VEGF Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Vascular endothelial growth factor (VEGF), also called VEGF-A and vascular permeability factor (VPF), is a secreted homodimeric glycoprotein belonging to the VEGF family with a role in stimulating angiogenesis and vasculogenesis (1,2). More specifically, VEGF-A secretion from most cell types contributes to promoting endothelial cell proliferation and migration, inhibiting apoptosis, increasing vascular permeability, and wound healing (1). The VEGF family consists of several members including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placenta growth factor (PLGF) (1-4). As a result of alternative splicing of the eight exon VEGFA gene, there are several VEGF-A protein isoforms of 121, 145, 165, 183, 189, and 206 amino acids (aa) in length, with VEGF121 and VEGF165 being the two most expressed isoforms (1,5). Full length VEGF-A monomer has a 26 aa signal sequence plus a 206 aa (VEGF206) sequence, with a theoretic molecular weight (MW) of 27 kDa, containing VEGF receptor 1 (VEGFR1) and VEGR2 binding sites and heparin-binding domains (1-3,5,6). VEGF121 lacks heparin affinity and binds the receptor tyrosine kinases (RTKs) VEGFR1 and VEGFR2, whereas VEGF165 has moderate affinity for heparin and, in addition to being a ligand for VEGFR1 and VEGFR2, can also bind the co-receptors neuropilin 1 (NRP1) and NRP2 (1,5). Hypoxia and hypoxia-related genes such as HIF-1, EGF, and PDGF are major regulators angiogenesis and VEGF expression (1,3). VEGF signaling initiated by ligand binding to its receptors results in activation of different pathways including PI3K and MAPK and ultimately guides endothelial cell proliferation, migration, and survival (1,3). While VEGF plays an important role in promoting normal angiogenesis and blood vessel formation, its expression is often upregulated in tumors and other angiogenesis-related pathologies like osteroarthritis (OA) (1-5,7). Given its function, VEGF and its receptors have become a therapeutic target for treating cancer and blocking angiogenesis (4,5,7). A recombinant humanized monoclonal anti-VEGFA antibody called bevacizumab (Avastin) was first approved by the FDA in 2004 for the treatment of a number of cancers (1-3,5). Cancer patients may experience resistance to anti-VEGF antibodies and, as such, clinical studies are exploring combination treatment options with chemotherapies and immune-checkpoint inhibitors (3,5).

References

1. Melincovici CS, Bosca AB, susman S, et al. Vascular endothelial growth factor (VEGF) - key factor in normal and pathological angiogenesis. Rom J Morphol Embryol. 2018;59(2):455-467.

2. Shaik F, Cuthbert GA, Homer-Vanniasinkam S, Muench SP, Ponnambalam S, Harrison MA. Structural Basis for Vascular Endothelial Growth Factor Receptor Activation and Implications for Disease Therapy. Biomolecules. 2020;10(12):1673. https://doi.org/10.3390/biom10121673

3. Apte RS, Chen DS, Ferrara N. VEGF in Signaling and Disease: Beyond Discovery and Development. Cell. 2019;176(6):1248-1264. https://doi.org/10.1016/j.cell.2019.01.021

4. Matsumoto K, Ema M. Roles of VEGF-A signalling in development, regeneration, and tumours. J Biochem. 2014;156(1):1-10. https://doi.org/10.1093/jb/mvu031

5. Itatani Y, Kawada K, Yamamoto T, Sakai Y. Resistance to Anti-Angiogenic Therapy in Cancer-Alterations to Anti-VEGF Pathway. Int J Mol Sci. 2018;19(4):1232. Published 2018 Apr 18. doi:10.3390/ijms19041232

6. Uniprot (P15692)

7. Hamilton JL, Nagao M, Levine BR, Chen D, Olsen BR, Im HJ. Targeting VEGF and Its Receptors for the Treatment of Osteoarthritis and Associated Pain. J Bone Miner Res. 2016;31(5):911-924. https://doi.org/10.1002/jbmr.2828

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBL1-17712

• VEGF Antibodies
• VEGF ELISA Kits
• VEGF Lysates
• VEGF Proteins
• VEGF Proteins and Enzymes
• VEGF ELISAs