Low Density Lipoprotein Cholesterol Decreases the Expression of Adenosine A2A Receptor and Lipid Rafts-Protein Flotillin-1: Insights on Cardiovascular Risk of Hypercholesterolemia.

High blood levels of low-density lipoprotein (LDL)-cholesterol (LDL-C) are associated with atherosclerosis, mainly by promoting foam cell accumulation in vessels. As cholesterol is an essential component of cell plasma membranes and a regulator of several signaling pathways, LDL-C excess may have wider cardiovascular toxicity. We examined, in untreated hypercholesterolemia (HC) patients, selected regardless of the cause of LDL-C accumulation, and in healthy participants (HP), the expression of the adenosine A2A receptor (A2AR), an anti-inflammatory and vasodilatory protein with cholesterol-dependent modulation, and Flotillin-1, protein marker of cholesterol-enriched plasma membrane domains. Blood cardiovascular risk and inflammatory biomarkers were measured. A2AR and Flotillin-1 expression in peripheral blood mononuclear cells (PBMC) was lower in patients compared to HP and negatively correlated to LDL-C blood levels. No other differences were observed between the two groups apart from transferrin and ferritin concentrations. A2AR and Flotillin-1 proteins levels were positively correlated in the whole study population. Incubation of HP PBMCs with LDL-C caused a similar reduction in A2AR and Flotillin-1 expression. We suggest that LDL-C affects A2AR expression by impacting cholesterol-enriched membrane microdomains. Our results provide new insights into the molecular mechanisms underlying cholesterol toxicity, and may have important clinical implication for assessment and treatment of cardiovascular risk in HC.

Increased fecal ethanol and enriched ethanol-producing gut bacteria Limosilactobacillus fermentum, Enterocloster bolteae, Mediterraneibacter gnavus and Streptococcus mutans in nonalcoholic steatohepatitis.

Background: Non-alcoholic steatohepatitis (NASH) has become a major public health issue as one of the leading causes of liver disease and transplantation worldwide. The instrumental role of the gut microbiota is emerging but still under investigation. Endogenous ethanol (EtOH) production by gut bacteria and yeasts is an emerging putative mechanism. Microbial metagenomics and culture studies targeting enterobacteria or yeasts have been reported, but no culturomics studies have been conducted so far. Aim: To assess fecal EtOH and other biochemical parameters, characterize NASH-associated dysbiosis and identify EtOH-producing gut microbes associated with the disease, fecal samples from 41 NASH patients and 24 controls were analyzed. High-performance liquid chromatography (HPLC) was used for EtOH, glucose, total proteins, triglyceride and total cholesterol. Viable bacteria were assessed with microbial culturomics. Microbial genetic material was assessed using 16S metagenomics targeting the hypervariable V3V4 region. Results: Fecal EtOH and glucose was elevated in the stools of NASH patients (p < 0.05) but not triglyceride, total cholesterol or proteins. In culturomics, EtOH-producing Enterocloster bolteae and Limosilactobacillus fermentum were enriched in NASH. V3V4 16S rRNA amplicon sequencing confirmed the enrichment in EtOH-producing bacteria including L. fermentum, Mediterraneibacter gnavus and Streptococcus mutans, species previously associated with NASH and other dysbiosis-associated diseases. Strikingly, E. bolteae was identified only by culturomics. The well-known Lacticaseibacillus casei was identified in controls but never isolated in patients with NASH (p < 0.05). Conclusion: Elevated fecal EtOH and glucose is a feature of NASH. Several different EtOH-producing gut bacteria may play an instrumental role in the disease. Culturomics and metagenomics, two complementary methods, will be critical to identify EtOH-producing bacteria for future diagnostic markers and therapeutic targets for NASH. Suppression of EtOH-producing gut microbes and L. casei administration are options to be tested in NASH treatment.

Adenosinergic System and Neuroendocrine Syncope: What Is the Link?

