Abstract
The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair, and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation, and migration. Extracellular signal-regulated kinase (Erk) is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response, however, involves a Vegfr-dependent mechanism that initiates concomitantly with the wound inflammatory response. This work reveals a highly dynamic sequence of signalling events in regenerative angiogenesis and validates a new resource for the study of vascular Erk-signalling in real-time.
Highlights
The formation of new blood vessels from pre-existing vasculature is a fundamental process central in the formation of a viable embryo and in the pathogenesis of many diseases (Carmeliet and Jain, 2011; Chung and Ferrara, 2011; Potente et al, 2011)
With the intermediate dose of 4 mM SU5416, while the closest cell to the wound site still displayed Extracellular signal-regulated kinase (Erk) activity, as did the second cell from the wound site, the third and farthest cells from the wounded sites were inhibited (Figure 5F,H, Figure 5—figure supplement 1G–H’). These results suggest that there is a gradient of Vegfr/Erk-signalling activity in the ablated intersegmental vessels (ISVs) endothelial cells (ECs) resulting in higher Vegfr/Erk activity in ECs closer to the wounded site, which can only be inhibited with SU5416 at higher concentrations
To understand how dynamically Erk activity is regulated in developing vasculature, we generated the EC-EKC transgenic line and validated its use as a proxy readout of active Erk-signalling in vasculature
Summary
The formation of new blood vessels from pre-existing vasculature (angiogenesis) is a fundamental process central in the formation of a viable embryo and in the pathogenesis of many diseases (Carmeliet and Jain, 2011; Chung and Ferrara, 2011; Potente et al, 2011). The KTR system allows rapid quantifiable measurements of ERK activity based upon subcellular localisation of a fluorescent fusion protein and is more sensitive to phosphatase-mediated kinase activity downregulation when compared to other commonly used reporters This has been applied to enable dynamic ERK-signalling pulses to be analysed at single-cell resolution both in vitro and in vivo (Regot et al, 2014; de la Cova et al, 2017; Mayr et al, 2018; Goglia et al, 2020; Pokrass et al, 2020; De Simone et al, 2021), where it has been demonstrated to be of high utility. This work reports a unique resource for imaging of vascular signalling and further illuminates mechanisms of vascular regeneration following wounding
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
