Astrocyte Ca²⁺ signalling: an unexpected complexity.

Astrocyte Ca(2+) signalling has been proposed to link neuronal information in different spatial-temporal dimensions to achieve a higher level of brain integration. However, some discrepancies in the results of recent studies challenge this view and highlight key insufficiencies in our current understanding. In parallel, new experimental approaches that enable the study of astrocyte physiology at higher spatial-temporal resolution in intact brain preparations are beginning to reveal an unexpected level of compartmentalization and sophistication in astrocytic Ca(2+) dynamics. This newly revealed complexity needs to be attentively considered in order to understand how astrocytes may contribute to brain information processing

Pharmacological Therapy in the Heart as an Alternative to Cellular Therapy: A Place for the Brain Natriuretic Peptide?

The discovery that stem cells isolated from different organs have the ability to differentiate into mature beating cardiomyocytes has fostered considerable interest in developing cellular regenerative therapies to treat cardiac diseases associated with the loss of viable myocardium. Clinical studies evaluating the potential of stem cells (from heart, blood, bone marrow, skeletal muscle, and fat) to regenerate the myocardium and improve its functional status indicated that although the method appeared generally safe, its overall efficacy has remained modest. Several issues raised by these studies were notably related to the nature and number of injected cells, as well as the route and timing of their administration, to cite only a few. Besides the direct administration of cardiac precursor cells, a distinct approach to cardiac regeneration could be based upon the stimulation of the heart's natural ability to regenerate, using pharmacological approaches. Indeed, differentiation and/or proliferation of cardiac precursor cells is controlled by various endogenous mediators, such as growth factors and cytokines, which could thus be used as pharmacological agents to promote regeneration. To illustrate such approach, we present recent results showing that the exogenous administration of the natriuretic peptide BNP triggers "endogenous" cardiac regeneration, following experimental myocardial infarction

The role of endogenous and exogenous RasGAP-derived fragment N in protecting cardiomyocytes from peroxynitrite-induced apoptosis.

Peroxynitrite (PN) is a potent nitrating and oxidizing agent generated during various pathological situations affecting the heart. The negative effects of PN result, at least in part, from its ability to activate caspases and apoptosis. RasGAP is a ubiquitously expressed protein that is cleaved sequentially by caspase-3. At low caspase-3 activity, RasGAP is cleaved into an N-terminal fragment, called fragment N, that protects cells by activating the Ras/PI3K/Akt pathway. At high caspase-3 activity, fragment N is further cleaved and this abrogates its capacity to stimulate the antiapoptotic Akt kinase. Fragment N formation is crucial for the survival of cells exposed to a variety of stresses. Here we investigate the pattern of RasGAP cleavage upon PN stimulation and the capacity of fragment N to protect cardiomyocytes. PN did not lead to sequential cleavage of RasGAP. Indeed, PN did not allow accumulation of fragment N because it induced its rapid cleavage into smaller fragments. No situations were found in cells treated with PN in which the presence of fragment N was associated with survival. However, expression of a caspase-resistant form of fragment N in cardiomyocytes protected them from PN-induced apoptosis. Our results indicate that the antiapoptotic pathway activated by fragment N is effective at inhibiting PN-induced apoptosis (as seen when cardiomyocytes express a capase-3-resistant form of fragment N) but because fragment N is too transiently generated in response to PN, no survival response is effectively produced. This may explain the marked deleterious consequences of PN generation in various organs, including the heart

Three-dimensional Ca2+ imaging advances understanding of astrocyte biology.

Astrocyte communication is typically studied by two-dimensional calcium ion (Ca2+) imaging, but this method has not yielded conclusive data on the role of astrocytes in synaptic and vascular function. We developed a three-dimensional two-photon imaging approach and studied Ca2+ dynamics in entire astrocyte volumes, including during axon-astrocyte interactions. In both awake mice and brain slices, we found that Ca2+ activity in an individual astrocyte is scattered throughout the cell, largely compartmented between regions, preponderantly local within regions, and heterogeneously distributed regionally and locally. Processes and endfeet displayed frequent fast activity, whereas the soma was infrequently active. In awake mice, activity was higher than in brain slices, particularly in endfeet and processes, and displayed occasional multifocal cellwide events. Astrocytes responded locally to minimal axonal firing with time-correlated Ca2+ spots

P90Necrotic cardiomyocytes release soluble pro-inflammatory molecule(s) inducing il1r/myd88-dependent inflammatory responses in cardiac fibroblasts

