Safety and Immunogenicity of Respiratory Syncytial Virus Pre-fusion Maternal Vaccine Co-administered with Diphtheria-Tetanus-Pertussis Vaccine: A Phase 2 Study.

BACKGROUND: Respiratory syncytial virus (RSV) fusion protein stabilized in the prefusion conformation (RSVPreF3) was under investigation as a maternal vaccine. METHODS: This phase 2, randomized, placebo-controlled, single-dose, multicenter study enrolled healthy, non-pregnant women, randomized 1:1:1:1:1 to five parallel groups studying RSVPreF3 (60 or 120 µg) co-administered with diphtheria, tetanus, and acellular pertussis vaccine (dTpa) or placebo, and dTpa co-administered with placebo. Safety and humoral immune responses were assessed. An extension phase also assessed a RSVPreF3 120 µg vaccination 12-18 months post-first vaccination. RESULTS: The safety profile of RSVPreF3 was unaffected by dose or dTpa co-administration. Solicited and unsolicited adverse events (AEs) were evenly distributed across study groups. Injection-site pain was higher following the second vaccination vs the first vaccination. Medically attended AEs were rare (<5% overall). Both RSVPreF3 dose levels (alone and with dTpa) were immunogenic, increasing levels of RSV-A neutralizing antibody ≥8 fold and anti-RSVPreF3 IgG antibody ≥11 fold at 1 month post-vaccination, which persisted at 12-18 months post-vaccination; modest 2-fold increases were observed with a second RSVPreF3 vaccination. CONCLUSIONS: This study indicates RSVPreF3 co-administration with dTpa induces robust immune responses and is well tolerated, regardless of the RSVPreF3 dose level used. CLINICAL TRIALS REGISTRATION: NCT04138056.

Cardiac myocyte ß3-adrenergic receptors prevent myocardial fibrosis by modulating oxidant stress-dependent paracrine signaling.

Aims: Human and mouse cardiac beta3-adrenergic receptors (beta3AR) exert antipathetic effects to those of beta1-2AR stimulation. We examined their role in modulating myocardial remodelling, particularly fibrosis in response to haemodynamic stress. Methods and results: Mice with cardiac myocyte-specific expression of beta3AR (ADRB3-tg) or tamoxifen-inducible homozygous deletion (c-Adrb3-ko, with loxP-targeted Adrb3) were submitted to transaortic constriction. A superfusion assay was used for proteomic analysis of paracrine mediators between beta3AR-expressing cardiac myocytes and cardiac fibroblasts cultured separately. We show that cardiac beta3AR attenuate myocardial fibrosis in response to haemodynamic stress. Interstitial fibrosis and collagen content were reduced in ADRB3-tg, but augmented in c-Adrb3-ko. ADRB3 and collagen (COL1A1) expression were also inversely related in ventricular biopsies of patients with valve disease. Incubation of cardiac fibroblasts with media conditioned by hypertrophic myocytes induced fibroblast proliferation, myo-differentiation, and collagen production. These effects were abrogated upon ADRB3 expression in myocytes. Comparative shotgun proteomic analysis of the myocyte secretomes revealed a number of factors differentially regulated by beta3AR, among which connective tissue growth factor [CTGF (CCN2)] was prominently reduced. CTGF was similarly reduced in stressed hearts from ADRB3-tg, but increased in hearts from c-Adrb3-ko mice. CTGF expression was mediated by reactive oxygen species production which was reduced by ADRB3 expression in vitro and in vivo. This antioxidant and anti-fibrotic effect involved beta3AR coupling to the neuronal isoform of nitric oxide synthase (nNOS) in cardiac myocytes, as both were abrogated upon nNOS inhibition or Nos1 homozygous deletion. Conclusion: Cardiac beta3AR protect from fibrosis in response to haemodynamic stress by modulating nitric oxide and oxidant stress-dependent paracrine signaling to fibroblasts. Specific agonism at beta3AR may offer a new therapeutic modality to prevent cardiac fibrosis.

Reduced scar maturation and contractility lead to exaggerated left ventricular dilation after myocardial infarction in mice lacking AMPKα1.

