Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

Derivation and Validation of Thresholds Using Synthetic Data Methods for Single-Test Screening of Emergency Department Patients with Possible Acute Myocardial Infarction Using a Point-of-Care Troponin Assay.

BACKGROUND: Single-sample (screening) rule-out of acute myocardial infarction (AMI) with troponin requires derivation of a single-test screening threshold. In data sets with small event numbers, the lowest one or two concentrations of myocardial infarction (MI) patients dictate the threshold. This is not optimal. We aimed to demonstrate a process incorporating both real and synthetic data for deriving such thresholds using a novel pre-production high-precision point-of-care assay. METHODS: cTnI concentrations were measured from thawed plasma using the Troponin I Next (TnI-Nx) assay (i-STAT; Abbott) in adults on arrival to the emergency department with symptoms suggestive of AMI. The primary outcome was an AMI or cardiac death within 30 days. We used internal-external validation with synthetic data production based on clinical and demographic data, plus the measured TnI-Nx concentration, to derive and validate decision thresholds for TnI-Nx. The target low-risk threshold was a sensitivity of 99% and a high-risk threshold specificity of >95%. RESULTS: In total, 1356 patients were included, of whom 191 (14.1%) had the primary outcome. A total of 500 synthetic data sets were constructed. The mean low-risk threshold was determined to be 5 ng/L. This categorized 38% (95% CI, 6%-68%) to low-risk with a sensitivity of 99.0% (95% CI, 98.6%-99.5%) and a negative predictive value of 99.4% (95% CI, 97.6%-99.8%). A similarly derived high-risk threshold of 25 ng/L had a specificity of 95.0% (95% CI, 94.8%-95.1%) and a positive predictive value of 74.8% (95% CI, 71.5%-78.0%). CONCLUSIONS: With the TnI-Nx assay, we successfully demonstrated an approach using synthetic data generation to derive low-risk thresholds for safe and effective screening.

Distinguishing heart failure with reduced ejection fraction from heart failure with preserved ejection fraction: A phenomics approach.

AIM: Pathophysiological differences between patients with heart failure with preserved (HFpEF) and reduced (HFrEF) ejection fraction (EF) remain unclear. Therefore we used a phenomics approach, integrating selected proteomics data with patient characteristics and cardiac structural and functional parameters, to get insight into differential pathophysiological mechanisms and identify potential treatment targets. METHODS AND RESULTS: We report data from a representative subcohort of the prospective Singapore Heart Failure Outcomes and Phenotypes (SHOP), including patients with HFrEF (EF <40%, n = 217), HFpEF (EF ≥50%, n = 213), and age- and sex-matched controls without HF (n = 216). We measured 92 biomarkers using a proximity extension assay and assessed cardiac structure and function in all participants using echocardiography. We used multi-block projection to latent structure analysis to integrate clinical, echocardiographic, and biomarker variables. Candidate biomarker targets were cross-referenced with small-molecule and drug databases. The total cohort had a median age of 65 years (interquartile range 60-71), and 50% were women. Protein profiles strongly discriminated patients with HFrEF (area under the curve [AUC] = 0.89) and HFpEF (AUC = 0.94) from controls. Phenomics analyses identified unique druggable inflammatory markers in HFpEF from the tumour necrosis factor receptor superfamily (TNFRSF), which were positively associated with hypertension, diabetes, and increased posterior and relative wall thickness. In HFrEF, interleukin (IL)-8 and IL-6 were possible targets related to lower EF and worsening renal function. CONCLUSION: We identified pathophysiological mechanisms related to increased cardiac wall thickness parameters and potentially druggable inflammatory markers from the TNFRSF in HFpEF.

Multiple circulating forms of neprilysin detected with novel epitope-directed monoclonal antibodies.

Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1-8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57-60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.

Myeloperoxidase Inhibition Reverses Biomarker Profiles Associated With Clinical Outcomes in HFpEF.

