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C. J. Barnstable, L. Chen, S. S. M. Zhang, J. Tombran-Tink; Pedf Binds With High Affinity to the Extracellular Domain of VEGF-R1 Receptor. Invest. Ophthalmol. Vis. Sci. 2007;48(13):38.
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We have previously reported that PEDF leads to endothelial cell apoptosis by binding to the VEGF-R2 receptor and blocking its intracellular signaling pathways with an end result of caspsase activation and cell death. The purpose of the present experiments was to study the interaction of PEDF with other major VEGF mitogenic receptors on endothelial cells.
Purified recombinant extracellular domain of VEGF-R1 was used to coat ELISA plates and surface plasmon resonance (SPR) cuvettes. Purified PEDF was added and binding detected by anti-PEDF antibodies (ELISA) or optical signals (SPR). Immunoprecipitations of mixtures of purified VEGF-R1 and PEDF or primary cultures of human umbilical vein endothelial cell (HUVEC) lysates and PEDF were carried out using affinity purified anti-VEGF-R1 or anti-PEDF. VEGF-RI was knocked down in confluent HUVECs cultures maintained in medium containing 0.2% serum to measure the effects of VEGF and PEDF. Cells were treated with pooled VEGF-R1 siRNA sense strands (Santa Cruz Biotechnology, Santa Cruz, CA) and the responses of cells to VEGF and PEDF measured 72 hr later.
PEDF binding to the recombinant extracellular domain of VEGF-R1 was high-affinity and saturable, when measured in an ELISA assay. Measurement of this binding by SPR indicated an overall Kd of less than 1 nM. Anti-PEDF specifically precipitated VEGF-R1 from both mixtures of the purified proteins as well as from HUVEC lysates. Similarly, anti VEGF-R1 precipitated PEDF from both mixtures of purified proteins and from HUVEC lysates. Treatment of HUVECs with VEGF-R1 siRNA had no effect on the ability of PEDF to inhibit VEGF-R2 mediated signaling or induce apoptosis.
VEGF-R1 is a strong binding receptor for PEDF. The binding of PEDF to VEGF-R1 is independent of its binding to VEGF-R2 that we have previously described and does not influence the negative actions of PEDF on VEGF mitogenic signals HUVEC cell survival. PEDF antiangiogenic effects are most likely associated with its strong interaction with both VEGF-R1 and VEGF-R2.
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