Although very common, the precise mechanisms that explain the symptomatology of neuroendocrine syncope (NES) remain poorly understood. This disease, which can be very incapacitating, manifests itself as a drop in blood pressure secondary to vasodilation and/or extreme slowing of heart rate. As studies continue, the involvement of the adenosinergic system is becoming increasingly evident. Adenosine, which is an ATP derivative, may be involved in a large number of cases. Adenosine acts on G protein-coupled receptors with seven transmembrane domains. A1 and A2A adenosine receptor dysfunction seem to be particularly implicated since the activation leads to severe bradycardia or vasodilation, respectively, two cardinal symptoms of NES. This mini-review aims to shed light on the links between dysfunction of the adenosinergic system and NHS. In particular, signal transduction pathways through the modulation of cAMP production and ion channels in relation to effects on the cardiovascular system are addressed. A better understanding of these mechanisms could guide the pharmacological development of new therapeutic approaches.

Myogenesis in C2C12 Cells Requires Phosphorylation of ATF6α by p38 MAPK.

Activating transcription factor 6α (ATF6α) is an endoplasmic reticulum protein known to participate in unfolded protein response (UPR) during ER stress in mammals. Herein, we show that in mouse C2C12 myoblasts induced to differentiate, ATF6α is the only pathway of the UPR activated. ATF6α stimulation is p38 MAPK-dependent, as revealed by the use of the inhibitor SB203580, which halts myotube formation and, at the same time, impairs trafficking of ATF6α, which accumulates at the cis-Golgi without being processed in the p50 transcriptional active form. To further evaluate the role of ATF6α, we knocked out the ATF6α gene, thus inhibiting the C2C12 myoblast from undergoing myogenesis, and this occurred independently from p38 MAPK activity. The expression of exogenous ATF6α in knocked-out ATF6α cells recover myogenesis, whereas the expression of an ATF6α mutant in the p38 MAPK phosphorylation site (T166) was not able to regain myogenesis. Genetic ablation of ATF6α also prevents the exit from the cell cycle, which is essential for muscle differentiation. Furthermore, when we inhibited differentiation by the use of dexamethasone in C2C12 cells, we found inactivation of p38 MAPK and, consequently, loss of ATF6α activity. All these findings suggest that the p-p38 MAPK/ATF6α axis, in pathophysiological conditions, regulates myogenesis by promoting the exit from the cell cycle, an essential step to start myoblasts differentiation.

Adenosine and Adenosine Receptors: Advances in Atrial Fibrillation.

Atrial fibrillation (AF) is the most common arrhythmia in the world. Because the key to developing innovative therapies that limit the onset and the progression of AF is to fully understand the underlying molecular mechanisms of AF, the aim of the present narrative review is to report the most recent advances in the potential role of the adenosinergic system in the pathophysiology of AF. After a comprehensive approach describing adenosinergic system signaling and the mechanisms of the initiation and maintenance of AF, we address the interactions of the adenosinergic system's signaling with AF. Indeed, adenosine release can activate four G-coupled membrane receptors, named A1, A2A, A2B and A3. Activation of the A2A receptors can promote the occurrence of delayed depolarization, while activation of the A1 receptors can shorten the action potential's duration and induce the resting membrane's potential hyperpolarization, which promote pulmonary vein firing, stabilize the AF rotors and allow for functional reentry. Moreover, the A2B receptors have been associated with atrial fibrosis homeostasis. Finally, the adenosinergic system can modulate the autonomous nervous system and is associated with AF risk factors. A question remains regarding adenosine release and the adenosine receptors' activation and whether this would be a cause or consequence of AF.

