Comparison of three free T4 (FT4) and free T3 (FT3) immunoassays in healthy subjects and patients with thyroid diseases and severe non-thyroidal illnesses.

BACKGROUND: Free T4 (FT4) and free T3 (FT3) immunoassays exhibit wide inter-assay variations. We therefore established control values with three different immunoassays and compared their clinical performances in thyroidal diseases and severe/acute non-thyroidal illnesses (NTI). METHODS: The UniCel DxI 800, Architect i2000, and Elecsys 2010 assays were used. FT4 and FT3 reference ranges were established in 68 controls, without conditions interfering with thyroid function, with normal TSH (0.35-3.02 mU/L) and negative anti-thyroid peroxidase antibodies. Free hormones were determined in 60 patients with thyroid diseases (TSH: < 0.001-31.5 mU/L) and 45 NTI patients (TSH: 0.10-4.72 mU/L). Control values were normalized as Z-scores; patients' results were expressed as Z-scores, using the control values of each assay. Pairwise comparisons of the Z-scores were performed by Deming's regression. Classification of patients' results based on 95% control values were evaluated by kappa agreement statistics. RESULTS: Control values for FT4 (pmol/L; geometrical means; 95% confidence intervals) were: 11.1 (7.6-16.1), 12.3 (9.1-16.6), 15.6 (11.4-21.4) and for FT3: 4.8 (4.0-5.7), 4.0 (3.0-5.3), 4.3 (3.1-5.09), with DxI 800, Architect, and Elecsys, respectively. Pairs of control Z-scores correlated significantly, but with different strengths (FT4: r = 0.915, 0.740, 0.770; FT3: r = 0.615; 0.589; 0.790, for DxI 800 vs. Elecsys; DxI 800 vs. Architect; Elecsys vs. Architect; p < 0.001); slopes and intercepts of paired controls were 1.00 and zero. In thyroid diseases, slopes of FT4 Z-scores among assays differed slightly from 1.00 (1.11, 0.88, 0.87 for DxI versus Elecsys, DxI 800 versus Architect, Elecsys versus Architect, respectively; p < 0.05); slopes of FT3 Z-scores were consistent with unity, except for DxI 800 versus Elecsys (0.88; p < 0.05). In NTI patients, regression slopes were consistent with unity (p < 0.05). The agreement statistics showed moderate to very good inter-assay concordances for thyroid and NTI patients' results. CONCLUSIONS: FT4 and FT3 assays show moderate to very good agreement, in patients with thyroid diseases or NTI when compared pairwise to their control values. Slight quantitative differences between some pairs of assays are observed in thyroid diseases, after normalization as Z-scores.

C-terminal FGF23 is a strong predictor of survival in systolic heart failure.

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone involved in the regulation of phosphate and calcium metabolism. We have evaluated the levels of C-terminal FGF23 (Ct-FGF23) in 73 patients presenting heart failure with reduced ejection fraction (HF-REF) and assess their potential predictive value for long-term survival through a 6 years follow-up. Ct-FGF23 levels were markedly increased in HF-REF. In univariate proportional hazard model, survival was related to glomerular filtration rate (eGFR), intact parathyroid hormone (PTH), B-type natriuretic peptides (BNP) and Ct-FGF23. In a multivariate analysis including age, EF, PTH, BNP, Ct-FGF23, calcium, phosphorus and eGFR levels, Ct-FGF23 is the strongest predictor of long term CV death.

Impact of growth hormone (GH) treatment on circulating Nt-proBNP concentrations and on cardiac function in adult GH-deficient patients.

Severe adult growth hormone deficiency (AGHD) is associated with increased cardiovascular risk. We have therefore investigated levels of amino terminal pro-brain natriuretic peptide (Nt-proBNP), a well established biomarker for cardiac failure, in adult GHD patients before and after GH replacement therapy, and potential parallel variations in cardiac function. Nt-proBNP concentrations were determined at baseline and after GH treatment in two studies including 28 and 12 patients with severe AGHD, respectively. In the second study, a maximal exercise test and a doppler echocardiography were performed to assess cardiac functional parameters. At baseline, Nt-proBNP levels were higher in AGHD patients than in controls (median: 7.8 vs. 3.7 pmol/L; p < 0.01 in study 1; 8.4 vs. 4.1 pmol/L; p < 0.01 in study 2). Following GH treatment, Nt-proBNP levels decreased significantly in both studies. None of the AGHD patients had signs of cardiac dysfunction at baseline and no significant effect of GH replacement therapy was observed on cardiac functional parameters, independent of changes in Nt-proBNP. In conclusion, GH treatment markedly reduces Nt-proBNP concentrations in adult GHD patients without obvious parallel changes in cardiac functional parameters. These results suggest that Nt-proBNP may appear as a biomarker of GH status and GH treatment efficiency.

