This work was supported by the University of Massachusetts Lowell Co-op Scholars Program

Control of Vascular Endothelial Growth Factor Binding to Its Receptor

Surenna Pecchia, Divyabharathy Tsiros, Matthew A. Nugent, Ph.D.Department of Biological Sciences, University of Massachusetts Lowell

Objective

Background

Approach

Angiogenesis is the process of growing new blood vessels from pre-existing blood vessels2. This process involves the proliferation and maintenance of endothelial cells, and serves as the main method of transporting oxygen and nutrients to cells throughout the body1. The angiogenic signal plays a crucial role in the maintenance of homeostasis—a poor signal leads to deficiencies in regeneration and healing, while an excessive signal can serve to fuel tumor growth2 (Figure 1). Tumors require a constant blood supply in order to grow to a substantial size; therefore, tumors stimulate angiogenesis by either transmitting chemical signals, or by stimulating normal cells nearby to secrete angiogenesis signaling molecules1. One such signaling molecule is vascular endothelial growth factor (VEGF). VEGF is a key protein regulator of angiogenesis, and is present in both normal and cancerous cells2. Two VEGF receptors (VEGFR1 and VEGFR2) are located on the endothelial cell surface, and initiate an angiogenic signal upon the binding of VEGF to one of its receptors2 (Figure 2). The VEGF+VEGFR2 complex is made more secure by the additional binding of heparan sulfate proteoglycans (HSPGs), which are also located on endothelial cell surfaces3. These HSPGs consist of a core protein, with heparan sulfate molecules branching off3(Figure 3). Heparan sulfates are long, sugar-chain molecules with a variable structure, which allows for extensive protein binding sites on its surface2. HSPGs can modulate the transport and distribution of proteins bound to the heparan sulfate chains to various intracellular locations4. On the endothelial cell surface, HSPGs and VEGFR2 in close proximity can result in both complexes binding VEGF molecules to create a high affinity signaling complex4. Previous studies suggest that VEGF bound to both HSPGs and VEGFR2 induces a stronger angiogenic signal than that produced by VEGF-VEGFR2 complexes alone.

My research project was focused on understanding the interactions between several different molecules involved in angiogenesis. I explored how different combinations of vascular endothelial growth factor (VEGF), VEGF receptor 2, and heparin/heparan sulfates bound to each other, as well as which combinations yielded the strongest binding affinites. Another focus of mine was to explore the mechanism by which VEGF, VEGFR2, and heparin bound to each other.

References

A 96-well Heparin Binding Plate was used in each binding assay. The bottom of each well is pre-coated with positive charges in order to ensure the binding of the negatively charged heparin/heparan sulfates.

Heparin/Heparan sulfates are negatively-charge sugar chain molecules. They contain multiple binding sites and are good facilitators of proteins and other molecules into cells. Because they are negatively charged, they’re able to bind to the bottom of each well.

VEGF Receptor 2 was added to each well containing heparin.

VEGF molecules were also added to each well containing heparin and VEGF Receptor 2.

Binding occurred between VEGF, R2, and heparin.

Any molecules that were not bound to the plate were washed away with buffers.

A Donkey anti-human HRP-linked antibody was added to each well. It bound to the Fc region of the VEGFR2 chimera.

The antibody contains a linked HRP region, which interacted with the TMB substrate solution to create a yellow pigment.

TMB

substrateColor change

(yellow)

Conclusions

1. http://www.cancer.gov/about-cancer/treatment/types/immunotherapy/angiogenesis-inhibitors-fact-sheet2. Teran, M., & Nugent, M. A. (2015). Synergistic binding of vascular endothelial growth factor-A and its receptors to heparin selectively modulates complex affinity. Journal of Biological Chemistry, 290(26), 16451-16462.3. Lin, X. (2004). Functions of heparan sulfate proteoglycans in cell signaling during development. Development, 131(24), 6009-6021.4. Bernfield, M., Götte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., & Zako, M. (1999). Functions of cell surface heparan sulfate proteoglycans. Annual review of biochemistry, 68(1), 729-777.5. http://polysac3db.cermav.cnrs.fr/discover_GAGs.html6. Shibuya, M. (2003). Vascular endothelial growth factor receptor‐2: its unique signaling and specific ligand, VEGF‐E. Cancer science, 94(9), 751-756.

