Periodic Report Summary - ANGIOFISH (New molecular mechanisms involved in vascular maturation and myogenesis in vertebrate development) Summary: The vascular system is the first organ to form and function during embryonic development. Its correct development is essential for the proper formation of vertebrate embryos as well as for adulthood. The mechanisms controlling the different stages of blood vessel development are largely unknown. Objectives of this project are to identify new mechanisms involved in angiogenesis (formation of new vessels) and blood vessel myogenesis (maturation of vessels). To do so we focus on the reciprocal relationship between endothelial cells and their surrounding microenvironment using the embryological and genetic attributes of the zebrafish model system. The goal is to provide new molecular and cellular entry points in normal and pathological angiogenesis. Description of the work performed since the beginning of the project: We have been using a set of new cellular, molecular and genetic approaches as well as advanced microscopy techniques, to elucidate how endothelial and vascular mural cells cooperate to shape the vascular system (angiogenesis) and regulate vascular maturation and myogenesis. So far, the work we have performed has been carried out in three different research lines: 1. new molecular players involved in vascular development and homeostasis 2. uncovering the role of death receptor and nuclear factor kappa beta (NF-kB) signalling in vascular maturation 3. discovering the origin and function of zebrafish vascular mural cells. Description of the main results achieved so far: 1. taking advantage of the zebrafish model system and its powerful genetic tools (e.g. forward genetic) we have identified a new gene with important antioxidant features by analysing a null allele of zebrafish UbiA prenyltransferase domain-containing protein one (Ubiad1), called barolo (bar). Bar mutants show specific cardiovascular failure due to oxidative stress and reactive oxygen species (ROS) mediated cellular damage. Ubiad1 protects against oxidative damage caused by excessive ROS in the heart and endothelial cell. We have preliminary data indicating that Ubiad1 is a non-mitochondrial prenyltransferase that synthesises coenzyme Q10 (CoQ10) in the Golgi membrane compartment. Loss of Ubiad1 in cardiovascular cells reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation. These findings identify Ubiad1 as a new CoQ10 prenyltransferase with specific cardiovascular protective function. By buffering ROS signalling Ubiad1 may also play important functions in cell metabolism, aging and cancer. Since several Ubiad1 variants are associated with Schnyder crystalline corneal dystrophy (SCCD) we are currently investigating the functional role of these SCCD in rescuing the absence of Ubiad1 in vivo. We also expect to address the role for CoQ10 in SCCD. 2. we are investigating the role of death receptor signalling in vascular homeostasis. We previously showed that inhibitor of apoptosis proteins (IAPs) regulate survival versus apoptosis in endothelial cells. Our aim is here to identify new players among the tumour necrosis factor (TNF) signalling platform that may be involved in regulation of angiogenesis. Therefore, we screened by gene 'knock-down' in zebrafish embryos most of the elements of the IAPtosome platform. Among the most interesting genes we found receptor-interacting serine/threonine-protein kinase one (RIPK1). We are currently investigating the role of RIPK1 in vascular development and homeostasis. 3. the last project is related to one of the original objectives: identification of cellular mechanisms involved in vascular mural cells (SMCs) development and differentiation. Here we have generated a transgenic line in zebrafish expressing the fluorescent protein 'mcherry' in all smooth muscle cells, both vascular mural cells and visceral smooth muscle cells. This Tg line will be very important to further describe the role of genes and micro-ribonucleic acid (miRNA) in mural cells development. We recently discovered that miRNA-143 and -145 are enriched in zebrafish mural cells and, that they play important role for SMC development and differentiation. To study the developmental origin of smooth muscle cells in the last two years we have recently and successfully set up in the lab the 'Cre/loxP-Rainbow' technology in zebrafish. We are currently testing the effect of miRNA 143/145 in zebrafish mural cells development using the 'Cre/LoxP-Rainbow' technology. Expected final results: The long-term goal is to provide additional molecular entry points to further investigate angiogenesis and vascular mural cell development/differentiation in normal and pathological conditions using the zebrafish vertebrate system. From each of these projects we are expecting to have a publication in peer-review journals. The first project started with the positional cloning of barolo and the identification of Ubiad1 as a new antioxidant gene and is now in the last stage of revision for the journal Cell. Due to our success we are also planning to apply for an international patent application regarding the use of Ubiad1 to counteract the side effect of statins. The second project on smooth muscle cells is currently under investigation. We have already published a review on miRNA in zebrafish development. Another research article will be submitted within the next six-month on the role of miRNA-145 in smooth muscle cell differentiation. In addition, we are expecting that over the next two years and with this grant we will be able to better define the role of RIPK1 in zebrafish angiogenesis. All projects look quite promising and may possibly open up a new field of discovery for endothelial cell biology and cardiovascular homeostasis. In addition we are expecting to increase our collaborations with international laboratories focused on angiogenesis in different animal models. As soon as our own results are published I expect to be invited as a speaker in an array of international meetings. My expectation is that in the foreseeable future we will establish several international and national collaborations, which will allow my group to publish in important peer-review journals (e.g. Molecular Cell, PLOS One, Blood, Evolution & Development, etc.). Contribution to the pathogenesis and therapy of the SCCD, a rare autosomic genetic disease associated to mutations in the Ubiad1 gene in humans. As soon as we have patent approval I expect to be able to provide new grounds for socio-economic investments as transfer technology, e.g. generation of 'spin-off' companies from the University of Torino. The Molecular Biotechnology Centre has previous experiences and know-how in such a transfer technology aspect.
