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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.

SUBMITTER: Anderson SM

PROVIDER: S-EPMC3621282 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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

Anderson Sean M SM Shergill Bhupinder B Barry Zachary T ZT Manousiouthakis Eleana E Chen Tom T TT Botvinick Elliot E Platt Manu O MO Iruela-Arispe M Luisa ML Segura Tatiana T

Integrative biology : quantitative biosciences from nano to macro 20110808 9

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 occu ...[more]

PMID: 21826315

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Project description:Vascular endothelial growth factor-A (VEGF-A) acts via 2 vascular endothelial growth factor receptors, VEGFR-1 and VEGFR-2, that play important and distinct roles in tumor biology. We reasoned that selective imaging of these receptors could provide unique information for diagnostics and for monitoring and optimizing responses to anticancer therapy, including antiangiogenic therapy. Herein, we report the development of 2 first-in-class 89Zr-labeled PET tracers that enable the selective imaging of VEGFR-1 and VEGFR-2.MethodsFunctionally active mutants of scVEGF (an engineered single-chain version of pan-receptor VEGF-A with an N-terminal cysteine-containing tag for site-specific conjugation), named scVR1 and scVR2 with enhanced affinity to, respectively, VEGFR-1 and VEGFR-2, were constructed. Parental scVEGF and its receptor-specific mutants were site-specifically derivatized with the 89Zr chelator desferroxamine B via a 3.4-kDa PEG linker. 89Zr labeling of the desferroxamine B conjugates furnished scV/Zr, scVR1/Zr, and scVR2/Zr tracers with high radiochemical yield (>87%), high specific activity (≥9.8 MBq/nmol), and purity (>99%). Tracers were tested in an orthotopic breast cancer model using 4T1luc-bearing syngeneic BALB/c mice. For testing tracer specificity, tracers were coinjected with an excess of cold proteins of the same or opposite receptor specificity or pan-receptor scVEGF. PET imaging, biodistribution, and dosimetry studies in mice, as well as immunohistochemical analysis of harvested tumors, were performed.ResultsAll tracers rapidly accumulated in orthotopic 4T1luc tumors, allowing for the successful PET imaging of the tumors as early as 2 h after injection. Blocking experiments with an excess of pan-receptor or receptor-specific cold proteins indicated that more than 80% of tracer tumor uptake is VEGFR-mediated, whereas uptake in all major organs is not affected by blocking within the margin of error. Critically, blocking experiments indicated that VEGFR-mediated tumor uptake of scVR1/Zr and scVR2/Zr was mediated exclusively by the corresponding receptor, VEGFR-1 or VEGFR-2, respectively. In contrast, uptake of pan-receptor scV/Zr was mediated by both VEGFR-1 and VEGFR-2 at an approximately 2:1 ratio.ConclusionFirst-in-class selective PET tracers for imaging VEGFR-1 and VEGFR-2 were constructed and successfully validated in an orthotopic murine tumor model.

Project description:A versatile method is presented to form dendrimer superstructures by exploiting coacervate-core micelles as a template to confine and organize the hyperbranched macromolecules. First, complex coacervate-core micelles are formed from negative-neutral block copolymers and positively charged polyamidoamine dendrimers. The dendrimers inside the micellar core are then covalently cross-linked with each other upon addition of glutaraldehyde. After removal of the block copolymer from the assembly by increasing the salt concentration, consecutively, the formed Schiff bases cross-linking the dendrimers are reduced to amines, followed by a final dialysis step. This leads to well-defined covalently cross-linked nanostructures, coined dendroids, with a size of around 30 nm in diameter and a molecular weight of approximately 2.5 MDa. By incorporating dendrimer-encapsulated gold nanoparticles (AuDENs) into the micelle template strategy, the aggregation number of dendrimers inside the dendroids is determined by counting the nanoparticles in TEM micrographs. Furthermore, TEM performed at different tilt angles and AFM analysis corroborate formation of stable, covalently linked three-dimensional structures. Reconstruction of the TEM tilt series results in a tomogram further illustrating the 3D distribution of the gold nanoparticles, and hence the individual dendrimers, in the nanostructure. These dendroids appear to have a hard, poorly compressible core and a relatively soft outside. The versatility of the hierarchical building up of the supermolecules is illustrated by the controlled and synchronous incorporation of empty dendrimers and AuDENs into a single hybrid dendroid structure. The presented strategy allows for the preparation of a variety of classes of supermolecules, depending on the type of micellar-core macromolecule, e.g., dendrimer, cross-linker, and nanoparticles, used. Considering the broad interest in dendrimers as well as micelles in a plethora of research areas, e.g., (targeted) drug delivery, biomedical imaging, theragnostics, and catalysis, there is a great potential for dendroids and related classes of covalently linked macromolecules, viz., supermolecules.

Dataset Information

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

Publications

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

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