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CORDIS - Resultados de investigaciones de la UE

Investigating the crosstalk between Notch and YAP/TAZ in sprouting angiogenesis

Periodic Reporting for period 1 - Angio-NYT (Investigating the crosstalk between Notch and YAP/TAZ in sprouting angiogenesis)

Período documentado: 2020-09-01 hasta 2021-08-31

Angiogenesis is the process leading to the formation of new blood vessels from pre-existing ones. This phenomenon is crucial for health and diseases, such as cancer and ischemia. Regulation of angiogenesis is thus key for numerous medical treatments, but an optimal control still needs to be achieved. To this aim, a more complete understanding of the regulatory mechanisms is necessary.

Notch is a cell-cell signalling that strongly regulates angiogenesis; Notch spatiotemporal dynamics influences the vascular network topology that results from angiogenesis. Identifying new spatiotemporal regulators of Notch dynamics might lead to the development of new strategies to control angiogenesis and, thus, new medical treatments. In this project, by combining experiments and simulations, I aim to identify new techniques to spatiotemporally control angiogenesis, via Notch regulation. This is achieved by: (objective 1) experimentally characterizing Notch and its cross-talking signalling pathways; (objective 2) developing computational models of angiogenesis accounting for Notch with its cross-talking pathways; and (objective 3) validating the computational findings via experiments. In terms of professional development (PD), I aim to: (PD aim I) train in cell experiments and computational modelling of angiogenesis; (PD aim II) establish new international collaborations; and (PD aim iii) obtain a faculty position at a European university.
The first year (outgoing phase) was spent on extensive training and research at the Boston University (BU), in collaboration with the Eindhoven University of Technology (TU/e) and other international research groups. Below there is a summary of the activities, divided based on Work Packages (WP).

WP1: training and career development
At the Boston University, I trained in performing 2D and 3D in vitro experiments with fibroblasts and endothelial cells, using microfluidic devices custom-made in the lab of Prof. CS Chen (BU). Dr. K Bentley (BU and Francis Crick Institute) trained me in modelling angiogenesis, explaining the importance of capturing the spatiotemporal dynamics of Notch in this process. Transferrable skills were trained by giving group presentations and by writing research proposals. The training at the BU was successfully completed at the end of August 2022 (PD aim I). During this period, I have been part of international collaborations with Dr. B Larrivée from the University of Montreal and Dr. SP Herbert from the University of Manchester (PD aim II). Finally, in March 2021, I was appointed tenure-track assistant professor at the TU/e (PD aim III) and at the same time continued my training and research at the BU until the 31st of August 2021.

WP2: 2D experiments and simulations
Computational simulations of endothelial cell behaviour as influenced by Notch and Bmp9 (a signalling pathway involved in angiogenesis) were performed to investigate previously published experiments (Larrivée et al. Dev Cell 2012). The simulations suggested the existence of a previously uncovered link between Notch and Bmp9. In particular, the results suggested that Bmp9 upregulates Fringe, an enzyme that increases the rate of Notch activation by its ligands. Together with collaborators, we successfully validated (objective 3) these simulations with new 2D in vitro experiments on endothelial cells. Therefore, the discovery of a new feature characterizing the crosstalk between Notch and Bmp9 was confirmed (objective 1). A computational model accounting for Bmp9 and Fringe in angiogenesis was developed as a result (objective 2). New computational simulations predicted that Bmp9, acting on Notch via Fringe, decelerates the process of endothelial cell fate selection, thereby causing a decrease in the vascular density resulting from angiogenesis. Summarizing, these two work packages were successfully completed by performing (WP3) and experimentally validating (WP2) simulations which, together with the experiments, led us to establish that Bmp9 induces Notch activation by increasing Fringe expression. The simulations further suggested that this phenomenon decelerates the endothelial cell fate selection during angiogenesis, causing a decrease in the resulting vascular density.

WP3: computational model validation
The effects of Bmp9 and Fringe on endothelial cells and angiogenesis were validated by using a 3D in vitro angiogenesis bead assay. As predicted by the simulations, the experiments validated that Bmp9 reduces the density of sprouting and, importantly, knocking down Fringe can partially re-establish the physiological density (objective 3). Preliminary zebrafish experiments were performed to verify the computational findings in vivo.

WP4: spatial control of angiogenesis
By using a setup previously developed at the TU/e (Tiemeijer et al. Sci Rep 2018), endothelial cells were constrained during culture to form lines of cells, to mimic capillaries in 2D; thereafter, cells were exposed to microcontact printed lines of beads coated with two Notch ligands, Dll4 and Jag1. The experiments showed that, only lines of Dll4 can control the location of endothelial sprouting. To understand these results, simulations of Notch as affected by the Dll4 and Jag1 lines were performed. The simulations were able to qualitatively replicate the experimental results. Their analysis suggested that Dll4 and Jag1 have a different potential to control angiogenesis because they elicit different temporal dynamics of Notch and endothelial cell fate selection.

WP5: dissemination/outreach and exploitation
I am (co-)first author of the manuscript describing the experiments and simulations that uncovered the fact that engineered lines of Dll4 and Jag1 have different potential to control endothelial sprouting because they induce different temporal dynamics of Notch signalling (Tiemeijer & Ristori et al., iScience, 2022). Finally, I contributed to a review on Notch signalling mechanosensitivity in tissue engineering (Karakaya & van Asten et al. Biomech Model Mechanobiol 2022).
Overall, the project has so far determined that, to spatially control endothelial sprouting: (i) lines of Dll4 ligands are more effective compared to lines of Jag1 ligands; (ii) to increase the efficiency of the spatial control, the temporal dynamics of Notch signalling needs to be controlled; and (iii) the temporal dynamics of Notch can be influenced by Bmp9, via upregulation of the expression of Fringe. These novel findings are relevant not only for cell biology and tissue engineering, providing guidelines for the spatiotemporal control of endothelial sprouting, but also for clinical applications. In fact, Bmp9 and Notch signalling are both involved in hereditary hemorrhagic telangiectasia, an inherited disorder that causes arteriovenous malformations. The newly uncovered link between Bmp9 and Notch, mediated by Fringe, might lead to a deeper understanding of hereditary hemorrhagic telangiectasia, and provide inspiration for new medical therapies. The possible link between hereditary hemorrhagic telangiectasia and Fringe will be explored in the following part of the project. To disseminate the results, the studies will be published as open access and presented at international conferences and seminars.
Combining experiments and simulations of endothelial cells in angiogenesis led to new discoveries.

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