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Joint Tissue Protective and Immune-Modulating miRNA Landscape of Mesenchymal Stromal Cell-Derived Extracellular Vesicles under Different Osteoarthritis-Mimicking Conditions

ABSTRACT: In regenerative medicine related to orthopedic conditions, mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) have been proposed as innovative clinical options. The definition of EV-shuttled signals and their modulation under orthopedic settings, such as osteoarthritis (OA), is crucial for MSC-related research, both for basic science and for use in clinical settings, either as therapeutics or as producers of cell-free products such as EVs or secretome. The objective of this work is to compare the literature available on high-throughput EV-miRNA data obtained from adipose-derived MSCs (ASCs) in standard conditions or cultured in high levels of IFNγ, low-level inflammatory conditions mimicking OA synovial fluid (SF), and OA-SF. The first result was that both IFNγ and low-level inflammatory treatment led to an increase, whereas SF led to a reduction in EV release. Second, more than 200 EV-miRNAs were found to be shared across the different conditions. After a bioinformatics search through experimentally validated and OA-related targets, pathways and tissues, several miRNAs resulted in the restoration of cartilage and synovium stability and the homeostasis of inflammatory cells, including macrophages, promoting their switch towards an M2 anti-inflammatory phenotype. Third, IFNγ and especially SF culturing were able to modulate the overall EV-miRNA fingerprint, although the main molecular messages related to OA resulted conserved between treatments with the majority of modulations within 2-fold range. In conclusion, ASC EV-miRNAs may be modulated in their overall landscape by OA-related culturing conditions albeit resulted largely stable in their specific OA-protective signals allowing for a faster clinical translation of these new cell-free therapies for joint diseases.

SUBMITTER: Ragni E

PROVIDER: S-EPMC9321932 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionMesenchymal stromal cells (MSCs) play an important role in the prevention of cell and tissue fibrosis. Senescence may decrease the function of MSCs during recovery from tissue and organ damage. Extracellular vesicles (EVs) released from MSCs contribute to the repair of kidney injury. We explored the influence of senescence on EVs derived from MSCs (MSC-EVs) and detected the protective effects of MSC-EVs expressing low levels of miR-294/miR-133 derived from old rats against chronic kidney disease (CKD).MethodsThe effects of MSC-EVs derived from 3-month-old and 18-month-old male Fisher 344 rats on renal fibrosis were explored in a unilateral ureteral obstruction (UUO) model. pLV-miR-294/pLV-miR-133 mimic/inhibitor were injected into young and old rats before UUO to detect the effects of miR-294/miR-133, which were decreased in MSC-EVs and sera from old rats, on renal function in CKD. Transforming growth factor-β1 (TGF-β1)-induced human renal proximal tubular epithelial (HK2) cells were used to imitate the pathological process of renal fibrosis in vitro. Western blotting was used to assess the expression of epithelial/mesenchymal markers and phosphorylation of proteins in HK2 cells.ResultsThe inhibition of UUO-induced CKD by MSC-EVs was weaker in old rats than in young rats. Downregulation of miRNAs (miR-294 and miR-133) in both MSC-EVs and sera from old rats obviously attenuated UUO-induced renal injury in old rats. miR-294 and miR-133 overexpression mitigated TGF-β1-mediated epithelial-mesenchymal transition (EMT) in HK2 cells, and the obvious increase in the phosphorylation of both SMAD2/3 and ERK1/2 induced by TGF-β1 was prevented in miR-294- and miR-133-overexpressing HK2 cells.ConclusionsThe ability of MSC-EVs to inhibit renal fibrosis decreased with age. miR-294/miR-133 in MSC-EVs and sera had an important effect on renal fibrosis in old rats and on EMT in HK2 cells. Furthermore, miR-294/miR-133 overexpression prevented SMAD2/3 and ERK1/2 phosphorylation in HK2 cells during TGF-β1-mediated EMT. These findings show that miR-294/miR-133 may be therapeutic in renal fibrosis and related renal dysfunction in elderly individuals.

Project description:Human amniotic membrane and amniotic membrane-derived mesenchymal stromal cells (hAMSCs) have produced promising results in regenerative medicine, especially for the treatment of inflammatory-based diseases and for different injuries including those in the orthopedic field such as tendon disorders. hAMSCs have been proposed to exert their anti-inflammatory and healing potential via secreted factors, both free and conveyed within extracellular vesicles (EVs). In particular, EV miRNAs are considered privileged players due to their impact on target cells and tissues, and their future use as therapeutic molecules is being intensely investigated. In this view, EV-miRNA quantification in either research or future clinical products has emerged as a crucial paradigm, although, to date, largely unsolved due to lack of reliable reference genes (RGs). In this study, a panel of thirteen putative miRNA RGs (let-7a-5p, miR-16-5p, miR-22-5p, miR-23a-3p, miR-26a-5p, miR-29a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, miR-660-5p and U6 snRNA) that were identified in different EV types was assessed in hAMSC-EVs. A validated experimental pipeline was followed, sifting the output of four largely accepted algorithms for RG prediction (geNorm, NormFinder, BestKeeper and ΔCt method). Out of nine RGs constitutively expressed across all EV isolates, miR-101-3p and miR-22-5p resulted in the most stable RGs, whereas miR-423-5p and U6 snRNA performed poorly. miR-22-5p was also previously reported to be a reliable RG in adipose-derived MSC-EVs, suggesting its suitability across samples isolated from different MSC types. Further, to shed light on the impact of incorrect RG choice, the level of five tendon-related miRNAs (miR-29a-3p, miR-135a-5p, miR-146a-5p, miR-337-3p, let-7d-5p) was compared among hAMSC-EVs isolates. The use of miR-423-5p and U6 snRNA did not allow a correct quantification of miRNA incorporation in EVs, leading to less accurate fingerprinting and, if used for potency prediction, misleading indication of the most appropriate clinical batch. These results emphasize the crucial importance of RG choice for EV-miRNAs in hAMSCs studies and contribute to the identification of reliable RGs such as miR-101-3p and miR-22-5p to be validated in other MSC-EVs related fields.

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Joint Tissue Protective and Immune-Modulating miRNA Landscape of Mesenchymal Stromal Cell-Derived Extracellular Vesicles under Different Osteoarthritis-Mimicking Conditions

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