Growth differentiation factor-15 in patients with or at risk of heart failure but before first hospitalisation.

OBJECTIVE: Identification of patients at risk of adverse outcome from heart failure (HF) at an early stage is a priority. Growth differentiation factor (GDF)-15 has emerged as a potentially useful biomarker. This study sought to identify determinants of circulating GDF-15 and evaluate its prognostic value, in patients at risk of HF or with HF but before first hospitalisation. METHODS: Prospective, longitudinal cohort study of 2166 consecutive patients in stage A-C HF undergoing cardiovascular magnetic resonance and measurement of GDF-15. Multivariable linear regression investigated determinants of GDF-15. Cox proportional hazards modelling, Net Reclassification Improvement and decision curve analysis examined its incremental prognostic value. Primary outcome was a composite of first hospitalisation for HF or all-cause mortality. Median follow-up was 1093 (939-1231) days. RESULTS: Major determinants of GDF-15 were age, diabetes and N-terminal pro-B-type natriuretic peptide, although despite extensive phenotyping, only around half of the variability of GDF-15 could be explained (R2 0.51). Log-transformed GDF-15 was the strongest predictor of outcome (HR 2.12, 95% CI 1.71 to 2.63) and resulted in a risk prediction model with higher predictive accuracy (continuous Net Reclassification Improvement 0.26; 95% CI 0.13 to 0.39) and with greater clinical net benefit across the entire range of threshold probabilities. CONCLUSION: In patients at risk of HF, or with HF but before first hospitalisation, GDF-15 provides unique information and is highly predictive of hospitalisation for HF or all-cause mortality, leading to more accurate risk stratification that can improve clinical decision making. TRIAL REGISTRATION NUMBER: NCT02326324.

Treatment Adherence in a Randomized Controlled Trial of Pirfenidone in HFpEF: Determinants and Impact on Efficacy.

OBJECTIVES: Medication adherence in patients with heart failure with preserved ejection fraction is unclear. This study sought to evaluate treatment adherence in the Pirfenidone in Patients with Heart Failure and Preserved Left Ventricular Ejection Fraction (PIROUETTE) trial. METHODS AND RESULTS: Adherence was evaluated through pill counts and diary cards. Univariable and multivariable regression models were used to assess the relationship between adherence and baseline characteristics. Instrumental variable regression was used to estimate the causal effect of pirfenidone treatment duration on myocardial fibrosis. Complete adherence data were available in 54 of 80 participants completing the trial. Mean adherence to study medication was 94.7% and 96.9% in the pirfenidone and placebo groups, respectively. Each additional day of treatment with pirfenidone resulted in a significant decrease in myocardial extracellular volume (-0.004%; 95% confidence interval: -0.007% to -0.001%; P = 0.007). Associations with adherence included older age, higher symptom burden, lower body weight, and smaller right ventricular size. CONCLUSION: Adherence to study medication in the PIROUETTE trial was very high among patients for whom complete adherence data were available. Importantly, each additional day of treatment reduced myocardial fibrosis. Potential predictors of adherence were identified. Implementation of improved methods for assessing adherence is required.

Impact of Myocardial Fibrosis on Cardiovascular Structure, Function and Functional Status in Heart Failure with Preserved Ejection Fraction.

Myocardial fibrosis, measured using cardiovascular magnetic resonance extracellular volume (ECV), is associated with adverse outcome in heart failure with preserved ejection fraction, but the mechanisms by which myocardial fibrosis exerts this deleterious effect are unclear. We performed mediation analyses of data from the Pirfenidone in Patients with Heart Failure and Preserved Left Ventricular Ejection Fraction (PIROUETTE) trial to determine whether myocardial fibrotic regression causes changes in cardiovascular function and functional status following antifibrotic therapy. Regression of myocardial fibrosis correlated with improvements in 6-min walk test and KCCQ clinical summary score. The only outcome variable that demonstrated a treatment effect was an increase in left ventricular ejection fraction (LVEF). The estimated average causal mediation effects of myocardial ECV, absolute myocardial extracellular matrix volume and absolute myocardial cellular volume on LVEF were 6.1%, 21.5% and 13.7%, respectively, none of which was significant and therefore not mediated by myocardial fibrosis. (PIROUETTE; NCT02932566).

