Cardiomyocyte Protection by Hibernating Brown Bear Serum: Toward the Identification of New Protective Molecules Against Myocardial Infarction

Ischemic heart disease remains one of the leading causes of death worldwide. Despite intensive research on the treatment of acute myocardial infarction, no effective therapy has shown clinical success. Therefore, novel therapeutic strategies are required to protect the heart from reperfusion injury. Interestingly, despite physical inactivity during hibernation, brown bears (Ursus arctos) cope with cardiovascular physiological conditions that would be detrimental to humans. We hypothesized that bear serum might contain circulating factors that could provide protection against cell injury. In this study, we sought to determine whether addition of bear serum might improve cardiomyocyte survival following hypoxia-reoxygenation. Isolated mouse cardiomyocytes underwent 45 min of hypoxia followed by reoxygenation. At the onset of reoxygenation, cells received fetal bovine serum (FBS; positive control), summer (SBS) or winter bear serum (WBS), or adult serums of other species, as indicated. After 2 h of reoxygenation, propidium iodide staining was used to evaluate cell viability by flow cytometry. Whereas, 0.5% SBS tended to decrease reperfusion injury, 0.5% WBS significantly reduced cell death, averaging 74.04 +/- 7.06% vs. 79.20 +/- 6.53% in the FBS group. This cardioprotective effect was lost at 0.1%, became toxic above 5%, and was specific to the bear. Our results showed that bear serum exerts a therapeutic effect with an efficacy threshold, an optimal dose, and a toxic effect on cardiomyocyte viability after hypoxia-reoxygenation. Therefore, the bear serum may be a potential source for identifying new therapeutic molecules to fight against myocardial reperfusion injury and cell death in general

Isolated Mitochondria State after Myocardial Ischemia-Reperfusion Injury and Cardioprotection: Analysis by Flow Cytometry

Rationale: Mitochondria are key organelles involved in cell survival and death during the acute phenomena of myocardial ischemia-reperfusion (i.e., myocardial infarction). To investigate the functions of isolated mitochondria such as calcium retention capacity, oxidative phosphorylation, and reactive oxygen species (ROS) production, already established methods are based on extramitochondrial measurements of the whole mitochondria population. Objective: The aim of this study was to develop a reliable and well-characterized method for multiparametric analysis of isolated single mitochondrion by flow cytometry (FC) in the context of myocardial infarction. The advantage of FC is the possibility to give a simultaneous analysis of morphological parameters (side and forward scatters: SSC and FSC) for each mitochondrion, combined with intramitochondrial measurements of several biological markers, such as ROS production or membrane potential (Δφm), using specific fluorescent probes. Methods and Results: For this study, a rat model of ischemia-reperfusion and a protective approach of post-conditioning using low reperfusion pressure was used. Thanks to the use of specific probes (NAO, MTR, TMRM, DilC1, and DHR123) combined with flow cytometry, we propose a method: (i) to identify mitochondrial populations of interest based on quality criteria (NAO/TMRM double staining); (ii) to monitor their morphological criteria, especially during swelling due to calcium overload; and (iii) to compare mitochondrial functions (membrane potential and ROS production) in different experimental groups. Applied to mitochondria from ischemic hearts, these measurements revealed that individual mitochondria are altered and that cardioprotection by low-pressure reperfusion reduces damage, as expected. Conclusions: Our results highlight FC as a reliable and sensitive method to investigate changes in mitochondrial functions and morphology in pathological conditions that disrupts their activity such as the case in ischemia-reperfusion. This methodological approach can be extended to other pathologies involving mitochondrial dysfunctions. Moreover, FC offers the possibility to work with very small amounts of isolated mitochondria, a factor that may limit the use of classical methods

LIGNÉES DE CELLULES ENDOTHÉLIALES HUMAINES COMME MODÈLE DE L'ORGANO-SPÉCIFICITÉ : ÉTUDE DU RÔLE DES CHIMIOKINES ET APPLICATIONS À L'INFLAMMATION CUTANÉE

