Isabelle Ernens

Adenosine Inhibits Matrix Metalloproteinase-9 Secretion By Neutrophils: Implication of A2a Receptor and cAMP/PKA/Ca2+ Pathway

Matrix metalloproteinases (MMPs), and in particular MMP-9 secreted by neutrophils, are capable of degrading the matrix components of the heart and are thought to be the driving force behind myocardial matrix remodeling after infarction. Adenosine, a naturally produced nucleoside, has been shown to have cardioprotective effects and to inhibit secretion of various cytokines. The aim of our study was to determine the effect of adenosine on the secretion of MMP-9 by neutrophils. Neutrophils were isolated from healthy volunteers through Ficoll and Dextran sedimentation. Neutrophils were activated by N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence or absence of adenosine or adenosine analogs. Zymography and enzyme linked immunosorbent assay were used to measure MMP-9 secretion. Adenosine (1 &mgr;mol/L) decreased the fMLP-induced MMP-9 secretion by 30±2% (n=8, P<0.001). The effect was dose-dependent and was not specific to fMLP because adenosine also inhibited MMP-9 secretion by LPS- or H2O2-stimulated neutrophils. The effect of adenosine was mimicked by the adenosine A2a receptor agonist CGS21680 and was inhibited by both the A2a antagonist SCH5826 and A2a RNA silencing. The A3 agonist IB-MECA moderately decreased fMLP-induced MMP-9 secretion. Agonists and antagonists of the other types of adenosine receptors had no significant effect. Adenosine increased intracellular cAMP concentration and accelerated the return to baseline of the intracytoplasmic calcium peak. The inhibition of MMP-9 secretion by adenosine, as well as the calcium effect, was prevented by the protein kinase A inhibitor H-89. In conclusion, we show here that adenosine inhibits MMP-9 secretion by neutrophils. Our results suggest that this effect implies the A2a receptor and is mediated through the cAMP/PKA/Ca2+ pathway. Therefore, adenosine may represent a new approach to prevent matrix degradation and remodeling after myocardial injury.

Adenosine up-regulates vascular endothelial growth factor in human macrophages

It is known from animal models that the cardioprotective nucleoside adenosine stimulates angiogenesis mainly through up-regulation of vascular endothelial growth factor (VEGF). Since macrophages infiltrate the heart after infarction and because adenosine receptors behave differently across species, we evaluated the effect of adenosine on VEGF in human macrophages. Adenosine dose-dependently up-regulated VEGF expression and secretion by macrophages from healthy volunteers. VEGF production was also increased by blockade of extracellular adenosine uptake with dipyridamole. This effect was exacerbated by the toll-like receptor-4 ligands heparan sulfate, hyaluronic acid and lipopolysaccharide, and was associated with an increase of hypoxia inducible factor-1alpha expression, the main transcriptional inducer of VEGF in hypoxic conditions. The agonist of the adenosine A2A receptor CGS21680 reproduced the increase of VEGF and the antagonist SCH58261 blunted it. In conclusion, these results provide evidence that activation of adenosine A2A receptor stimulates VEGF production in human macrophages. This study suggests that adenosine is a unique pro-angiogenic molecule that may be used to stimulate cardiac repair.

Activation of the adenosine-A3 receptor stimulates matrix metalloproteinase-9 secretion by macrophages

