Pre-Conditioning Methods and Novel Approaches with Mesenchymal Stem Cells Therapy in Cardiovascular Disease.

Transplantation of mesenchymal stem cells (MSCs) in the setting of cardiovascular disease, such as heart failure, cardiomyopathy and ischemic heart disease, has been associated with good clinical outcomes in several trials. A reduction in left ventricular remodeling, myocardial fibrosis and scar size, an improvement in endothelial dysfunction and prolonged cardiomyocytes survival were reported. The regenerative capacity, in addition to the pro-angiogenic, anti-apoptotic and anti-inflammatory effects represent the main target properties of these cells. Herein, we review the different preconditioning methods of MSCs (hypoxia, chemical and pharmacological agents) and the novel approaches (genetically modified MSCs, MSC-derived exosomes and engineered cardiac patches) suggested to optimize the efficacy of MSC therapy.

Metformin Attenuates Postinfarction Myocardial Fibrosis and Inflammation in Mice.

Diabetes is a major risk factor for the development of cardiovascular disease with a higher incidence of myocardial infarction. This study explores the role of metformin, a first-line antihyperglycemic agent, in postinfarction fibrotic and inflammatory remodeling in mice. Three-month-old C57BI/6J mice were submitted to 30 min cardiac ischemia followed by reperfusion for 14 days. Intraperitoneal treatment with metformin (5 mg/kg) was initiated 15 min after the onset of reperfusion and maintained for 14 days. Real-time PCR was used to determine the levels of COL3A1, αSMA, CD68, TNF-α and IL-6. Increased collagen deposition and infiltration of macrophages in heart tissues are associated with upregulation of the inflammation-associated genes in mice after 14 days of reperfusion. Metformin treatment markedly reduced postinfarction fibrotic remodeling and CD68-positive cell population in mice. Moreover, metformin resulted in reduced expression of COL3A1, αSMA and CD68 after 14 days of reperfusion. Taken together, these results open new perspectives for the use of metformin as a drug that counteracts adverse myocardial fibroticand inflammatory remodeling after MI.

Low-energy electron beam sterilization of solid alginate and chitosan, and their polyelectrolyte complexes.

Polysaccharidic scaffolds hold great hope in regenerative medicine, however their sterilization still remains challenging since conventional methods are deleterious. Recently, electron beams (EB) have raised interest as emerging sterilization techniques. In this context, the aim of this work was to study the impact of EB irradiations on polysaccharidic macroporous scaffolds. The effects of continuous and pulsed low energy EB were examined on polysaccharidic or on polyelectrolyte complexes (PEC) scaffolds by SEC-MALLS, FTIR and EPR. Then the scaffolds' physicochemical properties: swelling, architecture and compressive modulus were investigated. Finally, sterility and in vitro biocompatibility of irradiated scaffolds were evaluated to validate the effectiveness of our approach. Continuous beam irradiations appear less deleterious on alginate and chitosan chains, but the use of a pulsed beam limits the time of irradiation and better preserve the architecture of PEC scaffolds. This work paves the way for low energy EB tailor-made sterilization of sensitive porous scaffolds.

Selective Cardiomyocyte Oxidative Stress Leads to Bystander Senescence of Cardiac Stromal Cells.

Accumulation of senescent cells in tissues during normal or accelerated aging has been shown to be detrimental and to favor the outcomes of age-related diseases such as heart failure (HF). We have previously shown that oxidative stress dependent on monoamine oxidase A (MAOA) activity in cardiomyocytes promotes mitochondrial damage, the formation of telomere-associated foci, senescence markers, and triggers systolic cardiac dysfunction in a model of transgenic mice overexpressing MAOA in cardiomyocytes (Tg MAOA). However, the impact of cardiomyocyte oxidative stress on the cardiac microenvironment in vivo is still unclear. Our results showed that systolic cardiac dysfunction in Tg MAOA mice was strongly correlated with oxidative stress induced premature senescence of cardiac stromal cells favoring the recruitment of CCR2+ monocytes and the installation of cardiac inflammation. Understanding the interplay between oxidative stress induced premature senescence and accelerated cardiac dysfunction will help to define new molecular pathways at the crossroad between cardiac dysfunction and accelerated aging, which could contribute to the increased susceptibility of the elderly to HF.

Kidney inflammaging is promoted by CCR2+ macrophages and tissue-derived micro-environmental factors.

