Identification of human circulating factors following remote ischemic conditioning (RIC): Potential impact on stroke.

Remote ischemic conditioning (RIC) is a procedure consisting of short cycles of ischemia applied in a limb that activates endogenous protection in distant organs, such as the brain. Despite the promising outcomes of RIC, the biochemical factors governing inter-organ communication remain largely unexplored, particularly in humans. A pilot study on 20 healthy humans was performed to identify potential circulating biochemical factors involved in RIC signalling. Blood was collected before and immediately, 4 and 22 h after the end of RIC. To characterize the responses triggered by RIC, a combination of biochemical and proteomic analysis, along with functional in vitro tests in human cells, were performed. RIC did not alter the levels of nitric oxide, bilirubin and cell-free mitochondrial DNA. In contrast, carboxyhaemoglobin levels increased following RIC at all time points and young subset, suggesting endogenous production of carbon monoxide that is a cytoprotective gasotransmitter. Additionally, the levels of glutathione and cysteinylglycine bound to proteins also increased after RIC, while glutathione catabolism decreased. Plasma proteomic analysis identified overall 828 proteins. Several steps of statistical analysis (Student's t-test, repeated measures ANOVA, with Holm corrected pairwise p-values <0.05 threshold and fold change higher or lower than 100 %) leaded to the identification of 9 proteins with altered circulating levels in response to RIC at 4h and 22h. All 9 proteins are from extracellular space or exosomes, being involved in inflammation, angiogenesis or metabolism control. In addition, RIC-conditioned plasma from young subjects protected microglial cell culture against inflammatory stimuli, indicating an anti-inflammatory effect of RIC. Nevertheless, other functional tests in neurons or endothelial cells had no effect. Overall, we present some evidence for RIC-induced anti-inflammatory and antioxidant responses in healthy human subjects, in particular in young subjects. This study is a first step towards the disclosure of signalling factors involved in RIC-mediated inter-organ communication.

Carbon monoxide and mitochondria: Cell energy and fate control.

Carbon monoxide (CO) is a ubiquitously produced endogenous gas in mammalian cells and is involved in stress response being considered as a cytoprotective and homeostatic factor. In the present review, the underlying mechanisms of CO are discussed, in particular CO's impact on cellular metabolism affecting cell fate and function. One of the principal signaling molecules of CO is reactive oxygen species (ROS), particularly hydrogen peroxide, which is mainly generated at the mitochondrial level. Likewise, CO acts on mitochondria modulating oxidative phosphorylation and mitochondria quality control, namely mitochondrial biogenesis (mitobiogenesis) and mitophagy. Other metabolic pathways are also involved in CO's mode of action such as glycolysis and pentose phosphate pathway. The review ends with some new perspectives on CO Biology research. Carboxyhemoglobin (COHb) formation can also be implicated in the CO mode of action, as well as its potential biological role. Finally, other organelles such as peroxisomes hold the potential to be targeted and modulated by CO.

Proteomics to Identify New Blood Biomarkers for Diagnosing Patients With Acute Stroke.

Background Blood biomarkers are a potential tool for early stroke diagnosis. We aimed to perform a pilot and exploratory study on untargeted blood biomarkers in patients with suspected stroke by using mass spectrometry analysis. Methods and Results This was a prospective observational study of consecutive patients with suspected stroke admitted within 6 hours of last being seen well. Blood samples were collected at admission. Patients were divided into 3 groups: ischemic stroke (IS), intracerebral hemorrhage (ICH), and stroke mimics. Quantitative analysis from mass spectrometry data was performed using a supervised approach. Biomarker-based prediction models were developed to differentiate IS from ICH and ICH+stroke mimics. Models were built aiming to minimize misidentification of patients with ICH as having IS. We included 90 patients, one-third within each subgroup. The median age was 71 years (interquartile range, 57-81 years), and 49 participants (54.4%) were women. In quantitative analysis, C3 (complement component 3), ICAM-2 (intercellular adhesion molecule 2), PLGLA (plasminogen like A), STXBP5 (syntaxin-binding protein 5), and IGHV3-64 (immunoglobulin heavy variable 3-64) were the 5 most significantly dysregulated proteins for both comparisons. Biomarker-based models showed 88% sensitivity and 89% negative predictive value for differentiating IS from ICH, and 75% sensitivity and 95% negative predictive value for differentiating IS from ICH+stroke mimics. ICAM-2, STXBP5, PLGLA, C3, and IGHV3-64 displayed the highest importance score in our models, being the most informative for identifying patients with stroke. Conclusions In this proof-of-concept and exploratory study, our biomarker-based prediction models, including ICAM-2, STXBP5, PLGLA, C3, and IGHV3-64, showed 75% to 88% sensitivity for identifying patients with IS, while aiming to minimize misclassification of ICH. Although our methodology provided an internal validation, these results still need validation in other cohorts and with different measurement techniques.

