Impact of Ligand Binding on VEGFR1, VEGFR2, and NRP1 Localization in Human Endothelial Cells

Introduction: Vascular endothelial growth factor receptors (VEGFRs) bind to cognate ligands to facilitate signaling pathways critical for angiogenesis, the growth of new capillaries from existing vasculature. Intracellular trafficking regulates the availability of receptors on the cell surface to bind ligands, which regulate activation, and the movement of activated receptors between the surface and intracellular pools, where they can initiate different signaling pathways. Targeting VEGF receptors to disrupt blood vessel growth in cancer and retinopathy has achieved limited success and over a dozen human clinical trials of VEGF delivery to increase vascular growth in patients with peripheral artery disease (PAD) and coronary artery disease (CAD) have failed. The inability to successfully bridge treatment from animals to humans demonstrates that our understanding of the VEGF system is far from complete. We sought to examine the dynamics of membrane-bound receptors (mVEGFRs), ligands (VEGF and PLGF isoforms), and co-receptors expressed on endothelial cells under physiological and pathological conditions. Here we create a computational model of the system and combine it with experimental measurements to quantify the stability and trafficking of VEGFR1, VEGFR2, and their coreceptor Neuroplin-1 in response to VEGF and PLGF treatment.

Materials and

Methods: We have developed and validated a molecularly-detailed computational model of mVEGFR1, mVEGFR2, and mNeuropilin-1 trafficking in endothelial cells to quantify how the binding of different VEGF ligands alters the trafficking of these VEGF receptors. Our model aims to demonstrate the consequences of receptor localization and ligand binding on the localization and dynamics of signal initiation complexes. The model uses coupled, nonlinear, deterministic ordinary differential equations simulating biophysical and biochemical reactions of receptor dimerization, ligand-receptor binding, and trafficking processes. We used the model to simulate in vitro experiments of endothelial cell culture treated with ligands, drugs, and targeted shRNAs that perturb trafficking. We include simulations of four different splice isoforms of VEGF-A and PLGF, each of which binds to different combinations of the VEGF receptors, and we use new experimental data for two of these ligands to parameterize and validate our model.

Results, Conclusions, and Discussions: We show that mVEGFR2 trafficking is altered in response to ligand binding, but that trafficking of mVEGFR1 is not; we also show that the altered trafficking can be explained by an effect on just a single mechanistic process: increased internalization of the mVEGFR2 receptor when bound to ligand; other processes are unaffected. We further show that even though the canonical view of receptor tyrosine kinases is of activation on the cell surface, most of the ligand-receptor signal-initiation complexes formed by both mVEGFR1 and mVEGFR2 are intracellular (Fig.1). We also explore the competition between the receptors for ligand binding, the so-called ‘decoy effect’, and show that while, for in vitro cell culture, minimal such effect would be observed on the cell surface, inside the cell the effect can be substantial and influence signaling. This is rendered all the more important by the intracellular bias of signal-initiation complex location. These results expand our understanding of receptor-ligand trafficking dynamics and are critical for the design of therapeutic agents targeting the VEGF-VEGFR system in angiogenesis. In conclusion, we examined mVEGFR1, mVEGFR2, and mNRP1 interactions and trafficking in endothelial cells in the presence and absence of ligands which is relevant to ischemia in PAD. This is the first computational model to include detailed trafficking dynamics of mVEGFR1, mVEGFR2, and mNRP1 with ligands and the first comprehensive set of experimental data on trafficking for these three proteins measured in the same experiments. The model also allows us to quantify the effects that different ligands binding to these receptors have on receptor activation, receptor trafficking, receptor stability, and downstream signaling.

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