Evaluation of alginate microspheres for mesenchymal stem cell engraftment on solid organ

Mesenchymal stem cells (MSCs) may be used as a cell source for cell therapy of solid organs due to their differentiation potential and paracrine effect. Nevertheless, optimization of MSC-based therapy needs to develop alternative strategies to improve cell administration and efficiency. One option is the use of alginate microencapsulation, which presents an excellent biocompatibility and an in vivo stability. As MSCs are hypoimmunogenic, it was conceivable to produce microparticles with [alginate-poly-L-lysine-alginate (APA) microcapsules] or without (alginate microspheres) a surrounding protective membrane. Therefore, the aim of this study was to determine the most suitable microparticles to encapsulate MSCs for engraftment on solid organ. First, we compared the two types of microparticles with 4 × 106 MSCs/ml of alginate. Results showed that each microparticle has distinct morphology and mechanical resistance but both remained stable over time. However, as MSCs exhibited a better viability in microspheres than in microcapsules, the study was pursued with microspheres. We demonstrated that viable MSCs were still able to produce the paracrine factor bFGF and did not present any chondrogenic or osteogenic differentiation, processes sometimes reported with the use of polymers. We then proved that microspheres could be implanted under the renal capsule without degradation with time or inducing impairment of renal function. In conclusion, these microspheres behave as an implantable scaffold whose biological and functional properties could be adapted to fit with clinical applications

Oxidative stress-dependent sphingosine kinase-1 inhibition mediates monoamine oxidase A-associated cardiac cell apoptosis

The mitochondrial enzyme monoamine oxidase (MAO), its isoform MAO-A, plays a major role in reactive oxygen species-dependent cardiomyocyte apoptosis and postischemic cardiac damage. In the current study, we investigated whether sphingolipid metabolism can account for mediating MAO-A- and reactive oxygen species-dependent cardiomyocyte apoptosis. In H9c2 cardiomyoblasts, MAO-A-dependent reactive oxygen species generation led to mitochondria-mediated apoptosis, along with sphingosine kinase-1 (SphK1) inhibition. These phenomena were associated with generation of proapoptotic ceramide and decrease in prosurvival sphingosine 1-phosphate. These events were mimicked by inhibition of SphK1 with either pharmacological inhibitor or small interfering RNA, as well as by extracellular addition of C(2)-ceramide or H(2)O(2). In contrast, enforced expression of SphK1 protected H9c2 cells from serotonin- or H(2)O(2)-induced apoptosis. Analysis of cardiac tissues from wild-type mice subjected to ischemia/reperfusion revealed significant upregulation of ceramide and inhibition of SphK1. It is noteworthy that SphK1 inhibition, ceramide accumulation, and concomitantly infarct size and cardiomyocyte apoptosis were significantly decreased in MAO-A-deficient animals. In conclusion, we show for the first time that the upregulation of ceramide/sphingosine 1-phosphate ratio is a critical event in MAO-A-mediated cardiac cell apoptosis. In addition, we provide the first evidence linking generation of reactive oxygen species with SphK1 inhibition. Finally, we propose sphingolipid metabolites as key mediators of postischemic/reperfusion cardiac injury

Vesicular monoamine transporter 1 mediates dopamine secretion in rat proximal tubular cells.

International audienceRenal dopamine, synthesized by proximal tubules, plays an important role in the regulation of renal sodium excretion. Although the renal dopaminergic system has been extensively investigated in both physiological and pathological situations, the mechanisms whereby dopamine is stored and secreted by proximal tubule cells remain obscure. In the present study we investigated whether vesicular monoamine transporters (VMAT)-1 and -2, which participate in amine storing and secretion, are expressed in rat renal proximal tubules, and we defined their involvement in dopamine secretion. By combining RT-PCR, Western blot, and immunocytochemistry we showed that VMAT-1 is the predominant isoform expressed in isolated proximal tubule cells. These results were confirmed by immunohistochemistry analysis of rat renal cortex showing that VMAT-1 was found in proximal tubules but not in glomeruli. Functional studies showed that, as previously reported for VMAT-dependent amine transporters, dopamine release by cultured proximal tubule cells was partially inhibited by disruption of intracellular H(+) gradient. In addition, dopamine secretion was prevented by the VMAT-1/VMAT-2 inhibitor reserpine but not by the VMAT-2 inhibitor tetrabenazine. Finally, we demonstrated that tubular VMAT-1 mRNA and protein expression were significantly upregulated during a high-sodium diet. In conclusion, our results show for the first time the expression of a VMAT in the renal proximal tubule and its involvement in regulation of dopamine secretion. These data represent the first step toward the comprehension of the role of this transporter in renal dopamine handling and its involvement in pathological situations

Mesenchymal Stem Cells Promote Matrix Metalloproteinase Secretion By Cardiac Fibroblasts And Reduce Cardiac Ventricular Fibrosis After Myocardial Infarction.

