VEGF internalization is not required for VEGFR-2 phosphorylation in bioengineered surfaces with covalently linked VEGF

Abstract

Vascular endothelial growth factor (VEGF) is known to activate proliferation, migration, and survival pathways in endothelial cells through phosphorylation of VEGF receptor-2 (VEGFR-2). VEGF has been incorporated into biomaterials through encapsulation, electrostatic sequestration, and covalent attachment, but the effect of these immobilization strategies on VEGF signaling has not been thoroughly investigated. Further, although growth factor internalization along with the receptor generally occurs in a physiological setting, whether this internalization is needed for receptor phosphorylation is not entirely clear. Here we show that VEGF covalently bound through a modified heparin molecule elicits an extended response of pVEGFR-2 in human umbilical vein endothelial cells (HUVECs) and that the covalent linkage reduces internalization of the growth factor during receptor endocytosis. Optical tweezer measurements show that the rupture force required to disrupt the heparin-VEGF-VEGFR-2 interaction increases from 3–8 pN to 6–12 pN when a covalent bond is introduced between VEGF and heparin. Importantly, by covalently binding VEGF to a heparin substrate, the stability (half-life) of VEGF is extended over three-fold. Here, mathematical models support the biological conclusions, further suggesting that VEGF internalization is significantly reduced when covalently bound, and indicating that VEGF is available for repeated phosphorylation events.

Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells

“…These studies suggest that VEGFR2 internalization is required for optimal phosphorylation of VEGFR2 and subsequent optimal activation of downstream signaling. However, this is in contrast to other work demonstrating that internalization is not required for optimal phosphorylation of VEGFR2 (54,55). Therefore, it is not precisely clear how receptor internalization couples VEGFR2 to the activation of downstream signaling pathways.…”

Design of growth factor sequestering biomaterials

“…Cell culture substrates containing covalently-immobilized VEGF promoted HUVEC proliferation for longer durations when compared to those cultured on substrates with non-specifically adsorbed VEGF 173 . This phenomenon was corroborated by a similar study demonstrating that KDR phosphorylation in HUVECs was prolonged when VEGF was covalently immobilized to the culture substratum, and the stability of VEGF was also enhanced 175 . The prolonged effects of covalently-immobilized VEGF can be attributed to the cell’s inability to endocytose and degrade VEGF-KDR complexes, a process which normally inactivates the VEGF-dependent signal transduction pathway and suppresses over-proliferation in response to VEGF 173 .…”

The use of covalently immobilized stem cell factor to selectively affect hematopoietic stem cell activity within a gelatin hydrogel

“…Similar results were observed by Anderson et al . [78] where internalization of VEGF was reduced in case of covalently bound VEGF and altered the downstream signaling profiles of the human umbilical vascular endothelial cells .Together, our results suggest it may likely be possible to co-optimize soluble and matrix-bound dosages of SCF to promote the expansion of the desired primitive HSC phenotype. While beyond the scope of this effort, ongoing work is examining relative activation of signaling pathways so as to identify optimal combinations of immobilized and soluble SCF.…”