The effect of spironolactone on cardiovascular function and markers of fibrosis in people at increased risk of developing heart failure: the heart 'OMics' in AGEing (HOMAGE) randomized clinical trial.

AIMS: To investigate the effects of spironolactone on fibrosis and cardiac function in people at increased risk of developing heart failure. METHODS AND RESULTS: Randomized, open-label, blinded-endpoint trial comparing spironolactone (50 mg/day) or control for up to 9 months in people with, or at high risk of, coronary disease and raised plasma B-type natriuretic peptides. The primary endpoint was the interaction between baseline serum galectin-3 and changes in serum procollagen type-III N-terminal pro-peptide (PIIINP) in participants assigned to spironolactone or control. Procollagen type-I C-terminal pro-peptide (PICP) and collagen type-1 C-terminal telopeptide (CITP), reflecting synthesis and degradation of type-I collagen, were also measured. In 527 participants (median age 73 years, 26% women), changes in PIIINP were similar for spironolactone and control [mean difference (mdiff): -0.15; 95% confidence interval (CI) -0.44 to 0.15 µg/L; P = 0.32] but those receiving spironolactone had greater reductions in PICP (mdiff: -8.1; 95% CI -11.9 to -4.3 µg/L; P < 0.0001) and PICP/CITP ratio (mdiff: -2.9; 95% CI -4.3 to -1.5; <0.0001). No interactions with serum galectin were observed. Systolic blood pressure (mdiff: -10; 95% CI -13 to -7 mmHg; P < 0.0001), left atrial volume (mdiff: -1; 95% CI -2 to 0 mL/m2; P = 0.010), and NT-proBNP (mdiff: -57; 95% CI -81 to -33 ng/L; P < 0.0001) were reduced in those assigned spironolactone. CONCLUSIONS: Galectin-3 did not identify greater reductions in serum concentrations of collagen biomarkers in response to spironolactone. However, spironolactone may influence type-I collagen metabolism. Whether spironolactone can delay or prevent progression to symptomatic heart failure should be investigated.

The association of catestatin and endocan with the effects of cardiac shock wave therapy: Biomarker sub-study of the randomized, sham procedure-controlled trial.

Introduction: Cardiac shock-wave therapy (CSWT) is a non-invasive regenerative treatment method based on low-frequency ultrasound waves, which stimulate angiogenesis. Current data about the effects of revascularization procedures on angiogenesis biomarkers is limited. Recently, an association of catestatin and endocan with coronary collateral development was shown in several trials. In this study, we aimed to evaluate the impact of CSWT on the dynamics of catestatin and endocan levels and to assess their correlation with parameters of myocardial perfusion and function. Methods: Prospective, randomized, triple-blind, sham procedure-controlled study enrolled 72 adult subjects who complied with defined inclusion criteria (NCT02339454). We measured biomarkers in 48 patients with stable angina (24 patients of CSWT group, 24 patients of sham-procedure group). Additionally, patients were divided into responders and non-responders according to improvement in myocardial perfusion and/or contractility assessed by myocardial scintigraphy and dobutamine echocardiography (30 and 13 patients, respectively). The blood samples were collected at baseline, after the last treatment procedure (9th treatment week) and at 6-month follow-up to evaluate biomarkers concentration and stored at -80° until analysis. Serum catestatin and endocan levels were determined by commercially available ELISA kits. Results: Serum catestatin concentration significantly increased in all patients. While endocan levels significantly decreased in the responders sub-group. The increase in catestatin levels at 9th week and 6 months was positively associated with improvement in summed difference score (rho = 0.356, p = 0.028) and wall motion score, WMS (rho = 0.397, p = 0.009) at 6 months in the whole study population. Meanwhile, the decrease in endocan levels over 6 months was positively correlated with improvement in WMS at 3- and 6- months (r = 0.378, p = 0.015 and r = 0.311, p = 0.045, respectively). ROC analysis revealed that a change at 6 months in catestatin and endocan levels significantly predicted improvement in myocardial perfusion and contractile function with 68.9% sensitivity and 75.0% specificity (p = 0.039) and 51.7% sensitivity, and 91.7% specificity (p = 0.017), respectively. Baseline endocan concentration and its change at 6 months predicted response to CSWT with 68.8% sensitivity and 83.3% specificity (p = 0.039) and 81.3% sensitivity and 100% specificity (p < 0.0001), respectively. Conclusion: This study demonstrates the association of increase in catestatin and decrease in endocan levels with the improvement of myocardial perfusion and contractile function. The potential predictive value of catestatin and endocan dynamics for the response to regenerative therapy is shown.

