Periodic Reporting for period 4 - AXIAL.EC (PRINCIPLES OF AXIAL POLARITY-DRIVEN VASCULAR PATTERNING) Reporting period: 2021-03-01 to 2022-02-28 Summary of the context and overall objectives of the project The formation of a functional patterned vascular network is essential for development, tissue growth and organ physiology. Several human vascular disorders arise from the mis-patterning of blood vessels, such as arteriovenous malformations, aneurysms and diabetic retinopathy. Although blood flow is recognised as a stimulus for vascular patterning, very little is known about the molecular mechanisms that regulate endothelial cell behaviour in response to flow and promote vascular patterning. Recently, we uncovered that endothelial cells migrate extensively in the immature vascular network, and that endothelial cells polarise against the blood flow direction (Franco PLoS Biol 2015, Franco eLIFE 2016). AXIAL.EC project aims at investigating how this newly discovered vascular plasticity is regulated and its relationship to vascular malformations. Vascular malformations include arteriovenous malformations, low-flow venous malformations, cavernous malformations, and aneurysms, are all vascular anomalies that arise from maladaptive connectivity of vascular networks. Vascular malformations have a great impact on human health, but so far have very poorly defined etiologies. AXIAL.EC integrative approach, based on high-resolution imaging and unique experimental models, will provide a unifying model defining the cellular and molecular principles involved in vascular adaptation and maladaptation in the context of vascular malformations. Identifying cellular and molecular mechanisms leading to formation, development, and regression of vascular malformations could lead to new clinical treatments.AXIAL.EC project aims at investigating how this newly discovered vascular plasticity is regulated and its relationship to vascular malformations. Vascular malformations include arteriovenous malformations, low-flow venous malformations, cavernous malformations, and aneurysms, are all vascular anomalies that arise from maladaptive connectivity of vascular networks.Vascular malformations have a great impact on human health, but so far have very poorly defined etiologies. Identifying cellular and molecular mechanisms leading to formation, development, and regression of vascular malformations could lead to new clinical treatments.AXIAL.EC project aims at understanding the dynamic behaviour of endothelial cells during vascular adaptation and maladaptation; controlling and restoring vascular adaptation by regulating flow-mediated polarisation of endothelial cells; identifying novel regulators of flow-dependent endothelial cell polarisation. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Within the ERC project, we have established a novel reporter mouse line to observe and manipulate endothelial polarity in vivo (Aim1a; Barbacena et al. Genesis 2019 and Bernabeu et al. Biophysic J 2018). We used this tool to investigate how polarisation and coordination of endothelial cells movements are orchestrated to generate vascular patterning (Narotamo et al. Annu Int Conf IEEE Eng Med Biol Soc. 2021; Park et al. Circulation 2021; Figueiredo et al. PNAS 2021; Edgar et al. Plos Comp Biol 2021). In addition, related to Aim2 and Aim3, we have manipulated cell polarity using Pard3 and Prkci models, and found that Par polarity complex is important for flow-dependent polarisation and prevents atherosclerosis (Takao et al. EMBO rep. 2018). At the molecular level, we found that mechanotransduction at adherens junctions promotes VEGF-dependent and inhibits flow-dependent collective polarity of endothelial cells in a competitive manner (Carvalho et al. eLIFE 2019; Barbacena et al. BioRxiv 2021). In addition, we published review articles highlighting the role of cell migration, polarity and flow responses in vascular morphogenesis (Fonseca et al. Vasc Biol 2020; Pena et al. Curr Opin Hematol. 2021; Ouarné M et al. Cell Dev. 2021).We deposited one patent, protecting the mouse tool generated within this work, and we are in the process of submitting a second patent protecting a novel microfluidic device enabling high-throughput screenings under high-shear conditions.Engagement with the society and scientific communities was regularly maintained, either through conferences, site visits, webinars, engagement with schools, and interviews for newspaper or TV channels. 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) AXIAL.EC integrative approach, based on high-resolution imaging and unique experimental models, will provide a unifying model defining the cellular and molecular principles involved in vascular adaptation and maladaptation in the context of vascular malformations. Identifying cellular and molecular mechanisms leading to formation, development, and regression of vascular malformations could lead to new clinical treatments polarized endothelial cell in a blood vessel