BACKGROUND: Systemic microvascular dysfunction and inflammation are postulated to play a pathophysiologic role in heart failure with preserved ejection fraction (HFpEF). OBJECTIVES: This study aimed to identify biomarker profiles associated with clinical outcomes in HFpEF and investigate how inhibition of the neutrophil-derived reactive oxygen species-producing enzyme, myeloperoxidase, affects these biomarkers. METHODS: Using supervised principal component analyses, the investigators assessed the associations between baseline plasma proteomic Olink biomarkers and clinical outcomes in 3 independent observational HFpEF cohorts (n = 86, n = 216, and n = 242). These profiles were then compared with the biomarker profiles discriminating patients treated with active drug vs placebo in SATELLITE (Safety and Tolerability Study of AZD4831 in Patients With Heart Failure), a double-blind randomized 3-month trial evaluating safety and tolerability of the myeloperoxidase inhibitor AZD4831 in HFpEF (n = 41). Pathophysiological pathways were inferred from the biomarker profiles by interrogation of the Ingenuity Knowledge Database. RESULTS: TNF-R1, TRAIL-R2, GDF15, U-PAR, and ADM were the top individual biomarkers associated with heart failure hospitalization or death, and FABP4, HGF, RARRES2, CSTB, and FGF23 were associated with lower functional capacity and poorer quality of life. AZD4831 downregulated many markers (most significantly CDCP1, PRELP, CX3CL1, LIFR, VSIG2). There was remarkable consistency among pathways associated with clinical outcomes in the observational HFpEF cohorts, the top canonical pathways being associated with tumor microenvironments, wound healing signaling, and cardiac hypertrophy signaling. These pathways were predicted to be downregulated in AZD4831 relative to placebo-treated patients. CONCLUSIONS: Biomarker pathways that were most strongly associated with clinical outcomes were also the ones reduced by AZD4831. These results support the further investigation of myeloperoxidase inhibition in HFpEF.

Augmentation of Natriuretic Peptide Bioactivity via Combined Inhibition of Neprilysin and Phosphodiesterase-9 in Heart Failure.

BACKGROUND: The natriuretic peptides (NPs) are potent natriuretic/diuretic and vasodilatory factors, and augmentation of their levels or signaling via inhibition of the enzymes neprilysin (NEP) and phosphodiesterase 9 (PDE9), respectively, has beneficial actions in heart failure (HF). OBJECTIVES: The authors investigated dual enhancement of NP bioactivity by combining PDE9 inhibition and NEP inhibition in HF using an ovine model. METHODS: Eight sheep with pacing-induced HF received on 4 separate days intravenous PDE9 inhibition (PF-04749982), NEP inhibition (SCH-32615), PDE9 inhibition + NEP inhibition (PI+NI), and vehicle control treatment. RESULTS: Compared with the control treatment, NEP inhibition significantly increased plasma NP concentrations with a corresponding rise in second messenger cyclic guanosine monophosphate (cGMP), whereas PDE9 inhibition increased circulating cGMP with a negligible effect on NP levels. Combined PI+NI elevated plasma NPs to an extent comparable to that seen with NEP inhibition alone but further increased cGMP, resulting in a rise in the cGMP-to-NP ratio. All active treatments reduced mean arterial pressure, left atrial pressure, pulmonary arterial pressure, and peripheral resistance, with combined PI+NI further reducing mean arterial pressure and left atrial pressure relative to either inhibitor separately. Active treatments increased urine volume and sodium, potassium and creatinine excretion, and creatinine clearance, in association with rises in urine cGMP levels. PI+NI induced a significantly greater natriuresis and increase in urinary cGMP relative to either inhibitor singly. CONCLUSIONS: The present study demonstrates for the first time that combined PI+NI has additional beneficial hemodynamic and renal effects when compared with either PDE9 inhibition or NEP inhibition alone. The superior efficacy of this 2-pronged augmentation of NP bioactivity supports PI+NI as a potential therapeutic strategy for HF.

Circulating levels and prognostic cut-offs of sST2, hs-cTnT, and NT-proBNP in women vs. men with chronic heart failure.

