Vesículas extracelulares derivadas de células madre mesenquimales de tejido adiposo como terapia biológica en células articulares osteoartríticas

La osteoartritis (OA) es la enfermedad articular más común en las personad de edad avanzada y se asocia con una pérdida progresiva de cartílago articular, sinovitis de bajo grado y varias alteraciones perjudiciales en el hueso subcondral y los tejidos periarticulares. En la OA hay un desequilibrio entre los procesos anabólicos y catabólicos de las articulaciones que se agrava por el estrés mecánico y la acumulación de mediadores inflamatorios. No existe un tratamiento eficaz para la OA, aunque se han investigado nuevas terapias, como el uso de células madre mesenquimales (MSC) o los productos que liberan en el espacio extracelular (secretoma). En esta tesis, hemos estudiado el uso potencial de las vesículas extracelulares (EV) del secretoma de MSC derivadas de tejido adiposo (ASCs) como una posible terapia biológica heteróloga sin células para las células de la articulación osteoartrítica. Se sabe que las MSC, que han mostrado propiedades inmunomoduladoras y regenerativas, liberan un conjunto heterogéneo de mediadores solubles y EVs cuyas interacciones con otras células y tejidos podrían explicar los efectos de las MSC. Las EVs son secretadas activamente por prácticamente todos los tipos de células y representan un nuevo mecanismo de comunicación intercelular tanto en condiciones fisiológicas como patológicas. En nuestro trabajo, hemos caracterizado con éxito dos subtipos vesiculares específicos en el medio condicionado (CM) de las ASC, de diferentes tamaños y composiciones, definidas como microvesículas (MV) y exosomas (EX). Su composición de proteínas se ha estudiado mediante espectrometría de masas y microscopía confocal, identificando varias proteínas con posibles efectos inmunomoduladores y protectores, como la anexina A1. La inflamación crónica explica muchas alteraciones en la OA. Los posibles efectos inmunomoduladores de MV y EX se han estudiado en condrocitos osteoartríticos primarios y explantes de cartílago estimulados con IL-1beta. Nuestros resultados indican que ambos tipos de EV replican o mejoran los efectos del CM del que provienen. Redujeron la liberación de citoquinas proinflamatorias tales como IL-6 y TNFalpha y promovieron la síntesis de IL-10 y colágeno tipo II. También inhibieron la producción de PGE2 y NO, la actividad enzimática de MMP, la transcripción de COX-2 y mPGES-1, y la activación de NF-kB y AP-1. El bloqueo de EV-anexina A1 con anticuerpos específicos revirtió la inhibición de la síntesis de IL-6 y la producción de colágeno tipo II. En la OA, el envejecimiento conduce a estrés oxidativo y senescencia. Hemos evaluado los efectos anti-senescentes de los EV en los osteoblastos osteoartríticos primarios estimulados con IL-1beta y encontramos que las EVs promueven la liberación de IL-10 e inhiben la producción de IL-6 y PGE2. Inhiben la peroxidación lipídica, restauran el potencial de membrana mitocondrial y reducen la actividad de la beta-galactosidasa asociada a la senescencia y la acumulación de focos yH2AX. Nuestros resultados indican que los EV son actores relevantes en el secretoma de ASC y representan una nueva estrategia terapéutica para el tratamiento de enfermedades articulares inflamatorias crónicas.Osteoarthritis (OA) is the most common joint disease in the elderly and it is associated with a progressive loss of articular cartilage, low-grade synovitis and several deleterious alterations in subchondral bone and periarticular tissues. In OA there is an imbalance between joint anabolic and catabolic processes that is aggravated by mechanical stress and accumulation of inflammatory mediators. There is no effective treatment for OA, although new therapies have been investigated, such as the use of mesenchymal stem cells (MSCs) or the products that they release into the extracellular space (secretome). In this thesis we have studied the potential use of extracellular vesicles (EVs) from the secretome of adipose tissue-derived MSCs (ASCs) as a possible heterologous cell-free biological therapy for osteoarthritic joint cells. It is known that MSCs, which have shown immunomodulatory and regenerative properties, release a heterogeneous set of soluble mediators and EVs whose interactions with other cells and tissues could explain the effects of MSCs. EVs are actively secreted by practically all cell types and represent a novel mechanism of intercellular communication both under physiological and pathological conditions. In our work, we have successfully characterized two specific vesicular subtypes in the conditioned medium (CM) of ASCs, of different sizes and compositions, defined as microvesicles (MVs) and exosomes (EXs). Their protein composition has been studied by mass spectrometry and confocal microscopy, identifying several proteins with potential immunomodulatory and protective effects such as annexin A1. Chronic inflammation explains many alterations in OA. The possible immunomodulatory effects of MVs and EXs have been studied in primary osteoarthritic chondrocytes and cartilage explants stimulated with IL-1beta. Our results indicate that both types of EVs replicate or improve the effects of the CM from which they come. They reduced the release of proinflammatory cytokines such as IL-6 and TNFalpha and promoted the synthesis of IL-10 and collagen type II. They also inhibited the production of PGE2 and NO, the MMP enzymatic activity, the transcription of COX-2 and mPGES-1, and the activation of NF-kB and AP-1. EV-Annexin A1 blockade with specific antibodies reversed the inhibition of IL-6 synthesis and the production of collagen type II. In OA, aging leads to oxidative stress and senescence. We have evaluated the anti-senescent effects of EVs in primary osteoarthritic osteoblasts stimulated with IL-1beta, and found that EVs promote the release of IL-10 and inhibit the production of IL-6 and PGE2. They inhibit lipid peroxidation, restore the mitochondrial membrane potential, and reduce senescence-associated beta-galactosidase activity and the accumulation of yH2AX foci. Our results indicate that EVs are relevant actors in the ASC secretome and represent a new therapeutic strategy for the treatment of chronic inflammatory joint diseases

