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Inhibition of Angiogenesis by Recombinant VEGF Receptor Fragments

Davoud Ahmadvand PhD, Fatemeh Rahbarizadeh PhD, Farnoush Jafari Iri-Sofla PhD student, Gholamreza Namazi PhD student, Sepideh Khaleghi MSc, Bita Geramizadeh PhD, Parvin Pasalar PhD, Hosein Karimi PhD, Seyed Hamid Aghaee Bakhtiari PhD student
DOI: http://dx.doi.org/10.1309/LMMH2WYRLP7B3HJN 417-422 First published online: 1 July 2010

Abstract

Background: Blocking vascular endothelial growth factor receptor (VEGFR) is a successful approach for inhibiting vascular endothelial growth factor (VEGF) signaling. Small molecules impairing the interaction of VEGF with VEGF receptors have been synthesized and evaluated in this research.

Methods: In this study, we amplified and cloned the cDNA of VEGFR fragments. After expression of the fragments in Escherichia coli (E. coli), they were purified by immobilized metal affinity chromatography (IMAC). The biological activity of recombinant KDR fragments was evaluated by human umbilical vein endothelial cells (HUVEC) proliferation assay.

Results: The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PSGE) and immune-blotting results confirmed the production and purification of recombinant VEGFR fragments. The purified VEGFR2-III domain, VEGFR2-II–III domains, and VEGFR1-II domain showed 24%, 36%, and 27% inhibition effect in HUVEC proliferation assay, respectively. The recombinant VEGFR2-II–III domains strongly inhibited formation of capillary-like structures (CLS).

Conclusions: The produced recombinant proteins will serve as small soluble molecules with an inhibitory effect against VEGF in anti-angiogenesis researches.

Keywords:
  • angiogenesis
  • VEGFR fragments
  • expression
  • Abbreviations

    VEGFR
    vascular endothelial growth factor receptor
    VEGF
    vascular endothelial growth factor
    E. coli
    Escherichia coli
    IMAC
    immobilized metal affinity chromatography
    HUVEC
    human umbilical vein endothelial cells
    SDS-PSGE
    sodium dodecyl sulfate polyacrylamide gel electrophoresis
    CLS
    capillary-like structures
    Flt-1
    fms like tyrosine kinase
    KDR
    kinase insert domain-containing receptor
    Flk-1
    fetal liver kinase receptor 1
    PLGF
    placenta growth factor
    Ig
    immunoglobulin
    HRP
    horseradish peroxidase
    FBS
    fetal bovine serum
    Ni+-NTA
    nickel nitrilo-triacetic acid
    HBSS
    Hank’s buffer saline solution
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