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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MTUB-ANGIO (Microtubule Dynamics during angiogenesis)

Teaser

Summary

Angiogenesis is a decisive process through which new blood vessels emerge and develop from a pre-existing vasculature. This dynamic process requires that the endothelial cells, the cells that line our blood vessels, undergo profound shape changes and reorganize their scaffolding cytoskeleton. The microtubule network is precisely one of the main cytoskeletal elements of eukaryotic cells, but surprisingly the role of microtubules during angiogenesis remains poorly characterized.
As angiogenesis is a process that plays crucial roles in physiology and pathology, especially during cancer development because tumor progression crucially need blood vessel recruitment, a better understanding of the regulation of angiogenesis constitutes an important health research area. Microtubules are particularly interesting to study in this context because targeting them is one of most frequently used anticancer treatment.
In order to extend our knowledge on the cellular mechanisms underlying microtubule regulation and function during angiogenesis, we wanted to address the role of microtubule regulatory proteins using advanced live imaging of 3D culture of endothelial cells mimicking angiogenesis.
Very interestingly, by setting-up such an approach, we found that microtubule organization and dynamics are crucial for the endothelial cells to sprout properly and build a functional vessel network. We particularly studied two aspects of microtubule: their processive growth and the symmetry of the network they are building. We assessed the role of these microtubule properties by identifying important regulators and showing their decisive angiogenic role after interfering with their function. By validating some results in vivo using zebrafish, we established a previously unexpected role for the organisation of the microtubule network in the regulation of angiogenesis.

Work performed

We developed a robust protocol for obtaining primary endothelial efficiently silenced for interesting microtubule regulators and established a 3D tubulogenesis test as routine assay to assess their functional role in angiogenesis. The results of this screening provided a list of potential microtubule regulator implicated in sprouting angiogenesis.
We focused on the function of two interesting microtubule regulators: the regulation of microtubule growth persistence by the SLAIN protein (also mimicked by a marine drug) and the stabilization of microtubules that are not anchored at the centrosome by the CAMSAP2 protein. We set up high resolution imaging techniques that allowed us to study the microtubule and actin dynamics and organization, vesicles trafficking, RhoGTPase activity and Golgi positioning in a context of 3D matrices. This allowed us to show that on one hand, microtubule persistent growth is needed for the process of endothelial cell sprouting. On the other hands, we investigate the role of microtubule organization on angiogenesis and we demonstrated that non-centrosomal microtubules stabilized by CAMSAP2 were required for Golgi positioning and trafficking during directional migration on 2D substrates and for the establishment of a polarized cell morphology with large persistent protrusions in soft 3D matrices.
To confirm our cellular data in vivo, we inactivated the zebrafish ortholog of CAMSAP2 and by measuring different parameters of venous vessel development, we demonstrated the crucial role of CAMSAP2 for the establishment of the vasculature in vivo.
Our results and methods were presented at several national and international meetings: Jacques Monot conference on actin and microtubule, Dutch biophysics meeting, EMBO/ASCB meeting, EMBO microtubule meeting.
In addition, one research and one review article were published : Martin M, Veloso A, Wu J, Katrukha EA and Akhmanova A. (2018). Control of endothelial cell polarity and sprouting angiogenesis by non-centrosomal microtubules. Elife, e33864. doi: 10.7554/eLife.33864 and Martin M and Akhmanova A. (2018). Coming into focus: Mechanisms of microtubule minus-end organization, Trends in Cell Biology, 28(7). doi: 10.1016/j.tcb.2018.02.011.

Final results

The technology development set up in this project allowed us to address in detail the previously underappreciated role of microtubules during angiogenesis and to change our understanding of how microtubules affect vessel development. By uniting the research areas of microtubule cytoskeleton and angiogenesis, combined with state-of-the-art imaging and quantification methodology, the project provided new knowledge about the cellular mechanisms governing angiogenesis participating in a better fundamental understanding of this process. In the same time, it has also developed an original line of research for the angiogenesis field that could be pursued in the future.
Further work could have an impact on human health, not least in cancer research. Indeed, angiogenesis plays key roles in both physiology and pathology and is implicated in several disorders, specifically in tumor progression. Microtubules are already a target for cancer therapy, so the knowledge coming from this project is a good opportunity to provide new entry points for the development of angiogenesis-based therapies and future work could help to optimize the use of existing drugs.