PBST-B (Blank)

One Hour

PBST-B (Blank)

PBST-B (Blank)

PBST-B (Blank)

One Hour

Treatment – First Addition

Treatment – Second Addition

VEGF Plays a Critical Role in the Binding of R2 to HeparinVEGF R2

Donkey anti-human HRP-linked secondary antibody

VEGF+R2

In order to investigate the mechanisms by which heparin, VEGF, and VEGFR2 bind to each other, the sequence by which these proteins were added to heparin-coated wells was varied. All wells were coated with heparin and then incubated with VEGF or R2 in PBST-B, or with PBST-B alone for 1 hour and then each solution was removed from the wells, the wells were washed, and the second addition of R2 or R2+VEGF were added and allowed to incubate for an additional hour. Of the wells that had been incubated with VEGF only, three were given R2 only, and three were given PBST-B. Of the wells that had been incubated with R2 only, three were given VEGF only, and three were given PBST-B. Three of the wells that had been previously incubated with PBST-B, were given VEGF only, three were given R2 only, three more were given VEGF+R2, and the rest were given PBST-B as a blank. The wells treated with PBST-B first, and VEGF+R2 second, or with VEGF first, and R2 second showed similar high levels of binding. These results suggest that in order for there to be a strong binding affinity, VEGF must bind to heparin first, and R2 can bind the VEGF afterwards.

In order to investigate the differences in binding affinities between VEGF and R2, heparin coated and uncoated wells within 96-well plates were exposed to solutions containing: VEGF (10nM), VEGFR2 (1 nM), or VEGF (10 nM) and R2 (1 nM) in triplicate. The amount of R2 bound was measured using an ELISA detecting the Fc portion of the VEGFR2-Fc chimera protein, and the average ± S.D. are shown for each condition. The greatest amount of R2 binding was observed when VEGF and R2 were incubated with heparin coated plates. There appeared to be a small amount of binding of R2 to heparin in the absence of VEGF. There was virtually no signal in heparin coated and uncoated wells when R2 was not included in the incubation (i.e., VEGF alone or binding buffer containing bovine serum albumin without any additions).

Modified Heparins Result in Different Binding Affinities with VEGF and R2

2O-DS: The sulfate on the 2-carbon ring is removed and replaced with a hydrogen molecule.

6O-DS: The sulfate on the 6-carbon ring is removed and replaced with a hydrogen molecule.DOS: The sulfates on

both the 2-carbon ring and the 6-carbon ring are removed and replaced with hydrogen molecules.

NDS: The sulfate on the nitrogen is removed and replaced with a hydrogen molecule.NAc: The sulfate on the nitrogen is removed and replaced with an acetyl-group.

• VEGFR2 alone shows very low binding to heparin; however, in the presence of VEGF, R2 shows greater binding to heparin.

• When heparin is treated with VEGF first, and R2 second, there is a very strong binding affinity, while heparin treated with R2 first and VEGF second results in relatively little binding. This indicates that VEGF is a necessary facilitator of R2 binding heparin.

• By understanding how VEGF and VEGFR2 interact with each other, we can investigate therapies to either stimulate or inhibit angiogenesis. Stimulating this process would likely allow for tissue repair, while inhibiting the process could slow, or stop tumor growth.

VEGF165

VEGFR-2Figure 2. Vascular endothelial growth factor (VEGF) is a protein dimer. VEGFR2, is a dimer as well, and exists as a transmembrane protein. This receptor is characterized by a tyrosine kinase structure, as well as several immunoglobin domains located in the extracellular matrix6. Modified from Teran, (2015) Boston U.

In this assay, several different modified heparins were used to coat a well plate and compared to heparin as a control. Unlike un-modified heparin, which contains a sulfate group at the N and 6-O position of the glucosamine residues and on the 2-O position of the uronic acid residues, the modified heparins have had specific sulfate groups selectively removed (Figure 4). Most of the conditions displayed a high binding affinity when treated with R2+VEGF, except for the DOS heparin, which is devoid in 6-O and 2-O sulfation. This indicates that O-sulfation is critical for binding to occur, even though both the 2-O and 6-O desulfated heparins (2OS and 6OS) were able to support a significant amount of binding. N-desulfated (NDS) and N-acetylated (NAc) heparin showed an intermediate binding response, indicating that sulfation on the N-group is somewhat important for binding to occur. Concentration dependent variability is shown with heparin and heparin derivatives in its ability to form ternary complexes with VEGF and VEGFR-2.

Modified from PolySac Database5

Figure 1. Angiogenesis differs from vasculogenesis in that the former is the process of growing new blood vessels from pre-existing ones. This process can be manipulated positively, for enhanced wound healing, or negatively, in the case of cancers and other diseases2.Modified from Teran, (2015) Boston U.

Heparin

Figure 4.

Figure 3. Heparan sulfate proteoglycans consist of a core trans-membrane protein, with sugar chains branching off into the extracellular matrix. Heparan sulfates contain many protein binding sites, which allows them to bind VEGF and aid in stimulating angiogenesis3.Modified from Teran, (2015) Boston U.