**5. Marinobufagenin and CV Diseases**

Most of the studies designed to evaluate the pathogenetic mechanisms by which MBG contributes to CV disease risk have been performed in animal models. Increased concentrations of circulating MBG (as a result of sodium loading or via infusion pump) caused several effects: vascular [24,59] and microcirculation [103] alterations, pressor changes [4,46,104,105] and cardiac and renal [8,9,58,61,91] fibrosis. Investigations in humans have shown elevated MBG plasma levels in many pathological conditions: heart failure [8], acute myocardial infarction (elevated urinary MBG levels) [106], primary aldosteronism [107], renal ischemia [108] and CKD [100,109].

There are several scientific pieces of evidence supporting a possible pathogenic role of bufadienolides in hypertensive conditions associated with hydro-saline retention. Firstly, increased plasma and urinary concentration levels of MBG were observed in volume expansion conditions and in hypertensive patients mediated by volume expansion, due to salt accumulation [46,104,105,110]. Secondly, the administration of bufadienolides in experimental animals causes hypertension [11,13,111]. Thirdly, in rats, the hypertension caused by the administration of deoxycorticosterone acetate and salt is reversed through the intraperitoneal injection of an MBG antagonist, resibufogenin (RBG) [12,13,112], which differs from MBG only in the absence of a hydroxyl group in the β 5 position. Finally, the use of anti-MBG antibodies in salt-loaded pregnant rats and in salt-sensitive hypertensive rats, results in the reduction in blood pressure values [113].

In recent years, several evaluations of whether MBG could be used as an early marker of CV risk have been performed. The first study designed to evaluate the possible asso-

ciation between blood pressure values and MBG plasma levels demonstrated an inverse relationship between diastolic blood pressure values and the urinary excretion (24 h) levels of MBG in the case of high sodium intake (16.32 g of salt per day). The natriuretic effects of MBG could represent a homeostatic mechanism to restore blood pressure values to normal, constituting a protective mechanism in healthy subjects [82]. The dietary intervention had a total duration of 12 days; therefore, that effect could represent a homeostatic mechanism in the short term. In contrast, another group has shown that MBG plasma levels are positively associated with systolic blood pressure values during the period of high sodium intake (5 weeks). In that case, it might reflect a long-term homeostatic response in which the vasoconstrictor activity of MBG could superimpose the natriuretic effect [114]. Fedorova et al. showed completely different responses from the above studies. In a cohort of men and women, there was an increase in systolic blood pressure values following dietary sodium loading without changes in both plasma and urinary MBG concentrations [81]. Therefore, the results on the relationship between blood pressure values and circulating and urinary MBG levels in humans are conflicting and require further evaluation.

Recent findings have revealed that in a state of inactivity, sodium can settle in the interstitium between the skin and organs [115]. The alteration of these deposits could also affect blood pressure values. We can say that high sodium intake correlates both with a higher production of MBG and with rigid large arteries, even in healthy subjects. These pieces of evidence were confirmed in laboratory models, as risk factors for dementia and CV events [17]. In addition to MBG, several factors act on peripheral vascular resistance, such as neurohormonal, baroreflexes and myogenic factors [49]. It is also known that sodium regulates the rigidity of endothelial cells and modifies the release of nitric oxide by altering the tone of blood vessels and blood pressure [116]. In the current state of the art, there is still not enough evidence to consider urinary MBG excretion as a predictive value of increased cardiovascular risk before the onset of the disease [24], although numerous studies have confirmed the correlation between plasma MBG levels, sodium and occurrence of arterial hypertension. Furthermore, these results proved to be more applicable to men than to women.

In other parts of the body, such as the arterial vascular smooth muscle cells, MBG generates an increase in cytosolic Ca2+ amount that causes vasoconstriction through the activation of an "ionic pathway" [4]. In addition to this "ionic pathway", MBG can activate different intracellular signaling pathways that trigger cellular effects, such as cell proliferation, ROS genesis or the stimulation of apoptosis, also via the activation of pathways with other molecules, such as Phospholipase C-*γ* isozyme (PLC-γ), Phosphatidylinositol 3-kinase (PI-3K), IP3 receptor type 3 (IP3R), and ankyrin [117].

Specifically, it has been reported that cardiotonic steroids activate a signal cascade, which is mediated through Src, Ras, ROS, and ERKs, and promote endocytosis of the plasmalemmal Na+/K+-ATPase [10,99,118–120]. The activation of this cascade requires that the Na+/K+-ATPase to be in caveolae in order to proceed [121,122]. This series of signals is known to cause changes in gene expression, which can be inhibited by antioxidant molecules [7,10,120].