Carboxyhemoglobin (COHb): Unavoidable Bystander or Protective Player?

Carbon monoxide (CO) is a cytoprotective endogenous gas that is ubiquitously produced by the stress response enzyme heme-oxygenase. Being a gas, CO rapidly diffuses through tissues and binds to hemoglobin (Hb) increasing carboxyhemoglobin (COHb) levels. COHb can be formed in erythrocytes or in plasma from cell-free Hb. Herein, it is discussed as to whether endogenous COHb is an innocuous and inevitable metabolic waste product or not, and it is hypothesized that COHb has a biological role. In the present review, literature data are presented to support this hypothesis based on two main premises: (i) there is no direct correlation between COHb levels and CO toxicity, and (ii) COHb seems to have a direct cytoprotective and antioxidant role in erythrocytes and in hemorrhagic models in vivo. Moreover, CO is also an antioxidant by generating COHb, which protects against the pro-oxidant damaging effects of cell-free Hb. Up to now, COHb has been considered as a sink for both exogenous and endogenous CO generated during CO intoxication or heme metabolism, respectively. Hallmarking COHb as an important molecule with a biological (and eventually beneficial) role is a turning point in CO biology research, namely in CO intoxication and CO cytoprotection.

Crosstalk between cilia and autophagy: implication for human diseases.

Macroautophagy/autophagy is a self-degradative process necessary for cells to maintain their energy balance during development and in response to nutrient deprivation. Autophagic processes are tightly regulated and have been found to be dysfunctional in several pathologies. Increasing experimental evidence points to the existence of an interplay between autophagy and cilia. Cilia are microtubule-based organelles protruding from the cell surface of mammalian cells that perform a variety of motile and sensory functions and, when dysfunctional, result in disorders known as ciliopathies. Indeed, selective autophagic degradation of ciliary proteins has been shown to control ciliogenesis and, conversely, cilia have been reported to control autophagy. Moreover, a growing number of players such as lysosomal and mitochondrial proteins are emerging as actors of the cilia-autophagy interplay. However, some of the published data on the cilia-autophagy axis are contradictory and indicate that we are just starting to understand the underlying molecular mechanisms. In this review, the current knowledge about this axis and challenges are discussed, as well as the implication for ciliopathies and autophagy-associated disorders.

Carbon Monoxide Stimulates Both Mitophagy And Mitochondrial Biogenesis to Mediate Protection Against Oxidative Stress in Astrocytes.

Astrocytes are key glial cells for the metabolic and functional support of the brain. Mitochondrial quality control (MQC), in particular the balance between mitophagy and mitochondrial biogenesis, is a major event for the maintenance of cellular homeostasis. Carbon monoxide (CO) is an endogenous gasotransmitter that inhibits cell death and inflammation by targeting mitochondria. It is well established that CO promotes cytoprotection by increasing mitochondrial population and metabolism (oxidative phosphorylation). Thus, it is hypothesized that CO-induced cytoprotection may also be mediated by the balance between mitophagy and mitochondrial biogenesis. Herein, the carbon monoxide releasing molecule-A1 (CORM-A1) was used in primary cultures of astrocytes to assess CO role on mitochondrial turnover. PINK1/Parkin-dependent mitophagy was stimulated by CORM-A1 following 1 h of treatment. While at 24 h after treatment, CORM-A1 increased mitochondrial population, which may indicate mitochondrial biogenesis. In fact, mitochondrial biogenesis was confirmed by the enhancement of PGC-1α expression that upregulates several mitochondrial transcription factors. Furthermore, inhibition of mitophagy by knocking down PINK1 expression reverted CO-induced mitochondrial biogenesis, indicating that mitochondrial turnover is dependent on modulation of mitophagy. Finally, CORM-A1 prevented astrocytic cell death induced by oxidative stress in a mitophagy-dependent manner. In fact, whenever PINK1 was knocked down, CORM-A1-induced cytoprotection was lost. In summary, CORM-A1 stimulates mitochondrial turnover, which in turn prevents astrocytic cell death. CO cytoprotection depends on increasing mitochondrial population and on eliminating dysfunctional mitochondria.