Characteristics Associated With Growth Differentiation Factor 15 in Heart Failure With Preserved Ejection Fraction and the Impact of Pirfenidone.

Background Growth differentiation factor 15 (GDF-15) is elevated in heart failure with preserved ejection fraction and is associated with adverse outcome, but its relationship with myocardial fibrosis and other characteristics remains unclear. We sought to evaluate the effect of pirfenidone, a novel antifibrotic agent, on GDF-15 in heart failure with preserved ejection fraction and identify characteristics that associate with GDF-15 and with change in GDF-15 over 1 year. Methods and Results Among patients enrolled (n=107) in the PIROUETTE (Pirfenidone in Patients With Heart Failure and Preserved Left Ventricular Ejection Fraction) trial, GDF-15 was measured at baseline and at prespecified time points in patients randomized (n=94) to pirfenidone or placebo. The response of GDF-15 to pirfenidone and the association with baseline patient characteristics were evaluated. Pirfenidone had no impact on circulating GDF-15 at any time point during the 52-week trial period. In multivariable analysis, male sex, diabetes, higher circulating levels of N-terminal pro-B-type natriuretic peptide, lower renal function, and shorter 6-minute walk test distance at baseline were associated with baseline log-GDF-15. Impaired global longitudinal strain at baseline was the strongest predictor of increased GDF-15 over 52 weeks. Conclusions In patients with heart failure with preserved ejection fraction, circulating levels of GDF-15 were unaffected by treatment with pirfenidone and do not appear to be determined by myocardial fibrosis. Circulating GDF-15 was associated with a spectrum of important heart failure characteristics and it may represent a marker of overall physiological disruption. Registration URL: https://clinicaltrials.gov/ct2/show/NCT02932566; Unique identifier: NCT02932566.

Predicting hospitalisation for heart failure and death in patients with, or at risk of, heart failure before first hospitalisation: a retrospective model development and external validation study.

BACKGROUND: Identifying people who are at risk of being admitted to hospital (hospitalised) for heart failure and death, and particularly those who have not previously been hospitalised for heart failure, is a priority. We aimed to develop and externally validate a prognostic model involving contemporary deep phenotyping that can be used to generate individual risk estimates of hospitalisation for heart failure or all-cause mortality in patients with, or at risk of, heart failure, but who have not previously been hospitalised for heart failure. METHODS: Between June 1, 2016, and May 31, 2018, 3019 consecutive adult patients (aged ≥16 years) undergoing cardiac magnetic resonance (CMR) at Manchester University National Health Service Foundation Trust, Manchester, UK, were prospectively recruited into a model development cohort. Candidate predictor variables were selected according to clinical practice and literature review. Cox proportional hazards modelling was used to develop a prognostic model. The final model was validated in an external cohort of 1242 consecutive adult patients undergoing CMR at the University of Pittsburgh Medical Center Cardiovascular Magnetic Resonance Center, Pittsburgh, PA, USA, between June 1, 2010, and March 25, 2016. Exclusion criteria for both cohorts included previous hospitalisation for heart failure. Our study outcome was a composite of first hospitalisation for heart failure or all-cause mortality after CMR. Model performance was evaluated in both cohorts by discrimination (Harrell's C-index) and calibration (assessed graphically). FINDINGS: Median follow-up durations were 1118 days (IQR 950-1324) for the development cohort and 2117 days (1685-2446) for the validation cohort. The composite outcome occurred in 225 (7·5%) of 3019 patients in the development cohort and in 219 (17·6%) of 1242 patients in the validation cohort. The final, externally validated, parsimonious, multivariable model comprised the predictors: age, diabetes, chronic obstructive pulmonary disease, N-terminal pro-B-type natriuretic peptide, and the CMR variables, global longitudinal strain, myocardial infarction, and myocardial extracellular volume. The median optimism-adjusted C-index for the externally validated model across 20 imputed model development datasets was 0·805 (95% CI 0·793-0·829) in the development cohort and 0·793 (0·766-0·820) in the external validation cohort. Model calibration was excellent across the full risk profile. A risk calculator that provides an estimated risk of hospitalisation for heart failure or all-cause mortality at 3 years after CMR for individual patients was generated. INTERPRETATION: We developed and externally validated a risk prediction model that provides accurate, individualised estimates of the risk of hospitalisation for heart failure and all-cause mortality in patients with, or at risk of, heart failure, before first hospitalisation. It could be used to direct intensified therapy and closer follow-up to those at increased risk. FUNDING: The UK National Institute for Health Research, Guerbet Laboratories, and Roche Diagnostics International.