Urotensin II and urocortin trigger the expression of myostatin, a negative regulator of cardiac growth, in cardiomyocytes.

Urotensin II (UII) and urocortin (UCN) are potent contributors to the physiopathology of heart failure. Our study investigated the effects of UII and UCN on the expression of myostatin (Mstn) in primary culture of adult cardiomyocytes. Adult rat cardiomyocytes were stimulated for 48 h with UII and UCN. Cell size and protein content were determined. Mstn gene expression was determined by real time quantitative polymerase chain reaction. Treatment with UII and UCN stimulates hypertrophy of adult cardiomyocytes. This effect was associated with a twofold increase of Mstn gene expression. We have established for the first time that the two hypertrophic peptides UII and UCN stimulate the expression of Mstn.

Head-to-head comparison of the prohormone proBNP1-108 with BNP and Nt-proBNP in patients admitted to emergency department.

OBJECTIVES: B-type natriuretic peptide (BNP) and amino-terminal proBNP (Nt-proBNP) are derived from a common precursor, the proBNP(1-108) (proBNP), synthesized by cardiomyocytes. We determined proBNP concentrations in patients admitted to ED and suspected of CHF. DESIGN AND METHODS: One hundred fifty six consecutive patients admitted to ED were included. ProBNP, BNP and Nt-proBNP levels were determined at admission. RESULTS: In this ED population, assays for proBNP, BNP and Nt-proBNP were positively and significantly correlated. Circulating levels of proBNP were higher in patients admitted to ED for CHF than in patients admitted to ED other reasons. Applying receiver operating characteristic curve (ROC) analysis for the diagnosis of CHF, the area under the curve (AUC) was 0.92 for proBNP. CONCLUSIONS: Our study demonstrated that proBNP testing, the precursor of BNP and Nt-proBNP, appears as a relevant tool to assist the diagnosis of CHF in patients admitted to ED.

Urocortin-induced cardiomyocytes hypertrophy is associated with regulation of the GSK-3ß pathway.

Urocortin-1 (UCN), a member of the corticotropin-releasing factor, is a cardioprotective peptide, and is also involved in cardiac hypertrophy. The involvement of GSK-3ß, a pivotal kinase in cardiac hypertrophy, in response to UCN is not yet documented. Cardiomyocytes from adult rats were stimulated for 48 h with UCN. Cell size, protein, and DNA contents were determined. Phosphorylated and total forms GSK-3ß and the total amount of ß-catenin were quantified by Western immunoblots. The effects of astressin, a UCN competitive receptor antagonist, were also evaluated. UCN increased cell size and the protein-to-DNA ratio, in accordance with a hypertrophic response. This effect was associated with increased phosphorylation of GSK-3ß and marked accumulation of ß-catenin, a downstream element to GSK-3ß. All these effects were prevented by astressin and LY294002, an inhibitor of the phosphatidyl-inositol-3-kinase. UCN-induced cardiomyocytes hypertrophy is associated with regulation of GSK-3ß, a pivotal kinase involved in cardiac hypertrophy, in a PI3K-dependent manner. Furthermore, the pharmacological blockade of UCN receptors was able to prevent UCN-induced hypertrophy, which leads to inhibition of the Akt/GSK-3ß pathway.

Impact of vitamin D-related serum PTH reference values on the diagnosis of mild primary hyperparathyroidism, using bivariate calcium/PTH reference regions.

