Abstract
Vascular endothelial growth factor-A165 (VEGF-A165) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A165 and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A165 stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A165 on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A165 have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.
Highlights
Vascular endothelial growth factor A (VEGF-A) is a heparin-binding dimeric protein belonging to the vascular endothelial growth factor (VEGF) gene family together with VEGF-B, VEGF-C, VEGF-D, and the placental growth factor
The resulting protein has been designated Fibroblast Growth Factor (FGF)-2M. Both VEGF-A165 and FGF-2M were designed and expressed with the N-terminal 8 amino acid substrate sequence for Factor XIIIa (NQEQVSPL), which can be used for covalent attachment of the growth factors into a fibrin network used for coating cardiovascular prostheses or implants [45]
Our study presents a new and cost-effective method for extracellular expression of VEGF-A165 and fibroblast growth factor-2 (FGF-2) in a eukaryotic system of P. pastoris
Summary
Vascular endothelial growth factor A (VEGF-A) is a heparin-binding dimeric protein belonging to the VEGF gene family together with VEGF-B, VEGF-C, VEGF-D, and the placental growth factor. VEGF-A165 has been widely used for experimental work in vitro, e.g., creation of vascularized tissue-engineered constructs, intended for bone regeneration [12] or functionalization of a decellularized pericardial matrix, intended for cardiovascular tissue engineering, in order to promote its recellularization with endothelial and stem cells [13]. In this context, it is worth mentioning that VEGF-A165 immobilized on cell cultivation substrates can act as an extracellular adhesion molecule, binding to integrin adhesion receptors on cells [14,15]
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