Predictors of myocardial fibrosis and response to anti-fibrotic therapy in heart failure with preserved ejection fraction.

Myocardial fibrosis, measured using magnetic resonance extracellular volume (ECV), associates with adverse outcome in heart failure with preserved ejection fraction (HFpEF). In the PIROUETTE (The Pirfenidone in Patients with Heart Failure and Preserved Left Ventricular Ejection Fraction) trial, the novel anti-fibrotic agent pirfenidone reduced myocardial fibrosis. We sought to identify baseline characteristics that associate with myocardial fibrotic burden, the change in myocardial fibrosis over a year, and predict response to pirfenidone in patients with HFpEF. Amongst patients enrolled in the PIROUETTE trial (n = 107), linear regression models were used to assess the relationship between baseline variables and baseline myocardial ECV, with change in myocardial ECV adjusting for treatment allocation, and to identify variables that modified the pirfenidone treatment effect. Body mass index, left atrial reservoir strain, haemoglobin and aortic distensibility were associated with baseline ECV in stepwise modelling, and systolic blood pressure, and log N-terminal pro B-type natriuretic peptide were associated with baseline ECV in clinically-guided modelling. QRS duration, left ventricular mass and presence of an infarct at baseline were associated with an increase in ECV from baseline to week 52. Whilst QRS duration, presence of an infarct, global longitudinal strain and left atrial strain modified the treatment effect of pirfenidone when considered individually, no variable modified treatment effect on multivariable modelling. Baseline characteristics were identified that associate with myocardial fibrosis and predict change in myocardial fibrosis. No variables that independently modify the treatment effect of pirfenidone were identified (PIROUETTE, NCT02932566).

Pirfenidone in heart failure with preserved ejection fraction: a randomized phase 2 trial.

In heart failure with preserved ejection fraction (HFpEF), the occurrence of myocardial fibrosis is associated with adverse outcome. Whether pirfenidone, an oral antifibrotic agent without hemodynamic effect, is efficacious and safe for the treatment of HFpEF is unknown. In this double-blind, phase 2 trial ( NCT02932566 ), we enrolled patients with heart failure, an ejection fraction of 45% or higher and elevated levels of natriuretic peptides. Eligible patients underwent cardiovascular magnetic resonance and those with evidence of myocardial fibrosis, defined as a myocardial extracellular volume of 27% or greater, were randomly assigned to receive pirfenidone or placebo for 52 weeks. Forty-seven patients were randomized to each of the pirfenidone and placebo groups. The primary outcome was change in myocardial extracellular volume, from baseline to 52 weeks. In comparison to placebo, pirfenidone reduced myocardial extracellular volume (between-group difference, -1.21%; 95% confidence interval, -2.12 to -0.31; P = 0.009), meeting the predefined primary outcome. Twelve patients (26%) in the pirfenidone group and 14 patients (30%) in the placebo group experienced one or more serious adverse events. The most common adverse events in the pirfenidone group were nausea, insomnia and rash. In conclusion, among patients with HFpEF and myocardial fibrosis, administration of pirfenidone for 52 weeks reduced myocardial fibrosis. The favorable effects of pirfenidone in patients with HFpEF will need to be confirmed in future trials.

Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary inflammation.

Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1ß production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.

The Role of Source-Sink Dynamics in the Assessment of Risk to Nontarget Arthropods from the Use of Plant Protection Products.

The concept of source-sink dynamics as a potentially important component of metapopulation dynamics was introduced in the 1980s. The objective of the present review was to review the considerable body of work that has been developed, to consider its theoretical implications as well as to understand how source-sink dynamics may manifest under field conditions in the specific case of nontarget arthropods in the agricultural environment. Our review concludes that metapopulation dynamics based on field observations are often far more complex than existing theoretical source-sink models would indicate, because they are dependent on numerous population processes and influencing factors. The difficulty in identifying and measuring these factors likely explains why empirical studies assessing source-sink dynamics are scarce. Furthermore, we highlight the importance of considering the spatial and temporal heterogeneity of agricultural landscapes when assessing the population dynamics of nontarget arthropods in the context of the risk from the use of plant protection products. A need is identified to further develop and thoroughly validate predictive population models, which can incorporate all factors relevant to a specific system. Once reliable predictive models for a number of representative nontarget arthropod species are available, they could provide a meaningful tool for refined risk evaluations (higher tier level risk assessment), addressing specific concerns identified at the initial evaluation stages (lower tier level risk assessment). Environ Toxicol Chem 2021;40:2667-2679. © 2021 ERM, FMC, Syngenta, Bayer AG, BASF SE, Corteva agriscience. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

IL-10 production by ILC2s requires Blimp-1 and cMaf, modulates cellular metabolism, and ameliorates airway hyperreactivity.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are the dominant innate lymphoid cell population in the lungs at steady state, and their release of type 2 cytokines is a central driver in responding eosinophil infiltration and increased airway hyperreactivity. Our laboratory has identified a unique subset of ILC2s in the lungs that actively produce IL-10 (ILC210s). OBJECTIVE: Our aim was to characterize the effector functions of ILC210s in the development and pathology of allergic asthma. METHODS: IL-4-stimulated ILC210s were isolated to evaluate cytokine secretion, transcription factor signaling, metabolic dependence, and effector functions in vitro. ILC210s were also adoptively transferred into Rag2-/-γc-/- mice, which were then challenged with IL-33 and assessed for airway hyperreactivity and lung inflammation. RESULTS: We have determined that the transcription factors cMaf and Blimp-1 regulate IL-10 expression in ILC210s. Strikingly, our results demonstrate that ILC210s can utilize both autocrine and paracrine signaling to suppress proinflammatory ILC2 effector functions in vitro. Further, this subset dampens airway hyperreactivity and significantly reduces lung inflammation in vivo. Interestingly, ILC210s demonstrated a metabolic dependency on the glycolytic pathway for IL-10 production, shifting from the fatty acid oxidation pathway conventionally utilized for proinflammatory effector functions. CONCLUSION: These findings provide an important and previously unrecognized role of ILC210s in diseases associated with ILC2s such as allergic lung inflammation and asthma. They also provide new insights into the metabolism dependency of proinflammatory and anti-inflammatory ILC2 phenotypes.

The utility of cardiovascular imaging in heart failure with preserved ejection fraction: diagnosis, biological classification and risk stratification.

Heart failure with preserved ejection fraction (HFpEF) does not exist as a singular clinical or pathological entity but as a syndrome encompassing a wide range of clinical and biological phenotypes. There is an urgent need to progress from the unsuccessful 'one-size-fits-all' approach to more precise disease classification, in order to develop targeted therapies, personalise risk stratification and guide future research. In this regard, this review discusses the current and emerging roles of cardiovascular imaging for the diagnosis of HFpEF, for distilling HFpEF into distinct disease entities according to underlying pathobiology and for risk stratification.

