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

Despite prophylaxis and attempts to select a therapy, the frequency of preeclampsia does not decrease and it still takes the leading position in the structure of maternal mortality and morbidity worldwide. In this review, we present a new theory of the etiology and pathogenesis of preeclampsia that is based on the interaction of Na/K-ATPase and its endogenous ligands including marinobufagenin. The signaling pathway of marinobufagenin involves an inhibition of transcriptional factor Fli1, a negative regulator of collagen synthesis, followed by the deposition of collagen in the vascular tissues and altered vascular functions. Moreover, in vitro and in vivo neutralization of marinobufagenin is associated with the restoration of Fli1. The inverse relationship between marinobufagenin and Fli1 opens new possibilities in the treatment of cancer; as Fli1 is a proto-oncogene, a hypothesis on the suppression of Fli1 by cardiotonic steroids as a potential anti-tumor therapeutic strategy is discussed as well. We propose a novel therapy of preeclampsia that is based on immunoneutralization of the marinobufagenin by monoclonal antibodies, which is capable of impairing marinobufagenin-Na/K-ATPase interactions.

Cardiotonic Steroids Induce Vascular Fibrosis Via Pressure-Independent Mechanism in NaCl-Loaded Diabetic Rats.

Endogenous cardiotonic steroid, marinobufagenin (MBG), induces Fli1-dependent tissue fibrosis. We hypothesized that an increase in MBG initiates the development of aortic fibrosis in salt-loaded rats with type 2 diabetes mellitus (DM2) via pressure-independent mechanism. DM2 was induced by a single intraperitoneal administration of 65 mg/kg streptozotocin to neonatal (4-5 days) male Wistar rats. Eight-week-old DM2 rats received water or 1.8% NaCl (DM-NaCl) solution for 4 weeks (n = 16); half of DM-NaCl rats were treated with anti-MBG monoclonal antibody (mAb) (DM-NaCl-AB) during week 4 of salt loading; control intact rats received water (n = 8/group). Blood pressure, MBG, erythrocyte Na/K-ATPase activity, aortic weights, levels of fibrosis markers (Fli1, protein kinase Cδ, transforming growth factor-ß1, receptors of the transforming growth factor beta5, fibronectin, collagen-1), and sensitivity of the aortic explants to the vasorelaxant effect of sodium nitroprusside were assessed. No changes in systolic blood pressure were observed while erythrocyte Na/K-ATPase was inhibited by 30%, plasma MBG was doubled, and aortic markers of fibrosis became elevated in DM-NaCl rats versus control. Treatment of DM-NaCl rats with anti-MBG mAb activated Na/K-ATPase, prevented increases in aortic weights, and the levels of fibrosis markers returned to the control levels. The responsiveness of the aortic rings from DM-NaCl rats to the relaxant effect of sodium nitroprusside was reduced (half maximal effective concentration (EC50) = 29 nmol/L) versus control rings (EC50 = 7 nmol/L) and was restored by anti-MBG mAb (EC50 = 9 nmol/L). Our results suggest that in salt-loaded diabetic rats, MBG stimulates aortic collagen synthesis in a pressure-independent fashion and that 2 profibrotic mechanisms, Fli1 dependent and transforming growth factor-ß dependent, underlie its effects.

Antibody to Marinobufagenin Reverses Placenta-Induced Fibrosis of Umbilical Arteries in Preeclampsia.

BACKGROUND: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. OBJECTIVES AND METHODS: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 ± 4 mmHg; age 29 ± 2 years; 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 ± 2 mmHg; controls) were enrolled in the clinical study. RESULTS: PE was associated with a higher placental (0.04 ± 0.01 vs. 0.49 ± 0.11 pmol/g; p < 0.01) and plasma MBG (0.5 ± 0.1 vs. 1.6 ± 0.5 nmol/L; p < 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 ± 0.2 vs. 1.5 ± 0.2 µmol Pi/mL/hr; p < 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae (p < 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo (p < 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo. CONCLUSION: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE.

Monoclonal antibody to an endogenous bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase inhibition and lowers blood pressure in NaCl-sensitive hypertension.

