Fig 1.
A. Biochemical Reactions. Interactions between VEGF, VEGFR2, NRP1, and an extracellular matrix proteoglycan (M) are summarized. VEGF can bind VEGFR2, NRP1, and M. NRP1 and M cannot be present in the same complex (as they bind to the same surface of VEGF), and VEGFR2 and NRP1 cannot form a complex without VEGF. B. Trafficking Pathways. Surface molecular complexes can be internalized with rate constant kintn. Rab4/5-resident molecular complexes can be degraded (rate constant kdegr), recycled (rate constant krec4), or transferred to the Rab11 compartment (rate constant k4to11). Rab11 endosome-resident complexes are recycled with rate constant krec11. New surface receptors are produced at rate s. C. Phosphorylation Reactions. Intracellular tyrosine residues Y951, Y1175, and Y1214 are phosphorylated and dephosphorylated independently. D. Overview of signaling pathways and cellular behaviors downstream of tyrosine residues Y951, Y1175, and Y1214 on VEGFR2.
Table 1.
Model parameters for biochemical reactions.
Table 2.
Model parameters for trafficking.
Table 3.
Model parameters for VEGFR2 phosphorylation.
Table 4.
Initial conditions and parameters that vary by study.
Fig 2.
VEGF presentation and trafficking control the distribution of ligated VEGFR2.
A. Visualization of fit trafficking parameters. Red dots indicate a representative coherent set of parameters used throughout the rest of this study. (See Methods) B. Tuning of kdegr values to match data in 2010 presentation study. Dotted lines: fits using kdegr values fit in PAECs; solid lines: fits after kdegr values are increased by a factor of 2.4 to minimize the least squared error between simulations and this experimental data. Soluble VEGF (Vs), blue lines; bound VEGF (Vb), green lines. C. Summary of the distribution of VEGF-bound (V·R2) and unbound VEGFR2 (Free R2) over time, shown for Vb. Y-axis is shown in terms of the percentage of the steady-state (no VEGF) total VEGFR2 population. Note that, due to degradation, the total amount of VEGFR2 decreases after addition of VEGF. [V] = 20 ng/mL. D. Decrease in total VEGFR2 upon stimulation with VEGF. Solid line, [V] = 2 ng/mL; dashed line, [V] = 20 ng/mL; dotted line, [V] = 200 ng/mL. E-M. Distribution of V·R2 on the cell surface (E-G), in Rab4/5 endosomes (H-J), and in Rab11 endosomes (K-M) following stimulation with Vs (left column) or Vb (middle column). The right column shows the ratio of the first two columns.
Fig 3.
Prediction of VEGFR2 binding and phosphorylation parameters.
A. Total surface Vs·R2 + internal VEGF, used to fit koff,M·V for the 2011 presentation study [8]. Lines indicate simulation results, dots indicate experiments. Soluble VEGF (Vs), blue; electrostatic bound VEGF (Ve), green; covalent bound VEGF (Vc), red. B-C. pY1175 and pY1214 data for 2011 presentation study [8], which was used to fit dephosphorylation rate constants, along with H. D-F. Phosphorylation data from the 2010 presentation study [29], which was used to confirm dephosphorylation rate fits. D-E. Western blot data. F. ELISA data. Soluble VEGF, blue; bound VEGF, green. G-J. Additional phosphorylated VEGFR2 (by residue) experimental data from 2010 presentation study with model predictions. K. Ratio of pR2 for Vb and Vs at various times, from 2010 presentation study.
Fig 4.
Validation of complete model with trafficking and phosphorylation parameters.
A. Validation of trafficking parameters by comparing model predictions for VEGFR2 in the absence of NRP1 in PAECs compared to Ballmer-Hofer data for V165b (Vb), which does not bind NRP1. Data is compared to the normal case including NRP1 in the model and data for V165a (Va), which does bind NRP1, at 30 min in the trafficking study [40]. The percent of internal VEGFR2 in Rab4/5 and Rab11 endosomes (left) and the ratio of total VEGFR2 in Rab4/5 endosomes to total VEGFR2 in Rab11 endosomes (right) is shown. Grey: model simulations; black, experimental data. B-D. Validation of complete model including trafficking and phosphorylation parameters. B) Data taken from Martino et. al. 2011 [61]. [V] = 50 ng/mL. Assumed VEGF and FN are pre-mixed. Soluble VEGF, blue; VEGF bound to wild type fibronectin, green; VEGF bound to rFNIII9-10/12-14 fragments, red (koff,M·V values from Wijelath et. al. 2006 [18]). C-D. Data taken from an additional Anderson et. al. 2011 study [59]. E-F. Validation of phosphorylation parameters by comparing model predictions to data in cells with perturbations to specific phosphatases. E. Impact of siRNA against VEPTP on pY951, pY1175, and pY1214. Experimental data (black) taken from Mellberg et. al. 2006 [60]. TIME cells, [V] = 50 ng/mL, measurements taken at t = 5 min. In the model, the experimentally observed decrease in VEPTP expression to 20% of control values with VEPTP siRNA was simulated by decreasing the dephosphorylation rate for Y951 and Y1175 by a factor of 5 on the cell surface. HUVEC receptor numbers were used in model. F. Impact of exposing HEK 293 cells transfected with VEGFR2 to a constitutively active form of TCPTP on pR2, pY1175, and pY1214. Data taken from Mattila et. al. 2008 [62]. In the model, we simulated the constitutively active TCPTP by increasing the dephosphorylation rate for Y951 and Y1214 to match the rate for unligated R2 (30 s-1) in all compartments. HUVEC receptor numbers were used in model.
