Background The bioactive steroid, marinobufagenin (MBG), is an endogenous Na/K-ATPase bufadienolide inhibitor that is synthetized by adrenocortical and placental cells. MBG binding to Na/K-ATPase initiates pro-fibrotic cell signaling, and heightened MBG levels are implicated in the pathogenesis of hypertension, preeclampsia and chronic kidney disease. Steroids are derived from cholesterol through the “traditional” steroidogenesis pathway initiated by enzyme CYP11A1, and via the “acidic” bile acid pathway, which is controlled by enzyme CYP27A1. The mechanism of MBG biosynthesis in mammals however remains unknown. Methods and Results Here we show that post-transcriptional silencing of the CYP27A1 gene in human trophoblast and rat adrenocortical cells reduced the expression of CYP27A1 mRNA by 70%, reduced total bile acids 2-fold, and MBG levels by 67%, compared to non-treated cells or cells transfected with non-targeting siRNA. In contrast, silencing of the CYP11A1 gene did not affect MBG production in either cell culture, but suppressed production of progesterone 2-fold in human trophoblast cells, and of corticosterone by 90% in rat adrenocortical cells, compared to cells transfected with non-targeting siRNA. In vivo, in a high salt administration experiment, male and female Dahl-S rats became hypertensive after 4 weeks on a high NaCl diet, their plasma MBG levels doubled, and adrenocortical CYP27A1 mRNA and protein increased 1.6-fold and 2.0-fold. Conclusions Therefore, the endogenous steroidal Na/K-ATPase inhibitor, MBG, is synthesized in mammalian placenta and adrenal cortex from cholesterol through the novel “acidic” bile acid pathway. These findings will help to understand the role of MBG in highly prevalent human cardiovascular diseases.

Objective Endogenous cardiotonic steroids (CTS), including marinobufagenin (MBG), stimulate vascular synthesis of collagen. Because mineralocorticoid antagonists competitively antagonize effect of CTS on the Na/K-ATPase, we hypothesized that spironolactone would reverse the pro-fibrotic effects of MBG. Methods Experiment 1. Explants of thoracic aortae and aortic vascular smooth muscle cells (VSMC) from Wistar rats were cultured for 24 hours in the presence of vehicle or MBG (100 nmol/L) with or without canrenone (10 µmol/L), an active metabolite of spironolactone. Experiment 2. In 16 patients (56 ± 2 yrs) with resistant hypertension (RH) on a combined (Lisinopril / amlodipine / hydrochlorothiazide) therapy, we determined arterial pressure, pulse wave velocity (PWV), plasma MBG, and erythrocyte Na/K-ATPase before and six months after addition of placebo (n=8) or spironolactone (50 mg/day; n=8) to the therapy. Results In rat aortic explants and in VSMC, pretreatment with MBG resulted in a two-fold rise in collagen-1, and a marked reduction in the sensitivity of the aortic rings to the vasorelaxant effect of sodium nitroprusside following endothelin-1-induced constriction (EC50=480±67 nmol/L vs. 23±3 nmol/L in vehicle-treated rings; P<0.01). Canrenone blocked effects of MBG on collagen synthesis and restored sensitivity of vascular rings to sodium nitroprusside (EC50 = 17±1 nmol/L). RH patients exhibited elevated plasma MBG (0.42 ± 0.07 vs. 0.24 ± 0.03 nmol/L; P=0.01) and reduced Na/K-ATPase activity (1.9 ± 0.15 vs 2.8 ± 0.2 µmol Pi/ml/hr, P<0.01) vs. 7 healthy subjects. Six-month administration of spironolactone, unlike placebo treatment, was associated with a decrease in PWV and arterial pressure, and with restoration of Na/K-ATPase activity in the presence of unchanged MBG levels. Conclusion MBG-induced vascular fibrosis is a likely target for spironolactone.

Pharmacological agents suggested for infarct size limitation have serious side effects when used at cardioprotective doses which hinders their translation into clinical practice. The solution to the problem might be direct delivery of cardioprotective drugs into ischemic-reperfused myocardium. In this study, we explored the potential of silica nanoparticles for passive delivery of adenosine, a prototype cardioprotective agent, into ischemic-reperfused heart tissue. In addition, the biodegradation of silica nanoparticles was studied both in vitro and in vivo. Immobilization of adenosine on the surface of silica nanoparticles resulted in enhancement of adenosine-mediated infarct size limitation in the rat model. Furthermore, the hypotensive effect of adenosine was attenuated after its adsorption on silica nanoparticles. We conclude that silica nanoparticles are biocompatible materials that might potentially be used as carriers for heart-targeted drug delivery.