Intercellular signaling and P2Y11 purinoceptor implication after myocardial ischemia/reperfusion injury

Les lésions d’Ischémie/Reperfusion (I/R) contribuent à la physiopathologie de l’infarctus du myocarde. Le stress induit par l’I/R entraîne la mort des cardiomyocytes, une forte réponse inflammatoire de type stérile et la mise en place d’un processus de réparation impliquant les fibroblastes cardiaques. Il a précédemment été montré au laboratoire que l’activation du récepteur purinergique P2Y11 par l’ATP diminuait la sécrétion d’IL-6 et d’IL- 12 par la cellule dendritique (DC), permettant une diminution de la polarisation vers une réponse adaptative Th1. Nous avons donc émis l’hypothèse que la signalisation purinergique pouvait également moduler la mortalité des cardiomyocytes et l’activation des fibroblastes cardiaques après I/R, en diminuant la mise en place de réponses cellulaires délétères à long terme pour l’organe. L’objectif de cette thèse a été de déterminer in vitro le rôle de la signalisation purinergique sur la réponse des cardiomyocytes et des fibroblastes cardiaques à l’Hypoxie/Réoxygénation (H/R). Nous avons pu montrer que l’activation des récepteurs purinergiques au moment de la réoxygénation, en particulier du récepteur P2Y11, permettait de réduire la mortalité des cardiomyocytes après H/R. Nous avons ensuite montré que la stimulation de P2Y11 au moment de la réoxygénation diminue la prolifération des fibroblastes cardiaques et leur switch phénotypique en myofibroblastes, mais aussi diminue leur sécrétion de facteurs pro-inflammatoires. Le sécrétome des fibroblastes cardiaques a également induit une diminution de la sécrétion d’IL-6 et d’IL-12 par les DC, ainsi qu’une diminution de la mortalité des cardiomyocytes soumis à une H/R. Ces effets immunomodulateurs et cardioprotecteurs étaient dépendants de l’activation du récepteur P2Y11 sur les fibroblastes cardiaques. Ces résultats suggèrent fortement que le récepteur P2Y11 est au centre des réponses cellulaires post-H/R, et que le cibler in vivo à la reperfusion pourrait améliorer le pronostic clinique des patients atteints d’infarctus du myocarde.Ischemia/Reperfusion injuries are involved in the pathophysiology of myocardial infarction. I/R-induced stress leads to massive cardiomyocyte death, an acute inflammatory response and the establishment of a repair process by cardiac fibroblasts. Previous work in the laboratory showed that P2Y11 purinergic receptor activation by ATP decreased IL-6 and IL-12 secretion by dendritic cells (DC), inducing a decrease in polarization towards Th1 response. We hypothesized that purinergic signaling could also modulate cardiomyocyte death and activation of cardiac fibroblasts responses to hypoxia/reoxygenation (H/R). We showed that the activation of purinergic receptors at the onset of reoxygenation, especially P2Y11 receptor, improved cardiomyocytes survival following H/R. We then showed that P2Y11 stimulation at the onset of reoxygenation decreased cardiac fibroblasts proliferation and their phenotypic switch into myofibroblasts, but also decreased their secretion of pro-inflammatory factors. Cardiac fibroblasts secretome reduced IL-6 and IL-12 secretion by DC, and cardiomyocyte mortality. These immunomodulatory and cardioprotective effects were dependent on P2Y11 receptor activation in cardiac fibroblasts. These results suggest that P2Y11 receptor is strongly involved in post- H/R cellular responses, and that targeting this receptor in vivo could improve the clinical prognosis of patients with myocardial infarction

Monitoring the Secretory Behavior of the Rat Adrenal Medulla by High-Performance Liquid Chromatography-Based Catecholamine Assay from Slice Supernatants

