Final Report Summary - EC METABOLISM (Understanding the metabolism of endothelial cells underlying physiological and pathological angiogenesis) In our original proposal, we began with the desire to understand the cellular metabolism of endothelial cells (ECs) underlying physiological and pathological angiogenesis. Our central hypothesis was that there should be differences in the basal metabolism of ECs of arterial, venous and lymphatic origin, owing to the different environmental concentrations of oxygen and nutrients, as well as their different physiological functions. In this proposal, we have identified that lymphatic ECs (LECs) have higher fatty acid beta-oxidation flux compared to venous or arterial ECs. Contextually, this is important, as the lymph has a higher lipid content than the blood (1), and it has been shown that the lymphatics are important in regulating reverse cholesterol transport in atherosclerosis (2, 3). Further, although LECs are present in low oxygen environments, we found that hypoxia does not alter FAO flux in LECs. As we discovered that FAO is increased in LECs, we sought to determine whether FAO may have a role in lymphatic development, in vivo, and utilized a zebrafish model of vascular development. Using morpholinos targeting the zebrafish isoforms of CPT1a, we determine that knockdown of CPT1a in the zebrafish impaired lymphatic development, as early as the lymphangiogenic precursor secondary sprouts, as well as in the formation of the thoracic duct, the first perfused lymphatic structure in the zebrafish.To further explore the role of FAO in LEC differentiation, we used the well-established model of Prox1-mediated LEC differentiation in vitro. Here, we provide the first evidence that the same genetic signal which induces LEC differentiation also directly specifies the increased FAO in LECs. Further, we have detailed that this occurs not through regulation of energy generation or redox homeostasis, but rather through the production of acetyl-CoA, which fuels histone acetylation, an epigenetic modification, which can promote the transcription of lymphatic genes. The results from this work has been presented in several international meetings, and is currently in revision in the journal Science (4). It has also facilitated the training of several junior and senior laboratory members, several national and international collaborations, and the application of an operating grant to fund further investigation in this research direction.Further, as part of this research proposal, we have generated several lymphatic-specific, inducible transgenic mouse lines to specifically inhibit FAO in vivo. We are currently phenotyping these mice as part of our revision experiments for our submission to Science.Finally, the recruited research has been an instrumental part of several key publications in the laboratory, including a series of three papers published in Cell (5), Cell Metabolism (6) and Cell Cycle (7), detailing the role of glycolysis in venous EC growth and function, and the therapeutic potential of transient and partial inhibition of glycolysis in pathological angiogenesis. These works are currently being further investigated to develop drug targets for clinical translation. As well, the recruited researcher was co-author on a review article in the journal Nature, which overviews the “Metabolism of stromal and immune cells in health and disease” (8), a publication which has already been viewed almost 17,000 times, despite having been published only since July 2014.Overall, this proposal has been a great success, where the recruited researcher has been able to not only contribute on ongoing research efforts to demonstrate the therapeutic efficacy of targeting metabolism in angiogenesis, but also develop a novel research direction around another metabolic pathway in cellular differentiation. As angiogenesis and lymphangiogenesis play crucial roles in both health and a number of diseases (such as cancer, atherosclerosis and diabetes), the future socioeconomic impact of this work is great.References1. Randolph GJ, Miller NE, Lymphatic transport of high-density lipoproteins and chylomicrons. J Clin Invest 124, 929-935 (2014).2. Lim HY et al., Lymphatic vessels are essential for the removal of cholesterol from peripheral tissues by SR-BI-mediated transport of HDL. Cell Metab 17, 671-684 (2013).3. Martel C et al., Lymphatic vasculature mediates macrophage reverse cholesterol transport in mice. J Clin Invest 123, 1571-1579 (2013).4. Wong BW et al., Role of fatty acid beta-oxidation in lymphatic endothelial cell differentiation. Science, (in revision). (2014).5. De Bock K et al., Role of PFKFB3-driven glycolysis in vessel sprouting. Cell 154, 651-663 (2013).6. Schoors S et al., Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis. Cell Metab 19, 37-48 (2014).7. Schoors S et al., Incomplete and transitory decrease of glycolysis: a new paradigm for anti-angiogenic therapy? Cell Cycle 13, 16-22 (2014).8. Ghesquiere B et al., Metabolism of stromal and immune cells in health and disease. Nature 511, 167-176 (2014).