T Cell-based RAS Activity and Insulin Levels in Obese Subjects with Low Grade Inflammation.

BACKGROUND: Obesity is a major contributor to inflammation and oxidative stress that are key underlying causes for insulin resistance (IR) and diabetes. Accumulated evidence suggest that RAS may serve as a strong link between IR and obesity. We investigated RAS activity in circulating T cells by obese subjects with and without angiotensin (Ang) II stimulation in presence or not of IR and of low-grade inflammation. METHODS: We studied 29 obese and 10 healthy subjects. After T-lymphocytes isolation, mRNAs for angiotensin converting enzyme (ACE) and angiotensin 1-receptor (AT1-R) were quantified by reverse transcription polymerase chain reaction (RT-PCR). High-sensitivity C-reactive protein (hs-CRP), insulin and inflammatory cytokines serum levels, plasma renin activity (PRA) and ACE activity in cell pellet and supernatant, and angiotensin (Ang) II T cell content were also measured. RESULTS: Under baseline conditions, RAS gene expressions, ACE activity and Ang II levels in T cells, but not PRA, of obese subjects with or without IR and with or without hs-CRP ≥3mg/dl were higher than in controls (p < 0.05). The increase in all parameters induced by Ang II was significantly higher in T cells from the obese subjects with hs-CRP ≥3 mg/dl than in controls or in the obese subjects with hs-CRP <3 mg/dl. In the obese subjects with low grade inflammation and IR, the cytokine serum levels and T cells RAS gene expression was inversely correlated with insulin serum concentration. CONCLUSIONS: Low grade inflammation amplifies the T cell RAS response to Ang II stimulation. T cell RAS gene expressions and serum levels of inflammatory cytokines were inversely related with insulin serum concentration. A protective role of insulin towards the development of inflammatory events can be hypothesized.

Multiple hormonal and metabolic deficiency syndrome in chronic heart failure: rationale, design, and demographic characteristics of the T.O.S.CA. Registry.

Recent evidence supports the concept that progression of chronic heart failure (CHF) depends upon an imbalance of catabolic forces over the anabolic drive. In this regard, multiple hormonal deficiency syndrome (MHDS) significantly has impacts upon CHF progression, and is associated with a worse clinical status and increased mortality. The T.O.S.CA. (Trattamento Ormonale nello Scompenso CArdiaco; Hormone Therapy in Heart Failure) Registry (clinicaltrial.gov = NCT02335801) tests the hypothesis that anabolic deficiencies reduce survival in a large population of mild-to-moderate CHF patients. The T.O.S.CA. Registry is a prospective multicenter observational study coordinated by "Federico II" University of Naples, and involves 19 centers situated throughout Italy. Thyroid hormones, insulin-like growth factor-1, total testosterone, dehydroepiandrosterone , and insulin are measured at baseline and every year for a patient-average follow-up of 3 years. Subjects with CHF are divided into two groups: patients with one or no anabolic deficiency, and patients with two or more anabolic deficiencies at baseline. The primary endpoint is the composite of all-cause mortality and cardiovascular hospitalization. Secondary endpoints include the composite of all-cause mortality and hospitalization, the composite of cardiovascular mortality and cardiovascular hospitalization, and change of VO2 peak. Patient enrollment started in April 2013, and was completed in July 2017. Demographics and main clinical characteristics of enrolled patients are provided in this article. Detailed cross-sectional results will be available in late 2018. The T.O.S.CA. Registry represents the most robust prospective observational trial on MHDS in the field of CHF. The study findings will advance our knowledge with regard to the intimate mechanisms of CHF progression and hopefully pave the way for future randomized clinical trials of single or multiple hormonal replacement therapies in CHF.

Relationship between hypertension, diabetes and proteinuria in rural and urban households in Yemen.

