Jules B. Puschett

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid that is involved in the inhibition of the sodium pump Na⁺/K⁺-ATPase. Increased plasma levels of MBG have been reported in patients with preeclampsia. MBG increases microvascular barrier permeability in an animal model of preeclampsia. However, the mechanism by which MBG impairs endothelial permeability is unknown. We utilized rat lung microvascular endothelial cells (RLMEC) to examine alterations in MBG-induced monolayer permeability and the effect of MBG on the phosphorylation status of ERK1/2, Jnk, and p38. Apoptosis was evaluated by examining alterations in caspases 3/7, 8, and 9 and annexin-V staining. We also examined the effect of MBG on the endothelial adherens junctions of the RLMEC monolayer. MBG inhibited the proliferation, and increased the monolayer permeability, of RLMEC. These actions of MBG were attenuated by ERK, p38, and pan caspase inhibition. MBG significantly decreased the phosphorylation of ERK1/2 and activated the phosphorylation of Jnk and p38. MBG also significantly increased the expression of caspases 3/7, 8, and 9, indicating the activation of apoptosis. MBG-induced apoptosis signaling was not observed in cells pretreated with a p38 inhibitor. MBG treatment induced the disruption of endothelial cell junctions. This effect was prevented by a pan caspase inhibitor. In conclusion, 1) MBG induced an impairment of RLMEC proliferation; 2) the bufadienolide also caused endothelial hyperpermeability; and 3) these effects of MBG were mediated by the downregulation of ERK1/2, the upregulation of Jnk and p38, by the activation of apoptosis, and by the disruption of endothelial cell junctions.

Background/Aims: Preeclampsia is a hypertensive disorder which develops de novo in women during pregnancy. The urinary excretion of the cardiotonic steroid, marinobufagenin (MBG), is increased prior to the development of hypertension. Preeclamptic patients are volume expanded but much of the excess salt and water appears to be located primarily in the interstitial space. Therefore, ‘capillary leak’ syndrome has been postulated in this disorder. Methods: We evaluated the vascular leakage in normal rats following MBG injection and in a rat model of human preeclampsia. We measured the changes in light intensity comparing that in the intravascular to the extravascular space by assessing ‘leak’ of fluorescein-labeled albumin (FITC-albumin) from mesenteric postcapillary venules. Results: FITC-albumin extravasation continued to increase in a time-dependent fashion after MBG infusion and was significant (p < 0.05) at 60 min of observation when compared to sham rats. We also observed a significant difference in ‘vascular leakage’ in preeclamptic rats compared to control non-pregnant and normal pregnant groups starting at 20 min after the FITC-albumin infusion. Conclusion: We propose that MBG is involved in the production of a ‘vascular leak’ in our rat model of preeclampsia.

The bufodienolides are natriuretic steroids, which also have the capacity to cause vasoconstriction, and are cardiac inotropes. Their mechanism of action appears to be related to their ability to inhibit Na+/K+ ATPase. The actions of one of these compounds, marinobufagenin (MBG), have been investigated in a rat model of preeclampsia, an example of volume expansion-mediated hypertension. The urinary excretion of MGB is increased in this model. Furthermore, this increment in its excretion occurs prior to the development of hypertension and proteinuria. The animals also demonstrate intrauterine growth restriction. Studies of the effect of MBG on cytotrophoblast cells reveal that MGB inhibits the migration, proliferation and invasion of these cells. We propose that MGB is an important etiologic factor in at least some forms of preeclampsia and that the level of its excretion in the urine may prove to be of diagnostic value.

MBG is an endogenous mammalian cardiotonic steroid that is involved in the inhibition of the sodium pump Na⁺/K⁺-ATPase. Increased plasma levels of MBG are associated with hypertension, renal failure, and preeclampsia. In an animal model of preeclampsia, we have shown that MBG levels are elevated prior to the development of hypertension, indicating that it may play a key role in the pathogenesis of preeclampsia, which is characterized by shallow placentation. We recently demonstrated that MBG impairs both the proliferation and growth factor-induced migration of first-trimester cytotrophoblasts (CTB), cells critical for placental development. The intracellular signaling mechanisms regulating MBG-induced inhibition of Na⁺/K⁺-ATPase and the impairment of CTB differentiation are unknown. Activation of MAP kinase signaling (such as ERK1/2, p38 and Jnk) is known to play a critical role in the regulation of cellular proliferation and differentiation. Thus, the aim of our study was to begin to elucidate the mechanisms by which MBG inhibits proliferation via down-regulation of MAP kinase signaling. Chinese hamster ovary (CHO) cells were used to elucidate intracellular signaling mechanisms. CHO cells have previously been used to study Na⁺/K⁺-ATPase transport activities. The phosphorylation of ERK1/2, p38, and Jnk1/2 was evaluated by ELISA and by immunoblotting for phosphorylated and total proteins from CHO cells stimulated with MBG (10 and 100 nM) at 0-, 10-, 30-, and 60-minute time points. Apoptosis was evaluated by caspase 3/7 activation. MBG inhibited the proliferation of CHO cells, indicating a Na⁺/K⁺-ATPase-dependent mechanism involving intracellular Ca²⁺. MBG stimulated a significant time-dependent decrease in the phosphorylation of both ERK1/2 and Jnk1/2 but not p38. In addition to inhibition of proliferation, MBG stimulated a significant increase in caspase 3/7, indicating the activation of apoptosis. We conclude that the MBG-induced impairment of CHO cell differentiation/proliferation occurs via a Na⁺/K⁺-ATPase-dependent down-regulation of ERK1/2 and Jnk1/2 activation and activation of apoptosis. Therapeutic targeting of the MBG signaling pathway may provide treatment paradigms for preeclampsia.