Arterial stiffness is well known to be related to increased CV risk and death in individuals of all ages [123], regardless of blood pressure values. Sodium intake also correlates with arterial stiffness regardless of hypertensive status [124,125], even in healthy subjects. Thus, a possible association between MBG and arterial stiffening was hypothesized. Jablonski et al. have demonstrated, in individuals with high or hypertensive blood pressures, that a positive association between MBG and carotid to femoral pulse wave velocity (cf-PWV) [114] is actually the gold standard measurement of large artery stiffness [126]. The same positive correlation has also been demonstrated in young healthy women, regardless of salt intake [80]. To date, the pathogenetic mechanisms through which MBG is able to determine arterial stiffness are unknown, although MBG has already been shown to promote the development of vascular fibrosis in the aorta of rats [59]. The mechanism by which MBG promotes collagen production and deposition was studied in cultured rat

smooth muscle cells and was always dependent on the inhibition of Na+K+-ATPase [58,59]. Collagen-1 production is secondary to the marked downregulation of transcription factor Friend leukemia integration-1 (Fli-1) [58,59]. In fact, it has been shown that MBG could also sub-regulate a negative regulator of collagen-1 synthesis, Fli-1. The phosphorylation of Fli-1 through the active form of Protein Kinase C Delta (PKC-d) induces the activation of a collagen gene promoter. In vitro, MBG was found to be an activator of the Fli-1 pathway in cultured fibroblasts and smooth rat muscle cells.

Another important predictor of both increased CV risk and mortality is the left ventricular mass (LVM) measured using the echocardiogram [127]. In the CARDIA study (Coronary Artery Risk Development in Young Adults), a possible positive association between LVM and sodium in 24h urine levels was shown in young adults, although this relationship was confounded by the presence of obesity [128]. From this observation, it was hypothesized that the increase in MBG plasma levels, induced by sodium intake, may be associated with an increase in LVM. Strauss et al. have observed a significant association between LVM and MBG in young adults: in this case, the relationship was independent on obesity and also on blood pressure values, suggesting a possible pathway through which MBG induces myocardial hypertrophy [25].

To further confirm the correlation between MBG plasma levels and fibrosis, it has been shown that rats with (experimental) renal impairment have increased MBG plasma values, along with cardiac and renal fibrosis. In these mouse models, MBG promotes procollagen-1 expression by cultured cardiac fibroblasts. As procollagen expression increases, collagen and procollagen-1 mRNA increase too. Considering this, MBG probably induces a direct increase in collagen expression by fibroblasts [9]. It has also been observed that in spontaneously hypertensive (SHR) and normotensive Wistar–Kyoto (WKY) mouse models, following a diet rich in sodium, hypertension and left and renal ventricular hypertrophy, due to fibrosis with the overexpression of TGF-β<sup>1</sup> mRNA, were recorded. This has resulted, in both glomerular and peritubular sites, in an increase in collagen type 1 [129]. Interestingly, in animal models characterized by prolonged salt intake, diet-related profibrotic effects were eliminated through treatment with anti-MBG antibodies, without generating hemodynamic effects [130]. The administration of specific antibodies against MBG reduced aortic fibrosis and favored relaxation at the level of aortic fibers. The differences that can be attributed to the vascular component without hemodynamic changes, indicate that the possible vascular stiffening is independent of blood pressure and that the profibrotic factor generated by MBG is responsible for it [131].

Even in laboratory models treated with antibodies to MBG and previously subjected to a diet high in sodium, there was a reduction in systolic blood pressure and also a reduction in the weight of both the heart and kidneys. In these mouse models, TGF-β expression, which had previously been increased, was also downregulated after treatment with anti-MBG antibodies. The immunoneutralization of MBG resulted in the downregulation of genes involved in profibrotic expression. In addition, the normalization of renal function through creatinine clearance was also observed, probably due to the reduction in renal fibrosis, as it was accompanied by a significant decrease in kidney weight for the reduction in type I-III-IV collagen amounts. In mouse models, it has also been observed that treatment with anti-MBG antibodies reduces the development of heart failure. This was established by estimating ventricular weight via ultrasound in hypertensive rats immunoneutralized for MBG, compared with non-immunoneutralized hypertensive rats.

Early vascular damage was also reduced after immunoneutralization. In these models, a reduction in the mRNA expression of TGF-β1, FN1, MAPK1, Col1a2, Col3a1 and Col4a1 was also noted compared with those not treated with antibodies of MBG. This demonstrated how MBG initiates TGFβ-1-signaling in cultured ventricular myocytes, through Na+/K+-ATPase signal transduction and other factors, such as tissue angiotensin II [132]. In humans, sodium restriction also reduces urinary MBG production and excretion, resulting in reduced blood pressure and aortic stiffness. Another factor may impact fibrosis, sodium

concentration and MBG plasma levels: it has been observed that the sensitivity of blood pressure dietary salt intake increases with increasing age [23,133,134].
