Introduction:: [Introduction] Mechanical stimulations from the extracellular matrix (ECM) modulate vascular differentiation, morphogenesis, and dysfunction. Tissue stiffening, and vascular dysfunction in aging, limit prospects for vascular morphogenesis and regeneration. Specifically, an increase in ECM stiffness correlates with a decrease in vascular density and an increase in vascular permeability. A stiffening matrix compromises vascular adherence junction, leading to the dysfunction of vascular networks. In our previous study, we discovered that the interactions between endothelial cells (ECs) and extracellular matrix (ECM) results in EC- stiffening of the ECM. Our current hypothesis is that this stiffening occurs due to alterations in the microstructure of the ECM, which creates stiffness gradients around the vascular network (Fig 1A). We established an ECM-particle based in vitro hydrogel system for 3D traction force microscopy and matrix mechanical mapping of live EC networks. This system can be adapted for studying cell-matrix mechanical interactions. Traction force from live ECs generates matrix gradients during growth in 3D, and the persistent movement of beads is associated with asymmetries in traction force distribution and collagen stiffness gradients.
Materials and Methods:: [Materials and Methods] Endothelial colony-forming cells (ECFCs, labeled by CellTracker) are encapsulated with a large volume of carboxylate-modified fluorescent beads in each hydrogel and cultured in endothelial cell growth medium media supplemented with vascular endothelial growth factor. The stiffening hydrogel (Col/HA) is fabricated by mixing methacrylate hyaluronic acid (HA-MA) with methacrylate collagen I (Col-MA). We utilized MA to allow dynamic stiffening. Adding HA increases the number of MA sites, which allows the optimization of a range of stiffness increases. For stiffening the hydrogels, on day 2, the MA groups are photo-crosslinked using a Ru photoinitiator and visible light. We use the 0 (soft) and 60 (stiffened) sec photo-crosslinking times that correspond to 100 Pa to 240 Pa stiffness, respectively. The confocal microscopy time-lapse imaging on live ECFCs (by Nikon Ti2-E) is conducted at three timing points: day 1, day 2 right after photo-crosslinking, and day 3 (Fig 1B). The collected images are processed and analyzed by TFMLAB and NIS Elements software.
Results, Conclusions, and Discussions:: [Results and Discussion] The dynamic stiffening of the ECM was controlled by the crosslinking time of Col-MA. In this work, we focused on the 0 and 60 sec photo-crosslinking times, which corresponded to ~100 Pa and ~240 Pa stiffness to mimic the two-fold change in stiffness observed in aging and diseased tissues Within the ECM-particle based in vitro hydrogel system, where vessel morphology significantly changed after ECM subjected to a stiffness increase to 240 Pa (Fig. 1C). ECFCs modulate their surrounding ECM density during vasculogenesis, where stiffening matrix shows a higher collagen fiber density (Fig. 1D). The persistent movement of beads are corresponding to the matrix deformation. The increasing ECM stiffness alter the beads movement where in stiffener matrix the beads move slower and to less distance around the networks compared with softer matrix (Fig. 1E), suggesting stiffener matrix has slower and less matrix deformation than softer matrix.
[Conclusions] Endothelial cells mediate ECM stiffening during vasculogenesis and alter fiber structure. The stiffening matrix amplifies the EC-based mechanical modulations of the ECM, especially the ECM that close to the cell-ECM interface, further augments the stiffness impact on compromising vascular function. The application of this ECM-particle based in vitro hydrogel system could lay the foundation for uncovering the mechanical interactions of the vasculature with the surrounding microenvironment, ultimately leading to the development of new biomechanical sensing strategies. Ongoing studies focus on quantifying how the stiffening matrix induces EC-based ECM modulation.