Katie Bentley

Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR–Dll4–Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.

Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD+-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.

Tipping the Balance: Robustness of Tip Cell Selection, Migration and Fusion in Angiogenesis

Vascular abnormalities contribute to many diseases such as cancer and diabetic retinopathy. In angiogenesis new blood vessels, headed by a migrating tip cell, sprout from pre-existing vessels in response to signals, e.g., vascular endothelial growth factor (VEGF). Tip cells meet and fuse (anastomosis) to form blood-flow supporting loops. Tip cell selection is achieved by Dll4-Notch mediated lateral inhibition resulting, under normal conditions, in an interleaved arrangement of tip and non-migrating stalk cells. Previously, we showed that the increased VEGF levels found in many diseases can cause the delayed negative feedback of lateral inhibition to produce abnormal oscillations of tip/stalk cell fates. Here we describe the development and implementation of a novel physics-based hierarchical agent model, tightly coupled to in vivo data, to explore the system dynamics as perpetual lateral inhibition combines with tip cell migration and fusion. We explore the tipping point between normal and abnormal sprouting as VEGF increases. A novel filopodia-adhesion driven migration mechanism is presented and validated against in vivo data. Due to the unique feature of ongoing lateral inhibition, ‘stabilised’ tip/stalk cell patterns show sensitivity to the formation of new cell-cell junctions during fusion: we predict cell fates can reverse. The fusing tip cells become inhibited and neighbouring stalk cells flip fate, recursively providing new tip cells. Junction size emerges as a key factor in establishing a stable tip/stalk pattern. Cell-cell junctions elongate as tip cells migrate, which is shown to provide positive feedback to lateral inhibition, causing it to be more susceptible to pathological oscillations. Importantly, down-regulation of the migratory pathway alone is shown to be sufficient to rescue the sprouting system from oscillation and restore stability. Thus we suggest the use of migration inhibitors as therapeutic agents for vascular normalisation in cancer.

VEGFRs and Notch: a dynamic collaboration in vascular patterning

ECs (endothelial cells) in the developing vasculature are heterogeneous in morphology, function and gene expression. Inter-endothelial signalling via Dll4 (Delta-like 4) and Notch has recently emerged as a key regulator of endothelial heterogeneity, controlling arterial cell specification and tip versus stalk cell selection. During sprouting angiogenesis, tip cell formation is the default response to VEGF (vascular endothelial growth factor), whereas the stalk cell phenotype is acquired through Dll4/Notch-mediated lateral inhibition. Precisely how Notch signalling represses stalk cells from becoming tip cells remains unclear. Multiple components of the VEGFR (VEGF receptor) system are regulated by Notch, suggesting that quantitative differences in protein expression between adjacent ECs may provide key features in the formation of a functional vasculature. Computational modelling of this selection process in iterations, with experimental observation and validation greatly facilitates our understanding of the integrated processes at the systems level. We anticipate that the study of mosaic vascular beds of genetically modified ECs in dynamic interactions with wild-type ECs will provide a powerful tool for the investigation of the molecular control and cellular mechanisms of EC specification.

A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfcum18 could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.

Integrated simulation with experimentation is a powerful tool for understanding diatom valve morphogenesis.

We present a number of promising examples of computational studies, which improve our understanding of the morphogenesis process of diatom cell walls. Each example considers a different physical scenario whereby computational and mathematical models are used to evaluate hypotheses pertaining to diatom valve formation; considering the roles of cytoskeletal elements, interactions between cell components that might generate patterned structures from the submicron (nanoscale) to cell level, and the effect of environmental variables. We propose that the complex, multiscale phenomenon of diatom valve morphogenesis requires better integration of computational/mathematical and experimental procedures if we are to untangle all the contributing processes. Finally we outline a plan for future directions, to achieve this integration and further the field of diatom morphogenesis research.

An empirical study of a deliberation dialogue system

We present an empirical simulation-based study of the use of value-based argumentation in two-party deliberation dialogues, investigating the impact that argumentation can have on the quality of the outcome reached. Our simulation allows us to vary the number of values, actions and arguments that appear in the system; we investigate how the behaviour of the system changes as these parameters vary. This parameter sensitivity analysis tells us whether a value-based deliberation dialogue system may be useful for a particular real-world application. We measure the quality of the dialogue outcome (i.e. the action that the agents agree to) against a global view of whether that action would be agreeable to each agent if all of the agents knowledge were taken into account. We compare the deliberation outcome with a simple consensus forming procedure (where no arguments are exchanged). Our results show that the deliberation dialogue system we present outperforms consensus forming.

Morphological plasticity: environmentally driven morphogenesis

This paper focuses on the environmental role in morphogenesis in dynamic morphologies (DM). We discuss the benefits of morphological plasticity (MP) and introduce our Environment-Phenotype Map (E-P Map) framework in order to investigate and classify the continual development in DMs and morphologically adaptive behaviour. We present our MP-capable system the Artificial Cytoskeleton (ArtCyto), housed within our DM the ‘Cellanimat’, with an E-P Map closely based on MP examples from cell physiology. We provide experimental results to demonstrate that with this single E-P Map a bifurcation in morphology can occur, caused only by a difference in the environment, mirroring evidence from physiological data of fibroblast cell chemotaxis and macrophage cell phagocytosis.

DIATOM COLONY FORMATION: A COMPUTATIONAL STUDY PREDICTS A SINGLE MECHANISM CAN PRODUCE BOTH LINKAGE AND SEPARATION VALVES DUE TO AN ENVIRONMENTAL SWITCH1

The morphological plasticity and adaptive behavior exhibited during diatom colony formation in Aulacoseira is explored through computer simulation to study how the interplay of mechanisms such as cytoskeletal‐driven membrane protrusions, silica deposition, and environmental factors may contribute to the generation of two distinct spine morphologies on linkage and separation valves. A multiscale agent‐based computational model was developed, which showed that a single cytoskeleton‐driven, competitive growth mechanism could generate either of the two characteristic phenotypes, given only a single switch in the environment (as might be experienced by a change in light regime). Hypotheses are formulated from the model, and predictions made for potential follow‐up experiments.

Do Endothelial Cells Dream of Eclectic Shape

Endothelial cells (ECs), which line our blood vessels, exhibit dramatic plasticity and diversity of form/behavior at the individual and collective cell level. They re-organize themselves in space and time to extend new blood vessel networks during development and during a huge array of diseases including cancer. Here we will describe, using examples from our integrated in silico/in vitro/in vivo research program, how the Artificial Life (ALife) perspective and approaches have been paramount in driving entirely new experimental biology understanding of the vasculature by capitalizing on the emergent, predictive capacity and testable nature of agent-based models in close combination with in vitro and in vivo experiments. Our agent-based simulations explicitly consider the role of individual EC embodiment, active perception, heterogeneous vs homogeneous collective dynamics, pattern formation and counter-intuitive emergence from feedback in controller networks and many more Alife centric concepts. We recently...