The Initial Cardiac Tissue Response to Cryopreserved Allogeneic Adipose Tissue-Derived Mesenchymal Stromal Cells in Rats with Chronic Ischemic Cardiomyopathy.

Mesenchymal stromal cells have proven capable of improving cardiac pump function in patients with chronic heart failure, yet little is known about their mode of action. The aim of the study was to investigate the short-term effect of cryopreserved allogeneic rat adipose tissue-derived stromal cells (ASC) on cardiac composition, cellular subpopulations, and gene transcription in a rat model of chronic ischemic cardiomyopathy (ICM). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. After 6 weeks, the rats were treated with ASCs, saline, or no injection, using echo-guided trans-thoracic intramyocardial injections. The cardiac tissue was subsequently collected for analysis of cellular subpopulations and gene transcription 3 and 7 days after treatment. At day 3, an upregulation of genes associated with angiogenesis were present in the ASC group. On day 7, increases in CCR2+ and CD38+ macrophages (p = 0.047 and p = 0.021), as well as in the CD4/CD8 lymphocyte ratio (p = 0.021), were found in the ASC group compared to the saline group. This was supported by an upregulation of genes associated with monocytes/macrophages. In conclusion, ASC treatment initiated an immune response involving monocytes/macrophages and T-cells and induced a gene expression pattern associated with angiogenesis and monocyte/macrophage differentiation.

Processing-independent proANP measurement for low concentrations in plasma: reference intervals and effect of body mass index and plasma glucose.

BACKGROUND: Decreased concentrations of pro-atrial-derived natriuretic peptides (proABP) in plasma have been associated with obesity and suggested as a predictor of type 2 diabetes. However, assays for measuring proANP are generally aimed to quantitate higher concentrations of proANP associated with cardiac disease. Therefore, we aimed to measure plasma proANP concentrations in a non-obese Scandinavian reference material and evaluate potential associations of plasma proANP with body mass index (BMI) and plasma glucose, respectively. METHODS: We report an optimized processing-independent assay (PIA) for proANP in the lower concentration range. The assay was optimized by raising the amount of radioactive tracer and modifying the mixing ratio of resuspended plasma and buffer. Blood samples from a Scandinavian plasma cohort of 693 healthy subjects were then analyzed and age and gender-specific reference intervals were determined. RESULTS: Simple linear regression analyses of proANP and both BMI and plasma glucose in fasting subjects displayed insignificant associations. Multiple regression analyses supported these findings. However, a higher median plasma concentration of proANP was noted among women <50 years compared to men, whereas no gender-specific differences were seen in other age groups. CONCLUSIONS: Our results show that in a healthy non-obese population, BMI and plasma glucose in fasting subjects do not affect plasma proANP concentrations. Our method should be considered for future studies on low proANP concentration studies, e.g. in obesity and diabetes.

[68Ga]Ga-NODAGA-E[(cRGDyK)]2 and [64Cu]Cu-DOTATATE PET Predict Improvement in Ischemic Cardiomyopathy.

An increasing number of patients are living with chronic ischemic cardiomyopathy (ICM) and/or heart failure. Treatment options and prognostic tools are lacking for many of these patients. Our aim was to investigate the prognostic value of imaging angiogenesis and macrophage activation via positron emission tomography (PET) in terms of functional improvement after cell therapy. Myocardial infarction was induced in rats. Animals were scanned with [18F]FDG PET and echocardiography after four weeks and randomized to allogeneic adipose tissue-derived stromal cells (ASCs, n = 18) or saline (n = 9). Angiogenesis and macrophage activation were assessed before and after treatment by [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE. There was no overall effect of the treatment. Rats that improved left ventricular ejection fraction (LVEF) had higher uptake of both [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE at follow-up (p = 0.006 and p = 0.008, respectively). The uptake of the two tracers correlated with each other (r = 0.683, p = 0.003 pre-treatment and r = 0.666, p = 0.004 post-treatment). SUVmax at follow-up could predict improvement in LVEF (p = 0.016 for [68Ga]Ga-RGD and p = 0.045 for [64Cu]Cu-DOTATATE). High uptake of [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE PET after injection of ASCs or saline preceded improvement in LVEF. The use of these tracers could improve the monitoring of heart failure patients in treatment.

N-terminal pro-atrial natriuretic peptide measurement in plasma suggests covalent modification.

