Daniel Cussac

New nanoplatform based on Gd2O2S:Eu3+ core: synthesis, characterization and use for in vitro bio-labelling

The synthesis of Gd2O2S:Eu3+ nanoparticles (NPs), from hydroxycarbonate precursor precipitation followed by sulfuration in a H2S/Ar atmosphere at 750 °C; is reported. It is the first time that the size of Gd2O2S:Eu3+ NPs can be finely tuned from 60 nm to more than 200 nm by controlling the precipitation medium, the maturation time and the sulfuration route. The oxysulfide Gd2O2S:Eu3+ NPs emit strong red luminescence at 624 nm when excited by near UV (363 nm) or by X-rays. They may thus be considered as nano-phosphors and nano-scintillators. Then, the surface of oxysulfide NPs has been modified by an amino-silica or by a mesoporous silica shell (thickness 10–15 nm). The luminescence intensity of europium in gadolinium oxysulfide NPs has been maintained in the amino-silica coated particles Gd2O2S:Eu3+@SiO2-APTMS, whereas it has been significantly enhanced in the mesoporous silica coated Gd2O2S:Eu3+@mSiO2. This versatile new nanoplatform can be easily internalized in living NIH3T3 mouse cells. It is not cytotoxic up to 1 mg mL−1 and can be easily imaged by epifluorescence microscopy with excitation in the NUV. Consequently this spherical monodispersed Gd2O2S:Eu3+ nanophosphor could be considered as a very promising new fluorescent probe for bio-labelling, better than the corresponding oxide or fluoride nanoparticles.

High tumorigenic potential of a constitutively active mutant of the cholecystokinin 2 receptor

The cholecystokinin 2 receptor (CCK2R) increases proliferation of normal and neoplastic gastrointestinal cells and activates various mitogenic signaling pathways when stimulated by gastrin. To study the incidence of permanent activation of this receptor in tumorigenicity, a constitutively active mutant was generated by replacing residue Glu151 in the conserved E/DRY motif by Ala. Expression of the E151A-CCK2R mutant in NIH-3T3 cells causes ligand-independent activation of phospholipase C and ornithine decarboxylase, two enzymes critical for mitogenesis. Strikingly, the constitutive activity of this mutant was associated with dramatic alteration of NIH-3T3 cell morphology, enhanced cell proliferation and invasion. Moreover, injection of cells expressing E151A-CCK2R in nude mice resulted in the development of large and rapidly growing tumors. By contrast, none of these effects was observed with cells expressing the wild-type CCK2R, indicating that the tumorigenic properties of the E151A-CCK2R mutant is the result of its constitutive activation. To date, this is the first report that provides evidence for the high tumorigenic effect of a constitutively active CCK2R mutant, thus raising a potential role of the CCK2R in human cancer.

Characterization of Monoamine Oxidases in Mesenchymal Stem Cells: Role in Hydrogen Peroxide Generation and Serotonin-Dependent Apoptosis

Early death of grafted bone marrow mesenchymal stem cells (MSCs) represents a major limit to their use in cell therapy of solid organs. It is well known that oxidative stress plays a major role in cell death. We have recently shown that the serotonin-degrading enzyme monoamine oxidase A (MAO-A) generates large amount of hydrogen peroxide (H2O2) responsible for cell apoptosis. Hydrogen peroxide generation requires 5-HT internalization into the cell and its degradation by MAO-A. In the present study, we investigated whether MAO-A is expressed in MSCs and we defined its role in serotonin-dependent MSCs apoptosis. RT-PCR analysis and western blots showed that the serotonin transporter (SERT) and the 2 MAO isoenzymes, A and B, are expressed in MSCs. As shown by enzyme assays using [14C]serotonin or [14C]β-phenylethylamine as selective MAO-A or MAO-B substrates, MAO-A is largely predominant in MSCs. Incubation of MSCs with the MAO substrate tyramine led to a time-dependent generation of H2O2 that was prevented by the MAO inhibitor pargyline. Finally, exposure of the cells to serotonin promoted an increase in MSCs apoptosis prevented by pargyline and the SERT inhibitor imipramine. The pro-apoptotic effect of serotonin was associated to a decrease in the expression of the anti-apoptotic factor Bcl-2. In conclusion, these results show for the first time that the 5-HT-degrading enzyme MAO-A is an important source of H2O2 in MSCs and plays a major role in 5-HT-dependent MSCs apoptosis.