AIMS: To define plasma concentrations, determinants, and optimal prognostic cut-offs of soluble suppression of tumorigenesis-2 (sST2), high-sensitivity cardiac troponin T (hs-cTnT), and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in women and men with chronic heart failure (HF). METHODS AND RESULTS: Individual data of patients from the Biomarkers In Heart Failure Outpatient Study (BIOS) Consortium with sST2, hs-cTnT, and NT-proBNP measured were analysed. The primary endpoint was a composite of 1 year cardiovascular death and HF hospitalization. The secondary endpoints were 5 year cardiovascular and all-cause death. The cohort included 4540 patients (age 67 ± 12 years, left ventricular ejection fraction 33 ± 13%, 1111 women, 25%). Women showed lower sST2 (24 vs. 27 ng/mL, P < 0.001) and hs-cTnT level (15 vs. 20 ng/L, P < 0.001), and similar concentrations of NT-proBNP (1540 vs. 1505 ng/L, P = 0.408). Although the three biomarkers were confirmed as independent predictors of outcome in both sexes, the optimal prognostic cut-off was lower in women for sST2 (28 vs. 31 ng/mL) and hs-cTnT (22 vs. 25 ng/L), while NT-proBNP cut-off was higher in women (2339 ng/L vs. 2145 ng/L). The use of sex-specific cut-offs improved risk prediction compared with the use of previously standardized prognostic cut-offs and allowed to reclassify the risk of many patients, to a greater extent in women than men, and for hs-cTnT than sST2 or NT-proBNP. Specifically, up to 18% men and up to 57% women were reclassified, by using the sex-specific cut-off of hs-cTnT for the endpoint of 5 year cardiovascular death. CONCLUSIONS: In patients with chronic HF, concentrations of sST2 and hs-cTnT, but not of NT-proBNP, are lower in women. Lower sST2 and hs-cTnT and higher NT-proBNP cut-offs for risk stratification could be used in women.

Ceramidas Plasmáticas na Estratificação de Risco das Doenças Cardiovasculares / Plasma Ceramides in Cardiovascular Disease Risk Stratification

Resumo A produção de ceramida ocorre em todo o corpo e desempenha um papel importante na manutenção da fisiologia normal. No entanto, os níveis de ceramidas são alterados em estados de doença, principalmente durante o desenvolvimento de diabetes e dislipidemia. A produção de ceramidas também está associada à instabilidade das placas ateroscleróticas. Estudos recentes revelam que pacientes com doença arterial coronariana instável apresentam níveis plasmáticos aumentados de ceramidas (principalmente C16, C18 e C24:1). Atualmente, são consideradas biomarcadores emergentes nas doenças cardiovasculares, sendo utilizadas na predição de instabilidade da placa aterosclerótica e eventos cardiovasculares adversos de forma independente aos fatores de risco tradicionais. Com o objetivo de descrever e discutir o papel das ceramidas na estratificação das doenças cardiovasculares, o desenvolvimento desta revisão narrativa contextualiza a importância desse biomarcador no cenário atual da cardiologia.

Abstract Ceramide production takes place throughout the body and plays a key role in the maintenance of normal physiology. However, ceramide levels are altered during disease states, particularly considering the development of diabetes and dyslipidemia. Ceramide production is also associated with atherosclerotic plaque instability. Recent studies revealed that patients with unstable coronary artery disease (CAD) presented increased plasma ceramide levels (especially C16, C18, and C24:1). These molecules are currently considered emerging biomarkers of cardiovascular diseases (CVD), being used for predicting atherosclerotic plaque instability and adverse cardiovascular events independently from traditional risk factors. With the aim of describing and discussing the role of ceramides in the stratification of cardiovascular diseases, this narrative review contextualizes the importance of this biomarker in the present cardiology scenario.

Finding a reliable assay for soluble neprilysin.

