Periodic Reporting for period 3 - AngioMature (Mechanisms of vascular maturation and quiescence during development, homeostasis and aging) Reporting period: 2021-08-01 to 2023-01-31 Summary of the context and overall objectives of the project The maintenance of a mature, quiescent, and organotypically-differentiated layer of endothelial cells (EC) lining the inside of all blood vessels is vital for human health. Indeed, vascular dysfunction is directly or indirectly associated with two third of all cases of human mortality. The study of vascular dysfunction has during the last decades primarily focused on activation mechanisms of vessel lining endothelial cells. Yet, unlike vascular activation, the mechanisms of vessel maturation and quiescence are much less well understood. The goal of the Angiomature project is to identify, validate, and implement novel mechanisms of vascular maturation and organotypic EC differentiation that are active during development, maintenance of vascular stability in adults, and undergo changes in aging. The project thereby aims at generating fundamental insight into the molecular mechanisms of vascular function to thereby pave the way for novel paradigms in prevention and therapy. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The Angiomature project is structured to pursue three interrelated research lines (RL). RL1 is aimed at performing bulk and single cell analyses of the transcriptional and epigenetic program(s) of vascular maturation and organotypic differentiation during adolescence and aging. RL2 focusses on the analysis of the functional consequences of program(s) and candidate molecules in cellular systems as well as in differentiating EC of adolescent mice and differentiated EC of adult mice and their adaptation to challenge. The most ambitious and logistically most challenging RL3 studies changes of maturation and differentiation program(s) and vascular responses during aging. All three RLs have in the reporting period yielded important and in part groundbreaking discoveries. In RL1, we could in preclinical mouse models deconvolute on the systems level EC transcriptional programs (i) in the pre-metastatic and metastatic niches of the lung, (ii) spatially resolved in the homeostatic and challenged liver, and (iii) in the lungs during systemic aging. These experimental approaches yielded comprehensive systems maps that were in the liver expanded by proteomics and phosphoproteomics analyses to enable comprehensive datamining for novel organotypically and challenge-specifically expressed EC molecules to feed the pipeline for functional validation and proof-of-concept exploitation, which is pursued in RL2. RL2 led to important discoveries of organotypic angiocrine EC functions, notably, we (i) identified and validated Tie1 function blocking antibody Tie1-39, (ii) used Tie1-39 to discover a functional Angpt/Tie/Wnt signaling axis controlling angiocrine signaling and functional metabolic zonation in the liver, (iii) discovered the TGFß pathway modifier LRG1 as critical EC-derived regulator of pre-metastatic niche conditioning, (iv) established conditional EC mutants of Smad6, Smad7 and DNMT3a. In RL3, we made the surprising finding that aged mice have significantly less metastases than young mice and that this differential can be mechanistically linked to the aging vasculature. 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) We were very fortunate that the Angiomature project got well off the ground since qualified and project-specifically trained manpower was readily available and many of the genetic tools and techniques had been in place at the start of the project. We thereby managed to rapidly make important discoveries that enabled the completion and publication of projects that had already been ongoing at the beginning of the project. Likewise, some of the experimental lines started at the beginning of the project on the basis of the project-specific preliminary data made very rapid progress. Notably, the lung and liver endothelial screening and functional validation work yielded groundbreaking discoveries that are at the time of this report already in advanced stages of the revision process and are expected to be open-access published later in 2021, notably the LRG1 project as well as the Angpt/Tie/Wnt signaling loop work. As for the most ambitious long-term goals, we have since inception of the project established all the necessary tools and genetic reagents based on which we now perform screenings and functional validation experiments (including TGFß pathway regulators and epigenetic modulators). These ongoing experiments define the perspective of the second 30-months period of the project. We will continue to generate, integrate and systems analyze the multiomics data generated under RL1 (including extensive exploitation of the spatial phosphoproteomics technology developed in the project). The ongoing functional experiments of RL2 will be continued as outlined. Most importantly, we hope to finish the RL3 aging vascular control of metastasis experiments until late 2022. Collectively, we are confident that the Angiomature project will live up to its high ambition and expectations to generate paradigm-changing and translationally-relevant molecular and mechanistic insight into blood vessel functions controlling health and disease over the lifespan.