The project was spent on extensive training and research at the Boston University (BU) and Eindhoven University of Technology (TU/e), in collaboration with other international research groups. Below there is a summary of the activities, divided based on Work Packages (WP).
WP1: training and career development
At BU, I performed 2D and 3D in vitro experiments with fibroblasts and endothelial cells (ECs), 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. Transferrable skills were trained by giving group presentations and by writing research proposals. Training at the BU was successfully completed at the end of August 2021 (PD aim I). At the TU/e, I have extended my knowledge on cell biology by participating to internal seminars, and via regular meetings with Prof. Bouten and Prof. Sahlgren. I improved my leadership skills by participating to the “Leadership course for Assistant Professors”. The training to be performed at the TU/e was successfully completed at the end of August 2022 (PD aim I). During the entire project, I have established 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. Therefore, all my PD aims were achieved.
WP2: 2D experiments and simulations
Computational simulations of EC 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 ECs. 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 EC fate selection, thereby causing a decrease in the vascular density resulting from angiogenesis. Moreover, at the TU/e, following the same computational-experimental approach, I uncovered that the Bmp9-mediated expression of Fringe potentiates the interaction between ECs and pericytes, known to be crucial for vessel stabilization.
WP3: computational model validation in 3D
The effects of Bmp9 and Fringe on ECs 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).
During the second year, further simulations (objective 2) showed that Fringe also regulates the rate of EC shuffling during sprouting angiogenesis, known as another determining factor of vessel density. Moreover, the simulations predicted that deletion of Fringe in a fraction of the ECs composing a vessel causes their clustering at the vessel tip. These computational findings were validated via in vivo zebrafish and murine experiments, respectively (objective 3). The murine experiments highlighted a possible link between Fringe and the Bmp9-related disease known as hereditary haemorrhagic telangiectasia, thereby providing possible clinical relevance to the research.
WP4: spatial control of angiogenesis
By using a setup previously developed at the TU/e (Tiemeijer et al. Sci Rep 2018), ECs 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 EC fate selection.
WP5: dissemination/outreach and exploitation
As a result of the activities of this project, I have published 4 peer-reviewed manuscripts, and one manuscript is in preparation. The results were presented at a total of six international conferences and universities. Dissemination articles were published on the ICMS magazine “Highlights” and on the InFlames Flagship blog.