Endogenous Ethanol and Triglyceride Production by Gut Pichia kudriavzevii, Candida albicans and Candida glabrata Yeasts in Non-Alcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) increases with fructose consumption and metabolic syndrome and has been recently linked with endogenous ethanol production, notably by high alcohol-producing Klebsiella pneumoniae (HiAlc Kpn). Candida yeasts are the main causes of auto-brewery syndromes but have been neglected in NASH. Here, the fecal ethanol and microbial content of 10 cases and 10 controls were compared. Ethanol was measured by gas chromatography-mass spectrometry. Species identification was performed by MALDI-TOF MS, and triglyceride production was assessed by a colorimetric enzymatic assay. The fecal ethanol concentration was four times higher in patients with NASH (median [interquartile range]: 0.13 [0.05-1.43] vs. 0.034 [0.008-0.57], p = 0.037). Yeasts were isolated from almost all cases but not from controls (9/10 vs. 0/10, p = 0.0001). Pichia kudriavzevii was the most frequent (four patients), while Candida glabrata, Candida albicans, and Galactomyces geotrichum were identified in two cases each. The concentration of ethanol produced by yeasts was 10 times higher than that produced by bacteria (median, 3.36 [0.49-5.60] vs. 0.32 [0.009-0.43], p = 0.0029). Using a 10% D-fructose restricted medium, we showed that NASH-associated yeasts transformed fructose in ethanol. Unexpectedly, yeasts isolated from NASH patients produced a substantial amount of triglycerides. Pichia kudriavzevii strains produced the maximal ethanol and triglyceride levels in vitro. Our preliminary human descriptive and in vitro experimental results suggest that yeasts have been neglected. In addition to K. pneumoniae, gut Pichia and Candida yeasts could be linked with NASH pathophysiology in a species- and strain-specific manner through fructose-dependent endogenous alcohol and triglyceride production.

Contribution of Adenosine in the Physiological Changes and Injuries Secondary to Exposure to Extreme Oxygen Pressure in Healthy Subjects.

Climbers and aviators are exposed to severe hypoxia at high altitudes, whereas divers are exposed to hyperoxia at depth. The aim of this study was to report changes in the adenosinergic system induced by exposure to extreme oxygen partial pressures. At high altitudes, the increased adenosine concentration contributes to brain protection against hypoxia through various mechanisms such as stimulation of glycogenolysis for ATP production, reduction in neuronal energy requirements, enhancement in 2,3-bisphosphoglycerate production, and increase in cerebral blood flow secondary to vasodilation of cerebral arteries. In the context of mountain illness, the increased level of A2AR expression leads to glial dysfunction through neuroinflammation and is involved in the pathogenesis of neurological disorders. Nonetheless, a high level of adenosine concentration can protect against high-altitude pulmonary edema via a decrease in pulmonary arterial pressure. The adenosinergic system is also involved in the acclimatization phenomenon induced by prolonged exposure to altitude hypoxia. During hyperoxic exposure, decreased extracellular adenosine and low A2A receptor expression contribute to vasoconstriction. The resulting decrease in cerebral blood flow is considered a preventive phenomenon against cerebral oxygen toxicity through the decrease in oxygen delivery to the brain. With regard to lung oxygen toxicity, hyperoxia leads to an increase in extracellular adenosine, which acts to preserve pulmonary barrier function. Changes in the adenosinergic system induced by exposure to extreme oxygen partial pressures frequently have a benefit in decreasing the risk of adverse effects.

Plasma A2AR Measurement Can Help Physicians Identify Patients Suspected of Coronary Chronic Syndrome: A Pilot Study.

The evaluation of suspected coronary artery disease (CAD) in the medical community is challenging. Patients with suspected coronary chronic syndrome (CCS) are referred by the medical community to be assessed by specialists for the performance of noninvasive tests that have high rates of false positives and false negatives. While troponins are the gold standard for evaluate myocardial injuries, there is no biomarker to assess myocardial ischemia in patient populations with negative electrocardiography or without an increase in troponin level. A2A adenosine receptors control the coronary blood flow through its vasodilating properties. It has been shown that patients with CAD have a lower A2AR expression on peripheral blood mononuclear cells, suggesting a link between A2AR production and the severity of CAD. Herein, we present a new and innovative method of inhibition ELISA for A2AR in the plasma of patients who permit the evaluation of the amount of soluble A2AR. For this analysis, the total study sample was 54, including 31 patients with CAD with stenosis > 50% and a significant fractional flow reserve (FFR < 0.8) (Group 1) and 23 patients with normal or non-obstructive coronary arteries (stenosis < 50% and nonsignificant FFR > 0.8) (Group 2). The % inhibition (which is linked to the presence of soluble receptors) with the plasma of patients with FFR < 0.8 was significantly lower than that of patients with FFR > 0.8 (median [range]: 68% [20.7−86.9] vs. 83% [67−88.4]; p < 0.001). The ROC curve indicated a good sensitivity/specificity ratio with a cut off of 72.5% and an area under the curve of 0.87. In conclusion, a rapid ELISA to assess soluble A2AR in the plasma shows promise to screen patients suspected of having CAD.

