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Background Central arterial stiffness (CAS) is associated with elevated arterial blood pressure (BP) and is likely associated with stiffening of cerebral artery walls, with attendant cerebral hypoperfusion, neuronal density loss and cognitive decline. Dahl salt-sensitive (Dahl-S) rats exhibit age-associated hypertension and memory loss, even on a normal salt intake.Method: We sought to explore whether central arterial pulse wave velocity (PWV), a marker of CAS, is associated with hippocampal cerebral blood flow (CBF) and neuronal density in hypertensive Dahl-S rats. We measured systolic BP (by tail-cuff plethysmography), aortic PWV (by echocardiography) and CBF and N-acetyl aspartate (NAA) (by magnetic resonance imaging) in 6 month-old male Dahl-S rats (n = 12). Results Greater PWV was significantly associated with lower CBF and lower NAA concentration in the hippocampus, supporting a role of CAS in cerebrovascular dysfunction and decline in cognitive performance with aging. Conclusion These findings implicate increased CAS in cerebral hypoperfusion and loss of neuronal density and function in the Dahl-S model of age-associated cardiovascular dysfunction.

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-β1, 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 (EC(50)) = 29 nmol/L) versus control rings (EC(50) = 7 nmol/L) and was restored by anti-MBG mAb (EC(50) = 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-β dependent, underlie its effects.

Background: Aged Dahl salt sensitive rats (DSS) rats represent a model of vascular dementia, i.e., they develop central arterial stiffness (CAS), hypertension, and cognitive decline. DSS rats have compromised renin-angiotensin system (RAS) which is activated with age and contributes to hypertension. This study examined whether angiotensin converting enzyme (ACE) inhibitor, lisinopril, affects CAS and cognitive functions in aged male DSS (n=26). Methods & Results: Following initial measurements at 16-mo of age (baseline; BL), DSS were administered lisinopril through their water (LISI; 15mg/kg/day, n=11) or control treatment (n=15) for 2-mo. Pulse wave velocity (PWV), a marker of CAS, and systolic blood pressure (SBP) were measured at BL and at 18-mo of age. Open field test (OFT) to assess anxiety-like behavior and Morris water maze (MWM) to assess spatial memory were performed at 18-mo, then aortae and hearts were collected and weighed. Aortic wall collagen abundance was estimated by histochemistry. Statistical analyses were performed by 2-way ANOVA mixed effects model and t-test. The data are presented as mean ± SEM. At 18-mo of age, control animals had high SBP similar to BL (187±4 vs. 184±7 mmHg), while LISI-treated DSS had lower SBP by 39±4 mmHg vs. BL (p Conclusion: Initiation of anti-hypertensive treatment in old age was effective in reducing aortic fibrosis and lowering SBP and PWV in DSS rats. Longer treatment may be needed to improve cognitive function in this model of vascular dementia. Supported by the NIH/NIA Intramural Research Program

Background Elevated levels of an endogenous Na/K‐ ATP ase inhibitor marinobufagenin accompany salt‐sensitive hypertension and are implicated in cardiac fibrosis. Immunoneutralization of marinobufagenin reduces blood pressure in Dahl salt‐sensitive (Dahl‐S) rats. The effect of the anti‐marinobufagenin monoclonal antibody on blood pressure, left ventricular ( LV ) and renal remodeling, and gene expression were investigated in hypertensive Dahl‐S rats. Methods and Results Dahl‐S rats were fed high NaCl (8%, HS ; n=14) or low NaCl (0.1%, LS ; n=14) diets for 8 weeks. Animals were administered control antibody (LS control antibody, LSC; HS control antibody, HSC; n=7 per group) or anti‐marinobufagenin antibody once on week 7 of diet intervention (n=7 per group). Levels of marinobufagenin, LV, and kidney mRNAs and proteins implicated in profibrotic signaling were assessed. Systolic blood pressure was elevated (211±8 versus 133±3 mm Hg, P P P P P Conclusions Immunoneutralization of heightened marinobufagenin levels in hypertensive Dahl‐S rats resulted in a downregulation of genes implicated in transforming growth factor‐β pathway, which indicates that marinobufagenin is an activator of profibrotic transforming growth factor‐β–dependent signaling in salt‐sensitive hypertension.