AIMS Matrix metalloproteinase-9 (MMP-9) plays an important role in ventricular remodelling after acute myocardial infarction (MI). The cardioprotectant adenosine (Ado) may be involved in ventricular remodelling. We have shown that Ado inhibits the secretion of MMP-9 by human neutrophils. This study investigated the effect of Ado on MMP-9 production by human macrophages. METHODS AND RESULTS Cells used in this study were monocytes of healthy volunteers, a human monocyte cell line, and leukocytes from patients following MI. Monocytes were differentiated into macrophages and treated with Ado. Ado enhanced MMP-9 secretion by human macrophages in a time- and dose-dependent manner. Increasing the level of endogenous Ado by inhibition of Ado deaminase or Ado transferase also increased MMP-9 secretion. Ado enhanced MMP-9 production when macrophages were activated by hypoxia or Toll-like receptor-4 ligands such as lipopolysaccharide, hyaluronan, and heparan sulfate. The effect of Ado was replicated by the A3 agonist IB-MECA and inhibited by silencing the A3 receptor. Ado improved monocyte capacity to migrate through a matrix of gelatin B, and this effect was blocked by inhibition of MMP-9 activity. The chemotactic capacity of macrophages was reduced by Ado through a loss of expression of the monocyte chemotactic protein-1 receptor. Finally, MMP-9 expression was higher in blood cells from patients with acute MI compared with healthy volunteers. CONCLUSION Adenosine activates MMP-9 secretion by macrophages through its A3 receptor. The effect is in contrast to that observed in neutrophils, where Ado inhibits MMP-9 secretion by the A2a receptor. These observations may have important implications for therapeutic strategies targeting Ado receptors in the setting of MI.

Adenosine stimulates angiogenesis by up-regulating production of thrombospondin-1 by macrophages

Increase of blood capillary density at the interface between normal and ischemic tissue after acute MI reduces infarct size and improves cardiac function. Cardiac injury triggers the production of the matricellular component TSP‐1, but its role in angiogenesis is not clear, as both anti‐ and proangiogenic properties have been reported. It is unknown whether TSP‐1 is modulated by other factors released during cardiac injury. Among these, Ado is a well‐known promoter of angiogenesis. This study determined whether Ado modulates TSP‐1 expression and the implication on angiogenesis. Ado dose dependently increased the production of TSP‐1 by human macrophages. With the use of agonists and antagonists of AdoRs, coupled to RNA interference, we observed that this effect is mediated via A2AR and A2BR. The Ado effect was reproduced by cholera toxin (Gs protein activator) and forskolin (adenylate cyclase activator) and blocked by the PKA inhibitor H89. Conditioned medium from Ado‐treated macrophages stimulated microvessel outgrowth from aortic ring explants by 400%, and induced vessel formation in matrigel plugs. Microvessel outgrowth and vessel formation were blocked completely by addition of anti‐TSP‐1 antibodies to conditioned medium. Chronic administration of Ado to rats after MI maintained long‐term expression of TSP‐1 in the infarct border zone, and this was associated with enhanced border‐zone vascularization. Ado up‐regulates TSP‐1 production by macrophages, resulting in stimulation of angiogenesis. The mechanism involves A2AR and A2BR and is mediated through the cAMP/PKA pathway. This information may be important when designing Ado‐based therapies of angiogenesis.

Adenosine modifies the balance between membrane and soluble forms of Flt‐1

VEGFR‐1 (or Flt‐1) exists under a sFlt‐1 or a mFlt‐1 form. sFlt‐1 is antiangiogenic, and mFlt‐1 is proangiogenic. The cardioprotective nucleoside Ado is proangiogenic, but its effects on Flt‐1 are unknown and were tested in this study. In primary human macrophages from healthy volunteers, Ado inhibited sFlt‐1 expression induced by LPS (–43%, P=0.006), HS, and IL‐1β but not hypoxia. This effect was also observed in macrophages from patients with acute MI (–33%, P<0.001). It was reproduced by the A2A Ado receptor agonist CGS21680 and abrogated by the A2A antagonist SCH58261. Conversely, Ado increased mFlt‐1 expression, thus switching sFlt‐1 from the soluble toward the membrane form. This switch was also present in macrophages from acute MI patients (P<0.001). Assessment of HIF‐1α nuclear translocation and activation together with siRNA experiments suggested that the effect of Ado on Flt‐1 involves HIF‐1α. In conclusion, Ado down‐regulates sFlt‐1 and up‐regulates mFlt‐1 production, an effect that indicates that Ado may be used to stimulate angiogenesis in the heart.