Cardiac fibroblasts (CF) are crucial in left ventricular (LV) healing and remodeling after myocardial infarction (MI). They are typically activated into myofibroblasts that express alpha-smooth muscle actin (α-SMA) microfilaments and contribute to the formation of contractile and mature collagen scars that minimize the adverse dilatation of infarcted areas. CF predominantly express the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1), while AMPKα2 is the major catalytic isoform in cardiomyocytes. AMPKα2 is known to protect the heart by preserving the energy charge of cardiac myocytes during injury, but whether AMPKα1 interferes with maladaptative heart responses remains unexplored. In this study, we investigated the role of AMPKα1 in modulating LV dilatation and CF fibrosis during post-MI remodeling. AMPKα1 knockout (KO) and wild type (WT) mice were subjected to permanent ligation of the left anterior descending coronary artery. The absence of AMPKα1 was associated with increased CF proliferation in infarcted areas, while expression of the myodifferentiation marker α-SMA was decreased. Faulty maturation of myofibroblasts might derive from severe down-regulation of the non-canonical transforming growth factor-beta1/p38 mitogen-activated protein kinase (TGF-ß1/p38 MAPK) pathway in KO infarcts. In addition, lysyl oxidase (LOX) protein expression was dramatically reduced in the scar of KO hearts. Although infarct size was similar in AMPK-KO and WT hearts subjected to MI, these changes resulted in compromised scar contractility, defective scar collagen maturation, and exacerbated adverse remodeling, as indicated by increased LV diastolic dimension 30days after MI. Our data genetically demonstrate the centrality of AMPKα1 in post-MI scar formation and highlight the specificity of this catalytic isoform in cardiac fibroblast/myofibroblast biology.

microRNA-122 down-regulation may play a role in severe myocardial fibrosis in human aortic stenosis through TGF-ß1 up-regulation.

miRNAs (microRNAs) have been shown to play a role in myocardial fibrosis. The present study was designed to analyse whether alterations in miRNA expression contribute to the progression of myocardial fibrosis in AS (aortic valve stenosis) patients through up-regulation of the pro-fibrotic factor TGF-ß1 (transforming growth factor-ß type 1). Endomyocardial biopsies were obtained from 28 patients with severe AS, and from the necropsies of 10 control subjects. AS patients presented increased myocardial CVF (collagen volume fraction) and TGF-ß1 compared with the controls, these parameters being correlated in all patients. Patients were divided into two groups by cluster analysis according to their CVF: SF (severe fibrosis; CVF >15%; n=15) and non-SF (CVF ≤15%; n=13). TGF-ß1 was increased in patients with SF compared with those with non-SF. To analyse the involvement of miRNAs in SF, the miRNA expression profile of 10 patients (four with non-SF and six with SF) was analysed showing that 99 miRNAs were down-regulated and 19 up-regulated in the SF patients compared with the non-SF patients. Those miRNAs potentially targeting TGF-ß1 were validated by real-time RT (reverse transcription)-PCR in the whole test population, corroborating that miR-122 and miR-18b were down-regulated in patients with SF compared with those with non-SF and the control subjects. Additionally, miR-122 was inversely correlated with the CVF, TGF-ß1 and the TGF-ß1-regulated PCPE-1 (procollagen C-terminal proteinase enhancer-1) in all patients. Experiments in human fibroblasts demonstrated that miR-122 targets and inhibits TGF-ß1. In conclusion, for the first time we show that myocardial down-regulation of miR-122 might be involved in myocardial fibrosis in AS patients, probably through TGF-ß1 up-regulation.

Role of nitric oxide and oxidative stress in a sheep model of persistent atrial fibrillation.