BACKGROUND, OBJECTIVE: An international consensus conference underlined the importance of defining upper parathyroid hormone (PTH) reference values based on 25-OH-vitamin D [25(OH)D] to diagnose mild primary hyperparathyroidism. We determined the importance of this factor in a Belgian population. DESIGN, PATIENTS, METHODS: Intact PTH and 25(OH)D were measured in 261 healthy controls (18-65 years, winter/summer). They were classified as 25(OH)D replete (50-153 nmol/l; n = 129) or deplete (8-50 nmol/l; n = 132). PTH was determined in 49 patients with surgically proven primary hyperparathyroidism. PTH thresholds for 95% specificities and corresponding sensitivities were computed from both 25(OH)D replete and deplete receiver operating characteristic (ROC) curves. The 95% bivariate reference ellipses, relating PTH to calcium for 25(OH)D replete and deplete controls, were compared to the PTH/calcium pairs of patients with primary hyperparathyroidism. RESULTS: Parathyroid hormone correlated with 25(OH)D (r = -0.3232; P < 0.0001). PTH normative values were 20% lower in 25(OH)D replete than deplete controls (P < 0.0001). PTH thresholds, providing 95% specificities for primary hyperparathyroidism diagnosis, were 7.6 pmol/l and 5.8 pmol/l, using ROC curves derived from 25(OH)D deplete or replete controls, respectively. Corresponding sensitivities were of 56%vs 88%, respectively (P < 0.05). The 95% PTH/calcium bivariate reference ellipses for?deplete and replete 25(OH)D controls differed, but the PTH/calcium pairs of patients with primary hyperparathyroidism did not overlap these ellipses. CONCLUSION: For a given specificity, primary hyperparathyroidism diagnostic parathyroid hormone thresholds were lower and sensitivities higher using ROC curves, derived from 25(OH)D replete vs deplete controls. The 25(OH)D status does not affect the efficiency of primary hyperparathyroidism diagnosis, using bivariate PTH/calcium reference density ellipses.

Biomarkers: optimizing treatment guidance in heart failure.

Heart failure is a frequent and life-threatening syndrome which is not only the result of myocardial injury or hemodynamic overload as commonly perceived, but appears to be the result of an interplay among genetic, neurohormonal, inflammatory, and biochemical factors, collectively referred to as biomarkers. Biomarkers can become risk factors in case their therapeutic modification results in an improvement of clinical outcomes. Among those markers identified in patients with heart failure, a number appears to have direct clinical relevance in aiding diagnosis, risk stratification, monitoring therapy, and treating to targets in order to improve clinical outcomes. These include brain natriuretic peptides (e.g., BNP, NT-proBNP), inflammatory markers (e.g., hsCRP), neurohormones (e.g., aldosterone), cardiorenal markers (e.g., cycstatin C), and novel markers (e.g., galectin-3). While their utility to indicate risk is mostly well established, there are less data to establish that a treatment using biomarkers as a guidance results in better outcomes than a more generalized intensified treatment of patients with heart failure. Future directions may involve larger platforms that facilitate to simultaneously analyze hundreds of biomarkers and may help to tailor heart failure therapy on a single patient basis, considering the specific pathogenesis and prognosis. Also from a therapeutic perspective there are data that a single intervention such as aldosterone blockade may affect multiple biomarkers at the same time. Taken together the data indicate that biomarkers are evolving into a valuable addendum to the diagnostic and therapeutic armamentarium.

Increased plasma myostatin in heart failure.

AIMS: Myostatin (Mstn), a member of the transforming growth factors (TGF)-ß family that regulate skeletal muscle growth, has been identified as a regulator of cardiomyocyte growth. The aim of our study was to measure Mstn plasma concentrations in patients with congestive heart failure (CHF) and to evaluate their relationship with other neurohormones released in CHF. METHODS AND RESULTS: concentrations of Mstn were measured using a competitive immunoassay, in 76 CHF patients who were receiving optimal treatment and 60 healthy controls. Circulating levels of other neurohormones N-terminal pro-atrial natriuretic peptide (NT-proANP), B-type natriuretic peptide (BNP), N-terminal pro-B-type natriuretic peptide (NT-proBNP), and Big ET-1 were also measured. Plasma Mstn was higher in CHF patients than in controls (63.0 vs. 43.0 ng/mL; P < 0.001). In CHF, Mstn levels correlated positively with NT-proANP (r=0.25; P=0.03), BNP (r=0.33; P<0.01), NT-proBNP (r=0.32; P<0.01), and Big ET-1 (r=0.26; P=0.02). No significant correlations were observed with age and creatinine. CONCLUSION: Our results demonstrate that plasma concentrations of Mstn are significantly increased in CHF patients and that Mstn correlates with biomarkers related to HF severity. Our study confirms the activation of Mstn in patients with heart failure.