International audienceCatecholamine (CA) secretion from the adrenal medullary tissue is a key step of the adaptive response triggered by an organism to cope with stress. Whereas molecular and cellular secretory processes have been extensively studied at the single chromaffin cell level, data available for the whole gland level are much scarcer. We tackled this issue in rat by developing an easy to implement experimental strategy combining the adrenal acute slice supernatant collection with a high-performance liquid chromatography-based epinephrine and norepinephrine (NE) assay. This technique affords a convenient method for measuring basal and stimulated CA release from single acute slices, allowing thus to individually address the secretory function of the left and right glands. Our data point that the two glands are equally competent to secrete epinephrine and NE, exhibiting an equivalent epinephrine:NE ratio, both at rest and in response to a cholinergic stimulation. Nicotine is, however, more efficient than acetylcholine to evoke NE release. A pharmacological challenge with hexamethonium, an α3-containing nicotinic acetylcholine receptor antagonist, disclosed that epinephrine-and NE-secreting chromaffin cells distinctly expressed α3 nicotinic receptors, with a dominant contribution in NE cells. As such, beyond the novelty of CA assays from acute slice supernatants, our study contributes at refining the secretory behavior of the rat adrenal medullary tissue, and opens new perspectives for monitoring the release of other hormones and transmitters, especially those involved in the stress response

The feeder layer-mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3

Primary cultured epithelial cells that are used for basic research are often cultivated on plastic whereas those used for clinical purposes are usually cultured in the presence of a feeder layer. Here, we examined the influence of a feeder layer on the expression, affinity and DNA binding ability of the transcription factors, Sp1 and Sp3 in primary cultures of human skin keratinocytes. Co-culturing both newborn and adult skin keratinocytes with lethally irradiated 3T3 cells as a feeder layer contributed to maintain the cell's morphological and growth characteristics and delayed terminal differentiation in vitro. 3T3 also stabilized the DNA binding properties of Sp1 without altering its transcription. Stimulation of Sp1/Sp3 expression appears to be mediated through cell–cell interactions and by factors secreted by 3T3. Thus, we propose that the feeder layer delay terminal differentiation of primary cultured skin keratinocytes by preventing extinction of transcription factors, like Sp1 and Sp3, which play pivotal functions in the cell cycle

Modulation of Purinergic Receptors is Protective Against Hypoxia/Reoxygenation Injury in AC16 Cardiomyocytes

It has been reported that extracellular ATP concentrations during postischemic reperfusion activate cardiomyocyte purinergic receptors thus modulating both cardiac function and survival. This study is purported to assess the effects of purinergic modulation on the viability of a adult human ventricular derived cell line (AC16 cardiomyocytes) viability after hypoxia/reoxygenation (H/R). Cultured AC16 cardiomyocytes were subjected to 5 hours of hypoxia in a hypoxic chamber or by mineral oil layering. During the 1 h reoxygenation cells received no additional intervention or were treated with increasing ATP doses (10mM, 100mM and 1 mM) in the presence vs. the absence of either 100mM or 300mM suramin (a purinergic inhibitor). Cell viability was evaluated by MTT colorimetric assay. Administration of 100mM ATP (but not with 10mM) induced a significant improvement of cellular relative viability (RV) in experiments performed in the hypoxic chamber, an effect that was completely inhibited by suramin. Treatment with 1mM ATP and 1mM ATP (but not with 10mM and 100mM), elicited a significant increase of cellular RV equally in cardiomyocytes belonging to the control group and the ones exposed to both H/R protocols, regardless the presence or the absence of the purinergic inhibitor (100mM or 300mM). In conclusion, purinergic stimulation elicits cardioprotection in the settings of H/R injury, the effect being dependent both on the ATP concentration and the type/severity of the hypoxic insult

P2Y11 Agonism Prevents Hypoxia/Reoxygenation- and Angiotensin II-Induced Vascular Dysfunction and Intimal Hyperplasia Development

Vascular dysfunction in cardiovascular diseases includes vasomotor response impairments, endothelial cells (ECs) activation, and smooth muscle cells (SMCs) proliferation and migration to the intima. This results in intimal hyperplasia and vessel failure. We previously reported that activation of the P2Y11 receptor (P2Y11R) in human dendritic cells, cardiofibroblasts and cardiomyocytes was protective against hypoxia/reoxygenation (HR) lesions. In this study, we investigated the role of P2Y11R signaling in vascular dysfunction. P2Y11R activity was modulated using its pharmacological agonist NF546 and antagonist NF340. Rat aortic rings were exposed to angiotensin II (AngII) and evaluated for their vasomotor response. The P2Y11R agonist NF546 reduced AngII-induced vascular dysfunction by promoting EC-dependent vasorelaxation, through an increased nitric oxide (NO) bioavailability and reduced AngII-induced H2O2 release; these effects were prevented by the use of the P2Y11R antagonist NF340. Human vascular SMCs and ECs were subjected to AngII or H/R simulation in vitro. P2Y11R agonist modulated vasoactive factors in human ECs, that is, endothelial nitric oxide synthase (eNOS) and endothelin-1, reduced SMC proliferation and prevented the switch towards a synthetic phenotype. H/R and AngII increased ECs secretome-induced SMC proliferation, an effect prevented by P2Y11R activation. Thus, our data suggest that P2Y11R activation may protect blood vessels from HR-/AngII-induced injury and reduce vascular dysfunctions. These results open the way for new vasculoprotective interventions