Spironolactone effect on the blood pressure of patients at risk of developing heart failure: an analysis from the HOMAGE trial.

AIMS: Uncontrolled blood pressure (BP) increases the risk of developing heart failure (HF). The effect of spironolactone on BP of patients at risk of developing HF is yet to be determined. To evaluate the effect of spironolactone on the BP of patients at risk for HF and whether renin can predict spironolactone's effect. METHODS AND RESULTS: HOMAGE (Heart OMics in Aging) was a prospective multicentre randomized open-label blinded endpoint (PROBE) trial including 527 patients at risk for developing HF randomly assigned to either spironolactone (25-50 mg/day) or usual care alone for a maximum of 9 months. Sitting BP was assessed at baseline, Months 1 and 9 (or last visit). Analysis of covariance (ANCOVA), mixed effects models, and structural modelling equations was used. The median (percentile25-75) age was 73 (69-79) years, 26% were female, and >75% had history of hypertension. Overall, the baseline BP was 142/78 mmHg. Patients with higher BP were older, more likely to have diabetes and less likely to have coronary artery disease, had greater left ventricular mass (LVM), and left atrial volume (LAV). Compared with usual care, by last visit, spironolactone changed SBP by -10.3 (-13.0 to -7.5) mmHg and DBP by -3.2 (-4.8 to -1.7) mmHg (P < 0.001 for both). A higher proportion of patients on spironolactone had controlled BP <130/80 mmHg (36 vs. 26%; P = 0.014). Lower baseline renin levels predicted a greater response to spironolactone (interactionP = 0.041). CONCLUSION: Spironolactone had a clinically important BP-lowering effect. Spironolactone should be considered for lowering blood pressure in patients who are at risk of developing HF.

Proteomic and Mechanistic Analysis of Spironolactone in Patients at Risk for HF.

OBJECTIVES: This study sought to further understand the mechanisms underlying effect of spironolactone and assessed its impact on multiple plasma protein biomarkers and their respective underlying biologic pathways. BACKGROUND: In addition to their beneficial effects in established heart failure (HF), mineralocorticoid receptor antagonists may act upstream on mechanisms, preventing incident HF. In people at risk for developing HF, the HOMAGE (Heart OMics in AGEing) trial showed that spironolactone treatment could provide antifibrotic and antiremodeling effects, potentially slowing the progression to HF. METHODS: Baseline, 1-month, and 9-month (or last visit) plasma samples of HOMAGE participants were measured for protein biomarkers (n = 276) by using Olink Proseek-Multiplex cardiovascular and inflammation panels (Olink, Uppsala, Sweden). The effect of spironolactone on biomarkers was assessed by analysis of covariance and explored by knowledge-based network analysis. RESULTS: A total of 527 participants were enrolled; 265 were randomized to spironolactone (25 to 50 mg/day) and 262 to standard care ("control"). The median (interquartile range) age was 73 years (69 to 79 years), and 26% were female. Spironolactone reduced biomarkers of collagen metabolism (e.g., COL1A1, MMP-2); brain natriuretic peptide; and biomarkers related to metabolic processes (e.g., PAPPA), inflammation, and thrombosis (e.g., IL17A, VEGF, and urokinase). Spironolactone increased biomarkers that reflect the blockade of the mineralocorticoid receptor (e.g., renin) and increased the levels of adipokines involved in the anti-inflammatory response (e.g., RARRES2) and biomarkers of hemostasis maintenance (e.g., tPA, UPAR), myelosuppressive activity (e.g., CCL16), insulin suppression (e.g., RETN), and inflammatory regulation (e.g., IL-12B). CONCLUSIONS: Proteomic analyses suggest that spironolactone exerts pleiotropic effects including reduction in fibrosis, inflammation, thrombosis, congestion, and vascular function improvement, all of which may mediate cardiovascular protective effects, potentially slowing progression toward heart failure. (HOMAGE [Bioprofiling Response to Mineralocorticoid Receptor Antagonists for the Prevention of Heart Failure]; NCT02556450).