MARTIN Olivier, Professeur, Université d'Orléans : Président du jury KIEDA Claudine, Directeur de recherche, CNRS, CBM Orléans : Directeur de thèse OPDENAKKER Ghislain, Professeur, Université de Louvain : Rapporteur CHOUAIB Salem, Directeur de recherche,Institut Gustave Roussy : Rapporteur LECLERE Sophie, Ingénieur de recherche, Société BioEurope :Examinateur LOCKER Daniel, Professeur, Université d'Orléans : ExaminateurThe cell circulation, from blood to tissues, is oriented by a set of specific molecules expressed on endothelial cells of blood vessels. These specific molecules depend on the organ and the microenvironment. From organospecific endothelial cell lines, we studied how the chemokines and their receptors can participate to the endothelium organospecificity. We demonstrated that some chemokines have clearly restricted-action towards one endothelial cell line in term of lymphocyte recruitment and formation of vessels in vitro (angiogenesis). We have elaborated an inflammatory model based on the skin endothelial cells which permits the screening of potential anti-inflammatory molecules towards the skin endothelium in collaboration with the BioEurope Society.La circulation des cellules, du sang vers les tissus, est guidée par des molécules exprimées spécifiquement au niveau des cellules endothéliales constituant les vaisseaux sanguins. Les molécules conférant la spécificité de la localisation sont différentes selon l'organe et le microenvironnement considérés. A partir de lignées endothéliales spécifiques d'organes, les études menées sur le rôle des chimiokines et de leurs récepteurs dans la spécificité de l'endothélium ont montré que l'activité biologique de certaines chimiokines est restrictive à une lignée endothéliale en terme de recrutement des lymphocytes et par leur capacité à augmenter la formation de vaisseaux sanguins (angiogénèse) in vitro. De plus, notre modèle d'inflammation cutanée basé sur les cellules endothéliales de la peau, a permis des études plus appliquées mettant en évidence des molécules anti-inflammatoires vis-à-vis de l'endothélium de la peau en collaboration avec la Société BioEurope du groupe Solabia

Lignées de cellules endothéliales humaines comme modèle de l'organo-spécificité (étude du rôle des chimiokines et applications à l'inflammation cutanée)

La circulation des cellules, du sang vers les tissus, est guidée par des molécules exprimées spécifiquement au niveau des cellules endothéliales constituant les vaisseaux sanguins. Les molécules conférant la spécificité de la localisation sont différentes selon l organe et le microenvironnement considérés. A partir de lignées endothéliales spécifiques d organes, les études menées sur le rôle des chimiokines et de leurs récepteurs dans la spécificité de l endothélium ont montré que l activité biologique de certaines chimiokines est restrictive à une lignée endothéliale en terme de recrutement des lymphocytes et de leur capacité à augmenter la formation de vaisseaux sanguins (l angiogénèse) in vitro. De plus, notre modèle d inflammation cutanée basé sur les cellules endothéliales de la peau, a permis des études plus appliquées mettant en évidence des molécules anti-inflammatoires vis-à-vis de l endothélium de la peau en collaboration avec la Société BioEurope du groupe Solabia.ORLEANS-BU Sciences (452342104) / SudocSudocFranceF

Chemokine-glycosaminoglycan interactions participate to the endothelium organospecificity and cellular addressing

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A bibliometric analysis: Ca2+ fluxes and inflammatory phenotyping by flow cytometry in peripheral blood mononuclear cells

