes can be recapitulated by inactivation of Notch receptors in endothelial cells. Methods and Results:: Mice lacking ADAM10 and ADAM17 in endothelial cells ( Adam10/Adam17 ΔEC), which survived postnatally with organ-specific vascular defects, resembled Adam10 ΔEC mice. In contrast, Adam10 ΔEC mice generated with the Tie2Cre transgene previously used to inactivate endothelial Notch ( Adam10 ΔEC ^Flv ) died by E10.5. Quantitative polymerase chain reaction analysis demonstrated that Cre-mediated recombination occurs earlier in Adam10 ΔEC ^Flv mice than in the previously described Adam10 ΔEC mice. Finally, mice lacking endothelial Notch1 ( Notch1 ΔEC) share some organ-specific vascular defects with Adam10 ΔEC mice, whereas Notch4 ^−/− mice lacking endothelial Notch1 ( Notch1 ΔEC /Notch4 ^−/− ) had defects in all vascular beds affected in Adam10 ΔEC mice. Conclusions:: Our results argue against a major role for ADAM17 in endothelial Notch signaling and clarify the difference in phenotypes of previously described mice lacking ADAM10 or Notch in endothelial cells. Most notably, these findings uncover new roles for Notch signaling in the development of organ-specific vascular beds.

The vasculature is a remarkably interesting, complex, and interconnected organ. It provides a conduit for oxygen and nutrients, filtration of waste products, and rapid communication between organs. Much remains to be learned about the specialized vascular beds that fulfill these diverse, yet vital functions. This review was prompted by the discovery that Notch signaling in mouse endothelial cells is crucial for the development of specialized vascular beds found in the heart, kidneys, liver, intestines, and bone. We will address the intriguing questions raised by the role of Notch signaling and that of its regulator, the metalloprotease ADAM10, in the development of specialized vascular beds. We will cover fundamentals of ADAM10/Notch signaling, the concept of Notch-dependent cell fate decisions, and how these might govern the development of organ-specific vascular beds through angiogenesis or vasculogenesis. We will also consider common features of the affected vessels, including the presence of fenestra or sinusoids and their occurrence in portal systems with two consecutive capillary beds. We hope to stimulate further discussion and study of the role of ADAM10/Notch signaling in the development of specialized vascular structures, which might help uncover new targets for the repair of vascular beds damaged in conditions like coronary artery disease and glomerulonephritis.

The membrane-anchored metalloproteinase a disintegrin and metalloprotease 10 (ADAM10) is required for shedding of membrane proteins such as EGF, betacellulin, the amyloid precursor protein, and CD23 from cells. ADAM10 is constitutively active and can be rapidly and post-translationally enhanced by several stimuli, yet little is known about the underlying mechanism. Here, we use ADAM10-deficient cells transfected with wild type or mutant ADAM10 to address the role of its cytoplasmic and transmembrane domain in regulating ADAM10-dependent protein ectodomain shedding. We report that the cytoplasmic domain of ADAM10 negatively regulates its constitutive activity through an ER retention motif but is dispensable for its stimulated activity. However, chimeras with the extracellular domain of ADAM10 and the transmembrane domain of ADAM17 with or without the cytoplasmic domain of ADAM17 show reduced stimulated shedding of the ADAM10 substrate betacellulin, whereas the ionomycin-stimulated shedding of the ADAM17 substrates CD62-L and TGFα is not affected. Moreover, we show that influx of extracellular calcium activates ADAM10 but is not essential for its activation by APMA and BzATP. Finally, the rapid stimulation of ADAM10 is not significantly affected by incubation with proprotein convertase inhibitors for up to 8 h, arguing against a major role of increased prodomain removal in the rapid stimulation of ADAM10. Thus, the cytoplasmic domain of ADAM10 negatively influences constitutive shedding through an ER retention motif, whereas the cytoplasmic domain and prodomain processing are not required for the rapid activation of ADAM10-dependent shedding events.

Notch signaling is critical for controlling a variety of cell fate decisions during metazoan development and homeostasis. This unique, highly conserved signaling pathway relies on cell-to-cell contact, which triggers the proteolytic release of the cytoplasmic domain of the membrane-anchored transcription factor Notch from the membrane. A disintegrin and metalloproteinase (ADAM) proteins are crucial for Notch activation by processing its S2 site. While ADAM10 cleaves Notch1 under physiological, ligand-dependent conditions, ADAM17 mainly cleaves Notch1 under ligand-independent conditions. However, the mechanism(s) that regulate the distinct contributions of these ADAMs in Notch processing remain unclear. Using cell-based assays in mouse embryonic fibroblasts (mEFs) lacking ADAM10 and/or ADAM17, we aimed to clarify what determines the relative contributions of ADAM10 and ADAM17 to ligand-dependent or ligand-independent Notch processing. We found that EDTA-stimulated ADAM17-dependent Notch1 processing is rapid and requires the ADAM17-regulators iRhom1 and iRhom2, whereas the Delta-like 4-induced ligand-dependent Notch1 processing is slower and requires ADAM10. The selectivity of ADAM17 for EDTA-induced Notch1 processing can most likely be explained by a preference for ADAM17 over ADAM10 for the Notch1 cleavage site and by the stronger inhibition of ADAM10 by EDTA. The physiological ADAM10-dependent processing of Notch1 cannot be compensated for by ADAM17 in Adam10-/- mEFs, or by other ADAMs shown here to be able to cleave the Notch1 cleavage site, such as ADAMs9, 12, and 19. Collectively, these results provide new insights into the mechanisms underlying the substrate selectivity of ADAM10 and ADAM17 towards Notch1.

Purpose: To determine the feasibility of using telemedicine consultations in the evaluation of recovered donor corneas for transplant suitability. Methods: This study aims to establish and test the minimum imaging requirements for telemedical consultations of corneal tissue by remote eye bank medical directors. Digital images from the slit lamp, optical coherence tomography, and/or specular microscope were assembled into telemedical consults and emailed to 4 eye bank medical directors (M.A.T., J.W., C.S.S., N.K.R.). Feedback on the minimum image requirements for each corneal finding was collected. After establishing a standardized imaging and presentation protocol, test cases were presented to the medical directors to examine the validity of these remote consults. To establish a benchmark for the study's parameters, one medical director (J.W.) examined each case in person after his initial remote review. Examiners were masked to each other's responses. Results: Minimum image requirements for determination of corneal findings were defined and were specific to each anatomic layer of the cornea (epithelial, stromal, or endothelial). Using a defined set of digital images for a set of common corneal findings, the rate of agreement between remote evaluators, eye bank staff, and the in-person evaluator was 100% (11 of 11 examples). For ambiguous test cases, remote evaluators agreed on 80% of the cases (4 of 5). Conclusions: Results from this pilot study suggest that telemedical review of corneal tissue using high-quality digital images may be adequate for accurate identification of specific corneal findings commonly encountered by eye banks.