Raised plasma urotensin II in type 2 diabetes patients is associated with the metabolic syndrome phenotype.

Urotensin II (UII) exerts multiple effects on the cardiovascular system, acts as a diabetogenic agent, and may also contribute to the development of the metabolic syndrome (MetS). The aim of this study was to determine circulating UII in patients with type 2 diabetes mellitus (T2DM) and its relationship with MetS. A total of 360 consecutive patients with T2DM were included. MetS presence/absence (MetS [+]/[-]) was defined according to American Heart Association/National Heart, Lung and Blood Institute criteria. Plasma concentrations of UII were determined by radioimmunoassay. UII levels were significantly higher in MetS (+) than in MetS (-) T2DM patients (0.97 pg/mL [0.93-1.01], n=294 vs 0.82 pg/mL [0.75-0.88] pg/mL, n=66, respectively; P<.001). Multiple logistic regression analysis showed that UII was significantly associated with MetS (+) (odds ratio, 6.41 [95% confidence interval, 1.21-16.04]; P=.02). UII plasma concentrations are significantly higher in T2DM patients presenting with MetS. Therefore, circulating UII may participate in the worsening course of some T2DM patients and may provide novel therapeutic perspectives.

Extracellular cardiac matrix biomarkers in patients with acute myocardial infarction complicated by left ventricular dysfunction and heart failure: insights from the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) study.

BACKGROUND: Aldosterone stimulates cardiac collagen synthesis. Circulating biomarkers of collagen turnover provide a useful tool for the assessment of cardiac remodeling in patients with congestive heart failure and left ventricular systolic dysfunction after acute myocardial infarction. METHODS AND RESULTS: In a substudy of the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS), which evaluated the effects of the selective aldosterone receptor antagonist eplerenone versus placebo, serum levels of collagen biomarkers were measured in 476 patients with congestive heart failure after acute myocardial infarction complicated with left ventricular systolic dysfunction. The combination of the type I collagen telopeptide and brain natriuretic peptide levels above median at baseline was associated with all-cause mortality and the composite end point of cardiovascular death or heart failure hospitalization, with hazard ratios of 2.49 (P=0.039) and 3.03 (P=0.002), respectively. During follow-up, levels of aminoterminal propeptide of type I and type III procollagen were found to be consistently lower in the eplerenone group and significantly lower beginning at 6 months. CONCLUSIONS: Changes in biomarkers of collagen synthesis and degradation suggest that extracellular matrix remodeling is an active process in patients with congestive heart failure and left ventricular systolic dysfunction after acute myocardial infarction. High type I collagen telopeptide and high brain natriuretic peptide serum levels are associated with the highest event rate. Eplerenone suppresses post-acute myocardial infarction collagen turnover changes.

Multimarker panel in patients admitted to emergency department: a comparison with reference methods.

OBJECTIVES: Point of care testing and multimarker panels are rapidly expanding in emergency departments. We determined the reliability of Short-of-Breath SOB panel in patients admitted for acute dyspnea and/or chest pain. DESIGN AND METHODS: SOB D-dimer, BNP, cTnI, CK-MB and myoglobin assays were compared with references in 97 outpatients. RESULTS: The correlation between SOB and references methods was acceptable, but with limited precision and accuracy. CONCLUSIONS: Diagnostic performances and cut-off values should be further validated before clinicians replace traditional cardio-respiratory biomarkers by the new SOB panel.

Accuracy of N-terminal-pro-atrial natriuretic peptide in patients admitted to emergency department.