Background: Inflammation comes out to be a critical biological process in the pathophysiology of myocardial infarction (MI). We hypothesize that this inflammation is triggered by necrotic cardiomyocytes (Cmc) that release a set of endogenous molecules (DAMPs: danger-associated molecular patterns) activating inflammatory responses in cardiac fibroblasts. Aim: Analyze in vitro the immune activation of cardiac fibroblasts exposed to necrotic Cmc conditioned media. Methods: Primary neonatal murine cardiac fibroblasts and Cmc were obtained by digestion of neonatal hearts and differential plating technique allowing a selection for cardiomyocytes and cardiac fibroblasts. Cmc were killed by necrotic stimuli including oxidants (hydrogen peroxide) and mechanic stresses (freeze-thaw). Necrosis was assessed using Hoechst/PI stainings. Fibroblasts were exposed to necrotic Cmc conditioned media and mRNA expression of inflammatory genes was measured by real-time PCR and ELISA. Activation of signaling pathways was analyzed by western blot. We used cardiac cells from Myd88-/-, Trif-/- and Nlrp3-/- animals to evaluate the contribution of TLRs/IL1-R and NLRP3 inflammasome in the sensing of necrotic DAMPs. Results: mRNA expression of chemokines such as MCP-1, MIP-2 and IP-10 were induced in fibroblasts exposed to necrotic Cmc conditioned media. Alternatively, fibroblasts exposed to necrotic fibroblasts conditioned media showed a lower increase in mRNA expression of these chemokines. In addition, in fibroblasts from Myd88-/- mice, response to Cmc conditioned media was fully abrogated whereas no difference was observed in Trif-/- and Nlrp3-/- fibroblasts. Conclusion: Cardiac fibroblasts are able to produce a rapid and specific inflammatory response to necrotic Cmc conditioned media involving the expression of neutrophil and monocyte chemoattractants. The dependence on MyD88 adaptor protein strongly suggests that this response relies on TLR/IL-1R signaling. These results engage cardiac fibroblasts as key players in post-MI inflammatory responses as they are able to sense DAMPs from necrotic Cmc and possibly recruit inflammatory cells. Research supported by the Swiss National Science Foundation, Grant n° 310030_135394/

P619Role of Toll-like receptor 5 in the development of post-myocardial infarction inflammation

Background: Inflammatory processes play a key role in the pathophysiology of myocardial infarction (MI). Genetic deletion of toll-like recpetors (TLRs), especially TLR2 and TLR4 have shown protective role in murine models of MI. The role of other TLRs remains unknown. We have previously shown that cardiomyocytes express TLR5 and that the ligand of TLR5, flagellin, activates the NF-kappaB and MAPK pathways in cardiomyocytes. We also have shown that injection of flagellin induces acute systolic dysfunction in vivo in mice. Aim: Determine the role of TLR5 in the development of post-MI inflammation. Methods: A murine model of myocardial infarction was done by a 30 minutes ligation of the left anterior descending coronary artery followed by 2 hours of reperfusion. Infarct size was measured by standard Evans blue/TTC staining. Plasma creatine kinase (CK) was quantified as a read out of myocardial necrosis. Tissue and plasma cytokines (MIP-2, MCP-1, IL-6) were quantified by ELISA. To determine the extent of tissue lipid peroxidation we used malondialdehyde and 4-hydroxynonenal-HIS adduct assays. Tissue protein oxidation was tested by protein carbonyl ELISA kit. Phosphorylation of MAPK was analyzed by western blot. Results: Genetic suppression of TLR5 induced a significant increase of myocardial infarct size and plasma CK, of biochemical markers of myocardial oxidative stress, and cytokine levels in the heart and the plasma after MI. These effects were associated with a marked enhancement of p38 phosphorylation in the heart from TLR5 KO mice. Conclusion: TLR5 protects from acute myocardial injury and reduces local and systemic inflammation during myocardial infarction. The mechanisms may involve reduced p38 signaling, decreased oxidative stress and attenuated cytokine expression. Research supported by the Swiss National Science Foundation, Grant n° 310030_135394/

Natriuretic Peptide Receptor B modulates the proliferation of the cardiac cells expressing the Stem Cell Antigen-1.