The incidence of disorders associated with low inflammatory state, such as chronic kidney disease, increases in the elderly. The accumulation of senescent cells during aging and the senescence-associated secretory phenotype, which leads to inflammaging, is known to be deleterious and account for progressive organ dysfunction. To date, the cellular actors implicated in chronic inflammation in the kidney during aging are still not well characterized. Using the DECyt method, based on hierarchical clustering of flow cytometry data, we showed that aging was associated with significant changes in stromal cell diversity in the kidney. In particular, we identified two cell populations up-regulated with aging, the mesenchymal stromal cell subset (kMSC) expressing CD73 and the monocyte-derived Ly6C+ CCR2+ macrophage subset expressing pro-inflammatory cytokines. Aged CD73+ kMSCs depicted senescence associated features with low proliferation rate, increased DNA damage foci and Ccl2 expression. Using co-cultures experiments, we showed that aged CD73+ kMSC promoted monocyte activation and secretion of inflammatory cytokines albeit less efficiently than young CD73+ kMSCs. In the context of ageing, increased frequency of CD73+ kMSC subpopulations could provide additional niche factors to newly recruited monocytes favoring a positive regulatory loop in response to local inflammation. Interfering with such partnership during aging could be a valuable approach to regulate kidney inflammaging and to limit the risk of developing chronic kidney disease in the elderly.

Extracellular vesicles of MSCs and cardiomyoblasts are vehicles for lipid mediators.

Recent works reported the relevance of cellular exosomes in the evolution of different pathologies. However, most of these studies focused on the ability of exosomes to convey mi-RNA from cell to cell. The level of knowledge concerning the transport of lipid mediators by these nanovesicles is more than fragmented. The role of lipid mediators in the inflammatory signaling is fairly well described, in particular concerning the derivatives of the arachidonic acid (AA), called eicosanoïds or lipid mediators. The aim of the present work was to study the transport of these lipids within the extracellular vesicles of rat bone marrow mesenchymal stem cells (BM-MSC) and the cardiomyoblast cell line H9c2. We were able to characterize, for the first time, complete profiles of oxilipins within these nanovesicles. We studied also the impact on these profiles, of the polyunsaturated fatty acids (PUFAs) know to be precursors of the inflammatory signaling molecules (AA, eicosapentaenoic acid EPA and Docosahexaenoic acid DHA), at physiological concentrations. By growing the progenitor cells under PUFAs supplementation, we provide a comprehensive assessment of the beneficial effect of ω-3 PUFA therapy. Actually, our results tend to support the resolving role of the inflammation that stromal cell-derived extracellular vesicles can have within the cardiac microenvironment.

Multimodal gadolinium oxysulfide nanoparticles for bioimaging: A comprehensive biodistribution, elimination and toxicological study.

For some years now, gadolinium oxysulfide nanoparticles (NPs) appear as strong candidates for very efficient multimodal in vivo imaging by: 1) Magnetic Resonance (MRI), 2) X-ray Computed Tomography (CT) and 3) photoluminescence imaging. In this paper, we present a selection of results centered on the evaluation of physico-chemical stability, toxicity, bio-distribution and excretion mechanisms of Gd2O2S:Ln3+ nanoparticles intravenously injected in rats. Two formulations are here tested with a common matrix and different dopants: Gd2O2S:Eu3+5% and Gd2O2S:Yb3+4%/Tm3+0.1%. The NPs appear to be almost insoluble in pure water and human plasma but corrosion/degradation phenomenon appears in acidic conditions classically encountered in cell lysosomes. Whole body in vivo distribution, excretion and toxicity evaluation revealed a high tolerance of nanoparticles with a long-lasting imaging signal associated with a slow hepatobiliary clearance and very weak urinary excretion. The results show that the majority of the injected product (>60%) has been excreted through the feces after five months. Experiments have evidenced that the NPs mainly accumulate in macrophage-rich organs, that is mainly liver and spleen and to a lesser extent lungs and bones (mainly marrow). No significant amounts have been detected in other organs such as heart, kidneys, brain, intestine and skin. Gd2O2S:Ln3+ NPs appeared to be very well tolerated up to 400 mg/kg when administered intravenously. STATEMENT OF SIGNIFICANCE: Since 2011, we have focused our work on Gd2O2S nanoparticles (NPs) for multimodal bioimaging using fluorescence, Magnetic Resonance Imaging (MRI) and Computed Tomography with very efficient results already published. However, since the European Medicines Agency has concluded its review of gadolinium contrast agents, confirming recommendations to restrict the use of some linear gadolinium agents used in MRI, a particular attention must be paid to any new contrast media containing gadolinium. Therefore, we present in this paper a compilation of studies about toxicity, bio-distribution and excretion mechanisms of Gd2O2S:Ln3+ NPs intravenously injected into rats. We also present an in vitro kinetic study of NPs degradation in aqueous and biological media to provide some information on chemical and biological stability.