Extracellular vesicles: A new therapeutic strategy for joint conditions

[EN] Extracellular vesicles (EVs) are attracting increasing interest since they might represent a more convenient therapeutic tool with respect to their cells of origin. In the last years much time and effort have been expended to determine the biological properties of EVs from mesenchymal stem cells (MSCs) and other sources. The immunoregulatory, anti-inflammatory and regenerative properties of MSC EVs have been demonstrated in in vitro studies and animal models of rheumatoid arthritis or osteoarthritis. This cell-free approach has been proposed as a possible better alternative to MSC therapy in autoimmune conditions and tissue regeneration. In addition, EVs show great potential as biomarkers of disease or delivery systems for active molecules. The standardization of isolation and characterization methods is a key step for the development of EV research. A better understanding of EV mechanisms of action and efficacy is required to establish the potential therapeutic applications of this new approach in joint conditions.This work has been funded by grants SAF2013-48724R (MINECO,FEDER, Spain) and PROMETEOII/2014/071 (Generalitat Valenciana, Spain).Tofiño-Vian, M.; Guillen Salazar, MI.; Alcaraz Tormo, MJ. (2018). Extracellular vesicles: A new therapeutic strategy for joint conditions. Biochemical Pharmacology. 153:134-146. https://doi.org/10.1016/j.bcp.2018.02.004S13414615

Microvesicles from Human Adipose Tissue-Derived Mesenchymal Stem Cells as a New Protective Strategy in Osteoarthritic Chondrocytes

[EN] Background/Aims: Chronic inflammation contributes to cartilage degeneration during the progression of osteoarthritis (OA). Adipose tissue-derived mesenchymal stem cells (ADMSC) show great potential to treat inflammatory and degradative processes in OA and have demonstrated paracrine effects in chondrocytes. In the present work, we have isolated and characterized the extracellular vesicles from human AD-MSC to investigate their role in the chondroprotective actions of these cells. Methods: AD-MSC were isolated by collagenase treatment from adipose tissue from healthy individuals subjected to abdominal lipectomy surgery. Microvesicles and exosomes were obtained from conditioned medium by filtration and differential centrifugation. Chondrocytes from OA patients were used in primary culture and stimulated with 10 ng/ml interleukin(IL)-1 beta in the presence or absence of AD-MSC microvesicles, exosomes or conditioned medium. Protein expression was investigated by ELISA and immunofluorescence, transcription factor-DNA binding by ELISA, gene expression by real-time PCR, prostaglandin E-2 (PGE(2)) by radioimmunoassay, and matrix metalloproteinase (MMP) activity and nitric oxide (NO) production by fluorometry. Results: In OA chondrocytes stimulated with IL-1 beta, microvesicles and exosomes reduced the production of inflammatory mediators tumor necrosis factor-alpha, IL-6, PGE(2) and NO. The downregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 would lead to the decreased PGE(2) production while the effect on NO could depend on the reduction of inducible nitric oxide synthase expression. Treatment of OA chondrocytes with extracellular vesicles also decreased the release of MMP activity and MMP-13 expression whereas the production of the anti-inflammatory cytokine IL-10 and the expression of collagen II were significantly enhanced. The reduction of inflammatory and catabolic mediators could be the consequence of a lower activation of nuclear factor-kappa B and activator protein-1. The upregulation of annexin A1 specially in MV may contribute to the anti-inflammatory and chondroprotective effects of AD-MSC. Conclusions: Our data support the interest of AD-MSC extracellular vesicles to develop new therapeutic approaches in joint conditions. (C) 2018 The Author(s) Published by S. Karger AG, BaselThis work was supported by grants SAF2013-4874R (MINECO, FEDER) and PROMETEOII/2014/071 (Generalitat Valenciana), Spain.Tofiño-Vian, M.; Guillen Salazar, MI.; Perez Del Caz, M.; Silvestre, A.; Alcaraz Tormo, MJ. (2018). Microvesicles from Human Adipose Tissue-Derived Mesenchymal Stem Cells as a New Protective Strategy in Osteoarthritic Chondrocytes. Cellular Physiology and Biochemistry. 47(1):11-25. https://doi.org/10.1159/000489739S112547

