hBMSC-Derived Extracellular Vesicles Attenuate IL-1β-Induced Catabolic Effects on OA-Chondrocytes by Regulating Pro-inflammatory Signaling Pathways

Abstract

Background: Human bone marrow-derived mesenchymal stromal cells (hBMSCs) provide a promising therapeutic approach in the cell-based therapy of osteoarthritis (OA). However, several disadvantages evolved recently, including immune responses of the host and regulatory hurdles, making it necessary to search for alternative treatment options. Extracellular vesicles (EVs) are released by multiple cell types and tissues into the extracellular microenvironment, acting as message carriers during intercellular communication. Here, we investigate putative protective effects of hBMSC-derived EVs as a cell-free approach, on IL-1β-stimulated chondrocytes obtained from OA-patients.Methods: EVs were harvested from the cell culture supernatant of hBMSCs by a sequential ultracentrifugation process. Western blot, scanning electron microscopy (SEM), and nanoparticle tracking analysis (NTA) were performed to characterize the purified particles as EVs. Intracellular incorporation of EVs, derived from PHK26-labeled hBMSCs, was tested by adding the labeled EVs to human OA chondrocytes (OA-CH), followed by fluorescence microscopy. Chondrocytes were pre-stimulated with IL-1β for 24 h, followed by EVs treatment for 24 h. Subsequently, proliferation, apoptosis, and migration (wound healing) were analyzed via BrdU assay, caspase 3/7 assay, and scratch assay, respectively. With qRT-PCR, the relative expression level of anabolic and catabolic genes was determined. Furthermore, immunofluorescence microscopy and western blot were performed to evaluate the protein expression and phosphorylation levels of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB as components of pro-inflammatory signaling pathways in OA-CH.Results: EVs from hBMSCs (hBMSC-EVs) promote proliferation and reduce apoptosis of OA-CH and IL-1β-stimulated OA-CH. Moreover, hBMSC-EVs attenuate IL-1β-induced reduction of chondrocyte migration. Furthermore, hBMSC-EVs increase gene expression of PRG4, BCL2, and ACAN (aggrecan) and decrease gene expression of MMP13, ALPL, and IL1ß in OA-CH. Notably, COL2A1, SOX9, BCL2, ACAN, and COMP gene expression levels were significantly increased in IL-1β+ EV groups compared with those IL-1β groups without EVs, whereas the gene expression levels of COLX, IL1B, MMP13, and ALPL were significantly decreased in IL-1β+ EV groups compared to IL-1β groups without EVs. In addition, the phosphorylation status of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling molecules, induced by IL-1β, is prevented by hBMSC- EVs.Conclusion: EVs derived from hBMSCs alleviated IL-1β-induced catabolic effects on OA-CH via promoting proliferation and migration and reducing apoptosis, probably via downregulation of IL-1ß-activated pro-inflammatory Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling pathways. EVs released from BMSCs may be considered as promising cell-free intervention strategy in cartilage regenerative medicine, avoiding several adverse effects of cell-based regenerative approaches.

The Role of Extracellular Vesicles in the Pathogenesis, Diagnosis, and Treatment of Osteoarthritis

“…A large number of studies have shown that BMMSC-EVs can inhibit the adverse effects of inflammatory mediators on cartilage homeostasis [ 25 , 26 ], which may be achieved by downregulating the pro-inflammatory Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling pathways [ 27 , 28 ]. Chen et al found that the expression of E74-like factor 3 (ELF3) increased and miR-136-5p decreased in clinical samples of traumatic OA cartilage tissue.…”

Extracellular vesicles derived from mesenchymal stromal cells mediate endogenous cell growth and migration via the CXCL5 and CXCL6/CXCR2 axes and repair menisci

“…EV transplantation may orchestrate cellular processes, such as migration, cell growth, and matrix synthesis, which are associated with tissue regeneration [ 39 , 42 ]. Previous studies have shown that EVs can increase the cell growth and migration of chondrocytes and regenerate cartilage in the proteomic avascular joint resembling the meniscus [ 21 , 43 ]. In the current study, EVs promoted the migration and cell growth of synovial MSCs and chondrocytes and meniscus regeneration.…”

“…Studies have investigated the mechanism of EV-induced tissue regeneration. Possible modes of action include enhanced cell viability and growth [ 20 ], reduced apoptosis [ 21 ], and modulation of inflammatory responses [ 22 ]. However, these mechanisms of action have not been fully elucidated [ 23 ] and therefore need to be clarified in order to optimize the clinical use of EVs.…”

Small Noncoding RNAs in Knee Osteoarthritis: The Role of MicroRNAs and tRNA-Derived Fragments

“…Multiple in vitro and in vivo studies have reported miRNA involvement in OA onset and progression, by targeting cartilage-associated genes [ 64 ]. It is now known that miRNAs regulate the expression of genes involved in pathways altered in OA chondrocytes, such as apoptosis [ 65 ], expression levels of MMPs and ADAMTS [ 66 ] and chondrocyte signaling [ 67 ]. To date, it has been reported that about 80 miRNAs are involved in the pathology of OA and many excellent reviews have already presented miRNA function in OA, e.g., [ 68 , 69 , 70 ].…”