Fig 5.
VEGF presentation mode affects VEGFR2 phosphorylation more than VEGFR2 ligation.
The time-dependent response to soluble VEGF (Vs, blue, left column) and bound VEGF (Vb, green, middle column) of: VEGF-ligated VEGFR2 (V·R2, A-C); all phosphorylated VEGFR2 (pR2, D-F); and site-specific phosphorylated VEGFR2, pY1175 (G-I) and pY1214 (J-L). The ratios of responses to bound and soluble VEGF are shown at the right. Time-scale ends at 30 minutes, but pR2 curves are relatively flat after this time. Solid line, [V] = 2 ng/mL; dashed line, [V] = 20 ng/mL; dotted line, [V] = 200 ng/mL.
Fig 6.
Increased total VEGFR2 activation with immobilized VEGF is driven by the change in surface VEGFR2.
All panels show area under the curve (AUC), a measure of total VEGFR2 activation, for the first 60 minutes after stimulation with soluble (Vs- blue) or immobilized (Vb- green) VEGF at a concentration of 2 ng/mL. AUCs are shown for surface VEGFR2 (A-D), Rab4/5 VEGFR2 (E-H), and total VEGFR2 (I-L). Activation on any considered tyrosine residue (pR2, 1st column), Y1175 (2nd column), and Y1214 (3rd column) are compared. The last column shows the AUC for the curve pY1214/pY1175 (total pY1214/pY1175 curves shown in Fig 8F and 8G) for surface VEGFR2 (D), Rab4/5 VEGFR2 (H), and total VEGFR2 (L). Note that the difference in total pY1214/pY1175 for Vs and Vb emerges from the altered VEGFR2 distribution, not altered pY1214/pY1175 in each subcellular location.
Fig 7.
Neuropilin-1 and phosphatases modulate site-specific VEGFR2 phosphorylation.
A-C. Distribution of VEGF-bound VEGFR2 (sum of V·R2, V·N1·R2, and M·V·R2) in HUVECs. Solid lines: Baseline case with NRP1 present; dotted lines: no NRP1 present. Soluble VEGF (Vs), blue lines; bound VEGF (Vb), green lines. For all lines, [V] = 20 ng/mL, HUVEC receptor numbers. D-G. Total VEGFR2 phosphorylated on at least one of Y951, Y1175, and Y1214 (pR2, D), pY1175 (E), pY1214 (F), and total VEGFR2 (G) with NRP1 (solid lines) and without NRP1 (dotted lines). H-I. Model predictions for site-specific VEGFR2 phosphorylation under perturbation of phosphatase activity. H. Model predictions for the experiment in Fig 4F (siRNA to VEPTP), with the addition of a constitutively active TCPTP (as described in Fig 4G). I. Model predictions for exposure of HUVECs to a cell-surface phosphatase that dephosphorylates Y951, Y1175, and Y1214, similar to DEP-1. The impact of increasing phosphatase expression by a factor of 2, 5, or 10 is shown.
Fig 8.
Relative activation pY1175 and pY1214 varies as a function of VEGF immobilization and concentration.
A-C. Total VEGFR2 phosphorylated on at least one of Y951, Y1175, and Y1214 (pR2, A) and site-specific phosphorylation (pY1175, B and pY1214, C) at 5 min with varying koff,V·M. Note quantities of VEGFR2 phosphorylated on any residues in Rab11 endosomes are negligible. D. Ratio of pY1214 to pY1175 at 5 minutes with varying koff,V·M. E. Percent of surface V·R2 complexes that are bound to the matrix at t = 5 minutes. F-H. The ratio of pY1214-VEGFR2 to pY1175-VEGFR2 for Vs (F) and Vb (G). The ratio of this quantity for bound VEGF and soluble VEGF (Vb/Vs) is shown in (H). Solid line, [V] = 2 ng/mL; dashed line, [V] = 20 ng/mL; dotted line, [V] = 200 ng/mL. Soluble VEGF (Vs), blue lines; bound VEGF (Vb), green lines. For all lines, HUVEC receptor numbers were used.
Fig 9.
Differences in molecular interactions of VEGF isoforms are predicted to account for changes in observed vascular phenotype.
A. VEGF121 does not bind NPR1 or extracellular matrix proteins (M), leading to slow VEGFR2 ligation (due to lack of NRP1-mediated VEGF-VEGFR2 binding), fast internalization (no immobilization of VEGF121), and a lack of recycling via the Rab11 pathway. B. VEGF165 binds both NRP1 and extracellular matrix species, leading to faster VEGFR2 ligation, but slower internalization of VEGFR2 compared to stimulation with VEGF121. The Rab11 recycling pathway is accessible to VEGF165-NRP1-VEGFR2 complexes. C. VEGF189 binds to extracellular matrix species and NRP1 more strongly than VEGF165. This results in moderate VEGFR2 ligation speed (NRP1 must compete with matrix species (M) for VEGF) and slow VEGFR2 internalization.