Pilot study in human healthy volunteers on the mechanisms underlying remote ischemic conditioning (RIC) - Targeting circulating immune cells and immune-related proteins.

Remote ischemic conditioning (RIC) is a novel promising therapy for treatment of neurological diseases, including ischemic stroke. RIC consists of short cycles of ischemia in a distant non-vital organ that may protect other organs against ischemia. Extensive experimental data and some few clinical trials support the neuroprotective role of RIC in ischemic stroke. Nevertheless, the circulating factors involved in this inter-organ communication and neuroprotection are not clarified. This pilot study in humans characterized the innate and adaptive circulating immune cell populations following RIC. This analysis has a particular focus at 24 h after RIC to avoid circadian influence. In silico functional analysis of mass spectrometry data identified 15 immune-related proteins. Our results reveal an immune response following RIC.

Carbon Monoxide Modulation of Microglia-Neuron Communication: Anti-Neuroinflammatory and Neurotrophic Role.

Microglia, the 'resident immunocompetent cells' of the central nervous system (CNS), are key players in innate immunity, synaptic refinement and homeostasis. Dysfunctional microglia contribute heavily to creating a toxic inflammatory milieu, a driving factor in the pathophysiology of several CNS disorders. Therefore, strategies to modulate the microglial function are required to tackle exacerbated tissue inflammation. Carbon monoxide (CO), an endogenous gaseous molecule produced by the degradation of haem, has anti-inflammatory, anti-apoptotic, and pro-homeostatic and cytoprotective roles, among others. ALF-826A, a novel molybdenum-based CO-releasing molecule, was used for the assessment of neuron-microglia remote communication. Primary cultures of rat microglia and neurons, or the BV-2 microglial and CAD neuronal murine cell lines, were used to study the microglia-neuron interaction. An approach based on microglial-derived conditioned media in neuronal culture was applied. Medium derived from CO-treated microglia provided indirect neuroprotection against inflammation by limiting the lipopolysaccharide (LPS)-induced expression of reactivity markers (CD11b), the production of reactive oxygen species (ROS) and the secretion of inflammatory factors (TNF-α, nitrites). This consequently prevented neuronal cell death and maintained neuronal morphology. In contrast, in the absence of inflammatory stimulus, conditioned media from CO-treated microglia improved neuronal morphological complexity, which is an indirect manner of assessing neuronal function. Likewise, the microglial medium also prevented neuronal cell death induced by pro-oxidant tert-Butyl hydroperoxide (t-BHP). ALF-826 treatment reinforced microglia secretion of Interleukin-10 (IL-10) and adenosine, mediators that may protect against t-BHP stress in this remote communication model. Chemical inhibition of the adenosine receptors A2A and A1 reverted the CO-derived neuroprotective effect, further highlighting a role for CO in regulating neuron-microglia communication via purinergic signalling. Our findings indicate that CO has a modulatory role on microglia-to-neuron communication, promoting neuroprotection in a non-cell autonomous manner. CO enhances the microglial release of neurotrophic factors and blocks exacerbated microglial inflammation. CO improvement of microglial neurotrophism under non-inflammatory conditions is here described for the first time.

Carbon Monoxide-Neuroglobin Axis Targeting Metabolism Against Inflammation in BV-2 Microglial Cells.