Background The immune system, composed of organs, tissues, cells, and proteins, is the key to protecting the body from external biological attacks and inflammation. The latter occurs in several pathologies, such as cancers, type 1 diabetes, and human immunodeficiency virus infection. Immunophenotyping by flow cytometry is the method of choice for diagnosing these pathologies. Under inflammatory conditions, the peripheral blood mononuclear cells (PBMCs) are partially activated and generate intracellular pathways involving Ca 2+ -dependent signaling cascades leading to transcription factor expression. Ca 2+ signaling is typically studied by microscopy in cell lines but can present some limitations to explore human PBMCs, where flow cytometry can be a good alternative. Objective In this review, we dived into the research field of inflammation and Ca 2+ signaling in PBMCs. We aimed to investigate the structure and evolution of this field in a physio-pathological context, and then we focused our review on flow cytometry analysis of Ca 2+ fluxes in PBMCs. Methods From 1984 to 2022, 3865 articles on inflammation and Ca 2+ signaling in PBMCs were published, according to The Clarivate Web of Science (WOS) database used in this review. A bibliometric study was designed for this collection and consisted of a co-citation and bibliographic coupling analysis. Results The co-citation analysis was performed on 133 articles: 4 clusters highlighted the global context of Ca 2+ homeostasis, including chemical probe development, identification of the leading players in Ca 2+ signaling, and the link with chemokine production in immune cell function. Next, the bibliographic coupling analysis combined 998 articles in 8 clusters. This analysis outlined the mechanisms of PBMC activation, from signal integration to cellular response. Further explorations of the bibliographic coupling network, focusing on flow cytometry, revealed 21 articles measuring cytosolic Ca 2+ in PBMCs, with only 5 since 2016. This final query showed that Ca 2+ signaling analysis in human PBMCs using flow cytometry is still underdeveloped and investigates mainly the cytosolic Ca 2+ compartment. Conclusion Our review uncovers remaining knowledge gaps of intracellular players involved in Ca 2+ signaling in PBMCs, such as reticulum and mitochondria, and presents flow cytometry as a solid option to supplement gold-standard microscopy studies

Suppression of hypoxia-induced HIF-1α and of angiogenesis in endothelial cells by myo-inositol trispyrophosphate-treated erythrocytes

Allosteric regulation of oxygen delivery by RBCs may have significant effects on tumor growth. Indeed, angiogenesis, the formation of new blood vessels, is induced in growing tumors by low oxygen partial pressure. Hypoxia-inducible genes are switched on, among which are the VEGF gene and its receptors. Most important, under hypoxia, hypoxia-inducible factor 1α has a significantly prolonged half-life and up-regulates a number of hypoxia genes. Human microvascular endothelial cells (MECs), when subjected in vitro to hypoxia, align to form vessel-like structures as in the angiogenic process. We report here that, when cultured in hypoxic conditions in the presence of human RBCs loaded with a new membrane-permeant allosteric effector of Hb, myo-inositol trispyrophosphate (ITPP), endothelial cells (ECs) do not align, i.e., do not form “vessel”-like structures, because the “loaded” RBCs are capable of releasing under hypoxia more oxygen than their “normal” counterparts. Levels of VEGF and of hypoxia-inducible factor 1α, elevated in the human MECs under hypoxia, were dramatically reduced or even suppressed in the presence of the ITPP-loaded RBCs. Treatment of these ECs directly with free ITPP at different concentrations had no effect on their ability to undertake angiogenesis. Incubation with ITPP enhances the capacity of Hb to release bound oxygen, leading to higher oxygen tension in the hypoxic environment, thus inhibiting hypoxia-induced angiogenesis. These observations are suggestive of a potential in vivo role of ITPP-loaded, “low-O(2)-affinity” RBCs in cancer therapy

CTACK a new pro-angiogenic factor for dermal endothelial cells

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CTACK a new pro-angiogenic factor for dermal endothelial cells

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Measurement of fish freshness: Flow cytometry analysis of isolated muscle mitochondria

International audienceMitochondria are real sensors of the physiological status of tissues. After the death of an animal, they maintain physiological activity for several days. This activity is highly dependent on the availability of nutrients in the tissue. In this study, flow cytometry was used to measure the membrane potential of mitochondria isolated from European seabass (Dicentrarchus labrax) red muscle stored in ice for seven days in order to characterize fish freshness. Two probes, TMRM and Rhodamine 123, were used to measure mitochondrial potential. During the first few days (D0 to D3), isolated mitochondria maintained high potential, and then lost their potential (from D3 to D5), but were always re-polarizable after addition of substrates (glutamate, malate and succinate). From D7, the mitochondria were more strongly depolarized and were difficult to repolarize by the substrates. Using flow cytometry, we demonstrated that mitochondria were an excellent marker to confirm seabass freshness