Comparison of Neurocognitive Testing and the Measurement of Marinobufagenin in Mild Traumatic Brain Injury: A Preliminary Report.

The evaluation of concussed athletes, including testing to determine if and when they may return to play, has become an important task of athletic trainers and team physicians. Currently, concussion protocols are in place, which depend largely upon assessments based upon neurocognitive testing (NCT). The authors have evaluated the use of a biomarker of brain trauma, marinobufagenin (MBG), and compared its application in concussed athletes with the performance of NTC. We found a disparity between these two testing procedures. In this communication, the findings of these comparative data are presented. We noted that athletes whose NCT evaluations had returned to baseline and who were allowed to again participate in play then showed a recurrence of elevated urinary MBG excretion. These observations raise concern as to the processes currently in effect with regard to the decision as to returning athletes to the full activity. They suggest a need for further evaluation.

Differing effects of resibufagenin on cinobufatalin- versus marinobufagenin-induced preeclampsia in a rodent model.

OBJECTIVE: Previous studies from this laboratory have demonstrated that the bufodienolide, marinobufagenin, causes a syndrome in the pregnant rat that resembles human preeclampsia. Furthermore, marinobufagenin urinary excretion is elevated in approximately 85% of preeclamptic patients. Resibufagenin, an antagonist to marinobufagenin, completely prevents the syndrome (hypertension, proteinuria, and intrauterine growth restriction) if given from early pregnancy. STUDY DESIGN: We investigated the effects of another bufodienolide, cinobufatalin, to determine if it, likewise, could induce the rat "preeclamptic" syndrome, which it did. We then examined whether resibufagenin could prevent the syndrome due to cinobufatalin. RESULTS: Resibufagenin improved hypertension but not proteinuria, and did not prevent uterine growth restriction. CONCLUSION: We conclude that more than one bufodienolide may induce the preeclamptic syndrome and that each may require a specific antagonist to prevent (or treat) the syndrome.

Marinobufagenin regulates permeability and gene expression of brain endothelial cells.

Marinobufagenin (MBG) is a cardiotonic steroid that increases in the circulation in preeclampsia. Preeclampsia and eclampsia are associated with cerebral edema. Therefore, we examined the effects of MBG on human brain microvascular endothelial cells (HBMEC) in vitro. MBG enhanced the permeability of HBMEC monolayers at 1-, 10-, and 100-nM doses, but had no effect at 0.1 nM. Agilent Human Gene Expression microarrays were utilized in these studies. MBG treatment (10 nM for 12 h) downregulated concentrations of the soluble VEGFR transcript sFLT by 59% but did not alter those of FLTv3 mRNA (determined by quantitative PCR). When treated and control HBMEC transcriptomes were interrogated on microarrays, 1,069 genes appeared to be regulated by MBG. Quantitative RT-PCR confirmed that MBG treatment upregulated ENKUR mRNA concentrations by 57%. Its protein product interacts with calmodulin and calcium channel proteins. MBG treatment downregulated several genes whose protein products are involved in cell adhesion (ITGA2B, FERMT1, CLDN16, and TMEM207) and cell signaling (GRIN2C, SLC8A1, and ESR1). The level of downregulation ranged from 22 to 66%. Altogether, MBG actively enhanced the permeability of HBMEC monolayers while downregulating genes involved in adhesion. MBG treatment had variable effects on ENKUR, GRIN2C, and SLC8A1 genes, all associated with calcium transport. These studies provide the basis for future investigations of MBG actions in normal physiology and disease.

Marinobufagenin predicts and resibufogenin prevents preeclampsia: a review of the evidence.

OBJECTIVE: The purpose of this review is to provide information detailing the existing evidence with regard to the hypothesis that marinobufagenin (MBG) is an important etiologic and predictive factor in preeclampsia (PE). In addition, evidence describing the role of the antagonist to MBG, resibufogenin (RBG), in the prevention and/or treatment of this disorder is provided. STUDY DESIGN: The studies outlined were performed in an animal model of PE, in in vitro experiments, and in human studies. RESULTS: Data have been obtained that strongly support the hypothesis that ~60 to 70% of PE patients demonstrate elevations in urinary and serum MBG levels. In the animal model, the entire syndrome can be prevented by the administration of RBG beginning early in pregnancy. CONCLUSION: Expanded human trials of MBG as a predictor of the later development of PE are warranted as are studies of the efficacy and safety of RBG as a preventative/therapy.

Resibufogenin administration prevents oxidative stress in a rat model of human preeclampsia.

