Silencing of PKG1 Gene Mimics Effect of Aging and Sensitizes Rat Vascular Smooth Muscle Cells to Cardiotonic Steroids: Impact on Fibrosis and Salt Sensitivity.

Background Marinobufagenin, NKA (Na/K-ATPase) inhibitor, causes vasoconstriction and induces fibrosis via inhibition of Fli1 (Friend leukemia integration-1), a negative regulator of collagen synthesis. In vascular smooth muscle cells (VSMC), ANP (atrial natriuretic peptide), via a cGMP/PKG1 (protein kinase G1)-dependent mechanism, reduces NKA sensitivity to marinobufagenin. We hypothesized that VSMC from old rats, due to downregulation of ANP/cGMP/PKG-dependent signaling, would exhibit heightened sensitivity to the profibrotic effect of marinobufagenin. Methods and Results Cultured VSMC from the young (3-month-old) and old (24-month-old) male Sprague-Dawley rats and young VSMC with silenced PKG1 gene were treated with 1 nmol/L ANP, or with 1 nmol/L marinobufagenin, or with a combination of ANP and marinobufagenin. Collagen-1, Fli1, and PKG1 levels were assessed by Western blotting analyses. Vascular PKG1 and Fli1 levels in the old rats were reduced compared with their young counterparts. ANP prevented inhibition of vascular NKA by marinobufagenin in young VSMC but not in old VSMC. In VSMC from the young rats, marinobufagenin induced downregulation of Fli1 and an increase in collagen-1 level, whereas ANP blocked this effect. Silencing of the PKG1 gene in young VSMC resulted in a reduction in levels of PKG1 and Fli1; marinobufagenin additionally reduced Fli1 and increased collagen-1 level, and ANP failed to oppose these marinobufagenin effects, similar to VSMC from the old rats with the age-associated reduction in PKG1. Conclusions Age-associated reduction in vascular PKG1 and the resultant decline in cGMP signaling lead to the loss of the ability of ANP to oppose marinobufagenin-induced inhibition of NKA and fibrosis development. Silencing of the PKG1 gene mimicked these effects of aging.

Effect of Cardiotonic Steroid Marinobufagenin on Vascular Remodeling and Cognitive Impairment in Young Dahl-S Rats.

The hypertensive response in Dahl salt-sensitive (DSS) rats on a high-salt (HS) diet is accompanied by central arterial stiffening (CAS), a risk factor for dementia, and heightened levels of a prohypertensive and profibrotic factor, the endogenous Na/K-ATPase inhibitor marinobufagenin (MBG). We studied the effect of the in vivo administration of MBG or HS diet on blood pressure (BP), CAS, and behavioral function in young DSS rats and normotensive Sprague-Dawley rats (SD), the genetic background for DSS rats. Eight-week-old male SD and DSS rats were given an HS diet (8% NaCl, n = 18/group) or a low-salt diet (LS; 0.1% NaCl, n = 14-18/group) for 8 weeks or MBG (50 µg/kg/day, n = 15-18/group) administered via osmotic minipumps for 4 weeks in the presence of the LS diet. The MBG-treated groups received the LS diet. The systolic BP (SBP); the aortic pulse wave velocity (aPWV), a marker of CAS; MBG levels; spatial memory, measured by a water maze task; and tissue collection for the histochemical analysis were assessed at the end of the experiment. DSS-LS rats had higher SBP, higher aPWV, and poorer spatial memory than SD-LS rats. The administration of stressors HS and MBG increased aPWV, SBP, and aortic wall collagen abundance in both strains vs. their LS controls. In SD rats, HS or MBG administration did not affect heart parameters, as assessed by ECHO vs. the SD-LS control. In DSS rats, impaired whole-heart structure and function were observed after HS diet administration in DSS-HS vs. DSS-LS rats. MBG treatment did not affect the ECHO parameters in DSS-MBG vs. DSS-LS rats. The HS diet led to an increase in endogenous plasma and urine MBG levels in both SD and DSS groups. Thus, the prohypertensive and profibrotic effect of HS diet might be partially attributed to an increase in MBG. The prohypertensive and profibrotic functions of MBG were pronounced in both DSS and SD rats, although quantitative PCR revealed that different profiles of profibrotic genes in DSS and SD rats was activated after MBG or HS administration. Spatial memory was not affected by HS diet or MBG treatment in either SD or DSS rats. Impaired cognitive function was associated with higher BP, CAS, and cardiovascular remodeling in young DSS-LS rats, as compared to young SD-LS rats. MBG and HS had similar effects on the cardiovascular system and its function in DSS and SD rats, although the rate of change in SD rats was lower than in DSS rats. The absence of a cumulative effect of increased aPWV and BP on spatial memory can be explained by the cerebrovascular and brain plasticity in young rats, which help the animals to tolerate CAS elevated by HS and MBG and to counterbalance the profibrotic effect of heightened MBG.

Monoclonal Antibody to Marinobufagenin Downregulates TGFß Profibrotic Signaling in Left Ventricle and Kidney and Reduces Tissue Remodeling in Salt-Sensitive Hypertension.