Little information is available on the meanings of proteinuria in low-resource settings. A population-based, cross-sectional survey was performed in Yemen on 10 242 subjects aged 15-69 years, stratified by age, gender and urban/rural residency. Hypertension is defined as systolic blood pressure (BP) of 140 mm Hg and/or diastolic BP of 90 mm Hg, and/or self-reported use of antihypertensive drugs; diabetes is diagnosed as fasting glucose of 126 mg dl(-1) or self-reported use of hypoglycaemic medications; proteinuria is defined as +1 at dipstick urinalysis. Odds ratios (ORs) for associations were determined by multivariable logistic regression models. Prevalence (weighted to the Yemen population aged 15-69 years) of hypertension, diabetes and proteinuria were 7.5, 3.7 and 5.1% in urban, and 7.8, 2.6 and 7.3% in rural locations, respectively. Proteinuria and hypertension were more prevalent among rural dwellers (adjusted ORs 1.56; 95% confidence limit (Cl) 1.31-1.86, and 1.23; 1.08-1.41, respectively), diabetes being less prevalent in rural areas (0.70; 0.58-0.85). Differently from hypertension and diabetes, proteinuria was inversely related with age. Most importantly, 4.6 and 6.1% of urban and rural dwellers, respectively, had proteinuria in the absence of hypertension and diabetes. The approach of considering kidney damage as a consequence of hypertension and diabetes might limit the effectiveness of prevention strategies in low-income countries.

The impact of fine-tuning of optical recognition system on database reliability.

Although optical reading systems are important tools to transfer data from a paper form to electronic databases, the impact of system fine-tuning on the final error rate is not usually considered. At the end of a multi-step process involving paper form design training of operators, and fine-tuning procedure, the final rate of error can be reduced from 0.65% to 0.05%. Fine-tuning should be introduced as a standard procedure while using optical reading systems.

Impairment of cardiopulmonary receptor sensitivity in the early phase of heart failure.

OBJECTIVES: To characterise the efficiency of the cardiopulmonary baroreflex system in the early phase of heart failure and its relation to limitation of physical activity. DESIGN: Forearm blood flow (venous occlusion plethysmography), vascular resistance, and central venous pressure (CVP), estimated from an antecubital vein, were measured in the supine position at baseline and 15 minutes after application of lower body negative pressure at -7 and -14 mm Hg (receptor downloading) or leg raising (receptor loading). SUBJECTS: Heart failure patients without limitation (NYHA class I; n = 18) or with slight limitation of physical activity (NYHA class II; n = 13), and 11 healthy controls. RESULTS: The efficiency of the cardiopulmonary baroreflex function, expressed by the slope of the relation between CVP changes and the corresponding changes of calculated forearm vascular resistance (gain), was reduced both in NYHA class I patients (mean (SD) -1.99 (0.83) v -2.78 (0.66) in controls; p < 0.05) and NYHA class II patients (-1.29 (0.5); p<0.001 v controls). However, change in peripheral vascular resistance during preload increase was similar in controls (-3.3 (0.9) units) and in NYHA class I patients (-3.3 (2.1) units; NS v controls), and was significantly reduced only in NYHA class II patients (-1.6 (1.3) units, p < 0.03 v controls). The gain in the cardiopulmonary reflex was related to the distance walked during the six minute corridor test. CONCLUSIONS: A reduced tonic efficacy of the cardiopulmonary reflex system is already detectable in the early phase of heart failure, the impairment in acute response to preload increase being detectable only in symptomatic patients.

Possible role of acylphosphatase, Bcl-2 and Fas/Fas-L system in the early changes of cardiac remodeling induced by volume overload.