BACKGROUND: Lack of validation and standardization of research-use-only (RUO) immunoassays brings with it inherent threats to authenticity and functional quality. Poor correlation between different commercial neprilysin RUO immunoassays is concerning and discordant findings need to be resolved. We seek to identify and validate reliable neprilysin immunoassays to strengthen the scientific rigor and reproducibility of neprilysin-related investigation and of biomarker research in general. METHODS: Soluble neprilysin (sNEP) concentrations were determined in cohorts (n = 532) from Spain (Cohort 1), New Zealand (NZ, Cohort 2) and Singapore (Cohort 3), using commercial kits from six vendors. Apparent sNEP concentrations were correlated between different assays and with plasma neprilysin activity. Assay reliability was further validated by performance verification, MS analysis and cross-reactivity tests. RESULTS: sNEP in Cohorts 1 and 2 measured concurrently in Spain and NZ showed significant inter-laboratory correlation only for the Aviscera Bioscience sNEP ELISA SK00724-01. Neprilysin concentrations obtained with the R&D systems and SK00724-01 ELISAs correlated with each other but not with neprilysin activity. In Cohort 3, sNEP concentrations from the Perkin Elmer AlphaLISA and Biotechne ELLA assays agreed (r = 0.89) and both correlated with neprilysin activity (r = 0.87, 0.77 respectively). MS analysis detected authentic neprilysin in the AlphaLISA kit calibrator and in antibody pull-down material from human plasma. The AlphaLISA assay performed within acceptable limits (spike and recovery, dilutional linearity, inter- and intra-assay CV) and showed no cross-reactivity against neprilysin substrates and closely-related analogues. CONCLUSION: AlphaLISA and ELLA assays provide reliable measures of sNEP concentrations. Reliability of other commercial neprilysin assays remains in question.

Novel predictive role for mid-regional proadrenomedullin in moderate to severe aortic stenosis.

OBJECTIVE: We investigated the prognostic significance of selected known and novel circulating biomarkers in aortic stenosis (AS). METHODS: N-terminal pro-BNP (NT-proBNP), high-sensitivity troponin-T (hsTnT), growth differentiation factor-15 (GDF-15), suppression of tumorigenicity-2 (ST2), mid-regional proadrenomedullin (MR-proADM) and mid-regional proatrial natriuretic peptide (MR-proANP) were measured in patients with moderate to severe AS, New York Heart Association (NYHA) class I-II and left ventricular ejection fraction ≥50%, recruited consecutively across five centres from 2011 to 2018. Their ability to predict both primary (all-cause mortality, heart failure hospitalisation or progression to NYHA class III-IV) and secondary (additionally incorporating syncope and acute coronary syndrome) outcomes was determined by competing risk analyses. RESULTS: Among 173 patients with AS (age 69±11 years, 55% male, peak transaortic velocity (Vmax) 4.0±0.8 m/s), the primary and secondary outcomes occurred in 59 (34%) and 66 (38%), respectively. With aortic valve replacement as a competing risk, the primary outcome was determined consistently by the comorbidity index and each selected biomarker except ST2 (p<0.05), independent of NYHA class, Vmax, LV-global longitudinal strain and serum creatinine. MR-proADM had the highest discriminative value for both primary (subdistribution HR (SHR) 11.3, 95% CI 3.9 to 32.7) and secondary outcomes (SHR 12.6, 95% CI 4.7 to 33.5). Prognostic assessment of dual-biomarker combinations identified MR-proADM plus either hsTnT or NT-proBNP as the best predictive model for both clinical outcomes. Paired biomarker models were not superior to those including MR-proADM as the sole circulating biomarker. CONCLUSION: MR-proADM most powerfully portended worse prognosis and should be further assessed as possibly the biomarker of choice for risk stratification in AS.

Cardiac biomarkers retain prognostic significance in patients with heart failure and chronic obstructive pulmonary disease.

