Figure 1.
Schematics of VEGF Transport in Tumors, VEGF Receptor Binding, and Therapeutic Strategies
(A) Schematic of the in vivo model. Parenchymal cells secrete VEGF; VEGF121 is freely diffusible, but VEGF165 can be sequestered by proteoglycans in the ECM (light gray) and the basement membranes (dark gray). The isoforms bind to VEGF receptors on the endothelial cells.
(B) VEGF isoforms bind to VEGFR2 that transduces the angiogenic signal intracellularly. VEGF121 does not bind Neuropilin-1; VEGF165 may bind both VEGFR2 and Neuropilin-1 simultaneously. Thus there are two pathways for the binding of VEGF165 to the signaling VEGFR2 receptor: first by binding directly, and second by binding Neuropilin-1 and then diffusing laterally on the cell surface to couple to VEGFR2. VEGFR1, which modulates the signaling of VEGFR2, binds both isoforms of VEGF. VEGFR1 also binds directly to Neuropilin-1. This complex is permissive for VEGF121–VEGFR1 binding but not VEGF165–VEGFR1; thus, high levels of Neuropilin-1 displace VEGF165 from VEGFR1, making it available for VEGFR2 binding. Only VEGF165 binds directly to the ECM binding site (represented by GAG chains).
(C) By targeting Neuropilin-1, we can target specifically VEGF165-induced signaling. Three methods for targeting Neuropilin-1 are analyzed here: blockade of Neuropilin-1 expression (e.g., using siRNA); blockade of VEGF–Neuropilin binding (e.g., using a fragment of placental growth factor to occupy the binding site); and blockade of VEGFR–Neuropilin coupling (e.g., using an antibody for Neuropilin-1 that does not interfere with VEGF–Neuropilin binding).
Figure 2.
Interactions between VEGF121, VEGF165, and Their Cell Surface Receptors VEGFR1, VEGFR2, and Neuropilin-1, and Interstitial GAG Binding Sites
(A) VEGF121 binds to VEGFR2 but not Neuropilin-1. VEGF165 binds both receptors as well as GAG chains in the interstitial space. VEGF165 bound to Neuropilin-1 can diffuse laterally on the cell membrane and bind VEGFR2 (and vice versa), coupling these receptors together, even though the receptors themselves do not interact.
(B) VEGF121 and VEGF165 both bind VEGFR1. Neuropilin-1 and VEGFR1 interact directly, forming a complex that is permissive for VEGF121 binding but not VEGF165.
(C) Inhibition of Neuropilin-1 expression results in a decrease in the insertion rate of Neuropilin receptors into the cell membrane (sN).
(D) PlGF2Δ, a fragment of placental growth factor, competes with VEGF165 for the binding site on Neuropilin-1.
(E) An antibody to Neuropilin-1 that does not interfere with VEGF165 binding can block the coupling of VEGF165–Neuropilin to VEGFR2, resulting in sequestration of VEGF on nonsignaling Neuropilin.
Table 1.
Microgeometrical Parameters for Breast Cancer
Table 2.
Kinetic Parameters of VEGF System
Table 3.
VEGF Concentration and VEGFR Density
Figure 3.
VEGF Binding to VEGFR2 In Vivo Is Inhibited by Targeting Neuropilin-1
(A–C) The time course of VEGF–VEGFR2 complex formation on the endothelial cells following each of the three treatments. For blocking VEGF–Neuropilin-1 binding (B) and VEGFR–Neuropilin-1 coupling (C), this figure represents bolus intratissue protein delivery at time 0. For Neuropilin-1 expression blockade, insertion of Neuropilin-1 into the membrane decreases to the indicated level at time 0. The VEGF121–VEGFR2 binding curve is indistinguishable from the no-treatment line in each case. The tumor modeled here expresses 10,000 VEGFR2 and 100,000 Neuropilin per endothelial cell.
(D–F) Free (unbound) VEGF concentration in the interstitial space. *VEGF121 secreted at the same rate as VEGF165.