Copper exposure improves the upper thermal tolerance in a sex-specific manner, irrespective of fish thermal history.

Ectotherms can respond to climate change via evolutionary adaptation, usually resulting in an increase of their upper thermal tolerance. But whether such adaptation influences the phenotypic plasticity of thermal tolerance when encountering further environmental stressors is not clear yet. This is crucial to understand because organisms experience multiple stressors, besides warming climate, in their natural environment and pollution is one of those. Here, we studied the phenotypic plasticity of thermal tolerance in three-spined stickleback populations inhabiting spatially replicated thermally polluted and pristine areas before and after exposing them to a sublethal concentration of copper for one week. We found that the upper thermal tolerance and its phenotypic plasticity after copper exposure did not depend on the thermal history of fish, suggesting that five decades of thermal pollution did not result in evolutionary adaptation to thermal tolerance. The upper thermal tolerance of fish was, on the other hand, increased by ∼ 1.5 °C after 1-week copper exposure in a sex-specific manner, with males having higher plasticity. To our knowledge this is the first study that shows an improvement of the upper thermal tolerance as a result of metal exposure. The results suggest that three-spined sticklebacks are having high plasticity and they are capable of surviving in a multiple-stressor scenario in the wild and that male sticklebacks seem more resilient to fluctuating environmental conditions than female.

Hyperhomocysteinemia and cardiovascular diseases.

Homocysteine (Hcy) is a sulfhydryl-containing amino acid, which is not acquired through the diet, but rather synthesized as an intermediate metabolite in the methionine cycle. Hcy is present in plasma, with normal levels between 5 and 15 µmol/L, a slightly elevated level between 15 to 30 µmol/L, moderate from 30 to 100 µmol/L and a value > 100 µmol/L classified as severe hyperhomocysteinemia (HHcy). HHcy has been associated with inflammation and atherosclerosis and is considered an independent risk factor for cardiovascular diseases (CVD). Here, we review the main evidence showing the association and the possible involvement of HHcy in the most common CVD.

FGF10 promotes cardiac repair through a dual cellular mechanism increasing cardiomyocyte renewal and inhibiting fibrosis.

AIMS: Promoting cardiomyocyte renewal represents a major therapeutic approach for heart regeneration and repair. Our study aims to investigate the relevance of FGF10 as a potential target for heart regeneration. METHODS AND RESULTS: Our results first reveal that Fgf10 levels are up-regulated in the injured ventricle after MI. Adult mice with reduced Fgf10 expression subjected to MI display impaired cardiomyocyte proliferation and enhanced cardiac fibrosis, leading to a worsened cardiac function and remodelling post-MI. In contrast, conditional Fgf10 overexpression post-MI revealed that, by enhancing cardiomyocyte proliferation and preventing scar-promoting myofibroblast activation, FGF10 preserves cardiac remodelling and function. Moreover, FGF10 activates major regenerative pathways including the regulation of Meis1 expression levels, the Hippo signalling pathway and a pro-glycolytic metabolic switch. Finally, we demonstrate that elevated FGF10 levels in failing human hearts correlate with reduced fibrosis and enhanced cardiomyocyte proliferation. CONCLUSIONS: Altogether, our study shows that FGF10 promotes cardiac regeneration and repair through two cellular mechanisms: elevating cardiomyocyte renewal and limiting fibrosis. This study thus identifies FGF10 as a clinically relevant target for heart regeneration and repair in man.

A2 Adenosine Receptor Subtypes Overproduction in Atria of Perioperative Atrial Fibrillation Patients Undergoing Cardiac Surgery: A Pilot Study.