BACKGROUND: The N-terminal fragment of cardiac-derived pro-B-type natriuretic peptide is a glycosylated polypeptide. It is unknown whether N-terminal pro-atrial natriuretic peptide (proANP) fragments are also covalently modified. We therefore evaluated the clinical performance of 2 distinctly different proANP assays on clinical outcome. METHODS: We examined 474 elderly patients with symptoms of heart failure presenting in a primary healthcare setting. Samples were analyzed with an automated immunoluminometric midregion proANP (MR-proANP) assay and a new processing-independent assay (PIA) developed in our laboratory. The results were compared with Bland-Altman plots, and clinical performance was assessed by generating ROC curves for different clinical outcomes. RESULTS: Despite linear regression results indicating a good correlation (r = 0.85; P < 0.0001), the PIA measured considerably more proANP than the MR-proANP assay (mean difference, 663 pmol/L; SD, 478 pmol/L). In contrast, the clinical performances of the 2 assays [as assessed by the area under the ROC curve (AUC)] in detecting left ventricular dysfunction were similar [proANP PIA, 0.71 (95% CI, 0.63-0.79); MR-proANP assay, 0.74 (95% CI, 0.66-0.81); P = 0.32]. The prognostic ability to report cardiovascular mortality during a 10-year follow-up revealed AUC values of 0.66 (95% CI, 0.60-0.71) for the proANP PIA and 0.69 (95% CI, 0.63-0.74) for the MR-proANP assay (P = 0.08, for comparing the 2 assays). CONCLUSIONS: Our data suggest that N-terminal proANP fragments in patient plasma differ from the calibrator peptides used but that the difference does not affect ROC curves in an elderly cohort of patients with mild to moderate heart failure. We suggest that human N-terminal proANP fragments can be covalently modified.

Effect of insulin on natriuretic peptide gene expression in porcine heart.

Gut hormones affect cardiac function and contractility. In this study, we examined whether insulin affects the cardiac atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression and release of proANP-derived peptides in pigs. Anaesthetized pigs were included in an experimental study comparing the effect of hyperinsulinemia in 15 pigs submitted to two different protocols versus 11 control pigs receiving saline infusion. Phosphorylation of Akt on Thr308 was determined by western blotting with a pAkt-Thr308 antibody. The mRNA contents of ANP and BNP were determined with real-time PCR; plasma and cardiac tissue proANP was measured with an immunoluminometric assay targeted against the mid-region of the propeptide and a processing-independent assay. Insulin stimulation increased phosphorylation of Akt Thr308 in both left atrium and left ventricle of porcine hearts (p < 0.005). No change was observed in ANP and BNP mRNA contents in the right or left atrium. BNP mRNA contents in the left ventricle, however, decreased 3-fold (p = 0.02) compared to control animals, whereas the BNP mRNA content in the right ventricle as well as ANP mRNA content in the right and left ventricle did not change following hyperinsulinemia. Moreover, the peptide contents did not change in the four cardiac chambers. Finally, proANP concentrations in plasma did not change during the insulin infusion compared to the control animals. These results suggest that insulin does not have direct effect on atrial natriuretic peptide expression but may have a role in the left ventricle.

Cardiac procholecystokinin expression during haemodynamic changes in the mammalian heart.

Cardiac myocytes express the cholecystokinin gene (CCK) at propeptide level. We recently reported that cardiac CCK expression is acutely regulated by isoprenaline in a porcine model. The regulation of CCK expression after myocardial infarction, in exercise, and in severe heart failure is, however, unknown. Cardiac tissue was obtained from healthy new-born and adolescent farm pigs. Myocardial infarction was induced by coronary artery occlusion in adult minipigs. Healthy male subjects performed a 3-hour exercise test, and patients with severe heart failure referred for right heart catheterization were included. Extracts of porcine cardiac tissue and human plasma were analysed with specific proCCK radioimmunoassays. Cardiac proCCK expression shifted from the right atrium in new-born piglets to include the left atrium in adolescent pigs. Regional proCCK expression in the adolescent pig heart was mainly confined to the atria without different expression in sinus node tissue. In adult minipigs with myocardial infarction, no changes in overall left ventricular function or proCCK expression were observed after 8 weeks. In healthy adults, proCCK in circulation increased markedly during exercise in parallel with pro-B-type natriuretic peptide. Finally, patients with severe heart failure displayed markedly increased proCCK - but not CCK - concentrations in plasma. Taken together, our data shows that regional proCCK expression reflects haemodynamic changes in the mammalian heart. The data supports the notion that cardiac CCK expression resembles that of cardiac natriuretic peptides in atria. The ventricular content of proCCK, however, differs from natriuretic peptides and suggests a distinct secretory pathway in ventricular cardiomyocytes.

Cardiac Dysfunction in a Porcine Model of Pediatric Malnutrition.

