Vascular Endothelial Growth Factor (VEGF)-A165-induced Prostacyclin Synthesis Requires the Activation of VEGF Receptor-1 and -2 Heterodimer

We previously reported that vascular endothelial growth factor (VEGF)-A165 inflammatory effect is mediated by acute platelet-activating factor synthesis from endothelial cells upon the activation of VEGF receptor-2 (VEGFR-2) and its coreceptor, neuropilin-1 (NRP-1). In addition, VEGF-A165 promotes the release of other endothelial mediators including nitric oxide and prostacyclin (PGI2). However, it is unknown whether VEGF-A165 is mediating PGI2 synthesis through VEGF receptor-1 (VEGFR-1) and/or VEGF receptor-2 (VEGFR-2) activation and whether the coreceptor NRP-1 potentiates VEGF-A165 activity. In this study, PGI2 synthesis in bovine aortic endothelial cells (BAEC) was assessed by quantifying its stable metabolite (6-keto prostaglandin F1α, 6-keto PGF1α) by enzyme-linked immunosorbent assay. Treatment of BAEC with VEGF analogs, VEGF-A165 (VEGFR-1, VEGFR-2 and NRP-1 agonist) and VEGF-A121 (VEGFR-1 and VEGFR-2 agonist) (up to 10–9m), increased PGI2 synthesis by 70- and 40-fold within 15 min. Treatment with VEGFR-1 (placental growth factor and VEGF-B) or VEGFR-2 (VEGF-C) agonist did not increase PGI2 synthesis. The combination of VEGFR-1 and VEGFR-2 agonists did not increase PGI2 release. Pretreatment with a VEGFR-2 inhibitor abrogated PGI2 release mediated by VEGF-A165 and VEGF-A121, and pretreatment of BAEC with antisense oligomers targeting VEGFR-1 or VEGFR-2 mRNA reduced PGI2 synthesis mediated by VEGF-A165 and VEGF-A121 up to 79%. In summary, our data demonstrate that the activation of VEGFR-1 and VEGFR-2 heterodimer (VEGFR-1/R-2) is essential for PGI2 synthesis mediated by VEGF-A165 and VEGF-A121, which cannot be reproduced by the parallel activation of VEGFR-1 and VEGFR-2 homodimers with corresponding agonists. In addition, the binding of VEGF-A165 to NRP-1 potentiates its capacity to promote PGI2 synthesis.