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

Raman characterization of human skin aging

Skin aging is a complex biological process mixing intrinsic and extrinsic factors, such as sun exposure. At the molecular level, skin aging affects in particular the extracellular matrix proteins. Materials and Methods: Using Raman imaging, which is a nondestructive approach appropriate for studying biological samples, we analyzed how aging modifies the matrix proteins of the papillary and reticular dermis. Biopsies from the buttock and dorsal forearm of volunteers younger than 30 and older than 60 were analyzed in order to identify chronological and photoaging processes. Analyses were performed on skin section, and Raman spectra were acquired separately on the different dermal layers. Results: We observed differences in dermal matrix structure and hydration state with skin aging. Chronological aging alters in particular the collagen of the papillary dermis, while photoaging causes a decrease in collagen stability by altering proline and hydroxyproline residues in the reticular dermis. Moreover, chronological aging alters glycosaminoglycan content in both dermal compartments. Conclusion: Alterations of the papillary and reticular dermal matrix structures during photo-and chronological aging were clearly depicted by Raman spectroscopy

Thérapie cellulaire de l'insuffisance rénale et cardiaque

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Le réveil des cardiomyocytes adultes résidents

Les pathologies cardiaques, et en particulier l’infarctus du myocarde, conduisent inéluctablement à la mort des cardiomyocytes adultes favorisant le développement de l’insuffisance cardiaque. Les efforts en médecine cardiaque régénératrice se sont concentrés jusqu’à présent sur l’utilisation des cellules souches, laissant de côté les cardiomyocytes préexistants, considérés comme étant dans un état postmitotique. Néanmoins, des données récentes obtenues chez le poisson zèbre et les mammifères relancent le débat sur la capacité proliférative de ces cellules. Dans cette revue, nous proposons un état des lieux des connaissances de la capacité proliférative et régénératrice des cardiomyocytes résidents, et discutons certains aspects mécanistiques. Dans le futur, l’identification précise des mécanismes moléculaires permettant à ces cellules de reprendre leur prolifération devrait permettre d’ouvrir de nouvelles perspectives thérapeutiques en régénération cardiaque

Pro‐inflammatory activity of Cutibacterium acnes phylotype IA 1 and extracellular vesicles: An in vitro study

International audienceAcne is a chronic inflammatory skin condition that involves Cutibacterium acnes (C. acnes), which is classified into six main phylotypes (IA1, IA2, IB, IC, II and III). Acne development is associated with loss of C. acnes phylotype diversity, characterised by overgrowth of phylotype IA1 relative to other phylotypes. It was also shown that purified extracellular vesicles (EVs) secreted by C. acnes can induce an acne-like inflammatory response in skin models. We aimed to determine if the inflammatory profile of EVs secreted by C. acnes phylotype IA1 from an inflammatory acne lesion was different from C. acnes phylotype IA1 from normal skin, thus playing a direct role in the severity of inflammation. EVs were produced in vitro after culture of two clinical strains of C. acnes phylotype IA1, T5 from normal human skin and A47 from an inflammatory acne lesion, and then incubated with either human immortalised keratinocytes, HaCaT cells, or skin explants obtained from abdominoplasty. Subsequently, quantitative PCR (qPCR) was performed for human β-defensin 2 (hBD2), cathelicidin (LL-37), interleukin (IL)-1β, IL-6, IL-8, IL-17α and IL-36γ, and ELISA for IL-6, IL-8 and IL-17α. We found that EVs produced in vitro by C. acnes derived from inflammatory acne lesions significantly increased the pro-inflammatory cytokines and anti-microbial peptides at both transcriptional and protein levels compared with EVs derived from normal human skin. We show for the first time that C. acnes EVs from inflammatory acne play a crucial role in acne-associated inflammation in vitro and that C. acnes phylotype IA1 collected from inflammatory acne lesion and normal skin produce different EVs and inflammatory profiles in vitro

Role of endothelial AADC in cardiac synthesis of serotonin and nitrates accumulation.

Serotonin (5-HT) regulates different cardiac functions by acting directly on cardiomyocytes, fibroblasts and endothelial cells. Today, it is widely accepted that activated platelets represent a major source of 5-HT. In contrast, a supposed production of 5-HT in the heart is still controversial. To address this issue, we investigated the expression and localization of 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC) in the heart. We also evaluated their involvement in cardiac production of 5-HT. TPH1 was weakly expressed in mouse and rat heart and appeared restricted to mast cells. Degranulation of mast cells by compound 48/80 did not modify 5-HT cardiac content in mice. Western blots and immunolabelling experiments showed an abundant expression of AADC in the mouse and rat heart and its co-localization with endothelial cells. Incubation of cardiac homogenate with the AADC substrate (5-hydroxy-L-tryptophan) 5-HTP or intraperitoneal injection of 5-HTP in mice significantly increased cardiac 5-HT. These effects were prevented by the AADC inhibitor benserazide. Finally, 5-HTP administration in mice increased phosphorylation of aortic nitric oxide synthase 3 at Ser (1177) as well as accumulation of nitrates in cardiac tissue. This suggests that the increase in 5-HT production by AADC leads to activation of endothelial and cardiac nitric oxide pathway. These data show that endothelial AADC plays an important role in cardiac synthesis of 5-HT and possibly in 5-HT-dependent regulation of nitric oxide generation

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

TORC1 and TORC2 converge to regulate the SAGA co-activator in response to nutrient availability

International audienc