Abstract
Tube formation by endothelial cells (EC) is a one of the key factors involved in initial revascularization during the tissue regeneration process. Several previous studies suggested that EC tube formation can be regulated by the stiffness of the culture substrate and immobilization of vascular endothelial growth factor (VEGF). However, the roles of substrate stiffness and chemical signals involved in network formation are unclear. In this study, the effects of substrate rigidity and immobilized VEGF concentration on EC tube formation were examined. Human umbilical vein endothelial cells (HUVEC) were cultured on rigidity-controlled polyacrylamide (PAAM) gels coated by L-DOPA (3,4-dihydroxy-L-phenylalanine), or L-DOPA-Hep (heparin). The HUVECs were cultured on various amounts of immobilized VEGF on the L-DOPA-Hep-coated PAAM (5, 10, and 25 ng). We found that HUVECs formed more tube-like structures when they were cultured on the more rigid substrates, from 1.5 kPa to 144.1 kPa compared to the soft substrate. We also compared various amounts of VEGF immobilized on various rigidity substrates to determine if there was an effect on the tube area of HUVEC. Moreover, we determined an appropriate concentration of VEGF immobilized with various rigidity substrates for vascular tube formation. These findings suggest that there is a synergistic effect of substrate rigidity and chemical signals. Thus, it is possible to combine therapies to synergistically promote an in vitro model of revascularization.
Key words
Matrix rigidity, Human umbilical vein endothelial cells (HUVEC), VEGF, PAAM, tube formation