VEGF-A splice variants bind VEGFRs with differential affinities.

Vascular endothelial growth factor A (VEGF-A) and its binding to VEGFRs is an important angiogenesis regulator, especially the earliest-known isoform, VEGF-A165a. Yet several additional splice variants play prominent roles in regulating angiogenesis in health and in vascular disease, including VEGF-A121 and an anti-angiogenic variant, VEGF-A165b. Few studies have attempted to distinguish these forms from their angiogenic counterparts, experimentally. Previous studies of VEGF-A:VEGFR binding have measured binding kinetics for VEGFA165 and VEGF-A121, but binding kinetics of the other two pro- and all anti-angiogenic splice variants are not known. We measured the binding kinetics for VEGF-A165, -A165b, and -A121 with VEGFR1 and VEGF-R2 using surface plasmon resonance. We validated our methods by reproducing the known affinities between VEGF-A165a:VEGFR1 and VEGF-A165a:VEGFR2, 1.0 pM and 10 pM respectively, and validated the known affinity VEGF-A121:VEGFR2 as KD = 0.66 nM. We found that VEGF-A121 also binds VEGFR1 with an affinity KD = 3.7 nM. We further demonstrated that the anti-angiogenic variant, VEGF-A165b selectively prefers VEGFR2 binding at an affinity = 0.67 pM while binding VEGFR1 with a weaker affinity-KD = 1.4 nM. These results suggest that the - A165b anti-angiogenic variant would preferentially bind VEGFR2. These discoveries offer a new paradigm for understanding VEGF-A, while further stressing the need to take care in differentiating the splice variants in all future VEGF-A studies.

Author Correction: Discovery of High-Affinity PDGF-VEGFR Interactions: Redefining RTK Dynamics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Convergence of Cell-Based Surface Plasmon Resonance and Biomaterials: The Future of Quantifying Bio-molecular Interactions-A Review.

Cell biology is driven by complex networks of biomolecular interactions. Characterizing the kinetic and thermodynamic properties of these interactions is crucial to understanding their role in different physiological processes. Surface plasmon resonance (SPR)-based approaches have become a key tool in quantifying biomolecular interactions, however conventional approaches require isolating the interacting components from the cellular system. Cell-based SPR approaches have recently emerged, promising to enable precise measurements of biomolecular interactions within their normal biological context. Two major approaches have been developed, offering their own advantages and limitations. These approaches currently lack a systematic exploration of 'best practices' like those existing for traditional SPR experiments. Toward this end, we describe the two major approaches, and identify the experimental parameters that require exploration, and discuss the experimental considerations constraining the optimization of each. In particular, we discuss the requirements of future biomaterial development needed to advance the cell-based SPR technique.

Discovery of High-Affinity PDGF-VEGFR Interactions: Redefining RTK Dynamics.

Nearly all studies of angiogenesis have focused on uni-family ligand-receptor binding, e.g., VEGFs bind to VEGF receptors, PDGFs bind to PDGF receptors, etc. The discovery of VEGF-PDGFRs binding challenges this paradigm and calls for investigation of other ligand-receptor binding possibilities. We utilized surface plasmon resonance to identify and measure PDGF-to-VEGFR binding rates, establishing cut-offs for binding and non-binding interactions. We quantified the kinetics of the recent VEGF-A:PDGFRß interaction for the first time with KD = 340 pM. We discovered new PDGF:VEGFR2 interactions with PDGF-AA:R2 KD = 530 nM, PDGF-AB:R2 KD = 110 pM, PDGF-BB:R2 KD = 40 nM, and PDGF-CC:R2 KD = 70 pM. We computationally predict that cross-family PDGF binding could contribute up to 96% of VEGFR2 ligation in healthy conditions and in cancer. Together the identification, quantification, and simulation of these novel cross-family interactions posits new mechanisms for understanding anti-angiogenic drug resistance and presents an expanded role of growth factor signaling with significance in health and disease.

Synthesis, Chemical Characterization and Multiscale Biological Evaluation of a Dimeric-cRGD Peptide for Targeted Imaging of α V ß 3 Integrin Activity.

Cyclic peptides containing the Arg-Gly-Asp (RGD) sequence have been shown to specifically bind the angiogenesis biomarker α V ß 3 integrin. We report the synthesis, chemical characterization, and biological evaluation of two novel dimeric cyclic RGD-based molecular probes for the targeted imaging of α V ß 3 activity (a radiolabeled version, 64Cu-NOTA-PEG4-cRGD2, for PET imaging, and a fluorescent version, FITC-PEG4-cRGD2, for in vitro work). We investigated the performance of this probe at the receptor, cell, organ, and whole-body levels, including its use to detect diabetes associated impairment of ischemia-induced myocardial angiogenesis. Both versions of the probe were found to be stable, demonstrated fast receptor association constants, and showed high specificity for α V ß 3 in HUVECs (K d ~ 35 nM). Dynamic PET-CT imaging indicated rapid blood clearance via kidney filtration, and accumulation within α V ß 3-positive infarcted myocardium. 64Cu-NOTA-PEG4-cRGD2 demonstrated a favorable biodistribution, slow washout, and excellent performance with respect to the quality of the PET-CT images obtained. Importantly, the ratio of probe uptake in infarcted heart tissue compared to normal tissue was significantly higher in non-diabetic rats than in diabetic ones. Overall, our probes are promising agents for non-invasive quantitative imaging of α V ß 3 expression, both in vitro and in vivo.