Adenosine Reduces Cell Surface Expression of Toll-Like Receptor 4 and Inflammation in Response to Lipopolysaccharide and Matrix Products

Recent evidence suggests that Toll-like receptor 4 (TLR4) is not only involved in innate immunity but is also an important mediator of adverse left ventricular remodeling and heart failure following acute myocardial infarction (MI). TLR4 is activated by lipopolysaccharide (LPS) but also by products of matrix degradation such as hyaluronic acid and heparan sulfate. Although cardioprotective properties of adenosine (Ado) have been extensively studied, its potential to interfere with TLR4 activation is unknown. We observed that TLR4 pathway is activated in white blood cells from MI patients. TLR4 mRNA expression correlated with troponin T levels (R2 = 0.75; P = 0.01) but not with levels of white blood cells and C-reactive protein. Ado downregulated TLR4 expression at the surface of human macrophages (−50%, P < 0.05). Tumor necrosis factor-α production induced by the TLR4 ligands LPS, hyaluronic acid, and heparan sulfate was potently inhibited by Ado (−75% for LPS, P < 0.005). This effect was reproduced by the A2A Ado receptor agonist CGS21680 and the non-selective agonist NECA and was inhibited by the A2A antagonist SCH58261 and the A2A/A2B antagonist ZM241,385. In contrast, Ado induced a 3-fold increase of TLR4 mRNA expression (P = 0.008), revealing the existence of a feedback mechanism to compensate for the loss of TLR4 expression at the cell surface. In conclusion, the TLR4 pathway is activated after MI and correlates with infarct severity but not with the extent of inflammation. Reduction of TLR4 expression by Ado may therefore represent an important strategy to limit remodeling post-MI.

New dimensions in circadian clock function: the role of biological sex

Cardiovascular physiology and pathophysiology are both profoundly influenced by 24-h (circadian or diurnal) rhythms, ranging from daily heart rate and blood pressure variations to onset and outcome of myocardial infarction. With at least 10% of genes in the intact heart showing significant time-of-day dependent oscillations, it does not strike as a surprise that disrupting diurnal rhythms presents a major risk of developing cardiovascular disease. The importance of investigating 24-h rhythms in the cardiovascular context is emerging especially since the main cellular clockwork mechanism, including key clock genes Bmal1/2, Clock, Cry1/2 and Per1/2/3, has now been identified within all cardiovascular tissues. In this issue of Cardiovascular Research, Alibhai et al. bring a unique set of data to the field by exploring biological sex as an important player in the development of age-dependent cardiomyopathy in Clock mice. In these mutant mice the Clock gene is ubiquitously mutated resulting in a protein incapable of proper heterodimerization with BMAL and thereby lacking the capacity of transcriptional activation of Per. As such, the transcriptional-translational feedback loop is severely disturbed and mice with this mutation lack circadian clock function in all their cell types. In a previous study, the authors observed the development of agedependent cardiac dysfunction in male Clock mice, confirming a previous finding linking the circadian clock with aging. In this study Alibhai et al. tackled the intriguing question whether female sex protects against clock-mediated accelerated pathological remodelling of the aging heart. The authors investigated several metabolic changes and differences between the male and female wild-type and Clock mice. They describe that even with the mutated Clock, in comparison to male, female mice do not develop age-dependent cardiac disease until a very late age of 21 months, when they show milder signs of cardiac aging. The phenotype observed in male mice, comprising increased heart weight, cardiomyocyte hypertrophy, dilation, reduced myogenic responsiveness, impaired contractility and interstitial fibrosis, was mitigated by female biological sex. Ovariectomized female Clock mice on the other hand did develop cardiac dilation, glucose intolerance and reduced cardiac cytochrome c oxidase; i.e. the same phenotype as observed in male Clock mice, corroborating the protective role of the female sex hormones along with the importance of Clock in age-dependent cardiomyopathy. Several questions arise from the work of Alibhai et al. For example, the next interesting step of investigation would be to determine the exact mechanism of the protectiveness of female biological sex. Metabolism is likely to be a key player in the interaction with oestrogens. Zhu et al. showed that by interacting with the intrinsic circadian clock in adipose tissue, oestrogen prevents abnormal lipid accumulation caused by circadian disruption. However, the exact mechanisms of estrogen-protecting role are complex and not yet fully understood. Furthermore, along with exploring the mechanisms and signalling pathways responsible for the protective role of female biological sex in agedependent cardiomyopathy, it would be of interest to investigate whether the same phenomena can be observed in other heart diseases with known influence of circadian oscillations, such as myocardial infarction. Notably, this work reinforces the importance of studying sex-specific responses to internal or external factors influencing aging and organ function as recently reviewed by Ventura-Clapier et al. Thus, the use of animals of both sexes should be promoted in basic research to investigate deeper the contribution of hormonal and non-hormonal actors (sex-specific gene expression) underlying sex differences. In the present context, as the important roles of circadian rhythms in cardiovascular (patho)physiology are emerging, new platforms to analyse them are being developed. Embryonic stem cell-derived cardiomyocytes, stem cell antigen 1-positive (SCA1þ) cells (SPCs) and neonatal rat cardiomyocytes are relevant in vitro models regarding both molecular and functional influence of circadian rhythms in the heart. In vivo rhythmicity can therefore be recapitulated, allowing for the physiological and disrupted cardiac clock to be monitored. The influence of biological sex in clock-mediated cardiotropic mechanisms could be studied using these recently established in vitro and in vivo systems by taking into account the sex of the cell donor and mimicking hormonal influences in culture conditions. This will advance our understanding of sex-specific responses to circadian rhythm dysregulation affecting the cardiac system and pave the way to the gender-tailored therapy (Figure 1). In conclusion, this is the first time that interaction of ovarian hormones and circadian rhythms has been shown to influence cardiac aging.