Microglia are the immune competent cell of the central nervous system (CNS), promoting brain homeostasis and regulating inflammatory response against infection and injury. Chronic or exacerbated neuroinflammation is a cause of damage in several brain pathologies. Endogenous carbon monoxide (CO), produced from the degradation of heme, is described as anti-apoptotic and anti-inflammatory in several contexts, including in the CNS. Neuroglobin (Ngb) is a haemoglobin-homologous protein, which upregulation triggers antioxidant defence and prevents neuronal apoptosis. Thus, we hypothesised a crosstalk between CO and Ngb, in particular, that the anti-neuroinflammatory role of CO in microglia depends on Ngb. A novel CO-releasing molecule (ALF826) based on molybdenum was used for delivering CO in microglial culture.BV-2 mouse microglial cell line was challenged with lipopolysaccharide (LPS) for triggering inflammation, and after 6 h ALF826 was added. CO exposure limited inflammation by decreasing inducible nitric oxide synthase (iNOS) expression and the production of nitric oxide (NO) and tumour necrosis factor-α (TNF-α), and by increasing interleukine-10 (IL-10) release. CO-induced Ngb upregulation correlated in time with CO's anti-inflammatory effect. Moreover, knocking down Ngb reversed the anti-inflammatory effect of CO, suggesting that dependents on Ngb expression. CO-induced Ngb upregulation was independent on ROS signalling, but partially dependent on the transcriptional factor SP1. Finally, microglial cell metabolism is also involved in the inflammatory response. In fact, LPS treatment decreased oxygen consumption in microglia, indicating a switch to glycolysis, which is associated with a proinflammatory. While CO treatment increased oxygen consumption, reverting LPS effect and indicating a metabolic shift into a more oxidative metabolism. Moreover, in the absence of Ngb, this phenotype was no longer observed, indicating Ngb is needed for CO's modulation of microglial metabolism. Finally, the metabolic shift induced by CO did not depend on alteration of mitochondrial population. In conclusion, neuroglobin emerges for the first time as a key player for CO signalling against exacerbated inflammation in microglia.

Remote but not Distant: a Review on Experimental Models and Clinical Trials in Remote Ischemic Conditioning as Potential Therapy in Ischemic Stroke.

Stroke is one of the main causes of neurological disability worldwide and the second cause of death in people over 65 years old, resulting in great economic and social burden. Ischemic stroke accounts for 85% of total cases, and the approved therapies are based on re-establishment of blood flow, and do not directly target brain parenchyma. Thus, novel therapies are urgently needed. In this review, limb remote ischemic conditioning (RIC) is revised and discussed as a potential therapy against ischemic stroke. The review targets both (i) fundamental research based on experimental models and (ii) clinical research based on clinical trials and human interventional studies with healthy volunteers. Moreover, it also presents two approaches concerning RIC mechanisms in stroke: (i) description of the underlying cerebral cellular and molecular mechanisms triggered by limb RIC that promote neuroprotection against stroke induced damage and (ii) the identification of signaling factors involved in inter-organ communication following RIC procedure. Limb to brain remote signaling can occur via circulating biochemical factors, immune cells, and/or stimulation of autonomic nervous system. In this review, these three hypotheses are explored in both humans and experimental models. Finally, the challenges involved in translating experimentally generated scientific knowledge to a clinical setting are also discussed.

Microglia at the Centre of Brain Research: Accomplishments and Challenges for the Future.

Microglia are the immune guardians of the central nervous system (CNS), with critical functions in development, maintenance of homeostatic tissue balance, injury and repair. For a long time considered a forgotten 'third element' with basic phagocytic functions, a recent surge in interest, accompanied by technological progress, has demonstrated that these distinct myeloid cells have a wide-ranging importance for brain function. This review reports microglial origins, development, and function in the healthy brain. Moreover, it also targets microglia dysfunction and how it contributes to the progression of several neurological disorders, focusing on particular molecular mechanisms and whether these may present themselves as opportunities for novel, microglia-targeted therapeutic approaches, an ever-enticing prospect. Finally, as it has been recently celebrated 100 years of microglia research, the review highlights key landmarks from the past century and looked into the future. Many challenging problems have arisen, thus it points out some of the most pressing questions and experimental challenges for the ensuing century.

Assessment of Mitochondrial Protein Glutathionylation as Signaling for CO Pathway.

