This project aimed to study factors influencing vascular patterning in vivo.
Our first aim was to understand and manipulate endothelial flow-dependent polarity during vascular patterning and vascular homeostasis. To do so, we have been setting up a live imaging system using a 2-photon microscope and the Endo-GNrep mouse strain (allowing endothelial-specific activation of fluorescent reporters for Golgi apparatus, nucleus) and the mTmG strain to visualize the endothelial cell boundaries. Unfortunately, our original line did not provide a strong enough signal to perform the experiment planned. Thus, we are developing a new mouse strain (allowing endothelial-specific activation of stronger fluorescent reporters for Golgi apparatus, nucleus, and apical membrane protein ICAM-2). Live imaging protocols in adult mice (cranial window and ear model) have been optimized. Protocols to study stroke in this context and trigger blood flow variations are also optimized and ready to use as soon as the new reporter strain is made available.
Our second aim was to reveal the importance of endothelial flow-dependent polarity in arteriovenous malformations (AVMs). Briefly, we developed a new non-genetic model of AVMs that allowed us to study both the formation and regression of AVMs. We demonstrated that migration, cell size, and PI3K signaling are important for the formation of the shunts while proliferation is not. Interestingly, despite the role of cell migration, endothelial polarity in the shunts was not as affected as one could have thought and was close to the one observed in veins. The resolution of the shunts was also shown to be dependent on endothelial migration and not on apoptosis. These observations are currently being transposed into a common genetic model of AVMs before being published.
The third aim of this action was to identify activators and inactivators of endothelial flow-dependent polarity after developing a software for automatic 3D analysis of the retina vascular network including endothelial polarity, blood flow simulation, endothelial density, etc. This software has been partially released after the development of a machine learning script in collaboration with the Instituto Superior Tecnico of Lisbon (Barbacena et al., 2019; Narotamo et al. 2021). The final version will be released as soon as all the features are ready to be used. As the software development took longer than expected, we did not perform a screen of activators and inactivators of endothelial flow-dependent polarity yet. However, we identified an important competition between blood flow and VEGF for endothelial polarity (preprint, Barbacena et al., 2022) and excluded vesicular trafficking from affecting endothelial polarity (work in progress, Ouarné et al., 2022). We also participated in a study to perform better imaging of the retinal vascular network (Prahst et al., 2020).
Finally, in order to ease the access to vascular patterning study, we wrote two reviews about the methods currently available (Pena et al., 2021) and the current knowledge on vascular patterning (Ouarné et al, 2021).