Restoration of cardiac function after anaemia-induced heart failure in zebrafish

AIMS New therapeutic approaches are needed to fight against the growing epidemic of heart failure. Unlike mammals, zebrafish possess the incredible ability to regenerate cardiac tissue after acute trauma such as apical resection. Yet, the ability of zebrafish to recover after a chronic stress leading to heart failure has not been reported. The aim of this study was to test whether zebrafish can recover a normal cardiac function after anaemia-induced heart failure. METHODS AND RESULTS Eight- to ten-month-old zebrafish were treated with phenylhydrazine hydrochloride, an anaemia inducer, to generate heart failure. Treatment was stopped after 5 weeks and fish were followed-up for 3 weeks. Assessment of ventricular function by ultrasound at the end of the treatment revealed an increase in ventricle diameter (+47%) and a decrease in heart rate (-36%) and fractional shortening (-30%). A decrease in swim capacity was also observed (-31%). Tissue staining showed a thickening of the ventricular wall (5-fold), cell apoptosis and proliferation but no fibrosis. Expression of foetal genes, angiogenic factor and inflammation markers was increased, and β-adrenergic receptor-1 was decreased. Three weeks after phenylhydrazine hydrochloride withdrawal, all parameters returned to baseline and the fish recovered a normal cardiac function, tissue morphology and gene expression. CONCLUSIONS Zebrafish are able to completely recover from anaemia-induced heart failure. This model represents a unique opportunity to investigate the mechanisms of cardiac repair and may lead to the discovery of novel therapeutic targets of heart failure.

Atrial Structural Remodeling Gene Variants in Patients with Atrial Fibrillation

Atrial fibrillation (AF) is a common arrhythmia for which the genetic studies mainly focused on the genes involved in electrical remodeling, rather than left atrial muscle remodeling. To identify rare variants involved in atrial myopathy using mutational screening, a high-throughput next-generation sequencing (NGS) workflow was developed based on a custom AmpliSeq™ panel of 55 genes potentially involved in atrial myopathy. This workflow was applied to a cohort of 94 patients with AF, 76 with atrial dilatation and 18 without. Bioinformatic analyses used NextGENe® software and in silico tools for variant interpretation. The AmpliSeq custom-made panel efficiently explored 96.58% of the targeted sequences. Based on in silico analysis, 11 potentially pathogenic missense variants were identified that were not previously associated with AF. These variants were located in genes involved in atrial tissue structural remodeling. Three patients were also carriers of potential variants in prevalent arrhythmia-causing genes, usually associated with AF. Most of the variants were found in patients with atrial dilatation (n=9, 82%). This NGS approach was a sensitive and specific method that identified 11 potentially pathogenic variants, which are likely to play roles in the predisposition to left atrial myopathy. Functional studies are needed to confirm their pathogenicity.