DR3 stimulation of adipose resident ILC2s ameliorates type 2 diabetes mellitus.

Disturbances in glucose homeostasis and low-grade chronic inflammation culminate into metabolic syndrome that increase the risk for the development of type 2 diabetes mellitus (T2DM). The recently discovered group 2 innate lymphoid cells (ILC2s) are capable of secreting copious amounts of type 2 cytokines to modulate metabolic homeostasis in adipose tissue. In this study, we have established that expression of Death Receptor 3 (DR3), a member of the TNF superfamily, on visceral adipose tissue (VAT)-derived murine and peripheral blood human ILC2s is inducible by IL-33. We demonstrate that DR3 engages the canonical and/or non-canonical NF-κB pathways, and thus stimulates naïve and co-stimulates IL-33-activated ILC2s. Importantly, DR3 engagement on ILC2s significantly ameliorates glucose tolerance, protects against insulin-resistance onset and remarkably reverses already established insulin-resistance. Taken together, these results convey the potent role of DR3 as an ILC2 regulator and introduce DR3 agonistic treatment as a novel therapeutic avenue for treating T2DM.

PD-1 pathway regulates ILC2 metabolism and PD-1 agonist treatment ameliorates airway hyperreactivity.

Allergic asthma is a leading chronic disease associated with airway hyperreactivity (AHR). Type-2 innate lymphoid cells (ILC2s) are a potent source of T-helper 2 (Th2) cytokines that promote AHR and lung inflammation. As the programmed cell death protein-1 (PD-1) inhibitory axis regulates a variety of immune responses, here we investigate PD-1 function in pulmonary ILC2s during IL-33-induced airway inflammation. PD-1 limits the viability of ILC2s and downregulates their effector functions. Additionally, PD-1 deficiency shifts ILC2 metabolism toward glycolysis, glutaminolysis and methionine catabolism. PD-1 thus acts as a metabolic checkpoint in ILC2s, affecting cellular activation and proliferation. As the blockade of PD-1 exacerbates AHR, we also develop a human PD-1 agonist and show that it can ameliorate AHR and suppresses lung inflammation in a humanized mouse model. Together, these results highlight the importance of PD-1 agonistic treatment in allergic asthma and underscore its therapeutic potential.

Autophagy is critical for group 2 innate lymphoid cell metabolic homeostasis and effector function.

BACKGROUND: Allergic asthma is a chronic inflammatory disorder characterized by airway hyperreactivity (AHR) and driven by TH2 cytokine production. Group 2 innate lymphoid cells (ILC2s) secrete high amounts of TH2 cytokines and contribute to the development of AHR. Autophagy is a cellular degradation pathway that recycles cytoplasmic content. However, the role of autophagy in ILC2s remains to be fully elucidated. OBJECTIVE: We characterized the effects of autophagy deficiency on ILC2 effector functions and metabolic balance. METHODS: ILC2s from autophagy-deficient mice were isolated to evaluate proliferation, apoptosis, cytokine secretion, gene expression and cell metabolism. Also, autophagy-deficient ILC2s were adoptively transferred into Rag-/-GC-/- mice, which were then challenged with IL-33 and assessed for AHR and lung inflammation. RESULTS: We demonstrate that autophagy is extensively used by activated ILC2s to maintain their homeostasis and effector functions. Deletion of the critical autophagy gene autophagy-related 5 (Atg5) resulted in decreased cytokine secretion and increased apoptosis. Moreover, lack of autophagy among ILC2s impaired their ability to use fatty acid oxidation and strikingly promoted glycolysis, as evidenced by our transcriptomic and metabolite analyses. This shift of fuel dependency led to impaired homeostasis and TH2 cytokine production, thus inhibiting the development of ILC2-mediated AHR. Notably, this metabolic reprogramming was also associated with an accumulation of dysfunctional mitochondria, producing excessive reactive oxygen species. CONCLUSION: These findings provide new insights into the metabolic profile of ILC2s and suggest that modulation of fuel dependency by autophagy is a potentially new therapeutic approach to target ILC2-dependent inflammation.