Estudios bioquímicos y moleculares en el mutante C BF5 en levadura

[EN] Dyskeratosis congenita is a rare disease that is caused by defects in the telomere maintenance machinery, resulting in the presence of shortened telomeres in highly active regenerative tissues such as bone marrow or skin. Several DKC1 mutations are responsible for the appearance of the disease. The gene encodes the nucleolar protein DKC1 or dyskerin, which associates with snoRNAs (small nucleolar RNAs) in the H/ACA snoRNP complex. This gene has an ortholog in yeast called CBF5. Given the simplicity and potential in the management of the Saccharomyces cerevisiae yeast, we have used the haploinsuficient mutant cbf5 as a model of the disease. In past studies, we analyzed the functional transcriptome of a cbf5 mutant strain in order to obtain information that may be useful for future therapeutic approaches by designing specific nanotechnological molecular gates. The obtained results showed that our mutant presents a hyper-secretory phenotype, as well as a certain amount of resistance to endoplasmic reticulum stress. In this work, we have demonstrated the existence of an activation of several pathways related to nutrient stress response and reticulum stress response (Unfolded Protein Response). More studies of the altered signaling pathways in this model will allow identifying possible therapeutic targets which may work as molecular gates.[ES] La disqueratosis congénita es una enfermedad rara que se origina a través de defectos en la maquinaria de mantenimiento del telómero, resultando en la presencia de telómeros acortados en tejidos de alta actividad regenerativa como la médula ósea o la piel. Varias mutaciones en el gen DK1 son las responsables de la aparición de la enfermedad. El gen DKC1 codifica una proteína nucleolar, la disquerina, asociada a snoRNAs (small nucleolar RNAs) en el complejo H/ACA snoRNP. Este gen cuenta con un ortólogo en levadura denominado CBF5. Dada la sencillez y potencialidad en el manejo de la levadura Saccharomyces cerevisiae, hemos utilizado el haploinsuficiente cbf5 como modelo de estudio de la enfermedad. En pasados estudios, hemos realizado un análisis transcriptómico funcional de la cepa mutante cbf5 con el objetivo de obtener información que pueda ser útil para un posterior enfoque terapéutico mediante el diseño de puertas moleculares nanotecnológicas específicas. Los primeros resultados mostraron un fenotipo hiper-secretor, así como un posible fenotipo de resistencia a condiciones de estrés de retículo. En el presente trabajo, hemos demostrado la activación de las rutas de respuesta frente a estrés de nutrientes mediado por la ruta GCN, y así estrés de retículo (Respuesta a proteínas desplegadas). Un estudio más pormenorizado de las rutas de señalización alteradas en este modelo permitirá identificar posibles dianas terapéuticas que sirvan como puertas molecularesTofiño Vian, M. (2014). Estudios bioquímicos y moleculares en el mutante C BF5 en levadura. http://hdl.handle.net/10251/59654Archivo delegad

Extracellular Vesicles from Adipose-Derived Mesenchymal Stem Cells Downregulate Senescence Features in Osteoarthritic Osteoblasts

Osteoarthritis (OA) affects all articular tissues leading to pain and disability. The dysregulation of bone metabolism may contribute to the progression of this condition. Adipose-derived mesenchymal stem cells (ASC) are attractive candidates in the search of novel strategies for OA treatment and exert anti-inflammatory and cytoprotective effects on cartilage. Chronic inflammation in OA is a relevant factor in the development of cellular senescence and joint degradation. In this study, we extend our previous observations of ASC paracrine effects to study the influence of conditioned medium and extracellular vesicles from ASC on senescence induced by inflammatory stress in OA osteoblasts. Our results in cells stimulated with interleukin- (IL-) 1β indicate that conditioned medium, microvesicles, and exosomes from ASC downregulate senescence-associated β-galactosidase activity and the accumulation of γH2AX foci. In addition, they reduced the production of inflammatory mediators, with the highest effect on IL-6 and prostaglandin E2. The control of mitochondrial membrane alterations and oxidative stress may provide a mechanism for the protective effects of ASC in OA osteoblasts. We have also shown that microvesicles and exosomes mediate the paracrine effects of ASC. Our study suggests that correction of abnormal osteoblast metabolism by ASC products may contribute to their protective effects