International audienceRecent 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 (TIMPs) production by cardiac fibroblasts.In vitro experiments showed that conditioned medium from MSCs decreased viability, alpha-SMA expression and collagen secretion of cardiac fibroblasts. These effects were concomitant to the stimulation of MMP-2/MMP-9 activities and MT1-MMP expression. Experiments performed with fibroblasts from MMP2-/- mice demonstrated that MMP2 plays a preponderant role in preventing collagen accumulation upon incubation with conditioned-medium from MSCs. Interestingly, 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 post-ischemic 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 perspective for understanding of the mechanisms of action of MSCs and anticipate their potential therapeutic or side effects

Cardiac fibroblasts regulate sympathetic nerve sprouting and neurocardiac synapse stability.

International audienceSympathetic 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

Structural evidence for a new elaborate 3D-organization of the cardiomyocyte lateral membrane in adult mammalian cardiac tissues

International audienceAims: This study explored the lateral crest structures of adult cardiomyocytes (CMs) within healthy and diseased cardiac tissue.Methods and results: Using high-resolution electron and atomic force microscopy, we performed an exhaustive quantitative analysis of the three-dimensional (3D) structure of the CM lateral surface in different cardiac compartments from various mammalian species (mouse, rat, cow, and human) and determined the technical pitfalls that limit its observation. Although crests were observed in nearly all CMs from all heart compartments in all species, we showed that their heights, dictated by the subsarcolemmal mitochondria number, substantially differ between compartments from one species to another and tightly correlate with the sarcomere length. Differences in crest heights also exist between species; for example, the similar cardiac compartments in cows and humans exhibit higher crests than rodents. Unexpectedly, we found that lateral surface crests establish tight junctional contacts with crests from neighbouring CMs. Consistently, super-resolution SIM or STED-based immunofluorescence imaging of the cardiac tissue revealed intermittent claudin-5-claudin-5 interactions in trans via their extracellular part and crossing the basement membrane. Finally, we found a loss of crest structures and crest–crest contacts in diseased human CMs and in an experimental mouse model of left ventricle barometric overload.Conclusion: Overall, these results provide the first evidence for the existence of differential CM surface crests in the cardiac tissue as well as the existence of CM–CM direct physical contacts at their lateral face through crest–crest interactions. We propose a model in which this specific 3D organization of the CM lateral membrane ensures the myofibril/myofiber alignment and the overall cardiac tissue cohesion. A potential role in the control of sarcomere relaxation and of diastolic ventricular dysfunction is also discussed. Whether the loss of CM surface crests constitutes an initial and common event leading to the CM degeneration and the setting of heart failure will need further investigation

Crest maturation at the cardiomyocyte surface contributes to a new late postnatal development stage that controls the diastolic function of the adult heart

Abstract RATIONALE In addition to its typical rod-shape, the mammalian adult cardiomyocyte (CM) harbors a unique lateral membrane surface architecture with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) the role of which is still unknown. OBJECTIVE To investigate the development and functional role of CM crests during the postnatal period. METHODS AND RESULTS Electron/confocal microscopy and western-blot of left ventricular tissues from rat hearts indicated a late CM surface crest maturation, between postnatal day 20 (P20) and P60, as shown by substantial SSM swelling and increased claudin-5 cell surface expression. The P20-P60 postnatal stage also correlates with an ultimate maturation of the T-Tubules and the intercalated disk. At the cellular level, we identified an atypical CM hypertrophy characterized by an increase in long- and short-axes without myofibril addition and with sarcomere lateral stretching, indicative of lateral stretch-based CM hypertrophy. We confirmed the P20-P60 hypertrophy at the organ level by echocardiography but also demonstrated a transcriptomic program after P20 targeting all the cardiac cell populations. At the functional level, using Doppler echocardiography, we found that the P20-P60 period is specifically dedicated to the improvement of relaxation. Mechanistically, using CM-specific knock-out mice, we identified ephrin-B1 as a determinant of CM crest maturation after P20 controlling lateral CM stretch-hypertrophy and relaxation. Interestingly, while young adult Efnb1 CMspe−/− mice essentially show a relaxation impairment with exercise intolerance, they progressively switch toward heart failure with 100% KO mice dying after 13 months. CONCLUSIONS This study highlights a new late P20-P60 postnatal developmental stage of the heart in rodents during which the CM surface crests mature through an ephrin-B1-dependant mechanism and regulate the diastolic function. Moreover, we demonstrate for the first time that the CM crest architecture is cardioprotective

