Periodic Reporting for period 4 - rEnDOx (REDOX SIGNALING AND METABOLIC STATES IN ANGIOGENESIS IN HEALTH AND DISEASE) Reporting period: 2020-10-01 to 2022-03-31 Summary of the context and overall objectives of the project We generated new tools to visualize and quantify the dynamics of redox signaling and metabolic state occuring in blood vessels of living animals. We address the role of the different subcellular compartments such mitochondria, golgi, plasmamembrane, nuclei in endothelial redox and metabolic signaling. We also provide experimental evidences of differences in redox signaling and metabolic conditions of normal vs tumor vessels in vivo by defining a reference redox state and metabolic condition in human ECs and blood vessels. We have performed a global quantification of reduced and oxidized cysteine residues of all endothelial proteins by performing the first redox proteomic analysis in human ECs. New molecular targets involved in metabolism and redox homeostasis of endothelial cells has been identifies and will be investigated.We are investigating the role for Ubiad1 in pathological angiogenesis by define a novel role for Ubiad1 in blocking tumor growth by inhibiting tumor angiogenesis. We aim to provide experimental proofs that therapies based on redox enzymes (e.g. Ubiad1) inactivation/blockade are valuable alternatives strategies for anti-angiogenesis in cancer therapy. We are shedding lights on redoxome changes induced by lack of UBIAD1 and indication of its molecular function. We expect to identify known (e.g. VEGFR) but also unknown endothelial-specific enzymes/transducers whose function is regulated by UBIAD1-mediated redox signaling. A positive outcome for these experiments will give a strong rationale to identify UBIAD1 inhibitors by small molecule screening, ultimately offering the possibility for industrial valorisation.We demonstrate the existence of specific metabolites of the mevalonate pathways controlling angiogenesis, such as IPP and DMAPP. We generate and characterize zebrafish mutants and inducible cre/loxP mouse Tg lines for modelling mevalonate metabolism in physiological and pathological angiogenesis. We did that by identifying and manipulating IDI1 and FDPS metabolic enzymes (within the mevalonate pathway whose loss-of function has anti-angiogenic effects). We are working to provide evidences that manipulation of these enzymes of mevalonate pathway by loss-of-function approaches might be a new therapeutic way to block neoangiogenesis in tumors by improving anti-VEGF cancer therapy. Our data could offer a solid rationale to screen for new mevalonate pathway inhibitors, ultimately offering the possibility for industrial valorisation. The objectives of the project are those initially proposed. A better understand of the role of redox homoeostasis in endothelial cells as well as in cancer cells represent an urgent need for the society. The possibility to alter redox state with antioxidants and supplement need to be explored and fully comprehend. We aim to continue to do that in this grant. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Since the beginning of the project, we have been working on four research tasks: In Task 1) we generate new tools to visualize and quantify the dynamics of redox signaling and metabolic state occurring in blood vessels of living cells and animals. Address the role of the different subcellular compartments such mitochondria, golgi, plasma membrane, nuclei in endothelial redox and metabolic signaling (Panieri et al., 2017; Panier and Santoro, 2017). We had defined a reference redox state and metabolic condition in human ECs (Rupel et al., 2018, Stone et al., 2018). We provide experimental evidences of differences in redox signaling and metabolic conditions of normal vs tumor (Zulato et al., 2018). These results have been published in peer-review journals.In Task 2) we have set up and performed in ECs (HUVECs) a redox proteomic approach, called OXICAT, through which we define different angiogenic conditions for what we explore change in cysteine redox state. Among the target we identified G6PD and PGD, two key enzymes of the PPP pathway. The pentose phosphate pathway (PPP) has recently been shown to have a crucial role in cancer cell growth by providing both nucleotide precursors, needed for proliferation, and NADPH used for both intracellular ROS detoxification and catabolic metabolism. The role of PPP in angiogenesis has not been much studied. To this end we are aiming to test the role of PPP in the angiogenic processes in vitro and in vivo models. Recent insights establish how endothelial cells communicate with each other and with their environment to form a branched vascular network. We believe that a better understanding of these pathways might allow to identify new targets that could contribute to the development of new therapy in the treatment