(G–I) The concentration of VEGF inhibitor in the interstitial space, or density of Neuropilin, on the blood vessel endothelial cell surface.
Figure 4.
Average VEGF–VEGFR2 Signaling Inhibition
The average inhibition of VEGF–VEGFR2 complex formation over the 48 hours following blockade of Neuropilin-1 expression (A), or bolus intratissue protein delivery (B) of competitive binding inhibitor or VEGFR–Neuropilin-1 coupling blocker. Endothelial cells expressing 10,000 VEGFR2 and 100,000 Neuropilin-1.
Figure 5.
Tissue Expressing High VEGFR1 Levels Responds Differently to Treatment
(A–C) Formation of VEGF–VEGFR2 complexes over time following anti-Neuropilin treatment, for a tumor expressing 10,000 VEGFR1 per endothelial cell in addition to the VEGFR2 and Neuropilin-1 expression of Figures 3 and 4.
(D–F) VEGF–VEGFR1 complex formation.
(G–I) Free (unbound) interstitial VEGF concentration. *VEGF121 secreted at the same rate as VEGF165.
Figure 6.
Average VEGF–VEGFR2 Signaling Inhibition over the 48 Hours Following Treatment
Endothelial cells expressing 10,000 VEGFR1, 10,000 VEGFR2, and 100,000 Neuropilin-1.
Figure 7.
Tissue Specificity of Neuropilin-Targeted Inhibition of VEGF Signaling
Tissues that express low levels of Neuropilin-1 are insensitive to all Neuropilin-targeting treatments. The inhibition of VEGF–VEGFR2 signaling is directly proportional to Neuropilin-1 density (A–C), except at very high Neuropilin levels, which can overcome the inhibition. Tissues that express intermediate and high levels of Neuropilin-1 are further distinguished by the level of expression of VEGFR1. Blocking VEGFR–Neuropilin coupling is the most effective treatment to reduce VEGF–VEGFR2 signaling for tissues with any VEGFR1 expression level. However, in high VEGFR1 tissues, the other treatments are also quite effective. All three treatments significantly induce VEGF–VEGFR1 complex formation (D–F). The circles in each figure denote the conditions for Figures 3 and 4 (left, VEGFR1 actually zero in simulations) and Figures 5 and 6 (right) to compare the efficacy of the treatments for different tumors. The results shown are for a Neuropilin-1 expression knockdown to 1%, for 1 μM PlGF2Δ, and 1 μM AbNRP.
Figure 8.
VEGF Signaling Inhibition Is Effective for a Shorter Period of Time for a Tissue with a Higher Microvascular Density
(A–C) VEGF–VEGFR2 complex formation on the endothelial cells following each of the three treatments. The tumor modeled here expresses 10,000 VEGFR2 and 100,000 Neuropilin per endothelial cell. Gray lines represent the case of 2% vascular volume, as depicted in Figure 3; the black lines represent 4.2% vascular volume. Note that while the 103/cell, pM, and nM scales apply to both the gray and black lines, the pmol/L tissue scales apply only to the black lines; the normalization is different for the gray lines (see Figure 3 for the correct scales).
(D–F) Free (unbound) VEGF concentration in the interstitial space. *VEGF121 secreted at the same rate as VEGF165.
(G–I) The concentration of VEGF inhibitor in the interstitial space, or density of Neuropilin on the blood vessel endothelial cell surface.
Figure 9.
Average VEGF Signaling for the 48 Hours Following Treatment Inhibition Is Decreased at Higher Microvascular Density
The gray lines represent the case of 2% vascular volume (as depicted in Figure 4); the colored lines represent a 4.2% vascular volume. The peak inhibition due to the blockade of VEGFR–Neuropilin coupling is the same for both vascular densities; however, the peak for the other two treatments is lower. Endothelial cells expressing 10,000 VEGFR2 and 100,000 Neuropilin-1.