Poster V9 - VEGF-A and PDGFs: Discovery of high-affinity (nanomolar) interactions between cross-family ligand.

Introduction: Angiogenesis, the formation of new blood vessels, is indispensable for organ development and essential in the repair process. Therapeutic strategies targeting angiogenesis to address diseases such as obesity1, cancer2, and others 2,3 often result in temporary improvements and subsequent disease advancement. This is because angiogenesis is regulated by multiple pathways that can compensate for each other when single pathways are inhibited. Most angiogenesis research has focused on uni-family ligand-receptor binding, e.g., VEGFs bind to VEGF receptors, PDGFs bind to PDGF receptors, etc. However, the discovery of VEGF-PDGFRs binding4,5 challenges this convention and calls for the investigation of other cross-family interactions, including ligand interactions. Towards this goal, we sought to explore the possibility of cross-family ligand hetero-oligomeric interactions between VEGF-A and PDGF-AA, -AB, -BB, -CC, -DD, and ANG-4. We determine that these interactions do exist and report their affinities. Our discovery of angiogenic growth factor hetero-oligomerization offers a new dimension for controlling vascular development in health and disease.

Materials and

Methods: 1. Surface Plasmon Resonance (SPR): - We immobilized VEGF-A on the SPR (Reichert 4SPR, Reichert, Inc.) sensor chip (Planar Polyethylene Glycol/Carboxyl) and flowed 3 nM-50 nM of growth factor (e.g., PDGF-AA, -BB, -AB, -CC, -DD or ANG4) analyte solution for 3 min (association). Dissociation followed with 5 min of running buffer. The chip was regenerated between each sample, injecting the following in series: 1 min-100 mM H3PO4 and 1 min-10 mM NaOH. The resultant curve was reference subtracted, and fitted to the 1:1 Langmuir interaction model kinetics using TraceDrawer software, version 1.8.1. The data quality and fitting were assessed by the U-value and chi2 value (Table 1).

Results, Conclusions, and Discussions:

Results:
We determined the affinities of previously unexplored cross-family hetero-oligomeric interactions (Table 1). In order of binding strength, these were VEGF-A/PlGF (Kd = 296 pM), VEGF-A/PDGF-AA (Kd = 900 pM), VEGF-A/PDGF-BB (Kd = 2.2 nM), VEGF-A/PDGF-DD (Kd = 3.6 nM), and VEGF-A/PDGF-AB (Kd = 4.8 nM). Due to their comparatively low binding affinities, the interactions of VEGF-A/ANG-4 (KdVEGF-A/ANG-4 = 0.9 µM) and VEGF-A/PDGF-CC (KdVEGF-A/PDGF-CC = 1 µM) were deemed insignificant (Figure 1).

Discussion:
Our results show high-affinity hetero-oligomerization between VEGF-A and PlGF and four PDGFs (-AA, -AB, -BB, -DD). While VEGF-A (monomer)/PlGF (monomer) heterodimers naturally occur and influence tumor development, VEGF-A (dimer)/PlGF (dimer) hetero-oligomers have not been previously explored, so we offer new evidence. The discovery of high-affinity VEGF-A/PDGF hetero-oligomerization potentially explains the synergistic effects observed in vessel formation and the increased stability of vessels when VEGF-A and PDGFs are co-expressed. Altogether, VEGF-A/PlGF and VEGF-A/PDGF hetero-oligomerization offer new insights into angiogenesis regulation. These findings could lead to novel therapeutic strategies for vascular-related conditions by unveiling previously unexplored mechanisms in tumors and cardiovascular diseases. Further research is needed to determine the extent of these interactions in vivo and their effects on angiogenic signaling and function.

Acknowledgements (Optional): This material is based upon work supported by the National Science Foundation under Grant No. 1923151 and National Institute of Heart, Lung, and Blood of the National Institutes of Health under Award Number. 7R01HL159946-02. Any opinions, fndings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the
National Institutes of Health