#	Pagina
attuale pagina	/open-h2020/projects/199656/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - rEnDOx (REDOX SIGNALING AND METABOLIC STATES IN ANGIOGENESIS IN HEALTH AND DISEASE)

Teaser

Summary

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

This ERC proposal has been built on four research tasks: 1) imaging and measuring redox signaling in living blood vessels, 2) identifying the molecular mechanism through which UBIAD1 regulate redox state in ECs, 3) genetic inactivating UBIAD1 in mouse models of tumor angiogenesis, 4) address the function of the mevalonate pathway in developmental angiogenesis and in current anti-angiogenic therapies.
In line with description of action, I will here provide final results, ongoing progress, and improvement corresponding to the project achievements.

Task 1
This task has been successfully accomplished. 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 addition, we have started to use these sensors for detecting ROS signaling under laminar shear conditions. Endothelial cells (ECs) serve as the interface between blood and the vasculature and are subject to the frictional pressure acting upon the luminal surface of the vasculature, known as shear stress. Shear stress is highly important for the initiation of angiogenesis, promoting EC sprouting, alignment, migration, growth and signalling (Galie, 2014; Tzima, 2001; Franco, 2015). Further, fluid forces contribute to vessel maturation, by the promotion of endothelial paracrine signalling to mural cells (MCs), coordinating the recruitment, differentiation of MCs and their endothelial coverage (Armulik, 2011; Chen, 2017). Altogether, hemodynamic forces are essential for the transition from a primitive vascular plexus to an organised vascular network and play key role in many pathological cardiovascular conditions.
Several mechanical sensors for the detection of shear stress have been identified in ECs. Recent reports into endothelial cilia, piezo1 ion channels and the G-protein coupled receptor, GPR68, have highlighted their roles in shear-mediated angiogenesis (Norris and Santoro, 2018). Disruption of such sensors can lead to pathologies including haemorrhage, atherosclerosis and even embryonal lethality (Chen, 2017, Baratchi, 2017; Ranade, 2014; Xu, 2018). Surprisingly, whilst they are known to be crucial for EC function, there appears to be no mechanistic link between the numerous described mechanosensors and shear-induced ROS production.
Elucidating the mechanisms involved in the EC sensing of differential flow patterns and their effects on ROS generation at specific subcellular compartments, will inform us of the biochemical changes, redox targets and effectors required for angiogenesis and pathologies arising from aberrant flow.
The major objectives of this project are to:
â€¢ To develop a functional pump system to assess the effects of different patterns of shear stress and flow manipulation and on compartment-specific redox states in ECs.
â€¢ To determine the mechanosensors responsible for shear-induced changes in compartmental redox states.
â€¢ To elucidate the significance of changes in subcellular ROS with respect to EC function.

Endothelial cells have been shown to respond to shear stress through diverse range of signalling pathways, including PI3-Kinase/Akt, JNK and MAPK/ERK pathways, which orchestrates a range of cellular outcomes that are required for angiogenesis. In order to interrogate endothelial responses to shear stress in vitro, we established a working pump system a using peristaltic pump, a pulse dampener made in-house and Ibidi channel slides (VI

Final results

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.