Protein glutathionylation is a posttranslational process that regulates protein function in response to redox cellular changes. Furthermore, carbon monoxide-induced cellular pathways involve reactive oxygen species (ROS) signaling and mitochondrial protein glutathionylation. Herein, it is described as a technique to assess mitochondrial glutathionylation due to low concentrations of CO exposure. Mitochondria are isolated from cell culture or tissue, followed by an immunoprecipitation assay, which allows the capture of any glutathionylated mitochondrial protein using a specific antibody coupled to a solid matrix that binds to glutathione antigen. The precipitated protein is further identified and quantified by immunoblotting analysis.

The mito-QC Reporter for Quantitative Mitophagy Assessment in Primary Retinal Ganglion Cells and Experimental Glaucoma Models.

Mitochondrial damage plays a prominent role in glaucoma. The only way cells can degrade whole mitochondria is via autophagy, in a process called mitophagy. Thus, studying mitophagy in the context of glaucoma is essential to understand the disease. Up to date limited tools are available for analyzing mitophagy in vivo. We have taken advantage of the mito-QC reporter, a recently generated mouse model that allows an accurate mitophagy assessment to fill this gap. We used primary RGCs and retinal explants derived from mito-QC mice to quantify mitophagy activation in vitro and ex vivo. We also analyzed mitophagy in retinal ganglion cells (RGCs), in vivo, using different mitophagy inducers, as well as after optic nerve crush (ONC) in mice, a commonly used surgical procedure to model glaucoma. Using mito-QC reporter we quantified mitophagy induced by several known inducers in primary RGCs in vitro, ex vivo and in vivo. We also found that RGCs were rescued from some glaucoma relevant stress factors by incubation with the iron chelator deferiprone (DFP). Thus, the mito-QC reporter-based model is a valuable tool for accurately analyzing mitophagy in the context of glaucoma.

Response of the cerebral vasculature to systemic carbon monoxide administration-Regional differences and sexual dimorphism.

Previous studies about the modulation of the vasculature by CO were performed exclusively in male or sexually immature animals. Understanding the sex differences regarding systemic drug processing and pharmacodynamics is an important feature for safety assessment of drug dosing and efficacy. In this work, we used CORM-A1 as source of CO to examine the effects of this gasotransmitter on brain perfusion and the sex-dependent differences. Dynamic contrast-enhanced imaging (DCE)-based analysis was used to characterize the properties of CO in the modulation of cerebral vasculature in vivo, in adult C57BL/6 healthy mice. Perfusion of the temporal muscle, maxillary vein and in hippocampus, cortex and striatum was analysed for 108 min following CORM-A1 administration of 3 or 5 mg/kg. Under control conditions, brain perfusion was lower in females when compared with males. Under CO treatment, females showed a surprisingly overall reduced perfusion compared with controls (F = 3.452, p = .0004), while no major alterations (or even the expected increase) were observed in males. Cortical structures were only modulated in females. A striking female-dominated vasoconstriction effect was observed in the hippocampus and striatum following administration of CO, in this mixed-sex cohort. As these two regions are implicated in episodic and procedural memory formation, CO may have a relevant impact in learning and memory.

CO-mediated cytoprotection is dependent on cell metabolism modulation.

Carbon monoxide (CO) is a gasotransmitter endogenously produced by the activity of heme oxygenase, which is a stress-response enzyme. Endogenous CO or low concentrations of exogenous CO have been described to present several cytoprotective functions: anti-apoptosis, anti-inflammatory, vasomodulation, maintenance of homeostasis, stimulation of preconditioning and modulation of cell differentiation. The present review revises and discuss how CO regulates cell metabolism and how it is involved in the distinct cytoprotective roles of CO. The first found metabolic effect of CO was its increase on cellular ATP production, and since then much data have been generated. Mitochondria are the most described and studied cellular targets of CO. Mitochondria exposure to this gasotransmitter leads several consequences: ROS generation, stimulation of mitochondrial biogenesis, increased oxidative phosphorylation or mild uncoupling effect. Likewise, CO negatively regulates glycolysis and improves pentose phosphate pathway. More recently, CO has also been disclosed as a regulating molecule for metabolic diseases, such as obesity and diabetes with promising results.

Autonomic nervous system response to remote ischemic conditioning: heart rate variability assessment.