of vascular diseases.Task 3) is based on the identification of the molecular mechanism through which UBIAD1 regulate redox state in ECs and investigate the genetic inactivating UBIAD1 in mouse models of tumor angiogenesis. Unfortunately, we had several issues concerning the generation of Ubiad1 lox mice. Since these mice are not available in any repository we had to generate entirely in our lab. We did generate the construct for homologous recombination and generate several stable clone of ES cell line E14 derived from the inbred mouse strain 129. Unfortunately, although these clones were producing chimaeras, no founders were ever obtained. We recently received heterozygous C57BL/6 Ubiad1 lox/lox mice that were are crossing to homozygosity and then with endothelial specific Cre driver lines such as cdh5:CreERT2 and pdgf:CreERT2.Task 4) is based to address the function of the mevalonate pathway in developmental angiogenesis and in current anti-angiogenic therapies. Mevalonate (MVA) pathway is an essential anabolic metabolic pathway that produces sterols and isoprenoid metabolites (Buhaescu I. et al., 2007); however, its role in angiogenesis is still unclear. It has been described that high therapeutic concentrations of statins, inhibitors of the rate-limiting step in MVA pathway, inhibit angiogenesis by inhibition of isoprenylation (Weis M. et al., 2002). Indeed, geranylgeranyl pyrophosphate is required for the membrane localization and functionality of small GTP-binding proteins Rho family members. The Rho/ROCK pathway has been shown to be an integral part of VEGF-mediated angiogenesis (Chin VT. et al., 2015). In addition, an anti-angiogenic potential of nitrogen-containing bisphosphonates, inhibitions of farnesyl pyrophosphate synthase, it has been demonstrated (Coxon FP. et al., 2006). These drugs are able to inhibit EC growth and to induce apoptosis (Metcalf S. et al., 2011). The isomerization of un-reactive isopentenyldiphosphate (IPP) into its reactive isomer dimethyl-allylpyrophosphate (DMAPP) in the MVA pathway is the key rate-limiting step of the terpenoid biosynthesis and it is catalyzed by the enzyme isopentenyl-diphosphate isomerase 1 (IDI1)(Breitling R. et al., 2003). The product of the reaction, DMAPP, is used in the synthesis of MVA metabolites and to isopentenylate adenosine (A) residues in the anticodons of tRNA by TRIT1 (tRNA isopentenyltransferase 1), modification which is required to generate full expression of selenoproteins (Fradejas N. et al., 2013). Here we aim to investigate the role of IDI1 as possible targeting of angiogenesis. Specific experiments have been performed and are based on: a) to study the role of IDI1 in angiogenesis in vitro; b) to characterize the role of IDI1 in angiogenesis in vivo using a zebrafish model; and, c) to study developmental and pathological angiogenesis in IDI1 endothelial conditional knockout (KO) mice. Our preliminary results highlight the importance of IDI1 and isoprenoid pathway in endothelial homeostasis. In addition, we detected peculiar vascular defects in the full KO of IDI1 in zebrafish and an inhibition of retinal vasculature development in endothelial specific IDI1 KO. However, additional studies are required to clarify the role of this enzyme in pathological angiogenesis. 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 proposal is providing new perspectives by defining how the compartmentalized nature of cellular redox systems is linked to ECs fate and, therefore, understanding how subcellular redox signaling promotes disease development and progression. We will provide evidence that the UBIAD1 enzyme is a valuable target for clinical investigation. We are addressing the role of the mevalonate metabolic pathway in developmental and pathological angiogenesis. Last but not least we are establishing alternative strategies (or synergistic to existing ones) to cure tumor angiogenesis in human patients by acting on manipulation of endothelial cellular redox state, and consequently, on blood vessels metabolic states. Briefly, we develop new tools to identify, measure and regulate ROS in endothelial cell in vivo and in vitro.New molecular targets involved in metabolism and redox homeostasis of endothelial cells has been identifies and will be investigated. In particular focusing on their role in normal angiogenesis and in the context of pathological condition such tumor angiogenesis and cancer progression.We are expecting to achieve most of the aim proposed in the original proposal. We believe that our recent discovery on UBIAD1 and IDI1 enzyme and the genetic experiment characterized by a loss-of-function of those targets will provide clear evidences in a 5 year frame of the crucial role of redox signaling and metabolic state in angiogenesis opening to new exciting hypothesis on how to design redox-based therapies for pathological angiogenesis in humans.