Background Elevated levels of an endogenous Na/K-ATPase 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<0.01), marinobufagenin increased 2-fold in plasma (P<0.05) and 5-fold in urine (P<0.01), LV and kidney weights increased, and levels of LV collagen-1 rose 3.5-fold in HSC versus LSC. Anti-marinobufagenin antibody treatment decreased systolic blood pressure by 24 mm Hg (P<0.01) and reduced organ weights and level of LV collagen-1 (P<0.01) in hypertensive Dahl salt-sensitive rats with anti-marinobufagenin antibody versus HSC. The expression of genes related to transforming growth factor-ß-dependent signaling was upregulated in the left ventricles and kidneys in HSC versus LSC groups and became downregulated following administration of anti-marinobufagenin antibody to hypertensive Dahl-S rats. Marinobufagenin also activated transforming growth factor-ß signaling in cultured ventricular myocytes from Dahl-S rats. Conclusions Immunoneutralization of heightened marinobufagenin levels in hypertensive Dahl-S rats resulted in a downregulation of genes implicated in transforming growth factor-ß pathway, which indicates that marinobufagenin is an activator of profibrotic transforming growth factor-ß-dependent signaling in salt-sensitive hypertension.

Cardiotonic Steroids Induce Vascular Fibrosis Via Pressure-Independent Mechanism in NaCl-Loaded Diabetic Rats.

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-ß1, 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 (EC50) = 29 nmol/L) versus control rings (EC50 = 7 nmol/L) and was restored by anti-MBG mAb (EC50 = 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-ß dependent, underlie its effects.

Dietary Sodium Restriction Reduces Arterial Stiffness, Vascular TGF-ß-Dependent Fibrosis and Marinobufagenin in Young Normotensive Rats.

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-ß) 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⁻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 1α2, collagen 4α1 and TGF-ß, 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 ± 5 and 126 ± 6 vs. 128 ± 7 mmHg, HS4 and HS8 vs. BL, p > 0.05). HS increased MBG (164 ± 19 vs. 103 ± 19 pmol/24 h/kg, HS4 vs. BL, p < 0.05) and PWV (3.7 ± 0.2 vs. 2.7 ± 0.2 m/s, HS4 vs. NS4, p < 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 < 0.05). NS following HS downregulated HS-induced factors: in HS4/NS4, the MBG level was 91 ± 12 pmol/24 h/kg (twofold lower than HS8, p < 0.01), PWV was 3.7 ± 0.3 vs. 4.7 ± 0.2 m/s (HS4/NS4 vs. HS8, p < 0.05), aortic wall Tgfb1, Col1a2, Col4a1, Smad2, Smad3 mRNAs, and collagen abundance were reversed by salt reduction to the BL levels (p < 0.05). HS was associated with an activation of TGF-ß 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-ß. Thus, HS-induced aortic stiffness in normotensive animals occurred in the context of elevated MBG, which may activate SMAD-dependent TGF-ß 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.

Antibody to Marinobufagenin Reverses Placenta-Induced Fibrosis of Umbilical Arteries in Preeclampsia.

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

Aortic Fibrosis, Induced by High Salt Intake in the Absence of Hypertensive Response, is Reduced by a Monoclonal Antibody to Marinobufagenin.

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.

Synthesis of an Endogenous Steroidal Na Pump Inhibitor Marinobufagenin, Implicated in Human Cardiovascular Diseases, Is Initiated by CYP27A1 via Bile Acid Pathway.

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.

TRAIL, DR4 and DR5 are upregulated in kidneys from patients with lupus nephritis and exert proliferative and proinflammatory effects.

We have previously reported that TRAIL is upregulated on T cells from patients with lupus and that T cell associated TRAIL enhances autoimmune parameters in a murine model of lupus. Whether TRAIL/TRAIL-R interaction plays a role in organ involvement such as lupus nephritis has not yet been assessed. We demonstrate here that TRAIL, DR4 and DR5 are upregulated in proximal and distal tubules of patients with proliferative lupus nephritis. In vitro, expression of TRAIL, DR4 and DR5 on primary proximal tubular epithelial cells (PTEC) was induced by TNFalpha and IFNgamma. Functionally, TRAIL did not induce apoptosis but rather enhanced the proliferation of PTEC through activation of PI3 kinase/AKT and ERK1/2, increased IL-8 production and upregulated ICAM-1 expression. These data demonstrate that cytokine induced upregulation of TRAIL, DR4 and DR5 in tubules from patients with proliferative lupus nephritis may play a protective role by enhancing PTEC survival while also exerting a proinflammatory effect that may contribute to local inflammation and injury.

T cell TRAIL promotes murine lupus by sustaining effector CD4 Th cell numbers and by inhibiting CD8 CTL activity.