BACKGROUND AND OBJECTIVES: Marinobufagenin (MBG) is a cardiotonic steroid that is increased in preeclampsia. An analog of MBG, resibufogenin (RBG), prevents the development of preeclampsia in a rat model. Oxidative stress is a concomitant of endothelial dysfunction in the latter disorder. The objective of the current studies was to evaluate the status of oxidative stress in a rat model of preeclampsia. METHODS: We measured the aortic AT(1) receptor expression and urinary excretion of 8-isoprostane (8IP) in rats rendered "preeclamptic" and compared the findings to those obtained in normal pregnant animals, pregnant rats injected with MBG, and preeclamptic rats treated with RBG. RESULTS: Aortic AT(1) receptor expression and the urinary excretion of 8IP were significantly augmented in "preeclamptic" and MBG-injected pregnant rats compared to normal pregnant animals. RBG prevented evidence of oxidative stress in "preeclamptic" rats. CONCLUSION: MBG is involved in the causation of oxidative stress in our rat model and RBG attenuates this change.

Marinobufagenin levels in preeclamptic patients: a preliminary report.

Preeclampsia is a disorder resulting in significant fetomaternal complications with no definitive pharmacological intervention. A bufadienolide, marinobufagenin, has been implicated in the etiology of preeclampsia. We investigated both the blood and urine levels of marinobufagenin in preeclamptic and control subjects. Preeclamptic and normotensive pregnant women were recruited at various gestational age periods. Blood and urine specimens were obtained and analyzed for marinobufagenin levels and creatinine. The former determination was performed utilizing a new, novel chemifluorescent enzyme-linked immunosorbent assay. The marinobufagenin levels were higher in preeclamptics than in the controls in both serum and urine at various gestational age periods. Additionally, the mean level of marinobufagenin in the preeclamptic group was significantly greater than in controls in both blood and urine specimens ( P < 0.05). These data are consistent with a role for marinobufagenin in the etiology of preeclampsia. This study demonstrated comparable results in blood and urine samples. This suggests that subsequent studies on levels of marinobufagenin as a screening test for preeclampsia could be done utilizing urine samples, which are easier to obtain, less invasive, more cost-effective, and as accurate as the serological tests.

A chemifluorescent immunoassay for the determination of marinobufagenin in body fluids.

We describe here the development of a chemifluorescent competitive enzyme-linked immunosorbent assay (ELISA) that quantifies marinobufagenin (MBG) levels in biological fluids. Based on a polyclonal antibody raised against a novel MBG-bovine serum albumin conjugate, this assay achieved an MBG detection limit of less than 9 pg/mL. MBG levels in various rat urine and serum samples were effectively determined using this methodology. Interassay variability averaged 9.8%, while intra-assay variability averaged 1.9 and 2.5% in representative serum and urine samples, respectively. Recovery of exogenously added MBG averaged 106%, and parallelism data further established the accuracy of the assay. Employment of this assay to detect MBG abnormalities represents a powerful tool for the possible diagnosis, prevention and management of human hypertensive states, particularly preeclampsia.

Marinobufagenin is an upstream modulator of Gadd45a stress signaling in preeclampsia.

Preeclampsia (PE) is a hypertensive disorder of pregnancy, in which marinobufagenin (MBG), a circulating cardiotonic steroid, is increased. The Gadd45a stress sensor protein is an upstream modulator of the pathophysiological changes observed in PE. However, the effects of MBG on Gadd45a stress signaling remain unknown. We examined the expression of Gadd45a, the sFlt-1 receptor, and p38, as well as caspase 3 and 8 activities in placental samples from four groups of rats. These were: normal pregnant (NP, n=8); pregnant rats which received weekly injections of desoxycorticosterone acetate and 0.9% saline as their drinking water (PDS, n=9); normal pregnant rats injected with MBG (NPM, n=8); and PDS rats injected with resibufogenin (RBG), an in vivo antagonist of MBG (PDSR, n=8). Utilizing human cytotrophoblast (CTB) cells, we examined the effect of MBG on these stress signaling proteins in vitro. Placental Gadd45a expression, caspase 3 and 8 activities, sFlt-1 concentrations, and sFlt-1 receptor expression were significantly higher in PDS and NPM compared to NP and PDSR rats. Gadd45a protein was significantly upregulated in the CTB cells when MBG was present in concentrations ≥1nM. Treatment with MBG (≥1nM) also significantly arrested cell cycle progression and activated the expression of the Gadd45a-mediated stress signaling proteins. Inhibition of Gadd45a through RNAi-mediation attenuated MBG-induced CTB cell stress signaling. In conclusion, MBG is involved in the alteration in Gadd45a stress signaling both in vivo and in vitro and RBG prevents these changes when administered in vivo.