sST2 adds to the prognostic value of Gal-3 and BNP in chronic heart failure.

Background: The soluble form of the IL-33 receptor (sST2) and Galectin-3 (Gal-3) are fibrosis biomarkers with prognostic value in heart failure (HF). We investigated the prognostic capacity of sST2 when combined with Gal-3, and determined if the prognostic utility of sST2 is affected by mineralocorticoid receptor antagonist (MRA) therapy.Methods: sST-2 and Gal-3 were measured in 101 stable chronic HF (CHF) patients receiving MRA therapy and compared to 97 BNP and cardiovascular risk factor matched patients not treated with MRA. sST2 and Gal-3 levels were measured to determine the relationship with all-cause mortality at 6-year follow-up.Results: ROC curve cut-off points were defined as sST2 = 36.3 ng/mL, Gal-3 = 17.8 ng/mL, and BNP = 500 pg/mL, and had 6-year mortality hazard ratios (HR) of 7.3, 6.6 and 5.4, respectively. The combination of an elevated sST2 and Gal-3 had a HR = 4.4 [95% CI 1.9-8.9]. Combining sST2 and Gal-3 to a clinical model relevant for CHF prognosis allowed a significant reclassification of 1-year adverse outcome risk, even when BNP was included. Finally, prognostic prediction by sST2 was unaffected by MRA treatment.Conclusion: Simultaneous sST2 and Gal-3 elevation is associated with poorer prognosis compared to either alone, regardless of BNP levels, and the prognostic capacity of sST2 is independent of MRA therapy.

Insulin-like Growth Factor Binding Protein 2 predicts mortality risk in heart failure.

BACKGROUND: Insulin-like Growth Factor Binding Protein 2 (IGFBP2) showed greater heart failure (HF) diagnostic accuracy than the "grey zone" B-type natriuretic peptides, and may have prognostic utility as well. OBJECTIVES: To determine if IGFBP2 provides independent information on cardiovascular mortality in HF. METHODS: A retrospective study of 870 HF patients from 3 independent international cohorts. Presentation IGFBP2 plasma levels were measured by ELISA, and patients were followed from 1 year (Maastricht, Netherlands) to 6 years (Atlanta, GA, USA and Toulouse, France). Multivariate analysis, Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI) were performed in the 3 cohorts. The primary outcome was cardiovascular mortality. RESULTS: In multivariate Cox proportional hazards analysis, the highest quartile of IGFBP2 was associated with mortality in the Maastricht cohort (adjusted hazard ratio 1.69 (95% CI, 1.18-2.41), p = 0.004) and in the combined Atlanta and Toulouse cohorts (adjusted hazard ratio 2.04 (95%CI, 1.3-3.3), p = 0.003). Adding IGFBP2 to a clinical model allowed a reclassification of adverse outcome risk in the Maastricht cohort (NRI = 18.7% p = 0.03; IDI = 3.9% p = 0.02) and with the Atlanta/Toulouse patients (NRI of 40.4% p = 0.01, 31,2% p = 0.04, 31.5% p = 0,02 and IDI of 2,9% p = 0,0005, 3.1% p = 0,0005 and 4,2%, p = 0.0005, for a follow-up of 1, 2 and 3 years, respectively). CONCLUSION: In 3 international cohorts, IGFBP2 level is a strong prognostic factor for cardiovascular mortality in HF, adding information to natriuretic monitoring and usual clinical markers, that should be further prospectively evaluated for patients' optimized care.