AIMS: Oxidative stress can modulate nitric oxide (NO) signalling pathways. Both pathways have been shown to be involved in the pathophysiology of atrial fibrillation (AF), but data are conflicting. We aimed to characterize the NO-pathway and its relation to oxidative stress in persistent AF in a sheep model. METHODS AND RESULTS: Persistent AF was induced by rapid atrial pacing for a mean of 136.5 ± 21.7 days. Non-stimulated sheep served as controls. Nicotine adenine dinucleotide phosphate (NADPH) oxidase-stimulated superoxide production was significantly increased in the AF group (+51.3 ± 23.2%, P < 0.01). Although there were no changes in mRNA expression of the different NADPH oxidase subunits, the increased activity was associated with markedly increased protein expression of the NADPH oxidase activator, Rac1 (+26 ± 4.6%, P < 0.05). No differences were seen in superoxide dismutase activity, but glutathione peroxidase activity was lower in the AF group. There was a marked accumulation of 3-nitrotyrosine, a biomarker for peroxynitrite, in atrial tissue of AF animals, as demonstrated by immunohistochemical staining and dot blot analysis (+15.6 ± 1.8%, P < 0.05). Expression of atrial NOS3 mRNA was 24.9 ± 4.4% lower in the AF group vs. control (P < 0.05), while NOS1 and 2 were unchanged. Immunoblot analysis revealed no changes in protein expression. Nitrite/nitrate levels were significantly lower during AF (-24.8 ± 5.8%, P < 0.05). CONCLUSION: In a sheep model of persistent AF, NOS3 transcript levels are attenuated and circulating NOx levels decreased. Persistent AF is associated with increased oxidative stress, probably resulting from increased NADPH oxidase activity, without major changes in anti-oxidant capacity of the atrial tissue.

Role of lysyl oxidase in myocardial fibrosis: from basic science to clinical aspects.

Because of its dynamic nature, the composition and structure of the myocardial collagen network can be reversibly modified to adapt to transient cardiac injuries. In response to persistent injury, however, irreversible, maladaptive changes of the network occur leading to fibrosis, mostly characterized by the excessive interstitial and perivascular deposition of collagen types I and III fibers. It is now becoming apparent that myocardial fibrosis directly contributes to adverse myocardial remodeling and the resulting alterations of left ventricular (LV) anatomy and function present in the major types of cardiac diseases. The enzyme lysyl oxidase (LOX) is a copper-dependent extracellular enzyme that catalyzes lysine-derived cross-links in collagen and elastin. LOX-mediated cross-linking of collagen types I and III fibrils leads to the formation of stiff collagen types I and III fibers and their subsequent tissue deposition. Evidence from experimental and clinical studies shows that the excess of LOX is associated with an increased collagen cross-linking and stiffness. It is thus conceivable that LOX upregulation and/or overactivity could underlie myocardial fibrosis and altered LV mechanics and contribute to the compromise of LV function in cardiac diseases. This review will consider the molecular aspects related to the regulation and actions of LOX, namely, in the context of collagen synthesis. In addition, it will address the information related to the role of myocardial LOX in heart failure and the potential benefits of controlling its expression and function.

Author Correction: MicroRNA-19b is a potential biomarker of increased myocardial collagen cross-linking in patients with aortic stenosis and heart failure.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Myocardial fibrosis in chronic kidney disease: potential benefits of torasemide.

Interstitial and perivascular fibrosis is a constant finding in heart biopsies and necropsy studies in patients with chronic kidney disease and hypertension, namely in those with left ventricular hypertrophy. Fibrosis is the result of the unbalance between exaggerated collagen synthesis and unchanged or depressed collagen degradation. A number of factors linked to hypertension and the progressive deterioration of renal function may facilitate such an unbalance. Patients with chronic kidney disease and hypertension are prone to develop diastolic heart failure, and myocardial fibrosis has been suggested as a major determinant of disturbances in diastolic function in these patients. Thus, the therapeutic strategies aimed to reduce cardiac fibrosis may provide a particular cardioprotective benefit in patients with chronic kidney disease. In this regard, recent data suggest that the loop diuretic torasemide reduces myocardial fibrosis and ameliorates cardiac function in patients with chronic heart failure through local mechanisms beyond its effects on the renal excretion of fluid and electrolytes and systemic hemodynamics.

MicroRNA-19b is a potential biomarker of increased myocardial collagen cross-linking in patients with aortic stenosis and heart failure.