BACKGROUND: Remote ischemic conditioning (RIC) is a procedure applied in a limb for triggering endogenous protective pathways in distant organs, namely brain or heart. The underlying mechanisms of RIC are still not fully understood, and it is hypothesized they are mediated either by humoral factors, immune cells and/or the autonomic nervous system. Herein, heart rate variability (HRV) was used to evaluate the electrophysiological processes occurring in the heart during RIC and, in turn to assess the role of autonomic nervous system. METHODS: Healthy subjects were submitted to RIC protocol and electrocardiography (ECG) was used to evaluate HRV, by assessing the variability of time intervals between two consecutive heart beats. This is a pilot study based on the analysis of 18 ECG from healthy subjects submitted to RIC. HRV was characterized in three domains (time, frequency and non-linear features) that can be correlated with the autonomic nervous system function. RESULTS: RIC procedure increased significantly the non-linear parameter SD2, which is associated with long term HRV. This effect was observed in all subjects and in the senior (> 60 years-old) subset analysis. SD2 increase suggests an activation of both parasympathetic and sympathetic nervous system, namely via fast vagal response (parasympathetic) and the slow sympathetic response to the baroreceptors stimulation. CONCLUSIONS: RIC procedure modulates both parasympathetic and sympathetic autonomic nervous system. Furthermore, this modulation is more pronounced in the senior subset of subjects. Therefore, the autonomic nervous system regulation could be one of the mechanisms for RIC therapeutic effectiveness.

Carbon monoxide released by CORM-A1 prevents yeast cell death via autophagy stimulation.

Autophagy is an autodigestive process, promoting cytoprotection by the elimination of dysfunctional organelles, misfolded proteins and toxic aggregates. Carbon monoxide (CO) is an endogenous gasotransmitter that under low concentrations prevents cell death and inflammation. For the first time, the role of autophagy in CO-mediated cytoprotection against oxidative stress was evaluated in the model yeast Saccharomyces cerevisiae. The boron-based CO-releasing molecule, CORM-A1, was used to deliver CO. CORM-A1 partially prevented oxidative stress-induced cell death in yeast. Likewise, CORM-A1 activated autophagy under basal physiological conditions, which were assessed by autophagic flux and the expression of mCherry-Atg8 or GFP-Atg8. Inhibition of autophagy by knocking out key autophagic genes in yeast (ATG8 or ATG11) blocked CORM-A1 cytoprotective effect, indicating the critical role of autophagy in CO-induced cytoprotection. The CO-mediated cytoprotection via autophagy induction observed in yeast was validated in primary cultures of astrocytes, a well-characterized model for CO's cytoprotective functions. As in yeast, CORM-A1 prevented oxidative stress-induced cell death in an autophagy-dependent manner in astrocytes. Overall, our data support the cytoprotective action of CO against oxidative stress. CO promotes cytoprotection in yeast via autophagy, opening new possibilities for the study of molecular mechanisms of CO's biological functions using this powerful eukaryotic model.

CSRP3 mediates polyphenols-induced cardioprotection in hypertension.

Berries contain bioactive polyphenols, whose capacity to prevent cardiovascular diseases has been established recently in animal models as well in human clinical trials. However, cellular processes and molecular targets of berries polyphenols remain to be identified. The capacity of a polyphenol-enriched diet (i.e., blueberries, blackberries, raspberries, strawberry tree fruits and Portuguese crowberries berries mixture) to promote animal survival and protect cardiovascular function from salt-induced hypertension was evaluated in a chronic salt-sensitive Dahl rat model. The daily consumption of berries improved survival of Dahl/salt-sensitive rats submitted to high-salt diet and normalized their body weight, renal function and blood pressure. In addition, a prophylactic effect was observed at the level of cardiac hypertrophy and dysfunction, tissue cohesion and cardiomyocyte hypertrophy. Berries also protected the aorta from fibrosis and modulated the expression of aquaporin-1, a channel involved in endothelial water and nitric oxide permeability. Left ventricle proteomics analysis led to the identification of berries and salt metabolites targets, including cystein and glycin-rich protein 3 (CSRP3), a protein involved in myocyte cytoarchitecture. In neonatal rat ventricular cardiomyocytes, CSRP3 was validated as a target of a berries-derived polyphenol metabolite, 4-methylcatechol sulfate, at micromolar concentrations, mimicking physiological conditions of human plasma circulation. Accordingly, siRNA silencing of CSRP3 and 4-methylcatechol sulfate pretreatment reversed cardiomyocyte hypertrophy and CSRP3 overexpression induced by phenylephrine. Our systemic study clearly supports the modulation of CSRP3 by a polyphenol-rich berries diet as an efficient cardioprotective strategy in hypertension-induced heart failure.