Insulin like growth factor binding protein 2 (IGFBP-2) for risk prediction in patients with severe aortic stenosis undergoing Transcatheter Aortic Valve Implantation (TAVI).

INTRODUCTION: Severe aortic stenosis (AS) caused by degenerative calcification is the most frequent acquired valvular heart disease worldwide and mortality rates are considerably high. Transcatheter Aortic Valve Implantation (TAVI) is a well-established method for valve replacement in high risk patients with AS. However, there is a lack of reliable predictors for patients undergoing TAVI since commonly used scores were developed for surgical populations. MATERIALS AND METHODS: 208 patients subjected to TAVI were included in this study. Plasma samples were obtained before TAVI and were evaluated for IGFBP-2 using commercially available ELISA kits. IGFBP-2 levels were analyzed for their ability for risk prediction after TAVI. RESULTS: IGFBP-2 levels measured before TAVI correlated significantly with left ventricular ejection fraction, EUROSCORE and other functional and prognostic parameters like the 6-minute walking test. When patients were retrospectively divided in two groups with a cut-off of serum IGFBP-2 levels of 275 ng/ml, IGFBP-2 was a strong predictor for 30-day and one-year mortality (3% vs. 11%, p = 0.05 and 18.2% vs. 46.2%; p < 0.001 respectively). Compared to an EUROSCORE above 20 or an STS score cut-off above 8, IGFBP-2 plasma levels above 275 ng/ml outperformed the established risk score for prediction of one-year mortality as assessed by NRI (0.65 95% CI 0.37-0.94; p < 0.001 and 0.54 95% CI 0.25-0.82; p < 0.001, respectively). CONCLUSIONS: Our results indicate that IGFBP-2 could serve as new outcome predictor for patients undergoing TAVI procedure. By providing additional information to the commonly used EUROSCORE, IGFPB-2 analysis could further assist Heart Team decision making.

Adrenomedullin inhibits adipogenesis under transcriptional control of insulin.

We generated preadipocyte cell lines impaired in adrenomedullin production through integration of an adrenomedullin small interfering RNA expression vector. The reduction of adrenomedullin synthesis strongly accelerated adipose differentiation. These results were bolstered when overexpression of active adrenomedullin peptide led to delayed differentiation. Therefore, we propose that adrenomedullin is an antiadipogenic factor. Moreover, we checked whether insulin, a proadipogenic factor, regulates expression of adrenomedullin. We observed that insulin had an inhibitory effect on adrenomedullin expression in isolated human adipocyte cells. This response was dose dependent and was reversed by resistin, a new anti-insulin agent. We quantified circulating adrenomedullin in healthy obese patients and observed a threefold increase of adrenomedullin compared with lean patients. Furthermore, adrenomedullin plasma levels are negatively correlated to plasma insulin levels in these obese patients. The insulin inhibitory response was also observed in vivo in Sprague-Dawley rats but not in the insulin-resistant Zucker rat, suggesting that adrenomedullin expression is upregulated in insulin-resistant adipose cells. Using adrenomedullin promoter-luciferase reporter gene constructs, we have shown that the adrenomedullin response to insulin is mediated by insulin-responsive elements. These findings provide new insight into fat mass development and the relationship between obesity and elevated circulating adrenomedullin levels in diabetic patients.

Paradoxical Effect of Nonalcoholic Red Wine Polyphenol Extract, Provinols™, in the Regulation of Cyclooxygenases in Vessels from Zucker Fatty Rats (fa/fa).

