783: Protein-interaction pathways of VEGF receptor-2 in placentas from normal and preeclamptic pregnancies

Abstract

783 Protein-interaction pathways of VEGF receptor-2 in placentas from normal and preeclamptic pregnancies Kiran Rao, John Tsibris, Stanley Stevens, Thora Steffensen, Maja Okuka, Valerie Whiteman, William Spellacy University of South Florida College of Medicine, Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine, Tampa, FL, University of South Florida, The Florida Center of Excellence for Drug Discovery and Innovation, Tampa, FL, Tampa General Hospital, Department of Pathology, Tampa, FL OBJECTIVE: Suboptimal placental vascularization is a hallmark in the pathophysiology of preeclampsia. Our goal is to define the protein complexes of VEGFR-2 which regulate placental vascularization. STUDY DESIGN: Large complexes of VEGFR-2 tolerate tissue freezing, homogenization and extraction by detergent ASB-14, as shown by Blue-Native electrophoresis. Tissue homogenates were spun at 100,000g and the pellets were cross-linked with DSP, a primary amine-reactive water-insoluble disulfide reagent, to ensure capture of small proteins bound to VEGFR-2 and thus decrease false negatives. After treatment with carbonate pH 11 to remove adhering proteins, the pellets were extracted with 1% ASB-14. Extracts were first mixed with magnetic M-280 sheep anti-rabbit IgG Dynabeads (InVitrogen) to reduce false positives and then applied to M-280 charged with VEGFR-2 antibody 55B11 (Cell Signaling). Immunoprecipitated proteins were eluted with ASB-14 at pH 2.8, fractionated by SDS-PAGE and after digestion with trypsin and Glu-C were identified by mass spectrometry. RESULTS: Mitochondrial proteins prohibitins (PHB) and pyruvate dehydrogenase component E2 (PDC-E2) co-immunoprecipitated with VEGFR-2. Westerns blots (WB) of ASB-14 extracts (without DSP) showed that PHB levels (smaller-kDa band) were 25% lower, but PDC-E2 levels were 30% higher in preeclamptic than normal placentas (n 4-7 placentas each at 34-38 weeks gestation, p 0.05). A fraction of available Leptin, the top up-regulated gene in preeclampsia, and of VEGF-A and all of Notch-1 also co-immunoprecipitated with VEGFR-2, as revealed by WB of bead eluates. All proteins were eluted in two peaks, by 0.1% and 1% ASB-14, that may represent complexes of different affinity for VEGFR-2 and the plasma membrane and intracellular pools of VEGFR-2; in arterial morphogenesis, VEGFR-2 signals from the intracellular pool (Carmeliet and Jain, Nature 2011; 473:298). CONCLUSION: Sequential immunoprecipitations and membrane proteomics can identify the up-stream and down-stream members of the VEGFR-2 pathways and may provide new insights on their function and the etiology of preeclampsia.