BACKGROUND: Levels of marinobufagenin (MBG), an endogenous bufadienolide Na/K-ATPase (NKA) inhibitor, increase in preeclampsia and in NaCl-sensitive hypertension. METHODS: We tested a 3E9 monoclonal anti-MBG antibody (mAb) for the ability to lower blood pressure (BP) in NaCl-sensitive hypertension and to reverse the preeclampsia-induced inhibition of erythrocyte NKA. Measurements of MBG were performed via immunoassay based on 4G4 anti-MBG mAb. RESULTS: In hypertensive Dahl-S rats, intraperitoneal administration of 50 microg/kg 3E9 mAb lowered BP by 32 mmHg and activated the Na/K-pump in the thoracic aorta by 51%. NaCl supplementation of pregnant rats (n = 16) produced a 37 mmHg increase in BP, a 3.5-fold rise in MBG excretion, and a 25% inhibition of the Na/K-pump in the thoracic aorta, compared with pregnant rats on a normal NaCl intake. In eight pregnant hypertensive rats, 3E9 mAb reduced the BP (21 mmHg) and restored the vascular Na/K-pump. In 14 patients with preeclampsia (mean BP, 126 +/- 3 mmHg; 26.9 +/- 1.4 years; gestational age, 37 +/- 0.8 weeks), plasma MBG was increased three-fold and erythrocyte NKA was inhibited compared with that of 12 normotensive pregnant women (mean BP, 71 +/- 3 mmHg) (1.5 +/- 0.1 vs. 3.1 +/- 0.2 micromol Pi/ml/h, respectively; P < 0.01). Ex-vivo 3E9 mAb restored NKA activity in erythrocytes from patients with preeclampsia. As compared with 3E9 mAb, Digibind, an affinity-purified antidigoxin antibody, was less active with respect to lowering BP in both hypertensive models and to restoration of NKA from erythrocytes from patients with preeclampsia. CONCLUSION: Anti-MBG mAbs may be a useful tool in studies of MBG in vitro and in vivo and may offer treatment of preeclampsia.

Antibody to marinobufagenin lowers blood pressure in pregnant rats on a high NaCl intake.

OBJECTIVE: The pathogenesis of pre-eclampsia (PE), a major cause of maternal and fetal mortality, is not fully understood. Digitalis-like sodium pump ligands (SPLs) are believed to be implicated in PE, as illustrated by clinical observations that DIGIBIND, a digoxin antibody that binds SPLs, lowers blood pressure (BP) in PE. We recently reported that plasma levels of marinobufagenin (MBG), a vasoconstrictor SPL, are increased four-fold in patients with PE. In the present study, we tested whether a polyclonal antibody to MBG can lower BP in rats with pregnancy-associated hypertension. METHODS: Systolic BP (SBP), 24-h renal excretion of MBG and endogenous ouabain (EO), and sodium pump activity in the thoracic aortae were measured in virgin and pregnant Sprague-Dawley rats without and with NaCl supplementation (drinking 1.8% NaCl solution). RESULTS: NaCl supplementation of virgin rats stimulated renal excretion of MBG by 60%, but not that of EO, and did not change the BP. Compared with virgin rats, the last week of pregnancy in non-NaCl-loaded rats was associated with a decrease in SBP (106 +/- 2 versus 117 +/- 2 mmHg); a moderate increase in renal excretion of MBG (97.6 +/- 4.9 versus 57.4 +/- 7.0 pmoles/24 h) and EO (36.2 +/- 4.3 versus 24.1 +/- 3.2 pmoles/24 h). NaCl-loaded pregnant rats exhibited elevation in SBP (139 +/- 3 mmHg; P < 0.01 versus non-NaCl-loaded pregnant rats), in renal excretion of MBG (160.0 +/- 17.5 pmoles/24 h; P < 0.01 versus non-NaCl-loaded pregnant rats), but not in EO, and showed fetal growth retardation. Administration of the anti-MBG antibody to NaCl-loaded pregnant rats lowered SBP (111 +/- 2 mmHg; P < 0.01) and increased aortic sodium pump activity (144 +/- 3 versus 113 +/- 5 nmol Rb/g per min; P < 0.01 versus non-NaCl-loaded pregnant rats). CONCLUSIONS: These observations provide evidence that MBG contributes to BP elevation in pregnant rats rendered hypertensive by NaCl supplementation.

Central role for the cardiotonic steroid marinobufagenin in the pathogenesis of experimental uremic cardiomyopathy.

Patients with chronic renal failure develop a "uremic" cardiomyopathy characterized by diastolic dysfunction, cardiac hypertrophy, and systemic oxidant stress. Patients with chronic renal failure are also known to have increases in the circulating concentrations of the cardiotonic steroid marinobufagenin (MBG). On this background, we hypothesized that elevations in circulating MBG may be involved in the cardiomyopathy. First, we observed that administration of MBG (10 microg/kg per day) for 4 weeks caused comparable increases in plasma MBG as partial nephrectomy at 4 weeks. MBG infusion caused increases in conscious blood pressure, cardiac weight, and the time constant for left ventricular relaxation similar to partial nephrectomy. Decreases in the expression of the cardiac sarcoplasmic reticulum ATPase, cardiac fibrosis, and systemic oxidant stress were observed with both MBG infusion and partial nephrectomy. Next, rats were actively immunized against a MBG-BSA conjugate or BSA control, and partial nephrectomy was subsequently performed. Immunization against MBG attenuated the cardiac hypertrophy, impairment of diastolic function, cardiac fibrosis, and systemic oxidant stress seen with partial nephrectomy without a significant effect on conscious blood pressure. These data suggest that the increased concentrations of MBG are important in the cardiac disease and oxidant stress state seen with renal failure.