P2Y11 Agonism Prevents Hypoxia/Reoxygenation- and Angiotensin II-Induced Vascular Dysfunction and Intimal Hyperplasia Development

Vascular dysfunction in cardiovascular diseases includes vasomotor response impairments, endothelial cells (ECs) activation, and smooth muscle cells (SMCs) proliferation and migration to the intima. This results in intimal hyperplasia and vessel failure. We previously reported that activation of the P2Y11 receptor (P2Y11R) in human dendritic cells, cardiofibroblasts and cardiomyocytes was protective against hypoxia/reoxygenation (HR) lesions. In this study, we investigated the role of P2Y11R signaling in vascular dysfunction. P2Y11R activity was modulated using its pharmacological agonist NF546 and antagonist NF340. Rat aortic rings were exposed to angiotensin II (AngII) and evaluated for their vasomotor response. The P2Y11R agonist NF546 reduced AngII-induced vascular dysfunction by promoting EC-dependent vasorelaxation, through an increased nitric oxide (NO) bioavailability and reduced AngII-induced H2O2 release; these effects were prevented by the use of the P2Y11R antagonist NF340. Human vascular SMCs and ECs were subjected to AngII or H/R simulation in vitro. P2Y11R agonist modulated vasoactive factors in human ECs, that is, endothelial nitric oxide synthase (eNOS) and endothelin-1, reduced SMC proliferation and prevented the switch towards a synthetic phenotype. H/R and AngII increased ECs secretome-induced SMC proliferation, an effect prevented by P2Y11R activation. Thus, our data suggest that P2Y11R activation may protect blood vessels from HR-/AngII-induced injury and reduce vascular dysfunctions. These results open the way for new vasculoprotective interventions

Effect of elexacaftor / tezacaftor / ivacaftor on airway and systemic inflammation in cystic fibrosis

Treatment with elexacaftor/tezacaftor/ivacaftor (ETI) has been shown to improve lung function in people with cystic fibrosis (PWCF). However, its biological effects remain incompletely understood. Here we describe alterations in pulmonary and systemic inflammation in PWCF following initiation of ETI. To address this, we collected spontaneously expectorated sputum and matching plasma from PWCF (n=30) immediately prior to ETI therapy, then again at 3 and 12 months. Within 3 months, PWCF demonstrated reduced activity of neutrophil elastase, proteinase three and cathepsin G, and decreased concentrations of interleukin (IL)-1β and IL-8 in sputum, accompanied by decreased Pseudomonas burden and restoration of secretory leukoprotease inhibitor levels. Once treated with ETI, all airway inflammatory markers studied in PWCF had reduced to levels found in matched non-CF bronchiectasis controls. In PWCF with advanced disease, ETI resulted in decreased plasma concentrations of IL-6, C-reactive protein and soluble TNF receptor one as well as normalisation of levels of the acute phase protein, alpha-1 antitrypsin. These data clarify the immunomodulatory effects of ETI and underscore its role as a disease modifier. </p

A review of Alpha-1 Antitrypsin binding partners for immune regulation and potential therapeutic application

Alpha-1 antitrypsin (AAT) is the canonical serine protease inhibitor of neutrophil-derived proteases and can modulate innate immune mechanisms through its anti-inflammatory activities mediated by a broad spectrum of protein, cytokine, and cell surface interactions. AAT contains a reactive methionine residue that is critical for its protease-specific binding capacity, whereby AAT entraps the protease on cleavage of its reactive centre loop, neutralises its activity by key changes in its tertiary structure, and permits removal of the AAT-protease complex from the circulation. Recently, however, the immunomodulatory role of AAT has come increasingly to the fore with several prominent studies focused on lipid or protein-protein interactions that are predominantly mediated through electrostatic, glycan, or hydrophobic potential binding sites. The aim of this review was to investigate the spectrum of AAT molecular interactions, with newer studies supporting a potential therapeutic paradigm for AAT augmentation therapy in disorders in which a chronic immune response is strongly linked