OBJECTIVE: B-type natriuretic peptide (BNP) and N-terminal-pro-BNP (Nt-proBNP) are commonly used for the triage of patients in the emergency department (ED) with dyspnoea and/or chest pain. The aim of our study was to determine the accuracy of N-terminal-pro-ANP (Nt-proANP) in such patients. MATERIAL AND METHODS: Nt-proANP was measured by home-made radioimmunoassay in 137 ED patients admitted with cardiovascular and/or pulmonary disorders. BNP and Nt-pro-BNP were determined with automated assays. Final diagnosis was confirmed at discharge or after follow-up. RESULTS: Nt-proANP levels were significantly influenced by the diagnostic subgroups (ANOVA: p<0.001) and were [geometric mean (range)]: 19727 ng/L (5260-45200) in congestive heart failure (CHF, n=31), 6575 ng/L (1350-36000) in coronary artery disease (CAD, n=19), 5324 ng/L (1710-13150) in pulmonary embolism (PE, n=20), 5035 ng/L (1510-16600) in pulmonary diseases (PD, n=24) and 3001 ng/L (750-11860) in patients without cardiopulmonary diseases (n=43). Pairwise comparisons demonstrated that CHF patients had Nt-pro-ANP values higher than all other groups (p<0.05) and that patients without cardiopulmonary diseases had the lowest values (p<0.05). For diagnosis of CHF, the area under the ROC curve of Nt-proANP was 0.94 (95 % CI: 0.89-0.98) and was equivalent to Nt-proBNP (0.91; p=0.284) and BNP (0.93; p=0.572). CONCLUSIONS: The diagnostic accuracy of Nt-proANP was equivalent to BNP and Nt-proBNP in the present cohort of patients admitted to ED with dyspnoea and/or chest pain.

IGF-I does not prevent myotube atrophy caused by proinflammatory cytokines despite activation of Akt/Foxo and GSK-3beta pathways and inhibition of atrogin-1 mRNA.

Myofibrillar protein loss occurring in catabolic situations is considered to be mediated by the release of proinflammatory cytokines and associated with a decrease in circulating and muscle levels of insulin-like growth factor I (IGF-I). In this paper, we investigated whether the C(2)C(12) myotube atrophy caused in vitro by TNF-alpha/IFN-gamma cytokines might be reversed by exogenous IGF-I. Our results showed that, despite the presence of TNF-alpha/IFN-gamma, IGF-I retained its full ability to induce the phosphorylation of Akt, Foxo3a, and GSK-3beta (respectively, 16-fold, 9-fold, and 2-fold) together with a decrease in atrogin-1 mRNA (-39%, P < 0.001). Although this ubiquitin ligase has been reported to accelerate the degradation of MyoD, a myogenic transcription factor driving the transcription of myosin heavy chain (MHC), IGF-I failed to blunt the reduction of MyoD and MHC caused by TNF-alpha/IFN-gamma. Moreover, IGF-I only very slightly attenuated the myotube atrophy induced by TNF-alpha/IFN-gamma (TNF-alpha/IFN-gamma 15.48 mum alone vs. TNF-alpha/IFN-gamma/IGF-I 16.97 mum, P < 0.001). In conclusion, our data show that IGF-I does not reverse the myotube atrophy induced by TNF-alpha/IFN-gamma despite the phosphorylation of Foxo and GSK-3beta and the downregulation of atrogin-1 mRNA. Our study suggests therefore that factors other than IGF-I decrease are responsible for the muscle atrophy caused by proinflammatory cytokines.

Dipeptidyl-peptidase IV converts intact B-type natriuretic peptide into its des-SerPro form.

BACKGROUND: Analysis of plasma B-type natriuretic peptide (BNP) has suggested the in vivo formation of a truncated form, BNP (3-32), also called des-SerPro-BNP. The objectives of this study were to investigate (a) whether BNP and other natriuretic peptides are truncated by dipeptidyl-peptidase IV (DPP IV/CD26; EC 3.4.14.5) and (b) whether this truncation affects the susceptibility to cleavage by neutral endopeptidase (NEP; EC 3.4.24.11). METHODS: Human BNP (1-32), A-type natriuretic peptide 1-28 (ANP 1-28), and related peptides were incubated with purified DPP IV and with human plasma. In addition, BNP (1-32), BNP (3-32), and ANP (1-28) were subjected to hydrolysis by NEP. Cleavage products were analyzed by mass spectrometry. RESULTS: BNP (1-32) was cleaved by purified DPP IV with a specificity constant of 0.37 x 10(6) L.mol(-1).s(-1). The DPP IV activity in EDTA-plasma was able to truncate BNP (1-32) ex vivo. Addition of Vildagliptin, a specific DPP IV inhibitor, prevented this truncation in a concentration-dependent manner. Under in vitro circumstances in which ANP was hydrolyzed extensively, BNP (1-32) and BNP (3-32) were very resistant to NEP-mediated cleavage. CONCLUSIONS: DPP IV cleaves BNP (1-32) with an efficiency higher than or comparable to several known in vivo substrates of the enzyme. Even after loss of the amino-terminal dipeptide, BNP remains highly resistant to cleavage by NEP.