BACKGROUND: Half a million children die annually of severe acute malnutrition and cardiac dysfunction may contribute to the mortality. However, cardiac function remains poorly examined in cases of severe acute malnutrition. OBJECTIVE: To determine malnutrition-induced echocardiographic disturbances and longitudinal changes in plasma pro-atrial natriuretic peptide and cardiac troponin-T in a pediatric porcine model. METHODS AND RESULTS: Five-week old piglets (Duroc-x-Danish Landrace-x-Yorkshire) were fed a nutritionally inadequate maize-flour diet to induce malnutrition (MAIZE, n = 12) or a reference diet (AGE-REF, n = 12) for 7 weeks. Outcomes were compared to a weight-matched reference group (WEIGHT-REF, n = 8). Pro-atrial natriuretic peptide and cardiac troponin-T were measured weekly. Plasma pro-atrial natriuretic peptide decreased in both MAIZE and AGE-REF during the first 3 weeks but increased markedly in MAIZE relative to AGE-REF during week 5-7 (p ≤ 0.001). There was overall no difference in plasma cardiac troponin-T between groups. However, further analysis revealed that release of cardiac troponin-T in plasma was more frequent in AGE-REF compared with MAIZE (OR: 4.8; 95%CI: 1.2-19.7; p = 0.03). However, when release occurred, cardiac troponin-T concentration was 6.9-fold higher (95%CI: 3.0-15.9; p < 0.001) in MAIZE compared to AGE-REF. At week 7, the mean body weight in MAIZE was lower than AGE-REF (8.3 vs 32.4 kg, p < 0.001), whereas heart-weight relative to body-weight was similar across the three groups. The myocardial performance index was 86% higher in MAIZE vs AGE-REF (p < 0.001) and 27% higher in MAIZE vs WEIGHT-REF (p = 0.025). CONCLUSIONS: Malnutrition associates with cardiac dysfunction in a pediatric porcine model by increased myocardial performance index and pro-atrial natriuretic peptide and it associates with cardiac injury by elevated cardiac troponin-T. Clinical studies are needed to see if the same applies for children suffering from malnutrition.

Atrial natriuretic peptides in plasma.

Measurement of cardiac natriuretic peptides in plasma has gained a diagnostic role in the assessment of heart failure. Plasma measurement is though hampered by the marked instability of the hormones, which has led to the development of analyses that target N-terminal fragments from the prohormone. These fragments are stable in plasma and represent surrogate markers of the actual natriuretic hormone. Post-translational processing of the precursors, however, is revealing itself to be a complex event with new information still being reported on proteolysis, covalent modifications, and amino acid derivatizations. In this mini-review, we summarize measurement of the principal cardiac hormone, e.g. atrial natriuretic peptide (ANP) and its precursor fragments. We also highlight some of the analytical pitfalls and problems and the concurrent clinical "proof of concept". We conclude that biochemical research into proANP-derived peptides is still worthy of attention and that new biological insight may change our chemical perception of the markers.

Natriuretic peptides in cardiometabolic regulation and disease.

In the 30 years since the identification of the natriuretic peptides, their involvement in regulating fluid and blood pressure has become firmly established. Data indicating a role for these hormones in lifestyle-related metabolic and cardiovascular disorders have also accumulated over the past decade. Dysregulation of the natriuretic peptide system has been associated with obesity, glucose intolerance, type 2 diabetes mellitus, and essential hypertension. Moreover, the natriuretic peptides have been implicated in the protection against atherosclerosis, thrombosis, and myocardial ischaemia. All these conditions can coexist and potentially lead to heart failure, a syndrome associated with a functional natriuretic peptide deficiency despite high circulating concentrations of immunoreactive peptides. Therefore, dysregulation of the natriuretic peptide system, a 'natriuretic handicap', might be an important factor in the initiation and progression of metabolic dysfunction and its accompanying cardiovascular complications. This Review provides a summary of the natriuretic peptide system and its involvement in these cardiometabolic conditions. We propose that these peptides might have an integrating role in lifestyle-related metabolic and cardiovascular disorders.

ProANP plasma measurement predicts all-cause mortality in acutely hospitalised patients: a cohort study.