P2X7 Receptors Mediate CO-Induced Alterations in Gene Expression in Cultured Cortical Astrocytes-Transcriptomic Study.

Carbon monoxide (CO) is an endogenous gasotransmitter that limits inflammation and prevents apoptosis in several tissues, including the brain. Low concentrations of CO are cytoprotective in astrocytes, neurons, and microglia, but the underlying molecular mechanisms remain poorly understood. This work aims at identification of alterations in gene expression conferred by CO in primary cultures of cortical astrocytes, for further disclosure of the molecular mechanism of action of the gasotransmitter. Astrocytes were treated with the CO-releasing molecule CORM-A1 for 40 min, and transcriptional changes were analyzed using RNASeq. A total of 162 genes were differentially expressed in response to CO treatment, and 7 of these genes were selected for further analysis: FosB, Scand1, Rgs10, Actg1, Panx1, Pcbdh21, and Rn18s. The alterations in their expression were further validated using qRT-PCR. An increase in FosB protein expression was also observed after 40 min of CORM-A1 treatment, as determined by a western blot. CO-induced FosB expression and cytoprotection were both abrogated in the presence of the P2X7 receptor antagonist A-438079. Furthermore, CORM-A1 increased phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII), which is a downstream event of P2X7R activation. The functional importance of FosB in CO-induced survival was assessed by knocking down its expression with FosB siRNA. Astrocytes were challenged to death with oxidative stress and cell viability was assessed 24 h later. Downregulation of FosB did not prevent the effects of CO in the inhibition of astrocytic cell death. Nevertheless, the transcriptomic changes observed upon treatment of astrocytes with CO open new opportunities for further studies on CO cytoprotective pathways.

Phenolic Metabolites Modulate Cardiomyocyte Beating in Response to Isoproterenol.

Cardiovascular disease (CVD) is a public health concern, and the third cause of death worldwide. Several epidemiological studies and experimental approaches have demonstrated that consumption of polyphenol-enriched fruits and vegetables can promote cardioprotection. Thus, diet plays a key role in CVD development and/or prevention. Physiological ß-adrenergic stimulation promotes beneficial inotropic effects by increasing heart rate, contractility and relaxation speed of cardiomyocytes. Nevertheless, chronic activation of ß-adrenergic receptors can cause arrhythmias, oxidative stress and cell death. Herein the cardioprotective effect of human metabolites derived from polyphenols present in berries was assessed in cardiomyocytes, in response to chronic ß-adrenergic stimulation, to disclose some of the underlying molecular mechanisms. Ventricular cardiomyocytes derived from neonate rats were treated with three human bioavailable phenolic metabolites found in circulating human plasma, following berries' ingestion (catechol-O-sulphate, pyrogallol-O-sulphate, and 1-methylpyrogallol-O-sulphate). The experimental conditions mimic the physiological concentrations and circulating time of these metabolites in the human plasma (2 h). Cardiomyocytes were then challenged with the ß-adrenergic agonist isoproterenol (ISO) for 24 h. The presence of phenolic metabolites limited ISO-induced mitochondrial oxidative stress. Likewise, phenolic metabolites increased cell beating rate and synchronized cardiomyocyte beating population, following prolonged ß-adrenergic receptor activation. Finally, phenolic metabolites also prevented ISO-increased activation of PKA-cAMP pathway, modulating Ca2+ signalling and rescuing cells from an arrhythmogenic Ca2+ transients' phenotype. Unexpected cardioprotective properties of the recently identified human-circulating berry-derived polyphenol metabolites were identified. These metabolites modulate cardiomyocyte beating and Ca2+ transients following ß-adrenergic prolonged stimulation.