Alginate-chitosan PEC scaffolds: A useful tool for soft tissues cell therapy.

In this study we evaluate macroporous scaffolds made of alginate-chitosan polyelectrolyte complexes (PEC) as tools to optimize the results of soft tissues cell therapy. Cell therapy using mesenchymal stem cells (MSC) has become attractive for tissue repair and regeneration in a number of acute and chronic injuries. Unfortunately their low retention and/or survival after injection limit their beneficial effects. A biomaterial-assisted implantation, providing cells a three-dimensional (3D) microenvironment is a promising strategy. To this purpose, we designed a family of PEC scaffolds, and studied if they could meet the requirement of such application. Xray tomography showed that all PEC scaffolds present an interconnected macroporosity, and both rheology and tensile measurements reveal optimized mechanical properties (higher storage moduli and Young moduli) compared to alginate reference scaffolds. In vitro assays demonstrated their ability to allow MSC retention (higher than 90%), long-term viability and FGF2 secretion. Then, we used a skeletal muscle implantation model to assess the biological response to scaffolds graft, and showed that they support in vivo vascular formation within the implant-derived tissue. The combination of alginate/chitosan PEC scaffolds architecture and angiogenic potential make them appear as interesting tools to optimize MSC therapy results in soft tissues.

Aging induces cardiac mesenchymal stromal cell senescence and promotes endothelial cell fate of the CD90 + subset.

Aging is a major risk factor in the development of chronic diseases, especially cardiovascular diseases. Age-related organ dysfunction is strongly associated with the accumulation of senescent cells. Cardiac mesenchymal stromal cells (cMSCs), deemed part of the microenvironment, modulate cardiac homeostasis through their vascular differentiation potential and paracrine activity. Transcriptomic analysis of cMSCs identified age-dependent biological pathways regulating immune responses and angiogenesis. Aged cMSCs displayed a senescence program characterized by Cdkn2a expression, decreased proliferation and clonogenicity, and acquisition of a senescence-associated secretory phenotype (SASP). Increased CCR2-dependent monocyte recruitment by aged cMSCs was associated with increased IL-1ß production by inflammatory macrophages in the aging heart. In turn, IL-1ß induced senescence in cMSCs and mimicked age-related phenotypic changes such as decreased CD90 expression. The CD90+ and CD90- cMSC subsets had biased vascular differentiation potentials, and CD90+ cMSCs were more prone to acquire markers of the endothelial lineage with aging. These features were related to the emergence of a new cMSC subset in the aging heart, expressing CD31 and endothelial genes. These results demonstrate that cMSC senescence and SASP production are supported by the installation of an inflammatory amplification loop, which could sustain cMSC senescence and interfere with their vascular differentiation potentials.

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.

Elaboration and evaluation of alginate foam scaffolds for soft tissue engineering.

Controlling microarchitecture in polymer scaffolds is a priority in material design for soft tissue applications. This paper reports for the first time the elaboration of alginate foam-based scaffolds for mesenchymal stem cell (MSC) delivery and a comparative study of various surfactants on the final device performance. The use of surfactants permitted to obtain highly interconnected porous scaffolds with tunable pore size on surface and in cross-section. Their mechanical properties in compression appeared to be adapted to soft tissue engineering. Scaffold structures could sustain MSC proliferation over 14 days. Paracrine activity of scaffold-seeded MSCs varied with the scaffold structure and growth factors release was globally improved in comparison with control alginate scaffolds. Our results provide evidence that exploiting different surfactant types for alginate foam preparation could be an original method to obtain biocompatible scaffolds with tunable architecture for soft tissue engineering.

[Ageing: a matter of heart?]. / Vieillissement - Une question de cœur ?

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.

Sphingosine kinase 1 expressed by endothelial colony-forming cells has a critical role in their revascularization activity.