The aim of this work was to study the vascular effects of dietary supplementation of a nonalcoholic red wine polyphenol extract, Provinols, in Zucker fatty (ZF) obese rats. ZF or lean rats received diet supplemented or not with Provinols for 8 weeks. Vasoconstriction in response to phenylephrine (Phe) was then assessed in small mesenteric arteries (SMA) and the aorta with emphasis on the contribution of cyclooxygenases (COX). Although no difference in vasoconstriction was observed between ZF and lean rats both in SMA and the aorta, Provinols affected the contribution of COX-derived vasoconstrictor agents. The nonselective COX inhibitor, indomethacin, reduced vasoconstriction in vessels from both groups; however, lower efficacy was observed in Provinols-treated rats. This was associated with a reduction in thromboxane-A2 and 8-isoprostane release. The selective COX-2 inhibitor, NS398, reduced to the same extent vasoconstriction in aortas from ZF and Provinols-treated ZF rats. However, NS398 reduced response to Phe only in SMA from ZF rats. This was associated with a reduction in 8-isoprostane and prostaglandin-E release. Paradoxically, Provinols decreased COX-2 expression in the aorta, while it increased its expression in SMA. We provide here evidence of a subtle and paradoxical regulation of COX pathway by Provinols vessels from obese rats to maintain vascular tone within a physiological range.

The vasoactive peptide adrenomedullin is secreted by adipocytes and inhibits lipolysis through NO-mediated beta-adrenergic agonist oxidation.

Adipocytes are known to secrete a number of adipokines, but many adipocyte secretions and their functional importance remain to be characterized. This work shows that human white adipocytes and 3T3-F442A-derived adipocytes produce adrenomedullin (AM) and that AM acts in an autocrine/paracrine way on lipid metabolism by extracellular inactivation of isoproterenol, a beta-adrenergic agonist. AM is described as a counter-regulatory factor involved in the control of cardiovascular homeostasis. This peptide is believed to protect the heart from several complications implicated in obesity-linked cardiomorbidity, such as arterial hypertension, cardiac fibrosis, and decreased sinusal variability. The exact source of circulating AM remains a matter of debate, although endothelial and vascular smooth muscle cells seem to be important sites of production. We show that human adipose cells and 3T3-F442A-derived adipocytes express AM receptors and secrete AM. The function of this feature was investigated in 3T3-F442A cell line at the level of lipolysis regulation. AM inhibited beta-adrenergic-stimulated lipolysis by a nitric oxide (NO)-dependent mechanism, inducing a significant decrease in pD2 value for isoproterenol (8.6 +/- 0.2 vs. 9.8 +/- 0.1, P<0.001). This effect is cGMP-independent since it occurred in the presence of the NO-sensitive guanylate cyclase inhibitor ODQ. It is apparently mediated by a novel extracellular mechanism. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrated that AM-produced NO oxidized isoproterenol to generate its aminochrome, namely isoprenochrome. Isoprenochrome amounts were increased 3.62 +/- 1.13-fold in cell culture media (P<0.05). We describe for the first time that AM down-regulates lipolysis in adipocytes through the chemical modification of a beta-agonist.

Increased mean aliphatic lipid chain length in left ventricular hypertrophy secondary to arterial hypertension: A cross-sectional study.