AIMS: Chronic obstructive pulmonary disease (COPD) is a frequent comorbidity in patients with heart failure (HF). We assessed the influence of COPD on circulating levels and prognostic value of three HF biomarkers: N-terminal pro-B-type natriuretic peptide (NT-proBNP), high-sensitivity troponin T (hs-TnT), and soluble suppression of tumorigenesis-2 (sST2). METHODS: Individual data from patients with chronic HF, known COPD status, NT-proBNP and hs-TnT values (n = 8088) were analysed. A subgroup (n = 3414) had also sST2 values. RESULTS: Patients had a median age of 66 years (interquartile interval 57-74), 77% were men and 82% had HF with reduced ejection fraction. NT-proBNP, hs-TnT and sST2 were 1207 ng/l (487-2725), 17 ng/l (9-31) and 30 ng/ml (22-44), respectively. Patients with COPD (n = 1249, 15%) had higher NT-proBNP (P = 0.042) and hs-TnT (P < 0.001), but not sST2 (P = 0.165). Over a median 2.0-year follow-up (1.5-2.5), 1717 patients (21%) died, and 1298 (16%) died from cardiovascular causes; 2255 patients (28%) were hospitalized for HF over 1.8 years (0.9-2.1). NT-proBNP, hs-TnT and sST2 predicted the three end points regardless of COPD status. The best cut-offs from receiver-operating characteristics analysis were higher in patients with COPD than in those without. Patients with all three biomarkers higher than or equal to end-point- and COPD-status-specific cut-offs were also those with the worst prognosis. CONCLUSIONS: Among patients with HF, those with COPD have higher NT-proBNP and hs-TnT, but not sST2. All these biomarkers yield prognostic significance regardless of the COPD status.

Readmissions, Death and Its Associated Predictors in Heart Failure With Preserved Versus Reduced Ejection Fraction.

Background Data on rehospitalizations for heart failure (HF) in Asia are scarce. We sought to determine the burden and predictors of HF (first and recurrent) rehospitalizations and all-cause mortality in patients with HF and preserved versus reduced ejection fraction (preserved EF, ≥50%; reduced EF, <40%), in the multinational ASIAN-HF (Asian Sudden Cardiac Death in Heart Failure) registry. Methods and Results Patients with symptomatic (stage C) chronic HF were followed up for death and recurrent HF hospitalizations for 1 year. Predictors of HF hospitalizations or all-cause mortality were examined with Cox regression for time to first event and other methods for recurrent events analyses. Among 1666 patients with HF with preserved EF (mean age, 68±12 years; 50% women), and 4479 with HF with reduced EF (mean age, 61±13 years; 22% women), there were 642 and 2302 readmissions, with 28% and 45% attributed to HF, respectively. The 1-year composite event rate for first HF hospitalization or all-cause death was 11% and 21%, and for total HF hospitalization and all-cause death was 17.7 and 38.7 per 100 patient-years in HF with preserved EF and HF with reduced EF, respectively. In HF with preserved EF, consistent independent predictors of these clinical end points included enrollment as an inpatient, Southeast Asian location, and comorbid chronic kidney disease or atrial fibrillation. The same variables were predictive of outcomes in HF with reduced EF except atrial fibrillation, and also included Northeast Asian location, older age, elevated heart rate, decreased systolic blood pressure, diabetes, smoking, and non-usage of beta blockers. Conclusions One-year HF rehospitalization and mortality rates were high among Asian patients with HF. Predictors of outcomes identified in this study could aid in risk stratification and timely interventions. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT01633398.

Lipid profiles and outcomes of patients with prior cancer and subsequent myocardial infarction or stroke.

Patients with cancer are at increased risk of myocardial infarction (MI) and stroke. Guidelines do not address lipid profile targets for these patients. Within the lipid profiles, we hypothesized that patients with cancer develop MI or stroke at lower low density lipoprotein cholesterol (LDL-C) concentrations than patients without cancer and suffer worse outcomes. We linked nationwide longitudinal MI, stroke and cancer registries from years 2007-2017. We identified 42,148 eligible patients with MI (2421 prior cancer; 39,727 no cancer) and 43,888 eligible patients with stroke (3152 prior cancer; 40,738 no cancer). Median LDL-C concentration was lower in the prior cancer group than the no cancer group at incident MI [2.43 versus 3.10 mmol/L, adjusted ratio 0.87 (95% CI 0.85-0.89)] and stroke [2.81 versus 3.22 mmol/L, adjusted ratio 0.93, 95% CI 0.91-0.95)]. Similarly, median triglyceride and total cholesterol concentrations were lower in the prior cancer group, with no difference in high density lipoprotein cholesterol. Prior cancer was associated with higher post-MI mortality [adjusted hazard ratio (HR) 1.48, 95% CI 1.37-1.59] and post-stroke mortality (adjusted HR 1.95, 95% CI 1.52-2.52). Despite lower LDL-C concentrations, patients with prior cancer had worse post-MI and stroke mortality than patients without cancer.