This study analyzed the potential associations of 7 myocardial fibrosis-related microRNAs with the quality of the collagen network (e.g., the degree of collagen fibril cross-linking or CCL) and the enzyme lysyl oxidase (LOX) responsible for CCL in 28 patients with severe aortic stenosis (AS) of whom 46% had a diagnosis of chronic heart failure (HF). MicroRNA expression was analyzed in myocardial and blood samples. From the studied microRNAs only miR-19b presented a direct correlation (p < 0.05) between serum and myocardium. Compared to controls both myocardial and serum miR-19b were reduced (p < 0.01) in AS patients. In addition, miR-19b was reduced in the myocardium (p < 0.01) and serum (p < 0.05) of patients with HF compared to patients without HF. Myocardial and serum miR-19b were inversely correlated (p < 0.05) with LOX, CCL and LV stiffness in AS patients. In in vitro studies miR-19b inhibition increased (p < 0.05) connective tissue growth factor protein and LOX protein expression in human fibroblasts. In conclusion, decreased miR-19b may be involved in myocardial LOX up-regulation and excessive CCL, and consequently increased LV stiffness in AS patients, namely in those with HF. Serum miR-19b can be a biomarker of these alterations of the myocardial collagen network in AS patients, particularly in patients with HF.

Low-density lipoprotein-cholesterol-induced endothelial dysfunction and oxidative stress: the role of statins.

SIGNIFICANCE: Cardiovascular diseases (CVD) represent a major public health burden. High low-density lipoprotein (LDL)-cholesterol is a recognized pathogenic factor for atherosclerosis, and its complications and statins represent the most potent and widely used therapeutic approach to prevent and control these disorders. RECENT ADVANCES: A number of clinical and experimental studies concur to identify endothelial dysfunction as a primary step in the development of atherosclerosis, as well as a risk factor for subsequent clinical events. Oxidant stress resulting from chronic elevation of plasma LDL-cholesterol (LDL-chol) is a major contributor to both endothelial dysfunction and its complications, for example, through alterations of endothelial nitric oxide signaling. CRITICAL ISSUES: Statin treatment reduces morbidity and mortality of CVD, but increasing evidence questions that this is exclusively through reduction of plasma LDL-chol. The identification of ancillary effects on (cardio)vascular biology, for example, through their modulation of oxidative stress, will not only increase our understanding of their mechanisms of action, with a potential broadening of their indication(s), but also lead to the identification of new molecular targets for future therapeutic developments in CVD. FUTURE DIRECTIONS: Further characterization of molecular pathways targeted by statins, for example, not directly mediated by changes in plasma lipid concentrations, should enable a more comprehensive approach to the pathogenesis of (cardio)vascular disease, including, for example, epigenetic regulation and fine tuning of cell metabolism.

HMGCoA reductase inhibition reverses myocardial fibrosis and diastolic dysfunction through AMP-activated protein kinase activation in a mouse model of metabolic syndrome.

AIMS: The metabolic syndrome (MS) leads to myocardial fibrosis (MF) and diastolic dysfunction. Statins have proven beneficial effects in MS, but their impact on cardiac remodelling is uncertain. We examined the effects and mechanisms of chronic statin treatment on cardiac remodelling, e.g. fibrosis and diastolic properties. METHODS AND RESULTS: We used a mouse model deficient in leptin and the LDL-receptor (DKO) that reproduces this MS phenotype. DKO mice (12 weeks) were treated with rosuvastatin (R) for 6 months vs. placebo. Morphometric and echocardiographic measurements showed that R reduced cardiac mass and increased left-ventricular end-diastolic diameter despite unchanged cardiomyocyte dimensions. Similarly, R had no effect on the hypertrophic response to neurohormones in isolated cardiomyocytes. Conversely, R reversed the age-dependent development of MF as well as mRNA expression of TGF-ß1 and several pro-fibrotic markers (procollagen type I, its carboxy-terminal proteinase, Lysyl oxidase). R similarly inhibited the pro-fibrotic effects of TGF-ß1 on procollagen type I, alpha Smooth Muscle Actin expression and migratory properties of cardiac fibroblasts in vitro. In parallel, R increased the activation of AMP-activated protein kinase (AMPK), a known inhibitor of fibrosis, in vivo and in vitro, and the anti-fibrotic effects of R were abrogated in fibroblasts transfected with AMPKα1/α2 siRNA. The reversal of MF by R in DKO mice was accompanied with improved diastolic properties assessed by P-V loop analysis (slope of EDPVR, dP/dt min and cardiac output). CONCLUSION: In this model of MS, statin treatment reverses myocardial remodelling and improves ventricular relaxation through AMPK-mediated anti-fibrotic effects.

