Anti‐MBG antibodies attenuate MBG‐induced anti‐proliferative and anti‐angiogenic milieu in cytotrophoblast cell model

Abstract

Preeclampsia (PreE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. Recently, a digitalis‐like factor, marinobufagenin (MBG) has been implicated as a causative factor in preE. We demonstrated that MBG inhibits the proliferation, migration, and invasion of cytotrophoblast (CTB) cells. We have identified a novel human monoclonal antibody with higher specificity than Digibind for MBG. We assessed the attenuation of MBG‐induced CTBs dysfunction by three anti‐MBG antibodies: 206–208, H1L2, and 3e9.MethodsA panel of anti‐MBG antibodies with potential as human therapeutic agents was developed by Panorama Research, Inc.; Sunnyvale, CA (206–208, H1L2). H1L2 was a humanized version of previously described anti‐MBG murine antibody 3e9. 206–208 was identified in a human phage antibody library. Human CTBs were treated with DMSO (vehicle) or 0.1, 1, 10 or 100 nM of MBG for 48 h. Some cells were pretreated with 206–208, H1L2, or 3e9 for 2h, while others were co‐treated with these antibodies prior to MBG treatment. Culture media were collected for analysis of pro‐angiogenic and anti‐angiogenic factors. Cell viability was measured using a CellTiter Assay kit. Cell lysates were utilized to evaluate the expression of proliferating cell nuclear antigen (PCNA) and p38 MAPK phosphorylation by western blot. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms‐like tyrosine kinase‐1 (sFlt‐1), and soluble endoglin (sEng) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test.ResultsMBG down‐regulated PCNA and up‐regulated p38 phosphorylation in CTBs treated with ≥1 nM MBG compared to basal (DMSO treatment) (*p < 0.05 for each). Secretion of sFlt‐1 and sEng were increased, while VEGF and PIGF were decreased in CTBs treated with ≥1.0 nM MBG (*p < 0.05 for each). Both anti‐MBG antibodies pretreatment and co‐treatment significantly up‐regulated PCNA and down‐regulated p38 phosphorylation, and corrected the angiogenic profile of CTBs (p < 0.05 for each). The anti‐proliferative and anti‐angiogenic effects of MBG were not due to a cytotoxic effect, as evaluated by a cell viability assay. MBG at 0.1 nM had no effect.ConclusionsWe found that all 3 anti‐MBG antibodies attenuate MBG‐induced anti‐proliferative and anti‐angiogenic milieu in cultured CTBs. Here we describe for the first time two fully human anti‐MBG antibodies, which can be targeted as therapeutic agents for the development of innovative treatment strategies for preE and potentially other disorders involving aberrant MBG signaling.Support or Funding InformationNIH Small Business Innovation Research (SBIR) : 1R43HD079238‐01