T cells play an essential role in driving humoral autoimmunity in lupus. Molecules such as TRAIL exhibit strong T cell modulatory effects and are up-regulated in lupus, raising the possibility that they may influence disease severity. To address this possibility, we examined the role of TRAIL expression on pathogenic T cells in an induced model of murine lupus, the parent-into-F(1) (P-->F(1)) model of chronic graft-vs-host disease (GVHD), using wild-type or TRAIL-deficient donor T cells. Results were compared with mice undergoing suppressive acute GVHD. Although chronic GVHD mice exhibited less donor T cell TRAIL up-regulation and IFN-alpha-inducible gene expression than acute GVHD mice, donor CD4(+) T cell TRAIL expression in chronic GVHD was essential for sustaining effector CD4(+) Th cell numbers, for sustaining help to B cells, and for more severe lupus-like renal disease development. Conversely, TRAIL expression on donor CD8(+) T cells had a milder, but significant down-regulatory effect on CTL effector function, affecting the perforin/granzyme pathway and not the Fas ligand pathway. These results indicate that, in this model, T cell-expressed TRAIL exacerbates lupus by the following: 1) positively regulating CD4(+) Th cell numbers, thereby sustaining T cell help for B cells, and 2) to a lesser degree by negatively regulating perforin-mediated CD8(+) CTL killing that could potentially eliminate activated autoreactive B cells.

Increased expression and release of functional tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by T cells from lupus patients with active disease.

Increased expression of TRAIL in membrane-bound and soluble form in patients with systemic lupus erythematosus (SLE) has been previously reported. In this study, we characterized the upregulation of T-cell-associated and soluble TRAIL (sTRAIL) in vivo and the modulation of TRAIL expression and soluble protein release in vitro following T cell activation and IFNalpha exposure. The expression of membrane-bound TRAIL as determined by flow cytometry was higher on CD4(+) and CD8(+) T cells from lupus patients compared to controls, particularly on activated CD69(+)CD8(+) T cells. Similarly, sTRAIL levels determined by ELISA were significantly elevated in serum from patients with active SLE and correlated with levels of IFNalpha. In vitro, both T-cell-associated and sTRAIL were maximally induced by T cell activation plus IFNalpha in patients and controls. By Western blot analysis, sTRAIL was detected in sera in both the monomeric and multimeric, functional form. Both forms of TRAIL were functional in vitro as determined by Annexin V staining and (51)Cr release assay but the apoptotic activity of membrane TRAIL was 2.5-fold higher compared to that of sTRAIL. These results indicate that IFNalpha-induced enhancement of TRAIL expression and of TRAIL-mediated apoptosis may amplify the abnormal apoptotic process in SLE.

T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression.

Human mesenchymal stem cells (MSCs) were evaluated for their ability to activate allogeneic T cells in cell mixing experiments. Phenotypic characterization of MSCs by flow cytometry showed expression of MHC Class I alloantigens, but minimal expression of Class II alloantigens and costimulatory molecules, including CD80 (B7-1), CD86 (B7-2), and CD40. T cells purified from peripheral blood mononuclear cells (PBMCs) did not proliferate to allogeneic MSCs. Lack of response was not due to a deficiency of costimulation, since retroviral transduction of MSCs with either B7-1 or B7-2 costimulatory molecules did not result in lymphoproliferation. Although these results suggested that MSCs were immunologically inert or potentially tolerogenic, T cells cultured with MSCs produced IFN-gamma and displayed secondary kinetics to restimulation with PBMCs, indicating alloantigen priming rather than tolerance induction by the MSCs. To determine whether MSCs suppressed alloreactive T cells, MSCs were added to primary mixed lymphocyte reaction (MLR) cultures. MSCs suppressed cell proliferation when added at the initiation of culture or when added to an ongoing MLR culture. Suppression was dose-dependent, genetically unrestricted, and occurred whether or not MSCs were pretreated with IFN-gamma. MSCs in transwell chambers suppressed primary MLR cultures, indicating that suppression was mediated by soluble molecules. Analysis of cytokines in suppressed MLR cultures demonstrated up-regulation of IFN-gamma and IL-10, and down-regulation of TNF-alpha production relative to control cultures. We conclude that MSCs can initiate activation of alloreactive T cells, but do not elicit T cell proliferative responses due to active suppressive mechanisms.

Gene expression profiling in peripheral blood mononuclear cells from lupus patients with active and inactive disease.

Systemic lupus erythematosus (SLE) is characterized by periods of flare and remission. The search for parameters associated with disease activity has been an area of intense investigation. To identify genes that best differentiate patients with active from those with inactive disease, the expression pattern of 375 genes was analyzed in peripheral blood mononuclear cells (PBMC) from 12 patients with active and 14 patients with inactive disease. Using the "nearest shrunken centroids" method, 29 genes were found to best discriminate the two groups. Among these genes, 14 were upregulated and 15 were downregulated in patients with active compared to those with inactive disease. Fourteen of these genes also correlated with SELENA-SLEDAI with correlation coefficients ranging from 0.4 to 0.7. Most of these genes have not been previously associated with disease activity and belong to a variety of families such as adhesion molecules, proteases, TNF superfamily, and neurotrophic factors. Using a cross-validation method, the error rate for classifying samples in the two groups was 30%. These results highlight the potential use of microarray data in identifying genes associated with disease activity in SLE, which could become potential biomarkers or future therapeutic targets.