Objective: Although atrial fibrillation is a common cardiac arrhythmia in humans, the mechanism that leads to the onset of this condition is poorly elucidated. Adenosine is suspected to be implicated in the trigger of atrial fibrillation (AF) through the activation of its membrane receptors, mainly adenosine receptor (AR) subtypes A1R and A2R. In this study, we compared blood adenosine concentration (BAC), and A1R, A2AR, and A2BR production in right (RA) and left atrium (LA), and on peripheral blood mononuclear cells (PBMCs) in patients with underlying structural heart disease undergoing cardiac surgery with or without peri-operative AF (PeOpAF). Methods: The study group consisted of 39 patients (30 men and 9 women, mean age, range 65 [40-82] years) undergoing cardiac surgery and 20 healthy patients (8 women and 12 men; mean age, range 60 [39-72] years) as controls were included. Among patients, 15 exhibited PeOpAF. Results: Blood adenosine concentration was higher in patients with PeOpAF than others. A2AR and A2BR production was higher in PBMCs of patients compared with controls and was higher in PeOpAF patients than other patients. In LA and RA, the production of A2AR and A2BR was higher in patients with PeOpAF than in other patients. Both A2AR and A2BR production were higher in LA vs. RA. A1R production was unchanged in all situations. Finally, we observed a correlation between A1R, A2AR, and A2BR production evaluated on PBMCs and those evaluated in LA and RA. Conclusions: Perioperative AF was associated with high BAC and high A2AR and A2BR expression, especially in the LA, after cardiac surgery in patients with underlying structural heart disease. Whether these increases the favor in triggering the AF in this patient population needs further investigation.

Adenosine Receptor Reserve and Long-Term Potentiation: Unconventional Adaptive Mechanisms in Cardiovascular Diseases?

While the concept of a receptor reserve (spare receptors) is old, their presence on human cells as an adaptive mechanism in cardiovascular disease is a new suggestion. The presence of spare receptors is suspected when the activation of a weak fraction of receptors leads to maximal biological effects, in other words, when the half-maximal effective concentration (EC50) for a biological effect (cAMP production, for example) is lower than the affinity (KD) of the ligand for a receptor. Adenosine is an ATP derivative that strongly impacts the cardiovascular system via its four membrane receptors, named A1R, A2AR, A2BR, and A3R, with the A1R being more particularly involved in heart rhythm, while the A2AR controls vasodilation. After a general description of the tools necessary to explore the presence of spare receptors, this review focuses on the consequences of the presence of spare adenosine receptors in cardiovascular physiopathology. Finally, the role of the adenosinergic system in the long-term potentiation and its possible consequences on the physiopathology are also mentioned.

Hyperhomocysteinemia and Cardiovascular Disease: Is the Adenosinergic System the Missing Link?

The influence of hyperhomocysteinemia (HHCy) on cardiovascular disease (CVD) remains unclear. HHCy is associated with inflammation and atherosclerosis, and it is an independent risk factor for CVD, stroke and myocardial infarction. However, homocysteine (HCy)-lowering therapy does not affect the inflammatory state of CVD patients, and it has little influence on cardiovascular risk. The HCy degradation product hydrogen sulfide (H2S) is a cardioprotector. Previous research proposed a positive role of H2S in the cardiovascular system, and we discuss some recent data suggesting that HHCy worsens CVD by increasing the production of H2S, which decreases the expression of adenosine A2A receptors on the surface of immune and cardiovascular cells to cause inflammation and ischemia, respectively.

Adenosine and Its Receptors: An Expected Tool for the Diagnosis and Treatment of Coronary Artery and Ischemic Heart Diseases.

Adenosine is an endogenous nucleoside which strongly impacts the cardiovascular system. Adenosine is released mostly by endothelial cells and myocytes during ischemia or hypoxia and greatly regulates the cardiovascular system via four specific G-protein-coupled receptors named A1R, A2AR, A2BR, and A3R. Among them, A2 subtypes are strongly expressed in coronary tissues, and their activation increases coronary blood flow via the production of cAMP in smooth muscle cells. A2A receptor modulators are an opportunity for intense research by the pharmaceutical industry to develop new cardiovascular therapies. Most innovative therapies are mediated by the modulation of adenosine release and/or the activation of the A2A receptor subtypes. This review aims to focus on the specific exploration of the adenosine plasma level and its relationship with the A2A receptor, which seems a promising biomarker for a diagnostic and/or a therapeutic tool for the screening and management of coronary artery disease. Finally, a recent class of selective adenosine receptor ligands has emerged, and A2A receptor agonists/antagonists are useful tools to improve the management of patients suffering from coronary artery disease.

