316: Novel anti-MBG antibodies protect cytotrophoblast cells from a marinobufagenin-induced preeclampsia phenotype

Abstract

OBJECTIVE: Preeclampsia (PreE) is a hypertensive pregnancy disorder. 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. STUDY DESIGN: A panel of anti-MBG antibodies with potential as human therapeutic agents was developed by Panorama Research, Inc.; Sunnyvale, CA (206-208, H1L2). 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 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. RESULTS: MBG 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 antiangiogenic 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. CONCLUSION: We 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.

Preeclampsia: Сardiotonic Steroids, Fibrosis and a Hint to Cancerogenesis

“…A recent work also showed that incubation of explants of umbilical arteries obtained from the patients with PE with monoclonal antibodies against MBG led to a significant decrease in collagen-1 content [37]. Besides, incubation of healthy human umbilical arteries in the presence of low MBG concentrations for 24 hours led to decline in the Fli1 content and an increase in PKC-delta expression, while the level of pro-collagen-1 synthesis increased six-fold [95]. Another work has shown that the introduction of humanized monoclonal antibodies to MBG leads to phosphorylation of MAP kinase p38 in cytotrophoblast cells, indicating a possible therapeutic effect of these antibodies [96].…”

Preeclampsia: Cardiotonic Steroids, Fibrosis, Fli1 and Hint to Carcinogenesis

“…The incubation of healthy human umbilical arteries in the presence of low MBG concentrations for 24 h led to a decline in the Fli1 content and an increase in the PKC-delta expression while the level of procollagen-1 synthesis increased six-fold [ 37 ]. Another work has shown that the introduction of humanized anti-MBG monoclonal antibodies leads to phosphorylation of MAP kinase p38 in cytotrophoblast cells, indicating a possible therapeutic effect of these antibodies [ 57 ]. Moreover, a recent study has demonstrated that the silencing of Fli1 in human umbilical arteries mimics preeclamptic phenotypes through activating PKCδ and the activation of procollagen and collagen-1 synthesis [ 57 , 58 ].…”

“…Another work has shown that the introduction of humanized anti-MBG monoclonal antibodies leads to phosphorylation of MAP kinase p38 in cytotrophoblast cells, indicating a possible therapeutic effect of these antibodies [ 57 ]. Moreover, a recent study has demonstrated that the silencing of Fli1 in human umbilical arteries mimics preeclamptic phenotypes through activating PKCδ and the activation of procollagen and collagen-1 synthesis [ 57 , 58 ]. The antagonism of endogenous CS can be one of the new and fundamentally different possibilities of pharmacological therapy and prevention of vascular fibrosis.…”

“…A single administration of a monoclonal anti-MBG antibody in rats produced an anti-fibrotic effect; that is, restored Fli1 levels and a reduced collagen-1 abundance in the myocardium were observed [ 38 ]. Fli1 attracted attention primarily because of its contribution to different types of cancer including gastric cancer, Burkitt lymphoma, breast cancer, pancreatic ductal adenocarcinoma, small cell lung cancer and Ewing’s sarcoma [ 57 , 85 , 86 , 87 ]. We observed extremely high levels of MBG and low levels of Fli1 along with an extremely high level of collagen-1 in patients and experimental animals with preeclampsia, chronic renal failure and malignant hypertension [ 33 , 37 , 38 ].…”

Endogenous Bufadienolides, Fibrosis and Preeclampsia

“…Furthermore, in comparative study of the efficacy of specific antibodies with antidigoxin (DigiFab) and monoclonal antibody against MBG (3E9), it was shown that monoclonal antibodies restore the activity of PE-inhibited NKA in erythrocytes better than DigiFab, which suggested the involvement of MBG in the pathogenesis of PE [46]. In a recent study, it was shown that the administration of humanized monoclonal antibodies (206-208, H1L2) against MBG attenuate MBG-induced downregulated PCNA and upregulated p38 phosphorylation in cytotrophoblast cells, indicating a possible therapeutic action in PE [47]. erefore, immunoneutralization of MBG is a logical step in the treatment of PE.…”