Genetic variation in solute carrier genes is associated with preeclampsia.

OBJECTIVE: The objective of the study was to evaluate allelic variation in 26 members of the solute carrier (SLC) gene family for an association with preeclampsia. STUDY DESIGN: Preeclampsia cases were women with mild or severe preeclampsia. Controls were enrolled from women without hypertension-related complications who presented for delivery at term (≥37 weeks). The association between preeclampsia and SLC gene single-nucleotide polymorphisms (SNPs) and haplotypes was evaluated by logistic regression models. RESULTS: Rs4957061 in SLC9A3 was significantly associated with a reduced risk of preeclampsia in whites (T allele, P = .002; odds ratio [OR], 0.33; 95% confidence interval [CI], 0.16-0.67). For SLC4A1 in blacks, the G allele of rs2074107 (P = .004; OR, 1.56; 95% CI, 1.15-2.12) and the A allele of rs2857078 (P < .001; OR, 1.67; 95% CI, 1.27-2.17) were significantly associated with preeclampsia. Also in blacks, rs10066650 in SLCO4C1 (G allele, P = .002; OR, 1.72; 95% CI, 1.21-2.46) was significantly associated with increased risk. Sliding window haplotype analyses identified significantly associated haplotypes in these genes. CONCLUSION: SNPs and haplotypes in SLC9A3 in whites and SLC4A1 and SLCO4C1 in blacks are significantly associated with preeclampsia.

Emerging role of the bufadienolides in cardiovascular and kidney diseases.

The bufadienolides are a group of steroid hormones that circulate in blood and are excreted in urine. They have the ability to inhibit the adenosine triphosphatase sodium-potassium pump (Na(+)-K(+)-ATPase), with predilection for its alpha1 isoform. This capability enables them to share with other cardiac glycosides the facility to cause an increase in sodium excretion, produce vasoconstriction resulting in hypertension, and act as cardiac inotropes. Bufadienolides have been implicated in instances of volume expansion-mediated hypertension, syndromes in which they are considered capable of causing a vascular leak, interfering with cellular proliferation, and inhibiting cellular maturation. An antagonist to the most well-studied bufadienolide, marinobufagenin, is resibufogenin, a compound that provides promise for the treatment of disorders in which excessive levels of marinobufagenin are present and are etiopathogenetic.

Alterations in the renin-angiotensin system in a rat model of human preeclampsia.

BACKGROUND/AIMS: Preeclampsia is a hypertensive disorder unique to pregnancy in which elevated levels of marinobufagenin (MBG) have been reported. The renin-angiotensin system (RAS) may also play a role in the pathogenesis of preeclampsia. The aim of our study was to evaluate the status of the RAS in a rat model of preeclampsia characterized by hypertension, proteinuria, excessive weight gain and intrauterine growth restriction. METHODS: We evaluated the components of the RAS in 5 groups of animals: nonpregnant control; normal pregnant (NP); pregnant rats which received injections of desoxycorticosterone acetate and 0.9% saline as their drinking water (PDS); normal pregnant rats injected with MBG (NPM), and PDS rats to which resibufogenin (RBG) had been administered (PDSR). RBG is an antagonist of MBG differing in structure from MBG only in the absence of a hydroxyl group in the beta-5 position. RESULTS: Plasma levels of active renin, renin and Ang II were significantly lower in PDS and NPM compared to NP and PDSR rats (p < 0.05). However, placental levels of these components were increased significantly in PDS and NPM compared to NP and PDSR rats (p < 0.05). Placental AT(1) receptor expression was significantly higher in PDS and NPM compared to NP and PDSR rats (p < 0.05). CONCLUSIONS: (1) The peripheral RAS is downregulated, and the uteroplacental RAS is upregulated in this rat model of preeclampsia; (2) MBG is involved in the causation of these alterations, and (3) RBG prevents these changes.

Resibufogenin prevents the manifestations of preeclampsia in an animal model of the syndrome.

BACKGROUND AND OBJECTIVES: We have developed a rat model of preeclampsia which is based upon excessive volume expansion and includes hypertension, proteinuria and intrauterine growth restriction. In this model, the urinary excretion of the circulating steroid inhibitor of Na +/ K+ ATPase, marinobufagenin, is increased prior to the development of hypertension and proteinuria. An analogue of marinobufagenin, resibufogenin, successfully treats the hypertension and proteinuria. METHODS: We administered resibufogenin early in pregnancy in this model, prior to the development of the syndrome. RESULTS: We found that resibufogenin not only prevented the advent of hypertension and proteinuria, but also the development of intrauterine growth restriction. DISCUSSION: These results may have relevance to the human condition.

