Author:

Keywords:

Angiogenesis, Traction Forces, MAtrix - 308223;info:eu-repo/grantAgreement/EC/FP7/308223

Abstract:

“TRACTIONS, MORPHOLOGY AND MOTILITY DYNAMICS IN ENDOTHELIAL CELLS ON COMPLIANT SUBSTRATES” A. Izquierdo-Álvarez1, A. Jorge Peñas1, D.A. Vargas1, S. Ragunathan1, R. Subramani2 and H. Van Oosterwyck1,3. 1 Biomechanics Section, KU Leuven Celestijnenlaan 300C box 2419, 3001 Leuven. 2 Division of Mechatronics, Biostatistics and Sensors, KU Leuven, Kasteelpark Arenberg 30 box 2456, 3001 Leuven. 3 Prometheus, div. Skeletal Tissue Engineering, KU Leuven, Belgium A number of studies have shed light on the biochemical regulation of angiogenesis, but how mechanical properties modulate this process is still poorly understood. We have focused our attention on how substrate stiffness and adhesion proteins affect the morphology, traction and motility of human umbilical vein endothelial cells (HUVECs) and how they correlate to each other, both at the whole cell population as well as at single cell level. HUVECs were cultured on top of polyacrylamide surfaces of different stiffness, functionalized with fibronectin or collagen. For traction force microscopy (TFM), fluorescent beads contained in the substrate were used as fiducial markers. Time lapse TFM experiments were performed and displacement calculations were made using Free Form Deformation (FFD)-based image registration. We have previously demonstrated that this method calculates displacements more accurately and, consequently, leads to more reliable tractions [1]. In order to calculate migration parameters we have applied the Persistence Random Walk model previously described in Pei-Hsun et al. [2]. A series of morphological features and the minimal distance between cells where measured from the images of HUVECs. To test whether there is a relation between condition and behavior, we performed an analysis using the software SAPHIRE [3]. This analysis uses principal component analysis (PCA) and hidden Markov models (HMM) to define a series of cellular states for each cell incorporating temporal dependencies during model inference. This analysis provides a transition probability matrix that can be used to compare across conditions. Preliminary results show a direct correlation between the total force exerted by the cell and the stiffness of the substrate, being collagen the adhesion protein promoting higher tractions. Also, tracking of the local ‘hot spots’ of traction force with time, points to forces that are more persistent for cells on collagen than on fibronectin, especially in softer substrates. Furthermore, the PCA analysis revealed the length of cells’ minor axis as the morphological parameter that describes most variability within a cell population. Surprisingly, distance to other cells showed no correlation with cellular state. Finally, hierarchical clustering of transition probability matrices shows potential to distinguish among cellular responses to different substrate stiffness. In summary, the resulting data will shed light on the relation between the cell and its physical environment and how the mechanical cues modulate cell fate in angiogenesis. Acknowledgements: FP7/2007-2013)/ ERC StG n° 308223, FWO G.0821.13, KU Leuven internal funding (IDO 13/016) [1] Jorge-Peñas A et al. (2015) PLoS ONE 10(12): e0144184. [2] Pei-Hsun W et al. (2015) Nature Protocols 10, 517–527. [3] Gordonov et al. (2016) Integrative Biology 8(1):73-90.