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.

The cytotrophoblast (CTB) cells of the human placenta have membrane receptors that bind certain cardiotonic steroids (CTS) found in blood plasma. One of these, marinobufagenin, is a key factor in the etiology of preeclampsia. Herein, we used synthetic receptors (SR) to study their effectiveness on the angiogenic profile of human first trimester CTB cells. The humanextravillous CTB cells (Sw.71) used in this study were derived from first trimester chorionic villus tissue. Culture media of CTB cells treated with ≥1 nM SR level revealed sFlt-1 (Soluble fms-like tyrosine kinase-1) was significantly increased while VEGF (vascular endothelial growth factor) was significantly decreased in the culture media (* p < 0.05 for each) The AT2 receptor (Angiotensin II receptor type 2) expression was significantly upregulated in ≥1 nM SR-treated CTB cells as compared to basal; however, the AT1 (Angiotensin II receptor, type 1) and VEGFR-1 (vascular endothelial growth factor receptor 1) receptor expression was significantly downregulated (* p < 0.05 for each). Our results show that the anti-proliferative and anti-angiogenic effects of SR on CTB cells are similar to the effects of CTS. The observed anti angiogenic activity of SR on CTB cells demonstrates that the functionalized-urea/thiourea molecules may be useful as potent inhibitors to prevent CTS-induced impairment of CTB cells.

Diabetes in pregnancy is associated with microvascular complications and a higher incidence of preeclampsia. The regulatory signaling pathways involving nitric oxide, cGMP, and cGMP-dependent protein kinase (PKG) have been shown to be down-regulated under diabetic conditions and contribute to the pathogenesis of vascular complications in diabetes. The present study was undertaken to investigate how high glucose concentrations regulate PKG expression in cytotrophoblast cells (CTBs). Human CTBs (Sw. 71) were treated with 45, 135, 225, 495, or 945 mg/dL glucose for 48 h. Some cells were pretreated with a p38 inhibitor (10 μM SB203580) or 10 μM rosiglitazone. After treatment, the cell lysates were subjected to measure the expression of protein kinase G1α (PKG1α), protein kinase G1β (PKG1β), soluble guanylate cyclase 1α (sGC1α), and soluble guanylate cyclase 1 β (sGC1β) by Western blot. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test. The expressions of PKG1α, PKG1β, sGC1α, and sGC1β were significantly down-regulated (p < 0.05) in CTBs treated with >135 mg/dL glucose compared to basal (45 mg/dL). The hyperglycemia-induced down-regulation of cGMP and cGMP-dependent PKG were attenuated by the SB203580 or rosiglitazone pretreatment. Exposure of CTBs to excess glucose down-regulates cGMP and cGMP-dependent PKG, contributing to the development of vascular complications in diabetic mothers during pregnancy. The attenuation of hyperglycemia-induced down-regulation of PKG proteins by SB203580 or rosiglitazone pretreatment further suggests the involvement of stress signaling mechanisms in this process.

Objective: Marinobufagenin (MBG), a cardiotonic steroid, is distinctly elevated in preeclampsia (preE) and may directly contribute to pathogenesis, possibly through deleterious signaling in cytotrophoblast (CTB) cells. In this study, we evaluate the effects of anti-MBG human monoclonal antibodies on cellular signaling in CTB cells and in a rat model of preE. Methods: CTB cell proliferation in response to MBG with and without anti-MBG was measured using a Cell Titer assay. Pro-angiogenic factors (VEGF and PlGF) and anti-angiogenic factors (sFlt-1 and sEng) in response to MBG with and without antibody were measured. Finally, we evaluated the lead anti-MBG antibody in comparison with the parent murine antibody in a previously described rat model of preE which recapitulates the hypertension, proteinuria, and fetal growth effects of human preE. Results: CTB cells exposed to ≥ 1 nM MBG showed decreased ( p < 0.05) proliferation, decreased secretion of VEGF and PIGF, and increased secretion of sFlt-1 and sEng. Pretreatment with anti-MBG significantly ( p < 0.05) attenuated the MBG-induced modulation of cell proliferation and expression of VEGF, PIGF, sFlt-1, and sEng. In the rat model, anti-MBG treatment normalized blood pressure, reduced proteinuria, and eliminated fetal effects. Conclusions: MBG has the potential to be a causative agent for preE as it causes dysfunction in CTB cells due to the anti-angiogenic milieu. Our study suggests that anti-MBG antibodies can bind to MBG, neutralizing it, and preventing downstream signaling in vitro. In a saline-induced rat model of preE, treatment with anti-MBG antibody was effective at normalizing blood pressure, kidney function, and fetal birth weights. These data suggest that a human monoclonal antibody with high specificity and affinity for MBG has potential as a therapeutic for preE.

Background: Preeclampsia (PreE) is a pregnancy disorder characterized by new onset of hypertension and reduced fetal weight. We have previously shown that marinobufagenin (MBG) and hyperglycemia impairs cytotrophoblasts (CTBs) function. The potential therapeutic role of H2 relaxin in PreE was reported, a novel H2 relaxin B-chain-only peptide B7-33 and its lipidated derivative have recently been developed. This study evaluates whether B7-33 and its lipidated derivative improve CTBs function and preE phenotype. Methods: A palmitic acid was attached at the N-terminus of B7-33. Human CTBs were treated with DMSO (vehicle) or 0.1, 1, 10 or 100 nM of MBG or with 100, 150, 200, 300, or 400 mg/dL glucose for 48h and were co-treated either with B7-33 (25 nM) or its lipidated derivative (25 nM) in the presence and absence of either MBG or hyperglycemia exposure. Some cells were pretreated with relaxin antagonist (1.0 μM RXFP1) prior to MBG or hyperglycemia exposure. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and soluble fms-like tyrosine kinase-1 (sFlt-1) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: Lipidation of B7-33 results in a significant increase of half-life of B7-33 without altering its activity. Secretion of sFlt-1 was increased while VEGF and PIGF were decreased in CTBs treated with ≥1.0 nM MBG and ≥150 mg/dl of glucose (*p < 0.05 for each). Co-treatment with B7-33 (25 nM) and its lapidated derivative (25 nM) rescued CTBs from either MBG-induced and hyperglycemia-induced anti-angiogenic profile (p < 0.05 for each). B7-33 and its lapidated derivative cause an increase in the expression of VEGF in CTBs, however, they have no effect on other factors. The B7-33-induced upregulation of VEGF expression is attenuated by 1.0 μM RXFP1 antagonist. Conclusion: Both B7-33 and its lipidated derivative mitigate the MBG- and hyperglycemia-induced dysfunction of CTBs by attenuating anti-angiogenic phenotype similar to that seen in preE. Moreover, the B7-33 and its lipidated derivative induced effect on CTBs are attenuated by a relaxin antagonist. The preclinical study with B7-33 and its lipidated derivative are now underway.