About 77.9 million (1 in 4) American adults have high blood pressure. High blood pressure is the primary cause of left ventricular hypertrophy (LVH), which represents a strong predictor of future heart failure and cardiovascular mortality. Previous studies have shown an altered metabolic profile in hypertensive patients with LVH. The goal of this study was to identify blood metabolomic LVH biomarkers by H NMR to provide novel diagnostic tools for rapid LVH detection in populations of hypertensive individuals. This cross-sectional study included 48 hypertensive patients with LVH matched with 48 hypertensive patients with normal LV size, and 24 healthy controls. Two-dimensional targeted M-mode echocardiography was performed to measure left ventricular mass index. Partial least squares discriminant analysis was used for the multivariate analysis of the H NMR spectral data. From the H NMR-based metabolomic profiling, signals coming from methylene (-CH2-) and methyl (-CH3) moieties of aliphatic chains from plasma lipids were identified as discriminant variables. The -CH2-/-CH3 ratio, an indicator of the mean length of the aliphatic lipid chains, was significantly higher (P < 0.001) in the LVH group than in the hypertensive group without LVH and controls. Receiver operating characteristic curve showed that a cutoff of 2.34 provided a 52.08% sensitivity and 85.42% specificity for discriminating LVH (AUC = 0.703, P-value < 0.001). We propose the -CH2-/-CH3 ratio from plasma aliphatic lipid chains as a biomarker for the diagnosis of left ventricular remodeling in hypertension.

Transcriptional regulation of adipocyte hormone-sensitive lipase by glucose.

Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL mRNA was positively regulated by glucose in human adipocytes. Pools of stably transfected 3T3-F442A adipocytes were generated with human adipocyte HSL promoter fragments from -2,400/+38 to -31/+38 bp linked to the luciferase gene. A glucose-responsive region was mapped within the proximal promoter (-137 bp). Electromobility shift assays showed that upstream stimulatory factor (USF)-1 and USF2 and Sp1 and Sp3 bound to a consensus E-box and two GC-boxes in the -137-bp region. Cotransfection of the -137/+38 construct with USF1 and USF2 expression vectors produced enhanced luciferase activity. Moreover, HSL mRNA levels were decreased in USF1- and USF2-deficient mice. Site-directed mutagenesis of the HSL promoter showed that the GC-boxes, although contributing to basal promoter activity, were dispensable for glucose responsiveness. Mutation of the E-box led to decreased promoter activity and suppression of the glucose response. Analogs and metabolites were used to determine the signal metabolite of the glucose response. The signal is generated downstream of glucose-6-phosphate in the glycolytic pathway before the triose phosphate step.

Uncomplicated human obesity is associated with a specific cardiac transcriptome: involvement of the Wnt pathway.

A dramatic increase in obesity prevalence and cardiovascular morbidity is expected for the coming years. However, with relevance to the heart, little is known about the specific contribution of obesity on associated morbidity. Consequently, global analysis of gene regulations in human heart was undertaken to monitor molecular regulations related to obesity or to obesity-related hypertension. Transcriptome analysis using cDNA arrays was performed in right appendage biopsies from obese patients (n=5), from patients with arterial hypertension with (n=5) or without obesity (n=5), and from 5 leans. All biopsies came from patients that had cardiac surgery and coronary bypass. Statistical analysis of the data revealed 2686 differentially expressed genes out of 11,500 when compared with lean tissues. Differential expression was verified by real-time PCR in 84% of 50 randomly chosen genes. Among genes encountered, 397 were specifically regulated in obese, 1,299 in non-obese hypertensive, and 355 in obese hypertensive patients, respectively, whereas an additional set of 153 genes was differentially expressed in all these situations. Ontology analysis, hierarchical clustering, and molecular pathway analysis indicated that the heart molecular picture of obesity differs clearly from that observed for obesity-related hypertension or arterial hypertension. Clearly, the Wnt pathway known to be involved in cardiac hypertrophy mechanisms, showed opposite regulation in obese heart compared with hypertensive heart and potentially prevented the development of cardiac remodeling in obese patients. All over, this work shows that uncomplicated obesity has a strong impact on cardiac gene expression, which could be considered as precursor signs for future cardiac disease and also demonstrates that obesity-related hypertension generates a heart-molecular-distinct phenotype that cannot be predicted by a simple sum of the impact of obesity and arterial hypertension on gene expression.

Benefits of cardiac rehabilitation in heart failure patients according to etiology: INCARD French study.