IMPORTANCE: The association of natriuretic peptide measurement with all-cause mortality in a broad selection of acutely admitted patients has not yet been examined. OBJECTIVE: To test the risk association between pro-atrial natriuretic peptide (ANP) and short-term and long-term mortality and its predictive value in acutely hospitalised patients and compare this to N-terminal B-type natriuretic peptide (NT-proBNP). DESIGN, SETTING AND PATIENTS: Participants were selected from the Copenhagen Hospital Heart Failure Study (n=3644). Medical history, satisfactory echocardiography and blood samples were available on 2193 participants in 1998-1999 where NT-proBNP was measured. Vital status after discharge was obtained from national central data registers. A total of 1337 participants with eligible blood samples were selected in 2010-2011 for proANP measurement. Among these, 1255 (94%) were acutely hospitalised in 1998-1999. MAIN OUTCOME MEASURE(S): 1-year and long-term mortality. RESULTS: Median follow-up period was 11.5 years. At the end of follow-up, 926 patients had died, 239 during the first year. ProANP quartiles to 2-4 (median proANP levels 594 pmol/L, 990 pmol/L and 2052 pmol/L, respectively) associated with a stepwise increase in risk of 1-year and long-term mortality compared to the first quartile (336 pmol/L) in multivariable adjusted Cox proportional regression models (HR 1.53 95% CI 1.30 to 1.81 and HR 1.26 95% CI 1.17 to 1.36, respectively). An addition of NT-proBNP attenuated proANP's association with mortality in the models (HR 1.24 95% CI 1.01 to 1.53 and 1.14 95% CI 1.03 to 1.26, respectively). The increased risk was observed in participants with the highest proANP levels (fourth quartile). Similar results were observed in subgroups of participants with no evidence of cardiovascular disease (CVD). ProANP in quartiles improved discrimination when added to traditional risk factors in prediction models for 1-year (integrated discrimination improvement (IDI) 0.141 95% CI 0.085 to 0.197; C-index 0.753 95% CI 0.724 to 0.783, P for improvement 0.003) and long-term mortality (IDI 0.053 95% CI 0.032 to 0.074; C-index 0.736 95% CI 0.720 to 0.752, P for improvement <0.001) with similar results in subgroups. Discrimination was best in a combined model with proANP as well as NT-proBNP included. CONCLUSIONS AND RELEVANCE: High plasma proANP concentrations are associated with and predict short-term and long-term all-cause mortality in acutely hospitalised patients irrespective of CVD status at admission.

Next generation natriuretic peptide measurement.

Plasma measurement of natriuretic peptides is a "must" for clinical laboratories. For the next generation measurement, the unraveling of the molecular complexity of the peptides points toward a more qualitative assessment, as the posttranslational processing also changes with disease. Changes in the molecular heterogeneity could in itself contain valuable information of clinical status, and the time seems right for industry and dedicated researchers in the field to get together and discuss the next generation natriuretic peptide measurement. In such an environment, new strategies can be developed with the mutual aim of making already very good plasma markers even better.

Processing-independent analysis of peptide hormones and prohormones in plasma.

Peptide hormones are post-translationally matured before they reach a structure in which they can fulfill their biological functions. The prohormone processing may encompass a variety of endoproteolytic cleavages, N- and C-terminal trimmings, and amino acid derivatizations. The same prohormone can be variably processed in different cell types and, in addition, diseased cells often change the processing of a given precursor. The translational process is often either increased or decreased in diseased cells, which renders the ensuing modifications of the prohormone incomplete. Consequently, a variable mixture of precursors and processing-intermediates accumulates in plasma. In order to exploit disturbed posttranslational processing for diagnostic use and at the same time provide an accurate measure of the translational product, a simple analytical principle named "processing-independent analysis" (PIA) was designed. PIA-methods quantitate the total mRNA product irrespective of the degree of processing. PIA-methods have now been developed for a number of prohormones and proteins, and their diagnostic potential appears promising in diagnosis of cardiovascular disease and in several malignancies.

Measurement of the total proANP product in mammals by processing independent analysis.

The cellular processing of natriuretic propeptides is attenuated in heart disease, resulting in release of a mixture of unprocessed precursor, partially processed fragments, and the bioactive hormone. Here, we report a species-independent method for quantification of pro-atrial natriuretic peptide (proANP) and its products irrespective of variable post-translational processing. The processing-independent assay (PIA) was developed raising mono-specific antibodies against the C-terminus of sequence 1-16 in proANP. The assay procedure included plasma extraction followed by tryptic cleavage, which releases the assay epitope from the N-terminal region. The PIA was tested in elderly patients with symptoms of heart failure (n=450), in pigs with acute myocardial infarction (n=21), and in normal dogs and dogs with heart failure (n=77). The epitope specificity permitted reliable measurement in man, dog, cat and pig. In human plasma, the PIA correlated well with an established proANP analysis (r=0.86, P<0.0001) but with a 5.5-fold difference in plasma level (P<0.0001). In pigs, the PIA measured 9.2-fold higher concentrations compared to a human assay (804 versus 87pmol/L, P<0.0001). The basal proANP concentration was 396pmol/L in dogs: a dramatic increase was seen in canine heart failure. Our new processing- and species-independent proANP assay allows for the measurement of the total proANP product, irrespective of changes in post-translational maturation. We suggest that this tool should be used for comparative studies between human patients and porcine and canine models of human cardiac disease.