Respiratory-related heart rate variability in progressive experimental heart failure.

Heart failure is associated with autonomic imbalance, and this can be evaluated by a spectral analysis of heart rate variability. However, the time course of low-frequency (LF) and high-frequency (HF) heart rate variability changes, and their functional correlates during progression of the disease are not exactly known. Progressive heart failure was induced in 16 beagle dogs over a 7-wk period by rapid ventricular pacing. Spectral analysis of heart rate variability and respiration, echocardiography, hemodynamic measurements, plasma atrial natriuretic factor, and norepinephrine was obtained at baseline and every week, 30 min after pacing interruption. Progressive heart failure increased heart rate (from 91 +/- 4 to 136 +/- 5 beats/min; P < 0.001) and decreased absolute and normalized (percentage of total power) HF variability from week 1 and 2, respectively (P < 0.01). Absolute LF variability did not change during the study until it disappeared in two dogs at week 7 (P < 0.05). Normalized LF variability increased in moderate heart failure (P < 0.01), leading to an increased LF-to-HF ratio (P < 0.05), but decreased in severe heart failure (P < 0.044; week 7 vs. week 5). Stepwise regression analysis revealed that among heart rate variables, absolute HF variability was closely associated with wedge pressure, right atrial and pulmonary arterial pressure, left ventricular ejection fraction and volume, ratio of maximal velocity of early (E) and atrial (A) mitral flow waves, left atrial diameter, plasma norepinephrine, and atrial natriuretic peptide (0.45 < r < 0.65, all P < 0.001). In tachycardia-induced heart failure, absolute HF heart rate variability is a more reliable indicator of cardiac dysfunction and neurohumoral activation than LF heart rate variability.

Superiority of big endothelin-1 and endothelin-1 over natriuretic peptides in predicting survival in severe congestive heart failure: a 7-year follow-up study.

BACKGROUND: Plasma concentrations of atrial and brain natriuretic peptides (ANP, BNP), of their N-terminal pro-peptides, of endothelin-1 (ET-1), and big endothelin-1 (big ET-1) have diagnostic and prognostic significance in congestive heart failure (CHF). However, their respective values as a predictor of survival remain controversial and have never been directly compared in severe CHF. METHODS AND RESULTS: We analyzed, in 47 patients with severe CHF (New York Heart Association [NYHA] class III to IV; age 66 +/- 8 years, ejection fraction 20 +/- 6%), the prognostic performance of a panel of neurohormones and assays (N-terminal pro-ANP 1-25, 68-98 by radioimmunoassay [RIA], and 1-98 by enzyme-linked immunosorbent assay [ELISA], BNP by RIA and immunoradiometric assay [IRMA], N-terminal pro-BNP by Elisa, ET-1 by RIA, and big ET-1 by RIA and Elisa. Data were compared with 40 patients with mild to moderate CHF [NYHA I-II] and 30 healthy subjects. After a follow-up of 81 +/- 15 months, there were 34 deaths and 1 heart transplant. All neurohormones were significantly higher at baseline in patients with severe than in mild to moderate CHF or healthy subjects (all P < .001). Although all neurohormones but BNP IRMA were significant predictors of survival in univariate analysis, only big ET-1 RIA and ET-1 were independent predictors of survival (improvement chi(2): 7.5 and 4.6, P < .01 and P < .05). Using medians as cutpoints of big ET-1 RIA and ET-1, 2 severe CHF populations were defined with a different outcome (5-year survival: 55 versus 18%, P < .01). CONCLUSIONS: Big ET-1 and ET-1 are strong independent predictors of survival in patients with severe CHF and better for this purpose than natriuretic peptides or their pro-peptides. These markers allow easily to identify a population with a very high risk mortality eligible for more aggressive therapies.