Circulating heart failure biomarkers beyond natriuretic peptides: review from the Biomarker Study Group of the Heart Failure Association (HFA), European Society of Cardiology (ESC).

New biomarkers are being evaluated for their ability to advance the management of patients with heart failure. Despite a large pool of interesting candidate biomarkers, besides natriuretic peptides virtually none have succeeded in being applied into the clinical setting. In this review, we examine the most promising emerging candidates for clinical assessment and management of patients with heart failure. We discuss high-sensitivity cardiac troponins (Tn), procalcitonin, novel kidney markers, soluble suppression of tumorigenicity 2 (sST2), galectin-3, growth differentiation factor-15 (GDF-15), cluster of differentiation 146 (CD146), neprilysin, adrenomedullin (ADM), and also discuss proteomics and genetic-based risk scores. We focused on guidance and assistance with daily clinical care decision-making. For each biomarker, analytical considerations are discussed, as well as performance regarding diagnosis and prognosis. Furthermore, we discuss potential implementation in clinical algorithms and in ongoing clinical trials.

Acute Decompensated Heart Failure and the Kidney: Physiological, Histological and Transcriptomic Responses to Development and Recovery.

BACKGROUND Acute decompensated heart failure (ADHF) is associated with deterioration in renal function-an important risk factor for poor outcomes. Whether ADHF results in permanent kidney damage/dysfunction is unknown. METHODS AND RESULTS We investigated for the first time the renal responses to the development of, and recovery from, ADHF using an ovine model. ADHF development induced pronounced hemodynamic changes, neurohormonal activation, and decline in renal function, including decreased urine, sodium and urea excretion, and creatinine clearance. Following ADHF recovery (25 days), creatinine clearance reductions persisted. Kidney biopsies taken during ADHF and following recovery showed widespread mesangial cell prominence, early mild acute tubular injury, and medullary/interstitial fibrosis. Renal transcriptomes identified altered expression of 270 genes following ADHF development and 631 genes following recovery. A total of 47 genes remained altered post-recovery. Pathway analysis suggested gene expression changes, driven by a network of inflammatory cytokines centered on IL-1ß (interleukin 1ß), lead to repression of reno-protective eNOS (endothelial nitric oxide synthase) signaling during ADHF development, and following recovery, activation of glomerulosclerosis and reno-protective pathways and repression of proinflammatory/fibrotic pathways. A total of 31 dysregulated genes encoding proteins detectable in urine, serum, and plasma identified potential candidate markers for kidney repair (including CNGA3 [cyclic nucleotide gated channel subunit alpha 3] and OIT3 [oncoprotein induced transcript 3]) or long-term renal impairment in ADHF (including ACTG2 [actin gamma 2, smooth muscle] and ANGPTL4 [angiopoietin like 4]). CONCLUSIONS In an ovine model, we provide the first direct evidence that an episode of ADHF leads to an immediate decline in kidney function that failed to fully resolve after ≈4 weeks and is associated with persistent functional/structural kidney injury. We identified molecular pathways underlying kidney injury and repair in ADHF and highlighted 31 novel candidate biomarkers for acute kidney injury in this setting.

Impact of change in iron status over time on clinical outcomes in heart failure according to ejection fraction phenotype.

