Periodic Reporting for period 1 - ADAPT2FLOW (Adaptive and maladaptive endothelial cell dynamics during blood flow-driven vascular patterning) Reporting period: 2019-10-01 to 2021-09-30 Summary of the context and overall objectives of the project The formation of a functional patterned vascular network (well-defined arteries, veins, and capillaries) is essential for blood vessel functioning and global health. Several human vascular disorders arise from the mis-patterning of blood vessels, such as arteriovenous malformations, aneurysms, and diabetic retinopathy. Blood flow is recognized as the main inducer for vascular patterning, yet very little is known about the molecular mechanisms that vascular patterning. Endothelial cells (the cells bordering vessels) polarize and migrate against the blood flow direction. Yet, how this behavior contributes to the overall process of vascular patterning is completely unknown.It is essential to decipher how endothelial cell polarization and migration against blood flow contributes to vascular patterning as it will provide leads to produce new treatments. Indeed, those new therapies will allow improving the recovery of vascular diseases and might help to prevent some to occur like strokes in predisposed patients.This project aimed to study the dynamics of endothelial cells in vascular patterning during development, normal blood vessel functioning, and disease. Thanks to this action, we developed new tools to study endothelial cell dynamics in vascular patterning in developing and developed vascular networks. We identified an important competition between blood flow and VEGF (an essential factor of blood vessel development) in the control of these dynamics in the developing networks. More interestingly, we also developed a new model for a known vascular disorder that is arteriovenous malformations, and demonstrated that some cellular behaviors are essential to their formation and their resolution which could lead the scientific community to the production of pro-resolution treatments for patients with identified arteriovenous malformations. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 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). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This project allowed the development of new tools and models to study vascular patterning in vascular development, homeostasis, and disease (AVMs) as explained in the previous section. We have shown that some mechanisms not yet identified are essential in the formation and resolution of AVMs leading to new perspectives in the treatment of diseases involving these vascular malformations. We aim to complete this work in the future by using the tools created to study vascular diseases as stroke and developmental processes in order to identify more activators and inactivators of vascular patterning to be potential leads to new treatments of vascular pathologies. automatic analysis of endothelial polarity vascular regression