To identify early adaptive processes of cardiac remodeling (CR) in response to volume overload, we investigated the molecular events that may link intracellular Ca(2+) homeostasis alterations and cardiomyocyte apoptosis. In swine heart subjected to aorto-cava shunt for 6, 12, 24, 48 and 96 h sarcoplasmic reticulum (SR) Ca(2+) pump activity was reduced until 48 h (-30%), but a recovery of control values was found at 96 h. The decrease in SR Ca(2+)-ATPase (SERCA2a) expression at 48 h, was more marked (-60%) and not relieved by a subsequent recovery, while phospholamban (PLB) concentration and phosphorylation were unchanged at all the considered times. Conversely, acylphosphatase activity and expression significantly increased from 48 to 96 h (+40%). Bcl-2 expression increased significantly from 6 to 24 h, but at 48 h, returned to control values. At 48 h, microscopic observations showed that overloaded myocardium underwent substantial damage and apoptotic cell death in concomitance with an enhanced Fas/Fas-L expression. At 96 h, apoptosis appeared attenuated, while Fas/Fas-L expression was still higher than control values and cardiomyocyte hypertrophy became to develop. These data suggest that in our experimental model, acylphosphatase could be involved in the recovery of SERCA2a activity, while cardiomyocyte apoptosis might be triggered by a decline in Bcl-2 expression and a concomitant activation of Fas.

Altered Cx43 expression during myocardial adaptation to acute and chronic volume overloading.

Gap-junctions are specialized regions of intercellular contacts allowing electrical impulse propagation among adjacent cardiomyocytes. Connexin43 (Cx43) is the predominant gap-junction protein in the working ventricular myocardium and its reduced expression has been extensively implicated in the genesis of conduction abnormalities and re-entry arrhythmia of chronically hypertrophied hearts. In contrast, data on the role played by this protein during cardiac remodeling and early phases of developing hypertrophy are lacking. Therefore, in the present study, we investigated this issue using an experimental model of pig left ventricle (LV) volume overloading consisting in the creation of an aorto-cava fistula. At scheduled times (6, 24, 48, 96, 168 h, and 2, 3 months after surgery) echocardiographic and haemodynamic measurements were performed and myocardial biopsies were taken for the morphological and biochemical analyses. When faced with the increased load, pig myocardium underwent an initial period (from 6 up to 48 h) of remarkable tissue remodeling consisting in the occurrence of cardiomyocyte damage and apoptosis. After that time, the tissue developed a hypertrophic response that was associated with early dynamic changes (up-regulation) in Cx43 protein expression, as demonstrated by Western blot and confocal immunofluorescence analyses. However, an initial transient increase of this protein was also found after 6 h from surgery. With the progression of LV hypertrophy (from 168 hr up to 3 months), a reduction in the myocardial Cx43 expression was, instead, observed. The increased expression of Cx43 protein during acute hypertrophic response was associated with a corresponding increase in the levels of its specific mRNA, as detected by RT-PCR. We concluded that up-regulation of Cx43 gap-junction protein could represent an immediate compensatory response to support the new working conditions in the early stages of ventricular overloading.

Biochemical changes and their relationship with morphological and functional findings in pig heart subjected to lasting volume overload: a possible role of acylphosphatase in the regulation of sarcoplasmic reticulum calcium pump.

We evaluated the changes in sarcoplasmic reticulum (SR) function and the parallel hemodynamic and morphological modifications in a heart subjected to volume overload. We also determined the levels of acylphosphatase, a cytosolic enzyme, that could play a regulatory effect on SR Ca(2+) pump by hydrolyzing the phosphorylated intermediate of this transport system. For this, swine hearts were subjected to volume overload by aorta-cava shunt for 1, 2, or 3 months. Changes in heart contractility reflected modifications of SR function, whose reduction after 1 month of overload was followed by a gradual recovery. A decrease in SERCA2a protein and mRNA content was shown from 1 month and remained for the following 2 months. Phospholamban content and its phosphorylation status were not modified. Acylphosphatase was unchanged at 1 month, but at 2 months this enzyme exhibited an increased activity, protein and mRNA expression. Morphological alterations consisting of the cytoskeletal architectures, intermyofibrillar oedema, swollen mitochondria and abnormality of the membrane system (T-tubule and SR cisternae) were particularly evident after 1 month but almost disappeared after 3 months. These results suggest that our overloaded hearts underwent a substantial recovery of their structural and biochemical properties at 3 months after surgery. A possible involvement of acylphosphatase in the modification of SR function is discussed.

Increased cardiac sympathetic activity and insulin-like growth factor-I formation are associated with physiological hypertrophy in athletes.