AIMS: The importance of iron deficiency (ID) in heart failure with preserved ejection fraction (HFpEF) is unknown. In HF with reduced ejection fraction (HFrEF), ID is reported as an independent predictor of mortality in HF although not all published studies agree. Different definitions of ID have been assessed, and the natural history of untreated ID not established, which may explain the conflicting results. This study aimed to assess the relationship between ID and mortality in HFpEF, clarify which definition of ID correlates best with outcomes in HFrEF, and determine the prognostic importance of change in ID status over time. METHODS AND RESULTS: Analyses were conducted on data from 1563 patients participating in a prospective international cohort study comparing HFpEF with HFrEF. Plasma samples from baseline and 6 month visits were analysed for the presence of ID. Two ID definitions were evaluated: IDFerritin  = 'ferritin < 100 mcg/L or ferritin 100-300 mcg/L + transferrin saturation < 20%' and IDTsat  = 'transferrin saturation < 20%'. The risk of all-cause mortality and death/HF hospitalization associated with baseline ID (IDFerritin or IDTsat ) and change in ID status at 6 months (persistent, resolving, developing, or never present) was estimated in multivariable Cox proportional hazards models. Of 1563 patients, 1115 (71%) had HFrEF and 448 (29%) HFpEF. Prevalence of ID was similar in HFpEF and HFrEF (58%). Patients with ID were more likely to be female, diabetic, and have a higher co-morbid burden than patients without ID. ID by either definition did not confer independent risk for either all-cause mortality or death/HF hospitalization for patients with HFpEF [IDFerritin hazard ratio (HR) 0.65 (95% confidence interval 0.40-1.05), P = 0.08; IDTsat HR 1.16 (0.72-1.87), P = 0.55]. In the overall study cohort (HFrEF + HFpEF) and HFrEF subgroup, IDFerritin was inferior to IDTsat in prediction of all-cause mortality [overall cohort: HR 1.21 (0.95-1.53), P = 0.12 vs. HR 1.95 (1.52-2.51), P < 0.01; HFrEF: HR 1.12 (0.85-1.48), P = 0.43 vs. HR 1.57 (1.15-2.14), P < 0.01]. Persistence of IDTsat at 6 months was strongly associated with poor outcomes compared with never having IDTsat [HR 2.22 (1.42-3.46), P < 0.01] or having IDTsat at baseline self-resolve by 6 months [HR 1.40 (1.06-1.86), P = 0.02]. CONCLUSIONS: Iron deficiency is equally prevalent in HFpEF and HFrEF but is negatively prognostic only in HFrEF. The natural history of ID is important; persistent ID is strongly associated with mortality whereas resolution is not. IDTsat is the superior definition of ID and should inform future trials investigating the efficacy of intravenous iron replacement in patients with HFrEF.

Vascular endothelial growth factor-A promoter polymorphisms, circulating VEGF-A and survival in acute coronary syndromes.

BACKGROUND: Development of a competent collateral circulation in established coronary artery disease is cardio-protective. The vascular endothelial growth factor (VEGF) system plays a key role in this process. We investigated the prognostic performance of circulating VEGF-A and three genetic variants in the VEGFA gene in a clinical coronary cohort. METHODS AND RESULTS: The Coronary Disease Cohort Study (CDCS) recruited 2,140 patients, with a diagnosis of acute coronary syndrome (ACS), after admission to Christchurch or Auckland City Hospitals between July 2002 and January 2009. We present data for 1927 patients from the cohort genotyped for three SNPs in the VEGF-A gene, rs699947 (C-2578A), rs2010963 (C405G) and rs3025039 (C936T). Plasma VEGF-A concentrations were assayed in a subgroup (n = 550) of CDCS patients (geometric mean 36.6 [34.7-38.5] pg/ml). VEGF-A levels correlated with patient heart rate at baseline (p = 0.034). None of rs699947, rs3025039, nor rs2010963 genotypes were significantly associated with VEGF-A levels, but rs3025039 genotype was positively associated with collateral vessels perfusion according to the Rentrop classification (p = 0.01) and baseline natriuretic peptide levels (p<0.05). Survival in the CDCS cohort was independently associated with baseline VEGF-A levels and (in males) with rs699947 genotype. CONCLUSIONS: This study is strongly suggestive that VEGF-A levels have value as a prognostic biomarker in coronary heart disease patients and SNPs in VEGF-A deserve further investigation as prognostic markers and indicators of angiogenic potential influencing the formation of collateral circulation.

Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation.

High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. Antigenic peptides (13-24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets.