Dynamics of Endothelial Engagement and Filopodia Formation in Complex 3D Microscaffolds

Pierre Ucla,Sarah Baiz,Stéphane Germain,Catherine Monnot,Laurent Muller,Melisa Demircioglu,Sylvie Coscoy,Xingming Ju,Vincent Semetey

doi:10.3390/ijms23052415

Abstract

The understanding of endothelium–extracellular matrix interactions during the initiation of new blood vessels is of great medical importance; however, the mechanobiological principles governing endothelial protrusive behaviours in 3D microtopographies remain imperfectly understood. In blood capillaries submitted to angiogenic factors (such as vascular endothelial growth factor, VEGF), endothelial cells can transiently transdifferentiate in filopodia-rich cells, named tip cells, from which angiogenesis processes are locally initiated. This protrusive state based on filopodia dynamics contrasts with the lamellipodia-based endothelial cell migration on 2D substrates. Using two-photon polymerization, we generated 3D microstructures triggering endothelial phenotypes evocative of tip cell behaviour. Hexagonal lattices on pillars (“open”), but not “closed” hexagonal lattices, induced engagement from the endothelial monolayer with the generation of numerous filopodia. The development of image analysis tools for filopodia tracking allowed to probe the influence of the microtopography (pore size, regular vs. elongated structures, role of the pillars) on orientations, engagement and filopodia dynamics, and to identify MLCK (myosin light-chain kinase) as a key player for filopodia-based protrusive mode. Importantly, these events occurred independently of VEGF treatment, suggesting that the observed phenotype was induced through microtopography. These microstructures are proposed as a model research tool for understanding endothelial cell behaviour in 3D fibrillary networks.

Highlights

The fundamental understanding of endothelium–extracellular matrix (ECM) interactions during the formation of new blood vessels meets considerable medical needs
We investigated the role of complex microtopographies in the control of endothelial protrusive behaviour by using 3D microstructures generated by the two-photon polymerization of NOA resin (Norland Optical Adhesive)
We previously described simple 3D hexagonal lattices for the generation of deep epithelial protrusions strongly increasing cell basal surface area [37], and we were interested in testing the behaviour of endothelial cells on these scaffolds, referred to here as «closed» structures (Figure 1a)

Summary

Introduction

The fundamental understanding of endothelium–extracellular matrix (ECM) interactions during the formation of new blood vessels meets considerable medical needs. The microtopographies of three-dimensional scaffolds surrounding cells, with subcellular sizes in the nanometre to micrometre ranges, have been shown to elicit responses such as contact guidance [4–10], the modulation of migration or invasion [3,4,11–13] or the control of cell fate and differentiation [14–16]. In the field of bioengineering, a comprehensive range of methods has been developed, either to mimic the native microenvironment, or to create innovative microtopographies eliciting cell responses of medical interest. These techniques include photolithography [17], colloidal templating [14], electrospinning [18], moulding [19,20], 3D impression [21] or two-photon polymerization (TPP) [22–24]. The culture of endothelial cells on microstructures such as microgrooves or micropillars modulates their phenotype and their migratory properties [27–29]

Methods

Results

Conclusion