Physiological hypertrophy represents the adaptive changes of the heart required for supporting the increased hemodynamic load in regularly trained healthy subjects. Mechanisms responsible for the athlete's hypertrophy still remain unknown. In 15 trained competitive soccer players and in 15 healthy men not engaged in sporting activities (sedentary control subjects) of equivalent age, we investigated the relationship among cardiac growth factor formation, cardiac sympathetic activity, and left ventricular morphology and function. Cardiac formation of insulin-like growth factor (IGF)-I, endothelin (ET)-1, big ET-1, and angiotensin (Ang) II was investigated at rest by measuring artery-coronary sinus concentration gradients. Cardiac sympathetic activity was studied by [(3)H]norepinephrine (NE) kinetics. Cardiac IGF-I, but not ET-1, big ET-1, and Ang II, formation was higher in athletes than in control subjects (P<0.01). NE levels in arterial and peripheral venous blood did not differ between groups. In contrast, coronary sinus NE concentration was higher in athletes than in control subjects (P<0.01). Cardiac, but not total systemic, NE spillover was also increased in athletes (P<0.01), whereas cardiac [(3)H]NE reuptake and clearance were not different. Echocardiographic modifications indicated a volume overload-induced hypertrophy associated with increased myocardial contractility. Multivariate stepwise analysis selected left ventricular mass index as the most predictive independent variable for cardiac IGF-I formation and velocity of circumferential fiber shortening for cardiac NE spillover. In conclusion, increased cardiac IGF-I formation and enhanced sympathetic activity selectively confined to the heart appear to be responsible for the physiological hypertrophy in athletes performing predominantly isotonic exercise.

Activation of cardiac renin-angiotensin system in unstable angina.

OBJECTIVES: The aim of this study was to investigate the activity of the cardiac renin-angiotensin system (RAS) in unstable angina (UA). BACKGROUND: Angiotensin (Ang) II locally produced by continuously operating cardiac RAS may affect the pathophysiology of UA. METHODS: In 35 patients with UA, 32 with stable effort angina (SA) and 21 with atypical chest pain (controls), cardiac RAS was investigated during coronary angiography after five days of Holter monitoring by combining the measurement of aorta-coronary sinus gradient for Ang I and Ang II with the kinetics study of 125I-Ang I. Messenger RNAs (mRNA) for all the components of RAS were also quantified with the reverse transcriptase-polymerase chain reaction (RT-PCR) and localized by in situ hybridization in myocardial biopsy specimens from patients who underwent aorta-coronary bypass surgery. RESULTS: Cardiac Ang II generation was higher in patients with UA than it was in patients with SA or in controls (p < 0.001) due to increased de novo cardiac Ang I formation and its enhanced fractional conversion rate to Ang II. Messenger RNA levels for angiotensinogen (AGTN), angiotensin-converting enzyme (ACE) and Ang II type 1 (AT1) subtype receptors were higher in patients with UA (p < 0.01) than they were in patients with SA or in control hearts. Messenger RNAs for AGTN and ACE were almost exclusively expressed on endothelial and interstitial cells. Angiotensin II formation was correlated with ischemia burden (p < 0.001). However, the amount of Ang II formed and the expression levels of mRNAs for AGTN, ACE and AT1 were not related to the time that had elapsed since the last anginal attack. CONCLUSIONS: In patients with UA, cardiac RAS is activated, resulting in increased Ang II formation. Myocardial ischemia is essential for RAS activation, but it is unlikely to be a direct and immediate cause of RAS activation.

Cardiac angiotensin II formation in the clinical course of heart failure and its relationship with left ventricular function.