We investigated the impact of heart failure (HF) etiology on the outcome of cardiac rehabilitation (CR) assessed by functional and clinical parameters. Treatment of chronic HF requires multidisciplinary approaches with a recognized role for CR. INCARD is a French study aimed at evaluating the benefits of sustainable CR in coronary (C) and noncoronary patients (NC) treated and educated during a 24-month period of follow-up. Prospective, monocentric patients with HF underwent inpatient physical training followed by a home-based program. Evaluations were performed at inclusion, discharge, 3 months after discharge, and subsequently every 6 months over the 24 months of outpatient rehabilitation.A total of 147 HF patients with left ventricular ejection fraction (LVEF) <40 were admitted to the CR center, 63 accepted to join INCARD (29 C and 34 NC). Although the C participants C having both an echocardiographic LVEF and an initially lower peak VO2, inpatient rehabilitation improved all functional parameters. Only NC showed an improved LVEF during the first 3 months of outpatient-follow-up. The main outcome of the outpatient rehabilitation was a trend toward stabilization of clinical and laboratory parameters with no significant difference between C and NC. This study confirms the benefits of initial HF inpatient rehabilitation and encourages prolonged outpatient monitoring. The results on functional parameters suggest exercise training should be conducted regardless of the HF etiology.

The prognostic value of plasma galectin-3 in chronic heart failure patients is maintained when treated with mineralocorticoid receptor antagonists.

OBJECTIVE: Galectin-3 (Gal-3) is considered as a myocardial fibrosis biomarker with prognostic value in heart failure (HF). Since aldosterone is a neurohormone with established fibrotic properties, we aimed to investigate if mineralocorticoid receptor antagonists (MRAs) would modulate the prognostic value of Gal-3. METHODS: The IBLOMAVED cohort comprised 427 eligible chronic HF patients (CHF) with echocardiography and heart failure biomarkers assessments (BNP). After propensity score matching CHF patients for cardiovascular risk factors, to form balanced groups, Gal-3 levels were measured at baseline in plasma from patients treated with MRAs (MRA-Plus, n=101) or not (MRA-Neg, n=101). The primary end point was all-cause mortality with a follow-up of 3 years. RESULTS: Gal-3 in plasma from these patients were similar with median values of 14.0 ng/mL [IQR, 9.9-19.3] and 14.4 ng/mL [IQR, 12.3-19.8] (P = 0.132) in MRA-Neg and MRA-Plus, respectively. Patients with Gal-3 ≤17.8 ng/mL had an HR of 1 (reference group) and 1.5 [0.4-5.7] in MRA-Neg and MRA-Plus, respectively (p=0.509). Patients with Gal-3 ≥ 17.8 ng/mL had an HR of 7.4 [2.2-24.6] and 9.0 [2.9-27.8] in MRA-Plus and MRA-Neg, respectively (p=0.539) and a median survival time of 2.4 years [95%CI,1.8-2.4]. Multivariate Cox proportional hazard analysis confirmed that MRA and the interaction term between MRA treatment and Gal-3 >17.8 ng/mL were not factors associated with survival. CONCLUSIONS: MRA treatment did not impair the prognostic value of Gal-3 assessed with a 17.8 ng/mL cut off. Gal-3 levels maintained its strong prognostic value in CHF also in patients treated with MRAs. The significance of the observed lack of an interaction between Gal-3 and treatment effect of MRAs remains to be elucidated.

Kinetic analysis of cardiac transcriptome regulation during chronic high-fat diet in dogs.