Marinobufagenin causes endothelial cell monolayer hyperpermeability by altering apoptotic signaling.

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.

Vascular leak in a rat model of preeclampsia.

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.

Vascular effects of the bufodienolides.

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.

Examination of the cellular mechanisms by which marinobufagenin inhibits cytotrophoblast function.

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid involved in the inhibition of Na(+)/K(+)-ATPase. Increased plasma levels have been reported in patients with volume expansion-related hypertension. We have recently demonstrated that MBG impairs first trimester cytotrophoblast (CTB) cell proliferation, migration, and invasion, which may play a role in the development of preeclampsia. However, whether apoptosis contributes to altered CTB cell function by MBG remains unknown. Using the human extravillous CTB cell line SGHPL-4, we examined the effect of MBG and a similar Na(+)/K(+)-ATPase inhibitor, ouabain, on the phosphorylation status of Jnk, p38, and Src. Additionally, we measured apoptosis by caspase 9 and 3/7 activity and by annexin-V staining. We also investigated interleukin-6 (IL-6) secretion with or without p38 and Jnk inhibition. MBG significantly increased the phosphorylation of Jnk, p38, and Src and increased the expression of caspase 9 and 3/7 indicating the activation of apoptosis. MBG treatment also stimulated the expression of the early apoptosis marker, annexin-V, which was prevented by Jnk and p38 inhibition. MBG also stimulated the secretion of IL-6, which was attenuated by p38 inhibition. Ouabain had similar effects to those of MBG, suggesting that the apoptotic effects on CTB cells may be mediated by inhibition of Na(+)/K(+)-ATPase. In conclusion, the MBG-induced impairment of CTB function occurs via activation of Jnk, p38, and Src leading to increased apoptosis and IL-6 secretion. These observations may have clinical applicability with respect to the therapy of preeclampsia.

Uncoupled endothelial nitric oxide synthase and oxidative stress in a rat model of pregnancy-induced hypertension.

BACKGROUND: Preeclampsia is a human pregnancy-associated syndrome associated with hypertension, proteinuria, and endothelial dysfunction. We tested whether increased reactive oxygen species (superoxide and peroxynitrite) production and decreased bioavailability of the endothelial nitric oxide (NO) synthase (eNOS) cofactor tetrahydrobiopterin (BH4) contributes to maternal endothelial dysfunction in rats with pregnancy-induced hypertension and several characteristics of preeclampsia. METHODS: Nonpregnant (DS) and pregnant (PDS) rats were treated with deoxycorticosterone acetate and 0.9% saline for approximately 3 weeks and nonpregnant (Con) and pregnant (P) rats received tap water. Blood pressure, urinary protein levels, mesenteric vascular reactivity, aortic protein expression, and aortic reactive oxygen species levels were compared between the four groups. RESULTS: The PDS rats had significantly decreased mesenteric endothelium-dependent relaxation responses and aortic NO production compared to Con, DS, and P rats despite increased aortic eNOS expression. Aortic superoxide and peroxynitrite levels were increased in PDS rats compared with Con, DS, and P rats. Scavenging of reactive oxygen species or increasing tetrahydrobiopterin levels normalized mesenteric endothelium-dependent relaxation responses, aortic NO production, and aortic superoxide and peroxynitrite levels in PDS rats. CONCLUSIONS: These data suggest that increased superoxide production by NADPH oxidase, peroxynitrite degradation of BH4, and uncoupled eNOS contribute to endothelial dysfunction in a rat model of pregnancy-induced hypertension.

Marinobufagenin interferes with the function of the mineralocorticoid receptor.

Marinobufagenin (MBG) is a cardiotonic steroid of the bufadienolide class of compounds which has the ability to inhibit the ubiquitous enzyme, Na+/K+-ATPase, resulting in natriuresis. The involvement of MBG in the pathogenesis of volume expansion-mediated forms of hypertension has been suggested for some time, and we have proposed that MBG participates in the hypertension noted in preeclampsia. We examined the hypothesis that MBG might contribute to these forms of hypertension by promoting the activity of the mineralocorticoid receptor (MR). However, our data demonstrate that instead, MBG interferes with the functioning of the MR by inhibiting the transcriptional activity of the receptor, and this is reflected in a reduced interaction between the SRC-3 coactivator and the MR. Thus, the ability of MBG to cause a natriuresis may be due, not only to inhibition of Na+/K+-ATPase activity, but also to its ability to interfere with MR-dependent expression of the Na/K/H exchanger in the late distal nephron.