High level of α2‐adrenoceptor in rat foetal liver and placenta is due to α2B‐subtype expression in haematopoietic cells of the erythrocyte lineage

Rat foetal liver contains large amounts of α2‐adrenoceptors. The present work aimed to identify the receptor subtype and the cell type accounting for high expression and to clarify the mechanisms responsible for the sharp decrease in hepatic receptivity occurring during the late stage of foetal development. Binding experiments indicated that the density of α2‐adrenoceptors in the foetal liver (embryonic day 18; 615±155 fmol mg−1 of protein) is 18 fold higher than in newborn or adult (35.2±4.3 fmol mg−1). A high amount of receptor is also found in the placenta (443±53 fmol mg−1). In both tissues, the rank order of antagonists to inhibit radioligand binding matched the pharmacological profile of the α2B‐adrenoceptor and exclusively RNG transcripts were detected by RNase protection assays. Isolation of cell fractions from foetal liver showed that α2B‐adrenoceptor is primarily expressed by haematopoietic cells. Consistent with this view, the receptor is found to be abundant in foetal blood, carried by reticulocytes. The expression in blood gradually declines to zero at 3 weeks of age and it is not recovered following induction of reticulocytosis in adults. In foetal reticulocytes, a low proportion of the receptor population is coupled to G‐protein. The α2‐agonist UK14304 has a marginal effect on cyclic AMP level but significantly increases arachidonic acid release. The function of the receptor remains to be elucidated. However, together with observations on α2B‐knockout mice, the current finding strongly suggests a role for α2B‐adrenoceptor during foetal haematopoiesis in rodents.

Cardiac Fibroblasts Regulate Sympathetic Nerve Sprouting and Neurocardiac Synapse Stability

Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic innervation and neurocardiac synapse stability. They also suggest that neurocardiac synapse functionality relies on a triptych with tight interaction between sympathetic nerve endings, cardiomyocytes and fibroblasts. Deregulations of this triptych may be involved in pathophysiology of cardiac diseases.

[Ageing: a matter of heart?].

Ageing is considered as a major risk factor for the development of chronic diseases. Among these, heart failure seems to be particularly important for both triggering and accelerating pathological ageing. In the present review, we give a general overview of the most relevant results concerning the mechanism of normal and premature senescence of cardiomyocytes and cardiac stromal cells. In particular, we will address the role of telomere dysfunction, DNA damage response, impairment of mitochondrial function, miRNAs and secretome of senescent cells in cardiac ageing and failure.

0148: Intramyocardial transplantation of mesenchymal stromal cells for chronic myocardial ischemia and decreased left ventricular function: 1-year results of the MESAMI phase I clinical trial

Our aim was to investigate the safety and feasibility of transendocardial injections guided by 3-dimensional NogaStar XP mapping of autologous bone marrow-derived mesenchymal stromal cells (MSC) in patients with chronic myocardial ischemia and left ventricular dysfunction. The MESAMI 1 trial is a bicentric phase 1 study, including 10 patients with chronic myocardial ischemia. The inclusion criteria were: NYHA Class II-IV and/or angina pectoris CCS Class III or IV, chronic ischemic cardiomyopathy with LVEF ≤35%, optimal medical and revascularization therapies, and reversible perfusion defect by SPECT. Bone marrow was obtained by aspiration from the iliac crest and MSC were expanded in culture for 17 days. Transendocardial injections (n=14-16) of autologous bone marrow MSC were made into viable muscle in border zones of left ventricular scar. Bone marrow volume was 16.8±2.0ml, and patients received 61.5* 10 6 MSC. After 1-month follow-up (FU), the primary end-point of safety and feasibility was met since all patients tolerated the procedure with no adverse events due to the procedure and the cell therapy product. Secondary endpoints between baseline and 3-month FU showed a significant decreased of summed stress score measured by SPECT from 34.1±8 to 25.3±9.5(p 2 peak, and Quality of life (Qol) showed a trend toward improvement at 3 months compared to baseline and up to 12 months except for Qol. We demonstrated the safety and the feasibility of this innovative treatment. A randomized, double blind, multicenter, placebo-controlled clinical trial (MESAMI 2) will evaluate the efficiency of this procedure in a larger population.Clinical Trial Registration: NCT01076920