In the present study, we investigated, using custom dog cDNA arrays, the time course of transcriptional changes in the left ventricle of dogs fed a normal diet or a high-fat diet (HFD) for 9-24 wk. Array hybridizations were performed with complex probes representing mRNAs expressed in left ventricles from obese hypertensive and lean control dogs. We identified 63 differentially expressed genes, and expression of 17 of 20 randomly chosen genes was confirmed by real-time PCR. Transcripts were categorized into groups involved in metabolism, cell signaling, tissue remodeling, ionic regulation, cell proliferation, and protein synthesis. Hierarchical clustering indicated that the pattern of coregulated genes depends on duration of the HFD, suggesting that HFD-induced obesity hypertension is associated with continuous cardiac transcriptome adaptation despite stability of both body weight and blood pressure. GenMAPP analysis of the data pointed out the crucial importance of the ventricle TGF-beta pathway. Our results suggest that this system may be involved in molecular remodeling during HFD and in changes observed in the transcription profile, reflecting functional and morphological abnormalities that arise during prolonged HFD. These results also suggest some novel regulatory pathways for cardiac adaptation to obesity.

An interplay of Sp1, GKLF and CREB-2 controls human Pre-alpha-Inhibitor gene (ITIH3) transcription.

Pre-alpha-Inhibitor is a plasma protease inhibitor and a heterodimeric molecule whose one polypeptide chain is encoded by the ITIH3 gene. In order to understand the expression of this protein that is regulated in health and disease, we have analyzed the 5' flanking region of ITIH3, specifically focussing on its proximal promoter. A combination of methods including wild-type (wt) or mutant promoter linked to a reporter cat gene, co-transfections of cat constructs with expression plasmids for nuclear factors and electrophoretic mobility shift assays revealed that two antagonistic sets of regulatory elements and nuclear proteins are critical for the activity of this promoter. Indeed, several overlapping Sp1/Sp3-binding sites are required for a sustained activity. However, a tripartite complex including CREB-2 and two molecules of the gut-enriched, Krüppel-like factor cooperate to bind to an upstream area whose 3' end overlaps the Sp1-binding sites. The resulting competition between this tripartite complex and Sp1 results in impaired occupancy of Sp1-binding sites by Sp1 and a consequent reduction in ITIH3 transcription. Competition between Sp1 and a Krüppel-like factor for GC-rich sites has been previously reported, but this is the first description of an elaborate tripartite cooperation of two Krüppel-like factors and CREB as a key step in such a competition.

Adrenomedullin upregulates M2-muscarinic receptors in cardiomyocytes from P19 cell line.

1. The effects of AM on expression of muscarinic (M) receptors from P19-derived cardiomyocytes were examined. 2. RT-PCR experiments revealed expression of M(1)-M(4) receptor genes. Immuno-histochemistry indicated that M(2) expression is restricted to contractile cells. Carbachol inhibition of isoprenaline-induced increase in beating rate was prevented by atropine and methoctramine (pA(2): 8.1). Inhibition of [(3)H]-NMS binding by atropine (pK(i): -8.4+/-0.2) and methoctramine (pK(i): -8.3+/-0.2) suggests that M(2) is the functional expressed isoform. 3. [(3)H]-NMS binding and semiquantitative RT-PCR studies showed a dome shaped time course of M(2) expression with a maximum at 7 days of differentiation followed by a progressive decline. 4. AM concentration-dependently upregulated M(2) receptor mRNA during late differentiation stages in P19 cells but also in rat atrial cardiomyocytes. This effect was potentiated by factor H. AM (100 nM) plus factor H (50 nM) treatment of P19 cells for 24 h significantly increased [(3)H]-NMS-specific binding (B(max): 81+/-7 vs 31+/-6 fmol mg(-1) prot). The effect of AM on mRNA levels was prevented by AM receptor antagonist AM(22-52) (1 micro M) but not by CGRP antagonist, CGRP(8-37) (1 micro M). 5. The mRNA levels encoding CRLR receptor declined with culture duration, whereas those encoding L1/G10D receptor remained stable. 6. Our findings demonstrate that AM regulates M(2) receptors expression in cardiomyocytes probably through a mechanism involving L1/G10D receptors. The 'in vivo' significance of this phenomenon remains to be demonstrated.