AIMS: Cell therapy based on endothelial colony-forming cells (ECFCs) is a promising option for ischaemic cardiovascular diseases. A better understanding of the mechanisms by which these cells promote revascularization remains a critical challenge to improving their therapeutic potential. We aimed to identify the critical mechanisms involved in the revascularization activity of ECFCs by using the paracrine properties of mesenchymal stem cells (MSC). METHODS AND RESULTS: Conditioned medium from human bone marrow-derived MSCs (MSC-CM) increased the angiogenic activity of cord blood ECFCs in vitro (proliferation, migration, and pseudo-tube formation), the survival of ECFCs in mice (Matrigel Plug assay), and the capacity of ECFCs to promote the recovery of blood perfusion in mice with hindlimb ischaemia. Furthermore, the capillary density in ischaemic gastrocnemius muscle was significantly increased in mice transplanted with the ECFCs pre-treated with the MSC-CM. The enhancement of ECFCs activity involved the up-regulation of sphingosine kinase 1 (SphK1) expression and activity. The inhibition of SphK1 in ECFCs by using an inhibitor or a siRNA knockdown of SphK1 prevented the stimulation of the ECFCs induced by the MSC-CM. The improvement of ECFC activity by MSC-CM also involved the up-regulation of sphingosine-1-phosphate receptor 1 (S1P1) and a S1P/S1P1/3-dependent mechanism. Finally, we showed that the stimulation of ECFCs with exogenous S1P increased angiogenesis and promoted blood perfusion in hindlimb ischaemia. CONCLUSION: The up-regulation of SphK1 and S1P-dependent pathways is critical for the angiogenic/vasculogenic activity of ECFCs. The identification of this pathway provides attractive targets to optimize cell-based therapy for revascularization in ischaemic diseases.

Evaluation of polyelectrolyte complex-based scaffolds for mesenchymal stem cell therapy in cardiac ischemia treatment.

Three-dimensional (3D) scaffolds hold great potential for stem cell-based therapies. Indeed, recent results have shown that biomimetic scaffolds may enhance cell survival and promote an increase in the concentration of therapeutic cells at the injury site. The aim of this work was to engineer an original polymeric scaffold based on the respective beneficial effects of alginate and chitosan. Formulations were made from various alginate/chitosan ratios to form opposite-charge polyelectrolyte complexes (PECs). After freeze-drying, the resultant matrices presented a highly interconnected porous microstructure and mechanical properties suitable for cell culture. In vitro evaluation demonstrated their compatibility with mesenchymal stell cell (MSC) proliferation and their ability to maintain paracrine activity. Finally, the in vivo performance of seeded 3D PEC scaffolds with a polymeric ratio of 40/60 was evaluated after an acute myocardial infarction provoked in a rat model. Evaluation of cardiac function showed a significant increase in the ejection fraction, improved neovascularization, attenuated fibrosis as well as less left ventricular dilatation as compared to an animal control group. These results provide evidence that 3D PEC scaffolds prepared from alginate and chitosan offer an efficient environment for 3D culturing of MSCs and represent an innovative solution for tissue engineering.

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.

Alginate scaffolds for mesenchymal stem cell cardiac therapy: influence of alginate composition.

Despite the success of alginate scaffolds and mesenchymal stem cells (MSCs) therapy in cardiac failure treatment, the impact of the physicochemical environment provided by alginate matrices on cell behavior has never been investigated. The purpose of this work was double: to determine the alginate composition influence on (1) encapsulated rat MSC viability, paracrine activity, and phenotype in vitro and (2) cardiac implantability and in vivo biocompatibility of patch shape scaffolds. Two alginates, differing in composition and thus presenting different mechanical properties when hydrogels, were characterized. In both cases, encapsulated MSC viability was maintained at around 75%, and their secretion characteristics were retained 28 days postencapsulation. In vivo study revealed a high cardiac compatibility of the tested alginates: cardiac parameters were maintained, and rats did not present any sign of infection. Moreover, explanted hydrogels appeared surrounded by a vascularized tissue. However, scaffold implantability was highly dependent on alginate composition. G-type alginate patches, presenting higher elastic and Young moduli than M-type alginate patches, showed a better implantation easiness and were the only ones that maintained their shape and morphology in vivo. As a consequence of alginate chemical composition and resulting hydrogel structuration, G-type alginate hydrogels appear to be more adapted for cardiac implantation.

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]ß-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.