Homocysteine concentration and adenosine A2A receptor production by peripheral blood mononuclear cells in coronary artery disease patients.

Hyperhomocysteinemia is associated with coronary artery disease (CAD). The mechanistic aspects of this relationship are unclear. In CAD patients, homocysteine (HCy) concentration correlates with plasma level of adenosine that controls the coronary circulation via the activation of adenosine A2A receptors (A2A R). We addressed in CAD patients the relationship between HCy and A2A R production, and in cellulo the effect of HCy on A2A R function. 46 patients with CAD and 20 control healthy subjects were included. We evaluated A2A R production by peripheral blood mononuclear cells using Western blotting. We studied in cellulo (CEM human T cells) the effect of HCy on A2A R production as well as on basal and stimulated cAMP production following A2A R activation by an agonist-like monoclonal antibody. HCy concentration was higher in CAD patients vs controls (median, range: 16.6 [7-45] vs 8 [5-12] µM, P < 0.001). A2A R production was lower in patients vs controls (1.1[0.62-1.6] vs 1.53[0.7-1.9] arbitrary units, P < 0.001). We observed a negative correlation between HCy concentration and A2A R production (r = -0.43; P < 0.0001), with decreased A2A R production above 25 µM HCy. In cellulo, HCy inhibited A2A R production, as well as basal and stimulated cAMP production. In conclusion, HCy is negatively associated with A2A R production in CAD patients, as well as with A2A R and cAMP production in cellulo. The decrease in A2A R production and function, which is known to hamper coronary blood flow and promote inflammation, may support CAD pathogenesis.

Compromised thermal tolerance of cardiovascular capacity in upstream migrating Arctic char and brown trout-are hot summers threatening migrating salmonids?

Heat waves are threatening fish around the world, leading sometimes to mass mortality events. One crucial function of fish failing in high temperatures is oxygen delivery capacity, i.e. cardiovascular function. For anadromous salmonids, increased temperature could be especially detrimental during upstream migration since they need efficiently working oxygen delivery system in order to cross the river rapids to reach upstream areas. The migration also occurs during summer and early autumn exposing salmonids to peak water temperatures, and in shallow rivers there is little availability for thermal refuges as compared to thermally stratified coastal and lake habitats. In order to shed light on the mechanisms underpinning the capacity of migrating fish to face high environmental temperatures, we applied a physiological and molecular approach measuring cardiovascular capacities of migrating and resident Arctic char (Salvelinus alpinus) and brown trout (Salmo trutta) in Northern Norway. The maximum cardiovascular capacity of migrating fish was significantly lower compared to the resident conspecifics. The onset of cardiac impairment started only 2°C higher than river temperature, meaning that even a small increase in water temperature may already compromise cardiac function. The migrating fish were also under significant cellular stress, expressing increased level of cardiac heat shock proteins. We consider these findings highly valuable when addressing climate change effect on migrating fish and encourage taking action in riverine habitat conservation policies. The significant differences in upper thermal tolerance of resident and migrating fish could also lead changes in population dynamics, which should be taken into account in future conservation plans.

Hsp70s transcription-translation relationship depends on the heat shock temperature in zebrafish.

Virtually all organisms respond to heat shock by transcription of genes encoding for heat shock proteins (HSPs), but the mechanisms behind post-transcriptional regulation are not known in detail. When we exposed zebrafish to 5 and 7 °C above normal rearing temperature for 30 min, hsp70 mRNA expression was 28 and 150 -fold higher than in control, respectively. Protein expression, on the other hand, showed no significant change at the +5 °C and a 2-fold increase at the +7 °C exposure. This suggests that the transcription of hsp70 gene does not immediately correspond to translation to related proteins under certain stress temperatures, but, when the temperature is higher, and potentially detrimental, transcription and translation are intimately coupled. Those results confirm that temperature is an important abiotic factor involved in heat shock post-transcriptional regulation mechanisms in fish. However, further studies are needed to determine the relationship between this environmental factor and post-transcriptional regulation mechanisms. Earlier, the coupling/uncoupling of hsp transcription and translation has only been studied using cold-water fish, or zebrafish embryos. With current findings, we suggest this mechanism might be present even in adult warm water fish like the zebrafish.