Multimodal gadolinium oxysulfide nanoparticles: a versatile contrast agent for mesenchymal stem cell labeling

Despite a clear development of innovative therapies based on stem cell manipulation, the availability of new tools to better understand and follow stem cell behavior and improve their biomedical applications is not adequate. Indeed, an ideal tracking device must have good ability to label stem cells as well as complete neutrality relative to their biology. Furthermore, preclinical studies imply in vitro and in vivo approaches that often require several kinds of labeling and/or detection procedures. Consequently, the multimodality concept presented in this work may present a solution to this problem as it has the potential to combine complementary imaging techniques. Spherical europium-doped gadolinium oxysulfide (Gd2O2S:Eu3+) nanoparticles are presented as a candidate as they are detectable by (1) magnetic resonance (MRI), (2) X-ray and (3) photoluminescence imaging. Whole body in vivo distribution, elimination and toxicity evaluation revealed a high tolerance of nanoparticles with a long-lasting MRI signal and slow hepatobiliary and renal clearance. In vitro labeling of a wide variety of cells unveils the nanoparticle potential for efficient and universal cell tracking. Emphasis on mesenchymal stromal cells (MSCs) leads to the definition of optimal conditions for labeling and tracking in the context of cell therapy: concentrations below 50 μg mL-1 and diameters between 170 and 300 nm. Viability, proliferation, migration and differentiation towards mesodermal lineages are preserved under these conditions, and cell labeling appears to be persistent and without any leakage. Ex vivo detection of as few as five thousand Gd2O2S:Eu3+-labeled MSCs by MRI combined with in vitro examination with fluorescence microscopy highlights the feasibility of cell tracking in cell therapy using this new nanoplatform.

058 The ongoing MESAMI translational research program

Purpose Despite the improvement of pharmacological and surgical therapies, the mortality related to ischemic heart failure remains high. During the last years, bone marrow-mesenchymal stem cell (BM-MSC) therapy has been proposed as a novel approach for the prevention and therapy of heart failure. Intramyocardial injection allows concentration of grafted cells within the injured zone. However, a major problem of with intraparenchymal route of administration is the early death of most of grafted cells. The goal of the MESAMI program is to evaluate the effect of intramyocardial administration of BM-MSC preconditioned or not with the pineal hormone melatonin in ischemic cardiomyopathy. Methods and Results Our preclinical investigations have designed a preconditioning strategy of BM-MSCs with the melatonin that significantly increases survival and efficacy of grafted cells in animal models of myocardial ischemia. Melatonin treatment significantly ameliorates the beneficial effects of BM-MSC on the recovery of cardiac function. In the mean time, we started a phase I clinical trial in patients with severe ischemic cardiomyopathy and no option of revascularization, using the NOGA XP system to guide injections into the myocardium. Based on our basic research results, we are developing a multicenter phase II trial on the effects of intramyocardial administration of melatonin-preconditioned BM-MSC in patients with chronic ischemic cardiomyopathy. Conclusion The ongoing MESAMI program is representative of a translational research program in France.

Alpha2-Adrenergic Receptors in Intestinal Epithelial Cells: Mechanisms of Signaling, Role, and Regulation

Studies on different animal species have demonstrated the predominant importance of α2-adrenergic receptors (a2-ARs) in mediating the effects of catecholamines on intestinal functions. Several clinical trials have also pointed out the interest of proabsorptive and antisecretory properties of clonidine for the treatment of diabetic or cancer therapy-related diarrhea. Binding experiments and RNase protection assays showed that epithelial cells of human intestinal mucosa express a2A-AR subtype and that this receptor is abundant in immature and proliferative cells from the crypts. Although a2-agonists cause inhibition of cAMP production, the mechanisms whereby transepithelial transport of water and electrolytes is affected are not completely understood. The aim of this presentation is to review studies carried out on human cancerous intestinal cell-lines naturally expressing the a2A-AR or permanently transfected with the a2C10 gene. Beside confirming the inverse relationship between enterocytic differentiation and receptor expression, data obtained on these in vitro models provide insights into the signaling pathways and regulation of a2A-ARs in epithelial cells. They also support a role for this receptor in the control of proliferative and absorptive activity of intestinal epithelium.