Background: Independent of a high salt intake, Dahl Salt-Sensitive (DSS) rats demonstrate an age-associated increase in blood pressure (BP), and central arterial stiffness estimated by pulse wave velocity (PWV) accompanied by cognitive decline. We hypothesized, that anti-hypertensive treatment Lisinopril, in addition to BP decrease, will lower PWV and improve cognition of adult DSS. Methods: Twenty 6-mo old male DSS were kept on normal salt diet for the duration of the study; ten of them were continuously treated with Lisinopril (15 mg/kg/day) for 3 months. Systolic BP (SBP) and PWV (by doppler echocardiography) were measured, and open field test (OFT) was performed to assess locomotor and anxiety-like behavior at baseline prior to treatment and repeated in 3 months in 9-mo old rats. In a sub-cohort of DSS rats SBP was measured in 3-mo-old DSS. The data are presented as mean ± standard deviation and analyzed by 2-way ANOVA and T-test. Results: Prior to treatment, all DSS rats displayed hypertension and BP elevation with age (SBP: 170±11 vs. 142±5 mmHg, 6-mo vs. 3-mo; P Conclusion: Lowering of BP and PWV is associated with reduced anxiety-like behavior in DSS, which indicate the connection of cardiovascular disorder and cognitive function in adult hypertensive DSS. Whether Lisinopril has an additional direct effect on brain structure and function in DSS rats will be further studied. This research was supported entirely by the Intramural research Program of the NIH, National institute on Aging

Background: Hypertension and cardiovascular disease may be significant contributors to dementia and Alzheimer’s disease. However, the mechanism of this process is poorly understood. Central arterial stiffness (CAS) is accelerated by systemic hypertension and may be accompanied by increased stiffness in the cerebral arteries. Methods & Results: We used Dahl salt sensitive rats (Dahl-S) which develop CAS and hippocampal memory decline as they develop variable degrees of hypertension on a 0.5% salt diet with age. At 22 weeks, systolic blood pressure (SBP), pulse wave velocity (PWV), open field tests (OFT), hippocampal N-acetylaspartate (NAA) concentrations and blood flow (BF) data were all collected. The rats displayed moderate hypertension as compared to a 3 months baseline (SBP: 165±16 vs. 142±5 mmHg; P Conclusion: In the Dahl-S model of hypertension, greater PWV, a marker for CAS, was associated with decreased hippocampal perfusion. Relationship between CAS and hippocampal BF was as hypothesized. Likewise, hippocampal BF correlates with hippocampal NAA concentration as expected. The positive correlation between hippocampal BF and increased central rearing duration suggests that higher cerebral BF is associated with a lower anxiety level. Supported entirely by the Intramural research Program of the NIH, National institute on Aging.

High salt (HS) intake stimulates the production of marinobufagenin (MBG), an endogenous steroidal Na/K-ATPase ligand, which activates profibrotic signaling. HS is accompanied by a blood pressure (BP) increase in salt-sensitive hypertension, but not in normotensive animals. Here, we investigated whether HS stimulates MBG production and activates transforming growth factor-beta (TGF-&beta, ) profibrotic signaling in young normotensive rats, and whether these changes can be reversed by reducing salt to a normal salt (NS) level. Three-month old male Sprague&ndash, Dawley rats received NS for 4 and 8 weeks (0.5% NaCl, NS4 and NS8), or HS for 4 and 8 weeks (4% NaCl, HS4 and HS8), or HS for 4 weeks followed by NS for 4 weeks (HS4/NS4), n = 8/group. Systolic BP (SBP), pulse wave velocity (PWV), MBG excretion, aortic collagen 1&alpha, 2, collagen 4&alpha, 1 and TGF-&beta, Smad2, Smad3, Fli-1 mRNA, and total collagen abundance were measured at baseline (BL), and on weeks 4 and 8. Statistical analysis was performed using one-way ANOVA. SBP was not affected by HS (125 &plusmn, 5 and 126 &plusmn, 6 vs. 128 &plusmn, 7 mmHg, HS4 and HS8 vs. BL, p &gt, 0.05). HS increased MBG (164 &plusmn, 19 vs. 103 &plusmn, 19 pmol/24 h/kg, HS4 vs. BL, p &lt, 0.05) and PWV (3.7 &plusmn, 0.2 vs. 2.7 &plusmn, 0.2 m/s, HS4 vs. NS4, p &lt, 0.05). HS8 was associated with a further increase in MBG and PWV, with an increase in aortic Col1a2 80%), Col4a1 (50%), Tgfb1 (30%), Smad2 (30%) and Smad3 (45%) mRNAs, and aortic wall collagen (180%) vs. NS8 (all p &lt, 0.05). NS following HS downregulated HS-induced factors: in HS4/NS4, the MBG level was 91 &plusmn, 12 pmol/24 h/kg (twofold lower than HS8, p &lt, 0.01), PWV was 3.7 &plusmn, 0.3 vs. 4.7 &plusmn, 0.2 m/s (HS4/NS4 vs. HS8, p &lt, 0.05), aortic wall Tgfb1, Col1a2, Col4a1, Smad2, Smad3 mRNAs, and collagen abundance were reversed by salt reduction to the BL levels (p &lt, 0.05). HS was associated with an activation of TGF-&beta, signaling, aortic fibrosis and aortic stiffness accompanied by an MBG increase in the absence of SBP changes in young normotensive rats. The reduction of dietary salt following HS decreased MBG, PWV, aortic wall collagen and TGF-&beta, Thus, HS-induced aortic stiffness in normotensive animals occurred in the context of elevated MBG, which may activate SMAD-dependent TGF-&beta, pro-fibrotic signaling. This data suggests that a decrease in salt consumption could help to restore aortic elasticity and diminish the risk of cardiovascular disease by reducing the production of the pro-fibrotic factor MBG.

