Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - CAMVAS (Coordination And Migration of Cells during 3D Vasculogenesis)

Summary description of the project objectives:
Coordination And Migration during 3D Vasculogenesis (CAMVAS) aims to elucidate the mechanics of endothelial cells during the formation of microvascular networks. To this general objective, the project addresses the observation and quantification of the following mechanical quantities:

- Cellular forces and mechanical deformations occurring during the formation of capillaries, from the initial time, when cells are not interconnected and merely resuspended in the extracellular scaffold to the complete formation of a stable network
- Movements, in the sense of collective migrations of cells during the process.

The aim is to reach a deep understanding of the mechanics involved in this process, and ultimately build and validate a biophysical multiscale model of the vasculogenesis/angiogenesis process.

Description of the work performed since the beginning of the project:
The work performed so far has focused on the setting of suitable and controllable force experiments, and related data analyses, to enable the quantifications of the mechanics of endothelial cells during vasculogenesis. Microfluidics nanotechnologies developed at the outgoing host (Mechanobiology Laboratory at MIT, directed by Prof. Roger D. Kamm) have been adapted to this quantification. Existing imaging procedures in microfuidics, successfully used for vasculogenesis, have been modified to enable the visualization of mechanical deformations. The following steps have been taken:

- Development of experimental protocols for the use of fluorescent entities, or sensors (such as FRET sensors) in the microfluidics experiments of vasculogenesis, to infer on mechanical deformations and forces during the process.
- Development of computational protocols for the calculations of displacements and mechanical deformations in the extracellular matrix through tracking of fluorescent entities, or similarly, cross-correlation of the images to estimate deformations produced by the cellular forces.
- Development of computational protocols for the derivation of forces, from the deformation assessed experimentally. This step involves the definition of suitable constitutive theories for the mechanical behavior of the extracellular matrix material.
- Development of experimental protocols for assessment of cell migration during the dynamics of network formation.

Description of the main results achieved:
The main results achieved include the obtainment of deformations and forces during the early vasculogenesis process. This process typically lasts 3-5 days: at day 0, or time of seeding, cells are suspended in a suitable extracellular scaffold, for instance fibrin gel, filling a microfluidic chamber. After 24 to 48 hours, endothelial cells protrude and remodel the fibrin gel, to contact and eventually fuse, forming a primitive capillary network surrogate. After 48 hours, this microvascular networks grow and become perfusable, i.e. the capillaries remodel to create a lumen in which fluid can flow inside them. Microfluidic devices developed at the outgoing host at MIT have allowed the study of all these phases.
Considering the above-described process, the main results include:

- An evaluation of the matrix remodeling in 3D during the microvascular network formation that shows patterns of densification and degradation in the whole cell construct.
- Quantification, with the use of FRET, of a structural matrix protein (plasma fibronectin) deformation. This shows that fibronectin is increasingly unfolded as vasculogenesis progresses and the microvascular tissue forms.
- Immunostaining of basement membrane proteins, such as collagen I and laminin. The spatial organization of these proteins is confined to the extracellular space immediately adjacent to the cell membrane.
- Quantification of global cell construct contractility during vasculogenesis by tracking of fluorescent beads embedded and tied to the extracellular fibrin scaffold in 100 μm-sized regions. These experiments have shown that contractility increases as vasculogenesis progresses.
- Quantification of local stresses and strains exerted by endothelial cells in 3D during this microvascular morphogenesis. Preliminary analyses show that, although the fibrin remodels in a dishomogeneous fashion, local strain quantifications are possible. Strain increased homogenously as vasculogenesis occurs, through reduction and smoothing of peaks of strains. These peaks are instead active and intense if cells are not able to connect due to reduced density.
- Preliminary assessment of local stiffness changes in the extracellular space due to remodeling. These assessments include optical tweezers and Brillouin light scattering microscopy, and show that (i) densification due to cell remodeling produce a local increase of stiffness close to the cell membranes, and that (ii) overall stiffness increases over the time of vasculogenesis.

Expected final results and their potential impact and use
The expected final results could be summarized as:

- Development of a highly versatile microfluidic platform system for organ and disease modeling. This has a high socio-economic impact due to the importance of such nanotechnologies for research and experimentation with a limited use of animal models.
- Development of a biophysical multiscale model for vasculogenesis, including the extracellular matrix mechanics parameters. This modeling step is currently crucial in many areas in mechanobiology for the understanding and targeting of key parameters in organogenesis, tissue engineering and disease (such as cancer progression).
- Direct dynamic map of forces acting during vasculogenesis. Due to similarity, this will also be extrapolated in angiogenesis, and have implication for organ and disease in vitro modeling: future applications include mechanobiology-based treatment of the vascular tissue and the understanding of forces cancer metastasis and tumor extravasation steps.
- Dynamic maps of stiffness changes during morphogenesis of capillaries. This is in line with current efforts in biomedical research of exploiting cellular pathways related to the mechanobiology of stiffness sensing, fundamental for stem cell and tissue engineering research

Reported by

FUNDACIO INSTITUT DE BIOENGINYERIA DE CATALUNYA
Spain

Subjects

Life Sciences
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