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. 2011 Sep;3(9):887-96.
doi: 10.1039/c1ib00037c. Epub 2011 Aug 8.

VEGF internalization is not required for VEGFR-2 phosphorylation in bioengineered surfaces with covalently linked VEGF

Sean M Anderson  1 , Bhupinder Shergill, Zachary T Barry, Eleana Manousiouthakis, Tom T Chen, Elliot Botvinick, Manu O Platt, M Luisa Iruela-Arispe, Tatiana Segura
Affiliations

VEGF internalization is not required for VEGFR-2 phosphorylation in bioengineered surfaces with covalently linked VEGF

Sean M Anderson et al. Integr Biol (Camb). 2011 Sep.

Abstract

Vascular endothelial growth factor (VEGF) is known to activate proliferation, migration, and survival pathways in endothelial cells through phosphorylation of VEGF receptor-2 (VEGFR-2). VEGF has been incorporated into biomaterials through encapsulation, electrostatic sequestration, and covalent attachment, but the effect of these immobilization strategies on VEGF signaling has not been thoroughly investigated. Further, although growth factor internalization along with the receptor generally occurs in a physiological setting, whether this internalization is needed for receptor phosphorylation is not entirely clear. Here we show that VEGF covalently bound through a modified heparin molecule elicits an extended response of pVEGFR-2 in human umbilical vein endothelial cells (HUVECs) and that the covalent linkage reduces internalization of the growth factor during receptor endocytosis. Optical tweezer measurements show that the rupture force required to disrupt the heparin-VEGF-VEGFR-2 interaction increases from 3-8 pN to 6-12 pN when a covalent bond is introduced between VEGF and heparin. Importantly, by covalently binding VEGF to a heparin substrate, the stability (half-life) of VEGF is extended over three-fold. Here, mathematical models support the biological conclusions, further suggesting that VEGF internalization is significantly reduced when covalently bound, and indicating that VEGF is available for repeated phosphorylation events.

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Conflict of interest statement

Disclosure of conflicts of interest: The authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1
Covalent binding of VEGF to heparin surface extends phosphorylation of VEGFR-2 up to 60 minutes. (A) System set-up. Gold slides are functionalized with VEGF as outlined in materials and methods, and cells are plated on flexible PDMS sheets. First, the cells from the flexible PDMS sheets are flipped onto the cells to mediate contact between the cells and the VEGF on the gold surface. After the cells are exposed to the surface for the desired time, the flexible PDMS sheet is flipped back and cells are lysed with lysis buffer. The cells are then scraped in a petri dish and lysate is collected and analyzed. (B) Western blot data for Y1175 from 0 to 60 minutes. Blots are re-probed for total VEGFR-2. At 60 minutes, Vc sustains activation over Ve and Vs. (C) Quantification of pY1175 for Vc, Ve, and Vs. Vc leads to a higher phosphorylation level at 60 minutes (*p < 0.05). (D) Western blot data for Y1214 from 0 to 60 minutes. Blots are re-probed for total VEGFR-2. Vc and Ve show increased activation over Vs for all time points, and Vc shows extended activation over Ve at 45 and 60 minutes. (E) Quantification of pY1214 for Vc, Ve, and Vs. Vc leads to a higher phosphorylation level over Vs at all time points, and Ve at 45 and 60 minutes (*p < 0.05) (n = 3).
Figure 2
Figure 2
(A) cdc42 GLISA data for Vc and Vs treated cells after 60 minutes of treatment shows increased activation of this pathway by Vc (p < 0.001). (B) Western blot data for phospho and total p38 for various treatment conditions. Vc and Ve increase p38 activation more than two-fold over Vs at 60 minutes. (n = 3).
Figure 3
Figure 3
VEGFR-2 phosphorlation requires VEGFR-2 internalization, but not VEGF internalization. (A) Western blot data of phospho-VEGFR-2 (Y1175) under the different treatment conditions for treatment times of 5, 15, and 30 minutes, with and without addition of 80 μM dynasore 30 minutes before cell treatment. Dynasore inhibits receptor endocytosis and decreases Y1175 activation in all cases. Interestingly, Vc is still able to produce some phosphorylation even in the presence of dynasore. The dynasore time point is normalized to the corresponding non-dynasore time point. (B) Western blot data of phospho-VEGFR-2 (Y1214) under the different treatment conditions for treatment times of 5, 15, and 30 minutes, with and without addition of 80 μM dynasore 30 minutes before cell treatment. Interestingly, dynasore increases Y1214 activation in all cases. The dynasore time point is normalized to the corresponding non-dynasore time point.
Figure 4
Figure 4
(A) Optical tweezers method follows Kotlarchyk et al. First, the laser traps the bead. Second, the bead is placed next to the cell and begins interaction with cell. Third, the laser drags bead away from cell while interaction occurs. The displacement of the bead from the laser is proportional to the rupture force. Fourth, the system returns to step one. (B) Histogram shows Ve (dark) and Vc (gray) rupture force distributions. Ve has a range of 3–8 pN while Vc mode is shifted to the right, 6–12 pN, indicating an increase in rupture force required with introduction of the covalent bond.
Figure 5
Figure 5
(A) I125-VEGF data indicates covalently binding retards internalization of VEGF during receptor endocytosis. Ve can be removed from heparin surface, but more Vc remains on surface during receptor endocytosis. Model fits are from the chemical reaction kinetics model. Incorporation of the idea of non-internalizing ligand leads to fits of the bound VEGF data. (B) Receptor quenching experiment in which the cells are first exposed to growth factor surface for a few seconds or 30 minutes, and then followed with exposure to I125-VEGF for 30 minutes. The results indicate that receptor recycling is severely altered by covalently bound VEGF (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 6
Figure 6
Vc extends VEGF half-life. (A) After storage at 4°C and 37°C for the indicated number of days, Vc slides were exposed to cells for 5 minutes and then probed for detection of phospho-VEGFR-2 (Y1175). Vc stored at 37°C and Vs stored at 4°C have similar half-lives, but Vc stored at 4°C has an extended half-life. (B) Quantification of bands from (A) were fit to mathematical model derived from autocatalytic degradation. Vc stored at 37°C and Vs stored at 4°C have a half-life of 2 days. Vc stored at 4°C has a half-life of one week. (N is Vn, S is Vs, C is Vc, and the numbers indicate days in storage).
Figure 7
Figure 7
Vc, Ve, Vs, and Vn were exposed to multiple 30-minute rounds of cells, probed for detection of (A) phospho-VEGFR-2 (Y1175) and (B) phospho-VEGFR-2 (Y1214). Vc and Ve maintain phosphorylation of VEGFR-2 in both tyrosine residues through all four treatments. Vs begins to lose phosphorylation intensity during the third exposure for Y1175 and has lower activity for Y1214 overall.
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