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 &plusmn, 4 mmHg, age 29 &plusmn, 2 years, 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 &plusmn, 2 mmHg, controls) were enrolled in the clinical study. Results: PE was associated with a higher placental (0.04 &plusmn, 0.01 vs. 0.49 &plusmn, 0.11 pmol/g, p &lt, 0.01) and plasma MBG (0.5 &plusmn, 0.1 vs. 1.6 &plusmn, 0.5 nmol/L, 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 &plusmn, 0.2 vs. 1.5 &plusmn, 0.2 &micro, mol Pi/mL/hr, 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae (p &lt, 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 &lt, 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.

Background— The bioactive steroid, marinobufagenin, is an endogenous Na/K-ATPase bufadienolide inhibitor that is synthesized by adrenocortical and placental cells. Marinobufagenin binding to Na/K-ATPase initiates profibrotic cell signaling, and heightened marinobufagenin 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 marinobufagenin 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 marinobufagenin levels by 67% when compared with nontreated cells or cells transfected with nontargeting siRNA. In contrast, silencing of the CYP11A1 gene did not affect marinobufagenin 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 when compared with cells transfected with nontargeting siRNA. In vivo, in a high-salt administration experiment, male and female Dahl salt-sensitive rats became hypertensive after 4 weeks on a high-NaCl diet, their plasma marinobufagenin 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, marinobufagenin, 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 marinobufagenin in highly prevalent human cardiovascular diseases.

Immunoneutralization of elevated circulating levels of endogenous digitalis-like Na/K-ATPase inhibitors (i.e. cardiotonic steroids (CTS)) represents a novel approach in the treatment of preeclampsia (PE). Recently we demonstrated that DigiFab (Fab fragments of affinity-purified ovine digoxin antibody) restores PE-induced inhibition of Na/K-ATPase in erythrocytes ex vivo. Previously magnesium ions were shown to antagonize digitalis-induced toxicity, which is mediated by Na/K-ATPase inhibition. We hypothesized that magnesium sulfate would potentiate the effect of DigiFab in the reversal of CTS-induced Na/K-ATPase inhibition.To test this hypothesis, we studied the ex vivo effect of DigiFab on Na/K-ATPase activity in erythrocytes from patients with PE in the absence and in the presence of 3 mmol/L magnesium sulfate.Compared with 11 normotensive pregnant subjects (29 ± 1 years; gestational age = 39.0 ± 0.2 weeks; blood pressure = 111 ± 2/73 ± 2 mm Hg), the 12 patients with PE (30 ± 1 years; gestational age = 37.9 ± 0.3 weeks; blood pressure = 159 ± 5/99 ± 3 mm Hg) had plasma levels of marino-bufagenin increased 3-fold (1.38 ± 0.40 vs. 0.38 ± 0.10 nmol/L; P0.01) and activity of Na/K-ATPase in erythrocytes was inhibited (1.16 ± 0.11 vs. 2.80 ± 0.20 μmol Pi/ml/h; P0.01). Ex vivo, DigiFab (1 µg/ml) restored erythrocyte Na/K-ATPase activity (1.72 ± 0.13 µmol Pi/ml/h; P0.01), and 3 mmol magnesium sulfate potentiated the effect of DigiFab (2.30 ± 0.20 µmol Pi/ml/h; P0.01).Magnesium is capable of increasing the efficacy of immunoneutralization of marinobufagenin-induced Na/K-ATPase inhibition.