Brain Natriuretic Peptide (BNP) injections in adult "healthy" or infarcted mice led to increased number of non-myocyte cells (NMCs) expressing the nuclear transcription factor Nkx2.5. The aim of this study was to identify the nature of the cells able to respond to BNP as well as the signaling pathway involved. BNP treatment of neonatal mouse NMCs stimulated Sca-1 <sup>+</sup> cell proliferation. The Sca-1 <sup>+</sup> cells were characterized as being a mixed cell population involving fibroblasts and multipotent precursor cells. Thus, BNP treatment led also to increased number of Sca-1 <sup>+</sup> cells expressing Nkx2.5, in Sca-1 <sup>+</sup> cell cultures in vitro and in vivo, in the hearts of neonatal and adult infarcted mice. Whereas BNP induced Sca-1 <sup>+</sup> cell proliferation via NPR-B receptor and protein kinase G activation, CNP stimulated Sca-1 <sup>+</sup> cell proliferation via NPR-B and a PKG-independent mechanism. We highlighted here a new role for the natriuretic peptide receptor B which was identified as a target able to modulate the proliferation of the Sca-1 <sup>+</sup> cells. The involvement of NPR-B signaling in heart regeneration has, however, to be further investigated

Cytokine hemoadsorption with CytoSorb^® in post-cardiac arrest syndrome, a pilot randomized controlled trial.

Hemoadsorption (HA) might mitigate the systemic inflammatory response associated with post-cardiac arrest syndrome (PCAS) and improve outcomes. Here, we investigated the feasibility, safety and efficacy of HA with CytoSorb <sup>®</sup> in cardiac arrest (CA) survivors at risk of PCAS. In this pilot randomized controlled trial, we included patients admitted to our intensive care unit following CA and likely to develop PCAS: required norepinephrine (> 0.2 µg/kg/min), and/or had serum lactate > 6 mmol/l and/or a time-to-return of spontaneous circulation (ROSC) > 25 min. Those requiring ECMO or renal replacement therapy were excluded. Eligible patients were randomly allocated to either receive standard of care (SOC) or SOC plus HA. Hemoadsorption was performed as stand-alone therapy for 24 h, using CytoSorb <sup>®</sup> and regional heparin-protamine anticoagulation. We collected feasibility, safety and clinical data as well as serial plasma cytokines levels within 72 h of randomization. We enrolled 21 patients, of whom 16 (76%) had out-of-hospital CA. Median (IQR) time-to-ROSC was 30 (20, 45) minutes. Ten were assigned to the HA group and 11 to the SOC group. Hemoadsorption was initiated in all patients allocated to the HA group within 18 (11, 23) h of ICU admission and conducted for a median duration of 21 (14, 24) h. The intervention was well tolerated except for a trend for a higher rate of aPTT elevation (5 (50%) vs 2 (18%) p = 0.18) and mild (100-150 G/L) thrombocytopenia at day 1 (5 (50%) vs 2 (18%) p = 0.18). Interleukin (IL)-6 plasma levels at randomization were low (< 100 pg/mL) in 10 (48%) patients and elevated (> 1000 pg/mL) in 6 (29%). The median relative reduction in IL-6 at 48 h was 75% (60, 94) in the HA group versus 5% (- 47, 70) in the SOC group (p = 0.06). In CA survivors at risk of PCAS, HA was feasible, safe and was associated with a nonsignificant reduction in cytokine plasma levels. Future trials are needed to further define the role of HA after CA. Those studies should include cytokine assessment to enrich the study population. NCT03523039, registered 14 May 2018

The role of oxidative stress during inflammatory processes.

Abstract The production of various reactive oxidant species in excess of endogenous antioxidant defense mechanisms promotes the development of a state of oxidative stress, with significant biological consequences. In recent years, evidence has emerged that oxidative stress plays a crucial role in the development and perpetuation of inflammation, and thus contributes to the pathophysiology of a number of debilitating illnesses, such as cardiovascular diseases, diabetes, cancer, or neurodegenerative processes. Oxidants affect all stages of the inflammatory response, including the release by damaged tissues of molecules acting as endogenous danger signals, their sensing by innate immune receptors from the Toll-like (TLRs) and the NOD-like (NLRs) families, and the activation of signaling pathways initiating the adaptive cellular response to such signals. In this article, after summarizing the basic aspects of redox biology and inflammation, we review in detail the current knowledge on the fundamental connections between oxidative stress and inflammatory processes, with a special emphasis on the danger molecule high-mobility group box-1, the TLRs, the NLRP-3 receptor, and the inflammasome, as well as the transcription factor nuclear factor-κB