No abstract

Contact-dependent interactions between endothelial cells (ECs), as well as between ECs and mural cells, play a key role in the formation of a regular vascular system and the assembly of the vessel wall. Recent studies have identified ephrinB2 and EphB4 as markers and makers of arteriovenous differentiation during vascular development. On the basis of these findings,we hypothesized that Ephephrin interactions in the vascular system mediate distinct propulsive and repulsive effector functions that provide guidance signals for the proper spatial organization of the developing vasculature. Utilizing a set of specialized endothelial differentiation and angiogenesis assays, the present study was aimed at studying vascular morphogenic functions of endothelial EphB4 and ephrinB2 activation. EphrinB2-Fc acts anti-adhesively and induces detachment of ECs, as demonstrated by (1) inhibition of adhesion to ephrinB2-Fc-coated culture dishes, (2) detachment of ECs grown as differentiated 3D spheroids, and (3) endothelial denudation of explanted fragments of umbilical vein. Conversely, soluble ephrinB2-Fc inhibits lateral cell migration, vascular endothelial growth factor (VEGF) gradient-driven chemotaxis, capillary-like network formation and sprouting angiogenesis. In turn, soluble EphB4-Fc is pro-adhesive and stimulates EC migration and sprouting angiogenesis. EphrinB2-mediated repulsive signals are transduced by EphB4, as demonstrated by EphrinB2-Fc inhibition of sprouting angiogenesis of constitutively EphB4-overexpressing ECs. Confrontation experiments of EphB4-overexpressing ECs with ECs overexpressing full-length or truncated ephrinB2 that lacks the cytoplasmic catalytic domain demonstrated that forward EphB4 signaling with EphB4 tyrosine phosphorylation restricts intermingling of cells and supports cellular segregation. Taken together, these data identify distinct propulsive and repulsive effector functions of endothelial ephrinB2 and EphB4 that mediate spatial positional signals during angiogenesis and vessel assembly.

Conflict of interest: MH is an inventor on patents related to chimeric antigen receptor technologies that have been filed by the Fred Hutchinson Cancer Research Center

SummaryModels of the outer epithelia of the human body -namely the skin, the intestine and the lung -have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.