OBJECTIVE Endogenous cardiotonic steroids, including marinobufagenin (MBG), stimulate vascular synthesis of collagen. Because mineralocorticoid antagonists competitively antagonize effect of cardiotonic steroids on the Na/K-ATPase, we hypothesized that spironolactone would reverse the profibrotic effects of MBG. METHODS Experiment 1: Explants of thoracic aortae and aortic vascular smooth muscle cells from Wistar rats were cultured for 24 h 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 years) with resistant hypertension on a combined (lisinopril/amlodipine/hydrochlorothiazide) therapy, we determined arterial pressure, pulse wave velocity, plasma MBG, and erythrocyte Na/K-ATPase before and 6 months after addition of placebo (n = 8) or spironolactone (50 mg/day; n = 8) to the therapy. RESULTS In rat aortic explants and in vascular smooth muscle cells, 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 vs. 23 ± 3 nmol/l in vehicle-treated rings; P

BACKGROUND Marinobufagenin (MBG) is an endogenous Na/K-ATPase inhibitor, a natriuretic and a vasoconstrictor. MBG is implicated in salt-sensitive hypertension, cardiac hypertrophy, and initiate the pro-fibrotic signaling. Previously it was demonstrated that immunoneutralization of an endogenous MBG by 3E9 anti-MBG-antibody (mAb) in vivo lowered blood pressure (BP) and reversed cardiac fibrosis in salt-sensitive, and in partially nephrectomized rats. In the present study, we investigated whether mAb alleviates vascular remodeling induced in normotensive rats on high salt intake. METHODS Wistar rats (5 months old) received normal (CTRL; n = 8) or high salt intake (2% NaCl in drinking water) for 4 weeks ( n = 16). Rats from the group on a high salt intake were administered vehicle (SALT; n = 8) or mAb (50 µg/kg) (SALT-AB; n = 8) during the last week of high salt diet. BP, erythrocyte Na/K-ATPase activity, levels of MBG in plasma and 24-hour urine, and sensitivity of aortic explants to the vasorelaxant effect of sodium nitroprusside (SNP) were measured. Aortic collagen abundance was determined immunohistochemically. RESULTS In SALT vs. CTRL, heightened levels of MBG were associated with inhibition of erythrocyte Na/K-ATPase in the absence of BP changes. High salt intake was accompanied by a 2.5-fold increase in aortic collagen abundance and by a reduction of sensitivity of aortic explants to the vasorelaxant effect of SNP following endothelin-1-induced constriction. In the SALT-AB group, all NaCl-mediated effects were reversed by immunoneutralization of MBG. CONCLUSIONS High salt intake in young normotensive rats can induce vascular fibrosis via pressure-independent/MBG-dependent mechanisms, and this remodeling is reduced by immunoneutralization of MBG.

Background: Marinobufagenin (MBG), an endogenous cardiotonic steroid, is a Na/K-ATPase inhibitor and a vasoconstrictor. Previously it was demonstrated, that administration of 3E9 anti-MBG-antibody (mAb) reduced blood pressure (BP) and reversed left ventricular fibrosis in animal models of salt-sensitive hypertension and nephropathy. In the present study we investigated whether mAb alleviates BP and vascular remodeling in normotensive rats on a high salt intake. Methods: Wistar rats (5 months old) received normal salt diet (CTRL; n=8) or high salt intake (2% NaCl in drinking water) for 4 weeks. Rats on a high salt were administered vehicle (SALT; n=8) or mAb (50 ug/kg) (SALT-AB; n=8) 3 times during the last week of a high salt diet. BP was measured at baseline, after 3 and 4 weeks of experiment. Na/K-ATPase activity was measured in erythrocytes. Aortas were weighed, and were used to study sensitivity to the vasorelaxant effect of sodium nitroprusside (SNP), and for the histochemistry analysis of collagen deposition. Renal 24-hr MBG excretion was measured at week 4. Results: In SALT vs. CTRL, in the absence of BP changes, elevated levels of MBG (14.1±1.1 vs. 9.0±1.6 pmol/24hr, p Conclusion: These findings for the first time demonstrated that in normotensive rats on a high salt intake heightened MBG levels induced vascular fibrosis and impairment of vasorelaxation in the absence of blood pressure changes. Immunoneutralization of MBG reversed these changes. Thus, high dietary NaCl intake in normotensive animals can stimulate vascular fibrosis via pressure-independent/ MBG-dependent mechanisms, and this remodeling is reversible.

Michael Galagudza1, Dmitry Korolev1, Viktor Postnov2, Elena Naumisheva2, Yulia Grigorova3, Ivan Uskov1, Eugene Shlyakhto11Institute of Experimental Medicine, VA Almazov Federal Heart, Blood and Endocrinology Center, 2Chemical Faculty, St Petersburg State University, 3Department of Pathophysiology, IP Pavlov State Medical University, St Petersburg, Russian FederationAbstract: 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.Keywords: silica nanoparticles, targeted drug delivery, myocardium, ischemia, reperfusion