Signaling molecules in overcirculation-induced pulmonary hypertension in piglets: effects of sildenafil therapy.

BACKGROUND: The phosphodiesterase type-5 (PDE-5) inhibitor sildenafil has been reported to improve pulmonary arterial hypertension (PAH), but the mechanisms that account for this effect are incompletely understood. Severe pulmonary hypertension has been characterized by defects in a signaling pathway involving angiopoietin-1 and the bone morphogenetic receptor-2 (BMPR-2). We investigated the effects of sildenafil on hemodynamics and signaling molecules in a piglet overcirculation-induced model of early PAH. METHODS AND RESULTS: Thirty 3-week-old piglets were randomized to placebo or sildenafil therapy 0.75 mg/kg TID after anastomosis of the left subclavian artery to the pulmonary arterial trunk or after a sham operation. Three months later, the animals underwent a hemodynamic evaluation followed by pulmonary tissue sampling for morphometry, immunohistochemistry or radioimmunoassay, and real-time quantitative-polymerase chain reaction. Chronic systemic-to-pulmonary shunting increased pulmonary mRNA for angiopoietin-1, endothelin-1 (ET-1), angiotensin II, inducible nitric oxide synthase, vascular endothelial growth factor, and PDE-5. Pulmonary messenger RNA for BMPR-1A and BMPR-2 decreased. Pulmonary angiotensin II, ET-1, and vascular endothelial growth factor proteins increased. Pulmonary artery pressure increased from 20+/-2 to 33+/-1 mm Hg, and arteriolar medial thickness increased by 91%. The expressions of angiopoietin-1, ET-1, and angiotensin II were tightly correlated to pulmonary hypertension. Sildenafil prevented the increase in pulmonary artery pressure, limited the increase in medial thickness to 41%, and corrected associated biological perturbations except for the angiopoietin-1/BMPR-2 pathway, PDE-5, and angiotensin II. CONCLUSIONS: Sildenafil partially prevents overcirculation-induced PAH and associated changes in signaling molecules. Angiotensin II, PDE-5, and angiopoietin-1/BMPR-2 signaling may play a dominant role in the early stages of the disease.

Role of the insulin-like growth factor I decline in the induction of atrogin-1/MAFbx during fasting and diabetes.

In catabolic conditions, atrogin-1/MAFbx, a muscle-specific ubiquitin-ligase required for muscle atrophy, is increased, and concentrations of IGF-I, a growth factor known to have antiproteolytic action, are reduced. To define the relationship between the decline in IGF-I and the induction of atrogin-1/MAFbx, we studied the effect of IGF-I replacement on atrogin-1/MAFbx mRNA in rats fasted for 51 h and in rats made diabetic with streptozotocin (STZ). Fasting produced a 5.8-fold increase in atrogin-1/MAFbx (P < 0.001). This was attenuated to a 2.5-fold increase by injections of IGF-I (P < 0.05 vs. fasting). Animals with STZ-induced diabetes experienced a 15.1-fold increase in atrogin-1/MAFbx (P < 0.001). Normalization of their circulating IGF-I concentrations by IGF-I infusion blunted the induction of atrogin-1/MAFbx to 6.3-fold (P < 0.05 vs. STZ diabetes without IGF-I). To further delineate the regulation of atrogin-1/MAFbx by IGF-I, we studied a model of cultured muscle cells. We observed that IGF-I produced a time- and dose-dependent reduction of atrogin-1/MAFbx mRNA, with a 50% effective dose of 5 nm IGF-I, a physiological concentration. The degradation rate of atrogin-1/MAFbx mRNA was not affected by IGF-I, suggesting that the reduction of atrogin-1/MAFbx mRNA by IGF-I is a transcriptional effect. Exposure of muscle cells in culture to dexamethasone increased atrogin-1/MAFbx mRNA with a 50% effective dose of 10 nm, a pharmacological concentration. In the presence of dexamethasone, IGF-I at physiological concentrations retained its full inhibitory effect on atrogin-1/MAFbx mRNA. We conclude that IGF-I inhibits atrogin-1/MAFbx expression and speculate that this effect might contribute to the antiproteolytic action of IGF-I in muscle.