In 76 patients with heart failure (HF) (New York Heart Association [NYHA] classes I through IV) and in 15 control subjects, cardiac angiotensin II (Ang II) generation and its relationship with left ventricular function were investigated by measuring aorta-coronary sinus concentration gradients of endogenous angiotensins and in a part of patients by studying (125)I-labeled Ang I kinetics. Gene expression and cellular localization of the cardiac renin-angiotensin system components, the density of AT(1) and AT(2) on membranes and isolated myocytes, and the capacity of isolated myocytes for synthesizing the hypertrophying growth factors insulin-like growth factor-I (IGF-I) and endothelin (ET)-1 were also investigated on 22 HF explanted hearts (NYHA classes III and IV) and 7 nonfailing (NF) donor hearts. Ang II generation increased with progression of HF, and end-systolic wall stress was the only independent predictor of Ang II formation. Angiotensinogen and angiotensin-converting enzyme mRNA levels were elevated in HF hearts, whereas chymase levels were not, and mRNAs were almost exclusively expressed on nonmyocyte cells. Ang II was immunohistochemically detectable both on myocytes and interstitial cells. Binding studies showed that AT(1) density on failing myocytes did not differ from that of NF myocytes, with preserved AT(1)/AT(2) ratio. Conversely, AT(1) density was lower in failing membranes than in NF ones. Ang II induced IGF-I and ET-1 synthesis by isolated NF myocytes, whereas failing myocytes were unable to respond to Ang II stimulation. This study demonstrates that (1) the clinical course of HF is associated with progressive increase in cardiac Ang II formation, (2) AT(1) density does not change on failing myocytes, and (3) failing myocytes are unable to synthesize IGF-I and ET-1 in response to Ang II stimulation.

Renal endothelin in heart failure and its relation to sodium excretion.

BACKGROUND: Recent studies have shown that endothelin-1 (ET-1) antagonists increase sodium excretion and improve renal blood flow in experimental heart failure (HF). However, despite a number of investigations that have reported a significant increase in ET-1 plasma levels in patients with HF, it is still not known whether increased renal synthesis and urinary excretion of ET-1 occur. Our aim was to investigate renal ET-1 formation and its relation to sodium excretion in patients with HF. METHODS: One hundred forty-seven patients with HF, subdivided according to New York Heart Association (NYHA) functional classes, and 28 healthy controls were studied. ET-1 and big ET-1 were measured in plasma and in 24-hour urine by radioimmunoassay. Atrial and brain natriuretic peptide, arginine vasopressin, plasma renin activity, and hemodynamic variables were also investigated. RESULTS: Urinary ET-1 excretion was already increased in NYHA class II patients (P <.001 vs controls), whereas plasma ET-1 increased only in NYHA class III and IV patients (P <.001). In the 71 subjects who were not receiving diuretic treatment, urinary ET-1 was selected as the strongest predictor of sodium excretion by multivariate stepwise analysis. CONCLUSIONS: Urinary ET-1 excretion increases in an earlier phase of HF than plasma ET-1 and appears to be closely correlated with sodium excretion, indicating renal ET-1 is a target for ET-1 antagonists in patients with HF.

Early sequence of cardiac adaptations and growth factor formation in pressure- and volume-overload hypertrophy.

To investigate the time sequence of cardiac growth factor formation, echocardiographic and hemodynamic measurements were performed at scheduled times, and mRNAs for angiotensinogen, prepro-endothelin-1 (ppET-1), and insulin-like growth factor I (IGF-I) were quantified with RT-PCR and localized with in situ hybridization in pigs (fluothane anesthesia) by use of pressure or volume overload (aortic banding and aorta-cava fistula, respectively). Relative peptide formation was also measured by radioimmunoassay. In pressure overload, angiotensinogen and ppET-1 mRNA overexpression on myocytes (13 times vs. sham at 3 h and 112 times at 6 h, respectively) was followed by recovery (12 h) of initially decreased (0.5-6 h) myocardial contractility. In volume overload, contractility was not decreased, the angiotensinogen gene was slightly upregulated at 6 h (6.7 times), and ppET-1 was not overexpressed. IGF-I mRNA was overexpressed on myocytes (at 24 h) in both volume and pressure overload (14 times and 37 times, respectively). In the latter setting, a second ppET-1 overexpression was detectable on myocytes at 7 days. In conclusion, acute cardiac adaptation responses involve different growth factor activation over time in pressure versus volume overload; growth factors initially support myocardial contractility and thereafter induce myocardial hypertrophy.

