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

Resumen

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.