Dietary Fiber-Induced Microbial Short Chain Fatty Acids Suppress ILC2-Dependent Airway Inflammation.

Group 2 Innate lymphoid cells (ILC2) contribute significantly to allergic inflammation. However, the role of microbiota on ILC2s remains to be unraveled. Here we show that short chain fatty acids (SCFAs), such as butyrate, derived from fermentation of dietary fibers by the gut microbiota inhibit pulmonary ILC2 functions and subsequent development of airway hyperreactivity (AHR). We further show that SCFAs modulate GATA3, oxidative phosphorylation, and glycolytic metabolic pathways in pulmonary ILC2s. The observed phenotype is associated with increased IL-17a secretion by lung ILC2s and linked to enhanced neutrophil recruitment to the airways. Finally, we show that butyrate-producing gut bacteria in germ-free mice effectively suppress ILC2-driven AHR. Collectively, our results demonstrate a previously unrecognized role for microbial-derived SCFAs on pulmonary ILC2s in the context of AHR. The data suggest strategies aimed at modulating metabolomics and microbiota in the gut, not only to treat, but to prevent lung inflammation and asthma.

Considerations for Clinical Trials Targeting the Myocardial Interstitium.

The myocardial interstitium has emerged as a potential therapeutic target and as a biological entity to improve risk stratification and better guide existing interventions. Clinical trials focusing on the myocardial interstitium are required to establish causality and improve patient outcomes. This review will discuss issues around clinical trials targeting the myocardial interstitium, including antifibrotic therapies, efficacy outcome measurements, mechanistic outcome measurements and mediation analysis, sample size, trial duration, considerations for multicenter trials, stratifying trial recruitment according to the interstitium, and approaches to enrich recruitment, using examples of ongoing clinical trials.

Pirfenidone in Heart Failure with Preserved Ejection Fraction-Rationale and Design of the PIROUETTE Trial.

BACKGROUND: The PIROUETTE (PIRfenidOne in patients with heart failUre and preserved lEfT venTricular Ejection fraction) trial is designed to evaluate the efficacy and safety of the anti-fibrotic pirfenidone in patients with chronic heart failure and preserved ejection fraction (HFpEF) and myocardial fibrosis. HFpEF is a diverse syndrome associated with substantial morbidity and mortality. Myocardial fibrosis is a key pathophysiological mechanism of HFpEF and myocardial fibrotic burden is strongly and independently associated with adverse outcome. Pirfenidone is an oral anti-fibrotic agent, without haemodynamic effect, that leads to regression of myocardial fibrosis in preclinical models. It has proven clinical effectiveness in pulmonary fibrosis. METHODS: The PIROUETTE trial is a randomised, double-blind, placebo-controlled phase II trial evaluating the efficacy and safety of 52 weeks of treatment with pirfenidone in patients with chronic HFpEF (symptoms and signs of heart failure, left ventricular ejection fraction ≥ 45%, elevated natriuretic peptides [BNP ≥ 100 pg/ml or NT-proBNP ≥ 300 pg/ml; or BNP ≥ 300 pg/ml or NT-proBNP ≥ 900 pg/ml if in atrial fibrillation]) and myocardial fibrosis (extracellular matrix (ECM) volume ≥ 27% measured using cardiovascular magnetic resonance). The primary outcome measure is change in myocardial ECM volume. A sub-study will investigate the relationship between myocardial fibrosis and myocardial energetics, and the impact of pirfenidone, using 31phosphorus magnetic resonance spectroscopy. DISCUSSION: PIROUETTE will determine whether pirfenidone is superior to placebo in relation to regression of myocardial fibrosis and improvement in myocardial energetics in patients with HFpEF and myocardial fibrosis (NCT02932566). CLINICAL TRIAL REGISTRATION: clinicaltrials.gov (NCT02932566) https://clinicaltrials.gov/ct2/show/NCT02932566.