Selective upregulation of cardiac endothelin system in patients with ischemic but not idiopathic dilated cardiomyopathy: endothelin-1 system in the human failing heart.

Only scarce information is available on the activity and modifications of the cardiac endothelin (ET)-1 system in heart failure due to ischemic (ICM) or idiopathic dilated (DCM) cardiomyopathy. The activity of the ET-1 system was investigated by measuring cardiac ET-1 and big ET-1 formation and quantifying cardiac mRNA for prepro-ET-1 (ppET-1), ET-converting enzyme-1, and ET(A) and ET(B) receptors both in myocardium and in isolated myocytes using Northern blot, reverse transcription-polymerase chain reaction, and in situ hybridization in 22 patients with DCM and 20 with ICM who underwent cardiac transplantation and in 7 potential heart transplant donors (nonfailing hearts). Notwithstanding a similar increase of plasma ET-1 in the 2 groups, cardiac ET formation, mRNA levels for ppET-1, and ET(A) and ET(B) receptors were higher on both the myocardium and isolated myocytes from ICM than on those from DCM hearts (P<0.001 for all). ppET-1 and ET-converting enzyme-1 mRNAs were expressed on myocytes and endothelial and interstitial cells in ICM, whereas in DCM and nonfailing hearts they were mainly expressed on nonmyocyte cells. In both ICM and DCM, the ET(A) mRNA signal was expressed on both myocytes and nonmyocyte cells, whereas ET(B) mRNA was almost exclusively localized on nonmyocyte cells. ET(A)- and ET(B)-specific receptor binding was increased on both myocytes and cardiac membranes, showing a positive correlation with left ventricular ejection fraction in ICM (r=0.78 and 0.70) but not in DCM patients. The present results show that human ventricular myocytes express all of the components of the ET-1 system, which is selectively upregulated in ICM patients and appears to be functionally important in the maintenance of cardiac function.

Endothelin-1 urinary excretion, but not endothelin-1 plasma concentration, is increased in renovascular hypertension.

Animal experiments have shown an increase in prepro-endothelin-1 (prepro-ET-1) mRNA expression in the clipped kidney but none in the aortic and mesenteric arteries in 2-kidney, 1-clip Goldblatt hypertensive rats. The present study was aimed at investigating whether plasma and renal endothelin-1 (ET-1) systems are differently activated in patients with renovascular hypertension (RH). The plasma concentration and urinary excretion of ET-1 were measured in 5 patients with RH (before and after successful renal angioplasty), in 7 patients with essential hypertension (EH), and in 8 normotensive control subjects. Immediately before renal angioplasty, plasma samples for ET-1 and plasma renin activity (PRA) measurements were withdrawn from the aorta and both renal veins. Unlike the PRA, the plasma ET-1 concentration did not significantly differ between the involved and the uninvolved sides. The urinary ET-1 excretion level (Fig 1) was markedly increased in patients with RH (30+/-4 ng/g urinary creatinine (UC) vs. 2.5+/-0.2 ng/g UC and 2.6+/-0.5 ng/g UC in control subjects and patients with EH, respectively; P<.001), whereas the plasma ET-1 concentration was normal (0.8+/-0.2 pg/mL vs. 0.65+/-0.3 pg/mL and 0.8+/-0.2 pg/mL in control subjects and EH, respectively, not significant). Renal angioplasty was followed in all patients by normalization of blood pressure and PRA. One week after angioplasty, urinary ET-1 decreased to one fourth of baseline (8.04+/-5.23 ng/g UC, P<.001 vs. values before angioplasty and P<.04 vs. control subjects) and normalized 1 month thereafter (3.13+/-1.62 ng/g UC, not significant vs. control subjects), whereas plasma ET-1 remained steady. The present findings clearly indicate that in patients with RH, urinary ET-1 excretion is increased, whereas plasma ET-1 concentration remains normal. Successful percutaneous transluminal renal angioplasty induced a notable reduction in ET-1 urinary excretion, whereas it did not affect ET-1 plasma concentration.