Mesenchymal stem cells promote matrix metalloproteinase secretion by cardiac fibroblasts and reduce cardiac ventricular fibrosis after myocardial infarction.

Recent studies showed that mesenchymal stem cells (MSCs) transplantation significantly decreased cardiac fibrosis; however, the mechanisms involved in these effects are still poorly understood. In this work, we investigated whether the antifibrotic properties of MSCs involve the regulation of matrix metalloproteinases (MMPs) and matrix metalloproteinase endogenous inhibitor (TIMP) production by cardiac fibroblasts. In vitro experiments showed that conditioned medium from MSCs decreased viability, alpha-smooth muscle actin expression, and collagen secretion of cardiac fibroblasts. These effects were concomitant with the stimulation of MMP-2/MMP-9 activities and membrane type 1 MMP expression. Experiments performed with fibroblasts from MMP2-knockout mice demonstrated that MMP-2 plays a preponderant role in preventing collagen accumulation upon incubation with conditioned medium from MSCs. We found that MSC-conditioned medium also decreased the expression of TIMP2 in cardiac fibroblasts. In vivo studies showed that intracardiac injection of MSCs in a rat model of postischemic heart failure induced a significant decrease in ventricular fibrosis. This effect was associated with the improvement of morphological and functional cardiac parameters. In conclusion, we showed that MSCs modulate the phenotype of cardiac fibroblasts and their ability to degrade extracellular matrix. These properties of MSCs open new perspectives for understanding the mechanisms of action of MSCs and anticipate their potential therapeutic or side effects.

Ex vivo pretreatment with melatonin improves survival, proangiogenic/mitogenic activity, and efficiency of mesenchymal stem cells injected into ischemic kidney.

Bone marrow mesenchymal stem cells (MSCs) have shown great potential in cell therapy of solid organs. Approaches to improving the ability of grafted MSCs to survive and secrete paracrine factors represent one of the challenges for the further development of this novel therapy. In the present study, we designed a strategy of ex vivo pretreatment with the pineal hormone melatonin to improve survival, paracrine activity, and efficiency of MSCs. Using a rat model of acute renal failure, we showed that melatonin pretreatment strongly increased survival of MSCs after intraparenchymal injection. This effect was concomitant with overstimulation of angiogenesis, proliferation of renal cells, and accelerated recovery of renal function. To gain insight into the mechanisms involved in the effects observed in vivo, melatonin was tested in vitro on cultured MSCs. Our results show that through stimulation of specific melatonin receptors, melatonin induced an overexpression of the antioxidant enzyme catalase and superoxide dismutase-1 and increased the resistance of MSCs to hydrogen peroxide-dependent apoptosis. Compared with untreated cells, MSCs incubated with melatonin displayed a higher expression of basic fibroblast growth factor and hepatocyte growth factor. In addition, conditioned culture media from melatonin-treated MSCs stimulated tube formation by endothelial progenitor cells and proliferation of proximal tubule cells in culture. In conclusion, our results show that melatonin behaves as a preconditioning agent increasing survival, paracrine activity, and efficiency of MSCs. The use of this molecule for pretreatment of stem cells may represent a novel and safe approach to improving the beneficial effects of cell therapy of solid organs.

Proteomic analysis of anaplastic lymphoma cell lines: identification of potential tumour markers.

Anaplastic large-cell lymphomas (ALCL) are high grade lymphomas of T or null phenotype often associated with the t(2;5) translocation leading to the expression of a chimeric protein consisting of the N-terminal portion of nucleophosmin (NPM) and the intracellular domain of the anaplastic lymphoma kinase (ALK). Although ALCL are recognized as distinct clinical, biological and cytogenetic entities, heterogeneities persist in this group of tumours, which exhibit a broad spectrum of morphological features. Particularly, the common type tumour consisting in large cells contrast with the small cell variant that is sometimes associated with a leukemic phase. The ALK-negative ALCL is often associated with a poor prognosis. Here, we investigated the proteome of these subtypes of tumours using patient-derived cell lines. We compared the proteome of the cytosolic fraction of NPM-ALK-positive versus NPM-ALK-negative cells on one hand, and the proteome of common cell type versus small cell variant on the other hand. The identification of a set of proteins differentially expressed in the subtypes of ALCL points to new diagnosis/prognosis markers. This study also provides interesting information on the molecular mechanisms responsible for the different subtypes of ALCL.