Myocardial infarction is associated with immature sympathetic heart hyperinnervation.

<p>(<b>A</b>) Representative Western-blot analysis of NGF expression in the ischemic area of infarcted area (MI), compared to sham-operated rats (Ctl). (<b>B</b>) Tyrosine hydroxylase-positive nerve fibers (TH) in sham-operated animals (Ctl) and within the ischemic area of infarcted heart 15 days following myocardial infarction (MI). Values are mean ±SEM. ***, <i>p</i><0.001 vs. Ctl (n = 3–6 per rat lineage group). Scale bars  = 50 µm. (<b>C</b>) Immunofluorescent staining for PSA-NCAM (immature neurons marker) in myocardial sections of sham-operated animals (Ctl) and ischemic area (<i>I-area</i>) of infarcted hearts 15 days following myocardial infarction (MI). Scale bars  = 100 µm. (<b>D</b>) Cardiac tissue norepinephrine (NE) levels after myocardial infarction. Cardiac tissue norepinephrine was quantified by HPLC in sham-operated rats (Ctl) and in non-ischemic or ischemic areas of infarcted heart (MI), 15 days after myocardial infarction (n = 4–5 per rat group). (<b>E</b>) Representative and quantitative Western-blot analysis of cardiac tyrosine hydroxylase (TH) protein expression in sham-operated rats (Ctl) and in ischemic areas 15 days after myocardial infarction (MI). Values are means ±SEM. (n = 6 per rat group). *, p<0.05 vs. Ctl.</p

Neurotrophic factor gene expression profiling in isolated adult cardiomyocytes and cardiac fibroblasts.

<p><b>A</b> NGF, NT3, NT4, CNTF and BDNF gene expression were measured by real time qPCR using mRNA extracted from cardiomyocytes (CM) and cardiac fibroblasts (Fb) isolated from adult control heart. Values represent the mean ±SEM. (***, <i>p<0.001</i> vs. CM; **<i>p<0.001</i> vs. CM; <i>ND</i>: no detectable; n = 3–5). <b>B</b> NGF protein expression was evaluated by Western-blot experiments on primary cultures of adult cardiomyocytes (CM) and adult cardiac fibroblasts (Fb). Lower panel: Ponceau Red staining to control for equal protein loading between CM and Fb samples used for NGF blotting (upper).</p

Impact of cardiac fibroblasts on neurocardiac synapse structure/functionality.

<p>(<b>A</b>) Neurocardiac synapse function: norepinephrine secretion by NGF-differentiated PC12 was quantified by HPLC in the culture medium under 48 hours different culture conditions: PC12 cultured alone (PC12), fibroblasts/PC12 co-culture (PC12+Fb), PC12/cardiomyocytes co-culture (PC12+CM) or PC12/cardiomyocytes/fibroblasts tri-culture (PC12+Fb+CM). *, <i>p<0.05</i> vs. PC12; **, <i>p<0.01</i> vs. PC12; NS: no statistical difference (n = 3–4). (<b>B</b>) Neurocardiac synapse structure: photomicrographs of PC12 cells cultured alone or PC12/CM co-culture show neurite degeneration when compared with PC12+Fb co-culture and PC12+Fb+CM tri-culture, after 3 days in culture. (arrows: fibroblasts, asterisks: cardiomyocytes). Scale bars  = 100 µm. In all experiments, PC12 neuritogenesis was promoted by 8-days incubation in the presence of NGF (See materials and methods).</p