Blockade of TGF-ß 1 signalling inhibits cardiac NADPH oxidase overactivity in hypertensive rats.

NADPH oxidases constitute a major source of superoxide anion (·O(2)(-)) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-ß 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-ß 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR) to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-ß 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-ß 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-ß 1.

Cardiovascular translational medicine (III). Genomics and proteomics in heart failure research.

Heart failure is a complex syndrome and is one of the main causes of morbidity and mortality in developed countries. Despite considerable research effort in recent years, heart failure prevention and treatment strategies still suffer significant limitations. New theoretical and technical approaches are, therefore, required. It is in this context that the "omic" sciences have a role to play in heart failure. The incorporation of "omic" methodologies into the study of human disease has substantially changed biological approaches to disease and has given an enormous impetus to the search for new disease mechanisms, as well as for novel biomarkers and therapeutic targets. The application of genomics, proteomics and metabonomics to heart failure research could increase our understanding of the origin and development of the different processes contributing to this syndrome, thereby enabling the establishment of specific diagnostic profiles and therapeutic templates that could help improve the poor prognosis associated with heart failure. This brief review contains a short description of the fundamental principles of the "omic" sciences and an evaluation of how these new techniques are currently contributing to research into human heart failure. The focus is mainly on the analysis of gene expression microarrays in the field of genomics and on studies using two-dimensional electrophoresis with mass spectrometry in the area of proteomics.

Biochemical markers of myocardial remodelling in hypertensive heart disease.

The intricate mechanisms responsible for the structural remodelling of the myocardium that facilitates the evolution to heart failure in hypertensive patients, namely in those with left ventricular hypertrophy, requires from clinicians the utilization of a multibiomarker approach for short-term and long-term stratification as well as prognostication of patients. Biochemical markers may also help to identify patients with no clinical evidence of hypertensive heart disease, and provide information about the need for more aggressive therapy during different stages of the disease, and potentially provide valuable biochemical data for the specialist. Although there is a continuous and complex interplay between biochemical and imaging markers, perhaps their use will also have the potential to modify the medical management of patients with hypertensive heart disease and therapeutic decision-making by tailoring a targeted therapy according to the predominant mechanism of myocardial remodelling. This article will review in brief the most relevant information on a panel of circulating molecules that may accomplish the criteria required to be considered as biochemical markers of the cardiomyocyte and non-cardiomyocyte structural changes that occur in the hypertensive myocardium.

A synthetic peptide from transforming growth factor-beta1 type III receptor prevents myocardial fibrosis in spontaneously hypertensive rats.

AIM: We investigated whether P144, a synthetic peptide from transforming growth factor-beta(1) (TGF-beta(1)) type III receptor betaglycan, exhibits cardiac antifibrotic properties. METHODS AND RESULTS: The study was carried out in one group of 10-week-old normotensive Wistar-Kyoto rats treated with vehicle (V-WKY), one group of 10-week-old spontaneously hypertensive rats treated with vehicle (V-SHR), and one group of 10-week-old SHR treated with P144 (P144-SHR) for 12 weeks. Two more groups of 10-week-old untreated WKY and SHR were used to assess baseline values of the parameters tested. In addition, the effects of P144 on rat cardiac fibroblasts stimulated with TGF-beta(1) were also studied. Compared with V-WKY, V-SHR exhibited significant increases in the myocardial expression of phosphorylated Smad2, 38 and 42 kDa connective tissue growth factor (CTGF) isoforms, procollagen alpha1 (I) mRNA, and collagen type I protein, as well as in the expression of lysyl oxidase (LOX) mRNA and protein, collagen cross-linking and deposition. P144 administration was associated with significant reduction in all these parameters in P144-SHR. TGF-beta(1)-stimulated fibroblasts exhibited significant increases in phosphorylated Smad2, 38 and 42 kDa CTGF proteins, and procollagen alpha(1) (I) mRNA compared with control fibroblasts. No significant differences were found in these parameters between fibroblasts incubated with TGF-beta(1) and P144 and control fibroblasts. CONCLUSION: These results show that P144 inhibits TGF-beta(1)-dependent signalling pathway and collagen type I synthesis in cardiac fibroblasts. These effects may be involved in the ability of this peptide to prevent myocardial fibrosis in SHR.