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a) Time course of D_cell during a full cycle of hysteresis (blue). Standard deviation of D_cell, measured in two non-overlapping, expanding windows (σ_p for previous and σ_c for current windows) are shown in green and red, respectively. b) Zoomed-in time course of D_cell during a half-cycle of hysteresis, showing large D_cell fluctuations in the active state as the cell re-stabilises on each D_ext increment. Red lines: times of D_ext increment). c,d,e) Long D_cell time-courses of ECs exposed to fixed D_ext. Green line: rarest observed D_cell value, marking the position of the barrier between tip and stalk states (see Fig 3e). Red line: transitions between active/inhibited states. Left: D_ext= 500; EC restricted to the active state. Middle: D_ext= 850; bistable EC stochastically transitioning between active and inhibited states. Right: D_ext= 950; EC restricted to its inhibited state.

a)Violin plots showing that in completely uniform Vegf (no gradient) slows down selection (“time to pattern”) of the in silico for control (0.8) and LOF Vegf (0.4) concentrations compared to simulations with a vegf gradient (Fig 2b,c). Altering filopodial activity fails to rescue this. Blue shading corresponds to estimated probability density of timing values in that range. Upward arrows indicate pattern not achieved within the time range of the simulation for the corresponding parameter settings. Horizontal pink line indicates median value, horizontal black lines indicate middle quartiles, whiskers indicate upper and lower quartiles. N=100 simulations ran for 5000 timesteps.

The process of new blood vessel growth (angiogenesis) is highly dynamic, involving complex coordination of multiple cell types. Though the process must carefully unfold over time to generate functional, well-adapted branching networks, we seldom hear about the time-based properties of angiogenesis, despite timing being central to other areas of biology. Here, we present a novel, time-based formulation of endothelial cell behaviour during angiogenesis and discuss a flurry of our recent, integrated in silico/in vivo studies, put in context to the wider literature, which demonstrate that tissue conditions can locally adapt the timing of collective cell behaviours/decisions to drive different vascular network architectures. A growing array of seemingly unrelated ‘temporal regulators’ have recently been uncovered, including tissue derived factors (e.g. Semaphorins or the high levels of VEGF found in cancer) and cellular processes (e.g. asymmetric cell division or filopodia extension) that act to alter the speed of cellular decisions to migrate. We will argue that ‘temporal adaptation’ provides a novel account of organ/disease-specific vascular morphology and reveals ‘timing’ as a new target for therapeutics. We therefore propose and explain a conceptual shift towards a ‘temporal adaptation’ perspective in vascular biology, and indeed other areas of biology where timing remains elusive.This article is part of the themed issue ‘Systems morphodynamics: understanding the development of tissue hardware’.

Activation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by VEGF binding is critical for vascular morphogenesis. In addition, VEGF disrupts the endothelial barrier by triggering the phosphorylation and turnover of the junctional molecule VE-cadherin, a process mediated by the VEGFR2 downstream effectors T cell-specific adaptor (TSAd) and the tyrosine kinase c-Src. We investigated whether the VEGFR2-TSAd-c-Src pathway was required for angiogenic sprouting. Indeed, Tsad-deficient embryoid bodies failed to sprout in response to VEGF. Tsad-deficient mice displayed impaired angiogenesis specifically during tracheal vessel development, but not during retinal vasculogenesis, and in VEGF-loaded Matrigel plugs, but not in those loaded with FGF. The SH2 and proline-rich domains of TSAd bridged VEGFR2 and c-Src, and this bridging was critical for the localization of activated c-Src to endothelial junctions and elongation of the growing sprout, but not for selection of the tip cell. These results revealed that vascular sprouting and permeability are both controlled through the VEGFR2-TSAd-c-Src signaling pathway in a subset of tissues, which may be useful in developing strategies to control tissue-specific pathological angiogenesis.

The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2Y949F/Y949F leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2Y949F/Y949F mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2Y949F/Y949F mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms., Signals through VEGF receptor 2 (VEGFR2) increase vascular permeability, promoting cancer progression. Here the authors show that a point mutation in VEGFR2 preventing its auto-phosphorylation leads to reduced metastatic spread and improved response to chemotherapy in tumor-bearing mice, without affecting tumor inflammation.